text
stringlengths 0
1.67M
|
|---|
<SOH> SUMMARY OF THE INVENTION <EOH>Methods of enhancing the solubility of a fluorinated compound in an organic solvent are provided. In one embodiment, the solvent is non-halogenated. In one embodiment, the method comprises applying carbon dioxide gas to the solvent at a pressure effective to enhance the solubility of the fluorinated compound. The pressure of the carbon dioxide gas is, for example, in the range of 30-300 bar, or in the range of 40-90 bar. The organic solvent is or comprises, for example, pentane, hexane, heptane, octane, nonane, decane, cyclopentane, cyclohexane, dioxane, benzene, toluene, xylene, ether, diisopropylether, ethyl acetate, tetrahydrofuran, triethylamine, tripropylamine, N,N,N′,N′-tetramethylethylenediamine, methylene chloride, chloroform, chlorobenzene, acetone, nitrobenzene, acetonitrile, formamide, acetamide, dimethylformamide, dimethylacetamide, nitromethane, methanol, ethanol, propanol, butanol, isopropanol, sec-butanol, tert-butanol, ethylene carbonate, propylene carbonate, glyme, diglyme, dimethylsulfone, or dimethylsulfoxide, or mixtures thereof The method may further include recrystallizing the fluorinated compound by reducing the pressure of the carbon dioxide gas, for example over 24 hours or less, or over 4 hours or less. The fluorinated compound is for example a catalyst, such as an organic compound or an organometallic complex of a main group metal, main group semimetal, transition metal, actinide or lanthanide. The catalyst can include one or more highly fluorinated ligands or groups. Also provided are methods of recrystallizing a fluorinated compound in an organic solvent, such as a non-halogenated organic solvent, comprising: applying carbon dioxide gas to the solvent at a pressure effective to enhance the solubility of the fluorinated compound; and recrystallizing the fluorinated compound by reducing the pressure of the carbon dioxide gas, wherein the method further comprises providing a plurality of individual sample containers, each container comprising a fluorinated compound in the solvent, wherein carbon dioxide gas is applied to the solvent in the individual sample containers at a pressure effective to enhance the solubility of the fluorinated compound; and wherein the fluorinated compounds are crystallized in the individual sample containers by reducing the pressure of the carbon dioxide gas. The components of the individual samples can be varied. For example, the concentration or compound(s) or solvent(s) can be varied. The method may further include screening the recrystallized fluorinated compounds for crystallinity or purity. In another embodiment, there is provided a method of conducting a reaction using a fluorinated compound in an organic solvent, the method comprising applying carbon dioxide gas to the organic solvent at a pressure effective to solubilize the fluorinated compound during the reaction. In one embodiment, the solvent is non-halogenated. The pressure of the carbon dioxide gas is for example in the range of 40 to 90 bar. The fluorinated compound is for example a catalyst. Optionally, a plurality of reactions are conducted, wherein, for example, different reaction conditions, such as reagents, concentration, solvent, or pressure are varied. Methods of conducting a reaction in one embodiment comprise applying carbon dioxide pressure to an organic solvent comprising at least one substrate and a fluorinated catalyst, in an effective amount to solubilize the catalyst; and permitting the fluorinated catalyst to catalyze the reaction of the substrate to form a product. The carbon dioxide can be applied to the solvent at a pressure for example in the range of 40 to 90 bar. The method may include reducing the carbon dioxide gaseous pressure, thereby to cause precipitation of the catalyst, and optionally recovering the catalyst from the reaction product mixture. Examples of reactions include hydrogenation, hydroboration, hydroformylation, cyclopropanation, C—H insertion reactions, oxidation, hydroxylation, isomerization, coupling reactions, olefin metathesis, polymerization, hydrosilylation, hydrocyanation, epoxidation, and Diels-Alder reactions. The catalyst is, for example, an organic compound or an organometallic complex of a main group metal, main group semimetal, transition metal, actinide or lanthanide. In one embodiment, the solvent is non-halogenated. In one embodiment, a fluorous support or polymer is present in order to help trap the catalyst upon release of the CO 2 pressure. In one embodiment a method of conducting a reaction using a fluorinated compound in an organic solvent is provided, comprising applying carbon dioxide gas to an organic solvent, such as a non-halogenated organic solvent, comprising a fluorinated compound, at a pressure effective to solubilize the fluorinated compound during the reaction; permitting the catalyst to catalyze the reaction of the substrate to form a product; and reducing the carbon dioxide gaseous pressure, thereby to cause precipitation of the catalyst; wherein the method further comprises including a fluorinated support material in the organic solvent, wherein the fluorinated support material is capable of adsorbing the catalyst when the carbon dioxide pressure is reduced. The fluorinated support material is for example a fluorinated polymer, or an inorganic or organic support material comprising fluorinated organic groups that may be attached to the inorganic or organic support material. The method may comprise, for example, applying carbon dioxide pressure to a non-halogenated organic solvent comprising at least one substrate and a fluorinated organometallic catalyst, in an effective amount to solubilize the catalyst; providing a fluorinated support material in the solvent; permitting the catalyst to catalyze the reaction of the substrate to form a product; and reducing the carbon dioxide gaseous pressure, thereby to permit precipitation of the catalyst and adsorption of the fluorinated catalyst on the support material. The support material having the fluorinated catalyst thereon optionally may be separated from the solvent, and optionally reused to catalyze one or more additional reactions, thus enabling efficient recovery and recycling of the catalyst. The fluorinated catalyst may be adsorbed onto the support material prior to application of the carbon dioxide pressure, and released from the support material after application of the carbon dioxide pressure. The method may include comprises conducting a plurality of the reactions, wherein individual reactions vary in reaction conditions. The plurality of reactions may be done in plural different reaction containers, wherein individual reaction containers comprise an organic solvent, at least one reaction substrate, and a fluorinated organometallic catalyst, and wherein the method comprises: applying carbon dioxide pressure to the reaction containers in an effective amount to solubilize the catalyst; permitting the catalyst to catalyze the reaction of the substrate to form a product in the reaction containers; and reducing the carbon dioxide gaseous pressure, thereby to cause precipitation of the catalyst, and wherein optionally a fluorinated support material is provided in the solvent. The reaction containers may be provided within one or more sealable reaction vessels. The amount or identity of at least one of the organic solvent, the reaction substrate, or the catalyst may vary in the different reaction containers. The method may further include screening the reaction for a reaction product in the individual containers. |
Inspection reservation system |
An inspection reservation system used for reservation of inspection maintenance of a vehicle includes a first database for storing step count information indicating the number of steps required for inspection and maintenance of a vehicle, a second database for storing skill information containing a plurality of skill values indicating skills of maintenance staffs, and a processing apparatus. The processing apparatus calculates a total number of steps required for inspection and maintenance of a vehicle related to a reservation imputed to the system in accordance with the step count information. Furthermore, in accordance with the total number of steps and the skill information, the processing apparatus selects at least one maintenance staff who can perform the inspection and maintenance of the vehicle related to the reservation within a desired period of time. |
1. A check reserving system comprising: a first database for storing man-hour information representative of man-hours required for checking and maintaining vehicles; a second database for storing a skill information including a plurality of skill values respectively representative of skills of a plurality of mechanics; and a processor for calculating a total man-hour required for a check and a maintenance of a vehicle with respect to a reservation on the basis of said man-hour information, and selecting at least one mechanic who can carry out said check and said maintenance of said vehicle with regard to said reservation so as to satisfy a desired delivery date on the basis of said total man-hour and said skill information. 2. A check reserving system according to claim 1, wherein said first database further stores as a necessary skill value a skill value required to carry out said check item for each of check items included in said check and said maintenance of the vehicle, and said processor selects said at least one mechanic, from mechanics having a skill value equal to or higher than said necessary skill value defined for said check items included in said check and said maintenance of said vehicle with respect to said reservation. 3. A check reserving system according to claim 1, wherein said first database further stores place information representative of work places required for checking and maintaining vehicles, and said processor retrieves to reserve a work place required for said check and said maintenance of said vehicle with respect to said reservation so as to satisfy said desired delivery date of said vehicle with respect to said reservation, on the basis of said place information stored in said first database. 4. A check reserving system according to claim 1, wherein said first database further stores expense information representative of a check and maintenance expense of vehicles, and said processor further calculates and outputs an expense required for said check and said maintenance of said vehicle with respect to said reservation, on the basis of said expense information stored in said first database. 5. A check reserving system according to claim 1, wherein said first database further stores as a necessary skill value a skill value required to carry out said check for each of check items included in said check and said maintenance of the vehicle, wherein said processor selects said at least one mechanic, from mechanics having a skill value equal to or higher than said necessary skill value defined for said check items included in said check and said maintenance of said vehicle with respect to said reservation, wherein said first database further stores place information representative of work places required for checking and maintaining vehicles, and wherein said processor retrieves to reserve a work place required for said check and said maintenance of said vehicle with respect to said reservation so as to satisfy said desired delivery date of said vehicle with respect to said reservation, on the basis of said place information stored in said first database. 6. A check reserving system according to claim 5, wherein said first database further stores expense information representative of a check and maintenance expense of vehicles, and wherein said processor further calculates and outputs an expense required for said check and said maintenance of said vehicle with respect to said reservation, on the basis of said expense information stored in said first database. 7. A check reserving system according to claim 1, wherein said first database further stores as a necessary skill value a skill value required to carry out said check for each of check items included in said check and said maintenance of the vehicle, wherein said processor selects said at least one mechanic, from mechanics having a skill value equal to or higher than said necessary skill value defined for said check items included in said check and said maintenance of said vehicle with respect to said reservation, wherein said first database further stores expense information representative of a check and maintenance expense of vehicles, and wherein said processor further calculates and outputs an expense required for said check and said maintenance of said vehicle with respect to said reservation, on the basis of said expense information stored in said first database. 8. A check reserving system according to claim 1, wherein said first database further stores place information representative of work places required for checking and maintaining vehicles, wherein said processor retrieves to reserve a work place required for said check and said maintenance of said vehicle with respect to said reservation so as to satisfy said desired delivery date of said vehicle with respect to said reservation, on the basis of said place information stored in said first database, wherein said first database further stores expense information representative of a check and maintenance expense of vehicles, and wherein said processor further calculates and outputs an expense required for said check and said maintenance of said vehicle with respect to said reservation, on the basis of said expense information stored in said first database. 9. A check reserving system according to claim 1, further including a terminal through which the reservation may be inputted to the system. |
