The present invention concerns a nucleic acid which is capable of regulating the transcription of the ABC1 gene, which is a causal gene for pathologies linked to a dysfunctioning of cholesterol metabolism, inducing diseases such as atherosclerosis. The invention also relates to nucleotide constructs comprising a polynucleotide which encodes a polypeptide or a nucleic acid of interest, placed under the control a regulatory nucleic acid for the ABC1 gene. The invention also relates to recombinant vectors, transformed host cells and nonhuman transgenic mammals comprising a nucleic acid which regulates the transcription of the ABC1 gene or an abovementioned nucleotide construct, as well as methods for screening molecules or substances which are capable of modifying the activity of the regulatory nucleic acid for the ABC1 gene.

The present invention concerns a nucleic acid which is capable of regulating the transcription of the ABC1 gene, which is a causal gene for pathologies linked to a dysfunctioning of cholesterol metabolism, inducing diseases such as atherosclerosis.
The invention also relates to nucleotide constructs comprising a polynucleotide which encodes a polypeptide or a nucleic acid of interest, placed under the control of a regulatory nucleic acid for the ABC1 gene.
The invention also relates to recombinant vectors, transformed host cells and non-human transgenic mammals comprising a nucleic acid which regulates the transcription of the ABC1 gene or an above-mentioned nucleotide construct, as well as to methods for screening molecules or substances which are capable of modifying the activity of the regulatory nucleic acid for the ABC1 gene.
The invention also relates to methods for detecting an impairment in the transcription of the ABC1 gene in an at-risk individual.
A subject of the invention is also substances or molecules which modify the activity of the nucleic acid which regulates the transcription of the ABC1 gene, as well as pharmaceutical compositions containing such substances or such molecules.
High density lipoproteins (HDLs) are one of the four major classes of lipoprotein which circulate in the blood plasma.
These lipoproteins are involved in various metabolic pathways, such as lipid transport, bile acid formation, steroidogenesis or cell proliferation, and also interfere with plasmatic proteinase systems.
HDLs are perfect free cholesterol acceptors, and in combination with cholesterol ester transfer proteins (CETP), lipoprotein lipase (LPL), hepatic lipase (HL) and lecithin-cholesterol acyltransferase (LCAT), play a major role in the reverse transport of cholesterol, i.e. the transport of excess cholesterol in the peripheral cells to the liver, for its removal from the body in the form of bile acid.
It has been demonstrated that the HDLs generally play a central role in the transport of cholesterol from the peripheral tissues to the liver.
Various diseases linked to an HDL deficiency have been described, including Tangier disease, HDL deficiency and LCAT deficiency.
The deficiency involved in Tangier disease is linked to a cellular defect in the translocation of cellular cholesterol, which leads to a degradation of the HDLs.
In Tangier disease, this cellular defect leads to a disruption of lipoprotein metabolism. The HDL particles in Tangier disease, which do not incorporate cholesterol from the peripheral cells, and which are not able to be correctly metabolized, are rapidly eliminated from the body. The plasma HDL concentration in these patients is thus extremely reduced, and the HDLs no longer contribute to the return of cholesterol to the liver. This cholesterol accumulates in these peripheral cells and causes characteristic clinical manifestations such as the formation of orange-colored tonsils. Furthermore, other lipoprotein disruptions such as an overproduction of triglycerides and an increased synthesis and intracellular catabolism of phospholipids are generally observed.
Tangier disease, the symptoms of which have been described above, is classified among the familial conditions linked to metabolism of the HDLs which are commonly detected in patients affected by coronary diseases.
Numerous studies have shown that a reduced level of HDL cholesterol is a risk factor which is useful for detecting a coronary condition.
In this context, syndromes linked to HDL deficiencies have been of increasing interest for the past decade, since they make it possible to increase the understanding of the role of HDLs in atherogenesis.
Several mutations in the apo A-1 gene have been characterized. These mutations are rare and can lead to an absence of production of apo A-1.
Mutation in the genes encoding lipoprotein lipase (LPL) or its activator apoC-II are associated with severe hypertriglyceridemias and substantially reduced levels of HDL-c.
Mutations in the gene encoding the enzyme lecithin-cholesterol acyltransferase (LCAT) are also associated with severe HDL deficiency.
Furthermore, dysfunctions in the reverse transport of cholesterol might be induced by physiological deficiencies affecting at least one of the steps for transporting stored cholesterol from the intracellular vesicles toward the membrane surface, where it is accepted by the HDLs.
Recently, a study was carried out on the segregation of various allelic forms of 343 microsatellite markers distributed over the entire genome and distant from each other by 10.3 cM on average.
The linkage study was carried out on a family which had been well characterized over eleven generations, in which many members are affected by Tangier disease, the family comprising five consanguineous lines.
This study made it possible to identify a region located in the 9q31 locus of human chromosome 9 which is statistically linked to the condition (Rust S. et al., Nature Genetics Vol. 20, September 1998, pages 96-98).
However, the study by Rust et al. only characterizes a wide region of the genome in which impairments are likely to be associated with Tangier disease. The study simply stated that the relevant 9q31-34 region contains ESTs, but no known gene.
It has been shown that a region spanning 1 cM, situated in the 9q31 locus in humans, is generally associated with familial HDL deficiencies (Rust et al., 1999).
Furthermore, it has been shown that a gene encoding a protein of the family of ABC transporters, which is located precisely in the 1 cM region of the 9q31 locus, is involved in pathologies linked to a deficiency in the reverse transport of cholesterol.
For example, it has been shown that the gene encoding the ABC-1 transporter is mutated in patients with affected reverse transport of cholesterol, such as in patients suffering from Tangier disease.
The ABC ("ATP-binding cassette") transporter proteins constitute a family of proteins which are extremely conserved in evolution, from bacteria to humans.
The ABC transporter proteins are involved in the membrane transport of various substrates, for example, ions, amino acids, peptides, sugars, vitamins or steroid hormones.
The characterization of the complete amino acid sequence of some ABC transporters has made it possible to determine that these proteins have a common general structure, for example, two nucleotide-binding folds (Nucleotide Binding Fold or NBF) with moieties of Walker A type and Walker B type, as well as two transmembrane domains, each of the transmembrane domains consisting of six helices. The specificity of the ABC transporters for the various transported molecules appears to be determined by the structure of the transmembrane domains, whereas the energy required for the transport activity is provided by degrading ATP at the NBF fold.
Several of the ABC transporter proteins which have been identified in humans have been associated with various diseases.
For example, cystic fibrosis is caused by mutations in the CFTLR (cystic fibrosis transmembrane conductance regulator) gene.
Moreover, some multi-drug resistance phenotypes in tumor cells have been associated with mutations in the gene encoding the MDR (multi-drug resistance) protein which also has an ABC transporter structure.
Other ABC transporters have been associated with neuronal and tumor conditions (U.S. Pat. No. 5,858,719), or potentially implicated in diseases caused by an impairment of the homeostasis of metals, for example, the ABC-3 protein.
Similarly, another ABC transporter, referred to as PFIC2, seems to be involved in a form of progressive familial intrahepatic cholestasis, this protein being potentially responsible, in humans, for the export of bile salts.
In 1994, a cDNA encoding a novel mouse ABC transporter was identified and referred to as ABC1 (Luciani et al., 1994). This protein is characteristic of the ABC transporters in that it has a symmetrical structure comprising two transmembrane domains linked to a highly hydrophobic segment and to two NBF moieties.
In humans, a partial cDNA comprising the entire open reading frame of the human ABC1 transporter has been identified (Lanigmann et al., 1999).
It has also been shown that the gene encoding the human ABC1 protein is expressed in various tissues, and more particularly at high levels in the placenta, the liver, the lungs, the adrenal glands and the fetal tissues.
These authors have also shown that the expression of the gene encoding the human ABC1 protein is induced during the differentiation of monocytes into macrophages in vitro. Furthermore, the expression of the gene encoding the ABC1 protein is increased when human macrophages are incubated in the presence of acetylated low-density lipoproteins (AcLDLs).
The work of Rust S. et al., 1999, Brooks-Wilson A. et al., 1999, Bodzioch M. et al., 1999, Remaley A. et al., 1999 and of Marcil M. et al., 1999 has shown that patients suffering from Tangier disease and from HDL deficiencies (FHD; familial HDL deficiency) have a mutated ABC1 gene. Several mutations, which are distributed in various regions of the ABC1 gene, have been identified in the genome of various patients, for example, of patients with a severe form of the disease associated with coronary disorders. Moreover, diverse polymorphisms have been found, both in the exons and in the introns of the ABC1 gene, in patients suffering from milder forms of the disease, indicating that these patients carry specific alleles of the gene, which are distinct from the "wild-type" allele(s).
Although the expression of the human ABC1 gene seems to be regulated according to the type of cell or to the metabolic situation of a given cell type, the sequence(s) which make(s) it possible to regulate this gene were not known.
Thus, there exists a need in the state of the art to identify these regulatory sequences, for the two principal reasons below:
a) These sequences are likely to be mutated in patients suffering from a pathology linked to a deficiency in cholesterol transport, for example, in patients suffering from Tangier disease, or likely to develop such pathologies.
The characterization of the regulatory sequences of the human ABC1 gene would make it possible, firstly, to detect mutations in patients, and, for example, also to diagnose the individuals who belong to at-risk familial groups. In addition, the isolation of these regulatory sequences would make it possible to complement the mutated sequence with a functional sequence capable of overcoming the metabolic dysfunctions induced by the mutation(s) diagnosed, through the construction of targeted therapeutic means, such as means intended for gene therapy.
b) The characterization of the regulatory sequences of the ABC1 gene would place at the disposal of persons skilled in the art means capable of allowing the construction, by genetic engineering, and then the expression, of given genes in the cell types in which the ABC1 gene is expressed.
c) Moreover, some portions of the regulatory sequences of the ABC1 gene might constitute high expression-level constitutive promoter sequences, which are liable to enable the construction of novel means for expressing given sequences in cells, completing an already existing set of means.
To date, despite the efforts undertaken, the regulatory sequences of the ABC1 gene have remained totally unknown.
The inventors have henceforth isolated and then sequenced a genomic DNA comprising the first two exons of the ABC1 gene (respectively exon 1A and exon 1B), as well as a non-transcribed region of approximately 2.9 kb, which is located on the 5′ side of exon 1A, and which comprises regulation signals for the ABC1 gene.

