An polyampholyte is utilized in a condensed polynucleotide complex for purposes of nucleic acid delivery to a cell. The complex can be formed with an appropriate amount of positive and/or negative charge such that the resulting complex can be delivered to the extravascular space and may be further delivered to a cell.

Drug Delivery
A variety of methods and routes of administration have been developed to deliver pharmaceuticals that include small molecular drugs and biologically active compounds such as peptides, hormones, proteins, and enzymes to their site of action. Parenteral routes of administration include intravascular (intravenous, intraarterial), intramuscular, intraparenchymal, intradermal, subdermal, subcutaneous, intratumor, intraperitoneal, and intralymphatic injections that use a syringe and a needle or catheter. The blood circulatory system provides systemic spread of the pharmaceutical. Polyethylene glycol and other hydrophilic polymers have provided protection of the pharmaceutical in the blood stream by preventing its interaction with blood components and to increase the circulatory time of the pharmaceutical by preventing opsonization, phagocytosis and uptake by the reticuloendothelial system. For example, the enzyme adenosine deaminase has been covalently modified with polyethylene glycol to increase the circulatory time and persistence of this enzyme in the treatment of patients with adenosine deaminase deficiency.
The controlled release of pharmaceuticals after their administration is under intensive development. Pharmaceuticals have also been complexed with a variety of biologically-labile polymers to delay their release from depots. These polymers have included copolymers of poly(lactic/glycolic acid) (PLGA) (Jain, R. et al. Drug Dev. Ind. Pharm. 24, 703–727 (1998), ethylvinyl acetate/polyvinyl alcohol (Metrikin, D C and Anand, R, Curr Opin Ophthalmol 5, 21–29, 1994) as typical examples of biodegradable and non-degradable sustained release systems respectively.
Transdermal routes of administration have been effected by patches and ionotophoresis. Other epithelial routes include oral, nasal, respiratory, and vaginal routes of administration. These routes have attracted particular interest for the delivery of peptides, proteins, hormones, and cytokines, which are typically administered by parenteral routes using needles. For example, the delivery of insulin via respiratory, oral, or nasal routes would be very attractive for patients with diabetes mellitus. For oral routes, the acidity of the stomach (pH less than 2) is avoided for pH-sensitive compounds by concealing peptidase-sensitive polypaptides inside pH-sensitive hydrogel matrix (copolymers of polyethyleneglycol and polyacrylic acid). After passing low pH compartments of gastrointestinal tract such structures swells at higher pH releasing thus a bioactive compound (Lowman A M et al. J. Pharm. Sci. 88, 933–937 (1999). Capsules have also been developed that release their contents within the small intestine based upon pH-dependent solubility of a polymer. Copolymers of polymethacrylic acid (Eudragit S, Rohm America) are known as polymers which are insoluble at lower pH but readily solubilized at higher pH, so they are used as enteric coatings (Z Hu et al. J. Drug Target., 7, 223, 1999).
Biologically active molecules may be assisted by a reversible formation of covalent bonds. Quite often, it is found that the drug administered to a patient is not the active form of the drug, but is what is a called a prodrug that changes into the actual biologically active compound upon interactions with specific enzymes inside the body. In particular, anticancer drugs are quite toxic and are administered as prodrugs which do not become active until they come in contact with the cancerous cell (Sezaki, II., Takakura, Y., Hashida, M. Adv. Drug. Delivery Reviews 3, 193, 1989).
Recent studies have found that pH in solid tumors is 0.5 to 1 units lower than in normal tissue (Gerweck L E et al. Cancer Res. 56, 1194 (1996). Hence, the use of pH-sensitive polymers for tumor targeting is justified. However, this approach was demonstrated only in vitro (Berton, M, Eur. J. Pharm. Biopharm. 47, 119–23, 1999).
Liposomes were also used as drug delivery vehicles for low molecular weight drugs and macromolecules such as amphotericin B for systemic fungal infections and candidiasis. Inclusion of anti-cancer drugs such as adriamycin have been developed to increase their delivery to tumors and reduce it to other tissue sites (e.g. heart) thereby decreasing their toxicity. pH-sensitive polymers have been used in conjunction with liposomes for the triggered release of an encapsulated drug. For example, hydrophobically-modified N-isopropylacrylamide-methacrylic acid copolymer can render regular egg phosphatidyl chloline liposomes pH-sensitive by pH-dependent interaction of grafted aliphatic chains with lipid bilayer (O Meyer et al., FEBS Lett., 421, 61, 1998).
Gene and Nucleic Acid-Based Delivery
Gene or polynucleotide transfer is the cardinal process of gene therapy. The gene needs to be transferred across the cell membrane and enter the nucleus where the gene can be expressed. Gene transfer methods currently being explored included viral vectors and physical-chemical methods.
Viruses have evolved over millions of year to transfer their genes into mammalian cells. Viruses can be modified to carry a desired gene and become a "vector" for gene therapy. Using standard recombinant techniques, the harmful or superfluous viral genes can be removed and replaced with the desired normal gene. This was first accomplished with mouse retroviruses. The development of retroviral vectors were the catalyst that promoted current gene therapy efforts. However, they cannot infect all cell types very efficiently, especially in vivo. Other viral vectors based on Herpes virus are being developed to enable more efficient gene transfer into brain cells. Adenoviral and adenoassociated vectors are being developed to infect lung and other cells.
Besides using viral vectors, it is possible to directly transfer genes into mammalian cells. Usually, the desired gene is placed within bacterial plasmid DNA along with a mammalian promoter, enhancer, and other sequences that enable the gene to be expressed in mammalian cells. Several milligrams of the plasmid DNA containing all these sequences can be prepared and purified from the bacterial cultures. The plasmid DNA containing the desired gene can be incorporated into lipid vesicles (liposomes including cationic lipids such as Lipofectin) that then transfer the plasmid DNA into the target cell. Plasmid DNA can also be complexed with proteins that target the plasmid DNA to specific tissues just as certain proteins are taken up (endocytosed) by specific cells. Also, plasmid DNA can be complexed with polymers such as polylysine and polyethylenimine. Another plasmid-based technique involves "shooting" the plasmid DNA on small gold beads into the cell using a "gun". Finally, muscle cells in vivo have the unusual ability to take up and express plasmid DNA.
Gene therapy approaches can be classified into direct and indirect methods. Some of these gene transfer methods are most effective when directly injected into a tissue space. Direct methods using many of the above gene transfer techniques are being used to target tumors, muscle, liver, lung, and brain. Other methods are most effective when applied to cells or tissues that have been removed from the body and the genetically-modified cells are then transplanted back into the body. Indirect approaches in conjunction with retroviral vectors are being developed to transfer genes into bone marrow cells, lymphocytes, hepatocytes, myoblasts and skin cells.
Gene Therapy and Nucleic Acid-Based Therapies
Gene therapy promises to be a revolutionary advance in the treatment of disease. It is a fundamentally new approach for treating disease that is different from the conventional surgical and pharmaceutical therapies. Conceptually, gene therapy is a relatively simple approach. If someone has a defective gene, then gene therapy would fix the defective gene. The disease state would be modified by manipulating genes instead of their products, i.e. proteins, enzymes, enzyme substrates and enzyme products. Although, the initial motivation for gene therapy was the treatment of genetic disorders, it is becoming increasingly apparent that gene therapy will be useful for the treatment of a broad range of acquired diseases such as cancer, infectious disorders (AIDS), heart disease, arthritis, and neurodegenerative disorders (Parkinson's and Alzheimer's).
Gene therapy promises to take full-advantage of the major advances brought about by molecular biology. While, biochemistry is mainly concerned with how the cell obtains the energy and matter that is required for normal function, molecular biology is mainly concerned with how the cell gets the information to perform its functions. Molecular biology wants to discover the flow of information in the cell. Using the metaphor of computers, the cell is the hardware while the genes are the software. In this sense, the purpose of gene therapy is to provide the cell with a new program (genetic information) so as to reprogram a dysfunctional cell to perform a normal function. The addition of a new cellular function is provided by the insertion of a foreign gene that expresses a foreign protein or a native protein at amounts that are not present in the patient.
The inhibition of a cellular function is provided by anti-sense approaches (that is acting against messenger RNA) and that includes oligonucleotides complementary to the messenger RNA sequence and ribozymes. Messenger RNA (mRNA) is an intermediate in the expression of the DNA gene. The mRNA is translated into a protein. "Anti-sense" methods use an RNA sequence or an oligonucleotide that is made complementary to the target mRNA sequence and therefore binds specifically to the target messenger RNA. When this anti-sense sequence binds to the target mRNA, the mRNA is somehow destroyed or blocked from being translated. Ribozymes destroy a specific mRNA by a different mechanism. Ribozymes are RNA's that contain sequence complementary to the target messenger RNA plus a RNA sequence that acts as an enzyme to cleave the messenger RNA, thus destroying it and preventing it from being translated. When these anti-sense or ribozyme sequences are introduced into a cell, they would inactivate their specific target mRNA and reduce their disease-causing properties.
Gene therapy can be used as a type of vaccination to prevent infectious diseases and cancer. When a foreign gene is transferred into a cell and the protein is made, the foreign protein is presented to the immune system differently from simply injecting the foreign protein into the body. This different presentation is more likely to cause a cell-mediated immune response which is important for fighting latent viral infections such as human immunodeficiency virus (HIV causes AIDS), Herpes and cytomegalovirus. Expression of the viral gene within a cell simulates a viral infection and induces a more effective immune response by fooling the body that the cell is actually infected by the virus, without the danger of an actual viral infection.
Polymers for Drug and Nucleic Acid Delivery
Polymers are used for drug delivery for a variety of therapeutic purposes. Polymers have also been used in research for the delivery of nucleic acids (polynucleotides and oligonucleotides) to cells with an eventual goal of providing therapeutic processes. Such processes have been termed gene therapy or anti-sense therapy. One of the several methods of nucleic acid delivery to the cells is the use of DNA-polycation complexes. It has been shown that cationic proteins like histones and protamines or synthetic polymers like polylysine, polyarginine, polyomithine, DEAE dextran, polybrene, and polyethylenimine may be effective intracellular delivery agents while small polycations like spermine are ineffective. The following are some important principles involving the mechanism by which polycations facilitate uptake of DNA:
Polycations provide attachment of DNA to the cell surface. The polymer forms a cross-bridge between the polyanionic nucleic acids and the polyanionic surfaces of the cells. As a result the main mechanism of DNA translocation to the intracellular space might be non-specific adsorptive endocytosis which may be more effective then liquid endocytosis or receptor-mediated endocytosis. Furthermore, polycations are a convenient linker for attaching specific ligands to DNA and as result, DNA-polycation complexes can be targeted to specific cell types.
Polycations protect DNA in complexes against nuclease degradation. This is important for both extra- and intracellular preservation of DNA. Gene expression is also enabled or increased by preventing endosome acidification with NH4Cl or chloroquine. Polyethylenimine, which facilitates gene expression without additional treatments, probably disrupts endosomal function itself. Disruption of endosomal function has also been accomplished by linking to the polycation endosomal-disruptive agents such as fusion peptides or adenoviruses.
Polycations can also facilitate DNA condensation. The volume which one DNA molecule occupies in a complex with polycations is drastically lower than the volume of a free DNA molecule. The size of a DNA/polymer complex is probably critical for gene delivery in vivo. In terms of intravenous injection, DNA needs to cross the endothelial barrier and reach the parenchymal cells of interest. The largest endothelia fenestrae (holes in the endothelial barrier) occur in the liver and have an average diameter of 100 nm. The trans-epithelial pores in other organs are much smaller, for example, muscle endothelium can be described as a structure which has a large number of small pores with a radius of 4 nm, and a very low number of large pores with a radius of 20–30 nm. The size of the DNA complexes is also important for the cellular uptake process. After binding to the cells the DNA-polycation complex should be taken up by endocytosis. Since the endocytic vesicles have a homogenous internal diameter of about 100 nm in hepatocytes and are of similar size in other cell types, DNA complexes smaller than 100 nm are preferred.
Condensation of DNA
A significant number of multivalent cations with widely different molecular structures have been shown to induce condensation of DNA.
Two approaches for compacting (used herein as an equivalent to the term condensing) DNA:
1. Multivalent cations with a charge of three or higher have been shown to condense DNA. These include spermidine, spermine, Co(NH3)63+, Fe3+, and natural or synthetic polymers such as histone H1, protamine, polylysine, and polyethylenimine. Analysis has shown DNA condensation to be favored when 90% or more of the charges along the sugar-phosphate backbone are neutralized.
2. Polymers (neutral or anionic) which can increase repulsion between DNA and its surroundings have been shown to compact DNA. Most significantly, spontaneous DNA self-assembly and aggregation process have been shown to result from the confinement of large amounts of DNA, due to excluded volume effect.