<SOH> BACKGROUND ART <EOH>Vehicle maintenance workshops provide vehicle check services of checking vehicles in response to requests from clients. When receiving a request of a check from a client, a manager of a maintenance shop for providing vehicle check services is required to determine a date for the check of the vehicle considering the maintenance schedule of the maintenance shop. Typically, the manager of the maintenance workshop determines the date for the check of the vehicle as follows. At first, the manager adds up respective man-hours of a plurality of check items which are defined for each kind of the check, and calculates the total man-hour. Then, the manager looks for the dates when the check requested by the client can be executed from the calculated total man-hour and the maintenance schedule of the maintenance workshop, and selects the optimal date from the dates. The check of the vehicle is executed on the selected date. A storage management system for automatically preparing a schedule of a maintenance workshop in response to a request of a check from a client is disclosed in Japanese Laid Open Patent Application (Jp-A 2000-20581). The storage management system includes a terminal for operation by the client and a host computer. When the client enters a content of the maintenance and a desired storage condition from the terminal, the host computer calculates a period necessary for the maintenance on the basis of the maintenance content, and further detects a maintainable empty time based on the desired condition by the client, on the basis of a storage/delivery management table stored in a memory of the host computer. The optimal storage date in the period is automatically determined and displayed on the terminal. In accordance with the input of the determination of the client from the terminal, the optimal storage date and a delivery date determined from the storage date is automatically registered in the storage/delivery table to achieve a storage reservation. Consequently, the registration of the storage can be achieved by the client by himself. Because of the background of social situation in recent years, the temporal request from the client with regard to the check of the vehicle has been further severe. For example, more and more clients request the check to be completed in a short time, such as half an hour and 1 hour during commuting. For this reason, the realization of the check reserving system is desired which can satisfy the temporal request of the client as much as possible. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG. 1 is a block diagram showing a configuration of a check reserving system in an embodiment according to the present invention. FIG. 2 is a flowchart describing an operation of the check reserving system in the embodiment according to the present invention. detailed-description description="Detailed Description" end="lead"? |
Purification of lineage-specific cells and uses therefor |
A method for developing a population of substantially lineage-specific cells and their use inter alia in tissue replacement therapy, tissue augmentation therapy, diagnostic applications, for the identification of growth factors and other autocrine factors. Specifically, substantially homogeneous populations of mammalian cells of the astrocyte lineage are provided and selected on the basis of differential marker expression. |
1. A method for generating a substantially homogeneous population of lineage-specific cells from tissue of the central nervous system (CNS) of mammalian animals, said method comprising subjecting said CNS tissue to tissue disruptive means to provide a mixed population of cells comprising the lineage-specific cells to be isolated, subjecting the mixed population to cell separation discrimination means to generate a substantially homogeneous population of lineage-specific cells. 2. The method of claim 1 wherein the lineage-specific cells are astrocyte precursor cells (APCs). 3. The method of claim 1 wherein the lineage-specific cells are immature perinatal astrocytes (IPAs). 4. The method of claim 1 wherein the lineage-specific cells are mature perinatal astrocytes (MPAs). 5. The method of claim 1 wherein the lineage-specific cells are adult or aged astrocytes. 6. The method of claim 1 wherein the mammalian animal is at a prenatal stage. 7. The method of claim 1 wherein the mammalian animal is at a postnatal stage. 8. The method of claim 7 wherein the postnatal animal is an adult. 9. The method of claim 1 wherein the separation discrimination means is based on a different range of cell markers present at different developmental stages of the lineage-specific cells. 10. The method of claim 9 wherein the separation discrimination means is based on positive or negative selection of cell surface markers. 11. The method of claim 10 wherein the neuronal cells are removed by negative selection with N-CAM. 12. The method of claim 10 or 11 wherein glial cells are selected positively using one or more of A2B5, GD3, 3CB2, FGR3, PDGFRα or a combination thereof. 13. The method of claim 10 or 11 or 12 wherein oligodendrocytes are removed by negative selection with one or more of Gal-C, 01, 04, anti-Mog or NG2 or a combination thereof. 14. The method of claim 9 wherein the cells are sorted by recognition of cell markers by immunological reagents. 15. The method of claim 10 wherein the immunoglobulin reagents are antibodies. 16. The method of claim 2 wherein the cells isolated are immunohistochemically Pax2+ GFAP−. 17. The method of claim 2 wherein the cells isolated are immunohistochemically Pax2+ GFAP+. 18. The method of any one of claims 1 to 17 wherein the mammalian animal is a human. 19. The method of any one of claims 1 to 17 wherein the mammalian animal is a livestock animal, laboratory test animal or a companion animal. 20. A substantially homogeneous population of mammalian lineage-specific cells from the CNS, said cells made by the method comprising subjecting said CNS tissue to tissue disruptive means to provide a mixed population of cells comprising the lineage-specific cells to be isolated, subjecting the mixed population to cell separation discrimination means to generate a substantially homogeneous population of lineage-specific cells. 21. The population of mammalian cells of claim 20 wherein the lineage-specific cells are astrocyte precursor cells (APCs). 22. The method of claim 20 wherein the lineage-specific cells are immature perinatal astrocytes (IPAs). 23. The method of claim 20 wherein the lineage-specific cells are mature perinatal astrocytes (MPAs). 24. The method of claim 20 wherein the lineage-specific cells are adult or agedd astrocytes. 25. The method of claim 20 wherein the mammalian animal is at a prenatal stage. 26. The method of claim 20 wherein the mammalian animal is at a postnatal stage. 27. The method of claim 26 wherein the postnatal animal is an adult or aged astrocyte. 28. The method of claim 20 wherein the separation discrimination means is based on a different range of cell markers present at different developmental stages of the lineage-specific cells. 29. The method of claim 28 wherein the separation discrimination means is based on positive or negative selection of cell surface markers. 30. The method of claim 29 wherein the neuronal cells are removed by negative selection with N-CAM. 31. The method of claim 28 or 29 or 30 wherein glial cells are selected positively using one or more of A2B5, GD3, 3CB2, FGR3, PDGFRα or a combination thereof. 32. The method of claim 28 or 29 or 30 or 31 wherein oligodendrocytes are removed by negative selection with one or more of Gal-C, 01, 04, anti-Mog or NG2 or a combination thereof. 33. The method of claim 28 wherein the cells are sorted by recognition of cell markers by immunological reagents. 34. The method of claim 33 wherein the immunoglobulin reagents are antibodies. 35. The method of claim 20 wherein APCs are isolated by the immunological separation of a population of Pax2+ cells followed by removal of GFAP+ cells form the Pax2+ population. 36. The method of claim 20 wherein IPAs are isolated by the immunological separation of a population of vimentin+ cells and then isolating GFAP+ cells from said vimentin+ population. 37. The method of any one of claims 20 to 36 wherein the mammalian animal is a human. 38. The method of any one of claims 20 to 36 wherein the mammalian animal is a livestock animal, laboratory test animal or a companion animal. 39. A substantially homogeneous population of APCs or IPAs from the CNS such as brain including retina tissue prepared by the method comprising subjecting said CNS tissue or part thereof to tissue disruptive means and contacting said immobilized tissue with interactive molecules to a cell marker selectively present or absent on or in said APCs or IPAs to generate a population comprising at least APCs and/or EPAs and then contacting the isolated cells with an interactive molecule to at least one other cell marker specific for either said APCs or IPAs or specific for a cell marker absent from either APCs or IPAs to selectively remove or retain the desired cell type. 40. A method of cell replacement therapy in a mammalian animal, said method comprising generating a substantially homogeneous population of lineage-specific cells and introducing same into an organ or tissue requiring cells to be replaced or to another location from where the cells can migrate to an organ or tissue requiring cells wherein the introduced cells are subject to expansion or proliferation in vitro and/or in vivo by one or more growth factors. 41. The method of claim 40 wherein the lineage-specific cells are astrocyte precursor cells (APCs). 42. The method of claim 40 wherein the lineage-specific cells are immature perinatal astrocytes (IPAs). 43. The method of claim 40 wherein the lineage-specific cells are mature perinatal astrocytes (MPAs). 44. The method of claim 40 wherein the lineage-specific cells are adult astrocytes. 45. The method of claim 40 wherein the mammalian animal is at a prenatal stage. 46. The method of claim 40 wherein the mammalian animal is at a postnatal stage. 47. The method of claim 46 wherein the postnatal animal is an adult. 48. The method of claim 40 wherein the separation discrimination means is based on a different range of cell markers present at different developmental stages of the lineage-specific cells. 49. The method of claim 48 wherein the separation discrimination means is based on positive or negative selection of cell surface markers. 50. The method of claim 48 or 49 wherein the neuronal cells are removed by negative selection with N-CAM. 51. The method of claim 48 or 49 or 50 wherein glial cells are selected positively using one or more of A2B5, GD3, 3CB2, FGFR3, PDGFRα or a combination thereof. 52. The method of claim 48 or 49 or 50 or 51 wherein oligodendrocytes are removed by negative selection with one or more of Gal-C, Glc, 01, 04, anti-Mog or NG2 or a combination thereof. 53. The method of claim 48 wherein the cells are sorted by recognition of cell markers by immunological reagents. 54. The method of claim 53 wherein the immunoglobulin reagents are antibodies. 55. The method of claim 40 wherein the cells isolated are immunohistochemically Pax2+ GFAP−. 56. The method of claim 40 wherein the cells isolated are immunohistochemically Pax2+ GFAP+. 57. The method of claim 40 wherein IPAs are isolated by the immunological separation of a population of vimentin+ cells and then isolating GFAP+ cells from said vimentin+ population. 58. The method of any one of claims 48 to 57 wherein the mammalian animal is a human. 59. The method of any one of claims 40 to 58 wherein the mammalian animal is a livestock animal, laboratory test animal or a companion animal. 60. The method of claim 40 wherein the therapy is for a degenerative disorder. 61. The method of claim 60 wherein the degenerative disorder is Alzheimer's disease, Huntington's disease, HIV-associated dementia (HIV-D), a chronic neurological disorder, Parkinson's disease, epilepsy, stroke or alcoholism. 62. The method of claim 60 wherein the degenerative disorder is hypoxia or a spinal chord injury. 63. The method of claim 40 wherein the therapy is an acute brain injury or CNS dysfunction. 64. The method of any one of claims 40 to 63 wherein the lineage-specific cells are co-introduced with neural stem cells or neuronal cells. 65. The method of claims 40 to 63 wherein the lineage specific cells are from the same subject being treated. 66. The method of any one of claims 40 to 63 wherein the lineage-specific cells are from a different subject being tested. 