Transgenic non-mammalian animals, e.g. flies, that exhibit an adult onset neurodegenerative phenotype, as well as methods for preparing the same, are provided. Also provided are methods of using the subject transgenic non-mammalian animals to identify compounds having activity with respect to adult onset neurodegenerative diseases. Finally, kits for screening compounds for anti-neurodegenerative activity are provided.

1. Field of the Invention
The field of this invention is transgenic flies.
2. Background of the Invention
Adult onset neurodegenerative diseases are among some of the most devastating diseases currently afflicting mankind, at least in the developed nations of the world. Such diseases xe2x80x9care characterized by onset in adult life, chronic progressive course, distinct clinical phenotypes, specific cellular abnormalities involving subsets of neurons, and eventually fatal outcomes.xe2x80x9d Price et al., Science (Nov. 6, 1998) 282: 1079-1083. Examples of neurodegenerative diseases include: Alzheimer""s disease, Parkinson""s disease, amyotrophic lateral sclerosis, Huntington""s disease, and the like. Currently, treatment of such diseases is limited to disease management and there are no cures. As such, there is much ongoing research that is aimed at the identification and development of new therapeutic agents which can at least slow, if not reverse, the progression of neurodegenerative diseases.
Critical steps in the identification and development of new therapeutic agents are: (a) the generation of candidate agents; and (b) screening of the candidate agents for efficacy (and safety). With the advent of combinatorial chemistry protocols, large numbers of potential compounds, known as libraries, can be rapidly generated. Such libraries serve as a collection of potential therapeutic agents. Following generation of a library of potential therapeutic agents, the library must be screened to identity the promising candidates.
For screening purposes, a number of in vitro high throughput screening protocols have been developed. However, these in vitro screening assays must be followed by in vivo screening assays. Since it is undesirable to immediately screen compounds that show promise from in vitro assays in humans, an important step in the identification of therapeutic agents for such neurodegenerative diseases is the screening of potential therapeutic compounds in non-human animal models. As such, non-human animal models of neurodegenerative diseases play an important role in the discovery of therapeutic agents for such diseases.
One type of non-human animal model that can be used for screening purposes to identify therapeutic agents for use in treating neurodegenerative diseases is a non-human mammalian model, e.g. primates, mice, etc. However, primates are expensive, difficult to use, and require a significant period of time prior to developing adult onset neurodegenerative symptoms. These factors make the use of primates as adult onset neurodegenerative animal models prohibitive. Transgenic mice suffer from analogous disadvantages, i.e. expense, slow reproduction time, and generation of small numbers of offspring.
As such, there is a need for additional animal models of adult onset neurodegenerative diseases. Of particular interest would be the development of an animal model having a relatively short life span and a rapid reproduction cycle characterized by the production of large numbers of offspring. Preferably, such an animal model should also be relatively simple and economic to maintain.
Relevant Literature
Patents of interest include: U.S. Pat. No. 4,670,388. Methods of preparing transgenic Drosophila melanogaster are disclosed in: Spradling, A. C., and Rubin, G. M. (1982). Science 218, 341-347; Brand and Perrimon, Development (1993) 118: 401-415; and Phelps and Brand, Methods (April 1998) 14:367-379. See also, Spradling A C, P Element Mediated Transformation in Drosophila: A Practical Approach (ed. D. D. Roberts, IRL Press, Oxford) (1986) pp 175-179. Articles disclosing drosophila that exhibit neurodegenerative phenotypes include: Buchanan and Benzer, Neuron (May 1993) 10: 839-850; Jackson et al, Neuron (September 1998) 21: 633-642; Kretzchmar et al., J. Neuroscience (Oct. 1, 1997) 17: 7425-7432; Marfany et al., J. Neurogenetics (1998) 12: 41-54; Min and Benzer, Current Biology (1997) 7:885-888; Rogina et al., Proc. Nat""l Acad. Sci. USA (June 1997) 94: 6303-6306; and Warrick et al., Cell (Jun. 12, 1998) 93: 939-949.
Articles describing non-human animal models for neurodegenerative diseases include: Price et al., Science (Nov. 6, 1998) 282:1079-1083.
Transgenic non-mammalian animals, e.g. flies, that exhibit an adult onset neurodegenerative phenotype, as well as methods for preparing the same, are provided. The subject transgenic animals are characterized in that they have a transgene stably integrated into their genome the expression of which in embryonic neuroblasts results in the adult onset neurodegenerative phenotype. In preferred embodiments, the transgene is a myb gene. Also provided are methods of screening compounds for activity with respect to adult onset neurodegeneration, particularly compounds having therapeutic activity with respect to adult onset neurodegenerative disease conditions.
Non-mammalian transgenic animals, particularly insects, e.g. flies, that exhibit adult onset neurodegeneration, as well as methods for their preparation, are provided. The subject transgenic animals are further characterized in that they comprise a stably integrated transgene which is expressed in embryonic neuroblasts, where the expressed product results in the observed adult onset neurodegenerative phenotype. In preferred embodiments, the transgene is a myb gene. Also provided are methods of using the subject non-mammalian transgenic animals to screen for compounds having activity with respect to adult onset neurodegeneration, particularly compounds that are therapeutic for neurodegenerative diseases. In further describing the subject invention, the transgenic animals and methods for their production will be detailed first, followed by a discussion of the screening methods of the subject invention.
Animal Models
In the broadest sense, the invention provides non-mammalian animal models that exhibit an adult onset neurodegenerative phenotype, where the phenotype results from a non-adult expression event. In other words, the phenotype of the animal models of the subject invention results from an expression event that occurs in the non-adult animal. The phenotype may result from a variety of different non-adult expression events, where the expression event may be the expression of a mutated gene, the failure of expression of a normally expressed gene, the expression of a transgene, and the like.
In many preferred embodiments, the subject invention provides non-mammalian transgenic animals (i.e. multi-cellular non-plant organisms) that have an adult onset neurodegenerative phenotype. In other words, the subject transgenic animals exhibit one or more phenotypic traits that characterize neurodegenerative disorders and occur in the adult organism (e.g. during the adult stage of the organism""s life), where such traits include: impaired motor skills, impaired cognitive skills, reduced appetite, etc. As such, the subject animals are non-mammalian animal models for neurodegenerative disorders, particularly adult onset neurodegenerative disorders, such as: Parkinson""s Disease, Alzheimer""s Disease, Huntington""s Disease, stroke, and the like. The term transgenic animal is used broadly herein to refer to a non-plant multicellular organism at any stage of development, e.g. adult, fertilized eggs, embryos, larva, etc. Thus, a particular multicellular organism is a transgenic animal according to the subject invention no matter which stage of development it is at, so long as the animal exhibits the requisite adult onset neurodegenerative phenotypic characteristics that results from the correct spatial and temporal expression of the transgene stably integrated into its genome, as described in greater detail infra.
The transgenic animals of the subject invention are non-mammalian transgenic animals. Of particular interest are invertebrate transgenic animals, particularly members of the phylum arthropoda, and more particularly members of the class insecta. Of particular interest in many embodiments are transgenic flies. In many preferred embodiments, the transgenic flies are members of the family Drosophilidae, where the transgenic animal is often a Drosophila melanogaster. The subject invention is now further described in terms of transgenic flies.
Transgenic flies of the subject invention, e.g. Drosophila melanogaster, exhibit the following phenotypic characteristics: normal wild type phenotype through the initial hours of adult life, where by initial hours is meant at least the first 1 hour, usually at least the first 3 hours and more usually at least the first 5 hours, where in many embodiments the flies may exhibit a normal phenotype through the first 10 to 15 hours of adult life or longer, but in many instances the normal phenotype will cease by about 12 to 13 hours of adult life. Following the initial hours of adult life, the flies exhibit one or more phenotypic characteristic of neurodegeneration, including: progressive loss of neuromuscular control, e.g. of the wings; progressive degeneration of general coordination; progressive degenerative of locomotion; and progressive degeneration of appetite. The subject transgenic flies are further characterized in that death occurs at 4 to 6, usually 4 to 5 days of adult life.
A critical feature of the subject transgenic animals is that the animals harbor a stably integrated transgene that is spatially and temporally expressed in a manner sufficient to result in the desired adult onset neurodegenerative phenotype. The term xe2x80x9ctransgenexe2x80x9d is used herein to describe genetic material which has been or is about to be artificially inserted into the genome of a cell. With regards to spatial expression of the transgene, expression is generally limited to neuronal cells, specifically neuroblasts. With regards to temporal expression, the transgene is required to be expressed in the embryo. In other words, the transgenic expression occurs transiently in the embryonic neuroblasts.