The mechanism of DNA condensation is not clear. The electrostatic force between unperturbed helices arises primarily from a counterion fluctuation mechanism requiring multivalent cations and plays a major role in DNA condensation. The hydration forces predominate over electrostatic forces when the DNA helices approach closer then a few water diameters. In a case of DNA-polymeric polycation interactions, DNA condensation is a more complicated process than the case of low molecular weight polycations. Different polycationic proteins can generate toroid and rod formation with different size DNA at a ratio of positive to negative charge of two to five. T4 DNA complexes with polyarginine or histone can form two types of structures; an elongated structure with a long axis length of about 350 nm (like free DNA) and dense spherical particles. Both forms exist simultaneously in the same solution. The reason for the co-existence of the two forms can be explained as an uneven distribution of the polycation chains among the DNA molecules. The uneven distribution generates two thermodynamically favorable conformations.
The electrophoretic mobility of DNA-polycation complexes can change from negative to positive in excess of polycation. It is likely that large polycations don't completely align along DNA but form polymer loops that interact with other DNA molecules. The rapid aggregation and strong intermolecular forces between different DNA molecules may prevent the slow adjustment between helices needed to form tightly packed orderly particles.
As previously stated, preparation of polycation-condensed DNA particles is of particular importance for gene therapy, more specifically, particle delivery such as the design of non-viral gene transfer vectors. Optimal transfection activity in vitro and in vivo can require an excess of polycation molecules. However, the presence of a large excess of polycations may be toxic to cells and tissues. Moreover, the non-specific binding of cationic particles to all cells forestalls cellular targeting. Positive charge also has an adverse influence on biodistribution of the complexes in vivo.
Several modifications of DNA-cation particles have been created to circumvent the nonspecific interactions of the DNA-cation particle and the toxicity of cationic particles. Examples of these modifications include attachment of steric stabilizers, e.g. polyethylene glycol, which inhibit nonspecific interactions between the cation and biological polyanions. Another example is recharging the DNA particle by the additions of polyanions which interact with the cationic particle, thereby lowering its surface charge, i.e. recharging of the DNA particle U.S. Ser. No. 09/328,975. Another example is cross-linking the polymers and thereby caging the complex U.S. Ser. No. 08/778,657, U.S. Ser. No. 09/000,692, U.S. Ser. No. 97/24089, U.S. Ser. No. 09/070,299, and U.S. Ser. No. 09/464,871. Nucleic acid particles can be formed by the formation of chemical bonds and template polymerization U.S. Ser. No. 08/778,657, U.S. Ser. No. 09/000,692, U.S. Ser. No. 97/24089, U.S. Ser. No. 09/070,299, and U.S. Ser. No 09/464,871.
A problem with these modifications is that they are most likely irreversible rendering the particle unable to interact with the cell to be transfected, and/or incapable of escaping from the lysosome once taken into a cell, and/or incapable of entering the nucleus once inside the cell. A method for formation of DNA particles that is reversible under conditions found in the cell may allow for effective delivery of DNA. The conditions that cause the reversal of particle formation may be, but not limited to, the pH, ionic strength, oxidative or reductive conditions or agents, or enzymatic activity.
DNA Template Polymerization
Low molecular weight cations with valency, i.e. charge, <+3 fail to condense DNA in aqueous solutions under normal conditions. However, cationic molecules with the charge <+3 can be polymerized in the presence of DNA and the resulting polymers can cause DNA to condense into compact structures. Such an approach is known in synthetic polymer chemistry as template polymerization. During this process, monomers (which are initially weakly associated with the template) are positioned along template's backbone, thereby promoting their polymerization. Weak electrostatic association of the nascent polymer and the template becomes stronger with chain growth of the polymer. Trubetskoy et al used two types of polymerization reactions to achieve DNA condensation: step polymerization and chain polymerization (VS Trubetskoy, V G Budker, L J Hanson, P M Slattum, J A Wolff, L E Hagstrom. Nucleic Acids Res. 26:4178–4185, 1998) U.S. Ser. No. 08/778,657, U.S. Ser. No. 09/000,692, U.S. Ser. No. 97/24089, U.S. Ser. No. 09/070,299, and U.S. Ser. No. 09/464,871. Bis(2-aminoethyl)-1,3-propanediamine (AEPD), a tetramine with 2.5 positive charges per molecule at pH 8 was polymerized in the presence of plasmid DNA using cleavable disulfide amino-reactive cross-linkers dithiobis (succinimidyl propionate) and dimethyl-3,3′-dithiobispropionimidate. Both reactions yielded DNA/polymer complexes with significant retardation in agarose electrophoresis gels demonstrating significant binding and DNA condensation. Treatment of the polymerized complexes with 100 mM dithiothreitol (DTT) resulted in the pDNA returning to its normal supercoiled position following electrophoresis proving thus cleavage the backbone of the. The template dependent polymerization process was also tested using a 14 mer peptide encoding the nuclear localizing signal (NLS) of SV40 T antigen (CGYGPKKKRKVGGC SEQ ID 11), as a cationic "macromonomer". Other studies included pegylated comonomer (PEG-AEPD) into the reaction mixture and resulted in "worm"-like structures (as judged by transmission electron microscopy) that have previously been observed with DNA complexes formed from block co-polymers of polylysine and PEG (M A Wolfert, E H Schacht, V Toncheva, K Ulbrich, O Nazarova, L W Seymour. Human Gene Ther. 7:2123–2133, 1996). Blessing et al used bisthiol derivative of spermine and reaction of thiol-disulfide exchange to promote chain growth. The presence of DNA accelerated the polymerization reaction as measured the rate of disappearance of free thiols in the reaction mixture (T Blessing, J S Remy, J P Behr. J. Am. Chem. Soc. 120:8519–8520, 1998).
Caging of Polycation-Condensed DNA Particles.
The stability of DNA nanoassemblies based on DNA condensation is generally low in vivo because they easily engage in polyion exchange reactions with strong polyanions. The process of exchange consists of two stages: 1) rapid formation of a triple DNA-polycation-polyanion complex, 2) slow substitution of one same-charge polyion with another. At equilibrium conditions, the whole process eventually results in formation of a new binary complex and an excess of a third polyion. The presence of low molecular weight salt can greatly accelerate such exchange reactions, which often result in complete disassembly of condensed DNA particles. Hence, it is desirable to obtain more colloidally stable structures where DNA would stay in its condensed form in complex with corresponding polycation independently of environment conditions.
The complete DNA condensation upon neutralization of only 90% of the polymer's phosphates results in the presence of unpaired positive charges in the DNA particles. If the polycation contains such reactive groups, such as primary amines, these unpaired positive charges may be modified. This modification allows practically limitless possibilities of modulating colloidal properties of DNA particles via chemical modifications of the complex. We have demonstrated the utility of such reactions using traditional DNA-poly-L-lysine (DNA/PLL) system reacted with the cleavable cross-linking reagent dimethyl-3,3′-dithiobispropionimidate (DTBP) which reacts with primary amino groups with formation of amidines (V S Trubetskoy, A Loomis, P M Slattum, J E Hagstrom, V G Budker, J A Wolff. Bioconjugate Chem. 10:624–628, 1999) U.S. Ser. No. 08/778,657, U.S. Ser. No. 09/000,692, U.S. Ser. No. 09/070,299, and U.S. Ser. No. 09/464,871. Similar results were achieved with other polycations including poly(allylamine) and histone H1. The use of another bifucntional reagent, glutaraldehyde, has been described for stabilization of DNA complexes with cationic peptide CWK18 (R C Adam, K G Rice. J. Pharm. Sci. 739–746, 1999).
Recharging.
The caging approach described above could lead to more colloidally stable DNA assemblies. However, this approach may not change the particle surface charge. Caging with bifunctional reagents, which preserve positive charge of amino group, keeps the particle positive. However, negative surface charge would be more desirable for many practical applications, i.e. in vivo delivery. The phenomenon of surface recharging is well known in colloid chemistry and is described in great detail for lyophobic/lyophilic systems (for example, silver halide hydrosols). Addition of polyion to a suspension of latex particles with oppositely-charged surface leads to the permanent absorption of this polyion on the surface and, upon reaching appropriate stoichiometry, changing the surface charge to opposite one. This whole process is salt dependent with flocculation to occur upon reaching the neutralization point.
We have demonstrated that similar layering of polyelectrolytes can be achieved on the surface of DNA/polycation particles (V S Trubetskoy, A Loomis, J E Hagstrom, V G Budker, J A Wolff. Nucleic Acids Res. 27:3090–3095, 1999). The principal DNA-polycation (DNA/pC) complex used in this study was DNA/PLL (1:3 charge ratio) formed in low salt 25 mM HEPES buffer and recharged with increasing amounts of various polyanions. The DNA particles were characterized after addition of a third polyion component to a DNA/polycation complex using a new DNA condensation assay (V S Trubetskoy, P M Slattum, J E Hagstrom, J A Wolff, V G Budker. Anal. Biochem. 267:309–313, 1999) and static light scattering. It has been found that certain polyanions such as poly(methacrylic acid) and poly(aspartic acid) decondensed DNA in DNA/PLL complexes. Surprisingly, polyanions of lower charge density such as succinylated PLL and poly(glutamic acid), even when added in 20-fold charge excess to condensing polycation (PLL) did not decondense DNA in DNA/PLL (1:3) complexes. Further studies have found that displacement effects are salt-dependent. In addition, poly-L-glutamic acid but not the relatively weaker polyanion succinylated poly-L-lysine (SPLL) displaces DNA at higher sodium chloride concentrations. Measurement of ζ-potential of DNA/PLL particles during titration with SPLL revealed the change of particle surface charge at approximately the charge equivalency point. Thus, it can be concluded that addition of low charge density polyanion to the cationic DNA/PLL particles results in particle surface charge reversal while maintaining condensed DNA core intact. Finally, DNA/polycation complexes can be both recharged and crosslinked or caged U.S. Ser. No. 08/778,657, U.S. Ser. No. 09/000,692, U.S. Ser. No. 97/24089, U.S. Ser. No. 09/070,299, and U.S. Ser. No. 09/464,871.
The Use of pH-Sensitive Lipids, Amphipathic Compounds, and Liposomes for Drug and Nucleic Acid Delivery.
Cationic liposomes may deliver DNA either directly across the plasma membrane or via the endosome compartment. Regardless of its exact entry point, much of the DNA within cationic liposomes does accumulate in the endosome compartment. Several approaches have been investigated to prevent loss of the foreign DNA in the endosomal compartment by protecting it from hydrolytic digestion within the endosomes or enabling its escape from endosomes into the cytoplasm. They include the use of acidotropic (lysomotrophic), weak amines such as chloroquine that presumably prevent DNA degradation by inhibiting endosomal acidification (Legendre, J. & Szoka, F. Delivery of plasmid DNA into mammalian cell lines using pH-sensitive liposomes: Comparison with cationic liposomes. Pharmaceut. Res. 9, 1235–1242 (1992)). Viral fusion peptides or whole virus have been included to disrupt endosomes or promote fusion of liposomes with endosomes, and facilitate release of DNA into the cytoplasm (Kamata, H., Yagisawa, H., Takahashi, S. & Hirata, H. Amphiphilic peptides enhance the efficiency of liposome-mediated DNA transfection. Nucleic Acids Res. 22, 536–537 (1994). Wagner, E., Curiel, D. & Cotten, M. Delivery of drugs, proteins and genes into cells using transferrin as a ligand for receptor-mediated endocytosis. Advanced Drug Delivery Reviews 14, 113–135 (1994)).
Knowledge of lipid phases and membrane fusion has been used to design potentially more versatile liposomes that exploit the endosomal acidification to promote fusion with endosomal membranes. Such an approach is best exemplified by anionic, pH-sensitive liposomes that have been designed to destabilize or fuse with the endosome membrane at acidic pH (Duzgunes, N., Straubinger, R. M., Baldwin, P. A. & Papahadjopoulos, D. PH-sensitive liposomes. (eds Wilschub, J. & Hoekstra, D.) p. 713–730 (Marcel Deker INC, 1991)). All of the anionic, pH-sensitive liposomes have utilized phosphatidylethanolamine (PE) bilayers that are stabilized at non-acidic pH by the addition of lipids that contain a carboxylic acid group. Liposomes containing only PE are prone to the inverted hexagonal phase (HII). In pH-sensitive, anionic liposomes, the carboxylic acid's negative charge increases the size of the lipid head group at pH greater than the carboxylic acid's pKa and thereby stabilizes the phosphatidylethanolamine bilayer. At acidic pH conditions found within endosomes, the uncharged or reduced charge species is unable to stabilize the phosphatidylethanolamine-rich bilayer. Anionic, pH-sensitive liposomes have delivered a variety of membrane-impermeable compounds including DNA. However, the negative charge of these pH-sensitive liposomes prevents them from efficiently taking up DNA and interacting with cells; thus decreasing their utility for transfection. We have described the use of cationic, pH-sensitive liposomes to mediate the efficient transfer of DNA into a variety of cells in culture U.S. Ser. No. 08/530,598, and U.S. Ser. No. 09/020,566.
The Use of pH-Sensitive Polymers for Drug and Nucleic Acid Delivery
Polymers that pH-sensitive are have found broad application in the area of drug delivery exploiting various physiological and intracellular pH gradients for the purpose of controlled release of drugs (both low molecular weight and polymeric). pH sensitivity can be broadly defined as any change in polymer's physico-chemical properties over certain range of pH. More narrow definition demands significant changes in the polymer's ability to retain (release) a bioactive substance (drug) in a physiologically tolerated pH range (usually pH 5.5–8). All polyions can be divided into three categories based on their ability to donate or accept protons in aqueous solutions: polyacids, polybases and polyampholytes. Use of pH-sensitive polyacids in drug delivery applications usually relies on their ability to become soluble with the pH increase (acid/salt conversion), to form complex with other polymers over change of pH or undergo significant change in hydrophobicity/hydrophilicity balance. Combinations of all three above factors are also possible.

