The present invention relates to a PCR product cloning vector applicable directly to the cloning of a PCR product. More precisely, the present invention relates to a PCR product cloning vector designed to be effective in use for blue/white colony selection and produced based on the restriction enzyme treatment process to generate non-complementary unpaired single overhang and a method for producing the same. According to the method of the present invention, the PCR product cloning vector designed to be advantageous for blue/white cloning selection can be produced with improved efficiency compared with the convention method.

In molecular biology experiments, a gene carrier called "vector" is used for convenience in dealing with genes and for long term preservation. This vector is basically circular double-stranded DNA (dsDNA). According to the elements and use, the vector is classified into cloning vector, transcription vector and translation vector, etc.
When a gene is inserted into a vector, it is called "cloning". Various methods have been developed for cloning. Among them, the most common method is to use restriction enzyme treatment and ligation. In restriction enzyme treatment, restriction enzymes digest DNA into DNA fragments. In ligation, a ligase can insert the purified DNA fragment generated by precedent restriction enzyme treatment into a vector.
In this method, restriction enzymes digest DNA in the specific site which an experimenter wants to cut and ligase mediates ligation between ends of DNA fragments. In this method, DNA fragment is first prepared by digesting DNA using restriction enzymes and the vector is also digested by the same restriction enzyme to make DNA cloning site (sometimes, DNA and a vector are digested with different enzymes, which is not general, though). When DNA is digested with a restriction enzyme, a specific site of DNA cut by the restriction enzyme is called "restriction enzyme recognition sequence". This restriction enzyme recognition sequence is also called "restriction enzyme recognition site" and in some cases it is understood as a restriction enzyme catalytic site (cleavage site). This restriction enzyme recognition sequence differs among restriction enzymes. So, an experimenter needs to select a proper restriction enzyme for his experiment. DNA digested with a restriction enzyme has sticky-end or blunt-end.
To prepare a DNA fragment to be inserted into a vector, DNA is treated with restriction enzymes. The DNA fragment is then purified from restriction enzyme treated reaction mixture. To prepare a vector fragment, a vector is also treated with restriction enzymes and as a result a vector fragment having DNA cloning site is prepared (linearized structure). This vector fragment is also purified from restriction enzyme treated reaction mixture. The purification of DNA fragment and vector fragment from restriction enzyme treated reaction mixture can be performed by using a commercial DNA purification kit. And this purification can be performed easily by those in the art, so that precise description is not given herein.
To ligate a purified DNA fragment and a purified vector fragment, these two fragments are added to ligation buffer at a proper molar ratio, to which ligase is added, followed by inducing ligase reaction (ligation among DNA fragments). Accordingly, the end of DNA fragment is linked to the end of linearized vector fragment, resulting in a circular plasmid, suggesting the completion of cloning. The plasmid herein indicates the circular double-stranded DNA.
It is not easy and inefficient to separate and purify a cloning product alone from ligation reaction mixture to use it for the next step of experiment. Even if it is successfully separated and purified, the amount is not sufficient for the next step. Therefore, in most cases, transformation using Escherichia coli is performed to obtain enough amount of a cloning product. In general, transformation is performed by using heat-shock method. Sometimes, transformation using electroporation is performed but to transform E. coli using a small sized plasmid, heat-shock method is enough working. Transformation by heat-shock is performed as follows. 10 μl of ligation mixture was added to 100 μl of E. coli solution in which cell membranes of the cells are loosened by treating calcium chloride (generally called 'competent cell'). The mixture is then left in ice for 30 minutes to let the cloning product to be adhered on the surface of E. coli. 30 minutes later, the mixture is transferred quickly to a 42° C. bath and left therein for 1 minute. This is called as 'heat-shock'. At this time, the cloning product penetrates into E. coli. After heat-shock, the mixture is transferred again into ice quickly and left therein for 1 minute. After 1 minute of staying in ice, the mixture is taken out of ice, to which 800 μl of a proper culture medium is added. Generally, LB medium (trypton, 10 g/L; yeast extract, 5 g/L; sodium chloride, 10 g/L) is used in transformation. After adding a culture medium, the reaction mixture is incubated at 37° C. for one hour. During this incubation, cell membranes damaged by calcium chloride treatment become recovered. After one hour of incubation, the mixture is spread on a plate medium containing a proper antibiotic, followed by culture in an incubator until proper sized colonies are formed. At this time, a colony indicates E. coli cells proliferated from one E. coli cell. When colonies are formed big enough, it is considered that transformants are generated. And, the transformant is transferred into a liquid medium (generally LB medium), followed by further culture. From the cultured cells, enough amount of plasmid can be obtained.
To increase convenience and efficiency of cloning, diverse methods have been developed by modifying the conventional cloning method composed of a restriction enzyme treatment and ligation. One example is the method of using a specially designed vector capable of accepting a PCR-amplified product, which is related to the present invention.
PCR is a reaction using DNA polymerase, which produces massive amount of DNA amplicons from template DNA. PCR is a well-known, general technique used for biological study and for disease diagnosis. Cloning of such PCR product can also be achieved by the cloning method using a restriction enzyme and ligase as explained hereinbefore. However, to do so, an amplified PCR product has to contain a restriction enzyme recognition sequence. To introduce a restriction enzyme recognition sequence into a PCR product, a specifically designed PCR primer is required. The primer has to contain 6-10 nucleotides corresponding to a restriction enzyme recognition sequence selected by an experimenter in addition to the nucleotide sequence (priming sequence) binding to a template. A PCR product obtained by using a primer containing a selected restriction enzyme recognition sequence cannot be directly linked to a vector fragment because the cloning site is not exposed. So, after digesting a PCR product by a corresponding restriction enzyme and purification of the restriction enzyme treated PCR product, insertion into a vector fragment is performed.
However, the above method uses a PCR product as a cloning target instead of general DNA, which is the only difference from the conventional cloning method composed of a restriction enzyme treatment and ligation. Therefore, the method does not provide a special effect or convenience. Besides, a primer specially designed as the above contains additional nucleotides in addition to annealing sequence binding to nucleotide sequence of a target gene, suggesting that efficiency in template specific primer annealing decreases. A PCR product generated by the method contains restriction enzyme recognition sequences in both ends, suggesting that digestion efficiency by a restriction enzyme decreases. In general, when a restriction enzyme recognition sequence is located in terminal region of DNA molecule, digestion efficiency is lower than when it is located in the middle.
Therefore, many researchers tried to develop more simple method for cloning of PCR product and to improve efficiency of restriction enzyme treatment to a PCR product. As a result, they proposed the method in which restriction enzyme treatment step that has been always necessary according to the conventional cloning method is omitted. As an example, PCR product cloning method using topoisomerase I or a vector designed to be suitable for direct cloning of PCR product without restriction enzyme treatment was proposed.
The PCR product cloning method of the present invention is related to the latter, which is direct cloning of PCR product into a linearized vector fragment without restriction enzyme treatment. The linearized vector used for direct PCR product cloning without using a restriction enzyme is largely divided into two groups according to the type of terminal region of the PCR product used in cloning. One is the linearized vector fragment designed suitable for direct cloning of PCR product having blunt ends. The other is the linearized vector fragment designed suitable for direct cloning of PCR product having non-complementary unpaired single overhang in the end of only one chain of double strands.
A PCR product is a double-stranded DNA having a linearized structure in which two strands are complementarily combined. In general, the end form of PCR product is determined by DNA polymerase used in its amplification. That is, a PCR product can have either blunt ends or non-complementary unpaired single overhang ends according to the kind of DNA polymerase used. So, an experimenter can choose proper DNA polymerase considering the purpose of the experiment. For example, when Taq DNA polymerase frequently used for PCR as a thermophilic enzyme or Tth DNA polymerase is used, a unique PCR product having non-complementary unpaired single overhang at both 3′-ends is produced by template-independent addition. The nucleotide residue forming the non-complementary unpaired single overhang can be any of single deoxyadenosine monophosphate residue (dAMP; indicated as 'A' herein for convenience), single deoxythymidine monophosphate residue (dTMP; indicated as 'T' herein for convenience), single deoxyguanosine monophosphate (dGMP; indicated as 'G' herein for convenience), and single deoxycytidine monophosphate residue (dCMP; indicated as 'C' herein for convenience). Among them, the most frequent occurrence is A-overhang. It was reported that the type of overhang is determined by primer sequence (Hu, DNA Cell Biol. 12: 763, 1993; Magnuson et al., BioTechniques 21: 700, 1996).
As explained hereinbefore, a specially designed vector (mentioned as a vector for convenience, but exactly speaking it is a vector fragment) is required for direct cloning of a PCR product having blunt ends or overhang ends.
Basically, cloning of a PCR product having blunt ends (or even if a PCR product had overhang ends but the overhang ends have been eliminated by the treatment of nuclease, it is included in the group of PCR products having blunt ends) can be achieved only with a linearized vector fragment having blunt ends. So, this kind of cloning is theoretically simple. To prepare a linearized vector fragment having blunt ends, a restriction enzyme such as EcoR V, Dra I, or Hinc II can be used. A PCR product having blunt ends can be linked to a linearized vector fragment having blunt ends by ligase under the conventional DNA ligation conditions. However, according to this method, coupling between vector fragments or intra-molecularar circulization of vector fragments is dominant over real cloning, suggesting that cloning efficiency is reduced.
On the other hand, the production of a vector fragment for the direct cloning of such a PCR product having unpaired single overhang and the cloning of the same are rather complicated. For direct cloning of a PCR product having overhang ends, a linearized vector fragment having overhang ends matching to the overhang ends of a PCR product (complementary binding) is required. So, the overhang of a linearized vector fragment has to be located in the opposite strand to the overhang of a PCR product for complementary binding. To help understand the relative location of overhang, a schematic diagram illustrating the relative locations of terminal overhangs of a vector fragment and a PCR product is presented in FIG. 1. In PCR products, A-overhang is most frequently observed. So, T-overhang is most appropriate as terminal overhang of a vector fragment, but not necessarily limited thereto. Instead, a vector fragment can have any of A-overhang, C-overhang, and G-overhang. It is general to unite two overhangs of both ends of a vector fragment, but not always limited thereto. However, since A-overhang is most frequently observed in end of a PCR product, it is preferred for a vector fragment to have T-overhangs in both ends for complementary binding. Therefore, description hereinafter is focused on the vector fragment having T-overhang. It is only for convenience in explanation but cannot limit the present invention thereto.