Regulatory nucleic acid for the ABC1 gene, molecules modifying its activity and therapeutic uses


The present invention relates to an isolated polynucleotide coding for the lrp gene and to a process for the fermentative production of L-amino acids by altering the expression of this gene.

L-amino acids are used in animal nutrition, in the food industry, in human medicine and in the pharmaceuticals industry. It is known that these amino acids are produced by fermentation of strains of coryneform bacteria in particular Corynebacterium glutamicum. Due to their great significance, efforts are constantly being made to improve the production process. Improvements to the process may relate to measures concerning fermentation technology, for example stirring and oxygen supply, or to the composition of the nutrient media, such as for example sugar concentration during fermentation, or to working up of the product by, for example, ion exchange chromatography, or to the intrinsic performance characteristics of the microorganism itself.
The performance characteristics of these microorganisms are improved using methods of mutagenesis, selection and mutant selection. In this manner, strains are obtained which are resistant to antimetabolites, such as for example the lysine analogue S-(2-aminoethyl)cysteine, or are auxotrophic for regulatorily significant amino acids and produce L-amino acids. For some years, the methods of recombinant DNA technology have also been used for strain improvement of strains of Corynebacterium which produce L-amino acid.
LRP (leucine-responsive protein) is a global regulator, first described in Escherichia coli, which influences the transcription of a series of genes, the gene products of which are involved in the transport, biosynthesis and degradation of amino acids (Calvo et al, Microbiol. Rev. 58:466–490 (1994)). In recent years, similar genes have also been identified in other organisms, such as Bradyrhizobium japonicum (King, et al., J. Bacteriol. 179:1828–1831 (1997)), Klebsiella aerogenes (Janes, et al, J. Bacteriol. 181:1054–1058 (1999)), Sulfolobus acidocaldarius (Charlier et al., Gene 201:63–68 (1997)) and in the gram positive bacterium Bacillus subtilis (Belitsky, et al., J. Bacteriol. 179:5448–5457 (1997)).
In E. coli, the lrp protein regulates its own expression. Lrp also has an either negative or positive influence on many genes in E. coli. In general, it is the expression of gene products which are active in biosynthetic pathways which is stimulated. Gene products having a catabolic action are generally correspondingly negatively controlled. In some cases, the action of lrp is potentiated by the addition of L-leucine, but addition of L-leucine may also have a negative effect (Newman, et al., In: Neidhardt et al. Escherichia coli and Salmonella typhimurium: Cellular and molecular biology, American Society for Microbiology, Washington D.C., pp. 1513–1525 (1996)). In E. coli, lrp regulates a large number of genes and operons which play a central role in amino acid biosynthesis and amino acid catabolism. The following operons in E. coli are, for example, negatively controlled: livJ, which codes for a binding protein in a highly sophisticated uptake system for branched-chain amino acids (Haney, et al., J. Bacteriol. 174:108–115 (1992)) and lysU, which codes for lysine tRNA synthetase (Gazeau, et al., FEBS Letters 300:254–258 (1994)). Genes which have hitherto been known to be positively influenced in E. coli include, inter alia, ilvIH, gltBDF and leuABCD (Lin, et al., J. Bacteriol. 174:1948–1955 (1992)).
The last-stated operon is of fundamental interest in leucine biosynthesis and of particular interest for lysine biosynthesis in Corynebacterium glutamicum. It is suspected that there is an association between leucine auxotrophy and elevated lysine productivity values (Schrumpf et al. Appl. Microbiol. Biotech. 37:566–571 (1992)). Patek, et al. (Appl. Environ. Microbiol. 60:133–140 (1994)) have demonstrated that inactivating leuA in some lysine producers of C. glutamicum results in increased lysine yields. In a mutant of Brevibacterium lactofermentum, Tosaka, et al. (Agri. Biol. Chem. 43:265–270 (1979)) have been able to achieve a reduction in lysine formation with a simultaneous increase in threonine formation by addition of L-leucine.

Nucleotide sequence encoding corynebacterium glutamicum leucine response regulatory protein


A steerable distal support system for accessing stenosis, partial occlusions, or complete occlusions within a body lumen. The steerable distal support system generally includes an elongate member that comprises a proximal portion, a more flexible intermediate portion and a deflectable distal tip. The deflectable distal tip is at a distal end of the elongate body to facilitate directionality and positioning of the steerable distal support system to the target site. Optionally, an expandable centering assembly may be disposed on the steerable support assembly to center and anchor the steerable support assembly within the body lumen.

The present invention is generally related to medical devices and methods. More specifically, the present invention provides a steerable distal support system for accessing stenosis, partial occlusions, or total occlusions in a patient's body lumen.
Cardiovascular disease frequently arises from the accumulation of atheromatous material on the inner walls of vascular lumens, particularly arterial lumens of the coronary and other vasculature, resulting in a condition known as atherosclerosis. Atheromatous and other vascular deposits restrict blood flow and can cause ischemia which, in acute cases, can result in myocardial infarction or a heart attack. Atheromatous deposits can have widely varying properties, with some deposits being relatively soft and others being fibrous and/or calcified. In the latter case, the deposits are frequently referred to as plaque. Atherosclerosis occurs naturally as a result of aging, but may also be aggravated by factors such as diet, hypertension, heredity, vascular injury, and the like.
Atherosclerosis can be treated in a variety of ways, including drugs, bypass surgery, and a variety of catheter-based approaches which rely on intravascular widening or removal of the atheromatous or other material occluding the blood vessel. Particular catheter-based interventions include angioplasty, atherectomy, laser ablation, stenting, and the like. For the most part, the catheters used for these interventions must be introduced over a guidewire, and the guidewire must be placed across the lesion prior to catheter placement. Initial guidewire placement, however, can be difficult or impossible in tortuous regions of the vasculature. Moreover, it can be equally difficult if the lesion is total or near total, i.e. the lesion occludes the blood vessel lumen to such an extent that the guidewire cannot be advanced across.
For these reasons, it is desired to provide devices and methods which can access small, tortuous regions of the vasculature. In particular, it is desired to provide systems which can access partial occlusions, total occlusions, stenosis, blood clots, or thrombotic material.


Core/shell impact modifiers, useful in toughening poly(vinyl chloride) and other thermoplastics, are prepared at small particle sizes by an emulsion polymerization process, agglomerated by a novel technique which has little effect on emulsion solids, are further encapsulated by a final shell, and are isolated by spray-drying or coagulation. The isolated particles are readily re-dispersed to their original particle size in the matrix polymer.

It has been known for over 20 years in the field of impact modifiers for poly(vinyl chloride) (PVC) that larger particle rubber cores sometimes are desirable for improved impact in core/shell impact-modified PVC. Especially with butadiene-based cores, it is a lengthy process to enlarge the particle size of the core by further polymerization of more butadiene (or copolymer of butadiene with styrene) in the presence of the core under conditions where no new particles are formed (the "grow-out" process), although such processes are readily accomplished with acrylic rubber cores. An alternate method is to agglomerate the rubber particles by what is essentially a controlled partial coagulation by adjustment of pH, salt content, and the like, so as to give a cluster of particles, which then are used as a substrate onto which is polymerized one or more hard stages or shells. The so-agglomerated rubber behaves in imparting impact and in optical microscopy in a manner similar to grow-out particles of similar particle size.
However, there are also reasons in impact modification of PVC to prepare core/shell impact modifiers wherein the rubber is of relatively low particle size. These reasons include systems, i.e., processed PVC compounds where high clarity is desired, or where it is desired to pigment the system with avoidance of a washed-out or pearlescent appearance. These reasons further include the field of acrylic-based impact modifiers, where surprisingly particle sizes in the range of 50 to 100 nm are effective modifiers. However, such systems involving small particle sizes, especially if high in rubber content, are difficult to prepare at high solids, and are difficult to isolate by means such as coagulation or spray-drying. Post-addition approaches or co-spray-drying approaches have been utilized, but often dilute the efficacy of the impact modifier.
Further, there are other thermoplastics where smaller particle rubbers are preferred for a particular balance of impact properties, processing, and surface appearance, such as those polymers which do not craze on impact, such as polyesters.
Thus the art has sought a means for preparing core/shell impact modifiers for PVC and other thermoplastics wherein the rubbery core content is high, wherein the particle size of the modifier is small, wherein the material can conveniently and inexpensively be isolated in powdery form, and wherein the polymer particles readily redisperse to the initial small particle size.


