A method of constructing biologically active compounds which mimic the biological activity of the biologically active protein or which block the activity of the biologically active protein is disclosed. A method of identifying specific and discrete portions of pathogen antigens which either serve as epitopes for neutralizing antibodies or which are involved in pathogen binding to host cell receptors is disclosed. A method of constructing biologically active compounds which compete with cellular receptors for binding to either biologically active proteins or pathogen antigens is disclosed.

Protein binding or protein-protein interactions can be broadly defined as the discrete interaction of the surface of one protein with the surface of another protein. Such discrete interaction arises when residues of one protein are proximally located to residues of another protein and attractive forces between the residues such as vander Waals forces, ionic bonds and hydrogen bonds exist. Specific protein-protein interactions which occur in higher living organisms include but are not limited to those in which involve: a receptor-binding protein binding to a receptor; a pathogen antigen binding to a host cell receptor; protein interactions at cellular attachment sites; and, adhesion proteins interactions.
Examples of receptor-binding proteins, hereinafter also referred to as ligands, include cytokines, hormones and growth factors. These proteins bind to receptors on cells and cause changes in cellular activity or function. For example, cytokines are a variety of proteins which are cellular messengers, each cytokine having a specific effect upon a cell. Likewise, hormones and growth factors are also messengers which affect the function and activity of cells.
Pathogens are infectious organisms, such as bacteria, fungi, parasites, and viruses and, additionally, neoplasms, all of which express specific antigens. Such typically, there are specific sites on antigens, hereinafter referred to as binding epitopes or epitopes, which bind to a complementary portion of a cellular protein called a receptor site.
A great deal of effort has been expended in search of compounds which specifically either simulate, that is mimic, or block protein-protein interactions in cells.
With respect to cytokines, hormones and growth factors, a great deal of effort has been made to purify the natural proteins from natural sources or to synthetically produce them by chemical means or using recombinant DNA technology. While some success has been achieved, these molecules are quite large, difficult to handle and expensive to obtain. A great deal of effort is also directed at discovering synthetic ligands which either mimic the activity of natural proteins or which block the activity of natural proteins. Blocking natural protein activity can be achieved by either competing for the receptor with an inactive ligand (antagonist) or by having an agent bind to the natural protein and thereby prevent it from binding to the receptor.
There is a need for synthetic peptides and/proteins which mimic the activity of the natural biologically active proteins which interact with receptors. Such mimicking molecules would be useful as agents to affect the cells in the same way as the natural protein. Likewise, the discovery of antagonists, that is, molecules which block the receptor without having an effect on cellular function or activity would be useful. Furthermore, the discovery of agents which specifically interact with biologically active proteins and thereby render them unable to bind to receptors is also desirable. Molecules that prevent binding by a natural biologically active protein to its receptor in cases where the natural protein is believed to an agent associated with a disease condition or disorder are useful as drugs for preventing or treating such disease conditions or disorders.
A great deal of knowledge has been developed in the field of immunology, including at the molecular level. Advances in molecular biology have indicated that immunoglobulins, major histocompatibility complex antigens and T-cell receptors are all members of a family of molecules referred to as the immunoglobulin superfamily. During evolution, it is likely that a single, useful gene duplicated, and its copies diverged to create related molecules with distinct functions. Accordingly, immunoglobulins, which are agents of humoral immunity; T-cell receptors, which are associated with humoral as well as cellular immunity; and major histocompatibility complex molecules, involved in antigen presentation and the discrimination between self and nonself, all share homologies inherited from their common ancestor and exhibit related biological functions.
Of the members of the superfamily, the structure and function of immunoglobulins is best understood. Immunoglobulin molecules consist of a constant region and a variable region. The constant region is associated with cellular effector functions whereas the variable region participates in antigen recognition and binding.
Immunoglobulins of the most common class, IgG, consist of two heavy chains and two light chains linked together by noncovalent associations and also by covalent disulfide bonds. Each of the chains possesses a constant as well as a variable region. In the immunoglobulin molecule, the variable region is subdivided into framework regions, which are similar in structure among immunoglobulins, and hypervariable, complementarity determining regions (CDRs) which participate directly in antigen binding in the immunoglobulin active site.
X-ray crystallographic studies of purified immunoglobulin molecules have indicated that the active site is a crevice formed by the heavy and light chain variable regions, and that the dimensions of the active sites vary among immunoglobulin molecules consequent to amino acid sequence variations (Hood et al., 1978, in "Immunology," The Benjamin/Cummings publishing Co., Inc., Menlo Park, p. 208). Amino acid sequence, crystallographic structure, and specially designed hapten probes have been used in conjunction with computer analysis to elucidate the relationship between an immunoglobulin and the antigen which it recognizes.
Pathogens generally express antigens which are recognized by host immune systems as foreign and become the target of an immunological response to eliminate the infectious pathogen. Pathogen antigens often bind to cellular receptors on a host's cells as part of the process of infection of the host by the pathogen. In order to immunize the host and reduce the effectiveness of the pathogen to mount a challenge to the host, a number of vaccination strategies have been devised.
Several strategies have been employed to develop safe, effective vaccines against viral and bacterial pathogens. At present most vaccines in use consist of live attenuated pathogens, killed pathogens, components of a pathogen, or modified toxins (toxoids). See Institute of Medicine, "Vaccine Supply and Innovation", Washington, D.C.: National Academy Press (1985). While these preparations have been successfully used for many infectious diseases, many pathogens exist where these approaches have not worked or have not been applicable. Certain pathogens are potentially too dangerous to contemplate the use of attenuated or even inactivated preparations. The risk of developing cancer from immunization with certain retroviruses, or of developing acquired immunodeficiency syndrome (AIDS) from immunization with human immunodeficiency virus (HIV) underscores the drawbacks associated with the use of whole virus preparations for vaccination. In addition many pathogens display a marked antigenic heterogeneity that makes effective vaccination difficult. These considerations have led us to seek alternative method for effective immunization.
The idiotype network theory of N. K. Jerne, Ann. Immunol. (Paris) 125: 337-389, (1974), implies that an anti-idiotypic antibody raised against a neutralizing antibody specific for a pathogen would mimic that pathogen immunologically. Immunization with the anti-idiotype should result in the development of a significant anti-pathogen response with the elicitation of neutralizing antibodies and cell-mediated immunity. In recent years there have been several examples where this strategy has been effective, including reovirus type 3. See Sharpe, A. H., et al., J. Exp. Med. 160: 195-205 (1984); Kauffman, R. S., et al., J. Immunol., 131: 2539-2541, (1983); and Gaulton, G. N., et al., J. Immunol. 137: 2930-2936. With respect to Sendai virus, see Ertl, H. C. and Finberg, R. W., Proc. Natl. Acad. Sci. USA 81: 2850-2854 (1984). For report relating to rabies see Reagen, K. J. et al., J. Virol. 48: 660-666 (1983). This approach has been discussed in connection with polio virus in Uydeltaag, F.G.C.M. and Osterhaus, A.D.M.E., J. Immunol. 134: 1225-1229 (1985).
One of the key aspects of this approach is that a portion of the anti-idiotype mimics a portion of the pathogen antigen and induces a neutralizing response. Thus a potent anti-idiotype vaccine would seem to be an ideal immunogen in cases where intact pathogen could not be used or where irrelevant non-neutralizing epitopes dominate the immune response. However, the practical application of anti-idiotypes as vaccine has been limited by the difficulties in making human monoclonal antibodies and in the danger of producing serum sickness by using xenogeneic antibodies.
Another method currently under intensive investigation is the use of synthetic peptides corresponding to segments of the proteins from pathogenic microorganisms against which an immune response is directed. This approach has been successful in several instances including feline leukemia virus (Elder, J. H. et al., J. Virol. 61: 8-15, 1987), hepatitis B (Gerin, J. L., et al., Proc. Natl. Acad. Sci. USA, 80: 2365-2369 1983), Plasmodium falciparum (Cheung, A., et al., Proc. Natl. Acad. Sci. USA 83: 8328-8332, 1986), cholera toxin (Jacob, C. O., et al., Eur. J. Immunol. 16: 1057-1062, 1986) and others. When these peptides are capable of eliciting a neutralizing immune response they appear to be ideal immunogens. They elicit a specific response and typically do not lead to deleterious effects on the host. However, it can be difficult to predict which peptide fragments will be immunogenic and lead to the development of a neutralizing response.
It would be desirable to develop immunogens that elicit a response to specific neutralizing epitopes without causing responses to extraneous epitopes that could "dilute" the specific response or lead to harmful immune complex formation.
The present invention relates to a method of identifying specific linear and constrained discrete portions of a biologically active proteins involved in protein-protein interactions. By identifying such specific and discrete portions, biologically active peptides can be constructed which mimic the biological activity of the biologically active protein or which block the activity of the biologically active protein. Thus, biologically active peptides can be constructed which act as ligands that act on mammalian cells by binding to the receptor sites of those cells to alter or affect their function or behavior, or to prevent the binding of the natural biologically active protein to the cellular receptor, thereby preventing the biologically active protein from affecting the cell.
The present invention relates to a method of identifying specific linear and constrained discrete portions of pathogen antigens which either serve as epitopes for neutralizing antibodies or which are involved in pathogen binding to host cell receptors. By identifying discrete portions of pathogen antigens which are neutralizing epitopes, biologically active peptides can be constructed which are useful as components of vaccines against the pathogen. An effective neutralizing immune response will be elicited in a vaccinated individual. By identifying discrete portions of pathogen antigens which are involved in pathogen binding to host cell receptors, biologically active peptides can be constructed which are useful as agents which block pathogen attachment to cellular receptors. Additionally, by identifying discrete portions of pathogen antigens which are involved in pathogen binding to host cell receptors, biologically active peptides can be constructed which mimic pathogen antigens and act on mammalian cells by binding to the receptor sites of those cells to alter or affect their function or behavior, or which prevent or alter the effect which pathogen antigens would otherwise have upon those cells.
The present invention relates to the field of biologically active peptides which have some shared and/or similar amino acid sequences to the amino acid sequences of cellular receptor sites and thereby compete with such cellular receptors for binding to either biologically active proteins or pathogen antigens. In addition, the invention relates to the field of biologically active peptides which have some shared and/or similar amino acid sequences to the amino acid sequences of the ligand surface that attaches to a cellular receptor site. The ligand mimetic peptide can be used as a stimulant or inhibitor of that receptor. Where the biologically active peptide competes in pathogen/receptor binding, the biologically active peptides are useful to prevent pathogen attachment and thereby prevent infection. Where the biologically active peptide competes in biologically active protein/receptor binding, the biologically active peptides are useful to prevent ligand/receptor binding and thereby prevent the effect on cellular function or behavior normally associated with the biologically active protein/receptor binding.