67. A method for assessing the level of healthy tissue in a CNS biopsy such as a brain biopsy in an adult subject said method comprising determining in said biopsy presence of Pax2+ astrocyte cells wherein the presence of said Pax2+ cells is indicative of a reversion in the maturation of said astrocytes. 68. A composition of astrocyte precursor cells such as APCs or IPAs in substantially homogeneous form, said composition optionally further comprising one or more pharmaceutically-acceptable carriers and/or diluents. 69. Conditioned medium from the in vitro culture of astrocyte precursor cells such as APCs or IPAs wherein said conditioned medium comprises one or more growth factors or autocrine factors. 70. A growth or autocrine factor obtainable from conditioned medium of an in vitro cell culture of astrocyte precursors such as APCs or IPAs. 71. A method for generating a substantially homogenous population of mammalian cells of the astrocytic lineage, said method comprising isolating cell suspension from an adult brain or embryonic brain and removing neural precursor cells by a negative selection using N-CAM or functional equivalent and then selecting positively for glial cells using one or more of A2B5, GD3, 3CB2, FGFR3, PDGFRα or a combination thereof or a functional equivalent thereof; culturing the resulting cells in a serum free medium together with a growth factor and removing by negative selection oligodendrocytes using markers Glc, Gal-C, 01, 04, anti-Mog or a combination thereof or a functional equivalent thereof and then inducing differentiation along the maturation pathway by culturing cells in the presence of one or more of CNTF, LIF, BMP such as BMP4, cAMP, TGFβ and/or EGF or functional equivalents thereof. 72. The method of claim 71 wherein the serum free medium is DMEM/F-12. 73. The method of claim 72 wherein the DMEM/F-12 medium further comprises a growth factor selected from bFGF and chick embryo extract. 74. Cells of the astrocytic lineage isolated by the method of any one of claims 71 to 73. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country. Multipotent stem cells are undifferentiated cells which are capable of differentiation and proliferation into multiple cell lineages and types and have the ability of self-renewal. During development of the central nervous system (CNS), multipotent stem cells which have the capacity of generating many types of neurons and glia, give rise to precursor cells that are progressively more restricted in differentiation potential. Although substantial progress has been made in understanding the development of oligodendrocytes and Schwann cells, much less is known about astrocyte development. Furthermore, whereas several types of multipotent stem cells and lineage-restricted precursor cells have been characterized and applied clinically in recent years, the lack of knowledge of the sequence of events that underlies astrocyte development has limited the success of such applications. It has been shown that a glial-restricted precursor (GRP) cell isolated from the spinal cord of mice on embryonic day (E) 13.5 is capable of giving rise to oligodendrocytes and both type-1 and type-2 astrocytes in culture. However, the intermediate stages of differentiation between the GRP cell and mature, differentiated astrocytes present in the adult CNS are not well characterized. There is a need, therefore, to increase the understanding of the developmental biology of cells of the astrocytic lineage. Early studies provided evidence for the existence of astrocyte precursor cells (APCs) that give rise only to astrocytes (Raff et al., Devel. Biol. 106: 53-60, 1984; Fok-Seang, J. and Miller, R. H., J. Neurosci. 12: 2751-2764, 1992; Davis, A. A. and Temple, S., Nature 372: 263-266, 1994; Levison, S. W. and Goldman, J. E., J. Neurosci. Res. 48: 83-94, 1997; Mi. J. and Barres. B. A., J. Neurosci. 19: 1049-1061, 1999). Such cells present in cultures of neonatal rat spinal cord were characterized as highly migratory as well as positive for the A2B5 antigen and vimentin and negative for glial fibrillary acidic protein (GFAP) and galactocerebroside (Fok-Seang and Miller, 1992, supra). Such cells are also present in neonatal rat optic nerve and were characterized as positive for Pax2, A2B5, C5, Ran-2 and Vimentin and negative for GFAP, S100β, and weakly positive for nestin (Ni and Barres, 1999, supra). Little is known of the characteristics of APCs in vivo. Until the advent of the present invention, the existence of an APC that gives rise only to astrocytes in the developing human CNS and adult CNS has not previously been demonstrated. Furthermore, immunohistochemical and in situ hybridization analyses have shown that, in the mouse cerebellum, Pax2 (Mi and Barres, 1999, supra) is not expressed by cells of the astrocytic or oligodendrocytic lineages, but is rather localized to γ-aminobutyric acid-containing interneurons and deep cerebellar nuclei (Maricich, S. M. and Herrup, K., J Neurobiol. 41: 281-294, 1999). There is an apparent discrepancy, therefore, in Pax2 gene expression data following in vitro and in vivo studies of CNS development. Furthermore, until the advent of the present invention, there was a lack of in vivo studies of APC differentiation in human fetal tissue. The Pax2 gene is a member of the Pax gene family which encodes transcription factors, all of which are DNA-binding proteins that contain a paired-box domain. Each member of the Pax family is expressed in a spatially and temporally restricted manner, suggesting that these proteins contribute to the control of tissue morphogenesis and pattern formation. Pax2 is implicated in organogenesis of the kidney, eye, ear, and the CNS. Heterozygous mutations in the Pax2 gene result in failure of the optic groove to form in the mouse optic nerve (Otteson et al., Devel. Biol. 193: 209-224, 1998) and are associated in humans and mice with optic nerve coloboma (Sanyanusin et al., Nature Genetics 9: 358-364, 1995; Favor et al., Proc. Natl. Acad. Sci. USA 93: 13870-13875, 1996), a condition characterized by enlargement and blurring of the margin of the optic disk. Homozygous mutations in the Pax2 gene result in retinal coloboma as a consequence of failure of the retinal fissure to close (Torres et al., Development 122: 3381-3391, 1996). Pax2 expression during ocular development has been studied in mice, rats, and humans. Transcripts of the Pax2 gene are first apparent in the developing mouse eye on embryonic day (E) 9 and are initially restricted to the ventral optic cup and stalk (Nornes et al., Development 109: 797-809, 1990; Otteson et al., 1998, supra). By E16.5, these transcripts have disappeared from the ventral retina and are present in a ring of cells around the optic nerve head (ONH) and in the parenchyma of the optic nerve. At E18, Pax2 mRNA is apparent on the vitreal surface of the posterior retina, consistent with the timing and topography of astrocyte migration into the mouse retina. Pax2 mRNA was not detected in the retina, optic disk, or optic nerve of adult mice (Otteson et al., 1998, supra). In the rat optic nerve, Pax2 expression is already widespread at E17 (Mi and Barres, 1999, supra). Although the pattern of Pax2 expression during development of the rat optic nerve is consistent with the observations in the mouse, Pax2 expression persists at a low level in the adult rat nerve. Limited observations in humans have shown that, between 6 and 8 weeks of gestation (WG), Pax2 is expressed in the region of the optic disk and nerve (Terzic et al., Int. J. Dev. Biol. 42: 701-707, 1998). Despite the documentation of Pax2 expression during early embryonic development of the optic nerve and eyecup until the advent of the present invention, the relation between Pax2 expression and differentiation of the astrocytic lineage during the later stages of retinal development has been unknown. Various studies have examined the development of GFAP + astrocytes in vivo. In the human retina, astrocytes with two distinct morphologies and locations have been described: those with parallel processes closely associated with nerve fiber bundles (NFBs), and star-shaped astrocytes present in the ganglion cell layer (GCL) that often ensheath blood vessels (Wolter, J., Am. J: Ophthal 40: 88-99, 1955; Ogden, T. E., Invest. Ophthalmol. Vis. Cii. 17: 499-510, 1978; Ramirez et al., Vis. Res. 34: 1935-1946, 1994; Trevino et al., Vis. Res. 37: 1707-1711, 1997; Provis et al., Exp. Eye Res. 65: 555-568, 1997; Hughes et al., Invest. Ophthalmol. Vis. Sci. 41: 1217-1228, 2001). Astrocytes first appear in the monkey retina around the optic disk and spread peripherally, reaching the edge of the retina before birth (Gariano et al, Invest. Ophthalmol. Vis. Sci. 37: 2367-2375, 1996); vimentin and GFAP immunohistochemical analysis of retinal sections revealed that immature spindle-shaped astrocytes precede the developing vasculature. Astrocytes also precede the formation of blood vessels by a small margin in the human (Chan-Ling et al., Proc. Aust. Neurosci. Soc. 7: 48, 1996; Provis et al., 1997 , supra; Hughes et al., 2001 , supra), cat (Ling, T. and Stone, J., Dev. Brain Res. 44: 73-85, 1988; Chan-Ling, T. and Stone, J., J. Comp. Neurol. 303: 387-399, 1991), and rat (Ling et al., J. Comp. Neurol. 286: 345-352, 1989) retina and are thought to secrete vascular endothelial growth factor (VEGF), which mediates hypoxia-induced angiogenesis (Chan-Ling et al., Invest. Ophthalmol. Vis. Sci. 36: 1201-1214, 1995; Stone et al., J. Neurosci. 15: 4738-4747, 1995; Hughes et al, 2000, supra). In work leading to the present invention, the present inventor investigated the relationship between Pax2 expression and cells of the astrocytic lineage in the human retina and the optic nerve head (ONH), and characterized the time course of appearance and topography of spread of APCs and perinatal astrocytes in the human retina. The subject inventor identified positive and negative markers which were specific for particular developmental stages during maturation of astrocytic lineage. In accordance with the present invention, these markers in combination with other in vitro markers, are used to selectively enrich or generate populations of APCs or immature perinatal astrocytes (IPAs) or other astrocyte cells such as mature perinatal astrocytes. Furthermore, in accordance with the present invention, APCs are identified in the adult human brain. The ability to generate such populations permits their developmental expansion for use in tissue replacement and augmentation therapy and to identity factors involved in their proliferation and differentiation. The identification of immature APCs in the adult human brain is particularly significant in terms of a source of cells for autologous therapy. Furthermore, the present invention encompasses antagonists and agonists of these factors as well as naturally occurring molecules which inhibit proliferation, differentiation and/or growth of these cells. |
<SOH> SUMMARY OF THE INVENTION <EOH>Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. The present invention identifies astrocyte cell markers which are capable of distinguishing between developmental stages. From multipotent stem cells, lineage-specific APCs are formed expressing Pax2 and vimentin but not either glial fibrillary acid protein (GFAP) or S-100. The next developmental stage is the formation of IPAs which express all four of the above markers. Mature perinatal astrocytes (MPAs) lose the ability to express vimentin and then adult astrocytes further lose Pax2 expression as a function of physiologic aging. The ability to selectively enrich cultures of cells for APCs or IPAs permits their use in tissue replacement and augmentation therapy. Importantly, APCs have been identified in accordance with the present invention in adult brain as well as the retina and, hence, this aspect represents a source of APCs for autologous therapy as well as for heterologous therapy. Furthermore, homogeneous populations of APCs or IPAs can be used to isolate particular growth or autocrine factors for use in conjunctive therapy to tissue replacement and augmentation therapy or to induce repair or regeneration of endogenous tissue. The markers further permit mixed populations of astrocytes in various stages of development to be identified and this has diagnostic and therapeutic applications. Accordingly, one aspect of the present invention contemplates a method for generating a substantially homogeneous population of lineage-specific cells from tissue of the central nervous system (CNS) of mammalian animals, said method comprising subjecting said CNS tissue to tissue disruptive means comprising the lineage-specific cells to be isolated, subjecting the mixed population to cell separation discrimination means to generate a substantially homogeneous population of lineage-specific cells. Preferably, the lineage-specific cells are APCs or IPAs from tissue of the CNS such as brain including retina tissue. The present invention contemplates, therefore, a method of generating a substantially homogeneous population of APCs from tissue of the CNS such as from brain or parts thereof including the retina or parts thereof, said method comprising subjecting said CNS tissue to