Compositions and methods for drug delivery using pH sensitive molecules


A blood glucose meter having a compact housing, a display unit disposed on the housing, the display unit including a display light source to illuminate the display unit, an input unit disposed on the housing, the input unit configured to provide input functions for the blood glucose meter, and a power source provided within the housing for providing power to the blood glucose meter, where the housing includes a port integrated on said housing configured to receive a blood glucose test strip, and corresponding methods of measuring blood glucose meter is provided.

The present invention relates to data monitoring systems. More specifically, the present invention relates to portable analyte monitoring systems. More specifically, the present invention relates to a compact, blood glucose meter for use in a glucose monitoring system to self-monitor glucose level in whole blood, as an aid to disease management, and also by healthcare professionals as an aid to monitor the effectiveness of diabetes treatment.

Method and apparatus for providing power management in data communication systems


The technology relates to cargo vehicles. National, regional and/or local regulations set requirements for operating cargo vehicles, including how to distribute and secure cargo, and how often the cargo should be inspected during a trip. However, such regulations have been focused on traditional human-driven vehicles. Aspects of the technology address various issues involved with securement and inspection of cargo before a trip, as well as monitoring during the trip so that corrective action may be taken as warranted. For instance, imagery and other sensor information may be used to enable proper securement of cargo before starting a trip. Onboard sensors along the vehicle monitor the cargo and securement devices/systems during the trip to identify issues as they arise. Such information is used by the onboard autonomous driving system (or a human driver) to take corrective action depending on the nature of the issue.