Linearized vector fragments having overhang ends can be produced by two different methods. Both use a circular parental vector (indicating a starting vector for the production of a vector fragment, and in this invention, it is called as 'parental vector'). One of the methods is the method using restriction enzymes capable of producing blunt ends. In this method, a circular parental vector is digested with a restriction enzyme capable of producing blunt ends. Then, the prepared linearized vector fragment having blunt ends is purified. In the next stage, an overhang is added to the blunt ends of the linearized vector fragment, followed by purification again. At this time, a restriction enzyme capable of producing blunt ends is exemplified by EcoR V, Dra I, and Hinc II. To form an additional overhang, Taq DNA polymerase (Marchunk, D. et al., Nucleic Acid Res. 19: 1154. 1991) or terminal deoxynucleotidyl transferase (Holton, T. A. et al., Nucleic Acid Res. 19: 1156, 1991) is used in the presence of excess amount of Mg2+. According to this method, efficiency of addition of overhang to the end of a vector fragment depends on the activity of the selected modifying enzyme. So, there is still a high chance for a vector fragment to remain as an incomplete vector fragment that does not contain an additional overhang. Such an incomplete vector fragment without having an overhang can form intra-molecular circulizated molecule (self-ligation) during ligation and cannot be used for cloning. Therefore, this causes the decrease of cloning efficiency.
The other method is the method of using restriction enzymes capable of producing a linearized vector fragment having overhang ends without any additional treatment (Yoshikazu, I. et al., Gene 130: 152, 1993; David A. M., et al., Bio/Technology 9: 65, 1991). A restriction enzyme used at this time is exemplified by Ahd I, BciV I, Bfi I, Bfu I, Bmr I, BspOV I, Dri I, Eam1105 I, EclHK I, Hph I, Mbo I, Mnl I, Ncu I, NruG I, Taa I, Tsp4C I and Xcm I. Particularly, a parental vector having 1-2 restriction enzyme recognition sequences capable of being used for producing overhang ends directly is treated with a proper restriction enzyme, and as a result, a vector fragment having non-complementary unpaired single overhang at the end is prepared. It is not preferred but is possible for a parental vector to have several restriction enzyme recognition sequences capable of being used for producing overhang ends in several different regions. If that is the case, effective restriction enzyme recognition sequences are two sequences that are located outmost from the DNA fragment cut out of the parental vector.
To perform the cloning of a PCR product having overhang ends into a linearized vector fragment having overhang ends, these two fragments and ligase are added to ligation buffer, followed by ligation. To obtain massive cloning products after ligation, E. coli is transformed with the ligation mixture. At this time, other plasmids effective in transformation can be included in addition to the cloned plasmid in ligation mixture. The parental vector used for the production of a linearized vector fragment having overhang ends or the recirculized plasmid of the incompletely digested parental vector (one of the two restriction enzyme recognition sequences is digested but recirculized as a parental vector from ligation) can be included, although the amount is small. It is theoretically possible to eliminate these vectors completely by intensive separation and purification after restriction enzyme treatment. However, such complete elimination is not easy and some of the vectors are included generally. When a parental vector is digested with a restriction enzyme to produce a linearized vector fragment having overhang ends, a released DNA fragment is too small. So, on agarose gel electrophoresis, difference in mobility of each the vector digested completely and the vector digested incompletely is not so great. Thus, no matter how badly want to separate and purify a completely digested vector fragment alone using gel-extraction, an incompletely digested vector fragment or non-digested parental vector can still be included, which has to be overcome.
For the conventional transformation, a selection medium containing antibiotics is generally used. So, E. coli that does not have such a plasmid containing an antibiotic resistant gene cannot be growing on that medium. E. coli that has not been transformed with the plasmid containing an antibiotic resistant gene is fundamentally eliminated from the selection. Even if such E. coli that is not transformed with the plasmid containing an antibiotic resistant gene in the selection medium is eliminated first, E. coli transformed with a recirculized plasmid generated from a incompletely digested parental vector fragment, a parental vector itself, or a cloning product can have the plasmid containing an antibiotic resistant gene, suggesting that the E. coli transformed with these plasmids can grow in the selection medium containing antibiotics. So, the selection of the transformant having a cloning product using antibiotics is not possible.
To isolate only the transformant having a cloning product, blue/white colony selection method is proposed. The blue/white colony selection method uses a special medium and E. coli transformed with a proper cloning product can be determined by its apparent color. Accordingly, it is possible to distinguish a colony having a plasmid successfully cloned from a colony which does not. E. coli used for blue/white colony selection is genetically modified to fit for blue/white selection method, which is well known to those in the art, so the explanation thereof is omitted herein. If E. coli does not have a plasmid or has a plasmid but not expressing alpha-peptide, a white colony is formed in blue/white colony selection. E. coli which does not have any plasmid cannot be growing in the selection medium containing antibiotics because it does not contain any antibiotic resistant gene. So, there is no need to worry about such E. coli. In general, a transformant having a recirculized plasmid generated from an incompletely digested parental vector fragment or a parental vector appears as a blue colony in blue/white colony selection, while a transformant having a cloning product appears as a white colony in blue/white colony selection. That is, E. coli having the unproper cloning product can express alpha-peptide and appears a blue colony. If E. coli is transformed with a proper cloning product, E. coli cannot express alpha-peptide and appears as a white colony.
Blue/white colony selection is based on the action of alpha-peptide. So a plasmid applicable for blue/white colony selection has to contain a gene encoding alpha-peptide. Also, factors and sequences necessary for the expression of alpha-peptide (corresponding promoter, etc) have to be included in the plasmid as well. Alpha-peptide is N-terminal region of beta-galactosidase and the gene encoding alpha-peptide is represented by SEQ. ID. NO: 1. Alpha-peptide is an enzyme converting 5-Bromo-4-chloro-3-indolyl β-D-galatopyranoside (X-Gal) into a blue-colored material. To perform blue/white colony selection, alpha-peptide has to be expressed from the plasmid used for the transformation. To do so, isopropyl β-D-1-thiogalactopyranoside (IPTG) is added to the medium as expression inducer.
Therefore, alpha-peptide gene sequence has to be included in a plasmid used for the transformation for blue/white colony selection and the alpha-peptide sequence included in plasmid is provided by a cloning vector used in construction of the plasmid. Some parts of alpha-peptide sequence can be modified. In many commercial cloning vectors, some parts of alpha-peptide sequence have been modified from an original sequence to introduce cloning region within the alpha-peptide gene. Such modification is of course limited and has to be confirmed by experiments. Examples of acceptable modification have been known, based on which diverse modification attempts in the sequence have been made. Relevant references are easily obtained by those in the art, so the precise explanation is omitted in this invention.
In order for a transformant having a recirculized plasmid generated from an incompletely digested parental vector fragment or a parental vector to form a blue colony, alpha-peptide has to be expressed from these plasmids. To do so, reading frame of alpha-peptide is composed of a contiguous and non-overlapping set of three-nucleotide codons. Even if a part of alpha-peptide sequence is modified, complete open reading frame has to be in-frame. In order for a transformant to form a white colony, the cloned gene is located in the inside of alpha-peptide gene. That is, the cloning has to be able to knockout the alpha-peptide gene. In general, when a big size gene is inserted within alpha-peptide gene, alpha-peptide gene is knockout. So, blue/white colony selection explained above has been widely used to isolate colonies having cloning products.
As explained hereinbefore, distinguishment of a parental vector and an incompletely digested parental vector fragment from a completely digested parental vector is very difficult. They are usually mixed. Precisely, a transformant having a parental vector, a transformant having a recirculized plasmid, and a transformant having a cloning product can be distinguished in blue/white colony selection, so it causes no big confusion. Significant problem caused by inefficiency of a restriction enzyme is that the amount of effective vector fragments obtainable from equal amount of a parental vector is much less, resulting in inefficiency in production process. If cloning is performed with such vector fragments including a parental vector or an incompletely digested parental vector, which means as the amount of effective vector fragment used for real cloning becomes less, cloning efficiency will be decreased. Even if it is so difficult to distinguish a completely digested parental vector fragment alone from an incompletely digested parental vector fragment or a parental vector, the problem can be minimized by maximizing efficiency of a restriction enzyme during the production of a vector fragment. If the distance between restriction enzyme recognition sequences is too close, the functions of those restriction enzymes are interrupted, resulting in incomplete digestion. This problem is only caused by too close distance between restriction enzyme recognition sequences in the parental vector used for vector fragment production, so it can be improved by extending the distance. And the extension of the distance between restriction enzyme recognition sequences in the parental vector can be achieved by inserting an additional gene in between the restriction enzyme recognition sequences. The insertion of an additional gene can increase efficiency of a restriction enzyme and facilitates separation and recovery of products by taking advantage of difference in size of products after the treatment of restriction enzyme. However, such insertion of an additional gene in between the restriction enzyme recognition sequences present within the alpha-peptide gene may knockout alpha-peptide gene necessary for blue/white colony selection. This is consistent with that a transformant transformed with a cloning product having gene insertion in alpha-peptide sequence appears as a white colony in blue/white colony selection. That is, the insertion of an additional gene in between the restriction enzyme recognition sequences brings another problem of making selection of colonies having a parental vector or a recirculized parental vector by color impossible because all transformants having a cloning product and a parental vector appear as white colonies in blue/white colony selection.
The present inventors completed this invention by establishing a method for producing a PCR product cloning vector fragment which can overcome the problems of the conventional method such as inefficient digestion in restriction enzyme treatment and difficulty in separation of a restriction enzyme treated vector fragment and at the same time facilitates distinguishment of each transformant respectively transformed with a parental vector and a recirculized plasmid generated from an incompletely digested parental vector fragment by making them presented as blue colonies in blue/white colony selection.