The present application is directed to methods and devices for controlling charge buildup on a toner image as the toner image passes through one or more transfer nips. Charge buildup may be reduced by laterally offsetting a transfer roller from a photoconductor drum. The transfer roller may be constructed of an essentially non-compressible conductive material. AC current may be used to generate an electrical field between the photoconductor drum and the transfer roller.

The present application relates generally to electrophotographic image forming devices, and in particular to a toner transfer apparatus to control charge buildup in a toner image as the toner image passes through one or more image transfer stations.
Electrophotographic image forming devices, such as laser printers, facsimile machines, copiers, all-in-one devices, etc, are well known in the art. Color electrophotographic image forming devices may form a plurality of latent electrostatic images, develop each color plane image with toner particles, and ultimately transfer the color plane images to a media sheet and then fuse them to the media sheet using heat and pressure. Color electrophotographic image forming devices may be divided into two types by considering how toner is transferred to the media sheet. In a direct to media (DTM) type image forming device, the developed toner image of each color plane is successively transferred directly to the media sheet. In an intermediate transfer mechanism (ITM) type image forming device, the developed toner image of each color plane is successively transferred to an intermediate transfer mechanism, such as a belt, and then the full-color image is transferred to a media sheet at a secondary transfer location.
One known problem that particularly affects ITM type image forming devices is charge buildup on the developed toner on the ITM as the toner passes successively through high-voltage image transfer stations. Toner which has passed through multiple image transfer stations may be at a different charge than toner which has not passed through any additional image transfer stations. When the toner image is transferred to the media sheet at the secondary transfer location, the toner that is less charged may transfer at a lower voltage than more highly charged toner. In order to transfer the entire toner image, a voltage high enough to affect the transfer of the most highly charged toner is used. High transfer voltages may create a phenomenon known as Paschen breakdown. In Paschen breakdown, toner particles reverse polarity and their placement becomes unpredictable. The toner particles may even backtransfer from the media sheet to the ITM. Backtransfer detrimentally impacts image quality.

Methods and devices to transfer toner in an image forming device to control charge buildup on a toner image


A sharpening apparatus having at least one guide slot, a drive pulley drivingly coupled to a frame and at least one idler pulley rotatably coupled to a support member. The support member is pivotably coupled to the frame. The apparatus includes an endless abrading belt rotatable in a path over the drive pulley and the at least one idler pulley and a biasing member coupled to the frame for biasing the support member toward an engaged position. Pivotal movement of the support member adjusts tension in the belt. A tool is traversed into the guide slot for sharpening a cutting edge thereof. The belt and support member respond to application and release of a force applied to the belt by the cutting edge to decrease and increase, respectively, belt speed to preclude overheating of the cutting edge and damage to the belt and cutting edge.

Knife sharpening apparatuses of the type having an abrasive belt and means for supporting and driving the belt along an endless path are well known. It is also well known to use such machines for sharpening knives and other tools having elongated cutting edges. For a knife or the like to be optimally sharpened with such machines the cutting edge must engage the abrasive belt at an appropriate angle and with an appropriate amount of force applied to keep the cutting edge in contact with the belt. During the sharpening procedure heat is generated at and near an area of engagement between the cutting edge of the knife and the abrasive belt. If the applied force is too great, the cutting edge overheats resulting in a change in the microstructure of the cutting edge and a reduction of the surface hardness of the cutting edge. In addition, excessive force applied to the belt by the cutting edge can cause accelerated belt wear. On the other hand, if the applied force is inadequate to keep the cutting edge in contact with the belt, the sharpening procedure may require an undue amount of time. The cutting edge is an intersection of opposing faces of the knife disposed at a predetermined angle from one another. To optimize performance of the cutting edge, the angle between the opposing faces of the knife must be held within close tolerances at the cutting edge. Therefore, angular positioning of the knife relative to the belt affects sharpening quality.
To aid in the proper positioning of and the application of proper force to a knife being sharpened it is known to use a rest having a guide surface inclined with respect to the path of travel of the belt. It is also known that the guide surface includes an index mark established, usually by trial and error, on the surface indicating the point to which a knife is to be moved for proper sharpening. However, this does not preclude the possibility of the knife being accidentally or intentionally moved beyond the index mark, creating overheating of the cutting edge, belt damage and other problems.
U.S. Pat. No. 5,036,626 proposes the use of plastic unloader discs positioned on opposing sides of the belt of an abrading type knife sharpening machine to carry the knife edge away from the belt when excessive force is applied by the user to the knife and belt. However, the unloader discs can wear and therefore become less effective.
Some knife sharpeners of the belt abrading type have belts which travel at a relatively constant speed regardless of the force applied by the knife to the belt during a sharpening operation. In such knife sharpeners, overheating of and damage to the cutting edge can occur quickly when excessive force is applied, because the belt does not slow or stop relative to the force applied.
Belt abrading knife sharpeners have been provided in enclosed housings to protect the user's clothing, fingers or other objects from being entangled with the moving parts of the knife sharpener. However, some housings are comprised of two housing portions rotatably connected by a hinge so that opposing housing portions can be rotated relative to each other to access internal parts of the knife sharpener, for example, for changing the belt. If the two housing portions are rotated apart from one another while the belt is operating, rotating parts thereof are exposed and a safety hazard can result.
Many knife sharpeners are large, heavy and suitable primarily for commercial uses, for example, in a butcher's shop, grocery store or the like. Such knife sharpeners have not been frequently used by individual home owners because the size and weight of the knife sharpener makes it impractical for home countertop use. Moreover, such units generally operate on 120 volt A/C power which requires the knife sharpener to be stationed within access to a suitable power supply and requires use of relatively expensive motors.
Belt grinders or sanders of the general type having a belt drive pulley, at least one driven or idler pulley and an endless abrasive or other belt trained over the pulleys are known in the art, as evidenced by U.S. Pat. Nos. 3,497,336 and 4,294,044. During the operation of such a machine, it is desirable that the belt track is aligned properly over the pulleys. That is, it is desirable that the belt maintain a position substantially laterally centered on the belt engaging faces of the pulleys. However, slight alignment errors in the relation of the axes of the pulleys to one another or slight variations in the lengths of the edges of the belt can cause the belt to move off track by shifting laterally to one side or the other from the desired centered position. In some instances, the belt may run completely off of the pulleys or rub against a wall or other structure of the machine located adjacent one or both of the pulleys or the belt, resulting in the shortening of belt and/or machine life.
To allow for correction of the running path of the belt, it has been common practice in belt abrading machines to provide a means for adjusting the inclination of an idler pulley axis relative to a drive pulley axis. Such adjustment means have however tended to pose various problems of their own, including being of a complex, expensive construction and being difficult to operate or fine tune during operation.
Thus there is a need to provide a knife sharpening machine that accurately sharpens a knife without overheating or damaging the cutting edge, that is light weight, compact, inexpensive, does not require belt alignment and does not pose a safety risk. Prior art methods and systems for addressing these needs are either too expensive, too complicated, ineffective or a combination of all of these. Based on the foregoing, it is the general object of the present invention to improve upon prior art knife sharpening machines and methods and overcome the problems and drawbacks thereof.


A scalable flow virtual learning neurocomputer system and appratus with a scalable hybrid control flow/data flow employing a group partitioning algorithm, and a scalable virtual learning architecture, synapse processor architecture mapping, inner square folding and array separation, with capability of back propagation for virtual learning. The group partitioning algorithm creates a common building block of synapse processors containing their own external memory. The processor groups are used to create a general purpose virtual learning machine which maintains complete connectivity with high performance. The synapse processor group allows a system to be scalable in virtual size and direct execution capabilities. Internal to the processor group, the synapse processors are designed as a hybrid control flow/data flow architecture with external memory access and reduced synchronization problems.

Virtual learning implies the ability to modify the virtual connection weights and the virtual neural state values stored in an external memory. This function would be desireable for an environment which demands the complete scalability of other functions developed in co-pending applications. This function and an apparatus for achieving it has not been developed.

Scalable flow virtual learning neurocomputer


A test method and apparatus for determining the content of carboxylic acid-based corrosion inhibitors in a coolant or heat exchange fluid. The amount of acidic reagent added to the coolant or fluid between two pH equilibrium points is measured to determine the amount of inhibitors. The refractive index can be measured to determine the amount of freezing point depressant.