Autonomous vehicles, such as vehicles that do not require a human driver, can be used to aid in the transport of cargo or passengers from one location to another. Such vehicles may operate in a fully autonomous mode, or a partially autonomous mode where a person may provide some driving input. There are various rules and regulations involving the transportation of cargo in order to promote safety. Different requirements may apply to different types of vehicles and different types of cargo. Regardless of vehicle or cargo type, it may be very challenging for a cargo vehicle operating in autonomous mode to meet certain regulatory requirements due to the reduced role or complete absence of a driver in the vehicle.

Cargo inspection, monitoring and securement in self-driving trucks


The present application describes systems and methods for composing and sending facsimile messages using a digital pointing instrument. In further configurations, a user utilizes a pen to indicate a reference to a document that is appended to the outgoing facsimile. In another further configuration, the user utilizes a form having pre-defined regions that are used to correlate a pen stroke with a desired action.

The illustrative embodiments disclosed in the present application are useful in systems including those for processing facsimile messages and more particularly are useful in systems including those for sending facsimile messages using a digital pen.
Traditional facsimile machines are typically found in small to large corporate offices and are often found in the small office/home office (SOHO) settings. Facsimile machines may be found in small retail businesses, hotels, schools and in stores providing such services to individuals as a retail service. Such typical facsimile machines include a chassis having a connection to an analog telephone line, an analog modem for communication and a keypad for dialing the telephone number of a receiving facsimile machine. Such typical facsimile machines include a monochromatic (black and white), one bit per pixel scanner for facsimile output and a printer for output. The printer technology utilized in traditional facsimile machines include thermal printing, inkjet printing and laser printing. The scanner input and printing engine are typically limited to 8.5-inch wide paper. Accordingly, the size of documents that may be sent using a traditional facsimile machine is limited. The traditional facsimile machine is limited in the size of documents that may be printed, although incoming documents may in some cases be scaled to fit the available paper size.
The traditional facsimile machine receives documents that may be typed or handwritten. The documents are scanned into a facsimile transmission format that is typically compressed. The sender inputs the recipient telephone number, the facsimile machines negotiate a connection and protocol and the facsimile is transmitted as a compressed monochromatic bitmap representation of the original.
More recently, facsimile service systems have become available for general-purpose computers such as the Windows/Intel personal computer platforms in which a user may send a computer file such as a word processing file to a recipient facsimile machine. In such a system, the file is typically converted to a facsimile format file such as TIFF and the computer uses its analog modem to send the facsimile as if it had been scanned into a facsimile machine. These systems are useful only when the document to be sent is available in some digital form and are not useful for sending handwritten information such as a signed letter. Certain other systems utilize a general-purpose scanner and general-purpose computer to provide the functionality of a traditional facsimile machine.
In using a traditional facsimile machine, a user may handwrite a cover sheet having the number of pages and the origin and destination information written on it. The user will then send the facsimile cover sheet with the attached letter to the intended recipient by dialing the recipient facsimile machine number and starting the transmission.
Accordingly, user must have access to the attached letter at the traditional facsimile machine or on the general-purpose computer of the facsimile service system. Such a user may be required to walk to the neatest facsimile machine and may also be required to wait in line for an available facsimile machine or scanner for a facsimile service system. Similarly, the user of a facsimile service system must have access to the general-purpose computer to process a facsimile and may also have to wait for an available scanner. A reference entitled method and apparatus for pen-based faxing is shown in U.S. Pat. No. 6,201,903 issued Mar. 13, 2001 to Wolff, et al. and is incorporated herein by reference. The reference describes a system for designating a recipient of a facsimile using strokes made with a pen.

Method and system for creating and sending a facsimile using a digital pen


A device for three-dimensional measurement of an object, with a stereomicroscope of the telescope type, which has a common objective and a first and a second tube lens, a first image sensor, which is assigned to the first tube lens, a second image sensor (8), which is assigned to the second tube lens, and a control unit, which determines the three-dimensional shape of the object from the images obtained with the two image sensors and carries out a predetermined three-dimensional measurement, wherein the stereomicroscope is designed such that the images are obtained with object-side telecentric conditions of the stereomicroscope.

In the area of quality assurance, three-dimensional measurement of objects or workpieces is an everyday task. Contactless methods are of particular interest, because these practically rule out any damage to the workpiece and permit a fast cycle time. Optical methods of triangulation may be mentioned in particular. These use two optical images of the object to be measured, which were taken from different positions. The three-dimensional shape of the object to be measured can be obtained by comparing the two images.
However, these methods require precise knowledge of the imaging positions and of the optical channels with which the images were obtained. An arrangement with two identical objectives, aligned parallel to one another, would be particularly advantageous. In practice, however, this is very expensive, because owing to manufacturing tolerances no identical objectives are available.
On this basis, the problem to be solved by the invention is to provide a device for three-dimensional measurement of an object, with which three-dimensional measurement can be carried out with the desired accuracy at the lowest possible cost. Moreover, a corresponding method for three-dimensional measurement of an object is to be provided.

Device and method for the three-dimensional measurement of an object


Soft errors generated at an active time are reduced by adding a small-scale circuit to a high performance LSI, such as a processor without reducing the performance of the circuit. The processor has individual logic circuits each having a plurality of stages of logic gates for outputting true signals and complement signals for the individual logic gates. A latch circuit latches the true and complement signals of the logic circuits separately and a compare circuit detects for an error by comparing the true and complement output signals of the logic circuits to determine if they are at the same logical signal level or not, just upstream of the latch in which the individual true and complement output signals of the final logic circuit stages are individually latched. When the compare circuit detects an error because the true and complement output signals are at the same logical signal level, a recovery process is executed.

The present invention relates to a logic circuit including a logic LSI such as a processor that has a resistance of soft errors at an active time and, more particularly, to a high-performance microprocessor having error detection logic.
A logic LSI having high-speed action can be realized by a dynamic logic circuit. The dynamic logic circuit determines the potential of an output signal by holding or discharging a charge, which is stored in a dynamic node by a precharge, in accordance with a logic function. As a result, no short current flows at the active time so that the dynamic logic circuit can act at a high speed.
However, the charge, as stored in the dynamic node, may be erroneously discharged by noise caused, for example, by the incidence of .alpha. rays. At such time, the result of the logic function cannot be correctly reflected in the output of the dynamic logic circuit thereby to cause an error. This type of error will occur not steadily but only with the occurrence of noise such as the incidence of .alpha. rays, so that it is called "soft error".
A technique for making the high-speed action possible while resisting the aforementioned soft error at a single gate level is disclosed in Japanese Patent Laid-Open No. 168724/1990 (hereinafter the "First Prior Art"). This technique is realized by noise suppression means for holding the charge of a dynamic node after the precharge action, and means for precharging the internal node of an n-MOS network constructing the logic function. If the noise suppression means for holding the charge of the dynamic node is strengthened, the soft error can be sufficiently suppressed. On the other hand, however, the short current at the logic action time is increased to obstruct the high-speed action. By precharging the internal node of the n-MOS network constructing the logic function, therefore, the noise is prevented from occurring due to the charge share, and the noise suppression means for holding the charge of the dynamic node suppresses only the noise due to the incidence of .alpha. rays. As a result, the strength of the noise suppression means can be reduced to a minimum necessary for suppressing the occurrence of noise due to the incidence of .alpha. rays, so that the short current at the logic action time can be reduced to allow a high-speed action at some level.
An example of attaining resistance to soft error not at the single gate level but for a logic block of considerable size is disclosed on pp. 54 to 55 of "1982 IEEE International Solid-State Circuits Conference DIGEST OF TECHNICAL PAPERS" (hereinafter the "Second Prior Art"). According to the disclosed method, an error is detected in data by adding parity to the data and detecting an error in a logic circuit by duplexing a logic circuit to compare its outputs. Thus, there is provided means for discriminating between normal and abnormal actions, not by suppressing the occurrence of a noise due to the incidence of .alpha. rays, but by detecting the soft error due to noise directly.
A method of constructing a logic circuit having an effect similar to that of duplex logic circuits is disclosed on pg. 21 of "1996 IEEE International Solid-State Circuits Conference TUTORIAL#4" (hereinafter the "Third Prior Art"). The dynamic logic circuit forming the single gate is a circuit for generating both a true output and a complement output. Because of the dynamic circuit, both the output signals take a low potential at the precharge time, and one of the output signals is changed to a high potential at the logic action time. Since both the output signals intrinsically never take high potential at the same time, the error can be detected if both the output signals should simultaneously take the high potential.

Logic circuit having error detection function and processor including the logic circuit


Devices, systems, and methods for spread spectrum clock generation are disclosed. The devices, systems, and methods generate a clock signal at a frequency and generate a voltage output based on the frequency of the clock signal, wherein the generated voltage output is indicative of the frequency of the generated clock signal. The devices, systems, and methods also compare the frequency of the clock signal generated to a desired frequency output by comparing the generated voltage output to a voltage reference and adjust the frequency of the clock signal generated based on the results of the comparison.

High frequency oscillators may be used to generate clocks for electronic circuits. High frequency oscillators, for example, may be used for spread spectrum clock generation. Some example electronic circuits that may use such clock generation include integrated circuits (ICs), which may be used to implement, for example, systems-on-a-chip, processors, and chipset chips. Spread spectrum clock generation techniques may reduce adverse effects of electro-magnetic interference (EMI).
Spread spectrum clock generation techniques may use variable frequencies that vary between a maximum frequency value and a minimum frequency value to generate different oscillator wave forms such as sine waves, triangle waves, or other shaped waves. Spread spectrum clock generation may be performed using a phase-locked loop (PLL), such as by modulating a voltage controlled oscillator (VCO) control voltage or by modulating a feedback divider ratio.