The transgene encodes a product that, when expressed in embryonic neuroblasts, gives rise to the adult onset neurodegenerative phenotype. Generally, the transgene encodes a transcription factor or mimetic thereof having the desired result. Suitable transgenes are those that encode products which activate transcription and can bind to a Myb DNA binding sequence present in the genome of the transgenic animal. Of interest in many embodiments is a myb gene, where the myb gene may be a naturally occurring myb gene or a gene that encodes a product having the desired structural and functional properties, e.g. DNA binding, of the product expressed by a naturally occurring myb gene. Thus, the transgene may comprise a nucleic acid having the sequence of a naturally occurring myb gene or a sequence that includes only a myb domain that governs sequence specific DNA binding (i.e. a myb DNA binding domain) and a transcriptional activation domain. As such, the transgene at least includes a myb DNA binding domain or a sequence that is substantially similar to a naturally occurring myb DNA domain to provide for the requisite DNA binding activity, where substantially similar means a nucleic acid sequence having a sequence identity with a naturally occurring myb DNA binding domain of at least about 50%, usually at least about 60% and more usually at least about 65%, where sequence identity is determined using the BLAST program at default settings. In many embodiments, the transgene comprises a naturally occurring myb gene or a sequence substantially similar thereto. Of particular interest as the transgene is a non-Drosophila melanogaster myb gene, preferably a chicken or viral myb gene. Thus, preferred transgenes found in the subject animals are viral or chicken myb genes that encode a product that activates transcription and binds to DNA at a Myb binding sequence. Chicken myb genes of interest include: A-myb and C-myb; while viral myb genes of interest include: v-myb-1151; v-myb-ds7, v-myb-1120 and v-myb-859 (Ganter, B. and J. S. Lipsick (1999) Adv. Cancer Res. (In Press); Ma, X. and B. Calabretta (1994) Cancer Res. 54: 6512-6516; J. S. Lipsick (1996) Oncogene. 13: 223-235; Katzen, A. L., et al., (1998). Genes Dev. 12: 831-843; Garrido, C., et al., (1992) J. Vir. 66: 6773-6776; Fu, S. L. and J. S. Lipsick (1997) Cell Growth Diff. 8: 35-45; Grasser, F. A., et al., (1992) Oncogene 7: 1005-1009; Latham, K. E., et al., (1996) Oncogene 13: 1161-1168; Lane, T., et al., (1990) Mol. Cell. Biol. 10: 2591-2598; Chen, R. H., Fields, S. and J. S. Lipsick (1995) Oncogene 11: 1771-1779; Chen, R. H. and J. S. Lipsick (1993) Mol. Cell. Biol. 13: 4423-4431; Fu, S. L. and J. S. Lipsick (1996) J. Vir. 70: 5600-5610).
As expression of the transgene is targeted to occur in a non-adult stage of the animal, the transgene is stably integrated into the genome of the animal in a manner such that its expression is controlled both spatially and temporally to the desired cell type and the correct developmental stage, i.e. to expression in embryonic neuroblasts. Specifically, the subject transgene is stably integrated into the genome of the animal under the control of a promoter that provides for expression in embryonic neuroblasts. The transgene may be under the control of any convenient promoter that provides for this requisite spatial and temporal expression pattern, where the promoter may be endogenous or exogenous, but will generally be endogenous. A suitable promoter is the promoter located in the Drosophila melanogaster genome at position 86E1-3.
The transgene may be integrated into the fly genome in a manner that provides for direct or indirect expression activation by the promoter, i.e. in a manner that provides for either cis or trans activation of gene expression by the promoter. In other words, expression of the transgene may be mediated directly by the promoter, or through one or more transactivating agents. Where the transgene is under direct control of the promoter, i.e. the promoter regulates expression of the transgene in a cis fashion, the transgene is stably integrated into the genome of the fly at a site sufficiently proximal to the promoter and in frame with the promoter such that cis regulation by the promoter occurs.
In yet other embodiments where expression of the transgene is indirectly mediated by the endogenous promoter, the promoter controls expression of the transgene through one or more transactivating agents, usually one transactivating agent, i.e. an agent whose expression is directly controlled by the promoter and which binds to the region of the transgene in a manner sufficient to turn on expression of the transgene. Any convenient transactivator may be employed, where the GAL4 transactivator system is particularly preferred in many embodiments of the subject invention.
In these preferred embodiments of the subject invention in which the transgenic fly comprises the GAL4 targeted expression system, a GAL4 encoding sequence is stably integrated into the genome of the animal in a manner such that it is operatively linked to the endogenous promoter that provides for expression solely in embryonic neuroblasts. Flies of line 31-1 (subsequently referred to as 1822), as disclosed in Brand and Perrimon, Development (1993) 118: 401-415 express GAL4 in this manner, and are known to those of skill in the art. The transgene is stably integrated into a different location of the genome, generally a random location in the genome, where the transgene is operatively linked to an upstream activator sequence, i.e. UAS sequence, to which GAL4 binds and turns on expression of the transgene. Transgenic flies having a UAS: GAL4 transactivation system are known to those of skill in the art and are described in Brand and Perrimon, Development (1993) 118: 401-415; and Phelps and Brand, Methods (April 1998) 14:367-379.
Methods of Producing the Subject Transgenic Flies
The subject transgenic flies can be prepared using any convenient protocol that provides for stable integration of the transgene into the fly genome in a manner sufficient to provide for the requisite spatial and temporal expression of the transgene, i.e. in embryonic neuroblasts. A number of different strategies can be employed to obtain the integration of the transgene with the requisite expression pattern. Generally, methods of producing the subject transgenic flies involve stable integration of the transgene into the fly genome. Stable integration is achieved by first introducing the transgene into a cell or cells of the fly, e.g. a fly embryo. The transgene is generally present on a suitable vector, such as a plasmid. Transgene introduction may be accomplished using any convenient protocol, where suitable protocols include: electroporation, microinjection, vesicle delivery, e.g. liposome delivery vehicles, and the like. Following introduction of the transgene into the cell(s), the transgene is stably integrated into the genome of the cell. Stable integration may be either site specific or random, but is generally random.
Where integration is random, the transgene is typically integrated with the use of transposase. In such embodiments, the transgene is introduced into the cell(s) within a vector that includes the requisite P element, terminal 31 base pair inverted repeats. Where the cell into which the transgene is to be integrated does not comprise an endogenous transposase, a vector encoding a transposase is also introduced into the cell, e.g. a helper plasmid comprising a transposase gene, such as pTURBO (as disclosed in Steller and Pirrotta, xe2x80x9cP Transposons Controlled by the Heat Shock Promoter,xe2x80x9d Mol. Cell. Biol. (1986) 6:1640-1649). Methods of random integration of transgenes into the genome of a target Drosophila melanogaster cell(s) are disclosed in U.S. Pat. No. 4,670,388, the disclosure of which is herein incorporated by reference.
In those embodiments in which the transgene is stably integrated in a random fashion into the fly genome, means are also provided for selectively expressing the transgene at the appropriate time during development of the fly. In other words, means are provided for obtaining targeted expression of the transgene. To obtain the desired targeted expression of the randomly integrated transgene, integration of particular promoter upstream of the transgene, as a single unit in the P element vector may be employed. Alternatively, a transactivator that mediates expression of the transgene may be employed. Of particular interest is the GAL4 system described in Brand and Perrimon, supra.
In this particular embodiment, the subject transgenic flies are produced by: (1) generating two separate lines of transgenic flies: (a) a first line that expresses GAL4 in embryonic neuroblasts, e.g. under the control the endogenous fly promoter located at position 86E1-3, described supra; and (b) a second line in which the transgene is stably integrated into the cell genome and is fused to a UAS domain; (2) crossing the two lines; and (3) screening the progeny for the desired phenotype, i.e. adult onset neurodegeneration. Each of the above steps are well known to those of skill in the art. See e.g. Brand and Perrimon, Development (1993) 118: 401-415; and Phelps and Brand, Methods (April 1998) 14:367-379. See also the Experimental Section, infra.
The above strategy is employed to obtain fertilized eggs that comprise the transgene stably integrated into the genome in a manner such that it is expressed in the correct spatial and temporal manner so that the eggs give rise to adult flies exhibiting the desired adult onset neurodegenerative phenotype. Generally, the fertilized eggs are grown at a temperature that results in the adult onset neurodegenerative phenotype, where the temperature ranges from about 24 to 30xc2x0 C., usually from about 27 to 29xc2x0 C. and more usually at about 28xc2x0 C.
Utility
The subject flies find use in a variety.of applications, including: as tools for use in the elucidation of genetic mechanisms involved in neurodegenerative disorders; as a screening tool that identifies therapeutic compounds for use in the treatment of neurodegenerative conditions (i.e. as animal models for human AON disease conditions); and as tools for use in the identification of adult onset neurodegeneration gene targets, i.e. genes whose expression can be modulated, e.g. enhanced or disrupted, in order to alleviate adult onset neurodegeneration. The subject transgenic flies find particular use in screening methods designed to identify therapeutic agents for use in the treatment of neurodegenerative diseases.
Screening Methods
As mentioned above, the subject transgenic flies find particular utility in screening assays designed to identify therapeutic compounds for neurodegenerative conditions, particularly adult onset neurodegenerative conditions. Through use of the subject transgenic flies (or cells derived therefrom depending on the particular screening assay), one can identify compounds that have activity with respect to an adult onset neurodegenerative disease. Compounds have activity with respect to an adult onset neurodegenerative disease if they modulate or have an effect on at least one parameter or symptom of the disease, such as loss of appetite, loss of motor coordination, etc., where the modulatory activity may be to reduce or enhance the magnitude of the symptom. Thus, the screening methods of subject invention can be used to identify compounds that modulate the progression of neurodegenerative disease, e.g. by binding to, modulating, enhancing or repressing the activity of a protein or peptide involved in the progression of the neurodegenerative disease, and/or compounds that ameliorate, alleviate or even remove the phenotypic symptoms of the disease, where such activity may or may not be the result of activity with respect to the underlying mechanism of the disease. Screening to determine drugs that lack effect on the neurodegeneration condition is also of interest. Assays of the invention make it possible to identify compounds which ultimately: (1) have a positive affect with respect to a neurodegenerative disease condition and as such are therapeutics, e.g. agents which arrest or reverse the neurodegeneration or ameliorate or alleviate the symptoms of such a condition; or (2) have an adverse affect with respect to the neurodegeneration disease and as such should be avoided as therapeutic agents.