Polymerase chain reaction product-cloning vector suitable to its easy production and method for producing the same


Methods and apparatus to load a valve packing are described. An example apparatus to load a valve packing includes a load assembly having a biasing element disposed between a valve packing flange and a stop flange to provide a load to a seal assembly. The packing flange is adjustable relative to the stop flange to adjust the load to be applied to the seal. A first guide member coupled to the packing flange or the stop flange provides a first predetermined distance between the packing flange and the stop flange. The first guide member provides an indication of a first predetermined load to be provided by the load assembly when the packing flange and the stop flange are spaced at the first predetermined distance provided by the first guide member.

Process control plants or systems often employ fluid valves, such as rotary valves, linear valves, etc., to control the flow of process fluids. In general, fluid valves typically include a fluid flow control member that is disposed in the fluid path and which is operatively coupled to an actuator such as, for example, a pneumatic actuator, a manual actuator, etc. A valve stem or shaft operatively couples the flow control member to the actuator, which moves the flow control member between an open position and a closed position to allow or restrict fluid flow between an inlet and an outlet of the valve. Typically, the valve stem or shaft extends between the flow control member and the actuator through a bonnet coupled to the valve body.
A valve packing or packing assembly is often employed to prevent undesired leakage of process fluid to the environment through the valve body along the valve stem or shaft. The valve packing may include a plurality of springs and/or packing seals disposed along a portion of the valve stem or shaft to provide a seal. Some known valve packings employ a high-spring rate loading device or spring assembly (e.g., Belleville springs) to provide a relatively high load over a relatively small range of deflection or compression. However, such known loading devices typically require very precise deflections and/or tightly held manufacturing tolerances to enable a desired packing stress to be applied to the packing seals.
Failure to provide a desired packing stress to the packing seals may cause an improper seal. For example, a packing stress that is too low may cause the process fluid to leak to the environment through the packing seals. A packing stress that is too large (e.g., greater than a maximum recommended packing stress) may cause some types of packing seals (e.g., graphite seals) to transfer material to a valve stem, thereby causing build-up of material on the valve stem and damaging the packing seal. Additionally or alternatively, packing stresses that are too high may increase packing friction between the packing seals and the valve stem or shaft, which may reduce valve performance and/or the operational life of the valve packing and/or the valve stem or shaft.


A guiding catheter and method of its use are presented wherein the catheter includes an elongate catheter shaft having a proximal region and a distal region and a length. The shaft defines a distal region and includes a distal tip. The distal region defines an arc of approximately 180 degrees and having a radius of 0.3 inches to 0.6 inches. The distal tip forms a half turn of a left-hand helix having a pitch of 0 inches to 0.4 inches. The unique shape of the distal region allows the distal tip be perpendicularly aligned with the septal wall of the right atrium at the His bundle location when the catheter is advanced therein.

Guiding catheters are well known devices used to locate and cannulate vessels for a variety of medical procedures. They are of particular use in cardiac access procedures such as those involved in the implantation of cardiac pacing leads. Cardiac pacing leads are flexible, and historically, stylets have been inserted into the lumen of hollow leads to stiffen and allow the lead to be bent to aid in lead placement. Stylets are still in common use but are inadequate to provide precise control to reach and place a lead at the small target His bundle. Typically, when right side approach is desired it involves accessing the heart via the left subclavian vein, the cephalic vein and more rarely the internal or external jugular vein, or femoral vein. For catheter lead placement, a guide wire is advanced into the heat from the access site. The guiding catheter is then advanced through the vasculature and into the heart over the guidewire; once in position the guidewire is removed. A pacing lead is then advanced through the guiding catheter to be deployed at various regions in the heart.
Typically, pacing leads are deployed to various locations in the heart depending on the nature of the heart condition necessitating the pacing procedure. Conventional ventricular pacing typically involves implanting a lead at the apex of the right ventricle. This placement is still often utilized today even in the face of published evidence of the deleterious effects of bypassing the His/Purkinje system, otherwise known as the cardiac conduction system.
Pacemaker lead electrodes have been placed in or on the heart in a position that bypasses the His/Purkinje system since the inception of pacing in 1957. Directly stimulating the myocardium is and has been the standard of care even though His bundle pacing has been known and tried occasionally. It is believed that His bundle pacing is not widely practiced because it presents a small target and is very hard to reach successfully. This increases "fluro time" which is a detriment to both patient and physician. Another factor is that there is no wide recognition of the value of His pacing. At present there is a paucity of catheters that can facilitate His bundle pacing. When, for various reasons, the pacemaker must be implanted on the patient's right side and right subclavian vein used to reach the heart the target is still the myocardium and not the His bundle. It should be noted for completeness that the His bundle is accessed on the atrial aspect of the annulus of the tricuspid valve, just above the attachment of the septal valve leaflet.
The present disclosure describes embodiments of a catheter and method for its use in delivering a pacing lead to the His bundle at the septal wall. The cardiac conduction system is comprised in part of His bundle which resides between the atrioventricular (AV) node, and the bifurcation of left bundle branch (LBB) and right bundle branch (RBB). This anatomic location is regarded as a difficult target to reach. Embodiments of the present invention have overcome this difficulty.

This patent is subject to a terminal disclaimer.


A force sensor for sensing the weight of a vehicle seat occupant for a vehicle's occupant protection system has a pressure sensor responsive to hydraulic pressure resulting from axial force applied to the force sensor. A constant force spring in the form of a diaphragm with two flanges simultaneous functions as a seal, a constant force bias spring, an armature support resistant to radial forces and a piston for converting force to hydraulic pressure. Two studs receive applied force. An electrical insulator is sealed to the walls of a passage inside of one of the studs to provide electrical communication while sealing against liquid leakage. A pressure sensor is mounted on the electrical insulator and connected to electrical conductors in the insulator by stitch bonding. The diaphragm resists radial movement while allowing axial movement thereby making the sensor insensitive to radial forces. By being a constant force spring the diaphragm makes the sensor insensitive to temperature variations.

It is well known to sense a force by using that force to generate hydraulic pressure which is measured to ascertain the magnitude of the force. Such devices typically include a piston in a cup with a seal on the diameter of the piston to prevent liquid leakage. A connection to the liquid filled chamber provides pressurized liquid to a pressure gage which is calibrated to read in units of force.
Semiconductor pressure sensors are manufactured in large quantities by micromachining silicon wafers. Many designs based on many technologies and many physical principles are known. Many of these sensors require additional circuitry to achieve a useful function. Typically, an integrated circuit complements the micromachined pressure sensing element. Certain of these sensors are suitable for operation submersed in liquid and operate by sensing the pressure in the liquid.
Force sensors that convert force to hydraulic pressure have the disadvantage that they are unresponsive to forces that tend to draw the piston away from the liquid and create a vacuum. To measure forces in both directions a bias force on the piston is required which has the consequence that in the case of zero applied force a substantial pressure is being measured with a potential for drift with temperature and time.
Welding and brazing are common methods of joining metals that provides good strength and sealing against leakage. Welding commonly means joining metals by melting the metals being joined at the joint with or without addition of more metal. Brazing commonly means applying a different metal at the joint that wets the metals and hardens to join the metals.
Belleville springs are washers formed to be slightly conical (dished). Certain Belleville springs are called "constant force springs" because the force required to compress the spring is approximately independent of the amount of compression over a range of compressions. Belleville springs having a dish height to thickness ratio of about 1.4 are constant force springs when they are compressed to flatness. Herein, the term "constant force spring" is defined to include Belleville springs and other springs comprising dished washers operating in a range of compressions at which the spring force or pressure the spring force causes in a fluid confined by the spring is approximately independent of the spring compression.
It is well known to reduce the number of wires required for connection to a sensor to two by the sensor simultaneously drawing power through the two wires to power itself and by drawing pulses of current over and above the current required to power itself and modulating the width or magnitude of the pulses of current to indicate the physical quantity being sensed.
A general object of this invention is to provide a force sensor that is particularly adapted for sensing force resulting from the weight of an occupant of a seat of a vehicle for informing occupant protection systems of automotive vehicles which also overcomes certain disadvantages of the prior art.