Biologically active compounds and methods of constructing and using the same

The Trustees of the University of Pennsylvania


A cosmetic applicator for applying a composition, the cosmetic applicator comprising a rod having a central longitudinal axis and a distal end of the rod is connected to an applicator head. The applicator head comprises a molded core and a plurality of tines projecting outwardly from the core. The core is elongated along a central longitudinal axis which does not coincide or intersect with the central longitudinal axis of the rod. The core further includes at least one longitudinal spline running along the central longitudinal axis of the core for entire length of the core, the at least one longitudinal spline is free of tines and wherein an end of the spline of the core contacts an outer edge of a transition portion of the applicator head.

Field of the Invention
Embodiments of the present disclosure relate to a cosmetic applicator for applying a cosmetic, makeup, or personal care composition e.g. mascara to eyelashes or eyebrows. More particularly, the disclosure, relates to an applicator including a molded applicator head with a core and a plurality of application elements that are carried by the core.
Description of the Related Art
An applicator such as a mascara applicator must perform several functions. First, the applicator must accumulate mascara within the bottle, and carry the mascara through the wiper, which removes the excess mascara from its bristles. The mascara applicator must then apply the mascara to the eyelashes. Preferably, the mascara applicator should be capable of one or more additional functions, including, curling or lifting the eyelashes, and/or combing and separating the eyelashes, as well as spreading the mascara relatively evenly thereon. Desirably the mascara applicator must be capable of more precise applications such as application of mascara to the smaller, bottom lashes, the corners of the lashes, and general touch-up. In all these functions, the mascara applicator must be comfortable for the consumer to use.
Conventional mascara applicators generally include a brush having a twisted wire core and a bristle portion. The bristle portion generally includes a number of uniformly sized bristles spaced evenly throughout its length. Although these applicators are generally capable of depositing mascara onto the eyelashes, they do not facilitate application of the composition to all eyelashes in an accurate or detailed manner. Therefore, a user must carry a variety of applicators or must manipulate the brush in an unnatural direction to cover the eyelashes. Due to the nature of production technique, the design options for forming and arrangement of the bristles have been limited.
In case of molded mascara applicators that are produced by injection molding, a very extensive variety of design options exists.
U.S. Pat. No. 8,042,555 discloses an applicator which is a single-piece molded unit formed of a single material and includes a slim, tapered head portion, having an arcuate configuration. The head portion comprises a support and at least two rows of projecting tines integrally molded therewith. The tines extend transversely away from one side of the support relative to the longitudinal axis of the head, the opposite side of the support being smooth and free from tines.
U.S. Pat. No. 8,448,650 discloses an applicator which includes an applicator element comprising a cylindrical molded plastic body and three sections—a large section, a medium section, and a small section. Each section comprises an array of bristles arranged radially in a generally ovoid or sphere-like pattern, with shorter bristles toward the end of each section and longer bristles toward the middle each section.
There is a need to further improve the applicators for applying a composition, in particular mascara, to the eyelashes or eyebrows, in order to improve their performance, in particular to give the applicator greater lengthening, curling and combing power, and to do so while enabling a satisfactory quantity of composition to be loaded onto the applicator, and requiring hand movements in use that are easy to perform.


Dielectric such as polyether ether ketone may be used in forming radio-frequency flexible printed circuits. Filler may be incorporated into the dielectric to adjust the coefficient of thermal expansion of the flexible printed circuit. One or more layers of the flexible printed circuit may be unfilled and one or more layers of the flexible printed circuit may be filled. Antennas may be formed from metal traces on the flexible printed circuit, metal electronic device housing structures, or other conductive structures. A transmission line on the flexible printed circuit may couple radio-frequency transceiver circuitry in an electronic device to an antenna. A flexible printed circuit may have a portion with enhanced bendability in a location that overlaps a bend region. The enhanced bendability region may have less filler than other portions of the flexible printed circuit.

Electronic devices may be provided with wireless communications capabilities. To handle wireless communications, electronic devices include communications circuitry. The communications circuitry may include antennas, transmission lines, and transceiver circuitry operating at radio frequencies. Radio-frequency circuits may be formed on substrates such as flexible printed circuits.
Challenges can arise in forming radio-frequency circuitry on flexible printed circuits. For example, some flexible printed circuit materials such as extruded liquid crystal polymers that exhibit desirable radio-frequency properties such as low dielectric constant and low loss tangent values may be fragile. If care is not taken, the use of these flexible printed circuit materials may make a radio-frequency circuit susceptible to damage, potentially reducing device reliability and lowering manufacturing yields.

Devices with radio-frequency printed circuits


A controller of an internal combustion engine includes: a purge controller, operable to control a purge amount of the vaporized fuel purged to an air intake system; an exhaust air-fuel ratio detector; an air-fuel ratio feedback controller; a period determiner, operable to determine whether it is an estimation enabling period; and an alcohol concentration estimator, operable to estimate the alcohol concentration. When the purge of the vaporized fuel is cut during the estimation enabling period, the purge controller: performs control so that the purge is cut with a large degree of tailing, in an operation region where a load of the internal combustion engine is high and a rotation speed is high; and performs control so that the purge is cut with a smaller degree of tailing than the large degree of tailing, in an operation region where the load of the internal combustion engine is low and the rotation speed is low.

The present invention relates to a controller of an internal combustion engine.
There is an FFV (Flexible Fuel Vehicle) as a vehicle that can run on blended fuels of various compositions of gasoline and alcohol. With an FFV, an alcohol concentration in a blended fuel is detected by an alcohol concentration sensor, and operation parameters, such as a fuel injection amount of an engine, are controlled according to the detected alcohol concentration.
There is a related-art fuel property estimating device, with which, from an aspect of cost reduction, etc., an alcohol concentration sensor is not disposed, and the alcohol concentration is estimated from a detection value of an O2 sensor or other already-present sensor. For example, there is a related-art fuel property estimating device that estimates the alcohol concentration based on a feedback correction amount, determined from an exhaust air-fuel ratio detected by an O2 sensor, etc., only in a predetermined time in which predetermined enabling conditions are met (the conditions that fuel supplying has been performed and a cumulative fuel consumption amount after fuel supplying is no more than a fixed amount) (see JP-A-2004-251135 (Claim 1, FIG. 1, etc.)). This fuel property estimating device includes a plurality of concentration estimation enabling units, respectively corresponding to a plurality of disturbance factors influencing the exhaust air-fuel ratio, and is configured so that when a disturbance factor occurs, an enabling condition is not met at the corresponding concentration estimation enabling unit, corresponding to the disturbance factor that occurred, and estimation of concentration of a single component in a fuel is disabled. With this device, because the concentration estimation can be performed after the disturbance factor is eliminated, an estimated value can be improved in precision.
A purged fuel correction amount shall now be described. With a fuel controlling system of an engine, a purged fuel correction amount is set to compensate for an influence of purged vaporized fuel. A deviation of a feedback correction amount from a reference value during purging is deemed to be a contribution due to vaporized fuel and the purged fuel correction amount is determined based on this deviation. The purged fuel correction amount is reflected in feedback control only during purging. When the purge is cut, the purged fuel correction amount is decreased progressively in accordance with a degree of decrease in purging. In this case, if there is no change in fuel properties, because the feedback correction amount is a value (close to the reference value) from which the influence of the purged vaporized fuel is eliminated, the air-fuel ratio can be controlled with stability both during the vaporized fuel purge (that is, when the feedback correction amount and the purged fuel correction amount are used) and during the purge cut (that is, when just the feedback correction amount is used).
With an engine including a canister that stores vaporized fuel, the vaporized fuel is purged via a purge passage from the canister to an air intake system. The purged vaporized fuel contributes to combustion in a combustion chamber as a portion of the fuel and becomes a disturbance factor with respect to the correction amount of feedback control based on the exhaust air-fuel ratio. Thus, in a case where the alcohol concentration is estimated based on the feedback correction amount determined from the exhaust air-fuel ratio, even in a concentration estimation enabling period, in which concentration estimation enabling conditions are met, estimation of the alcohol concentration is disabled during the vaporized fuel purge. There is thus present in the concentration estimation enabling period, a concentration estimation executing period, in which estimation of the alcohol concentration is performed only when purging is cut and feedback control is operating.
On the other hand, during a period in which the vaporized fuel purge is performed, the estimation of the alcohol concentration is disabled and renewal of the estimated value is not performed because the period falls outside the concentration estimation executing period, the alcohol concentration may be changing greatly in actuality. In this case, when the vaporized fuel being purged is cut and the concentration estimation executing period is started, because renewal of the estimated value has not been performed at a point immediately after starting, feedback control is performed at a control amount (fuel injection amount) that is in accordance with the alcohol concentration estimated in a previous concentration estimation executing period. In this case, because the change of the alcohol concentration is reflected in the purged fuel correction amount but not reflected in the feedback correction amount, the alcohol concentration change amount (difference between the actual alcohol concentration and the alcohol concentration estimated in the previous concentration estimation executing period) is not reflected in the fuel injection amount immediately after the starting of the concentration estimation executing period accompanying the purge cut and an air-fuel ratio error occurs.
In particular, when the vaporized fuel purge is cut instantaneously, the air-fuel ratio changes rapidly in accompaniment. Although the changed air-fuel ratio must be converged quickly to a desired air-fuel ratio, the fuel injection amount for this can only be feedback corrected by a gain that is normally set for feedback control. It thus takes time to achieve convergence to the desired air-fuel ratio.

