D-enzyme compositions are described comprising an amino acid residue sequence that defines an polypeptide able to catalyze an enzymatic reaction. The D-enzyme has an amino acid residue sequence consisting essentially of D-amino acids

The biosphere is inherently chiral; each class of biological macromolecules is wade up of monomer molecules of uniform chirality (Mason, Chirality 3:223, 1991) and the biochemical interactions of biological macromolecules are inherently chiral.
Enzymes, for example, invariably act only on one enantiomer of a chiral substrate, or generate only one diastereomer from a prochiral substrate. Fersht, in "Enzyme Structure and Mechanism", W. H. Freeman and Company, San Francisco, 1977, pp. 75–81. This specificity can be related to the chiral structure of the enzyme molecule, including the three-dimensional folding of the polypeptide backbone and the orientation of the amino acid side chains in the folded protein molecule. Fersht, supra. To date only L-enzymes have been described in nature; this leaves the description of D-enzymes and their properties, which include folded structure, enzymatic activity, and chiral specificity, as unexplored questions.
Recently, Zawadzke et al., J. Am. Chem. Soc., 114:4002–4003, 1992, described the preparation of a small 45 amino acid residue polypeptide (D-rubrodoxin) using D-amino acids. L-rubrodoxin is found in clostridia and is the simplest iron-sulfur protein. It is believed to function in electron transport. However, it lacks an demonstrated enzymic activity.
Prior to the present invention, the largest L-protein known to be chemically synthesized in a conventional step-wise fashion is Preprogonadotropin Release Hormone (PreproGnRH). PreproGnRH has 93 amino acid residues. (Milton et al., Biochemistry, (1992) 31: 8800.) PreproGnRH inhibits prolactin release.
Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes (+) and (−) or d and l are employed to designate the sign of rotation of plane-polarized light by the compound, with (−) or l meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are mirror images of one another. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric or racemic mixture.
The property of optical activity is due to molecular asymmetry about carbon atoms that are linked to four different atoms or molecules. Where there is only one asymmetric carbon atom, or chiral center as it is sometimes called, there are two possible stereoisomers. Where there are n asymmetric carbons or chiral centers, the number of potential stereoisomers increases to 2n. Thus, a molecule with three chiral centers would have eight possible stereoisomers.
While the structural differences between stereoisomers are subtle and of little consequence in ordinary chemical reactions, they may be profound where biological systems are concerned, i.e., if the compounds are utilized in enzyme-catalyzed reactions. Thus, the L-amino acids are readily metabolized in humans but the corresponding D-analogs are not, and only D-glucose can be phosphorylated and processed into glycogen or degraded by the glycolytic and oxidative pathways of intermediary metabolism. Similarly, beta blockers, pheromones, prostaglandins, steroids, flavoring and fragrance agents, pharmaceuticals, pesticides, herbicides, and many other compounds exhibit critical stereospecificity. In the field of pesticides, Tessier [Chemistry and Industry, Mar. 19, 1984, p. 199] has shown that only two of the eight stereoisomers of deltamethrin, a pyrethroid insecticide, have any biological activity. The same statement concerning the concentration of bioactivity in a single isomer can be made about many other pesticides, including the phenoxypropionates and halopropionate derivatives, each containing one chiral center and existing in the form of two optical isomers.
Stereochemical purity is of equal importance in the field of pharmaceuticals, where 12 of the 20 most prescribed drugs exhibit chirality. A case in point is provided by naproxen, or (+)-S-2-(6-methoxy-2-naphthyl)-propionic acid, which is one of the two most important members of a class of 2-aryl-propionic acids with non-steroidal anti-inflammatory activity used, for instance, in the management of arthritis. In this case, the S(+) enantiomer of the drug is known to be 28 times more therapeutically potent that its R(−) counterpart. Still another example of chiral pharmaceuticals is provided by the family of beta-blockers, the L-form of propranolol is known to be 100 times more potent that the D-enantiomer.
Synthesis of chiral compounds by standard organic synthetic techniques generally leads to a racemic mixture which, in the aggregate, may have a relatively low specific bioactivity since certain of the stereoisomers in the mixture are likely to be biologically or functionally inactive. As a result, larger quantities of the material must be used to obtain an effective dose, and manufacturing costs are increased due to the co-production of stereochemically "incorrect" and hence, inactive ingredients.
In some instances, certain isomers may actually be deleterious rather than simply inert. For example, the D-enantiomer of thalidomide was a safe and effective sedative when prescribed for the control of morning sickness during pregnancy. However, its L-thalidomide counterpart was discovered to be a potent mutagen.