Force sensor for seat occupant weight sensor


A system for the transmission of data by seismic wave, the system having at least one transmitting unit, that at least one transmitting unit comprising a first processing unit encoding a first signal, the first processing unit being coupled to a digital to analog converter, an amplifier amplifying the first signal, and a seismic pressure wave inducer transmitting the first signal to a geological feature; and at least one receiving unit, the receiving unit comprising at least one receiver coupled to the geological feature for receiving the first signal, and an analog to digital converter whereby the first signal is transferred from the geophone and is conveyed to a second processing unit, the second processing unit being configured to identify and decode the first signal.

Often instruments and sensors are placed in locations where radio communications are impossible or impractical. Cave walls, walls of narrow gorges and other geological features may block radio transmissions which might otherwise be used to monitor or communicate with such instruments. Known techniques for monitoring such instruments require installation of cables. Often, the physical location of the instruments means that cables to such instruments must be long. Such installations are awkward and time consuming. Such long cables are vulnerable to damage.
In some fields, especially in energy prospecting, seismic charges and waves have been used to communicate information. These techniques often rely on time intervals between nearly identical seismic shots, in essence, these techniques are analogous to Morse Code, where the timing of separate signals conveys information. Other such techniques rely not on true seismic transmission of signals, but instead on the transmission through fluids, like slurry or water.
What is needed, therefore, are techniques for wirelessly communicating data through geological barriers.

Bae Systems Information and Electronic Systems Integration Inc.


A conventional land grid array (LGA) socket assembly uses the same socket contact in the power delivery area and the signal delivery area. Using low current socket contacts in the power delivery area may create self-heating and limit power delivery from a printed circuit board (PCB) to an IC package mounted in the socket. Embodiments of the present invention are directed to an LGA socket assembly that has a separate power delivery contact, which includes contact pins and contacts pads that are ganged using a cross beam to form a comb-shaped contact. In an alternative embodiment of the present invention, an LGA socket assembly has a shorter channel in the power delivery area than in known LGA socket assemblies. In still another embodiment, an LGA socket assembly has a shorter channel in the power delivery area in the signal delivery area.

1. Field
Embodiments of the present invention relate to sockets for coupling integrated circuits to printed circuit boards, and in particular to land grid array (LGA) sockets having separate geometries for power delivery and signal contacts.
2. Discussion of Related Art
A land grid array (LGA) socket is a common connector used to couple an integrated circuit (IC) package to a printed circuit board (PCB. FIGS. 1A through 1E show an exploded view of a typical known LGA IC package-socket-PCB assembly 100.
FIG. 1A shows a printed circuit board (PCB) 102, FIG. 1B shows the top side (1C package side) of an LGA socket body 104. LGA contacts 106 shown in FIG. 1C fit into the socket body 104. FIG. 1D shows the IC package 108 as viewed from above as though looking through the IC package 108. FIG. 1E shows details of a portion 110 of the PCB 102. The socket assembly (i.e., the socket body 104 with the contacts 106 inserted), which is shown as a bottom view, is soldered to the PCB 102, which is shown as a top view. The IC package 108 mounts to the top side of the socket body 104.
The contacts 106 fit into slots 112 on the socket 104. Each contact 106 has a contact pad 114 that is soldered to a corresponding solder pad 116 on the PCB 102. Each contact 106 also has a contact pin 118 that mates to corresponding lands 109 on the IC package 108.
The PCB 102 has a signal delivery area 120 and a power delivery area 122. The socket body 104 has a signal delivery area 124 and a power delivery area 126. The IC package 108 has a signal delivery 128 area and a power delivery area 130. Each of the signal delivery areas corresponds to the other signal delivery areas such that input/output (I/O) signals (such as data and address signals) coming from signal planes in the PCB are electrically coupled to the appropriate solder pad 116, contact 106, and socket body 104 land. Likewise, each of the power delivery areas corresponds to the other power delivery areas such that power (VCC) and ground (VSS) coming from power and ground planes in the PCB 102 are electrically coupled to the appropriate solder pad 116, contact 106, and socket body 104 land.
One characteristic of the conventional LGA IC package-socket-PCB assembly 100 is that the contact design in the power delivery areas is the same the contact design in the signal areas (i.e., the contact 106). The contacts in the power delivery areas do not have the same function as the contacts in the signal areas, however. For example, the contacts in the power delivery areas carry high currents and the contacts in the signal areas carry low currents. Increased current density through the contacts 106 positioned in the power delivery area 126 may create self-heating which may increase contact 106 resistance and limit power delivery to the IC package 108. As high performance components consume more power and current, electrical resistance of the contacts 106 may limit power delivery to such components.
An existing approach to reducing current density through the contacts 106 is to allocate more contacts 106 in the LGA IC package-socket-PCB assembly 100 for power delivery, i.e., increase the size of the power delivery area 126. Increasing the size of the power delivery area 126 reduces the size of the signal delivery area 126, however, which may mean that the number of contacts 106 available for signals is reduced.
Another existing approach is to add more contacts 106 to the assembly 100. More contacts 106 means that the socket assembly is larger, however, and that the IC package 108 is larger. Additionally, more contacts 106 generally requires greater IC package to socket insertion force in order to maintain an effective IC package to socket interface resistance. This greater insertion force can further complicate effective enabling design.
Another characteristic of the conventional LGA IC-socket-PCB assembly 100 is that the body of the socket 104 is made from injection-molded plastic and each slot 112 is injection molded. This introduces limitations on contact 106 spacing, which can also introduces limitations on PCB solder pad 116 spacing and IC package land 109 spacing. Also, if more pins are to be added, the individual parts of the LGA IC package-socket-PCB assembly 100 are more expensive.
The socket assembly portion 110 illustrates this point. The portion 110 includes channels 132. The channel 132 has a length 134 and a set of slots 112, lands 109, contacts 106, and solder pads 116. The channel 132 has a length of approximately 10.16 mm or more. Within that length the channel 132 has approximately eight to nine slots 112, lands 109, contacts 106, and solder pads 116. Also within that length the channel 132 has a pitch (e.g., slot 112-to-slot 112, land 109-to-land 109, and contact 106-to-contact 106) of approximately one millimeter (mm). Because of the slots 112 are injection molded individually, the LGA IC package-socket-PCB assembly 100 is so limited.

Comb-shaped land grid array (LGA) socket contact for improved power delivery


In a piezoelectric vibration type force sensor according to the present invention, vibration is restricted by a friction force between a piezoelectric body and a restricting member, and hence a range of sensing forces can be expanded compared with a case in which the vibration is restricted directly from a direction that is the same as an vibration direction. A conventional structure, in which a lead wire is soldered directly for electrically connecting the piezoelectric body to an external control circuit, causes a restriction of the vibration due to a solder attached to the piezoelectric body, resulting in narrowing the sensing range. Using the piezoelectric vibration type force sensor, the range of sensing forces can be expanded by making a state in which conducting portions of the piezoelectric body and the restricting member are not fixed but contact with each other for keeping electric conductivity.

As conventional force sensors, a strain gauge, a capacitive force sensors, a conductive rubber, and the like are developed. Each of the force sensors using those utilizes deformation of a member, and thus it is necessary to secure a sufficient amount of the deformation for realizing high sensitivity and a wider dynamic range of measurement. Therefore, if the sensor is downsized, sufficient deformation amount cannot be obtained. As a result, there arises a problem that an output signal is buried in noise to deteriorate accuracy.
As a countermeasure against the problem, there is provided a sensor utilizing a resonance phenomenon of a piezoelectric body as described in Japanese Patent Application Laid-Open No. S52-078483. The piezoelectric body has a property of vibrating in a specific direction when a voltage which temporally changes (e.g., alternating voltage) is applied, according to time variation of amplitude of the voltage.
According to the technique described in Japanese Patent Application Laid-Open No. S52-078483, an impedance 6 is connected in series to the piezoelectric body first, and a voltage having a fixed frequency that is not higher than a resonance frequency of a piezoelectric body 1 is applied from a power supply 7, whereby the piezoelectric body is vibrated. If a force is exerted on the piezoelectric body in this state, the impedance of the piezoelectric body increases so that the resonance frequency of the piezoelectric body is shifted to a high frequency side. In the shifted state, a value of the voltage applied to the piezoelectric body 1 is changed from the value before the force is exerted. According to this change of the voltage value, magnitude of the force that has exerted on the piezoelectric body can be quantitatively calculated (see FIG. 8).
The piezoelectric vibration type force sensor described in Japanese Patent Application Laid-Open No. S52-078483 has an advantage that its sensitivity is high with a low load because variation of the voltage amplitude is large. However, as an external force exerted on the sensor increases, the variation of the voltage amplitude decreases. Thus, its sensing range is narrow.
As a countermeasure against this problem, there are proposed methods for expanding the sensing range in Japanese Patent Application Laid-Open No. S60-187834 and Japanese Patent Application Laid-Open No. S60-222734.
First, Japanese Patent Application Laid-Open No. S60-187834 is characterized in that a structure in which two piezoelectric bodys are bonded to each other, i.e., a so-called bimorph type piezoelectric body is used. One end of this bimorph piezoelectric body is fixed while the other end is made to be a free end, whereby a force is exerted on the piezoelectric body. In addition, a frequency signal higher than a resonance point of the piezoelectric body is applied to the same. This sensor has the structure in which two piezoelectric bodys are bonded to each other, and hence amplitude thereof can be made larger, with the result that the range of sensing forces can be expanded.
Next, as to Japanese Patent Application Laid-Open No. S60-222734, a phase angle difference between the voltage applied to the piezoelectric body and a terminal voltage of the piezoelectric body, which is caused by the external force, is sensed to control the frequency so that the phase angle difference becomes a set value. This frequency variation is utilized for sensing the force. Thus, the sensitivity can be uniform and the sensing range can be made wider.
However, the invention of Japanese Patent Application Laid-Open No. S60-187834 uses the bimorph piezoelectric body. The bimorph piezoelectric body has the structure in which two piezoelectric elements are bonded to each other, and hence it is disadvantageous for cost reduction and downsizing.
In addition, the invention of Japanese Patent Application Laid-Open No. S60-187834 senses the phase angle difference between the voltage applied to the piezoelectric body and the terminal voltage of the piezoelectric body, which is caused by the external force, thereby controlling the frequency so that the phase angle difference becomes a set value. However, an additional phase angle sensing circuit and an additional phase angle setting circuit are necessary, resulting in complication of the circuit. Therefore, it is also disadvantageous for cost reduction and downsizing.