Controller of internal combustion engine

Mitsubishi Jidosha Kogyo Kabushiki Kaisha


A propeller system is formed on a shaft by at least one pair of axially spaced propeller blades each having a first and a second blade section extending at a selected acute (or obtuse) angle to the axis of the shaft, the first sections and second sections respectively of the pair of propeller blades are substantially parallel to each other and the tips of the first sections of each of the blades are connected to the tips of the second section of each of the blades respectively to form a box shape. The shaft is connected to the propeller system in a manner so that the axis of the shaft extends substantially diagonally of the box shape whereby the parallel sections of the blades move in opposite directions relative to the fluid in which they are contained when the shaft is rotated to facilitate improvement of the efficiency of the propeller system. The shaft preferably is mounted oriented to the direction of relative fluid flow between the propeller blades and the fluid so that each blade section extends substantially perpendicular to the direction of relative flow over a significant portion of its rotation and each blade section will be substantially parallel to the direction of fluid flow relative to the propeller blade during a further portion of the rotation of the propeller blades.

Many different propeller systems are used both for extracting energy from flowing fluid stream, or for example for driving a boat or airplane or the like through fluid.
Windmills may be classified into three basic classes; the horizontal axis lift type which is simply a propeller oriented with its axis of rotation substantially parallel to the wind direction and normally with its blades extending substantially perpendicular to the axis of rotation. These systems are conventionally used and are similar to propellers used to propel an airplane or as the screw for driving the ship or boat through the water.
It is also known to orient the axis of rotation of the windmill or the like substantially perpendicular to the direction of relative fluid flow. Such devices incorporating a drag type impeller are known as Savonious mills and those having a lift type impeller are known as Darrieus windmills.
Darrieus type windmills are shown in U.S. Pat. No. 1,835,018 issued Dec. 8, 1931 to Darrieus and U.S. Pat. No. 4,112,311 issued Sept. 5, 1978 to Theyse. U.S. Pat. No. 1,100,332 issued Jun. 16, 1914 to Smith appears to be a combination of the Savonious and Darrieus type windmills.
U.S. Pat. No. 4,390,148 issued Jun. 28, 1983 to Cudmore, discloses a rotary wind device wherein the axis of rotation of the rotor is oriented to be substantially parallel to the direction of intended movement but with a slight upward tilt to provide lift.
Some years ago Dr. Gunther Wagner built a prototype wind mill using two blade sections oriented at 90.degree. and mounted on a shaft the axis of which was at an angle of 45.degree. to the blade sections and oriented so that in one position one of the blade sections is horizontal while the other is vertical.


A method for the generation of Ca++ independent antibodies against blood coagulation factors is described wherein an antibody selection strategy based upon small peptides comprising target sequences for limited proteolysis is employed. These antibodies which distinguish between intact and cleaved species of haemostatic protein provide novel tools for the isolation of intact haemostatic proteins.