Methods are available for stereoselective synthesis that generally involve chemical synthesis and isolation steps that are lengthy, complex and costly. Moreover, a synthetic scheme capable of producing one specific enantiomer cannot be applied in a general way to obtain other optically active compounds. What is needed is a generalized approach to the resolution of racemic mixtures produced by ordinary chemical reactions, and a number of approaches have been used.
A widely used approach has been the selective precipitation of desired compounds from racemic mixtures. See, for example, Yoshioka et al. [U.S. Pat. No. 3,879,451], Paven et al. [U.S. Pat. No. 4,257,976], Halmos [U.S. Pat. No. 4,151,198], and Kameswaran [U.S. Pat. No. 4,454,344].
The above procedures successfully resolved racemic mixtures because treatment of the mixtures with optically pure reagents produced diastereomers which, unlike the initial racemic compounds, have different physical properties. Thus, fractional crystallization or other physical means may be employed to separate diastereomeric compounds.
Separation of diastereomers can also be carried out by chromatography. For example, Pollock et al. [J. Gas Chromatogr. 3: 174 (1965)] have resolved diastereomeric amino acids by gas chromatography. Mikes et al. [J. Chromatogr. 112:205 (1976)] have used liquid chromatography to resolve diastereomeric dipeptides. In most cases, the optically pure reagents have been in the stationary phase during chromatographic separation, but they may also be used in elutants. Hare et al. [U.S. Pat. No. 4,290,893] have used liquid chromatography to resolve racemic mixtures that were treated with aqueous elutants containing optically pure reagents and metal cations; resolution occurred because the resulting diastereomeric complexes had different partition coefficients in the chromatographic system.
All of the methods described to this point have relied upon the availability of suitable optically pure reagents, but such reagents are often not available or else their use is prohibitively expensive. In an alternative approach, enzymatic resolution techniques have been developed. Many different classes of enzymes have been used for the resolution of stereoisomers on a preparative scale, including hydrolases (especially the lipases and esterases such as chymotrypsin), lyases, and oxidoreductases (e.g., amino acid oxidases and alcohol reductases). Generally speaking, enzymes for use in resolutions should ideally exhibit broad substrate specificity, so that they will be capable of catalyzing reactions of a wide range of "unnatural" substrates, and a high degree of stereoselectivity for catalyzing the reaction of one isomer to the exclusion of others.
The hydrolases (e.g., lipases and esterases) are among the more attractive enzymes for use in resolutions, because they do not require expensive cofactors, and some of them exhibit reasonable tolerance to organic solvents. Additionally, chiral chemistry often involves alcohols, carboxylic acids, esters, amides, and amines with chiral carbons, and carboxyl hydrolases are preferred choices as stereoselective catalysts for reactions of such species. For instance, enzymatic treatment has been applied to the resolution of racemic mixtures of amino acid esters. Stauffer [U.S. Pat. No. 3,963,573] and Bauer [U.S. Pat. No. 4,262,092].
Separation of reaction products from enzymes has been facilitated by attaching the enzyme to a solid support which could be removed by centrifugation or packed into a column through which the racemic mixtures were passed.
Enzymes have also been explored for the resolution of classes of compounds other than the amino acids discussed above. Immobilized lipase in principal resolves mixtures by enzymatic hydrolysis or transesterification. In the case of a biphasic hydrolysis reaction, the differing solubility properties of the acids and esters involved required the dispersion and agitation of mixtures containing the immobilized solid-phase enzyme, an aqueous buffer, and the water-immiscible organic phase containing solvent and reactant—a relatively inefficient process.
Enzymes have been applied to the resolution of optical isomers of insecticides. For instance, Mitsuda et al. [Eur. Patent Appl'n. Publ. No. 0 080 827 A2] contacted a racemic acetic acid ester with stereoselective esterases of microbial and animal origin in biphasic systems (i.e., aqueous/organic dispersion). In related work on optically purified pyrethroids, Mitsuda et al. [U.S. Pat. No. 4,607,013] employed microbial esterases. Klibanov et al. [U.S. Pat. No. 4,601,987] resolved racemic 2-halopropionic acids by means of lipase-catalyzed esterification reactions conducted in organic media.
Additional examples can also be provided of the state-of-the-art enzyme-mediated resolution as applied to the production of optically purified pharmaceuticals. Sih [U.S. Pat. No. 4,584,270] has disclosed enzymatic means for the production of optically pure (R)-4-amino-3-hydroxybutyric acid, a key intermediate in the preparation of L-carnitine.
Until recently only naturally occurring L-enzymes could be described, and this left the presumed properties of D-enzymes, including their folded structures, enzymatic activity and chiral specificity, as unexplored questions. What was needed was sufficient progress in the chemical synthesis of proteins to make possible the total synthesis of the D-enantiomer of whole enzymes in sufficient quantity to form crystals and to perform other functions.

