Abstract: Van A and Van B vancomycin resistant enterococci detection media as well as a method of selectively detecting Van A and Van B vancomycin resistant enterococci clinically important in vancomycin resistant enterococci from testing microorganisms or specimens using the media. The media for selectively detecting Van A and Van B VRE from testing microorganisms and specimens are media where enterococci can grow where vancomycin, D-cycloserine and D-lactate are added. Preferably 32-256 ?g/ml of vancomycin, 1-64 ?g/ml of D-cycloserine, and 0.025-0.8 mol/l of sodium lactate are added to culture medium where enterococci can grow.

Abstract: Methods for producing high molecular weight poly-gamma-glutamic acid (PGA). The PGA is produced by fermentation, and purified by use of tangential flow filtration, followed by diafiltration, as necessary, to yield a product of the desired purity. Product obtained may be of very high purity using all the prescribed purification steps. Product of this purity is suitable for in vivo medical applications. Other applications, such as food or agricultural, may utilize lower purity levels, and hence do not require all the purification steps specified.

Abstract: The objective of the present invention is to provide a gene encoding novel afadin DIL domain-binding protein (ADIP) and use of the novel ADIP protein. The present inventors successfully identified a novel afadin-binding protein (ADIP) using yeast two-hybrid screening in order to identify how the nectin and afadin system organize tight junctions and adherens junctions at the cell-cell junctions. The novel protein is useful for evaluating actin cytoskeleton-controlling agents.

Abstract: The sequence of the disorazole polyketide synthase protein gene is disclosed. Domains of disorazole polyketide synthase and polynucleotides encoding them are provided. Methods to prepare disorazoles in pharmaceutically useful quantities are described, as are methods to prepare disorazole analogs and other polyketides using the polynucleotides encoding disorazole polyketide synthase domains or modifying enzymes.

Abstract: The invention relates to the field of biochemistry, molecular biology, pharmacology and diagnosis. More specifically the present invention relates to the production of proteins in a host cell. Even more specifically, the invention relates to a method for improving expression of two or more proteins in a (host) cell. The method is suited for production of, for example, recombinant antibodies that can be used in a pharmaceutical preparation or as a diagnostic tool. In one embodiment, the invention provides a method for obtaining a cell which expresses two or more proteins comprising providing the cell with two or more protein expression units encoding two or more proteins, characterized in that at least two of the protein expression units comprise at least one STAR sequence.

Abstract: The present invention relates to a method for the production of a stable biodegradable, high water absorbable ?-polyglutamic acid (?-PGA) hydrogel by directly cross-linking (A) a ?-polyglutamic acid (?-PGA), a ?-polyglutamate, or a mixture thereof, and optionally a polysaccharide containing a carboxylic and/or carboxylate group, an amino acid, or a mixture thereof; and/or (B) a microbial culture broth containing a ?-polyglutamic acid (?-PGA), a ?-polyglutamate, or a mixture thereof, and optionally a polysaccharide containing a carboxylic and/or carboxylate group, an amino acid, or a mixture thereof, with a cross-linker comprising a compound having three or more functional groups or a mixture of a compound having three or more functional groups and a compound having two functional groups, wherein each of the functional groups can react with a carboxylic group (—COOH), carboxylate group (—COO?), aldehyde group (—CHO), hydroxyl (—OH), carbonyl group (—CO), sulfone group (—SO2), amino group (—NH2) or nitro gr

Abstract: The invention relates to integrating at least two processes that use at least one acid, wherein one process utilizes an ion-exchange purification step.

Abstract: A process for biological production of ortho-aminophenols from nitroaromatic compounds using recombinant E. Coli strains. The process uses an enzyme system that makes use of a nitroreductase enzyme that initially reduces the nitroarene to the hydroxylaminoarene and a mutase enzyme that converts the hydroxylaminoarene to an ortho-aminophenol.

Abstract: A new class of enzymes that catalyze the conversion of a nitrile containing compound to the corresponding amine (such as a primary amine) are disclosed. Such enzymes are referred to herein as nitrile oxido-reductases. Methods of using the enzymes to reduce a nitrile to a amine, for example in vitro or in vivo, are provided. Such methods provide the first biocatalysis method for reducing nitrites to amines, and provides an alternative to currently used methods, which generally utilize harsh reaction conditions and the production of hazardous waste. While the hydrolysis of nitrites to amides and carboxylic acids via biocatalysis has found extensive use in industry, the lack of a known nitrile oxido-reductase has precluded the application of biocatalysis to nitrile reduction.