High frequency oscillator with spread spectrum clock generation


There are provided a composition for determining the efficacy of a c-Met antibody including marker genes and a method for determining the efficacy of a c-Met antibody using the marker genes.

1. Field
Provided are a composition for determining the effects of an anti-c-Met antibody including a marker gene, and a method for determining the effects of an anti-c-Met antibody using the marker gene.
2. Description of the Related Art
c-Met, a typical receptor tyrosine kinase (RTK) present at the surface of cells, binds to its ligand, hepatocyte growth factor (HGF) to promote intracellular signal transduction thereby promoting the growth of cells. Additionally, c-Met is overexpressed in cancer cells, such that it is widely implicated in cancer incidence, cancer metastasis, cancer cell migration, cancer cell penetration, angiogenesis, etc.
Anti-c-Met antibodies serve as anticancer drugs to suppress the increase of cancer cells. The suppression of the increase of cancer cells can be achieved when cell proliferation is delayed or apoptosis is facilitated. It has been reported that when EBC1 cells, a lung cancer cell line, were treated with an anti-c-Met antibody, their growth was reduced by the facilitation of apoptosis. In other words, anti-c-Met antibodies serve as anticancer drugs by facilitating apoptosis.
In anticancer therapy using an anti-c-Met antibody, it is important to monitor whether the anti-c-Met antibody that is being used for treatment properly exhibits cancer cell apoptosis effects for efficient anticancer treatment. Accordingly, there is a need for the development of a biomarker capable of monitoring the cancer cell apoptosis effects of an anti-c-Met antibody.

Marker for determining effects of anti-c-Met antibody and method of determining effects of anti-c-Met antibody using the marker


The present invention is urethane resin particles (C) for slush mold which contain a urethane resin (D) and an additive (N), wherein the particles have a shape factor SF1 of 101 to 200, a shape factor SF2 of 120 to 240, and a central particle diameter of 20 to 500 μm. It is preferred that the urethane resin (D) has a concentration of urea groups of 0.5 to 10% by weight, a total of the concentration of urethane groups and that of the urea groups of 4 to 20% by weight, a melting point of 160 to 260° C. and a glass transition temperature of −65 to 0° C.

In recent years, a slush molding method has widely been used for interior materials and the like of automobiles, since the molding makes it possible to mold easily into a product having a complicated form (such as an undercut or deep drawn form) and give an even wall thickness, and makes the yield percentage of the raw material good. Conventionally, in many cases, slush molding powdery products of a vinyl chloride-based resin have been used as skin materials of automobile interior components such as an instrument panel and a door trim.
However, when a vinyl chloride-based material is used over a long term, a plasticizer contained therein shifts to the surface thereof in accordance with a use environment thereof, so that the material is damaged in soft feeling. When an automobile is disposed and then subjected to incineration treatment, hydrogen chloride gas is generated in accordance with the incineration temperature, so that the incineration furnace may be corroded in some cases.
In order to solve these problems, developments are being advanced about a slush molding powdery material comprising a thermoplastic resin other than vinyl chloride. Use is made of, for example, a thermoplastic polyurethane elastomer having a number-average molecular weight of 20,000 to 50,000 and a glass transition temperature of −35° C. or lower (Patent Document 1).
Use is also made of a granular polyurethane resin composition made mainly of a thermoplastic urethane resin synthesized in a non-aqueous dispersion medium, a polymer of a vinyl monomer, and a thermally crosslinkable monomer (Patent Document 2). Furthermore, use is made of a thermoplastic elastomer composition powder having a particle diameter of 1 to 1000 μm and comprising an acrylic block copolymer and an acrylic polymer having, in a single molecule thereof, reactive functional groups the number of which is 1.1 or more (Patent Document 3).
In a material for slush molding, a colored material is used as a skin material of an automobile interior component in order to cause the component to exhibit high quality feeling. These materials for slush molding, each comprising a thermoplastic resin, are also colored by synthesizing uncolored particles, and then dusting a coloring agent such as an inorganic pigment or an organic pigment onto the surfaces thereof. In this coloring method, however, by shear at the time of stirring and mixing in the step of the coloring or in a step subsequent thereto, particles of the pigment which adhere onto the resin particle surfaces are slipped down from the surfaces, or the pigment particles are aggregated with each other on the particle surfaces. Thus, this method has a problem that the original color of the coloring agent is not expressed, a lump of the pigment is intermingled with the resultant product, or the like.
In order to solve such a problem, suggested is a method in which before a resin is granulated, a liquid prepolymer for the resin is mixed with a coloring agent, and subsequently the mixture is granulated (Patent Document 4).

This patent is subject to a terminal disclaimer.

Process for producing urethane resin particles for slush molding


A device (10) for rapidly filling sandbags (12) comprising a tray (14) having a plurality of apertures (16) therethrough. A plurality of funnel pails (18) are provided. Each funnel pail (18) is insertable through one of the apertures (16) in the tray (14). A structure (20) is for retaining upper portions of the funnel pails (18) in the apertures (16) of the tray (14). A plurality of empty sandbags (12) can be completely placed over the funnel pails (18) when the tray (14) is inverted. The tray (14) is then turned right side up, with the empty sandbags (12) on the funnel pails (18) placed upon the ground (22) and then sand (24) is shoveled into the funnel pails (18) to fill up the sandbags (12).

1. Field of the Invention
The instant invention relates generally to bag support holders and more specifically it relates to a device for rapidly filling sandbags. The device has the ability to fill the sandbags at three times the normal manual rate. It also prevents over and under filling of the sandbags. The device will also reduce fatigue among volunteers.
2. Description of the Prior Art
Numerous bag support holders have been provided in prior art. For example, U.S. Pat. Nos. 178,727 to Brown et al.; 4,842,228 to Kasper; 5,226,621 to Skoff; 5,397,085 to Spagnolo and 5,406,777 to Porto all are illustrative of such prior art. While these units may be suitable for the particular purpose to which they address, they would not be as suitable for the purposes of the present invention as heretofore described.


A circuit that includes a self-biased differential amplifier a level restore circuit. The output of the differential amplifier is coupled to the source pins of a p type transistor and an n type transistor. The output of the differential amplifier is further coupled to a first inverter circuit that provides feedback to the gates of the p and n type transistors. The first inverter also further amplifies the output voltage of the differential amplifier. When the differential amplifier outputs a high voltage, the p transistor attempts to drain the output and pull the output voltage down. When the differential amplifier begins switching from a high voltage to a low voltage, the p transistor accelerates the voltage swing and decreases the propagation delay of the buffer circuit. Likewise, when the differential amplifier swings from a low voltage to a high voltage, the n type transistor reduces the propagation delay of the voltage swing.

1. Field of the Invention
The present invention relates to a differential amplifier that can be used as an input buffer.
2. Description of Related Art
Data rates of communication busses continually increase with each new generation of processors. Present computer designs include a bus data rate of approximately 500 megabits per second (Mbits/sec). Data rates of 500 Mbits/sec are difficult to achieve using standard circuit designs because of the parasitic capacitance of the bus board. To overcome the physical limitations of the board, systems of such speed are typically designed to have a low voltage swing (the difference in voltage between a binary "1" and a binary "0"). The low voltage swing can be on the order of 600mV peak to peak.
Most memory and logic circuits operate in the 2-5 V range. Utilizing a high speed/low voltage swing bus with conventional electronic devices, typically requires an input buffer to increase the voltage of the signal from the bus to the devices.
Input buffers of the prior art have often incorporated a differential amplifier which amplifies the difference between the signal and a reference voltage. Most differential amplifier designs are particularly sensitive to fluctuations in temperature or voltages. Variations in the temperature or voltages, can lead to errors in the switching of the amplifier.
U.S. Pat. No. 4,937,476 issued to Bazes discloses an input buffer that utilizes a self-biased differential amplifier. The differential amplifier has a first pair of complementary n and p type FETs coupled to the input signal, and a second pair of complementary n and p type FETs coupled to a reference voltage. The first and second pair of FETs are coupled to a supply voltage that is controlled by a third pair of complementary FETs. The drain pins of the first set of FETs are connected to the gates of the third set of FETs, to create a negative feedback within the amplifier. The bias voltage of the first FETs is set at the midpoint of the active region. If the bias voltage moves from the midpoint, the feedback from the first FETs to the third FETs will vary the supply voltage so that the bias voltage is returned to the center of the active region. Any variations in temperature or voltage are therefore compensated through the self-biasing scheme incorporated into the Bazes design.
To provide an input buffer that can operate at high frequencies, it is desirable to minimize the propagation delays and increase the slew rate of the buffer. Additionally, the propagation delay from the high to low voltage swing is typically different from the propagation delay between the low to high voltage swing. The designer must typically compensate for the skew between the propagation delays. Although the Bazes circuit provides a self-biasing feature, the slew rate and skew between the propagation delays for the Bazes differential amplifier is somewhat unacceptable for usage with a data rate on the order of 500 Mbits/sec. It would therefore be desirable to have a circuit which can provide an input buffer that has an increased slew rate and minimal propagational skew, so that the circuit can be used with a high speed/low voltage swing bus.