D-enzyme compositions and methods of their use


A search engine is disclosed that utilizes natural language processing (NLP) techniques. The search engine utilizes meaning-based natural language processing using ontological semantics in analyzing the meaning of queries and the searched text. This system analyzes Web pages and queries. The NLP method produces equivalent meanings to a sequence of user initiated words, wherein relevance parsing of the original query produces a display of queries/questions as hot links to the next round of searching without additional typing by the user.

U.S. Pat. No. 5,794,050 to Dahlgren et al. provides for a Natural Language Understanding System. A naive semantic system that incorporates modules for text processing based upon parsing, formal semantics and discourse coherence, as well as relying on a naive semantic lexicon that stores word meanings in terms of a hierarchical semantic network is disclosed. Naive semantics is used to reduce the decision spaces of the other components of the natural language understanding system of. According to Dahlgren, naive semantics is used at every structure building step to avoid combinatorial explosion.
For example, the sentence "face places with arms down" has many available syntactic parses. The word "face" could be either a noun or a verb, as could the word places". However, by determining that "with arms down" is statistically most likely to be a prepositional phrase that attaches to a verb, the possibility that both words are nouns can be eliminated. Furthermore, the noun sense of "face" is eliminated by the fact that "with arms down" includes the concepts of position and body, and one sense of the verb "face" matches that conception. In addition to the naive semantic lexicon, a formal semantics module is incorporated, which permits sentences to be evaluated for truth conditions with respect to a model built by the coherence module. Coherence permits the resolution of causality, exemplification, goal, and enablement relationships. This is similar to the normal functionality of knowledge bases.
Natural language retrieval is performed by Dahlgren's system using a two-stage process referred to as digestion and search. In the digestion process, textual information is input into the natural language understanding module, and the NLU module generates a cognitive model of the input text. In other words, a query in natural language is parsed into the representation format of first-order logic and the previously described native semantics. The cognitive model is then passed to a search engine, that uses two passes: a high recall statistical retrieval module using unspecified statistical techniques to produce a long list of candidate documents; and a relevance reasoning module which uses first-order theorem proving, and human-like reasoning to determine which documents should be presented to the user. Generally, Dahlgren analyzes text based on sentence structure. The sentence is analyzed using a word-by-word analysis and a whole sentence analysis. Disclosed is a method for interpreting natural language input, wherein parsing and a naive semantic lexicon are utilized in conjunction to determine the plausibility of interpretative decisions, and wherein at least one entry identifying at least one sense of a word may be related to an ontological classification network, syntactic information, and a plurality of semantic properties.
Dahlgren system uses a semantic network similar to the ontologies employed in the system of present invention. However, it relies on a complicated grammatical system for the generation of formal structures, where complicated grammatical information is needed to eliminate possible choices in the parser. The concept based search engine system of the present invention provides an advantage in that it uses a simple grammatical system in which rule probabilities and conflicting ontological descriptions are used to resolve the possible syntactic parses of sentences. This greatly reduces the processing power required to index documents.
U.S. Pat. No. 6,675,159 to Lin et al. provides for a Concept-Based Search and Retrieval System. Disclosed is a concept-based method for searching text documents, wherein the method provides transforming a natural language query into predicate structures representing logical relationships between words in the natural language query; an ontology containing lexical semantic information about words; and means for ranking a set of matching natural language query predicate structures and equivalent text document predicate structures.
Lin's system imposes a logical structure on text, and a semantic representation is the form used for storage. The system provides logical representations for all of the content in a document and a semantic representation of comparable utility with significantly reduced processing requirements, and no need to train the system to produce semantic representations of text content. While training is needed to enable document categorization in the system, generation of the semantic representation is independent of the categorization algorithm.
U.S. Pat. No. 6,766,316 to Caudill et al. assigned to Science Application International Corporation, provides for a Method and System of Ranking and Clustering for Document Indexing and Retrieval. Disclosed is a relevancy ranking/clustering method and system for an information retrieval system which ranks documents based on relevance to a query and in accordance with user feedback. Additionally, a question and answering system further provides an answer formulation unit providing a natural language response to the input query.
U.S. Pat. No. 6,910,003 to Arnold, assigned to Discern Communications, Inc., discloses system and method for searching. Raw text is retrieved or input into the system. The raw text is parsed into components such as date, place, location, actors, and the like. The raw text is stored in topic specific information caches based on the individual components. In operation, a user enters a query. The system parses the user query and compares the parsed query to the topic specific information caches. Matching topic specific information caches are displayed to the user.
U.S. Patent Publication No. 2002/0059289 to Wenegrat et al. provides for Methods and Systems for Generating and Searching a Cross-Linked Keyphrase Ontology Database. Disclosed is a method of generating a cross-linked key-phrase ontology database, wherein the cross-linked key-phrase ontology database may be searched by parsing a natural language statement into a structured representation. The methods and systems of the invention involve the generation and use of a cross-linked keyphrase ontology database. A cross-linked keyphrase ontology database is created by: (a) defining at least one keyphrase; (b) representing the keyphrase by a keyphrase node in an ontology; (c) cross-linking the keyphrase node to at least one second keyphrase node, where the second keyphrase node represents a second keyphrase in a second ontology; and (d) repeating steps (b)-(c) for each keyphrase defined in step (a). The keyphrase in step (a) may be generated by parsing a text and can be selected from a group consisting of nouns, adjectives, verbs and adverbs. In one embodiment, the keyphrase in step (a) and the second keyphrase have at least one word in common. The text parsed may be in English or in any other written or spoken language
U.S. Patent Publication No. 2004/0103090 to Dogl et al. provides for a Document Search and Analyzing Method and Apparatus. Disclosed is a document search system having an ontology indexing function (ontology indexer 113), wherein search engine sub-system 125, in conjunction with indexer 113 and concept search engine 126, provides means for processing/parsing search queries to create a new entry for each word in a word index, and then associates it with a unique word ID, thereby allowing result builder 222 to create a two dimensional or three-dimensional graphical representation of the query data set or ontology (visualization model).
U.S. Patent Publication No. 2005/0125400 to Mori et al. provides for an Information Search System, Information Search Supporting System, and Method and Program for Information Search. Disclosed is an information search system having conversion means for decomposing a natural language sentence according to a dependence relationship between single words of the natural language and a corresponding ontology as means for generating an inquiry sentence.
Foreign patent WO/0235376 to Busch et al. provides for an Ontology-Based Parser for Natural Language Processing. Disclosed is a system and method for converting natural-language text into predicate-argument format by utilizing an ontology-based parser, wherein a sentence lexer may be utilized to convert a sentence into ontological entities tagged with part-of speech information.
U.S. Patent Publication No. 2002/0147578 to O'Neil et al., assigned to LingoMotors, Inc., provides for a Method and System for Query Reformulation for Searching of Information. Disclosed is a method for searching information using a reformulated query expression where a user inputs a query. The query is generally in a natural language form. The query is indicated as an input query. The input query is provided into an engine 103 to convert the natural language form into a logical form. The logical form is preferably one that has semantic information provided into the logical form. The logical form also has key terms of the query, among other information and is used for the query.