Abstract: The present invention is directed to a process of producing novel fatty acids in oleaginous yeast by producing oleaginous yeast by introducing into the yeast genes coding for enzymes selected from the group consisting of D5-desaturase, D6-desaturase, D12-desaturase, D15-desaturase and elongase; and culturing the yeast in the medium containing high levels of carbon sources. The present invention is further directed to a residue or fatty acid that is obtained from pressing the oleaginous yeast produced by the process of the invention.

Abstract: A process for preparing a hydroxylation catalyst by i) embedding a cystochrome P450 monooxygenase in a sol-gel matrix, ii) embedding an enzymatic NADPH-regenerating system in a sol-gel matrix, and combining the two components i) and ii) unless they were already mixed together before the embedding.

Abstract: The present invention provides a process for producing isoprenoid compounds or proteins encoded by DNA using DNA that contains one or more of the DNA encoding proteins having activity to improve efficiency in the biosynthesis of isoprenoid compounds effective in pharmaceuticals for cardiac diseases, osteoporosis, homeostasis, prevention of cancer, and immunopotentiation, health food and anti-fouling paint products against barnacles; the DNA; the protein; and a method for screening a substance with antibiotic and weeding activities comprising screening a substance inhibiting enzymatic reaction on the non-mevalonate pathway.

Abstract: The invention provides a method, apparatus and system for detecting electrochemical oxidoreduction activity mediated by a redox enzyme at a site remote from the enzyme. In one embodiment, the method comprises immobilizing the redox enzyme on a first region of a conductive surface and contacting a substrate capable of producing a detectable signal upon oxidation or reduction with a second region of the conductive surface. The second region is electrically coupled with the first region and the redox enzyme is not present in the second region. The method further comprises exposing the immobilized redox enzyme to conditions that effect oxidation or reduction of the enzyme, and detecting oxidation or reduction of the substrate at the second region. The invention can be adapted for detecting a plurality of analytes.

Abstract: The invention refers to the use of a p21-activated kinase (PAK) inhibitor for the treatment of a joint disease such as osteoarthritis or rheumatoid arthritis or for the treatment of a joint pain and the use of PAK as a target protein for the discovery of a PAK inhibitor as a medicament for the treatment of a joint disease.

Abstract: The subject invention is directed to the prevention and treatment of respiratory disorders by intratracheal administration of an effective amount of lysozyme, either alone or in combination with other therapeutic agents. Applicable respiratory disorders include, but are not limited to, pulmonary emphysema, asthma, bronchitis, pneumonia, respiratory distress syndrome, bronchopulmonary dysplasia, interstitial fibrosis, cystic fibrosis, and neoplasia. The method is intended for a variety of mammals, including humans ranging from premature neonates to adults.

Abstract: A catalytically active peptide comprising an imidazole function in position i flanked by at least one functional group to be amidated in position i+3+4k, where k is an integer equal to or higher than ?1 or in position i?4?4n, wherein n is an integer equal to or higher than 0, characterized in that it also comprises at least one activating group in position i+4+4n or i?3?4n, respectively, wherein n is as above.

Abstract: The invention provides a thermal tolerant (thermostable) cellulase, AviIII, that is a member of the glycoside hydrolase (GH) family. AviIII was isolated and characterized from Acidothermus cellulolyticus and, like many cellulases, the disclosed polypeptide and/or its derivatives may be useful for the conversion of biomass into biofuels and chemicals.

Abstract: A mutated alkaline cellulase derived from a cellulase having the amino acid sequence represented by SEQ ID NO: 1 or one having a homology of at least 90% therewith by deleting one or more amino acid residues from the 343- to 377-positions in SEQ ID NO: 1 or a region corresponding thereto and then inserting a peptide having from 2 to 15 amino acid residues into the deletion site; and a gene encoding the same. The above alkaline cellulase has an optimum pH value close to the pH value of laundry water and, therefore, is useful as an enzyme for detergents.