1. Field of the Invention
This invention relates to a method and apparatus for testing the quality of engine coolants and heat-exchange fluids. More particularly, this invention relates to a field test, requiring a minimum of operator interaction.
2. Related Art
Recently Organic Acid Technology (OAT) coolants and heat exchange fluids have been introduced commercially, and provide improved corrosion protection and long life. OAT corrosion inhibitor packages in aqueous and glycol concentrates are used in automotive, heavy duty, marine and industrial applications. OAT inhibitors are also used in secondary cooling systems and in a variety of industrial heat exchange fluids. The use of monocarboxylic or dicarboxylic acids, or the salts of such acids, as corrosion inhibitors in antifreeze/coolant and heat-exchange fluid compositions is disclosed for instance in EP-A-0 479 470, -0 564 721 and -0 573 287. Such compositions may also contain a variety of additives for special purposes, such as hydrocarbyl triazoles for corrosion protection of copper and copper alloys (EP-A-0 564 721).
There is a need to check the quality of the coolant solution regularly to ensure problem-free operation. For the development of OAT coolants, organic acids were neutralized with alkali metal hydroxides (preferably sodium hydroxide (NaOH) or potassium hydroxide (KOH)) to obtain the inhibitor salts used in the new organic formulations, see for example, U.S. Pat. Nos. 4,647,392; 4,851,145; and 4,759,864. It was soon realized that an acid titration of an OAT solution could provide information on the total carboxylic acid inhibitor content. U.S. Pat. No. 5,366,651 shows in FIG. 1 some titration curves for combinations of carboxylate inhibitors with an organic pH buffer such as imidazole.
In order to evaluate the OAT coolant quality in the field, the end user needs to be able to check the amount of contamination with other coolants, as well as carboxylate inhibitor content. The present invention provides an improved method and apparatus for field testing and analysis of coolants and heat exchange fluids.
According to one aspect of the invention, there is provided a test method for determining the content of carboxylic acid-based corrosion inhibitors in a coolant or heat exchange fluid which comprises:
(a) placing a sample comprising a predetermined amount of the material to be tested in a titration vessel,
(b) stirring or otherwise agitating the sample while adding an acidic reagent at a constant rate,
(c) measuring the pH of the stirred sample while the acidic reagent is added,
(d) determining a first equilibrium point (EP1) demonstrated by a first rapid drop in pH,
(e) continuing addition of acidic reagent and measuring the pH until a second equilibrium point (EP2), demonstrated by a second rapid drop in pH, is reached, and
(f) determining the amount of carboxylic acid-based corrosion inhibitor in the sample from the amount of acidic reagent added between EP1 and EP2.
Other aspects of the invention provide:
A method wherein the content of contaminant in the sample is determined from the amount of acidic reagent required to reach the first equilibrium point.
A method wherein the amount of freezing point depressant in the sample is determined from the refractive index of the sample.
A method wherein the pH is determined by means of a calibrated electrode in the titration vessel.
A method wherein at least one quantity selected from the observed values of pH, the amount of carboxylic acid corrosion inhibitor, the amount of contaminant, the refractive index, and the freezing point of the sample is displayed visually.
According to another aspect of the invention, there is provided an apparatus for conducting a test method as described above which comprises a titration vessel, first and second reservoirs for acidic reagent and waste neutralizing agents respectively, a first pump for transferring acidic reagent from the first reservoir to the titration vessel and a second pump for transferring waste neutralizing agent to the titration vessel from the second reservoir, a stirrer for stirring samples in the titration vessel, a pH electrode in the titration vessel, display means for displaying information determined by tests conducted in the apparatus, and electronic control means for controlling said apparatus.
The present invention is directed towards a field- or laboratory test technique to determine the quality of antifreeze/coolant or aqueous heat-transfer or heat-exchange fluids. An apparatus and method are described by means of which it is possible to measure the content of carboxylate corrosion inhibitors in OAT coolants or heat exchange fluids, such as Havoline Extended Life Coolant (XLC) (Havoline products available from Texaco Inc.), or heat exchange fluids containing Havoline Extended Life Corrosion Inhibitor (XLI) or mixtures thereof. In one preferred aspect of the invention, the method is used to also measure the contamination level in XLC contaminated by traditional borate or phosphate containing coolant or with the more recently introduced hybrid OAT-traditional coolants such as Havoline Antifreeze Coolant (AFC). Alternatively, it is of course possible to make calibrations for other OAT coolants.
In another aspect of the present invention, the measurement of contamination level, carboxylate corrosion inhibitor content is combined with a measurement of the pH of the test solution.
In yet another preferred aspect of the invention, the measurements of contamination level, total carboxylate inhibitor content and pH are combined with a refractometric determination of the freezing point of the test solution.

Field test apparatus and method for analysis of coolants and heat-exchange fluids


A system and method for placing cluster groupers in a visual display is provided. Groupers of document clusters are obtained. One of the groupers is placed into a center of a display. A further grouper is selected, and a size of the further grouper is determined. An angle for placement of the further grouper and a radius from the center of the display for placement of the further grouper are also determined based on the grouper size to minimize overlap with the placed grouper. The further grouper is positioned in the display in relation to the grouper based on the determined angle and radius.

Computer-based data visualization involves the generation and presentation of idealized data on a physical output device, such as a cathode ray tube (CRT), liquid crystal diode (LCD) display, printer and the like. Computer systems visualize data through graphical user interfaces (GUIs), which allow intuitive user interaction and high quality presentation of synthesized information.
The importance of effective data visualization has grown in step with advances in computational resources. Faster processors and larger memory sizes have enabled the application of complex visualization techniques to operate in multi-dimensional concept space. As well, the interconnectivity provided by networks, including intranetworks and internetworks, such as the Internet, enable the communication of large volumes of information to a wide-ranging audience. Effective data visualization techniques are needed to interpret information and model content interpretation.
The use of a visualization language can enhance the effectiveness of data visualization by communicating words, images and shapes as a single, integrated unit. Visualization languages help bridge the gap between the natural perception of a physical environment and the artificial modeling of information within the constraints of a computer system. As raw information cannot always be digested as written words, data visualization attempts to complement and, in some instances, supplant the written word for a more intuitive visual presentation drawing on natural cognitive skills.
Effective data visualization is constrained by the physical limits of computer display systems. Two-dimensional and three-dimensional information can be readily displayed. However, n-dimensional information in excess of three dimensions must be artificially compressed. Careful use of color, shape and temporal attributes can simulate multiple dimensions, but comprehension and usability become difficult as additional layers of modeling are artificially grafted into the finite bounds of display capabilities.
Thus, mapping multi-dimensional information into a two- or three-dimensional space presents a problem. Physical displays are practically limited to three dimensions. Compressing multi-dimensional information into three dimensions can mislead, for instance, the viewer through an erroneous interpretation of spatial relationships between individual display objects. Other factors further complicate the interpretation and perception of visualized data, based on the Gestalt principles of proximity, similarity, closed region, connectedness, good continuation, and closure, such as described in R. E. Horn, "Visual Language: Global Communication for the 21st Century," Ch. 3, MacroVU Press (1998), the disclosure of which is incorporated by reference.
In particular, the misperception of visualized data can cause a misinterpretation of, for instance, dependent variables as independent and independent variables as dependent. This type of problem occurs, for example, when visualizing clustered data, which presents discrete groupings of data, which are misperceived as being overlaid or overlapping due to the spatial limitations of a three-dimensional space.
Consider, for example, a group of clusters, each cluster visualized in the form of a circle defining a center and a fixed radius. Each cluster is located some distance from a common origin along a vector measured at a fixed angle from a common axis through the common origin. The radii and distances are independent variables relative to the other clusters and the radius is an independent variable relative to the common origin. In this example, each cluster represents a grouping of points corresponding to objects sharing a common set of traits. The radius of the cluster reflects the relative number of objects contained in the grouping. Clusters located along the same vector are similar in theme as are those clusters located on vectors having a small cosine rotation from each other. Thus, the angle relative to a common axis' distance from a common origin is an independent variable with a correlation between the distance and angle reflecting relative similarity of theme. Each radius is an independent variable representative of volume. When displayed, the overlaying or overlapping of clusters could mislead the viewer into perceiving data dependencies where there are none.
Therefore, there is a need for an approach to presenting arbitrarily dimensioned data in a finite-dimensioned display space while preserving independent data relationships. Preferably, such an approach would organize the data according to theme and place thematically-related clusters into linear spatial arrangements to maximize the number of relationships depicted.
There is a further need for an approach to selecting and orienting data clusters to properly visualize independent and dependent variables while compressing thematic relationships for display.

System and method for placing cluster groupers in a visual display


A minimal wear valve assembly which includes a fluid reservoir having an inlet port and an outlet port for passage of the fluid therethrough. The reservoir defines a valve seat about and downstream of the outlet port. The assembly includes a valve having a first substantially non-deformable valve surface. The valve is movable with respect to the valve seat from an open condition which permits flow of the fluid through the outlet port to a closed condition which prevents flow of the fluid through the outlet port. The valve supports a sealing member for movement with the valve. The sealing member has greater compressibility than the first valve surface and provides a seal between the first valve surface and the valve seat upon movement of the valve to the closed condition.