System and method for natural language processing and using ontological searches


A method for differentiating mesenchymal stem cells of bone marrow into neural cells comprises culturing the mesenchymal stem cells in a medium containing epidermal growth factor(EGF), basic fibroblast growth factor(bFGF) and hepatocyte growth factor(HGF), and the neural cells produced thereby can be employed for the treatment of a neural disease.

Stem cells have the ability to divide indefinitely in culture and give rise to specialized cells constituting a tissue upon stimulation by a specific differentiation stimulus.
Stem cells are divided into embryonic stem cells(ES cells) and tissue-specific stem cells depending on their differentiation potencies. ES cells are isolated from the inner cell mass(ICM) of embryos at the blastocyst stage and are pluripotent, i.e., they are capable of differentiating into virtually every type of cells found in an organism.
In contrast, tissue-specific stem cells appear at a stage of organ formation during the embryonic development and they are organ-specific and multipotent, i.e., they are generally committed to give rise to cells constituting a specific organ. These tissue-specific stem cells remain in most of adult organs and perform the critical role of continually replenishing the loss of cells occurring normally or pathologically. Representative tissue-specific stem cells include hematopoietic stem cells and mesenchymal stem cells present in bone marrow. Hematopoietic stem cells give rise to various blood cells such as erythrocytes and leukocytes; and mesenchymal stem cells, to the cells of connective tissues, e.g., osteoblasts, chondroblasts, adipocytes and myoblasts.
Recently, clinical applications of the stem cells have drawn an increasing interest since the successful isolation of human embryonic stem cell. The most noticeable potential application of the stem cells is their use as a perfect source of cell supply for a cell replacement therapy. Hardly curable diseases, e.g., neurodegenerative disease such as Parkinson's and Alzheimer's diseases, quadriplegia resulting from spinal cord injury, leukemia, apoplexy, juvenile-onset diabetes, cardiac infarction and liver cirrhosis, are caused by the disruption and permanent functional disorder of the cells constituting an organ, and the cell replacement therapy, wherein the loss of cells is replenished from the outside, has been presented as an effective remedy.
However, notwithstanding the obvious benefit of the cell replacement therapy, there exist many limitations in its clinical applications. Specifically, the conventional method, wherein fully differentiated cells isolated from the tissues of a donor are transplanted into a patient, has the problem that it is difficult to obtain a sufficient amount of cells to be supplied to the patient. In order to solve this problem, cells of a specific tissue differentiated from an isolated embryonic stem cell or differentiated cells from isolated and proliferated tissue-specific stem cells can be employed in a cell replacement therapy.
Hitherto, it has been proven that mouse embryonic stem cells can be differentiated on a culture dish into various cells such as hematopoietic cells, myocardial cells, insulin-secreting pancreatic cells and neural cells. Further, several reports have demonstrated that transplantation of the cells differentiated from stem cells is effective in the treatment of a disease caused by the loss of cells. For instance, synthesis of myelin in a mouse increased when myelin-synthesizing oligodendrocytes differentiated from an embryonic stem cell were transplanted into the mouse(Brustle et al., Science, 285: 754–756, 1999). Blood sugar level was regulated by transplanting insulin-secreting cells differentiated from an embryonic stem cell into a diabetes mouse model (Soria et al., Diabetes, 49: 157–162, 2000). Further, dyscinesia caused by spinal cord injury was remedied significantly by transplanting neural cells differentiated from an embryonic stem cell into a mouse having spinal cord injury(McDonald et al., Nat. Med., 5(12): 1410–1412, 1999).
However, since the human embryonic stem cell has been successfully isolated only recently and there is no report on the differentiation of the embryonic stem cell on a culture dish into other specific cells than neural cells, clinical use of specific tissue cells differentiated from an embryonic stem cell in a cell replacement therapy still remains on a level of possibility.
Further, since the efficiency of differentiation from an embryonic stem cell into target cells is low, there is a risk of an adverse side effect caused by other cells mixed with the target cells during transplantation. Accordingly, there exists a need for the development of a precise differentiation method for safer clinical application of the cells differentiated from embryonic stem cells.