This invention relates to the preparation of therapeutic compositions consisting of purified blood coagulation factors for the treatment of haemostatic disorders. Said compositions are obtained by affinity chromatography employing antibodies, more in particular monoclonal antibodies, that distinguish between intact and cleaved molecular species. Methods are disclosed to obtain such antibodies, which allow the isolation of vitamin K-dependent blood coagulation proteins, including Factor IX, Factor VII, Protein C or Protein S, as intact polypeptides, devoid of cleavage products representing activated or degraded species.
Inherited or acquired deficiencies of proteins of the blood coagulation system provide a major cause for the occurrence of haemostatic disorders. Even the lack or shortage of one single component of this system may be sufficient to disturb the delicate balance between procoagulant and anticoagulant pathways in a manner resulting in major clinical signs of bleeding or thrombosis. One of the most common bleeding disorders is Haemophilia A, which is due to deficiency or dysfunction of the coagulation Factor VIII. Less frequently occurring, but equally severe bleeding disorders include deficiencies of the haemostatic proteins Factor IX (Haemophilia B), Factor VII, or Factor X. On the other hand, thrombosis may occur as the result of even partial (heterozygous or acquired) deficiency of Protein C or Protein S, which are major components of a system that acts as an antagonist of the coagulation pathway (for reviews on haemostatic disorders see A. L. Bloom and D. P. Thomas (Eds.), Haemostasis and Thrombosis, 2nd edition, Churchill-Livingstone, Edinburgh, 1987, pp 393-436 and 452-464). Replacement therapy is considered as a powerful and effective means to restore the haemostatic balance in vivo. For instance, concentrates containing Factor IX have proven highly valuable blood products which are life-saving when used to control bleeding in patients suffering from Factor IX deficiency.
Commercially available Factor IX concentrates (so-called prothrombin complex concentrates) usually are prepared with ion exchange resins to separate Factor IX from the other plasma proteins. This technique however, yields Factor IX preparations that also contain a number of other, closely related haemostatic proteins. These include Factor VII, Factor X, Factor II, Protein C and Protein S, which all belong to the class of the vitamin K-dependent proteins. The term xe2x80x9cvitamin K-dependentxe2x80x9d is referring to the fact that these proteins contain glutamic acid residues that are carboxylated during biosynthesis in a vitamin K-dependent process. Carboxylation provides these proteins with unique Ca2+-binding sites that are obligatory for the biological activity of these proteins within the Ca2+-dependent haemostatic process. Due to these and other structural similarities (see B. Furie and B. C. Furie, Cell vol 53, 1988, pp 505-518), the vitamin K-dependent proteins are readily co-purified. Thus, most Factor IX concentrates are also containing other haemostatic proteins such as Factor VII, Protein C and Protein S, and consequently the same concentrates have been used for the treatment of deficiencies of those proteins as well. However, treatment of bleeding disorders with compositions containing anticoagulant proteins such as Protein C and Protein S is in fact highly undesired, as is treatment of thromboembolic disease with compositions containing Factor IX, Factor VII or other procoagulant components as major contaminants. Therefore, the ideal therapeutic composition to correct a deficiency of one specific haemostatic protein should consist of solely that single component in an intact conformation and nothing else except solvent, and sometimes an inert carrier. As a consequence, the purification strategies needed to achieve the desired degree of purity have become increasingly complex.
Along with the introduction of advanced, more complex purification protocols a novel problem of undesired proteolysis of the target species was encountered. Limited proteolysis is a key mechanism in the regulation of a number of biological systems (see H. Neurath and K. A. Walsh, Proc. Natl. Acad. Sci. USA vol 73, 1976, pp 3825-3832). Typical examples of such biological systems include the complement system, the fibrinolytic system, and the blood coagulation system. These biological cascade systems involve the sequential conversion of intact, inactive precursor proteins into active enzymes or cofactors by proteolysis of one or more specific peptide bonds. On the other hand, feedback mechanisms exist to maintain these processes under local control and lead to proteolytic inactivation of the target proteins. Accordingly, the components of the coagulation cascade are present in blood plasma in a precursor form lacking biological activity. With regard to replacement therapy, the presence of coagulation proteins that are no longer intact is troublesome, since after having been subject to limited proteolysis, such cleaved species may bypass the natural, local control of haemostasis in vivo. Although natural mechanisms effectively control proteolysis under physiological conditions, these can no longer be maintained when haemostatic proteins are isolated from their natural source. As such protease-sensitive sequences are exposed within the tertiairy structure of these proteins, they provide easily accessible targets for proteolysis in a non-physiological environment lacking natural control mechanisms. Therefore, it is virtually impossible to completely prevent partial proteolysis during purification. Uncontrolled proteolysis of these vulnerable proteins is not limited to purification from a natural source as human plasma or fractions thereof, but may equally occur when the same proteins are obtained by recombinant DNA technology from transformed cell lines in vitro, or from biological fluids, including milk, of transgenic animals in vivo.
The presence of cleaved species in therapeutic products is clearly not desired, because the presence of activated proteins may trigger thrombogenic responses of the haemostatic system, whereas the presence of inactivated proteins leads to products with suboptimal biological activity that may competitively inhibit the reactions to be corrected. Prothrombin complex concentrates contain activated species of virtually all vitamin K-dependent coagulation factors, and this has been established as a causative agent for the occurrence of thromboembolic complications since the 1970s is (G. C. White et al., Blood vol 49, 1977, pp 159-170; J. M. Lusher, Seminars in Hematology vol 28, suppl 6, 1991, pp 3-5). Theoretically, in particular those species that participate in the initiation phase of the coagulation system, and thereby are the most amplified in the cascade mechanism, are to be considered as the most potent in disturbing the physiological haemostatic balance. Indeed, in vivo studies employing purified activated coagulation factors have identified activated Factor IX (S. Gitel et al., Proc. Natl. Acad. Sci. USA vol 74, 1977, pp 3028-3032) and activated Factor VII (K. Mertens et al., Thromb. Haemostasis vol 64, 1990, pp 138-144) as thrombogenic even in extremely low dosage. This may raise particular concern for activated forms that are relatively resistent to inhibition in vivo. Most activated vitamin K-dependent coagulation factors are subject to almost instantaneous inhibition by the abundance of protease inhibitors in blood plasma. However, Factor IXa is only slowly inhibited, whereas Factor VIIa and activated Protein C have in vivo half-lives up to 2 hours (K. Mertens et al., Thromb. Haemostasis vol 64, 1990, pp 138-144; P. C. Comp, Hematology, McGraw-Hill, New York, N.Y., 1990, pp 1290-1303). Thus, upon infusing vitamin K-dependent haemostatic proteins into patients, it should be noted that even traces of activated forms may remain in the patients circulation sufficiently long to bypass physiological control. Therefore, Protein C and Factors VII and IX are among the proteins that should be prevented from being activated, or should be purified employing a strategy that is selective for their intact zymogens. On the other hand, other cleavages can occur that result in the inactivation of said proteins, thus reducing their therapeutic efficacy. For instance, Factor IX can be inactivated by the enzymes thrombin or elastase, and the cleavage product has no longer the potential of being converted into a form having Factor IXa activity (A. Takaki et al., J. Clin. Invest. vol 72, 1983, pp 1706-1715; W. Kisiel et al., Blood vol 66, 1985, pp 1302-1308). Similarly, the anticoagulant Protein S is readily cleaved by thrombin into a product that no longer has anticoagulant activity (for review see M. Hessing, Biochem. J. vol 277, 1991, pp 581-592). It thus appears highly important to avoid the occurrence of cleaved, non-intact species to reduce side-effects and to improve efficacy of therapeutic concentrates of these factors.
To better appreciate the structural differences between the intact zymogen species and their cleaved derivatives, the molecular events associated with limited proteolysis of these proteins are now described in more detail. Table I presents an overview of target sequences for limited proteolysis as they occur in a number of vitamin K-dependent proteins involved in the haemostatic system. These include:
(a) Human Factor VII: The intact zymogen is a single-chain glycoprotein of 406 amino acids, which is converted to Factor VIIa by the cleavage of a single bond between residues 152 and 153 (see Table I). This target sequence can be cleaved by a number of enzymes, including thrombin and Factors IXa, Xa and XIIa, which results in the activated species which consists of two polypeptide chains that are held together by a disulfide bond. This Factor VIIa provides a powerful thrombogenic trigger by activating a number of target substrates, including Factor X and Factor IX (B. Osterud and S. I. Rapaport, Proc. Natl. Acad. Sci. USA vol 74; 1977, pp 5260-5264).
(b) Human Factor IX: The intact zymogen is a single-chain glycoprotein of 415 amino acids, which is converted into Factor IXa by Factor XIa or Factor VIIa in two steps. The first step involves the cleavage between residues 145 and 146, which results in the formation of a two-chain inactive intermediate (called Factor IXxcex1) containing a light chain of residues 1-145 and a heavy chain of residues 146-415. In the second step, the heavy chain of Factor IXxcex1 is further cleaved between residues 180 and 181, resulting in a 35-residue (146-180) activation peptide and an active enzyme (Factor IXaxcex2) with the remaining heavy chain of residues 181-415. It should be noted that whereas the latter cleavage is required to develop the final Factor IXa activity, the prior cleavage of the 145-146 bond seems to be an obligatory step in Factor IX activation by Factor XIa or Factor VIIa (U. Hedner and E. W. Davie, in: R. W. Colman et al. (Eds), Hemostasis and Thrombosis, J. B. Lippincott, Philadelphia, 1987, pp 29-38; M. J. Griffith et al., J. Clin. Invest. vol 75, 1985, pp 4-10). Therefore, Factor IXxcex1 is even more vulnerable than the intact Factor IX zymogen with respect to cleavage at the 180-181 position and the concurrent formation of a thrombogenic Factor IXa species. In addition to this proteolytic activation, also inactivation may occur as the result of cleavage by thrombin or elastase. This proteolytic inactivation involves cleavage between residues 327 and 328, and between residues 338 and 339, resulting in derivatives that can no longer be converted to species with Factor IX procoagulant activity (see Table I).
(c) Human Protein C: The intact zymogen is a two-chain glycoprotein of 419 amino acids, consisting of a light chain of residues 1-155 and a heavy chain of residues 158-419. The zymogen is activated by a single cleavage in the heavy chain, between residues 169 and 170. The activated species is a powerful anticoagulant, which inactivates Factors Va and VIIIa in a manner requiring the presence of a vitamin K-dependent cofactor, called Protein S.
(d) Human Protein S: The intact species is a single-chain glycoprotein of 635 amino acid residues. The N-terminal portion of the protein contains two thrombin-sensitive bonds between residues 49-50 and 70-71. After cleavage of Protein S by thrombin, the N-terminal fragment still is connected to the molecule via a disulfide bond. However, only Protein S that is uncleaved within the thrombin-sensitive region is active as a cofactor for activated Protein C. Therefore, it is preferred to provide intact, uncleaved Protein S in a composition for effective treatment of heriditary or acquired Protein S deficiency.
In conclusion, a special need exists for purification methods that allow the selection of uncleaved, intact species from a source containing the intact proteins as well as proteolytic derivatives generated by cleavage at the target peptide bonds that are summarized in Table I.
The development of monoclonal antibody technology has radically changed the potential for purifying trace proteins from plasma to near homogeneity. Monoclonal antibodies can be prepared and selected in such a manner that they show complete specificity towards an individual plasma protein. When a monoclonal antibody specific for a plasma protein is immobilized on an inert matrix, it will absorb that plasma protein specifically from a mixture containing that plasma protein. After washing the matrix, the plasma protein may be eluted under fairly mild conditions in pure or virtually pure form. This approach has allowed the isolation of pure Factor IX from human blood plasma by immuno-affinity chromatography (A. H. Goodall et al., Protides of the Biological Fluids (Peeters, Ed.) vol 30; Pergamon Press, Oxford, 1982, pp 403-407; K. Mertens et al., Thromb. Haemostasis vol 50; 1983, p 249; K. Mertens, Ph.D. dissertation, State University of Leiden, 1985, pp 83-98). Similar methods of immuno-affinity chromatography have subsequently become available for purification of the other vitamin K-dependent haemostatic proteins.
In some cases, a more restricted specificity has been achieved, in that antibodies may specifically bind to their target vitamin K-dependent protein in the presence of Ca2+-ions. Under these conditions, the target proteins are in a biologically active conformation by virtue of the interaction of Ca2+-ions with the carboxylated glutamic acid residues that are unique to the vitamin K-dependent proteins. When applied in a purification process, such antibodies allow elution of the target protein under mild conditions by the Ca2+-binding agent EDTA, as has been described for Factor IX (H. A. Liebman et al., Proc. Natl. Acad. Sci. USA vol 82, 1985, pp 3879-3883; K. J. Smith, Blood vol 72, 1988, pp 1269-1277) as well as for Protein C (D. J. Stearns et al., J. Biol. Chem. vol 263, 1988, pp 826-832).
However, in spite of their high selectivity, those procedures have not been able to distinguish between the intact and activated or degraded coagulation factors, and as a consequence cleaved products are co-purified with the desired intact, non-activated target product. Moreover, antibodies that are Ca2+-dependent have the disadvantage that Ca2+-ions cannot be added to most source materials, including plasma or fractions thereof, without simultaneously triggering the Ca2+-dependent coagulation system that leads to proteolytic cleavage of the vitamin K-dependent target proteins. This represents a major drawback for the applicability of the final therapeutic product as it has been demonstrated that even small amounts of activated coagulation factors have been implicated as causative agents for disseminated intravascular coagulation and thromboembolism.
The present invention relates to methods for the generation and selection of Ca2+-independent, monospecific antibodies (either polyclonal or monoclonal) specific for defined epitopes covering intact (activation or degradation) cleavage sites of haemostatic proteins such as, for example, coagulation Factor IX and Protein S, for the isolation of these proteins as intact, uncleaved polypeptides. The strategy of this method is based on the notion that disruption of the primary sequence of the target protein leads to a number of changes within the protein, one being the exposition of the cleavage site itself. The present invention demonstrates that antibodies against epitopes covering intact cleavage sites may have different affinity for intact and cleaved forms of the epitope, thus allowing separation when applied in a chromatographic process. In order to be applicable in purification processes from a variety of sources, the antibodies described in the present invention are not only specific for a sequence that provides a target for limited proteolysis within the coagulation factor to be isolated, but are also Ca2+-independent. The method involves the selection of antibodies with appropriate specificity, immobilization thereof on a solid support, and then contacting a source material with the immobilized antibody to bind said protein. After washing and elution with an appropriate solution, the target protein is recovered in a highly purified form, in a non-activated, intact state that is of particular interest for use in a therapeutic composition for the treatment of haemostatic disorders.
The process of the present invention is a major breakthrough in the field of immuno-affinity chromatography in that it achieves unique selectivity for intact, non-cleaved target proteins. This allows for methods to obtain intact haemostatic proteins, which can thereafter be formulated into improved therapeutic blood products. These products provide safer and more effective agents than are available thus far for the treatment of patients encountering critical bleeding or clotting episodes.
Other objects and advantages of the present invention will become evident from the following detailed description of the invention.