tissue disruptive means to produce a population comprising APCs amongst other cells and subjecting said population of cells to a cell sorting methodology including such as subjecting cells to positive selection using surface markers GD3, A2B5, C3B2, FGFR3 and/or PDGFRα or a combination thereof, then subjecting the positively selective cells to negative selection using GlC, 01, 04, anti-Mog and/or NG2 or a combination thereof. The identity of the purified population of cells is confirmed using Pax2, vimentin, GFAP and S-100 immunohistochemically. Having obtained the APCs, a substantially homogenous population of IPAs or a mixed population of IPAs and APCs are included along the mature pathway is induced using one or a combination of inter alia CNTF, LIF, BMP (e.g. BMP4), TGFβ, cAMP and EGF. In a most preferred embodiment, the cells are purified as follows. A population of cells is selected and single cell suspensions prepared. Using negative selection such as N-CAM (also known as PSA-N-CAM neural cells are removed from their cell population. Glial cells are positively selected using markers such as A2B5, GD3, 3CB2, FGFR3, PDGFRα or a combination thereof. The cells are then cultured in a serum free medium such as DMEM/F-12 supplemented with growth factors such as bFGF and chick embryo extract. In the resulting population, oligodendrocytes are removed using markers such as GlC, 01, 04, Gal-C, anti-MOG and NG2. The resulting population is induced to differentiate along the maturation pathway using growth factors such as CNTF, LIF, BMP such as BMP4, cAMP, TGFβ and EGF. The cells can then be characterized immunohistochemically based on the markers presented in Table 1. The present invention provides, therefore, a substantially homogeneous population of mammalian lineage-specific cells from the CNS. The preferred mammalian lineage-specific cells are APCs or IPAs. Another aspect of the present invention contemplates a method of cell replacement therapy in a mammalian animal, said method comprising generating a substantially homogeneous population of lineage-specific cells and introducing same into an organ or tissue requiring cells to be replaced or to another location from where the cells can migrate to an organ or tissue requiring cells wherein the introduced cells are subject to expansion or proliferation in vitro and/or in vivo by one or more growth factors. Generally, the lineage-specific cells are APCs or IPAs. The APCs or IPAs or tissues derived therefrom may be from the subject being treated (i.e. autologous therapy) or from a suitably histocompatibility matched subject (i.e. heterologous therapy). Autologous therapy is preferred. Yet another aspect of the present invention provides a composition of astrocyte precursor cells such as APCs or IPAs in substantially homogeneous form, said composition optionally further comprising one or more pharmaceutical acceptable carriers and/or diluents. Still another aspect of the present invention contemplates a growth or autocrine factor obtainable from conditioned medium of an in vitro cell culture of astrocyte precursors such as APCs or IPAs. The growth or autocrine factor may be used in vitro to expand a population of lineage-specific cells or may be administered directly to the brain to facilitate or promote development of replacement cells. In a related embodiment, the present invention proposes the use of microarray technology and differential expression arrays to determine cell surface markers including differentially expressed cell surface markers at different stages of astrocyte cell development. Such studies assist in the identification of growth factor receptors for use in selecting growth and autocrine factors to promote proliferation and/or differentiation of particular astrocyte cells. A further aspect of the present invention contemplates the use of the purified astrocytes and in particular APCs and IPAs as gene therapy carriers. |
Novel promoters inducible by dna damaging conditions or agents and uses thereof |
The present invention relates a method of converting a promoter into a promoter which is inducible upon genotoxic compounds or conditions. The present invention further relates to a method of reducing the basal expression level of promoter which is inducible upon genotoxic compounds or conditions. These methods provides novel nucleotide sequences, vectors and host cells for the expression of proteins under the control of genotoxic conditons or compounds. The novel expression system has wide industrial applications into the field of recombinant protein production but has also clinical applications such as the controlled expression of therapeutic compounds in hypoxic tissues such as tumors. |
1. An isolated and purified polynucleotide comprising at least one first sequence element inserted in a second sequence element wherein the first sequence element is a repressor binding element of a promoter which is inducible by DNA damaging agents or conditions and wherein the second sequence element is a promoter sequence. 2-41. (cancelled). 42. The polynucleotide of claim 1, wherein the promoter sequence of the second sequence element is from a promoter which is not inducible by a DNA damaging agent or condition. 43. The polynucleotide of claim 1, wherein the promoter sequence of the second sequence element is from a promoter which is inducible by a DNA damaging agent or condition. 44. The polynucleotide of claim 1, wherein said polynucleotide is positioned 5′ to a nucleotide sequence suitable for the introduction of a third sequence element. 45. The polynucleotide of claim 1, wherein the insertion of a first sequence element occurs between about 46 base pairs and about 106 base pairs upstream of the ribosome binding site of the second sequence element. 46. The polynucleotide of claim 42, wherein the non-inducible promoter is a constitutive promoter or an inducible promoter. 47. The polynucleotide of claim 42, wherein the non-inducible promoter is a bacterial promoter. 48. The polynucleotide of claim 47, wherein the bacterial promoter is from gram positive bacteria. 49. The polynucleotide of claim 47, wherein the bacterial promoter is from gram negative bacteria. 50. The polynucleotide of claim 47, wherein the bacterial promoter is an EglA promoter of Clostridium sp. 51. The polynucleotide of claim 43, wherein the inducible promoter is a bacterial promoter. 52. The polynucleotide of claim 51, wherein the bacterial promoter is from gram positive bacteria. 53. The polynucleotide of claim 51, wherein the bacterial promoter is from gram negative bacteria. 54. The polynucleotide of claim 53, wherein the bacterial promoter is RecA. 55. The polynucleotide of claim 1, wherein the repressor binding element comprises a Cheo box consensus sequence as depicted in SEQ ID NO: 1. 56. The polynucleotide of claim 1, wherein the repressor binding element comprises a DinR box consensus sequence as depicted in SEQ ID NO: 2. 57. The polynucleotide of claim 1, wherein the repressor binding element comprises a sequence selected from the group of sequences depicted in SEQ ID 4 to SEQ ID 25. 58. The polynucleotide of claim 1, wherein the repressor binding element comprises a SOS box consensus sequence as depicted in SEQ ID NO3. 59. The polynucleotide of claim 1, wherein the repressor binding element comprises a sequence selected from the group of sequences depicted in SEQ ID 26 to SEQ ID 43. 60. The polynucleotide of claim 44, wherein the third sequence element encodes a protein with pharmaceutical properties or a protein which is able to convert an inactive compound into a pharmaceutically active compound. 61. The polynucleotide according to claim 60 wherein the protein with therapeutic properties is TNF-alpha (Tumour Necrosis Factor alpha). 62. A method of converting a promoter which is not inducible by DNA damaging agents or conditions into a promoter which is inducible by radiation, genotoxic compounds or DNA damaging compounds comprising the step of inserting at least one repressor binding element of a promoter which is inducible by a DNA damaging compound or condition into said non inducible promoter. 63. A method of increasing the induction level of a first promoter which is inducible by genotoxic compounds or conditions comprising the step of inserting at least one repressor binding element of said first promoter or of a second promoter which is inducible by a DNA damaging compound or condition into said first inducible promoter. 64. A method of decreasing the basal expression level of a first promoter which is inducible by genotoxic compounds or conditions comprising the step of inserting at least one repressor binding element of said first promoter or of a second promoter which is inducible by a DNA damaging compound or condition into said first inducible promoter. 65. A vector comprising a nucleotide sequence according to claim 1. 66. A bacterial host cell transfected with the vector of claim 65. 67. A bacterial host cell according to claim 66, wherein said cell is a facultative or obligate anaerobic bacterium. 68. A pharmaceutical composition comprising a cell according to claim 66 in admixture with at least one pharmaceutically acceptable carrier. 69. A pharmaceutical composition comprising a cell according to claim 67 in admixture with at least one pharmaceutically acceptable carrier. 70. A method for the in vitro production of recombinant proteins comprising the step of contacting a culture of host cells according to claim 66 with a DNA damaging compound or condition. |
<SOH> BACKGROUND OF THE INVENTION <EOH>In the search for new therapeutic modalities for cancer, gene therapy has gained enormous interest over the last years. Many strategies to apply gene therapy have been developed and even more vectors to deliver the gene of interest have been constructed. However, one of the major pitfalls of gene therapy is still the lack of specificity of gene delivery. Developing a good gene therapy protocol involves the use of a tumour-specific vector system and gene expression limited to the tumour only. This will result in a high therapeutic index: high local tumour control with low systemic side effects. Recently, the use of bacteria as tumour-specific protein transfer system has gained interest. Attenuated Salmonella (Pawelek, J. M. et al, 1997, Cancer Res. 54:4537-4544., Platt, J., S. Sodi et al, 2000, Eur. J. Cancer 36:2397-2402.), anaerobic Bifidobacterium (Zappe, H. et al, 1988, Appl. Environ. Microbiol. 54:1289-1292.) and apathogenic Clostridium (Fox, M. E. et al, 1996, Gene Ther. 3:173-178., Lambin, P. et al, 1998, Anaerobe 4:183-188; Lemmon, M. J. et al, 1997, Gene Ther. 4:791-765.) have shown to give selective colonisation in tumours without the presence of vegetative bacteria in the normal tissues (Lambin, ref supra). Moreover, the use of bacteria as protein transfer system is very safe since treatment can be stopped at any time by addition of the appropriate antibiotic (Theys, J. et al, 2001, FEMS Immunol. Med. Microbiol. 30:37-41.) The anaerobic gram positive bacterium Clostridium acetobutylicum was genetically engineered to express therapeutic proteins like mouse tumour necrosis factor α (mTNF-α) locally in the tumour under the control of a strong but constitutive promoter. (Theys, J. et al, 1999, Appl. Environ. Microbiol. 65:4295-4300.) Apart from temporal and spatial expression high levels of a therapeutic protein are desired. This would solve the problems associated with systemic administration of therapeutic proteins like TNF-α where hepatotoxicity and life-threatening hypotension occur as major side-effects (Old L J. 1985, Science 230:630-636.). Limiting expression of toxic agents to the tumour cell is extremely important if damage to the surrounding normal tissues is to be avoided. Anaerobic bacteria selectively colonise the hypoxic-necrotic areas of solid tumours which are absent in healthy normal tissues and genetically engineered bacteria will secrete therapeutic proteins locally in the tumour (Theys, J. et al, 2001, Cancer Gene Ther. 8, 247-297; Theys, J. et al, 1999, Appl. Environ. Microbiol. 