On the other hand, in case when tissue-specific stem cells are employed in a cell replacement therapy, there is the problem that lowering of the proliferating ability of the cells or differentiation into unfavorable cells may occur during a long-term culture. Further, transplantation of neural cells is required for the treatment of a neurodegenerative disease such as Parkinson's disease. Since it is difficult to obtain neural stem cells directly from the patient, they are generally obtained by culturing neural stem cells isolated from the brain tissue of dead fetus and differentiating them into neural cells. However, the use of fetal brain invites the ethical problem as well as is limited by insufficient supply, and may cause an immunological rejection. Further, most of neural stem cells are liable to differentiate into astrocytes rather than neurons.
Accordingly, if it is possible to differentiate mesenchymal stem cells in patient's own bone marrow into neural cells to be used in a cell replacement therapy, neural cells can be readily supplied and such problems as immunological rejection would not occur during treatment.
Hitherto, it has been considered that one kind of stem cells differentiate only into the cells of a tissue belonging to a specific system. It was reported that mesenchymal stem cells formed in vitro colonies in the presence of various growth factors such as platelet-derived growth factor, basic fibroblast growth factor(bFGF), transforming growth factor-β (TGF-β) and epidermal growth factor(EGF)(Kuznetsov et al., Br. J. Haematol., 97: 561, 1997; and van den Bos C. et al., Human Cell, 10:45, 1997), and about one-third of initially attached cells had a multipotency, thereby differentiating into connective tissue cells such as osteoblasts, chondroblasts and adipocytes(Pittenger M F et al., Science, 284: 143, 1999). Further, Ferrari G. et al. reported that bone marrow is a source of myogenic precursor cells that form new muscles(Science, 279: 1528, 1998).
Recent studies reported that mesenchymal stem cells can also differentiate into the cells of the neural system. For instance, Sanchez-Ramos et al. reported that mesenchymal stem cells differentiated into neurons and astrocytes upon culture in the presence of retinoic acid and brain-derived neurotrophic factor(BDNF)(Exp. Neurology, 164: 247–256, 2000). Dale Woodbury et al. reported that mesenchymal stem cells in bone marrow differentiated into neural cells in the presence of antioxidants such as β-mercaptoethanol and dimethyl sulfoxide(DMSO)(J. Neuro. Res., 61: 364–370, 2000). However, the use of strong differentiation-inducing agents such as DMSO may cause a problem in a clinical application.
The present inventors have endeavored to discover materials that are highly safe and capable of differentiating stem cells in bone marrow into neural cells, and have discovered that HGF promotes the differentiation of mesenchymal stem cells of bone marrow into neural cells, and also that the addition of EGF and bFGF to a culture medium, together with HGF, significantly enhances both the differentiation of stem cells into neural cells and the amplification of the resulting neural cells.
It has been reported that EGF and bFGF stimulate the differentiation of neural stem cells into neurons or astrocytes when they are added to a serum-free medium for culturing neural stem cells separated from the brain tissue (Melissa et al., Exp. Neurology, 158: 265–278, 1999).
HGF has been reported to enhance the viability of neurons in hippocampus and mesencephalon, and induce the growth of neurite in neocortical explant(Hamanoue M et al., J. Neurosci. Res., 43: 554–564, 1996). Further, in the peripheral nervous system, it functions as an existence factor for motoneurons(Ebens A et al., Neuron, 17: 1157–1172, 1996), and involves in the growth and existence of sensory neurons and parasympathetic neurons (Fleur Davey et al., Mol. Cell Neurosci., 15: 79–87, 2000).
However, it has never been reported that mesenchymal stem cells can be differentiated into neural cells by culturing them in a medium containing EGF, bFGF and HGF.

Method for differentiating mesenchymal stem cells into neural cells


A dynamic latch circuit or a dynamic flip-flop circuit of the present invention includes a transfer gate to be controlled by a clock and provided with a complementary configuration using a P-channel and an N-channel MOS (Metal Oxide Semiconductor) transistor. The transfer gate allows the individual node included in the circuit to fully swing between a high potential power source and a low potential power source. This causes a minimum of decrease to occur in an ON current for driving the respective node and thereby realizes high-speed operation. Further, the balance of the rising time and falling time of an output signal is improved, reducing the deviation of the duty of the output signal from 50%. The circuit is therefore operable with sufficient operation margins at positive- and negative-going edges. Consequently, the entire macrocircuit using the circuit of the present invention can have its operation frequency and therefore power consumption lowered.