Abstract: The present invention relates to microbial trypsin variants having chymotrypsin-like activity, comprising: (a) a one or more substitutions corresponding to positions 144, 193, 198, 201, 218, 223, 227, 228, 229, 230, and 231 of amino acids 25 to 248 of SEQ ID NO: 2, (b) one or more deletions corresponding to positions 192, 197, and 226 of amino acids 25 to 248 of SEQ ID NO: 2; and (c) an insertion between positions corresponding to positions 224 and 225 of amino acids 25 to 248 of SEQ ID NO: 2. The present invention further relates to nucleotide sequences encoding microbial trypsin variants having chymotrypsin-like activity; nucleic acid constructs, expression vectors, and recombinant host cells comprising such nucleotide sequences; and methods of producing microbial trypsin variants having chymotrypsin-like activity or a precursor thereof.

Abstract: The present invention provides recombinant and/or isolated infectious laryngotracheitis virus glycoproteins, including gD, gI, gG and gE.

Abstract: A prokaryotic recombinant host cell comprising a heterologous replication initiation protein that activates a conditional origin of replication and an extrachromosomal DNA molecule comprising a heterologous therapeutic gene and a conditional origin of replication whose functionality in the prokaryotic recombinant host cell requires a replication initiating protein which is foreign to the host cell is described. The host cell may comprise a pir gene having at least one mutation, which may occur in the pir gene copy number control region, the pir gene leucine zipper-like motif, or the pir gene DNA binding region.

Abstract: A body 300 having a cavity 310 for mounting a substrate 120 fabricated with probe sequences at known locations according to the methods disclosed in U.S. Pat. No. 5,143,854 and PCT WO 92/10092 or others, is provided. The cavity includes inlets 350 and 360 for introducing selected fluids into the cavity to contact the probes. Accordingly, a commercially feasible device for use in high throughput assay systems is provided.

Abstract: Individual and aggregate test strip arrays having a backing strip supporting a flexible array substrate which supports an addressable collection of probes are taught. Hybridization cells for use with the individual test strip arrays are also disclosed, and in particular embodiments the hybridization cells closely fit a portion or all of the individual test strip arrays. Array hybridization systems that include test strip arrays and hybridization cells are disclosed with methods for performing a hybridization assay on a sample solution.

Abstract: Apparatus and methods for carrying out biochemical processes directly on a substrate are provided. A substrate assembly includes a cartridge that removably supports a substrate in a fixed position, and a reaction containment member that is removably located on top of the substrate. The reaction containment member includes one or more cavities that form chambers directly above one or more target locations on the surface of the substrate. The chambers can be used to conduct biochemical processes directly over the substrate, as well as to perform thermal cycling of material contained inside the chamber using a heating element disposed directly on the substrate. The substrate assembly is preferably used in combination with a processing machine that dispenses materials into the chambers and that conducts biochemical reactions of materials contained within the chambers, without requiring the substrate assembly to be moved from one location to another location during the processes.

Abstract: The present invention relates to a detection and quantification method of different sets of target molecules (1, 3), being possibly present simultaneously in a biological sample by the use of a microarray present upon a solid support surface (6) and comprising different sets (A, B) of capture molecules (2, 4), present in different locations (8, 9) of the solid support surface (6).

Abstract: The present invention discloses a device for monitoring haptotaxis including a housing defining a chamber. The chamber comprises: a first well region including at least one first well, the first well configured to receive a test agent therein and further including biomolecules immobilized therein; a second well region including at least one second well, the second well region configured to receive a sample comprising cells therein and further being horizontally offset with respect to the first well region in a test orientation of the device; and a channel region including at a least one channel connecting the first well region and the second well region with one another, the channel region further including biomolecules immobilized therein.

Abstract: Methods of inducing differentiation of mammalian bone marrow stromal cells into cells of multiple embryonic lineages by contacting marrow stromal cells with precursor differentiation-inducing compounds followed by contacting the partially differentiated precursor cells with specific cell type differentiation-inducing compounds. In one embodiment, the MSC derived precursor cell cultures comprise cells, at least some of which simultaneously express markers that are characteristic of endodermal and ectodermal cell types. In another embodiment, the differentiated cells are insulin-secreting pancreatic islet cells. Precursor differentiation-inducing compounds of the invention include anti-oxidants such as, but not limited to, beta-mercaptoethanol, dimethylsulfoxide, butylated hydroxyanisole, butylated hydroxytoluene, ascorbic acid, dimethylfumarate, and n-acetylcysteine.