In the screening methods of the subject invention, a quantity of a candidate agent is generally orally administered to the fly. Following oral administration, the affect of the candidate agent on the adult onset neurodegenerative phenotype of the fly is determined, typically by comparison with a control (i.e. a transgenic fly to which the candidate agent has not been administered). The affect of the candidate agent is determined by determining whether one or more of the phenotypic characteristics of adult onset neurodegeneration are exacerbated or ameliorated in the test fly as compared to the control fly, where characteristics that are monitored include neuromuscular control of wings, general coordination, locomotion, appetite, and the like. The candidate agent is generally orally administered to the fly by mixing the agent into the fly nutrient medium and placing the medium in the presence of the fly, (either the larva or adult fly, usually the adult fly) such that the fly feeds on the medium. Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations of candidate agent. Typically, one of these concentrations serves as a negative control, i.e. no compound. In a preferred embodiment, a high throughput screening protocol is employed, in which a large number of candidate agents are tested in parallel using a large number of flies. By xe2x80x9clarge numberxe2x80x9d is meant a plurality, where plurality means at least 50, usually at least 100, and more usually at least 1000, where the number of may be 10,000 or 50,000 or more, but in many instances will not exceed 5000.
The subject methods find use in the screening of a variety of different potentially therapeutic candidate agents. Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. New potential therapeutic agents may also be created using methods such as rational drug design or computer modelling.
Screening may be directed to known pharmacologically active compounds and chemical analogs thereof, or to new agents with unknown properties such as those created through rational drug design. Candidate agents having therapeutic activity with respect to adult onset neurodegeneration can be identified based on their ability to at least ameliorate, if not completely alleviate or remove, one or more of the neurodegenerative phenotypes of the adult transgenic fly of the subject invention, such as loss of wing neuromuscular control, and the like, as described above.
The above screening methods may be part of a multi-step screening process of evaluating candidate therapeutic agents for their efficacy (and safety) in the treatment of neurodegenerative diseases in mammalian hosts, e.g. humans. In multi-step screening processes of the subject invention, a candidate compound or library of compounds is subjected to screening in a second in vivo model, e.g. a mouse model, following screening in the subject transgenic animal model. Following the initial screening in the non-mammalian transgenic animals of the subject invention, the positive compounds are then screened in non-human mammalian animal models, including transgenic non-human mammalian animal models. Transgenic mouse models of neurodegenerative diseases and methods for their use in screening assays are described in: U.S. Pat. Nos.: 5,777,194; 5,767,337; 5,720,936; 5,672,805; 5,612,486; 5,602,309; and 5,387,742, the disclosures of which are herein incorporated by reference. In addition, a pre in vivo screening step may be employed, in which the compound is first subjected to an in vitro screening assay for its potential as a therapeutic agent in the treatment of neurodegenerative conditions. Any convenient in vitro screening assay may be employed, where a variety of suitable in vitro screening assays are known to those of skill in the art.
Identification of Gene Targets
In addition to their use as animal models for screening candidate therapeutic agents, the subject transgenic flies also find use in the identification of adult onset neurodegeneration gene targets, i.e. genes whose expression can be beneficially modulated to treat adult onset neurodegeneration. Gene based therapies can be identified by doing traditional enhancer/suppressor analyses in the subject transgenic flies. In these analyses, genes in the subject transgenic flies are mutated to identify ones that either exacerbate or alleviate the adult onset neurodegeneration mutant phenotype. Methods of mutating genes and carrying out enhancer/suppressor analyses are well known to those of skill in the art (Hays, T S et al., Molecular and Cellular Biology (March 1989) 9(3):875-84; Deuring, R; Robertson, B; Prout, M; and Fuller, M T. Mol. Cell. Biol., 1989 9:875-84.; Fuller, M T et al., Cell Mot. Cyto. (1989) 14 :128-35; Rottgen G, Wagner T, Hinz U Mol. Gen. Genet. 1998 257:442-51).
Genes that mutate to enhance the adult onset neurodegeneration phenotype in a recessive manner yield potential protein therapeutics for adult onset neurodegenerative conditions, since elevating the normal gene product level of such genes potentially alleviates the neurodegenerative condition. Genes that mutate to suppress the adult onset neurodegeneration phenotype in a recessive manner yield gene targets for disruption to alleviate the neurodegenerative conditions, where disruption of these genes can be achieved using a variety of methods, ranging from deleting the DNA for the target gene to inhibiting its transcription, translation, or protein activity. For screening candidate agents, small molecule antagonists to these genes can be constructed and evaluated for efficacy in the fly model through oral administration. Alternatively, large molecular antagonists can be delivered by gene therapy, as described infra.
Kits
Also provided by the subject invention are kits for use in performing the subject screening methods. The subject kits include at least a plurality of transgenic flies of the subject invention, or a means for producing such a plurality of flies, e.g. a male and female transgenic fly of the subject invention, vectors carrying requisite genes, such as the transgene, a transposase gene, GAL4, etc. The flies may be housed in appropriate container(s), e.g. vials. The subject kits may also comprise a nutrient medium for the animals, e.g. drosophila medium.
Therapeutic Agents and Pharmaceutical Compositions
Also provided by the subject invention are therapeutic agents for use in treating a neurodegenerative condition, as well as pharmaceutical formulations thereof. The therapeutic agents of the subject invention are those agents identified using the screening methods described supra that show beneficial activity with respect to a neurodegenerative condition, particularly an adult onset neurodegenerative condition (or agents known to have an effect on the expression of a gene identified as modulating the phenotype of an adult onset neurodegenerative condition, where identification employs the use of the subject non-transgenic animals).
Also provided are pharmaceutical compositions of the subject therapeutic agents. In the pharmaceutical compositions or formulations of the subject invention, agents. described above are formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. In pharmaceutical dosage forms, the agents may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.
For oral preparations, the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose. derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
The agents can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
The agents can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
Furthermore, the agents can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
The term xe2x80x9cunit dosage form,xe2x80x9d as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
Where the agent is a polypeptide, polynucleotide, analog or mimetic thereof (as identified using the mutant screen analysis protocols described supra, it may be introduced into tissues or host cells by any number of routes, including viral infection, microinjection, or fusion of vesicles. Jet injection may also be used for intramuscular administration, as described by Furth et al. (1992), Anal Biochem 205:365-368. The DNA may be coated onto gold microparticles, and delivered intradermally by a particle bombardment device, or xe2x80x9cgene gunxe2x80x9d as described in the literature (see, for example, Tang et al. (1992), Nature 356:152-154), where gold microprojectiles are coated with the DNA, then bombarded into skin cells.
Those of skill in the art will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.
Kits with unit doses of the active agent, usually in oral or injectable doses, are provided. In such kits, in addition to the containers containing the unit doses will be an informational package insert describing the use and attendant benefits of the drugs in treating pathological condition of interest. Preferred compounds and unit doses are those described herein above.
Methods of Treating Neurodegenerative Conditions
Also provided are methods of treating neurodegenerative disease conditions, particularly adult onset neurodegenerative disease conditions, using the subject active agents. In the subject methods, an effective amount of the active agent of the subject invention is administered to the host to be treated. By xe2x80x9ceffective amountxe2x80x9d is meant a dosage sufficient to produce a desired result, where the desired result is generally an amelioration or alleviation, if not complete cessation, of one or more symptoms of the neurodegenerative disease being treated. Administration of the agents can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal,etc. A variety of hosts are treatable according to the subject methods. Generally such hosts are xe2x80x9cmammalsxe2x80x9d or xe2x80x9cmammalian,xe2x80x9d where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the hosts will be humans.
Validation of Non-mammalian Animal Models for a Human Disease Condition
Also provided are methods of validating non-mammalian animal models, such as the animal models of the subject invention, as animal models for human disease conditions. In the subject validation methods, a compound having a activity with respect to he human disease of interest is administered to a potential non-mammalian animal model for said disease. The potential animal model may be one that has been identified from nature or produced via human intervention, e.g. genetic engineering. The only limitation on the putative non-mammalian animal model for the human disease of interest is that it have a phenotype that is analogous to the phenotype suffering from the human disease, i.e. it has a phenotype that can be correlated to at least one symptom of the disease in a human. For example, the animal models of the present application have a phenotype that is analogous to the phenotype of a human suffering from an adult onset neurodegenerative disease condition.
Following administration of the compound to the putative non-mammalian animal model, the effect that the compound has on the phenotype of the non-mammalian animal phenotype observed. Based on the observation, a determination is made as to whether the compound has the same effect on the phenotype of the non-mammalian animal model as it does on the human disease phenotype. For example, where the compound improves the impaired appetite in a human suffering from the human disease and also improves the impaired appetite of the putative non-mammalian animal model, the compound has the same effect on the phenotype of the animal model as it does on the phenotype of a human suffering from the disease.
If the compound has the same effect on the phenotype of the putative non-mammalian animal model as it does on a human suffering from the disease, the non-mammalian animal model is validated as an animal model for the human disease condition.
The following examples are offered by way of illustration and not by way of limitation.