The present invention relates to a dynamic circuit for high-speed operation and, more particularly, to a dynamic flip-flop circuit and a dynamic latch circuit operable at a high speed while consuming a minimum of power.
A dynamic circuit of the kind described is used in the high-speed circuits art using a CMOS (complementary Metal Oxide Semiconductor) process. Various kinds of dynamic latch circuits and dynamic flop-flop circuits operable at high speeds are taught in "High-Speed CMOS Circuit Technique", IEEE Journal of Solid-State Circuits, Vol. 24, No. 1, February, 1989, pp. 62-70. However, the circuits taught in this document each has a problem that the rise time and fall time of its output signal are not balanced and cause the duty cycle to deviate from 50%, degrading the balance of circuit operation.

Dynamic circuit for high-speed operation


Disclosed are various embodiments for secure and anonymized electronic voting. A homomorphically encrypted vote and a digital signature of the homomorphically encrypted vote are received from a client device. Then, the digital signature of the homomorphically encrypted vote is validated. Next, the homomorphically encrypted vote is stored in a data store in response to validating the digital signature of the homomorphically encrypted vote. Finally, the homomorphically encrypted vote is recorded in a publicly available distributed ledger.

Voting is used for a variety of purposes. For example, consumer or customer polls may be used to identify popular products or product features. As another example, voting may be used to identify a most popular choice from a range of choices. For example, voting could be used to select a winning contestant in a performance or as a basis for providing an award to a candidate. Similarly, voting may be used to select policies or candidates at the ballot box.


A slave node for use with a Local Interconnect Network (LIN network) receives incoming messages from the LIN network in a manner that reduces operating current, power consumption and/or operating temperature. The slave node generally includes an input circuit, an electronic processing device, associated components such as an Integrated Glass Fog Sensor (IGFS) unit, and a transmitting device all mounted on a single circuit board. The input circuit, which can either be separate from or incorporated within the electronic processing device, pre-filters an incoming message from a LIN bus such that it only wakes the transmitting device out of its low-current sleep mode when required. Stated differently, the transmitting device is removed from an incoming message path so that it is only active when it is transmitting an outgoing message.

A LIN network is a single-wire serial communications protocol that is used in a number of industries, including the automotive industry. Reasons for its widespread use in the automotive industry include its scalability and its cost efficiency, the latter of which is at least partially attributed to the simplicity of Universal Asynchronous Receiver/Transmitter (UART) communication and the low cost of using a single-wire medium.
In general, a single LIN network usually includes a number of "slave nodes" that are periodically and/or intermittently addressed by a "master node." For example, a LIN network may include a single master node and up to sixteen separate slave nodes, and the slave nodes can be addressed by the master node at any given time and in a manner that requires no arbitration or collision management in the slave node itself. In such LIN networks, particularly where the number of slave nodes is large, current draw in each of the slave nodes can be a concern. This is particularly true in instances where it is an objective to reduce the power consumption and/or operating temperature in the individual slave nodes. In such situations, a combination of hardware and/or software techniques may be implemented to address the current draw issue, which in turn can affect power consumption and operating temperature.
In temperature and/or current sensitive applications, implementing a LIN network can present challenges due to the relatively high current draw of the slave nodes, and more specifically, of the LIN transceivers which are components of the slave nodes. According to the prior art circuit shown in FIG. 1, a typical LIN slave node 10 is connected to a LIN bus 12 and includes LIN transceiver 20, a microcontroller 22, and one or more additional component(s) 24 such as a sensor unit. The LIN transceiver 20 pre-filters and buffers all incoming signals from LIN bus 12, which causes the LIN transceiver to come out of a sleep mode every time there is a request or command placed on the LIN bus, even when it pertains to an entirely different slave node. Because LIN transceiver 20 is not a low current device, this can have a detrimental effect on the overall current being drawn by slave node 10. In certain temperature and/or current sensitive applications, the increased current draw caused by LIN transceiver 20 waking up each time a request is put on LIN bus 12 is undesirable.
An example of such a temperature and/or current sensitive application is disclosed in U.S. Pat. No. 6,422,062, which is assigned to the present assignee and is hereby incorporated by reference. This patent discloses an Integrated Glass Fog Sensor unit (hereafter referred to as an 'IGFS unit'), and is the type of sensor that could be used as sensor unit 24 in FIG. 1. Of course, the IGFS unit taught in the '062 patent is strictly being provided for exemplary purposes, as a number of other sensors and/or components could also be used in LIN slave node 10. The IGFS unit of this patent has a glass surface temperature sensor, an ambient air temperature sensor and a relative humidity sensor, and is used for predicting glass fog formation on a vehicle windshield.
Thus, it would be advantageous to provide a slave node for use in a LIN network where the slave node has a reduced operating current, power consumption and/or operating temperature. It is also desirable to provide a slave node for use with temperature and/or current sensitive applications such as, but not limited to, an IGFS unit.

Slave node and local interconnect network (LIN network) having same


When an image file exists in a folder determined to be the source of a file transfer according to the nth entry of file transfer data, a free space determining unit provided in an image processing device determines whether a work area on the hard disk meets or exceeds a required capacity. If the work area meets or exceeds the required capacity, a FTP client requests an FTP server to transfer the image file. As a result, an image file in a folder that is identified by the nth entry of the file transfer data to be the source of the transfer is transferred to a hot folder identified by the file transfer data to be the destination. Hence, by reliably allocating a work area required for image processing on the hard disk that accommodates the hot folder, it is possible to increase the stability of image processing without stalls and failures in the processing.