Valve assemblies are well known for their ability to regulate fluid flow through inlet and/or exhaust openings. One such valve is a poppet valve. Poppet valves are typically made from heat-resistant steel and include a circular head or valve. The valve includes a conical face which registers with a corresponding valve seat bounding the opening. In some cases the head may be disk shaped and seat over a port opening. In other cases, such as with a ball-poppet valve, the head may be a spherical member which closes the valve when positioned against an opening having a conical or tapering surface. A guided stem, by which the valve is lifted from its seated position, is movable under the urging of an actuator. When the conical face of the valve rests on the valve seat the fluid is sealed from flowing through the valve.
As the materials which form the poppet valve assembly are substantially incompressible, early failure of the valve assembly can result when the valve is employed to dispense fluids containing high concentrations of substantially incompressible and abrasive inorganic additives or fillers. Some fillers, such as Al.sub.2 O.sub.3, can cause frictional wear of the valve when flowing between the valve and the seat. Hertz-type stresses of the mating surfaces, which can cause flaking or shearing at the surfaces, are also known to occur when these solid fillers are pinched between a ball-poppet and the valve seat as the ball-poppet seals against the seat. As the ball-poppet reaches the final closed position there can be relative movement between a particle of the solid filler and the mating surfaces which can gouge the surface, creating a pathway between the mating surfaces through which the fluid may leak.
Furthermore, these solid fillers can become trapped between the mating surfaces as the poppet closes against the seat. Because the mating surfaces and the abrasive filler are all made from substantially incompressible materials, a particle of the filler becoming trapped between the mating surfaces can cause localized stress concentrations on the mating surfaces. Such localized stress concentrations can result in surface imperfections, distortions, and other irregularities, such as pitting or abrasive wear, which prevent sealing engagement between the valve and the seat, and thereby cause valve failure as the fluid will then have a pathway between the mating surfaces.
Some fillers may be crushed between the mating surfaces which can result in degradation of the material being dispensed. Some adhesives include fillers to provide a desired thermal or electrical characteristic to the adhesive. For example, glass spheres are within some dielectric adhesives to provide the desired insulative characteristics. These glass spheres can be about 1/5000 of an inch in diameter. These glass spheres could be crushed if trapped between two incompressible mating surfaces, which could then cause electrical shorts through the material and result in quality control problems for the user.
Currently, valves employed to dispense abrasive fluids employ poppet valves which seat a metal valve against a metal seat. Valve failure has been witnessed in these valves after 50,000 to 60,000 cycles of opening and closing. It would therefore be desirable to provide a valve assembly which provides suitable sealing characteristics between the valve and the valve seat and which minimizes the risk of failure when dispensing abrasive products.


A method includes receiving project information indicating a location of a worksite. The method also includes providing a first travel path to an electronic device associated with a mobile machine, wherein providing the first travel path to the electronic device causes at least part of the first travel path to be displayed via a display. The method further includes receiving location information indicating an initial location of the machine and one or more additional locations of the machine. Additionally, the method includes determining that the machine reached the worksite, and identifying, based at least in part on the location information, a second travel path extending from the initial location to the worksite. The method further includes determining whether the second travel path matches the first travel path, and storing at least one of the travel paths in a memory associated with a controller.

Machines, such as haul trucks, are often used to perform a variety of tasks associated with a worksite or project. For instance, in an example paving project, one or more haul trucks are typically used to transport paving material from a paving material plant to a worksite where the paving material will be distributed along a work surface of the worksite by one or more paving machines. One or more compaction machines following behind the paving machine can be used to compact the freshly-laid paving material to a desired density or stiffness. The operation of these machines is coordinated in order to perform paving operations in an efficient manner. For example, the quality of a mat of paving material deposited by the paving machine is improved when the paving machine is controlled to operate without periodic stoppages caused by an inadequate supply of paving material being delivered to the worksite by the one or more haul trucks described above. The quality of the mat is also improved when the paving material delivered to the worksite by the one or more haul trucks is maintained within a desired temperature range. However, in some situations, it is difficult to manage the delivery of paving material to the worksite. For instance, the various haul trucks delivering paving material often traverse different respective travel paths between the paving material plant and the worksite, which can lead to delays and inconsistencies in the delivery of paving material.
An example system for coordinating the activities of paving machines is described in U.S. Patent Application Publication No. 2013/0290062 (hereinafter referred to as the '062 reference). In particular, the '062 reference describes a system for implementing a computer-based method of coordinating activities associated with paving a roadway. The '062 reference describes, for example, a server configured to facilitate communication between system components. As explained in the '062 reference, the server receives a communication from a transport truck indicating that a batch of paving material has been delivered to the roadway, and such a communication is generated automatically by a truck computer system in conjunction with a global positioning system (GPS) receiver on the truck.
The '062 reference does not, however, describe determining or otherwise identifying an optimized haul truck travel path extending from the paving material plant to the worksite. For instance, the '062 reference does not describe identifying such a travel path based on a determination that the identified travel path results in a reduction in travel time for the haul truck. Nor does the '062 reference describe identifying such a travel path based on a determination that a number of additional haul trucks previously traveled the identified travel path. As a result, the paving the machines described in the '062 reference will be caused to stop periodically due to delayed or inconsistent delivery of paving material to the worksite. Such stoppages hinder the efficiency of the paving system, and reduce the consistency and quality of the paving material mat. Additionally, the temperature of paving material delivered to the paving machines described in the '062 reference may fall outside of a desired temperature range, further reducing the quality of the paving material mat.
Examples of the present disclosure are directed toward overcoming the deficiencies noted above.

Systems and methods for identifying machine travel paths


The present disclosure provides compositions and methods for regulating gene expression via RNA-mediated silencing. The present disclosure also provides compositions and methods to optimize the processing of a dsRNA molecule into small RNA duplexes. The present disclosure further provides compositions and methods to improve the efficiency of a dsRNA molecule in producing desired small RNAs and promoting the silencing of a gene of interest.

Non-transgenically produced, exogenous nucleic acid molecules, for example, double-stranded RNA (dsRNA) molecules, have been shown to trigger the silencing of a plant endogenous gene after being applied topically to a plant leaf or by soaking a seed with a solution containing the nucleic acid molecules. See U.S. Patent Publication Nos. 2011/0296556, and 2013/0318657 (both incorporated by reference in their entireties). Therefore, plant traits can be modified by introducing into a plant or seed dsRNA molecules that specifically regulate the expression of genes responsible for those traits.
RNA-mediated sequence-specific gene regulation, also called RNA interference (RNAi), starts with a dsRNA that comprises a RNA strand that complements the sequence of a gene of interest. The dsRNA molecule is then processed into shorter fragments of approximately 21-24 nucleotides by an RNase III-related enzyme (Dicer). These fragments, called small interfering RNAs (siRNAs), get incorporated into the RNA-induced silencing complex (RISC). After additional processing, the siRNAs are transformed into single-stranded RNAs that act as guide sequences to recognize and direct the cleavage of target gene transcripts.
Plant cells can produce dsRNAs. A tomato RNA-Dependent RNA Polymerase (RDR) has been reported to produce dsRNAs with 1-nt or 2-nt 3′-overhangs at both termini. See Schiebel et al., J. Biol. Chem. 263:11858-67 (1993); Rajeswaran et al., Nucleic Acid Res., 40(13):6241-54 (2012). It is desired to have a dsRNA processed into siRNAs, and eventually single-stranded RNAs, following a predictable and pre-programmed pattern. The instant application provides and discloses, among others, sequence and structural features that are incorporated into a dsRNA molecule to improve the predictability of the processing of a dsRNA into siRNAs functional in guiding the silencing of intended target genes. Therefore, the instant application provides nucleic acid molecules with higher efficacy in promoting gene regulation and trait modification.

Compositions and methods for regulating gene expression via RNA interference


According to embodiments of the present invention, a method for selectively managing a call connection between a caller and each of a first directory number associated with a subscriber and a second directory number associated with the subscriber includes causing at least the first directory number to be rung in response to a call request from the caller. In response to a determination that the rung first directory number has answered the call request: a call is automatically allowed to be completed between the caller and the first directory number if the first directory number is of a first type; and, if the directory number is of a second type, a prescribed additional response is required from the first directory number prior to allowing the call to be completed between the caller and the first directory number, and the second directory number is allowed to be rung in response to the call request until the prescribed additional response is provided.

Multiple communications systems have become ubiquitous. It is not uncommon for telecommunication service subscribers, typically individuals, to have a telephone system that includes more than one telephone terminal (e.g., both one or more wireline telephones and one or more wireless telephones). Subscribers' wireline telephones may or may not be co-located in the same building and the wireless telephones are, by design, highly mobile. This situation has resulted in a need for subscribers to maintain and distribute multiple telephone numbers that may be used to establish communication.
When a caller tries to reach a subscriber, the caller must dial the subscriber's first telephone number and wait to see if anyone answers the call. If the call goes unanswered, the caller must try the next number and again wait for an answer. This process must be repeated until one of the calls is answered or the caller has tried all of the subscriber's numbers without success. This situation can be frustrating for a caller because multiple telephone calls may have to be tried before successfully reaching the subscriber. Similarly, subscribers can be frustrated because the end result may be a missed call. Multiple line ring services have been offered to solve this problem for subscribers having multiple telephone numbers.
In a multiple line ring service, when a call is made to a subscriber's primary telephone number (typically a wireline), the service provides a ringing tone on the primary line and on designated secondary lines. As known in the art, such multiple line ring services may be extended to ring many different lines, wired or wireless, and to ring the lines at the same time (which may be referred to as a "simultaneous ring service") or to ring the lines in succession (which may be referred to as a "sequential ring service"). In some such services, the call is automatically connected or completed with the first line that answers. In other such services, when any line answers, the user at that line is requested to indicate acceptance of the call (e.g., by dialing "1"). The call will only be completed if the indication is provided, and the other lines may continue to ring pending such response from the user.