Non-mammalian transgenic animal having an adult onset neurodegenerative phenotype

The Board of Trustees of the Leland Stanford Junior University


A computer-implemented method of training a neural network comprises: identifying, by one or more processors, a set of parameters for a cost function for the neural network; applying, by the one or more processors, a gradient optimization of the cost function to generate a set of intermediate optimized parameter values for the cost function; initializing, by the one or more processors, a simulated annealing optimization of the cost function using the set of intermediate optimized parameter values for the cost function; applying, by the one or more processors, the simulated annealing optimization of the cost function to generate a set of final optimized parameter values for the cost function; assigning, by the one or more processors, the set of final optimized parameter values as weights for connections of the neural network; and using, by the one or more processors, the neural network to perform operations.

The cost function for neural networks may be non-convex. Gradient descent in non-convex cost functions may find a local minimum or a saddle point that is higher than the global cost minimum.
A cost function for a neural network is a measure of the quality of output of the neural network as a function of the parameters of the neural network. For example, a neural network used as a classifier may have a cost function that indicates the percentage of the input items that are incorrectly classified. In a non-convex cost function, there are multiple local minima, each of which may not be the global minimum. A saddle point is a point at which the derivative of the function is zero, but the point is not a local minimum.

Non-convex optimization by gradient-accelerated simulated annealing


A method and a device for establishing a trajectory for a vehicle is provided. The method includes the following steps: registering coordinates of objects using a sensor; calculating a cost map on the basis of the registered coordinates, wherein the cost map is subdivided into cells and a cost value is assigned to each cell, which cost value describes the presence and/or the vicinity of an object, calculating a cost space, which has a plurality of layers, wherein each layer is subdivided into cells and a cost value is assigned to each cell, wherein the cost values in the respective layers are calculated in each case for a specific orientation of the vehicle, and determining a trajectory that is as cost-effective as possible.

The invention relates to a method and a device for establishing a trajectory for a vehicle.
There are various driver assistance systems, which assist the driver of a motor vehicle with a partly automated or completely automated control of the motor vehicle. Such driver assistance systems include lane-change assist systems, braking assist systems, emergency or pedestrian avoidance assist systems or driver assistance systems for a fully automatic control of a motor vehicle.
Here, monitoring trajectories for collisions is very complicated and requires significant computational power in the motor vehicle.
Therefore, there is a significant need for a method and a device for establishing a trajectory for a motor vehicle, which can be carried out efficiently such that these can be calculated in real time where possible.
The invention is therefore based on the object of developing a method and a device for establishing a trajectory for a vehicle, in which efficient monitoring of the trajectory for collisions is possible.
This and other objects are achieved according to the invention.
The method according to the invention for establishing a trajectory in a vehicle comprises the following steps:
a) registering coordinates of objects using a sensor,
b) calculating an occupancy map or cost map on the basis of the registered coordinates, wherein the occupancy map or cost map is subdivided into cells and a cell value is assigned to each cell, which cell value describes the presence and/or the vicinity of an object,
c) calculating a cost space, which comprises a plurality of layers, wherein each layer is subdivided into cells and a cost value is assigned to each cell, wherein the cost values in the respective layers are calculated in each case for a specific orientation of the vehicle on the basis of the cell values, and
d) determining a trajectory that is as cost-effective as possible.
Using the method of the present invention, a cost space comprising a plurality of layers is calculated, wherein each layer is subdivided into cells and a cost value is assigned to each cell, wherein the cost values in the respective layers are calculated in each case for a specific orientation of the vehicle.
An occupancy map is subdivided into cells, wherein each cell represents a specific location. Binary cell values are assigned to the cells. The cell values specify whether an object is situated at the respective location or whether no object is present there.
A cost map is subdivided into cells, wherein each cell represents a specific location. Cost values are assigned to the cells.
A cost value specifies whether the location represented by the cell should be driven-on or avoided. A high cost value means that the location should be avoided. A low cost value means that the location can be driven-on or should be driven-on. The set of numbers for the cost values includes at least more than two numbers. Therefore, cost values are not binary values.
By way of example, cost values can be collision probabilities specifying the probability that a vehicle which drives over the location of the respective cell collides with an object. These collision probabilities specify the probability that an object is present, or not, at a specific location and can include any probability values between 0 and 1. This is particularly expedient if the measurement of the objects is afflicted by measurement uncertainties. The cost values can also contain further information. In particular, the collision costs contain information about the distance to the closest object.
An occupancy map merely specifies at which location an object is present or not present. Hence, such an occupancy map describes the location of the objects and therefore only contains information obtained when registering the coordinates of the objects. Such an occupancy map can be used as a cost map. Since the occupancy map describes the location of the objects using binary values, an occupancy map is not a cost map.
In principle, it is also possible to use an inverse cost system, in which a low cost value means that the location should be avoided and a high cost value means that the corresponding location can be driven-on or should be driven-on. In such an inverse cost system, a cost-effective trajectory is a trajectory with high costs. However, since cost systems generally evaluate high cost values as being negative, the following only considers examples in which a high cost value means that the location should be avoided and a low cost value means that the location can be driven-on or should be driven-on.
Motor vehicles and autonomously driving robots can be used as vehicles. Such robots can be domestic robots which are embodied, e.g., for cleaning floors or for mowing lawns, or else robots in manufacturing plants.
A cost space can be calculated very efficiently from a cost map which is divided into cells, wherein a cost value is assigned to each cell. In order to calculate the cost space, known, modified methods from image processing for expanding or dilating the cost map into the individual layers of the cost space can be used. Therefore, the cost space can be calculated quickly in real time.
The cost values in the cost space can be collision probabilities which can assume all values between 0 and 1 inclusive. The cost values of the cost space can also contain further information in addition to the collision probability or else can be linked to the collision probability. In particular, the collision costs contain information about the distance to the closest object. They can also contain information about the reliability of the coordinates of the objects registered by the sensor.
The method according to the invention preferably establishes a collision-free trajectory. If no collision-free trajectory should be present, a cost function establishes a trajectory that is as cost-effective as possible.
When calculating the occupancy map or the cost map, a distinction is preferably made between static and dynamic objects and the dynamic objects are eliminated or filtered out. By way of example, this is brought about by setting the probabilities for the presence of a dynamic object to zero.
Calculating a layer of a cost space on the basis of the cost map or the occupancy map is brought about, in particular, by virtue of the probabilities for the presence of an object in the occupancy map being expanded by a footprint of the vehicle. In particular, the expansion is performed by a dilation or a convolution.
Each footprint of the vehicle has an anchor or reference point, which, e.g., is the center-of-mass of the vehicle. A point which is arranged in the middle or centrally in the vehicle to the greatest possible extent is preferably selected as anchor. The cell of the layer of the cost space, which corresponds to the location of the cell of the occupancy map at which the anchor of the footprint is situated during expansion and which is referred to as an anchor cell below, is assigned either the sum of all probabilities contained in the cells of the cost map within the footprint or the maximum of all probabilities contained in the cells of the cost map within the footprint.
If a cost map is assumed during the calculation of a layer of a cost space, then the cost values of the cost map can be expanded by means of a binary footprint in order to obtain the cost values of the cost space. By contrast, if an occupancy map is used, then a footprint of the vehicle which, in addition to binary spatial information of the vehicle, also contains additional information, in particular distance information, should be used. By way of example, such a footprint can be a binary footprint which is surrounded by a cost neighborhood which has reducing costs with increasing distance from the center of the vehicle.
The expansion of the cost map to the cost space can be carried out by means of a convolution, wherein, for the purposes of calculating the convolution, the occupancy map and the footprint are preferably transformed into the frequency space. As a result of this, the convolution of each individual cell can be carried out using only a single multiplication.
Before expanding the cost map to the layers of the cost space, the objects are preferably surrounded by a cost neighborhood in each case, wherein the cells within the cost neighborhood in the cost map are assigned cost values which successively decrease to the outer edge of the cost neighborhood. This corresponds to a cost function with which a very small distance between an obstacle and the vehicle is occupied by high costs.
The expansion can be carried out by means of a convolution, wherein a predetermined number of cost values are discretized and an inverse imaging function (h(n)) is used to image the convolution results onto a cost dilation. As a result of this, a cost dilation is obtained by means of a convolution.
A footprint of the vehicle preferably has a rectangular or square embodiment. Identical symmetries of the footprint in a cost map are preferably only calculated once. In the case of a rectangular form, the number of calculations can thus be halved. In the case of a square form of the vehicle, the number of computation processes can be reduced to a quarter.
The dilation or the expansion of a two-dimensional footprint of the vehicle can be replaced by two dilations with in each case a one-dimensional footprint. This can significantly reduce computational complexity. The footprint is preferably aligned in the axial direction of the cost map. Here, in particular, use is made of the vHGW method.
The cost values of the individual cells, through which the trajectory extends, are preferably weighted by the length of the respective portion of the trajectory in the corresponding cell when determining a trajectory in the cost space. As a result of this, a correct overall value of the collision probability or of the costs is established with little computational complexity.
A device for establishing a trajectory for a vehicle comprises a sensor apparatus, a computer apparatus and an interface, wherein the computer apparatus has a computer program embodied to execute a method as mentioned above.
A trajectory with a risk of collision that is as low as possible can be provided at the interface. The interface can be coupled to a lane-change assist system, an automatic braking assist system, an emergency or pedestrian avoidance assist system or a completely autonomous driver assistance system.
The device can also be a component of a house or lawn robot, which automatically identifies obstacles and drives around them. Such a lawn robot, which may e.g. be embodied as a mowing robot, can also include a camera as a sensor apparatus, by means of which it identifies regions in which the grass has not yet been cut and assigns low costs to these regions such that the driving and mowing is preferably carried out in regions with uncut grass, wherein, preferably, the sensor apparatus additionally has a laser scanner for precisely detecting obstacles.
In principle, a sensor apparatus of these devices for vehicles and robots can be a laser scanner and/or a camera with the corresponding digital image evaluation apparatus and/or a radar.
Other objects, advantages and novel features of the present invention will become apparent from the following detailed description of one or more preferred embodiments when considered in conjunction with the accompanying drawings.