Self-biasing input stage for high-speed low-voltage communication


A process and apparatus are provided to enhance the recovery of petroleum from onshore and offshore reservoirs. The process includes the simultaneous stimulation of the formation by elastic sound waves, created by a sonic source installed at the oil well so that the elastic sonic waves which are superimposed reduce the adherence forces in the layer between oil/water and the rock formation, and by the oscillating electrical stimulation of the same layer, as from the same wells subject to sonic treatment. The electricity heats the formation by using resistive heating, and thus increases the pressure, thus eliminating the surface tensions between the faces of the fluid as a consequence of the oscillatory action of the ions in the surfaces of the fluid and in addition, reducing the viscosity of the fluids. The process is achieved as the petroleum is produced in the wells thus treated, and the flow of petroleum acts then as a cooling agent which removes the heat released by the well area and thus allows a larger input of energy than in any other method known so far.

Hydrocarbons known as crude oil are found in the world usually retained in sandstones of different porosities. The reservoirs lay from a few meters to several thousand meters below the earth surface and the seabottom, and vary largely in size and complexity, with respect to their fluid and gas contents, pressures and temperatures.
Petroleum is produced by means of wells drilled into the formations. The well itself is a complicated construction, including casings which protect the well bore against the formation itself and the pressures exerted by the reservoir fluids. Depending upon the depth, the casings are subjected to a stepwise reduction in diameter. In other words, pipe diameter decreases as depth increases. It is not unusual to have 50" (127 cm) casing in the upper regions and 7.5" (19,05 cm) casing in the lower ones.
Petroleum itself is drained from the productive formation by means of holes drilled in the casing, being, thereafter, lifted to the surface through which is referred to as production tubing. This tubing is centralized inside the casing by means of special centralizers, so that an annulus exists between the producting tubing and the casing.
Petroleum is initially produced due to the original reservoir pressure being higher than the complex forces of fluid adherence to the porous media. As pressure decreases in the course of production, a point of equilibrium is reached in which the adhesion forces are higher than the remaining pressure in place. At this point most part of the petroleum is still in the reservoir. It is estimated, in a global average, to be equal to nearly 85% of the petroleum which was there initially, but the recovery indexes vary largely from one reservoir to another. As an example we mention the Ekofish field, in the North Sea, where the primary recovery index was 17% of the original oil in place (OOIP), and the Statfjord, where said index is estimated in 45% of OOIP.
The object of all methods designed to improve petroleum recovery is, therefore, that of trying to overcome those adherences. The theoretical base to explain the cause of those adherences is as follows:
A--forces due to wettability PA0 B--forces due to permeability PA0 C--capillary forces PA0 D--adhesive and cohesive forces
It is convenient that the adherence forces dealt with in this invention be explained more in detail.

Process to increase petroleum recovery from petroleum reservoirs


With correct emotion classes selected as correct values of an emotion of an utterer of a first utterance from among a plurality of emotion classes C1, . . . , CK by listeners who have listened to the first utterance, as an input, the numbers of times ni that emotion classes Ci have been selected as the correct emotion classes are obtained, and rates of the numbers of times nk to a sum total of the numbers of times n1, . . . , nK or smoothed values of the rates are obtained as correct emotion soft labels tk(s) corresponding to the first utterance.

Recognition of an utterer's emotion from an utterance is an important technique. For example, at the time of counseling, it is possible to visualize a patient's emotion such as anxiety or sadness by recognizing the utterer's emotion, and deepening of a counselor's understanding and improvement of instruction quality can be expected. Further, by recognizing human emotions in a dialogue between a human being and a machine, it becomes possible to construct a more friendly dialog system that rejoices together when the human being rejoices and encourages the human being when the human being is sad. Hereinafter, a technique for, with a certain utterance as an input, estimating which of emotion classes (classified emotions, for example, normal, anger, joy and sadness) an utterer's emotion included in the utterance corresponds to is called emotion recognition.
Non-patent literature 1 discloses a conventional emotion recognition technique (hereinafter referred to as a conventional technique). In the conventional technique, an acoustic feature (for example, Mel-Frequency Cepstral Coefficients (MFCCs) or the like) corresponding to each short time is extracted from an utterance, and estimation of an emotion class is performed from an acoustic feature sequence, using a long short-term memory recurrent neural network (LSTM-RNN) which is a time-series model based on deep learning. At the time of learning an LSTM-RNN model, a set of pairs of an utterance and a correct emotion label of the utterance decided by a result of human listening are used. At this time, each correct emotion label is decided by, after a plurality of listeners listen to an utterance, a majority vote based on emotion classes that listeners feel correct, respectively (hereinafter referred to as listeners' respective correct emotion classes). Note that, if the correction emotion label is not decided by a majority vote (for example, in a case where all the listeners give different emotion classes, respectively), the utterance is regarded as having no correct emotion label and, therefore, cannot be used for model learning.

Label generation device, model learning device, emotion recognition apparatus, methods therefor, program, and recording medium

NIPPON TELEGRAPH AND TELEPHONE CORPORATION


Low power consumption protocol A telemetry unit (100) is provided for mounting inside a pneumatic tire, which includes a piezoelectric element (114) supported in a housing (112), with an actuator (136) arranged for contact with the element (114), to deflect the element (114) in response to external forces acting on the actuator (136) during rotation of the tire. For every rotation of the tire, cyclic pulses of electrical charge are generated by the deflection of the element (114). The charge is stored and utilised under a power consumption protocol including the steps of: initiating power to a data measurement circuit for measuring data from the environment local to the unit (100); disabling power to the data measurement circuit; initiating power to a data transmission circuit; transmitting data from the measurement circuit; and disabling power to the transmission circuit. The power consumption protocol therefore minimises consumption of the generated power, during measurement and transmission of data by the unit (100).

The present invention relates to a power consumption protocol or method for selectively controlling the power consumption of a telemetry unit having a power source. The invention is of particular advantage in controlling the consumption of power from a piezoelectric power generator, for supplying power to a remote telemetry apparatus for transmitting data from a rotatable body, for example from within a pneumatic tyre.
It is known to provide a tyre monitoring apparatus for measuring the pressure within vehicle tyres. The tyre monitoring apparatus may also measure other parameters within a tyre environment, such as the local temperature of a tyre. The measured data is transmitted, for example via a radio wave link, to the cabin of the vehicle where it is electronically processed before being displayed to the vehicle driver. This enables the recipient of the transmitted data to monitor changes in the condition of the tyre, for example to reduce damage to the tyre(s) of a vehicle, or to predict tyre failure. This is of particular advantage at high vehicle speeds, when the environment within a tyre is at its most hostile and the likelihood of damage to a tyre and, indeed, injury to the occupants of the vehicle, is at its greatest.
The majority of existing tyre monitoring apparatus use a battery as the power source, which is located on or within a wheel or tyre. Such arrangements have several undesirable limitations, for example limited battery life and size or weight which can be accommodated within a tyre. This can have a further undesirable knock on effect, in that if there is a limited power source available, for example as a result of weight implications, the number and frequency of data transmissions that can be relayed for processing is compromised.


A magnetic recording medium having a carbon protective layer for protecting a magnetic layer formed on a non-magnetic base includes a liquid lubrication layer formed by coating on the carbon protective layer. The liquid lubrication layer includes a hydrocarbon containing lubricant and a perfluoropolyether containing lubricant.