Antibodies specific for a haemostatic protein, their use for isolating protein, haemostatic compositions devoid of proteolytic cleavage products of the protein


Apparatus for determining stroke volume, cardiac output and systemic inflammation by fuzzy logic combination of features extracted from a voltage measured over the thorax, electrocardiogram and electroencephalogram comprising a) sensor for measuring electroencephalogram; b) sensor for measuring the electrocardiogram; c) calculating the heart rate variability from the electrocardiogram; d) two electrodes (1, 2) for measuring the voltage generated by a constant current generator in two other electrodes (1, 2); e) calculating the area under the curve of voltage for each heart-beat; f) calculating the derivative of the voltage curve; g) calculating the transfer entropy between EEG and ECG; h) combining at least 3 extracted parameters using an Adaptive Neuro Fuzzy inference system (ANFIS) or any other fuzzy reasoner into a final index of cardiac output; i) combining at least 3 extracted parameters using an Adaptive Neuro Fuzzy Inference System or any other fuzzy reasoner into a final index of systemic inflammation.

The present invention uses injection of a constant current at which the voltage is measured. However, as the current is constant measuring the voltage is proportional to the impedance, hence the concept of bio-impedance will be described in the following section.
Bioimpedance measurement on body parts was originally described in the U.S. Pat. No. 3,340,867, by Kubicek, patented Sep. 12, 1967. This invention describes impedance plethysmography, in particularly useful for determining cardiac output. The claims include position of electrodes being upper and lower part of the thorax. Later, bioimpedance measurement on body parts was described in the U.S. Pat. No. 3,835,840, Impedance plethysmography, apparatus and methods, priority date Sep. 27, 1973. This patent describes a method for using the electrical impedance as a correlate to blood flow in aorta or other arteries.
Sramek filed Jun. 29, 1982, the patent, Noninvasive Continuous Cardiac Outputmonitor, U.S. Pat. No. 4,450,527, where a modification of the original Nyboer equation has been made.
Combination of Parameters.
The device uses ANFIS models to combine the parameters, for the definition of the stroke volume, cardiac output and the inflammation index.
The parameters extracted from the two sensors and the demographic data of the patient are used as input to an Adaptive Neuro Fuzzy Inference System (ANFIS).
Overview of ANFIS
ANFIS is a hybrid between a fuzzy logic system and a neural network, it does not assume any mathematical function governing the relationship between input and output. ANFIS applies a data driven approach where the training data decides the behaviour of the system.
The five layers of ANFIS, shown in FIG. 6, have the following functions:                Each unit in Layer 1 stores three parameters to define a bell-shaped membership function. Each unit is connected to exactly one input unit and computes the membership degree of the input value obtained.        Each rule is represented by one unit in Layer 2. Each unit is connected to those units in the previous layer, which are from the antecedent of the rule. The inputs into a unit are degrees of membership, which are multiplied to determine the degree of fulfillment for the rule represented.        In Layer 3, for each rule there is a unit that computes its relative degree of fulfillment by means of a normalisation equation. Each unit is connected to all the rule units in Layer 2.        The units of Layer 4 are connected to all input units and to exactly one unit in Layer 3. Each unit computes the output of a rule.                    An output unit in Layer 5 computes the final output by summing all the outputs from Layer 4.
Standard learning procedures from neural network theory are applied in ANFIS. Back-propagation is used to learn the antecedent parameters, i.e. the membership functions, and least squares estimation is used to determine the coefficients of the linear combinations in the rules' consequents. A step in the learning procedure has two passes. In the first pass, the forward pass, the input patterns are propagated, and the optimal consequent parameters are estimated by an iterative least mean squares procedure, while the antecedent parameters are fixed for the current cycle through the training set. In the second pass, the backward pass, the patterns are propagated again, and in this pass back-propagation is used to modify the antecedent parameters, while the consequent parameters remain fixed. This procedure is then iterated through the desired number of epochs. If the antecedent parameters initially are chosen appropriately, based on expert knowledge, one epoch is often sufficient as the LMS algorithm determines the optimal consequent parameters in one pass and if the antecedents do not change significantly by use of the gradient descent method, neither will the LMS calculation of the consequents lead to another result. For example in a 2-input, 2-rule system, rule 1 is defined byif x is A and y is B then f1=p1x+q1y+r1
where p, q and r are linear, termed consequent parameters or only consequents. Most common is f of first order as higher order Sugeno fuzzy models introduce great complexity with little obvious merit.
Number of Classes.
The inputs to the ANFIS system are fuzzified into a number of predetermined classes. The number of classes should be larger or equal two. The number of classes can be determined by different methods. In traditional fuzzy logic the classes are defined by an expert. The method can only be applied if it is evident to the expert where the landmarks between two classes can be placed. ANFIS optimizes the position of the landmarks, however the gradient descent method will reach its minimum faster if the initial value of the parameters defining the classes is close to the optimal values. By default, ANFIS initial landmarks are chosen by dividing the interval from minimum to maximum of all data into n equidistant intervals, where n is the number of classes. The number of classes could also be chosen by plotting the data in a histogram and visually deciding for an adequate number of classes, by ranking as done by FIR, through various clustering methods or Markov models. The ANFIS default was chosen for this invention and it showed that more than 3 classes resulted in instabilities during the validation phase, hence either 2 or 3 classes were used.
Number of Inputs.
Both the number of classes and number of inputs add to the complexity of the model i.e. the number of parameters. For example, a system with 4 inputs, each fuzzified into 3 classes consists of 36 antecedent (non-linear) and 405 consequent (linear) parameters, calculated by the following two formulas:antecedents=number of classes×number of inputs×3consequents=number of classesnumber of inputs×(number of inputs+1)
The number of input-output pairs should in general be much larger, (at least a factor 10) than the number of parameters in order to obtain a meaningful solution of the parameters.
Stability Criteria.
Unfortunately there exists no definition of stability criteria for neuro-fuzzy systems. The most useful tool for ensuring stability is the experience obtained by working with a certain neuro-fuzzy system such as ANFIS in the context of a particular data set, and testing with extreme data for example obtained by simulation
Number of Epochs.
ANFIS uses a Root Mean Square Error (RMSE) to validate the training result and from a set of validation data the RMSE validation error can be calculated after each training epoch. One epoch is defined as one update of both the antecedent and the consequent parameters. An increased number of epochs will in general decrease the training error.
Other Terms.
The term "electroencephalography (EEG)" refers hereinafter to the recording of electrical activity along the scalp. EEG measures voltage fluctuations resulting from ionic current flows within the neurons of the brain. In clinical contexts, EEG refers to the recording of the brain's spontaneous electrical activity as recorded from multiple electrodes placed on the scalp. Diagnostic applications generally focus on the spectral content of EEG, that is, the type of neural oscillations that can be observed in EEG signals.
The term "electromyography (EMG)" refers hereinafter to a technique for evaluating and recording the electrical activity produced by skeletal muscles. EMG is performed using an instrument called an electromyograph, to produce a record called an electromyogram. An electromyograph detects the electrical potential generated by muscle cells when these cells are electrically or neurologically activated. The signals can be analyzed to detect medical abnormalities, activation level, or recruitment order or to analyze the biomechanics of human or animal movement.
The term "qCON" refers to an index of hypnotic effect derived from the EEG. The EEG is recorded from frontal electrodes (FIG. 1) (3), amplified (5) and digitized (7). Features are extracted from the EEG (8) from which the qCON is calculated. The qCON is defined as a scale from 0 to 99, where 80-99 indicates awake, 60-80 sedations, 40-60 adequate depth of anaesthesia and 0 to 40 deep anaesthesia. The qCON is further described in the article: Jensen E W, Valencia J F, López A, Anglada T, Ramos Y, Serra R, Jospin M, Pineda P, Gambus P Monitoring hypnotic effect and nociception with two EEG derived indices, qCON and qNOX, during general anaesthesia. Acta Anaesthesiol Scand. 2014 September; 58(8):933-41
The term "electrocardiography (ECG)" refers hereinafter to is a transthoracic (across the thorax or chest) interpretation of the electrical activity of the heart over a period of time, as detected by electrodes attached to the surface of the skin and recorded by a device external to the body. The recording produced by this noninvasive procedure is termed an electrocardiogram. An ECG picks up electrical impulses generated by the depolarization of cardiac tissue and translates into a waveform. The waveform is then used to measure the rate and regularity of heartbeats, as well as the size and position of the chambers, the presence of any damage to the heart, and the effects of drugs or devices used to regulate the heart, such as a pacemaker.
The term "fast Fourier transform (FFT)" refers hereinafter to an algorithm to compute the discrete Fourier transform (DFT) and its inverse. A Fourier transform converts time (or space) to frequency and vice versa; an FFT rapidly computes such transformations. As a result, fast Fourier transforms are widely used for many applications in engineering, science, and mathematics.
The term "RR intervals" refer to the time between successive R-peaks in the ECG. From the RR intervals the following parameters are extracted, as shown in table 1. These are calculated by the device. NNi refers to the ith RR interval.
TABLE 1Time and frequency domain variables.VariableDescriptionHRHeart rate, reciprocate of the mean of all RR intervals(bpm)RMSSDRoot mean square differences between successive RR intervals(ms)   RMSSD  =                    1        NN                                    ∑          i                                                                                (                                          NN                i                            -                              NN                                  i                  -                  1                                                      )                    2                     SDSDStandard deviation of differences between successive RR intervals(ms)pNN50Number of interval differences of successive RR intervals greater (%)than 50 ms divided by the total number of RR intervals., i.e:if (NNi − NNi−1) > 50 ms, count ++; count/n * 100;HFPower in high frequency range (0.15-0.4 Hz)(ms2)HFnHF power in normalized units, HFn = HF/(LF + HF) * 100(n.u)LFPower in low frequency range (0-0.14 Hz)