Methods, systems and computer program products for selectively managing telephone call connections


A pair of hook-and-loop fasteners are attached onto a carton for securing a lid thereof. First fasteners are provided spaced apart along a first exfoliation sheet. Second fasteners adapted to mate and engage with the first fasteners are provided spaced apart along a second exfoliation sheet. The first sheet is deflected over a first knife edge to peel-off a first fastener and engage it onto a second fastener to form a fastener pair. The second sheet is deflected over a second knife edge to peel-off the second fastener of the pair from the second sheet and adhesively stick the fastener pair onto the carton.

The present invention relates to a method or an apparatus for attaching a hook-and-loop fastener to a carton with an openable and closable lid to maintain a closing state of the lid relative to a carton body.
U.S. Pat. No. 6,892,878 shows a disk case for housing a photoelectromagnetic disk such as a CD (i.e. compact disk) or DVD (i.e. digital video disk). The disk case is typically comprised of a disk loading portion for loading a photoelectromagnetic disk and a sheet loading portion for loading a description sheet, which are openably and closably connected to each other via a hinge.
After a photoelectromagnetic disk and a description sheet are loaded into the disk case, the disk case is closed. Such a disk case is sometimes housed in a carton. Also, a carton with a lid has recently been used to house a disk case.
In such a carton with a lid, a closing state of a lid relative to a carton body needs to be maintained at the time of closure of the lid and also the lid needs to be easily disengaged from the carton body at the time of opening of the lid. Therefore, such a carton with a lid requires an engaging and disengaging mechanism for opening and closing a lid.
An object of the present invention is to provide a method and an apparatus for easily attaching a hook-and-loop fastener as an engageable and disengageable means on a carton with a lid to maintain a closing state of the lid relative to a carton body.

Method and apparatus for attaching a hook-and-loop fastener to a carton with a lid


A file server holding tag information representative of file contents and an access frequency of each file calculates a tag rating from the access frequency of each file and calculates a file rating from the tag rating affixed to the file. It controls to place a file having a high calculated file rating on a higher transfer performance layer, and place a file having a low calculated file rating on a lower transfer performance layer. Rapid accesses can be handled quickly by placing in advance a file whose access is anticipated to be increased after interest is increased although an access frequency is still low, on a layer having a short response time and high throughput, and by maintaining a file whose access is anticipated to be increased because of increased interest although an access frequency is lowered, on a layer having a short response time and high throughput.

The present invention relates to a storage system for managing a level at which a file is stored in accordance with tag information representative of file contents and file access frequency information and a file layer management method, respectively in layer storage environment for file levels.
Recently, demands for online storage such as content depots are increasing as storages for reposition/distribution of digital contents. Layer storage environment for file levels has been paid attention, such as relocating files in accordance with a file access frequency for the purposes of storage optimization.
In response to high momentum for protection of the global environment, information technology industry also increases the efforts against reduction in consumption power of apparatus. There is therefore increasing interest to reduction in a consumption power of a storage apparatus. Introduction of MAID (Massive Array of Inactive Disks) technology is progressed which can suppress a consumption power of a storage apparatus through spindown/spinoff of disks loaded in the storage apparatus. For example, JP-A-2000-293314 Publication (corresponding to U.S. Pat. No. 7,355,806B2, Hakamata et al.) discloses a power supply control method of making a host computer control a power supply in accordance with an access frequency to a storage apparatus.

Storage system and a file layer management method


In order to provide: a phosphite dehydrogenase protein having both improved solubility and improved heat stability; a method for producing a gene encoding the phosphite dehydrogenase protein; a method for producing the phosphite dehydrogenase protein; and use of the phosphite dehydrogenase protein, (i) a phosphite dehydrogenase protein having a specific amino acid sequence and (ii) a gene encoding the phosphite dehydrogenase protein are used.

A phosphite dehydrogenase protein (PtxD) is a protein which exists in some bacteria and is an enzyme which oxidizes phosphorous acid in an NAD+-dependent or NADP+-dependent manner to generate NADH or NADPH. The following are reaction formulae respectively corresponding to a case where phosphorous acid is oxidized in an NAD+-dependent manner and a case where phosphorous acid is oxidized in an NADP+-dependent manner.
[Chem. 1]
                Catalyst: PtxDHPO32−+NAD++H2O→HPO42−+NADH+H+  (Reaction formula 1)[Chem. 2]        Catalyst: PtxDHPO32−+NADP++H2O→HPO42−+NADPH+H+  (Reaction formula 2)
The above chemical reactions are expected as being highly potential because they allow efficient production of NADH or NADPH, each of which functions as a very important cofactor in production of a substance with use of a biological reaction. However, industrial use of the chemical reactions has not been developed yet. That is, at present, industrial mass-production of NADH or NADPH with use of phosphorous acid has not been realized (see, for example, Non-patent Literatures 1 and 2). Enzymes such as formic dehydrogenase, glucose dehydrogenase, alcohol dehydrogenase, and the like have conventionally been used for production of NADH and NADPH. However, since the enzymes use a highly reactive substrate, a reaction system thereof is unstable. Further, the enzymes yield a highly reactive product. This also makes the reaction system thereof unstable. Note, here, that a main cause for making the reaction system unstable is changes in pH. On the other hand, phosphite dehydrogenase is advantageous not only in that phosphite dehydrogenase uses a weakly reactive substrate and yields a weakly reactive product, but also in that the substrate used by phosphite dehydrogenase is inexpensive. Phosphite dehydrogenase therefore has a potential to be widely used in place of the above enzymes, if industrial use of phosphite dehydrogenase is realized. Specifically, in a case where phosphite dehydrogenase is used, a reaction system can be stabilized, since both phosphorous acid and phosphoric acid have a buffering action.
In order to produce NADH and NADPH in large quantities industrially, a large amount of the phosphite dehydrogenase protein is required. As such, it has conventionally been tried to obtain a large amount of the phosphite dehydrogenase protein by (i) forcibly causing expression of a heterologous organism-derived wild-type phosphite dehydrogenase protein in a host such as Escherichia coli and (ii) then purifying the wild-type phosphite dehydrogenase protein.
However, according to the technique, a large part of the wild-type phosphite dehydrogenase protein becomes insoluble in an aqueous solution when the wild-type phosphite dehydrogenase protein is forcibly expressed in E. coli or the like. This makes it impossible to collect the wild-type phosphite dehydrogenase protein. As such, there is a demand for a phosphite dehydrogenase protein which is highly soluble in an aqueous solution even in a case where the phosphite dehydrogenase protein is forcibly expressed.
Further, since a temperature of a reaction system rises in industrial production of NADH and NADPH in large quantities, it is necessary to use a phosphite dehydrogenase protein with high thermal stability. However, a conventional wild-type phosphite dehydrogenase protein has low thermal stability (specifically, many enzymes are denatured at 40° C.). As such, there is a demand for a phosphite dehydrogenase protein which can maintain high activity at high temperature.
Under such circumstances, it has been tried to screen for a phosphite dehydrogenase mutant which is improved in the above described properties.
For example, Non-patent Literature 3 discloses a technique in which a phosphite dehydrogenase protein, into which a mutation has been introduced, is forcibly expressed in E. coli, so that an amount of a mutated phosphite dehydrogenase protein contained in a soluble fraction is increased. Note that it cannot be determined from the data in Non-patent Literature 3 whether the increase in amount of the mutated phosphite dehydrogenase protein contained in the soluble fraction was caused by an increase in solubility of the mutated phosphite dehydrogenase protein or by an increase in expression level of the mutated phosphite dehydrogenase protein. Further, Non-patent Literatures 4 and 5 disclose mutated phosphite dehydrogenase proteins with high thermal stability.

Method for producing phosphite dehydrogenase protein and use thereof


Compositions and methods for use in methods and kits for the detection of an increased risk of pancreatic cancer are provided. Cyst fluid samples isolated from patients that are positive for at least three of the following markers, mucin 1, mucin 2, mucin 5AC, mucin 5B, mucin 6, CEA CAM 1, CEACAM 5, CEACAM 6, CEACAM 7, CEACAM 8, S100-A6, S100-A8, S100-A9 and S100-A11 indicate that such patients are at greater risk for development of pancreatic cancer when compared to cyst fluid samples isolated from patients lacking these markers.

Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Increasing use of high resolution computerized tomography and magnetic resonance imaging in clinical practice has resulted in detection of a growing number of pancreatic cysts (1). As a result, clinicians are frequently asked to determine the biological nature of these cystic lesions, and to make treatment recommendations accordingly. However, there are currently no diagnostic indicators that are consistently reliable, obtainable, and conclusive for diagnosing and risk-stratifying pancreatic cysts. The sensitivity of pancreatic cyst fluid cytology has been reported as only 27-64% in most series. Several studies have suggested that a variety of tumor markers (e.g., CEA(2), CA 19-9, CA 15-3) may distinguish mucinous from non-mucinous cystic lesions, and also may predict whether a cyst harbors areas of malignant transformation (3-5).
The biologic nature and histopathologic features of pancreatic cysts are varied (3, 6). Ten to twenty percent of pancreatic cysts are neoplastic, including neoplasms which grow as cystic structures (i.e., primary cystic neoplasms of the pancreas), and solid neoplasms that have undergone cystic degeneration. Serous cystadenomas (microcystic adenomas) account for approximately 32-39% of the primary cystic neoplasms and have very low malignant potential. Mucinous cystic neoplasms, which include mucinous cystadenomas and intraductal papillary mucinous neoplasms, are a subgroup of primary cystic neoplasms that have malignant potential. Nomenclature describing the evolution of these lesions, from benign to malignant, is provided elsewhere (7, 8). Mucinous cystic neoplasms and intraductal papillary mucinous neoplasms (IPMNs) account for approximately 10-45% and 21-33% of primary cystic neoplasms, respectively (6, 9-11). Two subtypes of IPMN have been described (1, 12), a main duct variant and a branch duct variant; the latter may have a more indolent course. There are other less common forms of primary cystic neoplasms of the pancreas, such as solid pseudopapillary tumors.
In the absence of reliable methods of quantifying the malignant potential of a suspected pre-malignant cystic neoplasm of the pancreas, if existing clinical parameters suggest the presence of one such lesion in a person that is otherwise an acceptable surgical risk, partial or total pancreatomy may be recommended but can result in significant morbidity and mortality (13). Alternatively, a conservative "watch-and-wait" approach (i.e., serial imaging over time) is advocated for some patients, but this strategy may be suboptimal due to incremental costs accrued during surveillance, and the possibility that malignant transformation may occur between surveillance time points.
In light of all the foregoing, it is clear that a more reliable method for identifying those patients at increased risk for developing pancreatic cancer is urgently needed.

Biomarkers for the diagnosis and treatment of pancreatic cancer


Systems and methods are provided for providing access to data on a personalized basis. A service operating on a server is identified, where data at the service is associated with a first user and other users. Data associated with the first user is extracted. A network location is spawned for the first user. The extracted data is transferred to the spawned network location to make the extracted data available to the first user in a read-only fashion by accessing the spawned network location. Additional network locations are spawned for second and third users, respectively, wherein data associated with the second and third users is transferred such that they are available to the second and third users by accessing their respective additional network locations.

Web services having interfaces that are known and accessible to multiple users are prone to abuse and attack. Disseminated knowledge of the location of the interface (e.g., an address) provides a gateway for unauthorized access attempts. Centralized storage of service data associated with multiple users (e.g., the users who are permitted to access the service) provides opportunities for abuse even by authorized users. For example, authorized users can access the service interface using their credentials and then can attempt to access service data to which they are not entitled (e.g., service data associated with other users) after entry to the system via their credentials.

This patent is subject to a terminal disclaimer.

Systems and methods for providing access control to web services using mirrored, secluded web instances


To realize an LED drive circuit capable of feeding equal currents to parallel-connected LED circuits with a simple arrangement, there are provided a current source generating a temporally altering current and a first smoothing capacitor and a second smoothing capacitor, and there are also provided: a first LED circuit, containing one LED or series-connected LEDs, which is connected in parallel with the first smoothing capacitor; and a second LED circuit, containing one LED or series-connected LEDs, which is connected in parallel with the second smoothing capacitor, the drive circuit comprising: a current-dividing coil containing two coils coupled at a tap so that the current generated in the current source flows into the tap; a first reverse current blocking diode connected between an end of the current-dividing coil and an electrode of the first smoothing capacitor; and a second reverse current blocking diode connected between another end of the current-dividing coil and an electrode of the second smoothing capacitor.

FIG. 9 is a circuit diagram for a conventional LED lighting device. As shown in the figure, the LED lighting device 801 includes an input power supply Vin (DC power supply), an LED drive circuit, and four LED circuits (803a to 803d). The LED drive circuit includes a PWM control IC 804, a switching element 805, a diode 806, a coil 807, resistors Ra to Rd, and a smoothing capacitor 808. The LED circuit 803a is made up of 10 LEDs connected in series between a node a1 and a node a2. Likewise, the LED circuit 803b is made up of 10 LEDs connected in series between a node b1 and a node b2; the LED circuit 803c is made up of 10 LEDs connected in series between a node c1 and a node c2; and the LED circuit 803d is made up of 10 LEDs connected in series between a node d1 and a node d2. The coil 807 has one of its ends connected to the positive terminal of the input power supply Vin. The other end of the coil 807, the anode of the diode 806, and the first conductive terminal (drain) of the switching element 805 are connected to each other. The control terminal (gate) of the switching element 805 is connected to the PWM control IC 804. The second conductive terminal (source) is connected to the negative terminal of the input power supply Vin. The cathode of the diode 806 is connected to the negative terminal of the input power supply Vin via the smoothing capacitor 808. The cathode of the diode 806, a first electrode of the smoothing capacitor 808, and the nodes a1 to d1 are connected to each other. The nodes a2 to d2 are connected to the negative terminal of the input power supply Vin via the respective resistors Ra to Rd. For example, the node a2 is connected to the negative terminal of the input power supply Vin via the resistor Ra. The node b2 is connected to the negative terminal of the input power supply Vin via the resistor Rb. Accordingly, the LED circuits 803a to 803d are connected in parallel between the cathode of the diode 806 and the negative terminal of the input power supply Vin. In addition, the node a2 is coupled also to a current feedback input of the PWM control IC 804.
The drive circuit provides a step-up converter (boost converter). The circuit accumulates energy in the coil 807 (coil) while the switching element 805 is on and applies electromotive voltage derived from that energy along with input voltage across the smoothing capacitor 808 and the LED circuits 803a to 803d while the switching element 805 is off. The current value through the LED circuit 803a is detected using the resistor Ra to make a current feedback to the PWM control IC 804. Based on the feedback, the duty ratio (on/off ratio) of the switching element 805 is controlled. Thus, the current flow through the LED circuit 803a is made constant.
Note that the LED circuits 803a to 803d are in parallel. Therefore, provided that the LED circuits 803a to 803d share the same forward current-forward voltage characteristic ("IF-VF characteristic"), the currents IFa to IFd through the respective LED circuits 803a to 803d are all constant and equal to each other.
FIG. 11 is a circuit diagram for another conventional LED lighting device. As shown in the figure, the LED lighting device 901 includes an input power supply Vin (DC power supply), an LED drive circuit, and LED circuits 903a to 903d. The LED drive circuit includes PWM control ICs 904a to 904d, switching elements 905a to 905d, diodes 906a to 906d, coils 907a to 907d, smoothing capacitors 908a to 908d, and resistors ra to rd. The LED circuit 903a is made up of 10 LEDs connected in series between a node a1 and a node a2. Likewise, the LED circuit 903b is made up of 10 LEDs connected in series between a node b1 and a node b2; the LED circuit 903c is made up of 10 LEDs connected in series between a node c1 and a node c2; and the LED circuit 903d is made up of 10 LEDs connected in series between a node d1 and a node d2.
Separate the FIG. 11 circuitry into four circuits A, B, C, and D. The circuit A consists of components 903a to 908a and ra. The circuit B consists of components 903b to 908b and rb. The circuit C consists of components 903c to 908c and rc. The circuit D consists of components 903d to 908d and rd. Circuits A to D have the same arrangement and operate in the same fashion.
Take the circuit A as an example. The coil 907a has one of its ends connected to the positive terminal of the input power supply Vin. The other end of the coil 907a, the anode of the diode 906a, and the first conductive terminal (drain) of the switching element 905a are connected to each other. The control terminal (gate) of the switching element 905a is connected to the PWM control IC 904a. The second conductive terminal (source) is connected to the negative terminal of the input power supply Vin. The cathode of the diode 906a is connected to the negative terminal of the input power supply Vin via the smoothing capacitor 908a. The cathode of the diode 906a, a first electrode of the smoothing capacitor 908a, and the node a1 are connected to each other. The node a2 is connected to the negative terminal of the input power supply Vin via the resistor ra. The circuitry accumulates energy in the coil 907a (coil) while the switching element 905a is on and applies electromotive voltage derived from that energy along with input voltage across the smoothing capacitor 908a and the LED circuit 903a while the switching element 905a is off. The current value through the LED circuit 903a is detected using the resistor ra to make a current feedback to the PWM control IC 904a. Based on the feedback, the duty ratio (on/off ratio) of the switching element 905a is controlled. Thus, the current flow through the LED circuit 903a is made constant.
The arrangement allows for individual control of the currents IFa to IFd through the circuits A to D. Therefore, even if the LED circuits 903a to 903d do not have exactly the same forward current-forward voltage characteristic ("IF-VF characteristic"), it is still possible to equalize the current flows through the LED circuits 903a to 903d, hence the emitted light intensity (luminance level) from the LED circuits 903a to 903d.
Related techniques are disclosed in the following publicly known documents: Japanese Unexamined Patent Publication 2004-319583 (Tokukai 2004-319583; published on Nov. 11, 2004), Japanese Unexamined Patent Publication 2002-8409 (Tokukai 2002-8409; published on Jan. 11, 2002), Japanese Unexamined Patent Publication 11-67471/1999 (Tokukaihei 11-67471; published on Mar. 9, 1999), Japanese Unexamined Patent Publication 2004-253364 (Tokukai 2004-253364; published on Sep. 9, 2004), Japanese Unexamined Patent Publication 2004-51014 (Tokukai 2004-51014; published on Feb. 19, 2004), Japanese Unexamined Patent Publication 8-194448/1996 (Tokukaihei 8-194448; published on Jul. 30, 1996), Japanese Unexamined Patent Publication 2000-12907 (Tokukai 2000-12907; published on Jan. 14, 2000), Japanese Unexamined Patent Publication 2004-335443 (Tokukai 2004-335443; published on Nov. 25, 2004), Japanese Unexamined Patent Publication 56-86495/1981 (Tokukaisho 56-86495; published on May 14, 1981), and Japanese Unexamined Patent Publication 59-108297/1984 (Tokukaisho 59-108297; published on Jun. 22, 1984).
Each of the above LED circuits contains 10 series-connected LEDs. These LEDs, although bearing the same product number, may have variations in the forward current-forward voltage characteristic. For example, the VF of LEDs of a specific product number vary from 2.2 volts (minimum) to 2.6 volts (standard) and 3.0 volts (maximum) for the same IF (30 mA) as shown in FIG. 10.
These variations between the LEDs in the forward current-forward voltage characteristic naturally cause variations between the LED circuits containing those LEDs in the forward current-forward voltage characteristic. In other words, in the LED lighting device 801 in FIG. 9, the LED circuits 803a to 803d do not share the same forward current-forward voltage characteristic ("IF-VF characteristic"). In such situations, the LED circuits 803a to 803d pass current of different values, hence emit light of different intensities (luminance levels). These defects can cause problems when the device is applied for illumination purposes and to the area light source in the backlight for large-scale liquid crystal displays. Variations in emitted light intensity can be a cause for uneven luminance. Different current values may lead to differing temperature rises and lifetimes. These problems in turn seriously impact product quality.
In contrast, in the LED lighting device 901 in FIG. 11, the LED circuits 903a to 903d pass current of the same values (IFa to IFd). The device 901 however has disadvantages, such as a high component count, corollary increased chances of malfunction (poor yield), and high manufacturing cost.