65:4295-4300). The use of bacterial host with a radio-inducible promoter would prevent expression in other necrotic tissues outside the tumour. In this manner, the combination of radiotherapy, one of the standard treatment modalities in cancer, and genetically engineered bacteria as tumour specific protein transfer system, enables the expression of therapeutic agents locally in the tumour due to both spatial and temporal control of protein expression. Preferably protein expression would only occur after radiotherapy, so gene expression will be switched on and physicians will know from what time on the therapeutic protein will be present. Hallahan, D. E. et al in (1995) Nature Med. 1:786-791 describe an adenoviral vector wherein TNF-alpha is positioned under the control of the radiation inducible Egr-1 promoter. It was earlier demonstrated that the recA promoter, belonging to the SOS-repair system of bacteria, is induced by radiotherapy, already at the clinically relevant dose of 2 Gy (Nuyts, S. et al, 2001. Anticancer Res. 21:.857-862; Nuyts, S. et al, 2001, Radiat Res. 155:716-726; Nuyts, S. et al, 2001, Gene Therapy, In press.). A single dose of 2 Gy significantly increased mTNF-α secretion by recombinant clostridia with 44%. Moreover, gene activation could be repeated with a second dose of 2 Gy, which makes it promising for clinical use, since in patient settings, daily fractions of 2 Gy are used (Nuyts, S. et al, 2001 cited supra) All genes belonging to the SOS-repair system are activated by the presence of DNA damage. In non-activated conditions, a repressor called LexA or DinR (for Bacillus subtilis ) binds on a specific operator sequence called respectively SOS-box (for Gram-negative bacteria) or Cheo box for Gram-positive bacteria. In addition to its role in homologous recombination, RecA functions as a coprotease for the LexA protein. In a healthy cell, LexA represses the expression of genes encoding DNA repair proteins (SOS genes). Upon injury of DNA, LexA catalyzes its own digestion, thereby allowing synthesis of necessary SOS proteins. However, LexA can only induce self-catalysis when activated by a ssDNA-RecA filament. A single filament will bind and activate several LexA proteins, each of which then cleaves other bound proteins. Thus, ssDNA-RecA, a product of DNA injury, stimulates DNA repair. through an increased transcription of the SOS-genes (Cheo, D. L. et al, 1991, J. Bacteriol. 173:1696-1703.; Miller, R., and T. Kokjohn, 1990, Annu. Rev. Microbiol. 44:365-394.). These genes will play a role in repairing the original DNA damage. Both LexA and DinR bind to their operator sequence as dimers (Kim, B., and J. W. Little. 1992, Science 255:203-205., Yazawa, K. et al, 2000, Cancer Gene Ther. 7:269-274.). The consensus sequence for the Cheo box in Gram-positive bacteria is 5′ GAACNNNNGTTC 3′ (cheo et al cited supra). This consensus sequence is positioned within promoter regions such that the regulatory molecule LexA bound at these sites could interfere with the initiation of transcription by RNA polymerase. Several genes can be found which have 2 or more putative Cheo boxes and for those, in which repressor binding is proven, the distance between the two boxes is 15 to 16 bp (yazawa cited supra ). A system similar as described for Clostridium is known for gram negative bacteria such as E. coli . When cells like E. coli are subject to excessive DNA damage, a system (the SOS response) that stops DNA synthesis and invokes massive DNA repair is triggered. The SOS system is regulated by RecA. If there is any DNA damage present during replication, RecA will associate with the single stranded DNA that is generated after DNA damage. RecA will also associate with a protein called LexA. LexA is a repressor that normally turns off a large group of genes associated with DNA repair, including recA, uvrA, uvrB and uvrD. Each of these genes has a similar consensus sequence called the SOS BOX (5′-CTGNNNNNNNNNNCAG-3′, where N can be any base). LexA binds to the SOS box, turning off genes with an SOS box in their promoters. However, when RecA interacts with single stranded DNA, RecA is “activated” such that RecA binds to LexA. LexA bound to RecA does not bind to the SOS box, and thus all the genes with an SOS box (mainly DNA repair genes) are turned on. The controlling factor in this system is the presence of single stranded DNA. Some genes with SOS boxes inhibit cell division. Thus when the LexA-RecA complex is formed, DNA repair is initiated and cell division is inhibited. When the damaged DNA is repaired, there will be no means to activate the RecA such that it binds to LexA, and thus LexA will again inhibit all genes with SOS boxes and related DNA repair will cease and cell division will continue. Despite the wide knowledge on DNA damage mediated expression of proteins and despite the variety of expression systems for proteins by anaerobic organisms in hypoxic tissues, there is still a need for DNA constructs, vectors and host cells which allow inducible expression with low levels of basal expression. There is also a need for DNA constructs, vectors and host cells which allow more regulated and higher expression of proteins than those known in the art. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to an isolated and purified polynucleotide comprising at least one first sequence element inserted in a second sequence element wherein the first sequence element is a repressor binding element of a promoter which is inducible by DNA damaging agents or conditions and wherein the second sequence element is a promoter sequence. The promoter can be not inducible by a DNA damaging agent or condition but also can be inducible by a DNA damaging agent or condition. The polynucleotide can be positioned 5′ to a nucleotide sequence suitable for the introduction of a third sequence element. The invention relates to a method of converting a promoter which is not inducible by DNA damaging agents or conditions into a promoter which is inducible by radiation, genotoxic compounds or DNA damaging compounds comprising the step of inserting at least one repressor binding element of a promoter which is inducible by a DNA damaging compound or condition into said non inducible promoter. The invention relates to a method of increasing the induction level of a first promoter which is inducible by genotoxic compounds or conditions comprising the step ofinserting at least one repressor binding element of a said first promoter or a second promoter which is inducible by a DNA damaging compound or condition into the first inducible promoter. The invention relates to a method of decreasing the basal expression level of a first promoter which is inducible by genotoxic compounds or conditions comprising the step of inserting at least one repressor binding element of a said first promoter or a second promoter which is inducible by a DNA damaging compound or condition into the first inducible promoter. The invention further relates to a vector comprising a nucleotide sequence of the present invention. The invention further relates to a bacterial host cell transfected with the vectors of the present invention. The invention further relates to a pharmaceutical composition comprising a cell of the present invention in admixture with at least one pharmaceutically acceptable carrier. The invention further relates to a method of expressing a therapeutic protein or a protein converting a precursor into a therapeutic compound comprising a first step of administering to an individual of the pharmaceutical composition and a second step of subjecting the person to a DNA damaging condition and/or administering to an individual a DNA damaging compound or a precursor thereof. The invention also relates to a method for the in vitro production of recombinant proteins comprising the step of contacting a culture of host cells of the present invention with a DNA damaging compound or condition. |
Inorganic ion exchangers for removing contaminant metal ions from liquid streams |
The invention relates to processes and compositions for at least partially removing cations of one or more metals selected from the group consisting of caesium, strontium, lead, silver, transition metals, lanthanides and actinides from a liquid containing the cations. The process comprises contacting the liquid with a sorbent material in an amount and for a time sufficient to at least reduce the concentration of the cations, wherein the sorbent material is a metal oxide composition having the empirical formula Ax/n[ByM1-yO3+δ]x.ZH2O where M is one or more metals of Group Vb or VIb of the Periodic Table; A is a cation selected from the group consisting of alkali metals, alkaline earth metals, silver, ammonium, hydrogen and mixtures thereof; n is the charge on the cation A; B is one or more elements selected from the group consisting of W, Zr, Mo, V, Ti, Fe, Ce, Sb, Nb, Mn, Co, Cr, Fe, Ta, Sn and Cu, provided that M and B are different; x represents the quantity of framework charge; y is a number in the range of 0 to about 0.5; Z is a number in the range 0 to 3; and 3+δ indicates the oxygen stoichiometry. Preferably, y is greater than zero. |
1. A process for at least partially removing cations of one or more metals selected from the group consisting of caesium, strontium, lead, silver, transition metals, lanthanides and actinides from an acidic liquid containing said cations, comprising contacting said liquid with an ion exchange/sorbent material in an amount and for a time sufficient to at least reduce the concentration of said cations in said liquid, wherein said material has a structure based on or closely related to a structure selected from the group consisting of hexagonal tungsten bronze, pyrochlore and mixtures thereof, and is a metal oxide composition having the empirical formula (I) Ax/n[ByM1-yO3+δ]x.ZH2O (I) where M is one or more metals of Group Vb or VIb of the Periodic Table; A is a cation selected from the group consisting of alkali metals, alkaline earth metals, silver, ammonium, hydrogen and mixtures thereof; n is the charge on said cation A; B is one or more elements selected from the group consisting of W, Zr, Mo, V, Ti, Fe, Ce, Sb, Nb, Mn, Co, Cr, Fe, Ta, Sn and Cu, provided that M and B are different; x represents the quantity of framework charge; y is a number in the range of 0 to about 0.5; Z is a number in the range >0 to 3; and 3+δ indicates the oxygen stoichiometry. 2. A process according to claim 1 wherein said metal M is selected from the group consisting of tungsten, niobium and mixtures thereof. 3. A process according to claim 1 wherein said acidic liquid is an aqueous liquid having a pH of less than about 4. 4. A process according to claim 1 wherein said sorbent material has a structure based on or closely related to a hexagonal tungsten bronze structure. 5. A process according to claim 1 wherein B is selected from the group consisting of Mo, Nb, V, Ce and Cu. 6. A process according to claim 1 wherein said sorbent material has a structure based on or closely related to a hexagonal tungsten bronze structure and y is in the range of from >0 to about 0.3. 7. A process according to claim 6 wherein B is Mo, M is W and y is from about 0.1 to about 0.2. 8. A process according to claim 6 or 7 wherein said at least partially removed cations are selected from the group consisting of caesium cations, strontium cations and mixtures thereof. 9. A process for selectively removing first cations of one or more metals selected from the group consisting of caesium, strontium, lead, silver, transition metals, lanthanides and actinides from a liquid containing said first cations and containing second cations of at least one other metal, comprising contacting said liquid with an ion exchange/sorbent material in an amount and for a time sufficient to at least reduce the concentration of said first cations, wherein said material has a structure based on or closely related to a structure selected from the group consisting of hexagonal tungsten bronze, pyrochlore and mixtures thereof, and is a metal oxide composition having the empirical formula (I) Ax/n[ByM1-yO3+δ]x.ZH2O (I) where M is one or more metals of Group Vb or VIb of the Periodic Table; A is a cation selected from the group consisting of alkali metals, alkaline earth metals, silver, ammonium, hydrogen and mixtures thereof; n is the charge on said cation A; B is one or more elements selected from the group consisting of W, Zr, Mo, V, Ti, Fe, Ce, Sb, Nb, Mn, Co, Cr, Fe, Ta, Sn and Cu, provided that M and B are different; x represents the quantity of framework charge; y is a number in the range of from 0 to about 0.5; Z is a number in the range >0 to 3; and 3+δ indicates the oxygen stoichiometry. 10. A process according to claim 9 wherein said metal M is selected from the group consisting of tungsten, niobium and mixtures thereof. 11. A process according to claim 9 wherein said material has a structure based on or closely related to a hexagonal tungsten bronze structure. 12. A process according to claim 9 wherein B is selected from the group consisting of Mo, Nb, V, Ce and Cu, and y is >0. 13. A process according to claim 9 wherein said sorbent material has a structure based on or closely related to a hexagonal tungsten bronze structure and y is in the range of from >0 to about 0.3. 14. A process according to claim 13 wherein B is Mo, M is W and y is from about 0.1 to about 0.2. 15. A process according to claim 13 or 14 wherein said first cations are selected from the group consisting of caesium cations, strontium cations and mixtures thereof. 16. A metal oxide composition having the empirical formula Ax/n[ByM1-yO3+δ]x.ZH2O where M is a metal of Group Vb or VIb of the Periodic Table; A is a cation selected from the group consisting of alkali metals, alkaline earth metals, silver, ammonium hydrogen and mixtures thereof; n is the charge on said cation; B is one or more elements selected from the group consisting of W, Zr, Mo, V, Ti, Fe, Ce, Sb, Nb, Mn, Co, Cr, Fe, Ta, Sn and Cu, provided that M and B are different; x represents the quantity of framework charge; y is a number in the range of >0 to about 0.5; Z is a number in the range 0 to 3; and 3+δ indicates the oxygen stoichiometry. 17. A composition according to claim 16 wherein M is selected from the group consisting of tungsten, niobium and mixtures thereof. 