1. Field of the Invention
The present invention relates to a data file transfer controlling device used in a data file processing system including data file servers and a data file processing device that are connected with each other and that can transfer and receive data files among each other over a network.
2. Description of Related Art
There is well known an image processing system including image file servers connected to and capable of communicating with an image processing device via a network. Such an image processing system is disclosed in Japanese unexamined patent application publication No. HEI-11-205379.
In this type of image processing system, image files are transferred from the image file servers to the image processing device and are processed on the image processing device. While the image files are being processed, it is impossible to process other image files. Accordingly, in order to process the other image files, it is necessary to wait until the current image process has completed. During this time, those image files waiting to be processed are temporarily stored in a storage area provided in the image processing device.


A new resin gas-assisted injection technique, which can achieve both bonding and surface modification of microfluidic devices. A sealing resin is injected with a surface modification agent into the microfluidic platform to fill the micron and submicron sized channels and reservoirs, as well as the gap between the platform and the lid. A gas (e.g., air or nitrogen) is then injected to replace most of the resin inside the channels and reservoirs. The remaining resin is cured (fully or partially) by ultraviolet light. By applying a masking technique, local modification of the channel surface can also be achieved through this method. Also provided are methods and structures related to micro-fluidic devices.

Microfluidic platforms have attracted a great deal of attention in recent years and extensive research has been conducted in this area. They are a core technology used in a number of miniaturized systems that have been developed for chemical, biological, and medical applications. A wide range of microfluidic devices such as pumps, valves, mixers, and flow sensors has been demonstrated. In the past, these devices have been fabricated almost exclusively in silicon, glass, or quartz by applying techniques available in the microelectronics industry. For many applications, particularly in the life sciences (e.g. analytical chemistry, drug discovery, clinical diagnostics, etc.), these materials and the associated production methods are too expensive, or the material properties often induce problems such as a lack of optical clarity, low impact strength, and poor bio-compatibility. Polymers as substrate materials, on the other hand, provide a wide range of physical and chemical properties. They also have the advantages of low cost, good processibility for mass production, and are biocompatible and recyclable. Polymer microfabrication techniques have attracted great interest in recent years. Various methods (e.g. LIGA, UV-LIGA) and molding technologies (e.g. reactive casting, microembossing, and injection molding) have been applied to fabricate polymer-based microfluidic devices.
After fabrication, these devices need to be sealed in order to perform microfluidic functions. Packaging (i.e. sealing a device with a lid) is a challenging issue in the fabrication of polymer-based microfluidic devices. Bonding between silicon and silicon or other materials (e.g., glass, metal, etc.) is well developed and can be achieved by different methods such as anodic bonding, fusion bonding, eutectic bonding, and adhesive bonding. Among these techniques, only adhesive bonding can be applied to polymer-based microfluidic devices. When using the adhesive bonding method, care needs to be taken in order to prevent the adhesive from flowing into the micro channels. Other techniques such as adhesive tape bonding (lamination), thermal (IR, hot-plate, laser) bonding, ultrasonic welding, and solvent bonding (i.e., partially dissolve the bonding surfaces, and evaporate the solvent) have also been tried. Lum and Greenstein prepared microdepressions on one substrate and microprojections on the other so that the substrates can be mated together to secure the relative position. A layer of monomer or pre-polymer was deposited on the microprojections before being mated and further polymerized to provide a bonding effect. Dreuth and Heiden applied a thin adhesive film on a substrate and the adhesive was then transferred to the elevated microstructures by stamp printing. Glasgow et al. introduced a solvent bonding technique in which a layer of polyimide precursor and solvent with dissolved precursor was placed in contact with patterned structures made of uncured polyimide precursor. The two halves were then cured with weights on top of the upper plate. They found that the bond quality was affected by the vent spacing for solvent evaporation, soft-bake duration, spin-coat speed during solvent application, and the concentration of the dissolved polyimide precursor in the solvent. Basically, these approaches alter the surface of the microdevices by using external forces (e.g. solvent, adhesive, ultrasonic, laser) and applying pressure to bring two halves together. They have problems with either blocking the microchannels or changing their dimensions and are mainly applicable for relatively large microchannels (several hundreds of microns to millimeters).
In most bioMEMS applications, biocompatibility is one of the main requirements for the substrate used due to protein adsorption and cell adhesion on the substrate surface. For instance, blood thrombosis starts with deposition and aggregation of platelets on the surface of the prosthesis. In order to prevent thrombosis, the platelets should be kept from adhering to the surface of the prosthesis. Factors that affect protein adsorption include protein concentration, surface energy/tension, surface roughness, crystallinity, surface charge, etc. The surface free energy has been believed to be the dominant factor in protein adhesion. The thermodynamic driving force for protein deposition is low if the interfacial free energy between the substrate and the bio-fluids is low enough. It is believed that an interfacial free energy with water on the order of 1–3 mJ/M^2 could be a criterion for a material being biocompatible. Generally, the interfacial free energy between polymeric materials and bio-fluids is too large to meet the biocompatibility criterion. Therefore, surface modification is necessary to lower the interfacial free energy to produce surfaces that are able to resist cell adhesion and protein adsorption.
A great deal of effort has been devoted to the surface modification of biomaterials by coating the substrate surface with a hydrophilic material, such as polyethylene oxide (PEO) and polyethylene glycol (PEG), or with a surfactant such as sodium dodecyl sulfate (SDS), to increase the surface free energy of the biomaterials. It has been found that a PEG coating can change the surface from hydrophobic to hydrophilic, and thereby reducing protein adsorption and cell adhesion. The two-phase deposition of a surfactant (SDS) on a PMMA surface can also increase the hydrophilicity of a hydrophobic surface.
It is therefore a goal of the present invention to provide a method that can be used to bond microfluidic devices together. It is a further goal of the present invention to provide a method for general surface modification of microchannels in micro-fluidic devices. It is a further goal of the present invention to provide a method for providing localized surface modification of microchannels in micro-fluidic devices.