Abstract: A chimeric gene comprising an isolated nucleic acid molecule encoding a delta 12-fatty acid epoxygenase enzyme and transgenic plants containing the chimeric gene are described. Expression of the chimeric delta 12-epoxygenase gene leads to altered levels of fatty acids in transformed cells.

Abstract: The invention relates to methods and compositions for site-specific recombinase-mediated mobilization of viral replicons and associated DNAs of interest from T-DNA. The methods of the invention comprise Agrobacterium-mediated transfer of T-DNA to a plant cell, wherein the T-DNA contains a viral replicon flanked by directly repeated target sites for a site-specific recombinase and optionally a DNA of interest linked to the viral replicon. The DNA of interest may also contain a non-identical target site for the recombinase. An expression cassette for the site-specific recombinase is present on the T-DNA or the plant genome, or is transiently introduced into the plant cell. Expression of the site-specific recombinase in the plant cell results in excision of the viral replicon and the associated DNA of interest. The viral replicon and DNA of interest are then replicated to high copy number in the host plant cell.

Abstract: The present invention relates to the discovery that the T1R receptors assemble to form functional taste receptors. Particularly, it has been discovered that co-expression of T1R1 and T1R3 results in a taste receptor that responds to umami taste stimuli, including monosodium glutamate. Also, it has been discovered that co-expression of the T1R2 and T1R3 receptors results in a taste receptor that responds to sweet taste stimuli including naturally occurring and artificial sweeteners. Also the present invention relates to the use of hetero-oligomeric taste receptors comprising T1R1/T1R3 and T1R2/T1R3 in assays to identify compounds that respectively respond to umami taste stimuli and sweet taste stimuli. Further, the invention relates to the constitutive of cell lines that stably or transiently co-express a combination of T1R1 and T1R3; or T1R2 and T1R3; under constitutive or inducible conditions.

Abstract: Methods and compositions are provided for diagnosing and treating Pseudoxanthoma elasticum (PXE) patients and PXE carriers. Methods and compositions are based on the discovery that PXE mutations are located in the MRP6 (ABCC6) gene.

Abstract: An article and method for high throughput and a high content investigation of compound interactions with live tissue or its substitutes under controlled conditions during compound absorption and related processes. In some variations of the illustrative embodiment, the article is a multi-chamber enclosure having at least two chambers separated by a membrane. Membranes can be prepared from live epithelial tissue or from an artificial material with or without attached cells from cell-line cultures. Each chamber is advantageously connected to a fluidic-control system by tubes that pass through a feed fitting. In addition to coupling the chambers with the fluidic-control system, the feed fitting, which is spring-biased, provides a sealing force to seal the enclosure. In some variations, one or more multi-chamber enclosures are installed in a mother chamber, which provides controlled environmental conditions.

Abstract: A method of single parameter and multiparameter characterizing of cells, particularly immunophenotyping of cells, is provided. The method preferably uses antibody-coated microspheres which are adapted to bind to specific types of cells. One or more sets of coated microspheres are contacted simultaneously or sequentially with a suspension of cells and bind the cells they are adapted to bind to form bead-cell complexes. Cells may bind to one or more microspheres. The bead-cell complexes are then separated from the suspension The complexes are preferably stained and then examined to characterize the cells, preferably the cells bound to the microspheres. A method of quantitating a specific cell type is provided. A kit and apparatus for performing the method are also provided.

Abstract: Methods and systems for sorting compounds and compound holders into batches for subsequent processing are provided.

Abstract: A method of separating at least a first non-fluorous compound from a mixture of compounds including at least the first non-fluorous compound and a second fluorous compound includes: charging the of compounds to a non-fluorous solid (stationary) phase and eluting with a fluorous eluting fluid (mobile phase). In one embodiment, the non-fluorous solid phase is polar in nature. The method can further include a second phase elution with a suitable organic solvent. A method conducting a chemical reaction, includes: mixing at least a first fluorous compound and a second compound, the first fluorous compound differing in fluorous nature from the second compound; exposing the first mixture to conditions to convert at least one of the first fluorous compound and the second compound to give a second mixture containing at least a third compound, charging the second mixture to a non-fluorous solid phase; and eluting with a fluorous fluid.