Piezoelectric vibration type force sensor


The present invention relates to novel antibodies, antibody fragments and antibody conjugates which display a high degree of selectivity for squamous cell carcinoma antigens including carcinomas of the lung, esophagus and cervix. The present invention relates to both in vivo and in vitro clinical screening methods for diagnosis or prognosis of carcinomas by means of detecting the expression of squamous cell carcinoma antigens in biological samples of the subject using the novel antibodies of the invention. The invention further provides for kits for carrying out the above described screening methods. Additionally, antibody conjugates may be used to efficiently deliver various agents which have anti-tumor effects to the tumor cell. The antibodies of the invention may also be administered to a patient in non-conjugated form to target ADCC to the tumor cell.

Lung cancer is one of the more common malignancy effecting the population. Once a tumor is diagnosed, the overall prognosis is poor, with an overall five year survival rate of only 13%. However, early detection and treatment of lung cancer can significantly improve 5 year survival rates. In those cases where the disease is detected early and surgical resection is feasible, the five year survival rate increases to 40%. For those at high risk for development of lung cancer intensive monitoring has not been effective in reducing the incidence of the disease or its outcome due in part to the inability to define early stages of the disease when transformation is first occurring within normal cell populations of the tracheobronchial tree.
Monoclonal antibodies to tumor-associated antigens provide useful reagents for diagnosis of cancer and for targeting of various anti-tumor agents such as radioisotopes, chemotherapeutic drugs and toxins to the site of the tumor. Many monoclonal antibodies reactive against carcinoma-associated antigens are known. These known antibodies bind to a variety of different carcinoma-associated antigens that include glycoproteins but for the most part bind to the carbohydrate moiety. For example, monoclonal antibodies that bind to glycoprotein antigens on specific types of carcinomas include those described in U.S. Pat. Nos. 4,737,579; 4,753,894; 4,579,827 and 4,713,352.
Since many tumor cells shed their membrane glycoproteins into surrounding body fluids such as serum, or bronchial secretions in the case of squamous cell carcinomas, the possibility of detecting shed antigen using monoclonal antibodies in an ELISA is a possible approach to early detection. This is the approach taken for detection of tumor markers such as PSA and CEA in serum. As an example, supernatants of the bronchial lavage could be used in immunoassays designed for early detection of transformed cells in bronchial epithelium where shed antigen appears in the supernatant fluid. To date, assays designed to define tumor cells in sputum samples have proven to be ineffective because of the unavailability of monoclonal antibodies capable of differentiating between normal and abnormal squamous cells or to be able to define genotypically altered cells before the phenotypic changes of malignancy are evident.
Most studies have employed epithelial markers such as cytokeratin antibodies which are non-specific and will react with most if not all epithelial cells. Thus, better monoclonal antibody based diagnostic and prognostic markers and more sensitive tests for use in defining clinical lung cancer are needed. Ideally the monoclonal antibodies will detect the expression of a specific tumor antigen at an early time when clinical disease is not obvious. Such a tumor antigen defined by a monoclonal antibody could be used as a target for immunotherapy directed against the specific tumor antigen expressed on the surface of the cell. Such antibodies may also be of prognostic value based their ability to identify markers expressed at different stages of disease or that function differently, i.e., e-capherin, growth factor or receptor.

Identification and development of specific monoclonal antibodies to squamous cell carcinoma


The present invention is a unitary pipe coupler having an internally mounted hollow tube stiffener which extends through the coupler. The stiffener serves as a guide over which ends of the pipes slide as the pipes are inserted into the coupler and provides communication within the coupler between the two pipes. As the pipes are inserted into the coupler, each pipe passes consecutively through an opening provided in the coupler, an O-ring, a plurality of rigid backward oriented gripper serrations provided on a gripper, a plurality of backward oriented flexible sealing serrations provided on a front boot seal, until the end of each pipe rests against a flexible boot seal which abuts a hub for mounting the stiffener to the coupler. The gripper serrations prevent the pipes from being pulled or pushed outwardly away from the coupler.

1. Field Of The Invention
The present invention relates to a stab-type coupler designed to couple together plastic pipes. The pipes are slid into the coupler around opposite ends of a hollow tube stiffener provided in the coupler, and once inside the coupler, the pipes are held in fluidtight connection therewith by means of boot seals and grippers provided within the coupler.
2. Description of the Related Art
Stab-type couplers are designed to secure together pipes, usually plastic or other non-metallic types of pipes. Often these types of couplers are used at a construction site to fasten together pipes that are being installed underground. In such work environments, it is desirable to have a coupler that employs a minimum number of separate parts that can be dropped or lost. Also, it is desirable that the seal created between the coupler and the pipes be easy to form, for example, by a workman wearing gloves that limit his manual dexterity. In addition, the seal between the pipes and the coupler must be fluidtight, i.e. sealed against leakage of either liquids or gases, and must be secure enough so that the pipes will not become detached once they are coupled.
The present invention addresses these needs by providing a unitary coupler that seals to a pipe simply by sliding an end of the pipe into the coupler, thus forming a fluidtight seal between the pipe and the coupler. Also, the gripping means provided with the present invention is designed so that when external pulling force is exerted onto the pipe in order to remove the pipe from the coupler, the gripping means tightens against the exterior surface of the pipe, thus preventing the pipe from being pulled from the coupler. The tightening action of the gripping means around the pipe functions in a manner similar to the way toy "Chinese Handcuffs" tighten around a child's fingers as the child attempts to pull his fingers out of the Chinese handcuffs; the harder the child pulls, the tighter the Chinese handcuffs tighten around the fingers.
Also, the gripping means of the present invention is designed so that increasing pressure from within the tubes will also cause the gripping means to increasingly tighten around the exterior surface of the pipe, thus preventing the pipe from being blown out of the coupler.


There is provided an intra-realm AAA (authentication, authorization and accounting) fallback mechanism, wherein the single global realm may be divided in one or more sub-realms. The thus presented mechanism exemplarily comprises detecting a failure of an authentication server serving at least one authentication client within a first sub-realm of a single-realm authentication system, and routing authentication messages of the at least one authentication client to a fallback authentication server within a second sub-realm of the single-realm authentication system, wherein routing may exemplarily comprise sub-realm based source routing.

In modern and future communication networks including fixed and mobile telecommunication networks (such as for example Internet Protocol-based networks, Global System for Mobile Communication (GSM), General Packet Radio Service (CPRS), Universal Mobile Telecommunication System (UMTS), Long-Term Evolution (LTE) or the like) security and trustworthiness issues play an increasingly important role. Therefore, AAA (authentication, authorization and accounting) infrastructure is being usually deployed in or on top of such communication networks.
According to current techniques in the AAA field, such AAA infrastructure is organized and managed based on so-called realms (sometimes also referred to as domains) representing administrative entities within an overall AAA system of a single operator or provider. One known approach resides in that an overall AAA system of a single operator or provider comprises a single realm. That is, the entire AAA infrastructure in such an overall AAA system is organized and managed in a single overall realm (i.e. domain).
An organization or management of AAA infrastructure within a single realm may, however, have drawbacks in view of server failures.
In a large realm (such as e.g. "company.com"), which is operated e.g. by a large mobile operator, there may be regional sites including one or more AAA servers serving one or more AAA clients residing in the topological or geographical area covered by the respective regional site. Such regional sites do not represent administrative entities, but may be regarded as autonomous in that they share the same "operator-global" subscription data which is completely or at least partly replicated in respective AAA servers (and related databases etc.) of the individual regional sites. Especially in a large network (i.e. a large realm), for example in a nation-wide or even continent-wide deployment (such as that of an American or Chinese operator) of AAA infrastructure, regional sites with AAA servers and AAA clients may be topologically and geographically far away from each other. In case of a server failure in a regional site, the clients are to be delegated to a remote regional site as a backup operation.
While there are known certain fallback mechanisms in the context of AAA infrastructure failures, such as e.g. Diameter fallback mechanisms when Diameter is used as an AAA protocol, the known fallback mechanisms do not perform well or even do not work properly in a single-realm AAA system. That is, in case of a failure of an AAA server in a regional site within a single-realm AAA system, the known fallback mechanisms are not capable of ensuring an appropriate delegation of clients of the failed server to a backup server as well as an appropriate (i.e. instant and reliable) traffic handling in this regard. While this drawback exists in any single-realm AAA system, it is particularly adverse in large single-realm AAA systems (especially those in which regional sites are interconnected over an intra-operator network) because the known fallback mechanisms do not scale well and are error prone. In this regard, continent wide intra-operator networks could be mentioned as a non-limiting example for large single-realm AAA systems in question.
On the other hand, there are no standardized fallback mechanisms in other authentication environments, such as e.g. when RADIUS is used as an AAA protocol.
Accordingly, there does not exist any feasible solution for an intra-realm AAA fallback mechanism, in particular a fallback mechanism in case of a server failure within a (large) single-realm AAA infrastructure.