Method and device for establishing a trajectory for a vehicle

Bayerische Motoren Werke Aktiengesellschaft


A method for maintaining data objects in memory introduces metrics for freshness and interestingness of the data object. A freshness estimate is defined as the length of time that the data contained within the data object is expected to remain correct from the date that the object was last known to be valid. The interestingness estimate is defined as the length of time that the data object is expected to retain its usefulness to the user from the time that the data object was last used. The freshness estimate and interestingness estimate are stored in the data object along with a reference date. The freshness estimate, interestingness estimate, and reference date are updated or confirmed each time the data object is accessed. A maintenance program is periodically invoked to determine whether the data objects are outdated by comparing the time estimates associated with each data object to that object""s reference date. Objects found to be outdated are candidates for removal from the system.

The present invention relates generally to an information management systems implemented by a computer and, more particularly, to a system for maintaining data objects stored in memory.
Upcoming communication devices, such as a personal digital assistants (PDA), combine the features of a mobile telephone and computer. These devices are designed to support communications and interactions among users. One way in which these devices facilitate interactions among users is by storing information about persons or entities with whom the user interacts. One simple example would be an address book that lists the telephone number and address for each contact. Such information can be stored in virtual business cards. In emerging communication devices, users have the ability to exchange virtual business cards among themselves. Thus, a user can accumulate a large number of business cards.
The accumulation of cards by a user presents several problems. First, the user may accumulate cards that, over time, become unimportant. In devices with limited memory, a card that is not actively used consumes precious memory. Secondly, the information contained in a business card may change over time so that the information becomes no longer reliable. There is not currently any method to identify stale or unused business cards so that they can be removed.
One way to manage business cards is for the user to manually remove those cards that become stale or uninteresting. Unfortunately, this method requires the user to take time to periodically review and remove unwanted cards. For the above reasons, relying on the user to purge records manually is not a good solution to the problem. Another technique for removing contacts is to remove each contact that has not been accessed within a certain time period, such as one month. However, this simplistic approach treats all contacts as having the same aging process, which is typically not the case. Contacts that are never accessed should become candidates for removal much sooner than files in which there is some kind of infrequent access.
Thus, there is a need for further improvements in information management software to make it easier and less cumbersome to update and maintain a database of contact information.
The present invention is a method for maintaining card objects in a computer system. According to the present invention, a plurality of card objects are stored in the memory of a computer. Each card object includes a field for storing a reference date. Each card object further includes one or more time constant fields for storing time estimates. The disclosed embodiment includes two time estimatesxe2x80x94the freshness estimate and the interestingness estimate. The freshness estimate is an estimate of the length of time that the data contained in the card object is expected to remain reliable or fresh since the date that it was last accessed. The interestingness estimate indicates the length of time that the card object is expected to remain relevant or interesting to the user from the date that it was last accessed.
When a card object in the database is accessed, an update procedure is invoked. The purpose of the update procedure is to update the freshness and interestingness estimates based upon a predetermined rule set. More particularly, the freshness estimate is increased when a new version of the card object is obtained and the elapsed time since the card object was last accessed is greater than the current freshness estimate. The freshness estimate is decreased when the data contained in the card object is unchanged and the elapsed time since the card object was last accessed is less than the current freshness estimate. The interestingness estimate is increased when the elapsed time since the card object was last accessed is greater than the current interestingness estimate.
A separate maintenance procedure is periodically invoked to iterate through all of the card objects in the database and to remove those card objects deemed to be stale or uninteresting. Further, this maintenance procedure may be invoked when available memory is running low. A card object is deemed to be stale if the elapsed time since the date on which the card object was last accessed is greater than the current freshness estimate. A card object is deemed to be uninteresting if the elapsed time since the card object was last accessed is greater than the current interestingness estimate. The preferred method for removing a card object is by moving it to a data archive. If the card object is subsequently requested, it can be restored to active memory.
Using the method of the present invention, stale or irrelevant objects can be moved from memory and archived automatically without human intervention. This method frees up memory for objects which are used most frequently, while less frequently used objects are stored in an archive. This invention is particularly useful in those situations where the primary memory for storing objects is somewhat limited. Stale and irrelevant objects can be moved from primary memory to a data archive and recalled when needed. The present invention also increases the speed of look-ups by reducing the number of objects in the memory.

Stored data object management and archive control


In a pressure controller, a drive piston and pressure control valve are freely movably supported in a housing, the drive piston is supported by being energized and the pressure control valve is supported by being energized to make a pressure chamber and a decompression chamber communicatively connectible, the drive piston and the pressure control valve are moved by an electromagnetic force of a solenoid via a ring piston to make a hyperbaric chamber and the pressure chamber communicatively connectible and also an external piston is freely movably provided facing the drive piston and by making a control pressure acting on the external piston from a rear pressure chamber on a drive piston side equal to or greater than an external pressure acting from outside from an input unit, pressure control can properly be performed by readily moving a drive valve by pressure from outside when a power supply is defective.