18. A composition according to claim 16 which has a structure based on or closely related to a structure selected from the group consisting of hexagonal tungsten bronze and pyrochlore. 19. A composition according to claim 16 wherein B is selected from the group consisting of Mo, Nb, V, Ce and Cu, and y is >0. 20. A composition according to claim 16 wherein said sorbent material has a structure based on or closely related to a hexagonal tungsten bronze structure and y is from about 0.05 to about 0.3. 21. A composition according to claim 20 wherein B is Mo, M is W and y is from about 0.1 to about 0.2. 22. A process for at least partially removing cations of one or more metals selected from the group consisting of caesium, strontium, lead, silver, transition metals, lanthanides and actinides from a liquid containing said cations, comprising contacting said liquid with a sorbent material in an amount and for a time sufficient to at least reduce the concentration of said cations, wherein said sorbent material is a metal oxide composition having the empirical formula Ax/n[ByM1-yO3+δ]x.ZH2O where M is one or more metals of Group Vb or VIb of the Periodic Table; A is a cation selected from the group consisting of alkali metals, alkaline earth metals, silver, ammonium, hydrogen and mixtures thereof; n is the charge on said cation A; B is one or more elements selected from the group consisting of W, Zr, Mo, V, Ti, Fe, Ce, Sb, Nb, Mn, Co, Cr, Fe, Ta, Sn and Cu, provided that M and B are different; x represents the quantity of framework charge; y is a number in the range of >0 to about 0.5; Z is a number in the range >0 to 3; and 3+δ indicates the oxygen stoichiometry. 23. A process according to claim 22 wherein M is selected from the group consisting of tungsten, niobium and mixtures thereof. 24. A process according to claim 22 wherein said metal oxide composition has a structure based on or closely related to a structure selected from the group consisting of hexagonal tungsten bronze and pyrochlore. 25. A process according to claim 22 wherein B is selected from the group consisting of Mo, Nb, V, Ce and Cu. 26. A process according to claim 22 wherein said sorbent material has a structure based on or closely related to a hexagonal tungsten bronze structure and y is from about 0.05 to about 0.3. 27. A process according to claim 26 wherein B is Mo, M is W and y is from about 0.1 to about 0.2. 28. A process according to any one of claims 22-27 wherein said at least partially removed cations are selected from the group consisting of caesium cations, strontium cations and mixtures thereof. 29. A process according to claim 11 or 22 wherein said liquid is an aqueous liquid. 30. A process according to claim 29 wherein said aqueous liquid is an acidic liquid. 31. A process according to claim 29 wherein said aqueous liquid is an alkaline liquid. 32. A process for increasing the ion exchange capacity of a metal oxide composition having a structure based on or closely related to a structure selected from the group consisting of hexagonal tungsten bronze and pyrochlore, said metal oxide composition having the empirical formula Ax/n[ByM1-yO3+δ]x.ZH2O where M is one or more metals in oxidation state (VI); A is a cation selected from the group consisting of alkali metals, alkaline earth metals, silver, ammonium, hydrogen and mixtures thereof; n is the charge on said cation A; B is one or more elements selected from the group consisting of W, Zr, Mo, V, Ti, Fe, Ce, Sb, Nb, Mn, Co, Cr, Fe, Ta, Sn and Cu, provided that M and B are different; x represents the quantity of framework charge; y is a number in the range of 0 to about 0.5; Z is a number in the range >0 to 3; and 3+δ indicates the oxygen stoichiometry; the process comprising the step of reducing at least some of the metal atoms M in said metal oxide composition to an oxidation state less than (VI). 33. A process according to claim 32, wherein said reducing step is a chemical reduction step. 34. A process according to claim 33, wherein said chemical reduction step comprises treating said metal oxide composition with hydrazine. 35. A process according to claim 32, wherein M is tungsten. 36. A process according to claim 32, wherein y is 0. 37. A ion exchange material prepared by the process of any one of claims 32-36. 38. A process for preparing a metal oxide composition according to claim 16, comprising preparing an aqueous solution comprising oxyanions of element(s) B and oxyanions of metal(s) M in relative molar proportions of y to (1-y); adjusting the pH to 1.5 or higher; heating the mixture at a temperature in the range of about 100-300° C. for at least 12 hours; cooling the mixture and separating the metal oxide composition from the aqueous phase. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Ion exchange treatment (defined as A+BX B+AX, where A and B are both cations or both anions) typically involves the interchange/sorption of ionic species between a liquid phase (containing the contaminant) and a suitable solid phase using batch or continuous flow processes, until the specific sites available for ion exchange/sorption in the solid phase become saturated, and in the case of the continuous process, the contaminant/toxic ions appear in the eluant. Treatment is then stopped, and the contaminant ions present on the solid phase are subsequently exchanged with other ions. In this way the contaminant ions are recovered and the exchanger is regenerated. Inorganic sorbents are used extensively in industry and in research areas such as radiochemistry, geochemistry, and radiopharmaceuticals. Many of their industrial applications are within the broad context of waste remediation, particularly in areas such as nuclear waste decontamination, mine tailings management, and various hydrometallurgical separations. A particular application of inorganic sorbents is as ion exchangers in the decontamination of liquid nuclear wastes, such as for example those formed during the dissolution of fuel elements using strong acids. These liquid nuclear wastes are usually stored in underground stainless steel tanks. Typically in legacy wastes, that is those wastes which have been stored for times typically exceeding about 5 years, 137 Cs and 90 Sr are the radionuclides responsible for most of the radioactivity (with minor contributions from 99 Tc and 106 Ru). Such wastes can result from weapons development and power generation activities. Being highly soluble, Cs and Sr can easily become mobilised and find their way into the biosphere representing a significant danger to public health. It is therefore highly desirable to remove such radioactive elements selectively from the nuclear waste streams, particularly at the pre-treatment stage, and thereby allow safe disposal of the bulk of the waste as low level waste. This pre-treatment option for nuclear waste management is being implemented by many governmental agencies and independent contractors responsible for waste minimisation and the safe disposal of nuclear wastes. Accordingly, a wide range of ion exchangers, both organic and inorganic, have been tested for potential use in pre-treatment of liquid nuclear waste. Numerous organic (polymeric) ion exchangers are known and have been utilised to treat nuclear waste streams, with a number of these being very selective for Cs and Sr. In general however, such polymeric ion exchangers have inherently low radiation stability and are consequently not suitable for interim storage. Because of this they are widely regarded as being less favourable than inorganic systems for nuclear waste remediation. Inorganic ion-exchangers are preferred for nuclear waste applications because inorganic materials have greater radiation stability and hence maintain their efficacy longer. However, despite the recognised advantages of using inorganic sorbents to pre-treat liquid radioactive wastes, particularly for the uptake of Cs + and Sr 2+ , there remains a need for inorganic sorbents that can simultaneously sorb practical quantities of Cs + and Sr 2+ from highly acidic aqueous streams. Such acidic waste streams which are generated through dissolution of spent nuclear fuel elements in nitric acid, are particularly relevant. Further, there is a need for reusable inorganic ion exchangers for the pre-treatment of acidic waste streams, including liquid nuclear waste, to selectively remove contaminant metal ions, thereby resulting in waste having a lower level of contamination. Such low-level waste could then be effectively disposed of with an enormous cost saving compared to the expense in disposing of non pre-treated liquid nuclear waste. Hydrous metal oxides, including those of niobium, antimony, and tungsten, are known to sorb various cations. Most of these compounds are either amorphous to X-rays and/or do not possess well defined microporosity or surface chemistry. Microporous oxides of the above named metals are, however, known to have structures containing well defined tunnels. Of particular relevance here are those with the so-called hexagonal tungsten bronze (HTB) and pyrochlore structures shown in FIG. 1 . It is important to note that in the term ‘hexagonal tungsten bronze’, ‘hexagonal’ refers to the symmetry of the unit cell while the term ‘bronze’ is not a structural one, but refers to the fact that the compounds often have metallic lustre. Thus, the term ‘hexagonal tungsten bronze’ as used herein refers to a class of compounds with the HTB or HTB-like structure which do not necessarily contain tungsten. Certain HTB compounds are well known and have been synthesised in various ways, ranging from DC magnetron sputtering, which produces materials with the formula WO 3 , to low temperature hydrothermal methods such as those used for zeolite preparations that yield hydrated fine particle materials with the general formula Na x WO 3 . ZH 2 O. The latter methods usually employ acidified sodium tungstate solutions which are heated to temperatures in the range 100-300° C. to effect crystallisation. It is here disclosed for the first time that certain HTB and pyrochlore compounds, including fine particle, hydrothermally-prepared HTB and pyrochlore compounds, are capable of selectively removing certain ions from solutions containing much higher concentrations of sodium ions, including acidic solutions. In particular it is disclosed that certain HTB and pyrochlore compounds are capable of selectively removing both Cs and Sr ions simultaneously from aqueous solutions. These properties make them useful in applications involving environmental decontamination. |
<SOH> SUMMARY OF THE INVENTION <EOH>A first aspect of the present disclosure concerns the use of a porous group V or VI metal oxide as an ion exchanger material. Thus, in a first embodiment the invention provides a process for at least partially removing cations of one or more metals selected from the group consisting of caesium, strontium, lead, silver, transition metals, lanthanides and actinides from an acidic liquid containing said cations, comprising contacting said liquid with an ion exchange/sorbent material in an amount and for a time sufficient to at least reduce the concentration of said cations in said liquid, wherein said material has a structure based on or closely related to a structure selected from the group consisting of hexagonal tungsten bronze, pyrochlore and mixtures thereof, and is a metal oxide composition having the empirical formula (I) in-line-formulae description="In-line Formulae" end="lead"? A x/n [B y M 1-y O 3+x ] δ .ZH 2 O (I) in-line-formulae description="In-line Formulae" end="tail"? where M is one or more metals of Group Vb or VIb of the Periodic Table; A is a cation selected from the group consisting of alkali metals, alkaline earth metals, silver, ammonium, hydrogen and mixtures thereof; n is the charge on said cation A; B is one or more elements selected from the group consisting of W, Zr, Mo, V, Ti, Fe, Ce, Sb, Nb, Mn, Co, Cr, Fe, Ta, Sn and Cu, provided that M and B are different; x represents the quantity of framework charge; y is a number in the range of 0 to about 0.5; Z is a number in the range >0 to 3; and 3+δ indicates the oxygen stoichiometry. In a second embodiment, the invention provides a process for selectively removing first cations of one or more metals selected from the group consisting of caesium, strontium, lead, silver, transition metals, lanthanides and actinides from a liquid containing said first cations and containing second cations of at least one other metal, comprising contacting said liquid with an ion exchange/sorbent material in an amount and for a time sufficient to at least reduce the concentration of said first cations, wherein said material has a structure based on or closely related to a structure selected from the group consisting of hexagonal tungsten bronze, pyrochlore and mixtures thereof, and is a metal oxide composition having the empirical formula (I) in-line-formulae description="In-line Formulae" end="lead"? A x/n [B y M 1-y O 3+δ ] x .ZH 2 O (I) in-line-formulae description="In-line Formulae" end="tail"? where M is one or more metals of Group Vb or VIb of the Periodic Table; A is a cation selected from the group consisting of alkali metals, alkaline earth metals, silver, ammonium, hydrogen and mixtures thereof; n is the charge on said cation