A known magnetic recording medium for use with a fixed magnetic disc drive is constructed as shown in FIG. 2 by way of example. To produce such a recording medium, a non-magnetic metal layer 12 is formed on a non-magnetic substrate 11, to provide a non-magnetic base 1, and a non-magnetic metal base layer 2 is laminated on the base 1. On the non-magnetic metal base layer 2 is then formed a thin film of magnetic layer 3 made of a ferromagnetic alloy, such as Co--Cr--Ta (cobalt--chromium--tantalum), or Co--Cr--Pt (cobalt--chromium--platinum). Further, a carbon protective layer 4 is formed on the magnetic layer 3, and a lubrication layer 5 made of a liquid lubricant is applied by coating onto the carbon protective layer 4, to thus form a magnetic disc.
The non-magnetic base 1 may be an alumite base, a glass base, or a ceramic base, or may be obtained by forming the non-magnetic metal layer 12 made of Ni--P on the non-magnetic substrate 11 made of an Al--Mg alloy by electroless plating. The base 1 may be ground, or formed with minute protrusions and recesses by texturing. The non-magnetic base 1 thus obtained is fed through a furnace under an Ar atmosphere, while being heated to about 200.degree. C., so that the non-magnetic metal base layer 2 made of Cr, magnetic layer 3, and carbon protective layer 4 made of amorphous carbon are successively formed on the base 1 by sputtering. The carbon protective layer 4 is then coated with a lubricant containing perfluoropolyether (perfluoro polyester).
When the above-described magnetic recording disc is installed in the fixed magnetic disc drive that employs a contact-start-stop (CSS) system to read and write information, the magnetic disc is repeatedly brought into contact with a recording head of the disc drive during the CSS operation. In this CSS system, the recording head is in contact with a surface of the magnetic disc while the disc drive is not operated, and, only when the drive is operated, the recording head floats slightly above the surface of the magnetic disc, so as to read or write information.
The carbon protective layer 4 and the lubrication layer 5 containing perfluoropolyesther are formed on the magnetic layer 3 so as to protect this layer 3 from friction due to such sliding movements of the magnetic disc relative to the recording head.
In the case of a magnetic disc having a relatively small diameter of not larger than 5 inches, the protective layer is generally made of a carbon material, which is formed into a film by sputtering under an Ar atmosphere. The carbon protective layer is favorably used since the amorphous carbon (a-C) layer formed by sputtering has relatively strong graphitic properties, and thus shows a relative low coefficient of friction under an atmosphere containing water, which is a typical property of graphite.
The carbon protective layer as described above has a sufficiently high wear resistance with respect to a conventional Mn--Zn ferrite head (Vickers hardness: about 650), and is highly resistant to the friction between the magnetic disc and the head during a CSS operation. This carbon protective layer, however, has a lower hardness than a ceramic material (Vickers hardness: about 2000), such as Al.sub.2 O.sub.3.Ti or CaTiO.sub.3, which is used for a slider of a thin-film head or MIG head employed in recent fixed magnetic disc drives. Thus, the protective layer tends to wear due to contact with the recording head formed of such a ceramic material, and the head may crash in some cases. To solve these problems, the protective layer used in recent years has developed properties similar to those of diamond having extremely high hardness, to provide a diamond-like carbon (DLC) protective layer in which the percentage of carbon atoms bonded in a diamond structure is higher than that of carbon atoms bonded in a graphite structure.
As a liquid lubricant, there has been used a perfluoropolyether containing lubricant having polar terminal groups, so as to prevent contaminants from being adsorbed onto adsorption sites (functional groups) appearing on the surface of the carbon (DLC) protective layer. If the molecular weight of perfluoropolyether is too low, the chain molecule has a small length, and the coefficient of friction is undesirably increased. If the molecular weight is too high, the chain molecule tends to have an excessively long free end, and the head tends to adhere to the lubrication layer. In view of these, the weight average molecular weight of the perfluoropolyether containing lubricant is favorably within the range of about 2000-5000 MW (molecular weight), and the length of the chain molecule is experimentally controlled to about 30 .ANG.. Since the use of a lubrication layer having a large thickness may induce the recording medium to absorb an excessive lubricant on the medium surface, the thickness of the lubrication layer is desirably controlled to several dozens of angstroms (.ANG.) that is about equal to the length of one molecule. If the carbon protective layer is coated with the lubricant, with a thickness substantially equal to the length of one molecule of the lubricant having a relatively large molecular weight, it is difficult to uniformly cover the entire area of the protective layer with the lubrication layer having such a thickness. As a result, uncovered areas or gaps are left between the macromolecules of the lubricant.
Further, the residue (on the side of the free end) of the molecule of the perfluoropolyether containing lubricant, other than the terminal group bonded to the adsorption site on the surface of the carbon protective layer, has a hydrophobic property. When the carbon protective layer is coated with the lubricant, therefore, the contact angle with water (as measured by a liquid-drop method for measuring the surface tension) is around 90.degree., namely, the coated surface of the recording medium has a large surface tension, and shows poor wettability on the surface of the carbon protective layer. In the presence of the above-described gaps between the molecules of the lubricant, the top surface of the magnetic recording medium, when closely observed, includes mutually isolated molecules of the liquid lubricant having hydrophobic surfaces, which are dispersed on the surface of the carbon protective layer.
In the meantime, recent magnetic disc drives using the magnetic recording medium as described above have a completely enclosed structure wherein the interior space is isolated or sealed from the exterior. Due to this structure, the interior space is easily filled up with gases generated from components inside the disc drive. When the magnetic disc drive is used under a condition of high humidity, in particular, the gases from the interior components are likely to be dissolved into water generated due to the high humidity or dew formation, to thereby produce acids. These gases can be easily identified by analyzing the components inside the magnetic disc drive by gas chromatography. As a result of such an analysis, gases of oxalic acid, formic acid, organic acids, such as acrylic acid, and inorganic acids, such as SO.sub.2 and Cl were detected.
If the magnetic recording medium is used under the atmosphere as described above, the acid gases are first adsorbed or attached to gap portions of the medium surface between the molecules of the lubricant, and are dissolved into adsorbed water under a condition of high humidity, to produce acids. Once the density of the acids becomes higher than a predetermined level, the acids act on ether linkages of the perfluoropolyether forming the lubricant, causing oxidation and reduction, whereby the chain molecules having a high molecular weight are decomposed into low molecules of oxalic acid or formic acid. As a result of the decomposition of the liquid lubricant into low molecules, the adhesiveness between the liquid lubricant and the medium surface is lowered or eliminated, and the lubricant is more likely to be transferred to the magnetic head. Consequently, the magnetic head is contaminated, and its floating characteristics are affected (the floating height is increased), with a result of deterioration of the reproducing capability of the head. Further, the lubricant thus formed into low molecules does not maintain desired lubricating characteristics any longer, and the magnetic head may crash due to its contact with the recording medium in the worst case.

Magnetic recording medium and method for manufacturing the same


An application program is run in a process supported by an operating system in a computer system. The application program is executed in a virtual machine, and the virtual machine is within the process. Loading a library for the application program includes determining whether this is the first library load instruction in the application program. If it is the first library load instruction, then the library is loaded in a first memory area, and a handle and link map to the first memory area is defined for the library. If the library load instruction is a second or subsequent library load instruction, the further library is loaded in a further memory area, also associated with the link map identifier, and a second handle to the further library is defined and stored.

In certain fields of application, e.g. the so-called "high-end" servers, there is a need to run a large number of applications, e.g. Java applications, with a good scalability. This in turn implies to ensure: (i) application level isolation, i.e. isolation between the individual Java applications; (ii) native code (e.g. C code) isolation at the level of the hosts, e.g. the JVMs.
In a conventional approach, each application is run via a respective JVM, which is in turn run within a respective process of the operating system. This intrinsically ensures isolation between the various JVMs and applications
The above indicated solutions give satisfactory results; however, they suffer drawbacks in terms of resource requirements, especially due to the fact that a number of JVM and/or OS resources have to be duplicated into a respective copy for each of the applications.
A proposal has been made by Greg CZAJKOWSKI, in a paper entitled "Application Isolation in the Java Virtual Machine", Proceedings of ACM, OOPSLA'00, Minneapolis, Minn., October 2000. This proposal tends to execute a plurality of applications in one or each JVM. However, it does not provide native code isolation.
A further proposal has been made by Greg CZAJKOWSKI, Laurent DAYNÈS, and Mario WOLCZKO, in "Automated and Portable Native Code Isolation", Sun Microsystem Technical Note SMLI TR-2001-96, April 2001. This further proposal suggests to execute native code in an operating system process different from that of the "safe language application", e.g. java language. This however induces a performance overhead, when crossing process boundaries. i.e. due to the need to use another process whenever usage of a function written in native code is desired.
Thus, although they give satisfactory results in many cases, the proposals of the prior art may not be adequate in the above indicated context of the "high-end" servers.

Native code isolation in a multi-tasking Java virtual machine


An optical pickup device includes a lens for projecting a light onto a recording medium, a lens holder elastically supported by a frame and holding said lens, an actuator mounted in and driving said lens holder, and a weight part balanced with a weight of said actuator.

1. Field of the Invention
The present invention generally relates to optical pickup devices, and more particularly to an optical pickup device having a lens for projecting a light onto a recording medium and optically reading information stored in the recording medium by controlling the orientation of the lens.
2. Description of the Related Art
In a disk unit loaded with a disk recording medium (hereinafter referred to as a disk), such as a CD-ROM apparatus, for instance, information stored in the disk is read by projecting a laser beam onto the disk. An optical pickup device incorporated into this type of disk unit has an objective lens for projecting the laser beam emitted from a laser diode onto the disk, and controls the orientation of the objective lens so that the laser beam projected through the objective lens is focused on a recording surface of the disk.
That is, in the optical pickup device, focus control and tracking control are performed so that the objective lens follows the surface fluctuation of the disk or the motion of a track due to eccentricity. Such control of the objective lens is performed by an actuator utilizing electromagnetic force. Generally, the actuator is composed of a combination of a coil and a magnet.
A lens holder holding the objective lens is reduced in size and weight so that focus control and tracking control can be performed easily. Further, the lens holder is supported by a support structure where four wire-like elastic supporters are provided in parallel so that a sensitive control operation of the objective lens is performed by a driving force from the actuator.
However, the conventional optical pickup device has its movable part such as the lens holder moved easily in order to increase the accuracy of the above-mentioned focus and tracking controls. Therefore, variations in component size, for instance, cause a deviation between the center of gravity of the movable part and the driving point position of the actuator, thus generating a turning moment in the movable part. This turning moment causes unnecessary vibrations to be generated in the movable part, thus destabilizing focus and tracking controls.
Further, insufficient stiffness of the movable part due to a thinner lens holder increases the magnitude of higher-order-harmonic resonance so that a servo system starts oscillating.
Moreover, if variations in component size or assembly errors are caused, the center of gravity of the movable part or the position of a driving point on which the driving force from the actuator is exerted is deviated. Therefore, unnecessary vibrations may be caused by a turning moment generated in the movable part.
In the optical pickup device, a natural frequency may be considered to be a factor of the generation of unnecessary vibrations. Since the natural frequency is determined by a component shape and/or material, it is required to increase or decrease the level of the natural frequency by changing such a factor (factors).
Further, it is also possible to eliminate the deviation of the center of gravity of the movable part or the driving point of the actuator by increasing the processing or assembly accuracy of components. However, increasing the processing or assembly accuracy of components causes a problem of an increase in the costs of mass production.
Furthermore, in order to manage the position of the center of gravity with high accuracy, it is required to be informed quantitatively of an error between calculated and actual positions of the center of gravity by repeating trial production and evaluation. Therefore, in the case of managing the position of the center of gravity, there has been a problem that a product development period becomes longer due to repetition of the trial production and evaluation.
In addition, in order to lower the level of the natural frequency of the movable part, it is required to increase the stiffness of an individual component. However, it has been difficult to increase component stiffness by increasing component thickness due to restriction of component interference. It is possible as a method of solving such a problem to increase stiffness by forming the lens holder with a metal strip being inserted therein by insert molding. However, this causes a problem of an increase in production costs.