One or more shaped axial bore extending from an accessed posterior or anterior target point are formed in the cephalad direction through vertebral bodies and intervening discs, if present, in general alignment with a visualized, trans-sacral axial instrumentation/fusion (TASIF) line in a minimally invasive, low trauma, manner. An anterior axial instrumentation/fusion line (AAIFL) or a posterior axial instrumentation/fusion line (PAIFL) that extends from the anterior or posterior target point, respectively, in the cephalad direction following the spinal curvature through one or more vertebral body is visualized by radiographic or fluoroscopic equipment. Preferably, curved anterior or posterior TASIF axial bores are formed in axial or parallel or diverging alignment with the visualized AAIFL or PAIFL, respectively, employing bore forming tools that can be manipulated from proximal portions thereof that are located outside the patient""s body to adjust the curvature of the anterior or posterior TASIF axial bores as they are formed in the cephalad direction. Further bore enlarging tools are employed to enlarge one or more selected section of the anterior or posterior TASIF axial bore(s), e.g., the cephalad bore end or a disc space, so as to provide a recess therein that can be employed for various purposes, e.g., to provide anchoring surfaces for spinal implants inserted into the anterior or posterior TASIF axial bore(s).

The present invention relates generally to spinal surgery, particularly methods and apparatus for forming one or more shaped axial bore through vertebral bodies in general alignment with a visualized, trans-sacral axial instrumentation/fusion (TASIF) line in a minimally invasive, low trauma, manner.
It has been estimated that 70% of adults have had a significant episode of back pain or chronic back pain emanating from a region of the spinal column or backbone. Many people suffering chronic back pain or an injury requiring immediate intervention resort to surgical intervention to alleviate their pain.
The spinal column or back bone encloses the spinal cord and consists of 33 vertebrae superimposed upon one another in a series which provides a flexible supporting column for the trunk and head. The vertebrae cephalad (i.e., toward the head or superior) to the sacral vertebrae are separated by fibrocartilaginous intervertebral discs and are united by articular capsules and by ligaments. The uppermost seven vertebrae are referred to as the cervical vertebrae, and the next lower twelve vertebrae are referred to as the thoracic, or dorsal, vertebrae. The next lower succeeding five vertebrae below the thoracic vertebrae are referred to as the lumbar vertebrae and are designated L1-L5 in descending order. The next lower succeeding five vertebrae below the lumbar vertebrae are referred to as the sacral vertebrae and are numbered S1-S5 in descending order. The final four vertebrae below the sacral vertebrae are referred to as the coccygeal vertebrae. In adults, the five sacral vertebrae fuse to form a single bone referred to as the sacrum, and the four rudimentary coccyx vertebrae fuse to form another bone called the coccyx or commonly the xe2x80x9ctail bonexe2x80x9d. The number of vertebrae is sometimes increased by an additional vertebra in one region, and sometimes one may be absent in another region.
Typical lumbar, thoracic and cervical vertebrae consist of a ventral or vertebral body and a dorsal or neural arch. In the thoracic region, the ventral body bears two costal pits for reception of the head of a rib on each side. The arch which encloses the vertebral foramen is formed of two pedicles and two lamina. A pedicle is the bony process which projects backward or anteriorly from the body of a vertebra connecting with the lamina on each side. The pedicle forms the root of the vertebral arch. The vertebral arch bears seven processes: a dorsal spinous process, two lateral transverse processes, and four articular processes (two superior and two inferior). A deep concavity, inferior vertebral notch, on the inferior border of the arch provides a passageway or spinal canal for the delicate spinal cord and nerves. The successive vertebral foramina surround the spinal cord. Articulating processes of the vertebrae extend posteriorly of the spinal canal.
The bodies of successive lumbar, thoracic and cervical vertebrae articulate with one another and are separated by intervertebral discs formed of fibrous cartilage enclosing a central mass, the nucleus pulposus that provides for cushioning and dampening of compressive forces to the spinal column. The intervertebral discs are anterior to the vertebral canal. The inferior articular processes articulate with the superior articular processes of the next succeeding vertebra in the caudal (i.e., toward the feet or inferior) direction. Several ligaments (supraspinous, interspinous, anterior and posterior longitudinal, and the ligamenta flava) hold the vertebrae in position yet permit a limited degree of movement.
The relatively large vertebral bodies located in the anterior portion of the spine and the intervertebral discs provide the majority of the weight bearing support of the vertebral column. Each vertebral body has relatively strong bone comprising the outside surface of the body and weak bone comprising the center of the vertebral body.
Various types of spinal column disorders are known and include scoliosis (abnormal lateral curvature of the spine), kyphosis (abnormal forward curvature of the spine, usually in the thoracic spine), excess lordosis (abnormal backward curvature of the spine, usually in the lumbar spine), spondylolisthesis (forward displacement of one vertebra over another, usually in the lumbar or cervical spine) and other disorders, such as ruptured or slipped discs, degenerative disc disease, fractured vertebra, and the like. Patients who suffer from such conditions usually experience extreme and debilitating pain and often neurologic deficit in nerve function.
Approximately 95% of spinal surgery involves the lower lumbar vertebrae designated as the fourth lumbar vertebra (xe2x80x9cL4xe2x80x9d), the fifth lumbar vertebra (xe2x80x9cL5xe2x80x9d), and the first sacral vertebra (xe2x80x9cS1xe2x80x9d). Persistent low back pain is attributed primarily to degeneration of the disc connecting L5 and S1. Surgical procedures have been developed and used to remove the disc and fuse the vertebral bodies together and/or to stabilize the intervertebral structures. Although damaged discs and vertebral bodies can be identified with sophisticated diagnostic imaging, the surgical procedures are so extensive that clinical outcomes are not consistently satisfactory. Furthermore, patients undergoing presently available fusion surgery experience significant complications and uncomfortable, prolonged convalescence.
A number of devices and techniques involving implantation of spinal implants to reinforce or replace removed discs and/or anterior portions of vertebral bodies and which mechanically immobilize areas of the spine assisting in the eventual fusion of the treated adjacent vertebrae have also been employed or proposed over the years In order to overcome the disadvantages of purely surgical techniques. Such techniques have been used effectively to treat the above described conditions and to relieve pain suffered by the patient. However, there are still disadvantages to the present fixation implants and surgical implantation techniques. The historical development of such implants is set forth in U.S. Pat. Nos. 5,505,732, 5,514,180, and 5,888,223, for example.
One technique for spinal fixation includes the immobilization of the spine by the use of spine rods of many different configurations that run generally parallel to the spine. Typically, the posterior surface of the spine is isolated and bone screws are first fastened to the pedicles of the appropriate vertebrae or to the sacrum and act as anchor points for the spine rods. The bone screws are generally placed two per vertebra, one at each pedicle on either side of the spinous process. Clamp assemblies join the spine rods to the screws. The spine rods are generally bent to achieve the desired curvature of the spinal column. Wires may also be employed to stabilize rods to vertebrae. These techniques are described further in U.S. Pat. No. 5,415,661, for example.
These types of rod systems can be effective, but require a posterior approach and implanting screws into or clamps to each vertebra over the area to be treated. To stabilize the implanted system sufficiently, one vertebra above and one vertebra below the area to be treated are often used for implanting pedicle screws. Since the pedicles of vertebrae above the second lumbar vertebra (L2) are very small, only small bone screws can be used which sometimes do not give the needed support to stabilize the spine. These rods and screws and clamps or wires are surgically fixed to the spine from a posterior approach, and the procedure is difficult. A large bending moment is applied to such rod assemblies, and because the rods are located outside the spinal column, they depend on the holding power of the associated components which can pull out of or away from the vertebral bone.
In a variation of this technique disclosed in U.S. Pat. Nos. 4,553,273 and 4,636,217, both described in U.S. Pat. No. 5,735,899, two of three vertebrae are joined by surgically obtaining access to the interior of the upper and lower vertebral bodies through excision of the middle vertebral body. In the ""899 patent, these approaches are referred to as xe2x80x9cintraosseousxe2x80x9d approaches, although they are more properly referred to as xe2x80x9cinterosseousxe2x80x9d approaches by virtue of the removal of the middle vertebral body. The removal is necessary to enable a lateral insertion of the implant into the space it occupied so that the opposite ends of the implant can be driven upward and downward into the upper and lower vertebral bodies. These approaches are criticized as failing to provide adequate medial-lateral and rotational support in the ""899 patent. In the ""899 patent, an anterior approach is made, slots are created in the upper and lower vertebrae, and rod ends are fitted into the slots and attached to the remaining vertebral bodies of the upper and lower vertebrae by laterally extending screws.