A; B is one or more elements selected from the group consisting of W, Zr, Mo, V, Ti, Fe, Ce, Sb, Nb, Mn, Co, Cr, Fe, Ta, Sn and Cu, provided that M and B are different; x represents the quantity of framework charge; y is a number in the range of from 0 to about 0.5; Z is a number in the range >0 to 3; and 3+δ indicates the oxygen stoichiometry. It will be appreciated that in the empirical formula (I), the framework charge x is determined by the valence of the principal framework element M and the element(s) B and the oxygen stoichiometry and typically takes values from 0 to 1, more typically in the range 0 to 0.5. Z is typically in the range >0 to 3, more typically in the range 0.5 to 1.5. The quantity 3+δ in the formula indicates the oxygen stoichiometry which may vary slightly from 3 owing to the presence of framework defects such as vacancies. In a third embodiment the invention provides a process for at least partially removing cations of one or more metals selected from the group consisting of caesium, strontium, lead, silver, transition metals, lanthanides and actinides from a liquid containing said cations, comprising contacting said liquid with a sorbent material in an amount and for a time sufficient to at least reduce the concentration of said cations, wherein said sorbent material is a metal oxide composition having the empirical formula in-line-formulae description="In-line Formulae" end="lead"? A x/n [B y M 1-y O 3+δ ] x .ZH 2 O in-line-formulae description="In-line Formulae" end="tail"? where M is one or more metals of Group Vb or VIb of the Periodic Table; A is a cation selected from the group consisting of alkali metals, alkaline earth metals, silver, ammonium, hydrogen and mixtures thereof; n is the charge on said cation A; B is one or more elements selected from the group consisting of W, Zr, Mo, V, Ti, Fe, Ce, Sb, Nb, Mn, Co, Cr, Fe, Ta, Sn and Cu, provided that M and B are different; x represents the quantity of framework charge; y is a number in the range of >0 to about 0.5; Z is a number in the range >0 to 3; and 3+δ indicates the oxygen stoichiometry. In a fourth embodiment the invention provides a metal oxide composition having the empirical formula in-line-formulae description="In-line Formulae" end="lead"? A x/n [B y M 1-y O 3+δ ] x .ZH 2 O in-line-formulae description="In-line Formulae" end="tail"? where M is a metal of Group Vb or VIb of the Periodic Table; A is a cation selected from the group consisting of alkali metals, alkaline earth metals, silver, ammonium, hydrogen and mixtures thereof; n is the charge on said cation; B is one or more elements selected from the group consisting of W, Zr, Mo, V, Ti, Fe, Ce, Sb, Nb, Mn, Co, Cr, Fe, Ta, Sn and Cu, provided that M and B are different; x represents the quantity of framework charge; y is a number in the range of >0 to about 0.5; Z is a number in the range 0 to 3; typically >0 to 3, and 3+δ indicates the oxygen stoichiometry. When Z is 0, the compound of empirical formula (I) must be rehydrated prior to use. In a fifth embodiment the invention provides a process for increasing the ion exchange capacity of a metal oxide composition having a structure based on or closely related to a structure selected from the group consisting of hexagonal tungsten bronze and pyrochlore, said metal oxide composition having the empirical formula in-line-formulae description="In-line Formulae" end="lead"? A x/n [B y M 1-y O 3+δ ] x .ZH 2 O in-line-formulae description="In-line Formulae" end="tail"? where M is one or more metals in oxidation state (VI); A is a cation selected from the group consisting of alkali metals, alkaline earth metals, silver, ammonium, hydrogen and mixtures thereof; n is the charge on said cation A; B is one or more elements selected from the group consisting of W, Zr, Mo, V, Ti, Fe, Ce, Sb, Nb, Mn, Co, Cr, Fe, Ta, Sn and Cu, provided that M and B are different; x represents the quantity of framework charge; y is a number in the range of 0 to about 0.5; Z is a number in the range >0 to 3; and 3+δ indicates the oxygen stoichiometry; the process comprising the step of reducing at least some of the metal atoms M in said metal oxide composition to an oxidation state less than (VI). In the processes and compositions of the invention, A, the extra framework cation, is typically H + , K + or Na + , and is more typically Na + . In the processes and compositions of the invention, B is typically selected from the group consisting of molybdenum, niobium, tantalum, antimony, vanadium, cerium and copper, and mixtures thereof. It is typical that the process of the first to third embodiments is a column-based ion exchange process or a batch process. It is typical that in the process of the first to third embodiments, the metal cation or cations is/are eluted from the sorbent material after they have been sorbed onto it, by contacting the sorbent material with a concentrated mineral acid or other suitable eluant. Typically, nitric acid is used as an eluant. In the processes of the first to third embodiments, the metal oxide composition is typically in the form of fine particles, more typically having a size in the range of about 0.1 to about 10 microns. Typically, the process of the first to third embodiments takes from between about 0.1 to about 100 contact hours. More typically, the contact time is from between about 0.1 to about 40 hours, most typically from between about 30 min to about 1 hour. In the processes of the invention, the metal ion that is removed from the liquid is typically selected from the group consisting of caesium ions, strontium ions, lead ions, silver ions, transition metal ions, lanthanide metal ions and actinide metal ions and mixtures thereof. More typically, the metal ion is selected from the group consisting of caesium ions, strontium ions and mixtures thereof. In particular, the processes of the first to third embodiments of the invention and the compositions of the fourth embodiment are capable of being used to remove caesium and strontium ions simultaneously from aqueous liquids, more particularly acidic aqueous liquids. Typically in the processes of the first to third embodiments the liquid is an aqueous or substantially aqueous based liquid. In the process of the first to third embodiments, the liquid is typically an acidic aqueous liquid, more typically having a pH of less than about 4. In the processes of the second and third embodiments the liquid may be an organic liquid. Metal oxides of the empirical formula (I) having a structure based on or closely related to the hexagonal tungsten bronze or pyrochlore structures may be prepared by a hydrothermal process. That is, they may be prepared by mixing solutions of the alkali metal salts (typically sodium salts) of one or more oxyacids of metals of Group Vb or Group VIb of the Periodic Table in relative molar amounts corresponding to y and (1-y), adjusting the pH to 1.5 or higher, and heating the mixture under pressure at a temperature of about 100-300° C. more typically 120-230° C., for at least 12 hours, followed by cooling and collecting the solid metal oxide composition by filtration. More usually, the pH will be adjusted in the range of from 1.5 to 2.0 if it is desired to obtain a hexagonal tungsten bronze material or in the range of about 3.5 to 4.5 if it is desired to obtain a pyrochlore material. Adjustment to a pH in the range of 2.0 to 3.5 will typically give a mixture of hexagonal and pyrochlore structures. Even more usually, when M is W, the pH will be adjusted in the range of about 1.5 to 2.0 to obtain essentially only a hexagonal tungsten bronze structure. Accordingly, the invention also provides a process for preparing a metal oxide composition of the empirical formula (I) as defined above, comprising preparing an aqueous solution comprising oxyanions of element(s) B and oxyanions of metal(s) M in relative molar proportions of y to (1-y); adjusting the pH to 1.5 opr higher, more typically in a range selected from (i) about 1.5 to 2.0, and (ii) about 3.5 to 4.5; heating the mixture at a temperature in the range of about 100-300° C., more typically 120-230° C., for at least 12 hours; cooling the mixture and separating the metal oxide composition from the aqueous phase. The group Vb or VIb metal oxide has a crystal structure based on or closely related to hexagonal tungsten bronze or pyrochlore. Examples of these are shown in FIG. 1 . These structures are characterized in that they have 4, 5 or 6 membered ring channels/tunnels capable of accommodating guest or exchangeable ions of suitable size. Each side of the quadrilateral, pentagonal or hexagonal channel is defined by the oxygen atoms that form part of the octahedral coordination polyhedron of the principal framework metal cation. The term “structure based on, or closely related to” as used herein will be understood by those skilled in the art to refer to a material or phase having an ideal crystal structure of the hexagonal tungsten bronze or pyrochlore shown in FIG. 1 , and to structures that are slight modifications of the ideal structures such that one skilled in the art is able to recognize in the modified structures the essential structural elements that confer membership to the ideal structural class. That is, the term “structure based on, or closely related to hexagonal tungsten bronze or pyrochlore” will be understood to mean materials of the hexagonal crystal system with a of 7.3 to 7.5 Å and c of 3.8 to 4.0 Å or to pyrochlores of the cubic crystal system with a of 10.2 to 10.6 Å. Exemplary of such microporous oxide phases are the hexagonal bronzes or pyrochlores of tungsten, niobium, tantalum, and molybdenum. Example 3 and associated FIG. 5 compare the uptake of Cs by a selection of niobate and tungstate sorbents. The following Table 1 also provides selectivity data for Cs and Sr in solutions containing 2 ppm Cs (0.0015 mM), 0.6 ppm Sr (0.0068 mM) and 0.7 M HNO 3 . These data were obtained by contacting 20 mL of the solution with 0.20 g of the sorbent at 25° C. over a 24 h period with occasional agitation. At the end of the contact time, an aliquot of the supernatant was extracted, filtered through 0.20 μm filter elements and analysed for Cs and Sr by Atomic Emission Spectroscopy. Values of the distribution coefficients (K d ) for the various cations, which give an indication of the selectivity of the exchanger, were calculated according to the formula K d = C i - C f C f × V m in which: V=volume of waste simulant (mL), m=mass of the exchanger (g), C i =initial concentration of cation in the supernatant (g/mL), C f =final concentration of cation in the supernatant (g/mL). The K d values were calculated using the weight of the sample as synthesized. TABLE 1 Sorbent K d (Cs)(mL/g) K d (Sr) (mL/g) Nb-pyrochlore 1013 0 W-pyrochlore 5867 20 Mo 0.5 W 0.5 -pyrochlore 2038 21 Mo 0.25 W 0.75 -pyrochlore 3480 16 W-HTB 17000 143 In Table 1, the terms “Nb-pyrochlore” and “W-pyrochlore” refer respectively to compounds having the pyrochlore structure which have the formula Na x NbO 3 .ZH 2 O and Na x WO 3 .ZH 2 O; the term “Mo y W (1-y) -pyrochlore” refers to compounds having the pyrochlore structure which have the formula Na x Mo y W (1-y) O 3 .ZH 2 O; and the term “W-HTB” refers to a compound having the hexagonal tungsten bronze structure which has the formula Na x WO 3 .ZH 2 O. Typically, the ion-exchange process of the first and second embodiments can employ undoped or doped crystalline microporous group Vb and VIb metal oxides such as tungsten and niobium oxides as the sorbent material. By “doped” in this context is meant a composition of empirical formula (I) in which y is non-zero. Typically, the doped microporous tungsten or niobium oxide is a crystalline, electroactive, microporous tungstate with a modified hexagonal tungsten bronze or pyrochlore crystal structure. More typically, the metal oxide is an optionally doped microporous tungsten oxide or optionally doped microporous niobium oxide, most typically optionally doped microporous tungsten oxide. Typically, the microporous tungsten oxide is characterized in that it has a crystal structure that is a derivative of the hexagonal tungsten bronze structure. More typically, the microporous tungsten oxide is a crystalline, electroactive, microporous tungstate with a modified hexagonal tungsten bronze crystal structure. In the compositions of empirical formula (I), it is typical that the value of x, and hence the quantity of contaminant sorbed, is controlled electrochemically. It is also typical that the value of x and hence the quantity of contaminant sorbed, is controlled chemically. For example, the value of x may be controlled chemically by a process of the fifth embodiment by treating a composition of empirical formula (I) with a chemical reducing agent such as hydrazine or other reducing agent either during its preparation or afterwards. Essentially any reducing agent may be used for this purpose. Examples of suitable reducing agents are oxalic acid, thiosulfates such as sodium thiosulfate, amines such as pyridine and hydroxylamine, and reducing gases such as hydrogen or carbon monoxide. Alternatively, the reducing agent may be generated in situ during the preparation of the metal oxide composition by including an amount of