LED drive circuit, LED lighting device, and backlight


The present invention relates to oligonucleotides useful for determining the presence of Mycoplasma pneumoniae and/or Mycoplasma genitalium in a test sample. The oligonucleotides of the present invention may be incorporated into hybridization assay probes, capture probes and amplification primers, and used in various combinations thereof.

Mycoplasmas are small prokaryotic organisms (0.2 to 0.3 μm) belonging to the class Mollicutes, whose members lack a cell wall and have a small genome size. The mollicutes include at least 100 species of Mycoplasma, 13 of which are known to infect humans. One of these species, M. pneumoniae, is a major cause of community-acquired pneumonia (non-pneumococcal bacterial pneumonia), a type of pneumonia which is responsible for up to 2 million respiratory tract infections in the United States annually. The incidence of mycoplasma non-pneumonic respiratory infection has been estimated to be 10 to 20 times higher than this number. See 2 GERALD L. MANDELL ET AL., PRINCIPLES AND PRACTICE OF INFECTIOUS DISEASES § D (5th ed. 2000); see also PATRICK R. MURRAY ET AL., MANUAL OF CLINICAL MICROBIOLOGY § V (6th ed. 1995).
While M. pneumoniae infections are generally mild, as many as 10% of those infected with the organism progress to bronchopneumonia, requiring treatment or hospitalization. The most common early presentation of an M. pneumoniae infection is tracheobronchitis. Radiological findings vary widely and often indicate a more severe condition than symptoms typically associated with an M. pneumoniae infection (e.g., flu-like dry cough, pharyngitis, fever, malaise, headache and nasal congestion) would suggest Recovery of the organism from extra-pulmonary sites is rare, although hematologic, musculoskeletal, cardiovascular, dermatologic and neurologic complications have been reported. See ELMER W. KONEMAN, COLOR ATLAS AND TEXTBOOK OF DIAGNOSTIC MICROBIOLOGY 862 (5th ed. 1997).
M. pneumoniae infections occur year round, especially in large populations, but typically peak in late fall or winter. The incidence of isolation of the organism increases with age, and it is second only to Streplococcus pneumoniae as a cause of pneumonia in the elderly. M. pneumoniae-related pneumonia is seen most commonly in children over 5 years of age and in young adults. Crowded military populations, camps and schools are particularly at risk for community-acquired pneumonia caused by M. pneumoniae infections. In one study, over 5% of pneumonias seen in young military recruits were associated with M. pneumoniae. See Gray et al., "Respiratory diseases among U.S. military personnel: countering emerging threats," Emerg. Infect. Dis., 3:379–387 (1999).
The incubation period for M. pneumoniae, which typically ranges from 2 to 3 weeks, is significantly longer than most viral respiratory infections. After clinical manifestation, symptoms associated with a M. pneumonia infection generally last from 3 to 10 days. Appropriate antibiotic treatments can significantly shorten the duration of the respiratory symptoms associated with a M. pneumoniae infection. However, since culture and many available diagnostic tests are difficult, time consuming and/or not readily available, ineffective antibiotic treatments are often prescribed, thereby unnecessarily prolonging the symptomatic period.
Diagnosis of a M. pneumoniae infection is often based solely on clinical signs and symptoms. Although culture remains the gold standard for diagnosing a M. pneumoniae infection, isolation, detection and identification of the fastidious M. pneumoniae organisms is difficult and can take weeks to complete. Diagnosis has also been based on demonstrating the presence of cold agglutinins. However, this test is a nonspecific indicator, as cold agglutinins may never develop in some patients infected with M. pneumoniae and have also been observed with lymphoma and a variety of viral diseases, including mononucleosis caused by Epstein-Barr virus and cytomegalovirus. Assaying for complement-fixing antibodies has also been used to confirm infection with M. pneumoniae, but is of little practical value in guiding diagnostic and therapeutic decisions, as the antibodies arise too late in the infection. An enzyme immunoassay has also been developed for detecting IgM and IgG directed against M. pneumoniae, but is limited in its usefulness since it does not become positive until 1 to 2 weeks into infection. In addition, an antigen-capture, indirect immunoassay has been used to detect M. pneumoniae antigens in sputum samples; however, the reagents of this assay cross-react with M. genitalium antigens. See, e.g., Bartlett et al., "Community-Acquired Pneumonia in Adults: Guidelines for Management," Clin. Infect. Dis., 26:811–838 (1998). Thus, a need exists for a sensitive and specific assay which can be used to determine the presence of M. pneumoniae in a test sample during a clinically relevant period.
Also of clinical relevance is the detection of M. genitalium in a test sample. M. genitalium, which is thought to be a cause of nongonococcal urethritis (NGU), a sexually transmitted disease, has been detected to a significantly greater extent in symptomatic males than in asymptomatic males. See Yoshida et al., "Phylogeny-Based Rapid Identification of Mycoplasma and Ureaplasmas from Urethritis Patients," J. Clin. Microbiol., 40:105–110 (2002). In addition to NGU, M. genitalium is thought to be involved in pelvic inflammatory disease, which can lead to infertility in women in severe cases. See JACK MANILOFF ET AL., MYCOPLASMAS: MOLECULAR BIOLOGY AND PATHOGENESIS 417 (ASM 1992). M. genitalium may also cause disease in the respiratory tract, making it important for some assays to distinguish between the presence of M. pneumoniae and M. gentialium. See LEE H. HILBORNE ET AL., A REVIEW OF THE SCIENTIFIC LITERATURE AS IT PERTAINS TO THE GULF WAR ILLNESSES, VOL.1: INFECTIOUS DISEASES CH. 3 (Rand 2000). Therefore, it would be of clinical importance to have an assay for specifically detecting the presence of M. genitalium in a test sample which is capable of distinguishing between M. genitalium and M. pneumoniae.

Probes, compositions and kits for determining the presence of


A system for positioning and maintaining in place an horizontal plate upon an horizontal surface such as the upper face of a rest block, typically when such plate and such block are elements of a modular furniture system, characterized in that the components of said positioning system are not visible on any of said plate surfaces, which enables said plate to be reversible.

1. Field of the Invention
This invention relates to a new and improved system for positioning and maintaining a horizontal plate upon a horizontal surface such as the upper face of a rest element, typically when such plate and such rest element are pieces of a modular furniture system, the main advantage of said positioning system of the invention being that its pieces do not affect the plate surfaces so that said plate can be used upside down at will without showing any element of the system.
2. Related Art
A modular furniture system of this kind is described in the EP-A-91440021.3 of the applicant. In such system it is common to position and maintain a plate upon its rest block with studs/cups couples provided respectively on each face of the plate and on the upper face of the rest block or with bolts passing through the plate to engage a housing in the block.
However, one advantage of this system is the possibility for an angular plate to be turned upside down to invert the orientation thereof, to obtain large working surfaces of sineous shape, but inasmuch said positioning means must be present on both faces, which affects the aesthetics of the assembly, the non-used means remaining visible on the upper face of the plate.
This invention eliminates this drawback with a new system for efficiently positioning and maintaining a plate upon a horizontal support by acting only laterally upon the plate, i.e. only the edge of the plate without affecting any part of the working surface, whatever its orientation.

System for positioning and maintaining a plate upon a surface