A number of disc shaped replacements or artificial disc implants and methods of insertion have been proposed as disclosed, for example, in U.S. Pat. Nos. 5,514,180 and 5,888,223, for example. A further type of disc reinforcement or augmentation implant that has been clinically employed for spinal fusion comprises a hollow cylindrical titanium cage that is externally threaded and is screwed laterally into place in a bore formed in the disc between two adjacent vertebrae. Bone grafts from cadavers or the pelvis or substances that promote bone growth are then packed into the hollow center of the cage to encourage bone growth (or ingrowth) through the cage pores to achieve fusion of the two adjacent vertebrae. Two such cage implants and the surgical tools employed to place them are disclosed in U.S. Pat. Nos. 5,505,732 and 5,700,291, for example. The cage implants and the associated surgical tools and approaches require precise drilling of a relatively large hole for each such cage laterally between two adjacent vertebral bodies and then threading a cage into each prepared hole. The large hole or holes can compromise the integrity of the vertebral bodies, and if drilled too posteriorly, can injure the spinal cord. The end plates of the vertebral bodies, which comprise very hard bone and help to give the vertebral bodies needed strength, are usually destroyed during the drilling. The cylindrical cage or cages are now harder than the remaining bone of the vertebral bodies, and the vertebral bodies tend to collapse or xe2x80x9ctelescope,xe2x80x9d together. The telescoping causes the length of the vertebral column to shorten and can cause damage to the spinal cord and nerves that pass between the two adjacent vertebrae.
Methods and apparatus for accessing the discs and vertebrae by lateral surgical approaches are described in U.S. Pat. No. 5,976,146. The intervening muscle groups or other tissues are spread apart by a cavity forming and securing tool set disclosed in the ""146 patent to enable endoscope aided, lateral access to damaged vertebrae and discs and to perform corrective surgical procedures.
A compilation of the above described surgical techniques and spinal implants and others that have been used clinically is set forth in certain chapters of the book entitled Lumbosacral and Spinopelvic Fixation, edited by Joseph Y. Margolies et al. (Lippincott-Raven Publishers, Philadelphia, 1996). Attention is directed particularly to Chapters 1, 2, 17, 18, 38, 42 and 44. In xe2x80x9cLumbopelvic Fusionxe2x80x9d (Chapter 38, by Prof. Rene P. Louis, MD) techniques for repairing a spondylolisthesis, in this case, a severe displacement of L5 with respect to S1 and the intervening disc, are described and depicted. An anterior lateral exposure of L5 and S1 is made, a discectomy is performed, and the orientation of L5 to S1 is mechanically corrected using a reduction tool, if the displacement is severe. A fibula graft or metal Judet screw is inserted as a dowel through a bore formed extending caudally through L5 and into S1. When the screw is used, bone growth material, e.g., bone harvested from the patient, is inserted into the bore alongside the screw, and the disc space is filled with bone sutured to the screw to keep it in place between the vertebral surfaces to act as a spacer implant occupying the extracted disc between L5 and S1. External bridge plates or rods are also optionally installed. The posterolateral or anterior lateral approach is necessitated to correct the severe spondylolisthesis displacement using the reduction tool and results in tissue injury. Because of this approach and need, the caudal bore and inserted the Judet screw can only traverse L5 and S1.
A similar anterior approach for treating spondylolisthesis is disclosed in U.S. Pat. No. 6,056,749. In this approach, a bore hole is formed in a cephalad vertebral body and extends through the intervening disc into a caudal vertebral body, the disc is removed, a disk cage is inserted laterally into the disc space, and an elongated, hollow threaded shaft is inserted into the bore and through a hole in the disc cage. The disk cage takes the place of the harvested bone disc inserts and its interlocking intersection with the shaft takes the place of the sutures employed to tie the harvested bone disc inserts to the screw in the technique described in the above-referenced Chapter 38 publication.
The above-described spinal implant approaches involve highly invasive surgery that laterally exposes the anterior or posterior portions of the vertebrae to be supported or fused. Extensive muscular stripping and bone preparation can be necessary. As a result, the spinal column can be further weakened and/or result in surgery induced pain syndromes. Thus, presently used or proposed surgical fixation and fusion techniques involving the lower lumbar vertebrae suffer from numerous disadvantages. It is preferable to avoid the lateral exposure to correct less severe spondylolisthesis and other spinal injuries or defects affecting the lumbar and sacral vertebrae and discs.
A less intrusive posterior approach for treating spondylolisthesis is disclosed in U.S. Pat. No. 6,086,589, wherein a straight bore is formed through the sacrum from the exposed posterior sacral surface and in a slightly cephalad direction into the L5 vertebral body, preferably after realigning the vertebrae. A straight, hollow, threaded shaft with side wall holes restricted to the end portions thereof and bone growth material are inserted into the bore. A discectomy of the disc between L5 and S1 is preferably performed and bone ingrowth material is also preferably inserted into the space between the cephalad and caudal vertebral bodies. Only a limited access to and alignment of S1 and L5 can be achieved by this approach because the distal ends of the straight bore and shaft approach and threaten to perforate the anterior surface of the L5 vertebral body.
A wide variety of orthopedic implants have also been proposed or clinically employed to stabilize broken bones or secure artificial hip, knee and finger joints. Frequently, rods or joint supports are placed longitudinally within longitudinal bores made in elongated bones, e.g., the femur. A surgical method is disclosed in U.S. Pat. No. 5,514,137 for stabilizing a broken femur or other long bones using an elongated rod and resorbable cement. To accomplish a placement of a rod into any single bone, an end of a bone is exposed and a channel is drilled from the exposed end to the other end. Thereafter, a hollow rod is inserted, and resorbable cement is injected through the hollow rod, so as to provide fixation between the distal end of the rod and the cancellous tissue that surrounds the rod. A cement introducer device can also be used for the injection of cement. A brief reference is made in the ""137 patent to the possibility of placing rods in or adjacent to the spine in the same manner, but no particular approach or devices are described.
Drilling tools are employed in many of the above described surgical procedures to bore straight holes into the vertebral bones. The boring of curved bores in other bones is described in U.S. Pat. Nos. 4,265,231, 4,541,423, and 5,002,546, for example. The ""231 patent describes an elongated drill drive shaft enclosed within a pre-curved outer sheath that is employed to drill curved suture holding open ended bores into bones so that the suture passes through both open ends of the bore. The ""423 patent describes an elongated flexible drill drive shaft enclosed within a malleable outer sheath that can be manually shaped into a curve before the bore is formed. The ""546 patent describes a complex curve drilling tool employing a pivotal rocker arm and curved guide for a drill bit for drilling a fixed curve path through bone. All of these approaches dictate that the curved bore that is formed follow the predetermined and fixed curvature of the outer sheath or guide. The sheath or guide is advanced through the bore as the bore is made, making it not possible for the user to adjust the curvature of the bore to track physiologic features of the bone that it traverses.
The preferred embodiments of the invention involve methods and apparatus for forming one or more axial bore through spinal vertebral bodies for performing surgical procedures for receiving spinal implants, or for other medical reasons wherein the axial bore is shaped with one or more recess adapted to anchor spinal implants or receive material dispensed therein or for other purposes.
The preferred embodiments of the present invention involve methods and apparatus including surgical tool sets for first forming an anterior or posterior TASIF axial bore(s) extending from a respective anterior or posterior target point of an anterior or posterior sacral surface through at least one sacral vertebral body and one or more lumbar vertebral body in the cephalad direction. Then, further bore enlarging tools are employed to enlarge one or more selected section of the anterior or posterior TASIF axial bore(s), e.g., the cephalad bore end or a disc space along the bore, so as to provide a recess therein. The recess can be employed for various purposes, e.g., to provide anchoring surfaces for spinal implants inserted into the anterior or posterior TASIF axial bore(s), or to received materials placed into the recess formed in a disc space or vertebral body.
To form an axial bore, the anterior target point on the anterior sacral surface is accessed using a percutaneous tract extending from a skin incision through presacral space. The posterior target point on the posterior sacral surface is accessed using a surgical exposure of the posterior sacral surface. An anterior axial instrumentation/fusion line (AAIFL) or a posterior axial instrumentation/fusion line (PAIFL) that extends from the anterior or posterior target point, respectively, in the cephalad direction following the spinal curvature through one or more vertebral body is visualized by radiographic or fluoroscopic equipment. Preferably, curved anterior or posterior TASIF axial bores are formed in axial or parallel alignment with the visualized AAIFL or PAIFL, respectively, although the invention is not confined to curved axial bores.
When a single anterior or posterior TASIF axial bore is formed, it can be formed in axial or parallel alignment with the visualized axial AAIFL and PAIFL. Similarly, multiple anterior or posterior TASIF axial bores can be formed all in parallel alignment with the visualized axial AAIFL and PAIFL or with at least one such TASIF axial bore formed in axial alignment with the visualized axial AAIFL and PAIFL.
Moreover, multiple anterior or posterior TASIF axial bores can be formed all commencing at the anterior or posterior target point and extending in the cephalad direction with each TASIF axial bore diverging apart from the other and away from the visualized axial AAIFL and PAIFL. The diverging TASIF axial bores terminate as spaced apart locations in a cephalad vertebral body or in separate cephalad vertebral bodies.
In certain embodiments, small diameter anterior and posterior TASIF axial bore forming tools can be employed in the same manner to form pilot holes extending in the cephalad direction through one or more sacral and lumbar vertebral bodies in alignment with the visualized AAIFL and PAIFL. The pilot holes can be used as part of anterior and posterior percutaneous tracts that are subsequently enlarged to form the anterior and posterior TASIF axial bores.
This summary of the invention and the objects, advantages and features thereof have been presented here simply to point out some of the ways that the invention overcomes difficulties presented in the prior art and to distinguish the invention from the prior art and is not intended to operate in any manner as a limitation on the interpretation of claims that are presented initially in the patent application and that are ultimately granted.