A poppet type or spool type electromagnetic valve is known as a general pressure controller and the electromagnetic valve blocks (or opens) the channel by energizing a valve element with energizing force of a spring in one direction and opens (or blocks) the channel by moving the valve element with energization of a solenoid in another direction against energizing force of the spring. Since such an electromagnetic valve cannot control pressure by moving a valve element when a power supply feeding a current value to a solenoid is defective, the electromagnetic valve has a so-called fail-safe function.
Electromagnetic valves having the fail-safe function include those described in Patent Documents 1 and 2 listed below.
An electromagnetic proportional decompression valve described in Patent Document 1 is provided with a spool for causing a control pressure port to generate a pressure in accordance with communicative connection between a supply port and a tank port and also provided with a first solenoid plunger for pressing against an end face of the spool and a second solenoid plunger for pressing against an end face of the first solenoid and, when a current is shut off, the fail-safe function under abnormal conditions is fulfilled by retaining the first solenoid plunger at a predetermined position by being pressed by the second solenoid plunger with energizing force of a spring to maintain a predetermined control pressure even when a solenoid exciting current is shut off.
Also, an electromagnetic valve described in Patent Document 2 can operate a spool with energizing force of a compression spring to communicate an input port and an output port and also makes a drain port and the output port communicable by operating the spool against energizing force of the compression spring with electromagnetic force and, when the spool does not operate due to a coil defect, the output port is made controllable to open/close even when a defect appears by supplying a predetermined oil pressure to an oil pressure supply mechanism to move the spool against energizing force of the compression spring to change the valve opening state of the output port.
Patent Document 1: Japanese Patent No. 3110861
Patent Document 2: Japanese Patent Application Laid-Open No. 2004-060806


Novel phenazine derivatives of formula (I)

Angiogenesis is a highly regulated process, whereby new blood vessels form from preexisting ones (Folkman J. Angiogenesis: an organizing principle for drug discovery Nat. Rev. Drug Discov. 2007; 6: 273-86). In adult mammals, physiologic angiogenesis is largely limited to the ovaries, uterus, and placenta, with the turnover rate of vascular endothelial cells being very low in most other tissues. Pathophysiologic angiogenesis is a characteristic of wound healing and diseased states, particularly cancer, where the number of proliferating endothelial cells increases significantly and the morphology of the vasculature is altered in multiple ways (Baluk P, Hashizume H, McDonald D M. Cellular abnormalities of blood vessels as targets in cancer. Curr. Opin. Genet. Dev. 2005; 15: 102-11). For many types of cancer, as tumor cells undergo dysregulated proliferation, the tumor mass initially expands beyond the support capacity of the existing vasculature, leading to decreased levels of oxygen and nutrients and the accumulation of metabolic wastes. Tumor cells respond to this deterioration of the tumor microenvironment by up-regulating several proangiogenic factors, including vascular endothelial growth factor (VEGF)-A, basic fibroblast growth factor, placental growth factor, and platelet-derived endothelial growth factor, which collectively activate quiescent endothelial cells and promote their migration into the tumor. This shift of the tumor microenvironment to an angiogenic state, or "angiogenic switch" (Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996; 86: 353-64), is an important rate-limiting factor in tumor development. Despite the active angiogenesis induced by tumor cell-derived proangiogenic factors, structural defects associated with the tumor vasculature often lead to inefficient blood perfusion in established tumors, which contributes to tumor hypoxia. Tumor metastasis is also regulated by angiogenesis, as well as by lymphangiogenesis, where new lymphatic vessels are formed from preexisting ones (Christofori G. New signals from the invasive front. Nature 2006; 441: 444-50). Tumor cell dissemination, the first step in tumor metastasis, requires access to both blood and lymphatic circulation. Once successfully extravasated, the survival and further colonization of the disseminated tumor cells is dependent on angiogenesis at the secondary site. Angiogenesis is thus a key factor in the development and metastasis of a variety of tumor types and is an important hallmark of malignant disease. Moreover, angiogenesis presents unique opportunities for therapeutic intervention in cancer treatment, as first proposed by the late Judah Folkman more than thirty five years ago (Folkman J. Tumor angiogenesis: therapeutic implications. N Engl. J. Med. 1971; 285: 1182-6).
Today, inhibition of angiogenesis is recognized as new modality of cancer treatments. The targets of anti-angiogenic treatments are the proliferating/migrating endothelial cells into any tumor. Because this tumor-driven phenotype of endothelial cells differs from the quiescent endothelial cells lining blood vessels of healthy tissues, anti-angiogenic treatments were initially anticipated to be highly specific and thus safe drugs.
Recently, however, safety concerns were raised about anti-angiogenic drugs including anti-VEGF antibody (Avastin) and a variety of Tyrosine Kinase Inhibitors (Verheul and Pinedo., Nature Rev. Cancer, 7, 475-485 52007) (including bleeding, gastric perforation, hypertension and thrombotic events). The other biological roles played by angiogenic mediators or growth factors (such as VEGF) in maintaining the cardiovascular homeostasis are very likely to be the major cause of these life-threatening side effects.
Drugs with a higher selectivity for the tumor vasculature are therefore avidly needed.
Pathophysiologic hypoxia is actually recognized as a hallmark of most tumor types and documented to trigger angiogenesis. Even though angiogenesis aims at decreasing hypoxia, proliferating and migrating endothelial cells forming new vessels are per se exposed to low pO2. In addition, two other forms of hypoxia directly impact endothelial cells in tumors. First, cyclic hypoxia arising from variations in red blood cell flux is reported to occur within tumor microvessels, thereby exposing at least intermittently tumor endothelial cells to a hypoxic environment (Martinive et al. Mol. Cancer Ther. 2006 June; 5(6):1620-7; Baudelet C. et al. NMR Biomed. 2006 February; 19(1): 69-76; Dewhirst et al. Nature Rev. Cancer 8, 425-437 (June 2008); Dewhirst Cancer Res. 2007 Feb. 1; 67(3): 854-5). Second, longitudinal hypoxia describes the O2 gradient developing from the vessels supplying the tumor, i.e. the deeper the vessel penetrates in the tumor mass, the lesser oxygen is left available to diffuse in the surrounding tumor tissue.
As a summary, there is still a stringent need for potent tumor selective anti-angiogenic agents. Therefore a goal of the present invention is to satisfy this urgent need by identifying efficient pharmaceutically active compounds having tumor selective anti-angiogenic and/or cytotoxic activity.

Substituted 2,3-dihydro-1H-benzo[a]pyrano[2,3-c]phenazines as anti-angiogenic and anti-cancer agents

Abstract: are disclosed. In particular, substituted 2,3-dihydro-1H-benzo[a]pyrano[2,3-c]phenazines are disclosed. The compounds can be used as anti-angiogenic and/or anti-tumor agents.


An imaging sensor using a novel bit line processing circuit, that circuit, and the method of processing the pixel outputs from an image sensor using that processing circuit are disclosed. The image sensor includes an array of pixel sensors, a signal digitizing circuit, and a digitizing controller. Each pixel sensor generates a voltage signal that is a function of a charge on the photodetector in that pixel sensor, and couples that voltage signal to a bit line in response to a first signal. The signal digitizing circuit is connected to the bit line, the digitizing circuit converting the voltage signal to a plurality of output digital values, the output digital values having selectable levels of digitization noise. The digitizing controller generates the level of noise based on the voltage signal. The signal digitizing circuit includes a variable gain amplifier and an ADC having a fixed number of bits.

Low cost imaging arrays fabricated in CMOS are utilized in numerous cameras. In digital photography, the image is recorded by an imaging array in which each pixel includes a photodetector that measures the amount of light that falls on some portion of the pixel area. Imaging arrays that have a high dynamic range are required for many applications, including scientific research photography. The dynamic range of an imaging array will be defined to be the ratio of the maximum signal for a pixel to the minimum signal that is above the noise.
Typically, the imaging arrays are constructed from a two-dimensional array of pixel sensors organized as a plurality of rows and columns of pixel sensors. All of the pixel sensors in a given column are connected to a common signal line. The specific pixel in a column that is currently being readout is determined by a row select signal that connects all of the pixel sensors in a given row to their respective signal lines.
To improve the readout time of the array, each signal line has a dedicated signal amplifier and readout circuit so that the entire row of pixel sensors can be readout in parallel. The readout amplifier and associated analog-to-digital converters (ADCs) must have a dynamic range that is commensurate with the dynamic range of the individual pixels and must provide a readout time for each pixel that is as short as possible, particularly in low light images, as the noise in each pixel value increases with the time between the end of an exposure and the time the pixel is readout. To increase the dynamic range of the readout circuit, schemes in which multiple readout amplifiers are used on each signal line have been proposed. For example, U.S. patent application Ser. No. 12/036,998 filed Feb. 25, 2008 describes a readout system in which digitization circuits having different gains are implemented for each readout line, the choice of digitization gain being determined by the signal on the readout line in question.
While multiple readout digitization circuits can provide improved dynamic range, the improvement comes with a significant cost, since the number of transistors that must be included in the processing circuitry for each signal line is substantially increased. Since there can be thousands of columns of pixels, hence, signal lines, each additional transistor represents a significant increase in the area of silicon required to implement the readout array, and hence, the cost of the image sensor.

Variable gain column amplifier adapted for use in imaging arrays


A suction strainer includes a hollow internal core tube and an external filtering surface built around the internal core tube. A plurality of openings are defined through the side wall of the internal core tube core. In some cases the openings through the side wall are constructed and arranged such that there is somewhat less open area near the downstream end than the upstream end of the internal core tube, and the amount of open area tapers between the upstream end and the downstream end. As a result, when liquid is drawn into the internal core tube through the plurality of openings, a substantially uniform inflow distribution may be defined along substantially the entire length of the internal core tube. The internal core tube functions as a rigid structural support for the external filtering surface, enabling the apparatus to withstand post-LOCA hydrodynamic forces. The size of the filtering surface is enlarged by virtue of the fact that the filtering surface defines a plurality of filtering disk assemblies that are connected to and extend radially from the internal core tube. A separation distance is defined between neighboring disk assemblies, and filtering inner walls connect between neighboring disk assemblies and extend around the internal core tube at a radius less than the outermost radius of the disk assemblies. The total flow surface area is increased within a limited geometric profile. This serves to maximize surface area while minimizing post-LOCA reactive forces on attachment ECCS piping in a BWR suppression pool.