Gas-assisted resin injection technique for bonding and surface modification in micro-fluidic devices


A device for introducing gases into liquids in the form of fine bubbles, especially for introducing air into waste water, which device (1) comprises gas distributors (2) consisting of a longitudinal chamber (20) covered by a porous foil (19) arranged beneath the liquid level and to which gas is fed through a supply duct (5) and emerges into the liquid through the foil in the form of fine bubbles. The gas distributors (2) are connected to supply pipes (5) each provided for a group of gas distributors (2). The gas distributors (2) have a base wall (18) of rigidly-stable material, preferably metal, forming one side of the chamber (20) covered with the porous foil (19). Furthermore, it is preferred that the gas distributors (2), in the direction of their longitudinal extension, are mounted on carriers (7, 7a) arranged on the bottom of a basin or container in which the liquid is present.

Devices for introducing gases into liquids serve to admix the liquids with certain gases so as to initiate or aid biological and/or chemical processes in the liquids under participation of the gases introduced. Thus, the introduction of gases such as, e.g., oxygen or air, may serve to supply these gases to microorganisms present in the gas-treated liquid. The introduction of gases into a liquid may, however, also have the effect that substances present in the liquid change over into the gaseous phase and flow off; this occurs when blowing out carbonic acid or ammonia, e.g. The introduction of gases into liquids has various effects which, depending of the respective individual case, may be judged as advantageous or as disadvantageous. Thus, e.g., the motion of a liquid associated with the introduction of gases into the respective liquid, which motion is caused by the movement of the gas bubbles in the liquid, may be considered advantageous in some instances, because such a motion of the liquid counteracts the formation of deposits in the respective container or basin in which gases are introduced into the liquid. Frequently, it is also the aim to incorporate in a liquid as large a percentage of the gases introduced as possible; this is favorably influenced by as small a size of the gas bubbles introduced into the respective liquid as possible. An introduction of gases into liquids in the form of fine bubbles can be attained if the gases to be introduced are fed into the liquid through a porous foil submerged beneath the surface of the liquid into which the gases are to be introduced. This is particularly provided for biological sewage treatment plants, to introduce oxygen from air into the activated sludge mixture present in a treatment basin. Devices have been known with large-area gas distributors, and such a large-area configuration of the gas distributors leads to increased structural expenditures due to the provision of measures for supporting the porous foil against the internal pressure prevailing in the chambers of the gas distributors and required for the introduction of gas into the liquid, and if there are larger distances between individual, large-area gas distributors, there also occurs an non-uniform distribution of the introduction of the gas into the liquid, and from the non-uniform introduction of gas there also result currents within the liquid. Such currents may reduce the dwell time of the gas bubbles in the liquid, and, correspondingly, a smaller percentage of the gases introduced into the liquid is accommodated by the liquid. The above-mentioned wider distances between the individual gas distributors mostly result from the economically-based desire to get by with as small a total area of gas distributors as possible.
In a known device of the initially defined type, the gas distributors are designed in the form of a flat air hose consisting of an elastic material, in particular rubber, which is provided with a plurality of air outlet openings at its upper wall, one end of this hose being connected to an air supply duct, cantilevering from this connection site, rod-shaped carrying elements being slid into this hose, the carrying elements also cantilevering from this connection site at the air supply duct. This construction is structurally complex and, due to the design of the gas distributors and the cantilevering structure of the same, it is restricted to a relatively short longitudinal extension of the gas distributors, so that also in this instance, for economical reasons and so as not to make an access to the basin difficult due to a great number of air supply ducts, care must be taken to get by with a small overall area of gas distributors and to put up with greater distances between the air supply ducts, which, however, as mentioned before, will lead to a non-uniform distribution of the gas introduction into the liquid and the disadvantages entrained thereby.

Device for introducing gases into liquids in fine bubbles

Dipl.-Ing. Udo Meyer Aquaconsult Ges. M.B.H.