Methods and apparatus for forming shaped axial bores through spinal vertebrae


A method is provided which includes on-chip identification of individual die. The first wafer sort includes the steps of programming a plurality of dice on a wafer, programming predetermined memory memory cells on each good die to identify the wafer on which that die is located, and storing the location of each good die in a file created for each wafer. Then, the plurality of dice are subjected to predetermined conditions. In the second wafer sort, predetermined memory cells on one die are accessed to determine the associated file of that die. The associated file is then loaded. Finally, the good dice are tested. In another embodiment, the first wafer sort includes identifying the first good die on the wafer. After the next good die on the wafer is found, that die is programmed to indicate the location of the proceeding good die. This programming step is repeated until the last good die on the wafer is programmed. Once again, the wafer is subjected to adverse conditions. During the second wafer sort, the last good die on the wafer is identified and tested. Then, the next die location indicated by the tested die is tested. These steps of indicating the next die and testing that die are repeated until the first good die is tested. The present invention significantly reduces valuable test time by an amount directly related to the initial yield. Moreover, the present invention reduces wear and tear on the test equipment itself, thereby increasing the longevity of this expensive piece of equipment.

1. Field of the Invention
This invention relates to testing of dice on a wafer, and in particular to reducing the testing of dice with on-chip identification of good dice.
2. Description of the Related Art
Wafer sorting is a well-known process performed after wafer fabrication. During wafer sorting, each die on the wafer is electrically tested for electrical performance and circuit functioning. Thus, wafer sorting provides information on the effectiveness of the wafer fabrication process in producing working dice (i.e. chips) and provides identification of working dice which will be used in a subsequent packaging process.
FIGS. 1A and 1B show a typical wafer sort process including testing of all dice during a second sort. As is well-known to those in the art, each die typically includes a plurality of memory cells, in this example electrically programmable read only memory (EPROM) cells. An EPROM cell includes a source and a drain region separated by a channel region. A floating gate is formed over the channel region, while a control gate is formed over the floating gate. Referring to FIGS. 1A and 1B, all memory cells on a die are programmed in step 11 by varying the charge on the floating gate, thereby changing the threshold voltage required to allow a current to flow between the source and drain regions.
In step 12, the programming of the memory cells on the die is verified with a probe to ensure all memory cells exhibit an appropriate threshold voltage. If any of the memory cells on the die fail to exhibit the appropriate threshold voltage, then a number in a counter increments to indicate that a programming attempt has occured. This number in the counter is compared to a predetermined, maximum number of attempts set by the tester. If the number in the counter is less than or equal to the predetermined maximum number, all the memory cells on the die are reprogrammed. For simplicity, this comparing and reprogramming is included in verification step 12. Step 12 is typically repeated until either all the memory cells are satisfactorily programmed, or until the predetermined maximum number of attempts has been exceeded. At this time, if the die is "good" (i.e. all the memory cells exhibit the appropriate threshold voltage) as determined in step 13, the die is electronically inked. Electronic inking is a well-known method in the art in which one or more memory cells on the die are programmed to indicate a particular testing pattern for the probe in the second wafer sort (described below). If the die is "bad" (as determined in step 13) or after the probe programs and verifies the electronic inking of the die (as performed in step 14), the probe determines in step 15 if that die is the last die on the wafer. If that die is not the last die, the probe proceeds to the next die in step 16. However, if that die is the last die, then the probe proceeds in step 18 to the next wafer (if identified in step 17). Steps 11-18 constitute the first wafer sort 10.
After first wafer sort 10 is completed, the wafers are placed under predetermined, adverse conditions 20 to accelerate charge loss, thereby simulating memory use over, for example, a twenty year period. Typically, these predetermined, adverse conditions include baking the wafers as shown in step 21.
Second wafer sort 30 begins in step 31 by reading a first die for electronic ink. If the die is electronically inked, as determined in step 32, the probe tests the memory cells on the die in step 33 to determine the degree of charge loss. If the charge loss of a particular die exceeds a predetermined level as determined in step 34, that die is physically inked in step 37 (indicating the die is rejected for packaging). If, on the other hand, the charge loss is less than or equal to that predetermined level (i.e. the die is "good" and is ready to be used in the subsequent packaging process), then the probe determines if another die is present in step 35. If another die is present, the probe proceeds to the next die in step 36. Steps 31-37 are repeated until the last die on the wafer is found. Then, the probe moves to the next wafer in step 39 (if determined to be present in step 38). Otherwise, the probe stops in step 40.
This conventional method includes unnecessary testing time because all dice, whether or not successfully programmed in first wafer sort 10 (i.e. determined to be good or bad) are read for electronic ink in second wafer sort 30. Therefore, a need arises for a method of reducing the test time of dice on a wafer.

Method of reducing dice testing with on-chip identification


A composite material comprising a metal matrix containing tungsten grain is roduced from tungsten powders formed by plasma rapid solidification. The powders comprise tungsten and up to 20 weight percent of a metal selected from the group consisting of molybdenum, tantalum, niobium, rhenium, and chromium. The surfaces of the powders are cleaned to reduce the surface oxide thereon, and the powders are coated with at least one metal selected from the group consisting of copper, nickel, cobalt, hafnium and tantalum. The coated powders are formed into a sintered preform which is less than fully dense, and the sintered preform is further consolidated to full density by a technique selected from hot isostatic pressing, rapid omin-directional compaction, and hot extrusion.

Tungsten heavy alloy materials are known in the art for use in various applications, including, among others, ballistic devices. In the past, tungsten heavy alloys have been fabricated by liquid phase sintering of mixed elemental powders. The material resulting from such liquid phase sintering generally comprises a two phase composite consisting of rounded tungsten grains dispersed in an alloy matrix. While resulting materials have exhibited sintered densities in excess of 99.5 percent of the theoretical density values, and high ductility and strength, it has been difficult to control the property uniformity and to consistently provide the maximum attainable properties.
Generally, the mechanical properties of the alloy materials are strongly dependent on the specific microstructural characteristics of the materials. The most prominent of these characteristics are the contiguity, the dihedral angle and the volume fraction of the tungsten phase. Ideally, for a given tungsten content, the optimal microstructure should exhibit low contiguity, a low dihedral angle, small grain size and strong tungsten-tungsten grain boundaries and tungsten-matrix interface. However, in practice, it appears that there is a clear tradeoff of these properties. For example, it appears that a low dihedral angle can be induced by higher tungsten solubility in the matrix phase which is possible through alloying or the use of higher sintering temperatures. Conversely, there is grain growth penalty owing to the use of higher sintering temperatures. Accordingly, it would be advantageous to provide a means for independently controlling the grain size, the dihedral angle and the contiguity.
Tungsten heavy alloy materials have also been produced using solid state sintering methods whereby finer microstructures, for example, of from two to three .mu.m tungsten particle size, are obtained as compared with the liquid phase sintered products, having tungsten particle sizes of from 30 to 50 .mu.m. However, solid state sintering provides materials having high contiguity and therefore the materials are extremely brittle. The use of very fine powders, for example, 0.1 .mu.m in diameter, results in materials having a microstructure exhibiting low ductility.
Thus, a need exists for improved tungsten heavy alloy materials which have both fine grain size and low contiguity, and therefore exhibit both high strength and good ductility, and methods for the production of such materials.

Methods are producing composite materials of metal matrix containing tungsten grain

The United States of America as represented by the Secretary of the Army


Disclosed is a method for eliminating access to data on removable storage media of a removable storage media cartridge. A key is stored on the removable storage media cartridge, such that data on the removable storage media is accessible with the key. Upon receiving a command to eliminate access to data on the removable storage media the key is shredded such that access to data on the removable storage media is eliminated.

Protecting and securing data is one of the primary concerns that must be addressed when designing an information management system, whether for a single user, small business or large scale data warehouse. Oftentimes, data may be continually archived on various removable storage medium, such as tape cartridges or optical disks. When archiving data on tape or other removable storage medium, one security concern is that the storage medium may be lost or stolen, especially during transport, and sensitive data on the storage medium may be discovered. Also, if the storage medium can be accessed through remote commands transmitted over a network, then there is a concern that someone may "hack" into the system, access the storage medium and then access the data.
Data on removable storage media, such as medical records, financial records or other archived information, must be kept for a certain retention period, however beyond that retention period the media that contains the data must be securely destroyed. Further, when it is detected that a particular piece of media is defective or unreliable (i.e. there are a certain number of errors detected during reading and/or writing by the data storage drive, library, or host computer), data on the defective media should be transferred to another piece of the media. The defective piece of media may still have some readable data and may need to be securely destroyed.
To protect confidential or sensitive data (e.g. credit card information, medical records, social security numbers, etc.) and to maintain customer privacy it is advantageous to perform a shredding process on certain storage media once it is no longer useful so that it is no longer accessible or recoverable. Shredding is defined herein as destroying or otherwise rendering data stored on a removable storage media permanently unreadable by any reasonable means.
Some customers may erase or overwrite the media to avoid unwanted access to the data contained on the media. To securely overwrite media, such as magnetic tape media, the entire tape should be overwritten one or more times utilizing a data pattern that contains one of logical ones, zeroes, or some combination thereof. Tape cartridges can hold up to a terabyte of information and thus, the process to erase or overwrite just one piece of media can take hours, consuming valuable resources. This burden on resources can be especially troublesome during bulk erase processes when large amounts of media must be erased.
Other customers may pay a 3rd party to properly destroy the media by a mechanical process such as by melting, burning, or degaussing. However, this option can be very costly as well as open the removable media up to aforementioned risks during transport. In view of the foregoing, there is a need in the art for a method to securely erase and eliminate access to data on removable storage media that is both inexpensive and does not usurp valuable resources.

Bulk data erase utilizing an encryption technique

International Business Machines Corporation