Abstract: The present invention relates to methods of inducing ovulation in a female host comprising the administration of a non-polypeptide cyclic adenosine monophosphate (cAMP) level modulator to the female host. In another aspect, the invention provides for specific administration of the phosphodiesterase inhibitor prior to the luteal phase of the host's ovulatory cycle. Preferred non-polypeptide cAMP level modulators include phosphodiesterase inhibitors, particularly inhibitors of phosphodiesterase 4 isoforms.

Abstract: The invention provides methods for labeling a molecule by contacting a sample molecule with a solid support coupled to a chemical group comprising a cleavable functional group, one or more functional groups, and a reactive group for the sample molecule, under conditions allowing the sample molecule to covalently bind to the reactive group; and cleaving the cleavable functional group, thereby releasing the sample molecule comprising the one or more functional groups, which can be a tag. The invention also provides a solid support covalently coupled to a chemical group comprising a cleavable functional group, a mass spectrometry tag and a reactive group for covalently attaching a sample molecule, wherein the cleavable functional group, the tag and the reactive group are positioned relative to each other to allow transfer of the tag to the sample molecule upon cleavage of the cleavable functional group.

Abstract: Gas flow is controlled to a feed gas consuming device depending on whether a contaminant gas is present. In one embodiment, hydrogen gas flow from a hydrogen gas generator to a hydrogen consuming device, such as a fuel cell, gas chromatograph or a flame ionization detector, is terminated when there is chemical contaminant breakthrough in the hydrogen gas flow. The apparatus relates to the use of a sensor for detecting a predetermined concentration of a chemical contaminant such as ammonia. In one embodiment the apparatus terminates the gas flow when a concentration of ammonia in the gas flow corresponds to a breakthrough (e.g., approximately in the range of 2.0% or greater). The apparatus prevents the ammonia-contaminated hydrogen from disabling such a hydrogen consuming device that would have otherwise received the contaminated gas flow.

Abstract: A method and apparatus for Flow Injection Analysis (FIA) into Atmospheric Pressure Ion sources (API) including Electrospray (ES) and Atmospheric Pressure Chemical Ionization (APCI) sources whereby the sampling and spray needles are one and the same. The sampling and spray needle configured with an autoinjector apparatus or used in manual injection is introduced directly into a mating ES or APCI probe configured in an API source. Such a sampling and spray needle eliminates the need for injector valves, transfer lines or additional fluid delivery systems in FIA into API sources interfaced to mass spectrometers or other chemical analyzers. The use of a sampling and spray needle configuration reduces component costs, liquid dead volume, sample dilution effects, and minimizes cross contamination effects, solvent consumption and waste while increasing sample throughput.

Abstract: The present invention provides a method and device for processing, sampling, and diluting a fluid and for detecting an analyte within the processed, sampled, and diluted fluid. In the method and device for processing, sampling, and diluting a fluid, an amount of fluid to be processed, sampled, and diluted is accepted by a porous membrane; a portion of the membrane saturated with the fluid is isolated thereby defining a predetermined sample volume of fluid; and the predetermined fluid volume sample is then released from the isolated membrane with a specified quantity of a fluid diluent to provide a diluted fluid sample. Analytes within the diluted fluid sample are detected by test strips in fluid contact with the diluted fluid sample.

Abstract: Fluid introduction is facilitated through the use of a port which extends entirely through a microfluidic substrate. Capillary forces can be used to retain the fluid within the port, and a series of samples or other fluids may be introduced through a single port by sequentially blowing the fluid out through the substrate and replacing the removed fluid with an alternate fluid, or by displacing the fluid in part with additional fluid. In another aspect, microfluidic substrates have channels which varying in cross-sectional dimension so that capillary action spreads a fluid only within a limited portion of the channel network. In yet another aspect, the introduction ports may include a multiplicity of very small channels leading from the port to a fluid channel, so as to filter out particles or other contaminants which might otherwise block the channel at the junction between the channel and the introduction port.

Abstract: Fluid introduction is facilitated through the use of a port which extends entirely through a microfluidic substrate. Capillary forces can be used to retain the fluid within the port, and a series of samples or other fluids may be introduced through a single port by sequentially blowing the fluid out through the substrate and replacing the removed fluid with an alternate fluid, or by displacing the fluid in part with additional fluid. In another aspect, microfluidic substrates have channels which varying in cross-sectional dimension so that capillary action spreads a fluid only within a limited portion of the channel network. In yet another aspect, the introduction ports may include a multiplicity of very small channels leading from the port to a fluid channel, so as to filter out particles or other contaminants which might otherwise block the channel at the junction between the channel and the introduction port.