Detailed Description of the Invention for Monitoring Cardiac Output.
The novelty of this patent is the combination of several parameters extracted from the voltage plethysmographic curve and the heart rate variability. The voltage plethysmographic curve is achieved by applying a constant current of 400 uA between the upper and lowest electrodes on the thorax, see FIG. 1, (1) and (2). The voltage plethysmographic curve is also referred to as the voltage plethysmogram (VP) or the voltage curve. The voltage curves are achieved for each heart beat, consecutive curves have normally a similar morphology. The voltage is measured between the electrodes adjacent to upper and lowest electrodes (inner electrodes). The current will seek the path with the lowest impedance, i.e. the blood filled aorta. Hence the more blood present the impedance will be lower and consequently the voltage as well. The voltage curve and the ECG are recorded from the same electrodes (1) (2), amplified (4), digitized (6). Features are extracted from the voltage curve and the ECG (9), those features are fed into ANFIS models (11) where the output is cardiac output (11) and the final inflammation index (12).
The voltage plethysmography of each heart-beat is a correlate to the stroke volume (SV). The voltage plethysmogram (VP) will show periodic fluctuations from which the heart rate (HR) can be detected. The HR could also be detected from the ECG and the two compared to assure correct performance. From SV and HR the cardiac output (CO) can be computed byCO=HR×SV.
FIG. 2 shows a typical example of the voltage curve. The variable, tc, is the opening time of the aortic valve. The area, A, is an initial correlate of the SV. A is calculated by integrating the voltage plethysmogram over the period tc. The start time of tc is defined as an increase in the VP in the 5-15% range while the ending time of tc is defined as the start of the interval where the VP is stable within changes of 1-5%.
The maximum positive amplitude and the maximum negative amplitude is assessed from the voltage curve. The maximum positive amplitude of the voltage curve (maxHpos) and the (maxHneg) is shown in FIG. 2. Additionally the derivative of the VP is calculated. The maximum of the derivative is termed
  max        (                  d                                                  V                    d                                                  t              )  and used as an input to the ANFIS1 model. From the dV/dt curve the left ventricular ejection time (LVET) can be estimated as time from 20-30% after the dv/dt starts to increase (B, on FIG. 3) until the minimum of dv/dt (X, on FIG. 3) within the period (RR interval).
FIG. 3 shows a schematic example of the derivative of the voltage plethysmogram, indicating the Left Ventricular Ejection Time (LVET). FIG. 4 shows a recording using the graphical user interface (GUI) of the invention showing the VP, while FIG. 5 shows the VP and the ECG using the GUI of the invention.
The complete processing system of the present invention for the CO part consists of 2 ANFIS models.
ANFIS1 (710) takes as input the parameters extracted from the voltage plethysmogram, in a preferred embodiment there are at least 3 inputs, as shown in FIG. 7, however more inputs could be included. As input for ANFIS2 (720) is also used a simple estimate of SV, termed SV cylinder estimate
      S                          V                          cylinder                          estimate    =            kL      2                  max                    (                              d                                                                          V                                d                                                                          t                          )                        t      c
where L is length of the trunk (could be the distance between the lower and upper pair of electrodes) and max(dV/dt) is the maximum of the derivative of the voltage plethysmogram. The constant, k, adjusts the SV to the normal range which is 50 to 150 ml.
The RR interval is an input to ANFIS 2 as well. The RR is the reciprocal of the HR hence CO can be calculated asCO=SV/RR.
The range of CO is 0 to 25 l/min, while the normal physiological range is 4 to 8 l/min. Finally, gender and age are added as well as input to ANFIS2. The output of ANFIS2 is the final estimate of cardiac output (CO). The training of the ANFIS models is carried out with a large amount of data where the CO is known for the patient. The training defines the parameters of the ANFIS model which can then predict CO when the input are presented to the model. The output of the ANFIS model defines the CO.
Background of the Inflammation Index.
The works of Tracey (Tracey, K. Understanding immunity requires more than immunology. Nat. Immunol. 2010; 11, 561-564.) have clearly defined the role of the vagus nerve as the afferent sensor from the point of aggression to the brain, and perhaps more important from a therapeutic point of view, in the efferent pathway of this reflex because its stimulation facilitate the process of the intensity of the inflammation. The vagus nerve, the tenth cranial nerve, carry the fibers of the neural pathways of the parasympathetic system branching out and reaching virtually every organ. Details of the alterations induced by the inflammatory response were published by (Huston J M, Tracey K J. The pulse of inflammation: heart rate variability, the cholinergic anti-inflammatory pathway and implications for therapy. J Intern Med. 2011 January; 269(1):45-53.).
The cholinergic anti-inflammatory pathway descends on the vagus nerve. The vagal stimulation of the heart leads to changes in heart rate variability (HRV). The study or HRV using mathematical techniques such as spectral analysis allowed to identify the frequencies that are associated with increased specificity to changes due to parasympathetic dominance, possibly with an estimate of vagal cholinergic activity by analyzing the rapid frequency component of HRV according to the ESC Task Force, 1996.
Mechanisms of inflammation induce changes in behavior and body constituting inflammatory syndrome associated with illness. Initially and under control, inflammation promotes healing through the molecular and cellular. But when not properly compensated, the inflammation can have serious consequences for the patient. The inflammatory cascade can be very aggressive towards the patient's own tissue leading to sepsis, even death (Tracey K, Nat Immunol, 2010).
In the case of the surgical aggression and neuroinflammation produced, lack of compensation perpetuate inflammatory neuronal involvement leading to cognitive impairment in the medium term as already demonstrated in experimental animal studies. This hypothesis could justify the high percentage of cognitive alterations detected with no apparent relation to other etiologies beyond increased incidence of higher age.
The patent application PCT/IB2013/054615 "METHOD AND SYSTEM FOR ASSESSING LIKELIHOOD OF SEPSIS" discloses a system for detection of sepsis, however the present invention differs by using ANFIS models for combining the parameters. The disclosed system PCT/IB2013/054615 uses a radio frequency input which is not used in the present invention.
The patent application PCT/CA2012/000243 "METHOD AND SYSTEM FOR DETERMINING HRV AND RRV AND USE TO IDENTIFY POTENTIAL CONDITION ONSET" discloses a system using HRV and respiratory rate variability (RRV) for detection of sepsis, though in infant patients, but it does not use an ANFIS model as done in the present invention.
The US patent application no 20050137484 "Method and apparatus for the early diagnosis of subacute, potentially catastrophic illness" is intended to be used on infants and newborns, and as such does not cover the complete age range as the present invention does and it does not use an ANFIS model as is done in the present application.
The US patent application US7941199 B2, disclose a method for the assessment of sepsis, but it does not use Heart Rate Variability nor ANFIS, hence the present invention differs significantly.
The US patent application US 20110137852 A1, "Sepsis Monitoring and Control" does include the usage of Heart Rate but not heart rate variability nor ANFIS, hence the present invention is substantially different.
Description of the Invention for the Calculation of the Inflammation Index.
The second objective of the present invention is to monitor the degree of inflammation, typically but not necessarily, of critically ill patients in the intensive care unit, by analyzing the heart rate variability and compensating these changes for the influence of the depth of anaesthesia as assessed by for example the qCON hypnotic effect index derived from the electroencephalogram (EEG).
The present invention records the electrocardiogram (ECG), extracts the R-R interval and other features from the ECG, such as intervals between p,q,r and t peaks. Furthermore an FFT of the RR interval is carried out, from which the Heart Rate Variability (HRV) is defined. From the HRV, different frequency bands are extracted for example HF and LF, see table 1.
Other parameters are extracted as well, such as RMSSD, SDSD, PNN50 as described in table 1.
An FFT is carried out directly on the ECG from which the overall variation of the heart rate can be detected in the spectrum. This is shown in FIG. 9 where the spectrum to the right (910) from an awake and healthy subject is much wider than the spectrum to the left (900), which is from a simulated ECG.
As shown in FIG. 8, the parameters derived from the ECG are fed into an ANFIS model (810) and in a second step they are combined with demographic data and the qCON (820) in order to define the final index of inflammation.
A novelty of this index is that the EEG derived index, the qCON, is used in the model to compensate for the fact that HRV is typically affected by the level of consciousness of the patient during sedation. HRV is typically decreased as an effect of the anaesthesia, but as the depth of anaesthesia is known from the value of the qCON, both the qCON and the initial inflammation index is fed into the ANFIS 4 model, where a fuzzy rule will be automatically generated in a such way that the final inflammation index is compensated for the depth of anaesthesia.
Another novelty of the invention is the application of transfer entropy from EEG to ECG. Symbolic transfer entropy measures the causal influence of source signal X on target signal Y, and is based on information theory. Here the EEG equal X while ECG equals Y. The information transfer from signal X to Y is measured by the difference of two mutual information values, I [YF; XP, YP] and I [YF; YP], where XP, YP, and YF are, respectively, the past of source and target signals and the future of the target signal. The difference corresponds to information transferred from the past of source signal XP to the future of the target signal YF and not from the past of the target signal itself. The article "Vicente R, Wibral M, Lindner M, Pipa G: Transfer entropy—A model-free measure of effective connectivity for the neurosciences. J Comput Neurosci 2011; 30:45-67" is incorporated here as a reference. The transfer entropy EEG to ECG is fed into the ANFIS3 (810) model which defines the initial inflammation index.
In a preferred embodiment the transfer entropy from EEG to ECG, is calculated from the frequency with the highest energy content in FFT spectrum of the EEG to the HF of the RR-interval from the ECG.
The output of ANFIS4 (820) is the final inflammation index, which is low, below 50, when the patient is septic and high, above 200, when the patient has recovered as shown in FIG. 10. The range of the final inflammation index is 0 to 300 while the index is unitless.