Optical pickup device with elastic supporters and with dynamic vibration absorbing balance weight bonded in concave part with adhesive agent


An air bearing slider including a recessed cavity surface to provide an adaptable bearing surface to compensate for thermal expansion or pole tip protrusion. The recessed cavity surface forms a leading edge cavity step to a raised bearing surface proximate to a trailing edge of the slider. The leading edge cavity step forms a rampless air bearing surface proximate to the trailing edge of the slider which is adaptable to form a ramp to pressurize a protruding surface of a transducer portion of the head.

Data storage devices store digitally encoded information on discs. Heads read data from or write data to discs which are supported for rotation relative to a base chassis by a spindle motor or drive. Heads include transducer elements, such as magnetoresistive, magneto-optical or inductive elements for read or write operations. An actuator assembly moves the head relative to select data tracks on the disc for read or write operations.
Typically the head includes an air bearing slider which is coupled to a head suspension assembly. Rotation of the disc creates an air flow along the air bearing slider to provide a lift force. The lift force of the air bearing slider is countered by a load force supplied via a load beam of the suspension assembly to define in part a fly height of the slider. The slider is coupled to the head suspension assembly via a gimbal spring so that the slider pitches and rolls to follow the topography of the disc surface. Typically the slider flies at a pitch angle so that a trailing edge of the slider defines a close point of the slider relative to the disc surface. During operation, the air bearing compensates for shock or vibration to maintain a stable fly height for the slider above the disc surface.
The transducer elements of the head are fabricated on the trailing edge of the slider. Typically the transducer elements are embedded in a thermally expandable substrate or body such as Alumina Al2O3. Thermal expansion of the transducer portion via changes in ambient drive temperature or via heat generated by write current can cause transducer elements to protrude (i.e. pole tip protrusion) below air bearing surfaces of the slider. Protrusion of the transducer elements or pole tip protrusion can compromise effect of the air bearing to maintain a stable fly height between the slider and the disc surface. Embodiments of the present invention provide solutions to these and other problems, and offer other advantages over the prior art.

Adaptable air bearing slider with trailing edge cavity


A composite pipeline sleeper features a rotationally molded outer shell, and an internal reinforcement structure that is disposed within, is materially distinct from, and is more rigid than, said rotationally molded outer shell. A topside of said pipeline sleeper comprises a cradle-shaped area for seated receipt of a pipe there atop.

Oil pipelines have been known to rely on timber sleepers, for example of the same type used as railway ties, to support the pipeline in elevated relation above the underlying ground surface. Shortcomings of timber sleepers include a short lifespan, heavy equipment and manpower requirements, environmental impact (creosote content), and degradation over time.
Accordingly, there remains a need for improvements and alternatives in the field of pipeline sleepers.

Composite pipeline sleeper with hollow outer shell and rigid internal reinforcement


The present invention relates to polynucleotide molecules for regulating gene expression in plants. In particular, the invention relates to promoters that are useful for regulating gene expression of heterologous polynucleotide molecules in plants. The invention also relates to expression constructs and transgenic plants containing the heterologous polynucleotide molecules.

1. Field of the Invention
The invention relates to the field of plant molecular biology and more specifically relates to polynucleotide molecules useful for the expression of transgenes in plants. The invention in particular relates to P-Dgat1 and P-Dgat2 promoters isolated from Arabidopsis thaliana and useful for expression of transgenes of agronomic importance in seeds of crop plants.
2. Background of the Invention
One of the goals of plant genetic engineering is to produce plants with agronomically desirable characteristics or traits. Advances in genetic engineering have provided the requisite tools to transform plants to contain and express foreign genes. The technological advances in plant transformation and regeneration have enabled researchers to take an exogenous polynucleotide molecule, such as a gene from a heterologous or native source, and incorporate that polynucleotide molecule into a plant genome. The gene can then be expressed in a plant cell to exhibit the added characteristic or trait. In one approach, expression of a gene in a plant cell or a plant tissue that does not normally express such a gene may confer a desirable phenotypic effect. In another approach, transcription of a gene or part of a gene in an antisense orientation may produce a desirable effect by preventing or inhibiting expression of an endogenous gene.
Promoters are polynucleotide molecules that comprise the 5′ regulatory elements, which play an integral part in the overall expression of genes in living cells. Isolated promoters that function in plants are useful for modifying plant phenotypes through the methods of genetic engineering. The first step in the process to produce a transgenic plant includes the assembly of various genetic elements into a polynucleotide construct. The construct includes a transcribable polynucleotide molecule (gene of interest) that confers a desirable phenotype when expressed (transcribed) in the plant cells by a promoter that is operably linked to the gene of interest. A promoter in a construct may be homologous or heterologous to the gene of interest also contained therein. The construct is then introduced into a plant cell by various methods of plant transformation to produce a transformed plant cell and the transformed plant cell is regenerated into a transgenic plant. The promoter controls expression of the gene of interest to which the promoter is operably linked and thus affects the characteristic or trait conferred by the expression of the transgene in plants.
For production of transgenic plants with various desired characteristics, it would be advantageous to have a variety of promoters to provide gene expression such that a gene is transcribed efficiently in the amount necessary to produce the desired effect. The commercial development of genetically improved germplasm has also advanced to the stage of introducing multiple traits into crop plants, often referred to as a gene stacking approach. In this approach, multiple genes conferring different characteristics of interest can be introduced into a plant. It is often desired when introducing multiple genes into a plant that each gene is modulated or controlled for optimal expression, leading to a requirement for diverse regulatory elements. In light of these and other considerations, it is apparent that optimal control of gene expression and regulatory element diversity are important in plant biotechnology.
A variety of different types or classes of promoters can be used for plant genetic engineering. Promoters can be classified on the basis of characteristics such as temporal or developmental range, levels of transgene expression, or tissue specificity. For example, promoters referred to as constitutive promoters are capable of transcribing operably linked genes efficiently and expressing those genes in multiple tissues. Different types of promoters can be obtained by isolating the upstream 5′ regulatory regions of genes that are transcribed and expressed in the desired manner, e.g., constitutive, tissue enhanced, or developmentally induced.
Numerous promoters, which are active in plant cells, have been described in the literature. These include the nopaline synthase (nos) promoter and octopine synthase (ocs) promoters carried on tumor-inducing plasmids of Agrobacterium tumefaciens and the caulimovirus promoters such as the Cauliflower Mosaic Virus (CaMV) 19S or 35S promoter (U.S. Pat. No. 5,352,605), CaMV 35S promoter with a duplicated enhancer (U.S. Pat. Nos. 5,164,316; 5,196,525; 5,322,938; 5,359,142; and 5,424,200), and the Figwort Mosaic Virus (FMV) 35S promoter (U.S. Pat. No. 5,378,619). These promoters and numerous others have been used in the creation of constructs for transgene expression in plants. Other useful promoters are described, for example, in U.S. Pat. Nos. 5,391,725; 5,428,147; 5,447,858; 5,608,144; 5,614,399; 5,633,441; 6,232,526; and 5,633,435, all of which are incorporated herein by reference.
Promoters are also needed for expression of genes in seeds for the production of plant oils and other traits. Diacylglycerol acyltransferase (referred to hereinafter as Dgat) is an integral membrane protein that catalyzes the final enzymatic step in the production of triacylglycerols in plants, fungi, and mammals (Harwood, Biochem. Biophysics. Acta, 13017-13056 (1996); Daum et al., Yeast, 16:1471-1510 (1998); and Coleman et al., Annu. Rev. Nutr., 20:77-103 (2000). Dgat is responsible for transferring an acyl group from acyl-coenzyme-A to 1,2-diacylglycerol (Dag) to form triacylglycerol (TAG). Nearly all commercially important fats and oils of plant origin consist of triacylglycerols. Triacylglycerols are hereafter referred to as "oils" or "plant oils." In plants, particularly in oilseeds, Dgat is associated with the membrane and lipid body fractions, and plays a key role in oil synthesis (Kennedy et al., J. Biol. Chem., 222:193 (1956); Finnlayson et al., Arch. Biochem. Biophys., 199:179-185 (1980)).
Two different families of Dgat proteins have been identified. The first family of Dgat proteins (referred to hereinafter as Dgat1) is related to the acyl-coenzyme A:cholesterol acyltransferase (ACAT) and has been described in the literature (see, e.g., U.S. Pat. Nos. 6,100,077 and 6,344,548). A second family of Dgat proteins (referred to hereinafter as Dgat2), unrelated to the previously identified family of Dgat1 proteins, is described in U.S. Published Application US20030028923. The present invention describes promoters associated with these Dgat families.
While previous work has provided a number of promoters useful to direct transcription in transgenic plants, there is still a need for novel promoters with beneficial expression characteristics. In particular, there is a need for promoters that are capable of directing expression of exogenous genes, for oil production, in seeds. Many previously identified promoters fail to provide the patterns or levels of expression required to fully realize the benefits of expression of selected seed-enhanced oil-associated genes in transgenic plants. There is, therefore, a need in the art of plant genetic engineering for novel promoters for use in oilseeds.