Abstract: For detecting a component of a substance (of liquid or solid) on a front surface of a substrate, the substrate with the substance thereon is transferred into a vaporizing section, the substance is heated in the vaporizing section so that the component is vaporized from the substance in the vaporizing section, the component vaporized is fed from the substance in the vaporizing section to a detecting section, and the vaporized component is detected in the detecting section.

Abstract: Colorimetric sensors comprising a receptor incorporated within polydiacetylene assemblies to form a transducer capable of indicating a color change when contacted with an analyte are disclosed. Methods of using the colorimetric sensor and a kit for the colorimetric detection of an analyte are also disclosed.

Abstract: Materials and methods for studying and modulating the interaction of carbohydrate-containing moieties with other species are described, in particular, small particles, e.g. clusters of metal or semiconductor atoms, which can be employed as a substrate for immobilizing a plurality of ligands comprising carbohydrate groups. These “nanoparticles” can then be used to study carbohydrate mediated interactions, e.g. with other carbohydrates or proteins, and as therapeutics and diagnostic reagents.

Abstract: A method for depositing gold on a substrate is provided. By this aspect nucleation centers are bound, deposited or formed at one or more sites on the substrate, the substrate is then contacted with a treatment composition which comprises soluble gold-providing agent from which gold is deposited onto a nucleation center to yield gold metal deposits. The method of the invention may be used for a variety of image enhancing techniques as well as diagnostic and analyte detection techniques.

Abstract: A method for separating biological materials and other substances is disclosed, wherein a mixture containing desired and undesired components are exposed to magnetic particles having ligands capable of binding to the desired and/or the undesired components to form a magnetic mixture, placing the magnetic mixture onto a substrate material; exposing the substrate coated with the magnetic mixture to a magnetic field of sufficient strength to cause the magnetic components to migrate across the substrate; and repeatedly increasing and decreasing the magnetic field in a pulsing manner with a frequency sufficient to cause the desired magnetic components to separate spatially from the undesired magnetic components. A device for separating biological materials capable of being operated to increase and decrease magnetic field in a pulse fashion is also disclosed.

Abstract: The present invention provides a recovery processing method to restore the substrate processing apparatus to an operating state after correcting an abnormality having occurred in the substrate processing apparatus in operation and having resulted in a stop in the operation, comprising a substrate retrieval step in which substrate salvage processing is first executed for a wafer W left in a chamber in the substrate processing apparatus in correspondence to the extent to which the wafer has been processed at the time of the operation stop and the substrate having undergone the substrate salvage processing is then retrieved into the cassette storage container and an apparatus internal state restoration step in which the states inside the individual chambers of the substrate processing apparatus are restored.

Abstract: A quantum computing method comprising constructing a dressing transformation V between a physical Hamiltonian H and an ideal Hamiltonian HID. The physical Hamiltonian H describes a physical quantum computer that comprises a plurality of qubits, including interactions between the plurality of qubits and a continuum. The ideal Hamiltonian HID describes the universal quantum computer that corresponds to the physical quantum computer. Each qubit in the plurality of qubits is initialized and quantum calculations are performed using the plurality of qubits. Measurement of the plurality of qubits is performed in the dressed state.

Abstract: An electroluminescence (EL) device and a method are provided for fabricating said device with a nanotip electrode. The method comprises: forming a bottom electrode with nanotips; forming a Si phosphor layer adjacent the nanotips; and, forming a transparent top electrode. The Si phosphor layer is interposed between the bottom and top electrodes. The nanotips may have a tip base size of about 50 nanometers, or less, a tip height in the range of 5 to 50 nm, and a nanotip density of greater than 100 nanotips per square micrometer. Typically, the nanotips are formed from iridium oxide (IrOx) nanotips. A MOCVD process forms the Ir bottom electrode. The IrOx nanotips are grown from the Ir. In one aspect, the Si phosphor layer is a SRSO layer. In response to an SRSO annealing step, nanocrystalline SRSO is formed with nanocrystals having a size in the range of 1 to 10 nm.