Methods and apparatus for the on-line and real time acquisition and analysis of voltage plethysmography, electrocardiogram and electroencephalogram for the estimation of stroke volume, cardiac output, and systemic inflammation


A casino board game method comprised of providing a board, a plurality of playing chips, dice, playing pieces, HOTEL CARDS, CASINOS CARDS, and playing money. A roulette wheel is located within the center of the board with rectangular spaces extending around the periphery of the board for movement of the playing pieces of the players.

1. Field of the Invention
The present invention relates to a casino board game apparatus and more particularly pertains to simulating a visit to casinos of Las Vegas with a casino board game apparatus.
2. Description of the Prior Art
The use of casino board games is known in the prior art. More specifically, casino board games heretofore devised and utilized for the purpose of accumulating money and real estate are known to consist basically of familiar, expected and obvious structural configurations, notwithstanding the myriad of designs encompassed by the crowded prior art which have been developed for the fulfillment of countless objectives and requirements.
By way of example, U.S. Pat. No. 5,228,698 to Dubarry, Jr. discloses a casino board game apparatus.
U.S. Pat. No. 5,169,154 to Borghi discloses a casino board game.
U.S. Pat. No. 4,930,789 to Harris et al. discloses a casino board game.
U.S. Pat. No. 5,129,652 to Wilkinson discloses a casino drawing/lottery game and case/prize management system.
U.S. Pat. No. 4,357,015 to Santora et al. discloses a roulette game.
U.S. Pat. No. Des. 298,557 to McNally discloses the ornamental design for an amusement roulette game table.
While these devices fulfill their respective, particular objective and requirements, the aforementioned patents do not describe a casino board game apparatus for simulating a visit to casinos of Las Vegas.
In this respect, the casino board game apparatus according to the present invention substantially departs from the conventional concepts and designs of the prior art, and in doing so provides an apparatus primarily developed for the purpose of simulating a visit to casinos of Las Vegas.
Therefore, it can be appreciated that there exists a continuing need for new and improved casino board game apparatus which can be used for simulating a visit to casinos of Las Vegas. In this regard, the present invention substantially fulfills this need.
In the view of the foregoing disadvantages inherent in the known types of casino board games now present in the prior art, the present invention provides an improved casino board game apparatus. As such, the general purpose of the present invention, which will be described subsequently in greater detail, is to provide a new and improved casino board game apparatus and method which has all the advantages of the prior art and none of the disadvantages.
To attain this, the present invention essentially comprises a playing board having an outer board area. The outer board area has a plurality of individual spaces around an outer periphery of the playing board. Each of the individual spaces defines a hotel or casino. One of the individual spaces is designated as TRY YOUR LUCK. One of the individual spaces is designated as GO TO JAIL. One of the individual spaces is designated as IN JAIL. One of the individual spaces is designated as START. The plurality of individual spaces define a path of travel for players. The playing board has an inner board area. The inner board area has a roulette table disposed thereon. The inner board area has designated areas for TRY YOUR LUCK cards. The game includes a plurality of playing chips for roulette around the playing board. The game includes a pair of playing dice to determine the amount of movement of the plurality of playing pieces around the outer board area. The game includes a plurality of HOTEL CARDS. Each of the HOTEL CARDS has the name of a particular hotel thereon corresponding to an individual playing space with that particular hotel designated thereon. The game includes a plurality of CASINOS CARDS. Each of the CASINO CARDS has the name of a particular casino thereon corresponding to an individual playing space with that particular casino designated thereon. The game includes a plurality of playing money.
There has thus been outlined, rather broadly, the more important features of the invention in order that the detailed description thereof that follows may be better understood, and in order that the present contribution to the art may be better appreciated. There are, of course, additional features of the invention that will be described hereinafter and which will form the subject matter of the claims appended hereto.
In this respect, before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting.
As such, those skilled in the art will appreciate that the conception, upon which this disclosure is based, may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.
It is therefore an object of the present invention to provide a new and improved casino board game apparatus which has all the advantages of the prior art casino board games and none of the disadvantages.
It is another object of the present invention to provide a new and improved casino board game apparatus which may be easily and efficiently manufactured and marketed.
It is a further object of the present invention to provide a new and improved casino board game apparatus which is of durable and reliable construction.
An even further object of the present invention is to provide a new and improved casino board game apparatus which is susceptible of a low cost of manufacture with regard to both materials and labor, and which accordingly is then susceptible of low prices of sale to the consuming public, thereby making such a casino board game apparatus economically available to the buying public.
Still yet another object of the present invention is to provide a new and improved casino board game apparatus which provides in the apparatuses and methods of the prior art some of the advantages thereof, while simultaneously overcoming some of the disadvantages normally associated therewith.
Even still another object of the present invention is to provide a new and improved casino board game apparatus for simulating a visit to casinos of Las Vegas.
Lastly, it is an object of the present invention to provide a new and improved casino board game apparatus comprised of a playing board. A plurality of playing chips are included for roulette-like movement around the playing board. The game includes a plurality of HOTEL CARDS. The game includes a plurality of CASINO CARDS. The game includes a plurality of playing money.
These together with other objects of the invention, along with the various features of novelty which characterize the invention, are pointed out with particularity in the claims annexed to and forming a part of this disclosure. For a better understanding of the invention, its operating advantages and the specific objects attained by its uses, reference should be had to the accompanying drawings and descriptive matter in which there is illustrated preferred embodiments of the invention.


A solution is proposed for accessing a database by an application client. A corresponding method comprises receiving a connection command for opening a user connection between a user client of the application client and the database from the application client, the connection command comprising an indication of a context of the user client, assigning an application connection between the application client and the database to the user client in response to the connection command, and switching a context of the application connection to the context of the user client.

The background of the present disclosure is hereinafter introduced with the discussion of techniques relating to its context. However, even when this discussion refers to documents, acts, devices and the like, it does not suggest or represent that the discussed techniques are part of the prior art or are common general knowledge in the field relevant to the present disclosure.
Databases are commonly used to store large amount of data in structured form (for example, organized in tables in relational databases). Usually, before each client may access a generic database to perform any operations thereon (for example, to retrieve selected data), the client connects to the database by opening a corresponding connection. This involves the completion of all the arrangements that are required to allow the client to communicate with the database and to allow the database to process any operative command submitted thereto. Each connection is associated with a context that defines how the operative commands of the client are to be processed (for example, the identity of the client, assigned privileges and permissions).
The opening of any connection requires several operations that are relatively complex and time-consuming (for example, for authenticating the client); this may adversely affect the performance, especially when a few operative commands (down to a single one) are submitted to the database for each connection. Moreover, the connections waste a relatively high amount of resources (for example, memory space), even when they are inactive; therefore, most databases limit the maximum number of connections that may be open at the same time.
Often, the clients of the databases are software applications (referred to as application clients); in turn, each application client may be used by multiple users, such as physical persons (referred to as user clients) that access the database through the application client.
In this case, the application client may connect to the database on behalf of all its user clients; for this purpose, the application client always connects to the database with a corresponding context (i.e., its identity and highest possible privileges and permission), with the different privileges and permissions of the user clients that are then managed by the application client. In this way, it is also possible to exploit connection pooling techniques. For this purpose, a pool of connections (all with the same context of the application client) is created; whenever a connection is to be opened an available connection is instead retrieved from the pool and whenever a connection is to be closed it is instead returned to the pool (for example, by means of a connection wrapper that makes this behavior completely opaque to the application client). This allows reducing repeated openings and closings of the connections. However, the database is completely agnostic about the user clients actually accessing it.
Conversely, a connection to the database should be opened for each user client (with the corresponding context) to make the database aware of the user clients actually accessing it (for example, for security or logging purposes); however, this reduces the performance of the application client.
Alternatively, as described in U.S. Pat. No. 6,986,060 (the entire disclosure of which is herein incorporated by reference), a security context may be shared among different sessions on a database server. For this purpose, a function call may be used to record a context for an application client with the database server (for example, an application session between a user and an application). Another function call may then be used to associate the application client with a database session between the database server and the application. Whenever a request is receiver by the database server through the database session, the corresponding context is retrieved. A database operation required to satisfy the request is performed thereby enforcing access rights associated with this context (for example, by rewriting a query). This allows selectively switching a large number of application sessions among a smaller number of database sessions for connection pooling purposes.
Moreover, U.S. Pat. No. 8,549,038 as well (the entire disclosure of which is herein incorporated by reference) describes the sharing of a session; in this case, the session has a common context and a pluggable context that directly depends on the end user, the application and/or the privilege level.
However, the management of the different contexts at the level of the database requires a quite complex rewriting of the client applications; moreover, the sharing of the same connection by multiple application clients (with different contexts) may involve security exposures.
In addition, the article "Context transfer method through SQL for database security", IPCOM000137107D, Publication Date: 8 Jun. 2006 (the entire disclosure of which is herein incorporated by reference) describes a technique for transferring context information to a database. For this purpose, a query is modified so as not to change its result; particularly, the context information is attached to the query as a predicate that is always true.