The present invention relates generally to the field of suction strainers, and more particularly to the field of suction strainers employed in the suppression pools of boiling water reactor (BWR) nuclear power plants.
A suction strainer employed in a suppression pool removes solids from a flow of liquid (e.g., water) being drawn into an emergency core cooling system (ECCS) pump. The flow of water is drawn through the suction strainer and then into the suction line of the ECCS pump. Employment of suction strainers is desirable because solid debris drawn into the suction line of a pump can degrade pump performance by accumulating in the pump or its suction or discharge lines, or by impinging upon and damaging internal pump components.
While almost any pump degradation can be characterized as being costly, the degradation of ECCS pump performance at BWR nuclear plants can be detrimental to safe plant shutdown following a loss of coolant accident (LOCA). At a BWR nuclear power plant following a LOCA, it is critical for the ECCS pumps to operate for an extended period of time in an undegraded fashion. In one mode of operation, the ECCS pumps are operated to recirculate water from the suppression pool back to the reactor core for the purpose of core cooling. A LOCA results from a high pressure pipe rupturing with such great force that large quantities of debris, such as pipe and vessel insulating material, and other solids, may be washed into the suppression pool. Conventional ECCS suction strainers currently installed in BWR plants would have a tendency to become clogged by such debris due to their small size and poor design. Also, when the large pressure pipes rupture with great force, suction strainers in the suppression pool are subjected to great hydrodynamic forces that can damage the suction strainers as well as subject the attachment recirculation piping to large reactive forces. These structural considerations, and space constraints, limit the size and shape of suction strainers in suppression pools.
Conventional BWR plant suction strainers are typically constructed and arranged in a manner such that, under full flow conditions, localized high entrance velocities are established through that portion of the suction strainer that is most proximate to the suction line of the pump, while low entrance velocities are established through that portion of the suction strainer that is more distant from the suction line of the pump. The high entrance velocities may draw more solid debris into contact with the suction strainer causing the portions of the suction strainer experiencing the high entrance velocities to experience higher head loss. As the portion of the suction strainer most proximate to the suction line collects debris, high entrance velocities are established at the portion of the suction strainer that is next closest to the suction line causing that portion to collect debris. This process often continues until the entire suction strainer has collected debris in varying quantities, resulting in a non-uniform build-up of debris on the outer surface of the strainer.
Localized high entrance velocities can be detrimental even when solids are not present in the liquid being pumped. For example, high entrance velocities can result in turbulent flow which tends to create greater pressure losses than laminar flow. Any such pressure losses reduce the net positive suction head (NPSH) available to a pump. As the NPSH available decreases, pump cavitation may occur. Similarly, localized high entrance velocities can cause vortexing. When a suction strainer is not sufficiently submerged, the vortexing can cause air ingestion which can severely degrade pump performance.
Attempts have been made to resolve certain of the problems associated with suction strainer-like devices in other applications. For example, cylindrical suction flow control pipes have been encircled with screen material and employed in water wells. Such wells typically employ a well pump above the ground surface and a riser pipe extending from the well pump to the water table. The suction flow control pipe is connected to the end of the riser pipe and extends further below the water table. Openings are defined through the side wall of the suction flow control pipe such that there is somewhat less open area near the riser pipe and somewhat more open area distant from the riser pipe. As a result, when water is drawn into the flow control pipe through the openings, a substantially uniform inflow distribution is defined along the length of the flow control pipe. While such suction flow control pipes offer some advantages, they are not suitable for all applications.
Attempts have been made, totally separate from flow control pipes, to increase filtering surface areas of BWR ECCS suction strainers in an effort to decrease pressure losses and thereby prevent pump cavitation. For example, such suction strainers may include a plurality of spaced, coaxial, stacked filtering disks. More particularly, such stacked disk suction strainers typically include an annular flange for attachment to the corresponding flange on the pump suction line. The stacked disk suction strainer provides an enhanced surface area and defines a longitudinal axis that is encircled by the attachment flange. A first disk is attached to the attachment flange. The first disk includes a pair of a radially extending, circular, disk walls, each of which encircle the longitudinal axis, and define a central hole. A first disk wall of the pair of disk walls is connected to the attachment flange. The first and second disk wall of the pair of disk walls face one another and are separated by a slight longitudinal distance. The first disk further includes an outer annular wall that encircles the longitudinal axis. The outer annular wall includes an annular first edge and an annular second edge. The entirety of the annular first edge of the outer annular wall is connected to the entire peripheral edge of the first perforated disk wall; and the entirety of the annular second edge of the outer annular wall is connected to the entire peripheral edge of the second perforated disk wall such that the pair of disk walls are connected at their periphery.
The stacked disk suction strainer further includes a plurality of inner annular walls that encircle the longitudinal axis, each of which includes an annular first edge and an annular second edge. The annular first edge of one of the inner annular walls is connected around the periphery of the central hole of the second disk wall. The annular second edge of that inner annular wall is connected around the periphery of the central hole of a disk wall of a second disk. The first and second disk walls, and the outer and inner annular walls are perforated and comprise the filtering surface of the stacked disk suction strainer. Additional perforated disks and inner annular walls are attached to one another in the above manner until the last disk is attached, wherein the outer disk wall of the last disk does not include a central hole. The stacked disk suction strainers may incorporate separate structural members to maintain the structural integrity of the stacked disk suction strainer. However, the conventional stacked disk suction strainers do not incorporate an internal core tube and related components, whereby the conventional stacked disk suction strainers are difficult to structurally reinforce and are susceptible to vortexing and the detrimental non-uniform localized entrance velocities discussed above.
There is, therefore, a need in the industry for an improved suction strainer.
Briefly described, the preferred embodiments of the of the present invention include a suction strainer that includes a filtering device with a strategically enlarged filtering surface and an internal core. The internal core is preferably in the form of an internal core tube, which is preferably an internal pipe with flow openings. In accordance with the preferred embodiments of the present invention, the internal core tube structurally reinforces the filtering device.
In accordance with the preferred embodiments of the present invention, the structural reinforcement provided by the internal core tube is enhanced by reinforcing structural members that extend radially from the internal core tube. The reinforcing structural members are preferably connected to and extend radially from and angularly around the internal core tube to structurally support the filtering surfaces of the external filtering structure. The internal core tube, in conjunction with the structural members, seeks to prevent air ingestion and vortexing. The suction strainer preferably extends away from the suction line of an ECCS pump to define a length, and in accordance with certain examples the preferred embodiments of the present invention, the internal core tube seeks to promote controlled inflow along the length to preclude the establishment of non-uniform localized entrance velocities through the filtering surface. In accordance with other examples of the preferred embodiments of the present invention, the internal core tube is not constructed to specifically promote such a uniform inflow along the length.
In accordance with the preferred embodiments of the present invention, the suction strainer is constructed in a manner that seeks to enlarge the filtering surface while minimizing the projected area of the suction strainer. The minimization of the projected area as well as structural reinforcement of the suction strainer enables the suction strainer to withstand high levels of hydrodynamic impact loading following a LOCA. The suction strainer also serves to minimize the bending moment and other reactive forces on the attachment ECCS piping in the BWR suppression pool.
In accordance with the preferred embodiments of the present invention, the filtering surface is defined by an external filtering structure that is attached to, extends from, and is built around the internal core tube and the reinforcing structural members. When the suction strainer is connected to the suction of a pump and submerged, a liquid flow path is established through the internal core tube and external filtering structure. The liquid originates exterior to the external filtering structure and is drawn through the filtering surfaces of the external filtering structure. The filtering surfaces separate solids from the liquid. The size of the filtering surface is enlarged by virtue of the fact that the filtering surface defines protrusions such that the distance that the filtering surface extends from the internal core tube alternates. The resulting enlarged filtering surface seeks to decrease average flow velocities through the filtering surface and thereby spread the collected solid debris in thinner layers, thereby decreasing overall pressure losses associated with the suction strainer. Once the liquid flows through the filtering surface, the liquid is drawn through the internal core tube and into the suction of the pump.
In accordance with the preferred embodiments of the present invention, the protrusions of the external filtering structure are in the form of a plurality of filtering plate assemblies that are connected to and extend radially from the internal core tube. Each plate assembly includes a pair of plate walls that face one another, define a distance therebetween, and are connected at their peripheries by an outer wall that surrounds the internal core tube. A separation distance is defined between neighboring plate assemblies. Inner walls connect between neighboring plate assemblies and extend around the internal core tube at a radius less than the radius of the outer walls. The outer and inner walls as well as the plate walls are perforated and comprise the filtering surfaces of the suction strainer. In accordance with first and second preferred embodiments of the present invention, the plurality of plate assemblies are preferably in the form of stacked disks that are spaced to defined troughs therebetween. In accordance with other embodiments, the plate assemblies are in other forms that increase the surface area of the suction strainer.
In accordance with preferred embodiments of the present invention, the internal core tube has a downstream end for connection to the pump suction flange and an upstream end distant from the downstream end. The internal core tube defines a longitudinal axis extending between the upstream and downstream ends. In accordance with the preferred embodiments of the present invention, a plurality of openings are defined through the side wall of the internal core tube. In accordance with certain examples of the preferred embodiments, the openings are constructed and arranged such that there is somewhat less open area near the downstream end than the upstream end, and the amount of open area tapers between the upstream end and the downstream end. As a result, when water flows into the internal core tube through the openings, a substantially uniform flow rate distribution is defined along substantially the entire length of the internal core tube.
It is therefore an object of the present invention to provide an improved BWR ECCS suction strainer.
Another object of the present invention is to increase safety by improving the operability of the ECCS of a BWR nuclear plant following a LOCA.
Yet another object of the present invention is to structurally reinforce a suction strainer sufficiently so that it can withstand the hydrodynamic forces following a LOCA in the suppression pool at a BWR nuclear plant.
Still another object of the present invention is to minimize reactive forces on the attachment ECCS piping following a LOCA.
Still another object of the present invention is to maximize the total strainer surface area within a limited geometric profile while providing a maximum strength strainer.
Still another object of the present invention is to simultaneously minimize both the thickness of collected debris on the strainer and the average entrance velocities to minimize the resultant NPSH of the ECCS following a LOCA.
Still another object of the present invention is to maximize the amount of time required to reach a particular head loss across the strainer.
Still another object of the present invention is to control the distribution of fluid flow over the strainer so as to collect debris uniformly, from disk to disk or from trough to trough, to allow scaling of the strainer for other flow rates with similar, but different size, strainers with different water flow rates.
Still another object of the present invention is to prevent vortexing and air ingestion.