This patent is subject to a terminal disclaimer.

International Business Machines Corporation


A resistor is connected in series with a vertical cavity surface emitting laser, and DC bias electric current is supplied thereto from a DC bias electric current supplying circuit by way of an inductor. Input data is inputted from a differential input line to a voltage amplifier by way of a PECL input buffer, for voltage amplification. The amplified signal is attenuated by an attenuator, transferred to a transfer line by way of a capacitor for AC coupling, and applied to the series resistance and the vertical cavity surface emitting laser. The voltage amplifier has a peak in its frequency characteristics and is structured such that the gain, the frequency band, and the peaking characteristics can be adjusted.

1. Field of the Invention
The present invention relates to a semiconductor laser driving circuit in which rise and fall time of waveform when a pulse modulation signal is provided to a vertical cavity surface emitting laser is improved.
2. Description of the Related Art
Conventionally, a modulation electric current driving circuit in a semiconductor laser driving circuit for high-speed optical communication is constructed as an electric current switch type in which a transistor or a field effect transistor (FET) has a differential construction.
FIG. 1 shows an example of the conventional structure of a driving circuit of a vertical cavity surface emitting laser (referred to as xe2x80x9cVCSELxe2x80x9d hereinafter). Transistors Tr1 and Tr2 constitute a differential switch circuit, and an emitter of Tr1 and Tr2 is connected to a constant electric power source which supplies a constant electric current Im. A collector of Tr1 is connected to the cathode of the VCSEL by way of a resistor R1 and a voltage VCC is supplied to the anode of the VCSEL. On the other hand, a collector of Tr2 is connected to a resistor R2 and a voltage VCC is supplied to the other terminal of the resistor R2. A DC bias electric current supplying circuit is connected to the VCSEL on the cathode side thereof by way of an inductor L1, so that DC bias electric current Idc can be controlled.
Data is inputted from differential inputs TX+ and TXxe2x88x92 by way of an input buffer. When the logic xe2x80x9c1xe2x80x9d is inputted to the TX+, Tr1 is turned xe2x80x9cONxe2x80x9d and modulation electric current Im is supplied to the VCSEL, resulting in emission of light. When the logic xe2x80x9c0xe2x80x9d is inputted to the TX+, Tr1 is turned xe2x80x9cOFFxe2x80x9d and modulation electric current Im is not supplied to the VCSEL, resulting in a non-emission state of light.
In general, in a semiconductor laser driving circuit including a VCSEL, DC bias electric current which is approximately of the level of the threshold electric current Ith is constantly supplied and pulse modulation electric current is coupled thereto, in order to improve the turn-on delay of the laser.
However, in the case of a VCSEL, due to the carrier accumulation effect in its active region, its rise time and fall time tend to become long when the modulation is carried out at a high speed. Accordingly, there arises a problem that required rise and fall time and required specifications of the eye pattern mask may not be achieved in a VCSEL.
For example, in a modulation electric current driving circuit of the electric current switching type as shown in FIG. 1, since the modulation electric current is coupled with the DC bias electric current by DC coupling, the modulation electric current becomes zero when the light emitting element stops emitting light (at the time of the logic xe2x80x9c0xe2x80x9d level). However, as shown in FIG. 2A, the driving electric current does not decrease to the value of the DC bias electric current or lower. Therefore, the waveform of light output of the VCSEL has, as shown in FIG. 2B, quite large values of rise time tr and fall time tf due to the carrier accumulation effect to an active region of the VCSEL. Rise time and fall time are normally specified from 20% to 80% of the logic High level. In the case of a VCSEL, a portion larger than 80% or so rises up quite moderately in the rising and a portion smaller than 20% or so falls down quite moderately in the falling. Due to these moderate rise time and fall time, as shown in FIG. 3, the resulting eye pattern cannot suffice the predetermined eye mask of standard level.
The object of the present invention is to provide a semiconductor laser driving circuit in which rise and fall time of waveform when a pulse modulation signal is provided to a vertical cavity surface emitting laser is improved such that the eye pattern can suffice the predetermined eye mask of standard level.
In order to achieve the aforementioned object, there is provided a semiconductor laser driving circuit comprising: a bias electric current supplying circuit for supplying a bias electric current to a vertical cavity surface emitting laser; and a modulation signal circuit for amplifying input data according to frequency characteristics having a peak in the high value region thereof and coupling the amplified signal as a pulse modulation signal with the bias electric current.
Due to the structure described above, an overshoot and an undershoot can be added to the rise portion and the fall portion of the pulse modulation signal, respectively.
In a preferred embodiment of the present invention, the modulation signal circuit has a peaking adjustment circuit for variably adjusting peaking in the frequency characteristics.
In a preferred embodiment of the present invention, the modulation signal circuit has, at its output end, a capacitor for AC coupling.
In a preferred embodiment of the present invention, the modulation signal circuit has an attenuator on the downstream side of a voltage amplifier.
In a preferred embodiment of the present invention, the modulation signal circuit has a gain adjusting circuit for variably adjusting gains of a voltage amplifier.
Further, in order to achieve the aforementioned object, there is provided a semiconductor laser driving circuit comprising: a bias electric current supplying circuit for supplying a bias electric current to a vertical cavity surface emitting laser; and a modulation signal circuit for inputting input data and coupling the input signal as a pulse modulation signal with the bias electric current; and a differentiating circuit for inputting the input data, generating a differential waveform according to the input data and coupling the differential waveform with a rise portion and a fall portion of the pulse modulation signal.
Due to the structure described above, an overshoot and an undershoot can be added to the rise portion and the fall portion of the pulse modulation signal, respectively.
In the preferred embodiment of the present invention, the differentiating circuit has a buffer at its output end.
In the preferred embodiment of the present invention, the semiconductor laser driving circuit further comprises a variable attenuator provided on the upperstream side or on the downstream side of the differentiating circuit.
The nature, principle and utility of the invention will become more apparent from the following detailed description when read in conjunction with the accompanying drawings.


A method and system for automatically generating a helpdesk ticket when a remote rendering device possesses a series of customer resolvable events within a time period. The helpdesk ticket can be moved into an active state when an alert associated with the customer resolvable events is triggered and the ticket can be moved into a passive state when the alert is no longer triggered for the predefined time period. The helpdesk ticket can be retained in the active state if the fault condition detected for a defined number of times without being out of that state for the predefined time period. A device management module sends a notification indicative on each change of the state associated with the rendering device to a helpdesk unit. The device management module can also reset the time period between alerts if the problem occurs infrequently and the ticket can be automatically closed.

Network printing systems are capable of interacting with an assemblage of varying rendering devices, client computers, servers, and other components that are connected to and communicate over a network. Rendering devices include components such as printers, scanners, faxes, copy machines, and so forth. Preventative maintenance can be performed with respect to such rendering devices to ensure that the device continues to function well with quality output and to protect the device from possible breakdowns while extending the lifetime of the device.
Typically, preventative maintenance is ensured by employing a technician off site in order to manage and maintain the rendering devices. Problems (e.g., paper jams) associated with such rendering devices, may be resolved by a customer without having to actually contact a technician to service the rendering device. These types of customer resolvable issues are often not tracked as problems requiring a technician to be dispatched on-site, as they are assumed to represent normal operations, fully curable by the customer. In some cases, however, when the rendering device encounters a recurring issue, it can be assumed that such problems are due to a machine malfunction and that service should be dispatched. Organization "helpdesks" typically are not aware of such issues until members of the organization and/or other users, such as employees and customers, report the problem(s).
Based on the foregoing it is believed that a need exists for an improved method and system for automatically generating a helpdesk ticket when a remote rendering device possesses a series of customer resolvable issues within a period of time. A need also exists for effectively managing the helpdesk ticket associated with the rendering device, as described in greater detail herein.

Method and system for maintenance of network rendering devices