Suction strainer with an internal core tube


This water-in-oil emulsion-type stick-shaped deodorant composition contains: (A) a volatile component having a boiling point of 250° C. or less, where the total amount of water and ethanol in the (A) component is 83-100 mass %; (B) component (B-1), component (B-2), or a combination of components (B-1) and (B-2), wherein component (B-1) is an antiperspirant and component (B-2) is a fungicide; (C) a crosslinked silicone surfactant; (D) a non-crosslinked silicone surfactant; and (E) an oily component which has a solid phase at 25° C.

Deodorant agents are compositions intended to prevent body odor. It is known that body odor is produced by the bacterial interaction on the body surface with sweat and sebum secreted from the human body. Therefore, the known means for preventing body odor include, for example, a method of using bactericides or fungicides to control bacteria on the body surface, a method of using antiperspirants such as metal oxides to control secretion of sweat, a method of using antioxidants such as plant extracts to control oxidation of sebum, a method of using basic substances such as zinc oxide to form complexes with lower fatty acids which can cause body odor, to control body odor, a method of using fragrant substances to mask body odor, and the like. See Non-Patent Document 1.
Since deodorant agents are typically applied to profusely sweating parts and frequently used in heavily sweating season, it is often required that the deodorant product gives a dry feeling when applied to the skin. Typical of the means for developing a dry feeling is blending of silicone having a high volatility rate. As the general-purpose highly volatile substance, octamethylcyclotetrasiloxane, decamethylcyclopentasiloxane and the like are selected. For the purpose of controlling the volatility rate, Patent Document 1 (WO 2010/080482) discloses a cosmetic composition comprising a blend of at least two cycloalkylmethicones having different volatility rates. The technology describes one exemplary non-aqueous solid antiperspirant. When it is intended to develop a dry feeling by blending a large amount of a highly volatile silicone as in Example, a strong dry feeling may be perceived when the deodorant agent is applied. When the deodorant agent is applied to the skin, quick volatilization of oily substances can occur so that the oil composition of the deodorant agent on the skin may change rapidly, that is, the relative ratio of non-volatile oil component may increase to invite a quick viscosity buildup, eventually causing an uncomfortable feeling like sticky feeling.
On the other hand, Patent Document 2: JP 6058201 proposes an emulsion type deodorant stick. The technology describes an emulsion type deodorant stick wherein purified water, distilled water, ion exchange water, hot spring water or deep ocean water is blended as the water component, preferably in an amount of up to 25% by weight based on the total weight of the deodorant. In all examples, decamethylcyclopentasiloxane is selected in order to develop a dry feeling.

Water-in-oil emulsion-type stick-shaped deodorant composition


Systems and methods are provided for managing resources. In one implementation, a method is provided in which a management server determines whether a condition related to one or more resources has occurred. The management server further determines at least one program instance to terminate. The at least one program instance executes on one of a plurality of servers. The management server further terminates the determined at least one program instance, which was used by an excess program execution capacity user.

Computer infrastructure services providers supply dynamically scalable and abstracted resources as services over the Internet, as well as maintain computers on which users can run applications and store data. Computer infrastructure services providers locate the computers that comprise such infrastructures in one or more facilities that are commonly referred to as data centers. A small data center may occupy a room or a floor of a building, while a large data center may occupy several floors or an entire building. A typical data center may house many computers, such as servers, along with other components (e.g., backup power generators, un interruptible power supplies, power distribution units, etc.) that support the resources of the data center. Data centers also are built in non-traditional ways, such as within shipping containers or on boats.
Power is a commodity that is essential to running a data center. Power also is the most constrained resource that is used in a data center. The power consumption of components within a data center varies with the utilization of the components. For example, as a server becomes more heavily loaded, it consumes more power. To avoid unexpected failures, it is thus necessary to provision sufficient power to drive every component of a data center, such as servers, at full utilization. However, because all components of a data center rarely operate at full utilization at all times, a data center typically will significantly over-provision power.
Over-provisioning power that is not used efficiently results in a cost increase for all resources within a data center. For example, a server that remains idle or that experiences a small load will nevertheless require a significant power budget in case its load suddenly increases. Increasing utilization of the power delivered to a data center can significantly reduce the cost of operating the data center.
While it is possible to operate individual resources in a data center at levels that are near or over capacity, a sudden utilization increase of some resources may drive overall power consumption beyond the data center's available supply. As a result, the data center may need to shut down certain resources or some resources may experience sudden failures. Resources that are shut down or fail unexpectedly prevent the computer infrastructure services provider from providing services to customers and can cause data loss or corruption. As a result of these considerations, on the one hand, a computer infrastructure services provider who is trying to maximize power utilization may oversubscribe power of resources to minimize costs, but risks having resources shut down when power consumption exceeds supply. On the other hand, the computer infrastructure services provider instead may over provision power, which increases power costs. The problem is further complicated by the fact that external environmental factors, such as an increase in temperature, may cause an increase in power consumption, such as by requiring additional cooling capacity to be brought online. Therefore, there is a need for improved systems and methods that provide power management for facilities, such as data centers.

This patent is subject to a terminal disclaimer.

Managing power consumption in a data center


A liquid crystalline polyester resin composition is provided, by which a molded article with an enhanced specific modulus can be produced. The liquid crystalline polyester resin composition comprises mica fillers in an amount of 15 to 30 parts by mass and carbon fibers with specific volume resistance of 108 Ωcm or more in an amount of 5 to 20 parts by mass with respect to 100 parts by mass of the liquid crystalline polyester.

1. Field of the Invention
The present invention relates to a liquid crystalline polyester resin composition which is suitable for being applied to optical pickup members such as a holder for optical pickup lenses, a base frame and the like, a molded article and a holder for optical pickup lenses.
2. Description of the Related Art
Hitherto, in response to a demand for a reduction in the weight of the optical pickup member of this kind, attempts to switch a constituent material of the pickup member from a metal material to a resin material were made. Particularly, since a liquid crystalline polyester resin composition containing glass fibers mixed is superior in mechanical properties, moldability, dimensional accuracy, heat resistance and a damping property among resin material, it is adopted as optical pickup members.
However, the demand for a damping property becomes severe with increases in capacity and speed of information which recent digital disk drive systems handle. As the countermeasure for this, it is required that a secondary resonance frequency of the optical pickup member is shifted to a high frequency side and a width of gain is enlarged, namely, that the rigidity of the optical pickup member is increased (resonance frequency is proportional to the half power of specific modulus; increases in a MD direction and a TD direction), it becomes clear that the conventional liquid crystalline polyester resin composition cannot response to this demand.
As the liquid crystalline polyester resin compositions which are suitable for these optical pickup members, a resin composition composed of liquid crystalline polyester and aluminum borate whiskers is disclosed (for example, JP-A-11-80517) and a resin composition composed of liquid crystalline polyester, glass fibers and spherical hollow bodies, which is aimed at reducing the weight, is disclosed (for example, JP-A-2004-143270).

Liquid crystalline polyester resin composition, molded article and holder for optical pickup lenses