the metal M in its elemental form in the acidic medium in which the metal oxide is prepared. Other metallic elements that can partially or completely dissolve in the acid medium to give an oxidisable species can also be used. Examples are Fe, Cr, Zn, Cu, Ni and Ti. It will be appreciated that when a different metal to M is used for this purpose, some of the metal can be expected to be incorporated into the metal oxide composition that is prepared, by substituting for the element M. An electrochemical reduction process according to the fifth embodiment may for example be carried out using an electrochemical cell consisting of the following: (a) a Ag/AgCl or calomel reference electrode; (b) a platinum or other counter electrode; and (c) a working electrode which consists of titanium metal containing a coating of the metal oxide composition sorbent. The working electrode may be prepared by standing a strip of titanium metal sheet upright in a hydrothermal reactor which is then charged with the required solutions, adjusted to the desired pH, for the preparation of the metal oxide composition as described herein. The hydrothermal reactor is heated between 150 and 200° C. and a coating of metal oxide composition is deposited on the titanium substrate. In an exemplary electrochemical process, the electrochemical cell is charged with a suitable simulant solution such as 50 mM CsNO 3 in 1.0 M HNO 3 solution. The potential of the cell is driven from 1000 mV, where the electrode material is in the oxidised state to close to 0 mV where the electrode material is in the reduced (charged) state. In the reduced state W(VI) is largely converted to W(V) and counterions (such as Cs + or perhaps H + ) are required to be inserted from the solution in order to maintain electroneutrality of the sorbent. The potential is held close to 0 mV for about 20 minutes and the sorbent may be observed to change colour. After 20 minutes the cell may be disconnected and after a further 20 minutes the electrode is removed from the simulant. SEM-EDS analysis of the electrode material shows that Cs has been sorbed. One way of modulating the ion exchange properties of the metal oxide sorbent material is by changing B. For instance the specific composition with A=Na, B=Mo and y=0.1 confers particularly high selectivity for Cs and Sr, and also particularly high capacity in acidic solutions. Accordingly, it is typical that the metal oxide used in the processes of the first, second and third embodiments of the present invention has the formula A x/n [B y M 1-y O 3+δ ] x .ZH 2 O where A is an extra framework exchangeable cation such as sodium or potassium, and B is a dopant element such as Mo, V, Ti. In the metal oxide sorbent materials of empirical formula (I), y is typically greater than 0, more typically from about 0.05 to about 0.3, even more typically from about 0.05 to 0.2, still more typically from about 0.1 to about 0.2. Typical embodiments of the invention employ metal oxide compositions of the above formula A x/n [B y M 1-y O 3+δ ] x .ZH 2 O in which B=Mo, A=Na and y=about 0.05 to about 0.3, more typically 0.5 to 0.2, still more typically about 0.1 to about 0.2. Typically, the metal oxide has an X-ray diffraction pattern with at least one broad peak at a d-spacing between 14 and 18 Å. One embodiment of the present invention relates to oxides of the empirical formula (I) where the dopant B has the effect of modifying the structure in such a way as to improve the ion-exchange properties. The nature of the structural modification can involve a distortion, and/or dislocation, and/or disordering, and/or the presence of a second minor phase which is intimately related to the major phase such as an intergrowth and/or changes in surface chemistry/charge. Accordingly, one aspect of the present disclosure relates to the modification of the hexagonal tungsten bronze structure so as to optimise the capacity and selectivity for sorption of cationic species, especially caesium and strontium, from aqueous waste streams, especially acidic ones. For instance, such modification may involve controlled reduction (eg induced chemically, electrochemically, optically or in a radiation field) and associated generation of reduced framework W(V) sites, by a process of the fifth embodiment. Alternatively, low-valency species (e.g. Ti(IV), Mn(II) etc) may be incorporated directly into the framework so as to increase the framework charge. The ability to elute the contaminant ions from the exchanger using strong acid is an important feature of the present invention. The present invention is based on the unexpected discovery that tungsten bronzes and pyrochlores can be applied to the decontamination of waste streams, especially nuclear waste streams. Improvement of the ion exchange properties of tungsten bronzes and pyrochlores resulting from a chemical modification of structural order and/or framework/surface chemistry brought about through compositional adjustment and/or framework reduction is also an important feature discovered by the present inventor on which the present invention is premised. |
Insect trap |
A trap (1) is described which is substantially cylindrical in shape having a base (2), side wall (3) and a removable upper portion (4) which has an upper surface (7) substantially concave in shape. An open-topped mesh basket (6) rests on a mesh (5) support within the trap (1). A light (14) is positioned just above the upper surface (7) of the upper portion (4) which, when energized, attracts the insect to be exterminated. The trap (1) is approximately two thirds filled with water (16) which is then pumped from within the body of the trap to create a substantially helical flow of liquid over the upper surface (7) of the upper portion (4) as it returns to the body of the trap (1). Insects attracted to the light (14) are entrained by the water (16) and are flushed down into the trap and retained within the mesh basket (6). As an alternative, the upper surface (7) can be substantially convex in shape and the water flows substantially radially over the upper surface (7) of the upper portion (4). |
1. A trap to attract and ensnare a flying insect such as a fly, mosquito, aphid, midge, moth or similar, said trap including: a container adapted to hold liquid, said container having a base, a curved upper portion substantially opposed to said base, and a side wall connecting said base and said upper portion; means to recirculate said liquid held in said container from at or near said base over an upper surface of said upper portion; and an attractant for said insect positioned at or near said upper surface of said upper portion; wherein said means to recirculate said liquid and said upper portion are adapted to allow said liquid to flow in a specific path over said upper surface. 2. A trap as defined in claim 1 wherein said liquid is water. 3. A trap as defined in claim 1 which further includes a mesh-like material removably positioned within said container and adapted to allow said liquid to pass through while preventing any said insect that may be within said container from passing through. 4. A trap as defined in claim 1 wherein said attractant is positioned above said upper surface of said upper portion. 5. A trap as defined in claim 1 wherein said attractant is a light source. 6. A trap as defined in claim 5 wherein said light source emits radiation at a wavelength which attracts a specific said insect. 7. A trap as defined in claim 1 wherein said insect is a moth. 8. A trap as defined in claim 7 wherein said moth is a heliathes moth. 9. A trap as defined in claim 1 wherein said means to recirculate said liquid and said portion upper portion are adapted to allow said liquid to flow in a substantially helical path over said upper surface. 10. A trap as defined in claim 9 wherein said upper surface is substantially concave in shape. 11. A trap as defined in claim 1 wherein said means to recirculate said liquid and said upper portion are adapted to allow said liquid to flow radially over said upper surface. 12. A trap as defined in claim 11 wherein said upper surface is substantially convex in shape. 13. A method of attracting and exterminating an insect selected from the group of a fly, mosquito, aphid, midge, moth or similar, said method including the use of a trap as defined claim 1. |
Medicament dispenser |
There is provided a medicament dispenser comprising a housing; a medicament container having a dispensing mechanism; a container seat for receipt of the container; an anchor station on the housing or connecting therewith; and drive means capable of moving the container seat relative to the anchor station to actuate the dispensing mechanism. The drive means is responsive to the application of non-mechanical energy thereto. The medicament dispenser further comprises adjusting means adapted to enable medicament containers of varying sizes to be received by the housing. |
1. A medicament dispenser comprising a housing; a medicament container having a dispensing mechanism; a container seat for receipt of the container; an anchor station on the housing or connecting therewith; and drive means capable of moving the container seat relative to the anchor station to actuate the dispensing mechanism, said drive means being responsive to the application of non-mechanical energy thereto, wherein the medicament dispenser further comprises adjusting means adapted to enable medicament containers of varying sizes to be received by the housing. 2. A medicament dispenser comprising a medicament container having a dispensing mechanism; a container seat for receipt of the container; a dispenser seat for receipt of the dispensing mechanism; and drive means capable of moving the container seat relative to the dispenser seat to actuate the dispensing mechanism, said drive means being responsive to the application of non-mechanical energy thereto, wherein the medicament dispenser further comprises adjusting means adapted to enable medicament containers of varying sizes to be received by the housing. 3. A medicament dispenser comprising a housing; a medicament container for containing medicament for release; an aerosol container having a dispensing mechanism; a container seat for receipt of the aerosol container; an anchor station on the housing or connecting therewith; and drive means capable of moving the container seat relative to the anchor station to actuate the dispensing mechanism to energise released medicament, said drive means being responsive to the application of non-mechanical energy thereto, wherein the medicament dispenser further comprises adjusting means adapted to enable aerosol containers of varying sizes to be received by the housing. 4. A medicament dispenser comprising a medicament container for containing medicament for release; an aerosol container having a dispensing mechanism; a container seat for receipt of the container; a dispenser seat for receipt of the dispensing mechanism; and drive means capable of moving the container seat relative to the dispenser seat to actuate the dispensing mechanism to energise released medicament, said drive means being responsive to the application of non-mechanical energy thereto, wherein the medicament dispenser further comprises adjusting means adapted to enable aerosol containers of varying sizes to be received by the housing. 5. A medicament dispenser according to claim 1, wherein the adjusting means comprises variable engaging means. 6. A medicament dispenser according to claim 5, wherein said the variable engaging means comprises a first engager located on a first part of the medicament dispenser and a second engager located on a second part of the medicament dispenser, wherein the first and second engager are engageable with the medicament container. 7. A medicament dispenser according to claim 5, wherein the adjusting means comprises a clutch and drive plate mechanism, which interconnects with a carriage shaft which houses the medicament container. 8. A medicament dispenser according to claim 5, wherein the adjusting means comprises a rack and wheel mechanism. 9. A medicament dispenser according to claim 5, wherein the adjusting means comprises a flexible bag containing a viscous fluid, suitably located in the base of the housing. 10. A medicament dispenser according to claim 5, wherein the adjusting means comprises a drive wedge mechanism. 11. A medicament dispenser according to claim 1, wherein the dispensing mechanism is selected from the group consisting of a valve, pump or plunger mechanism. 12. A medicament dispenser according to claim 1, wherein the drive means comprises a motor. 13. A medicament dispenser according to claim 1, wherein the drive means comprises a coupling, which deforms, or undergoes a phase transition in response to the application of non-mechanical energy thereto. 14. A medicament dispenser according to claim 13, wherein the coupling comprises a shape memory alloy. 15. A medicament dispenser according to claim 14, wherein the shape memory alloy is a nickel-titanium alloy. 16. A medicament dispenser according to claim 1, wherein the drive means additionally comprises gear means to gear up the torque provided thereby. 17. A medicament dispenser according to claim 16, wherein said gear comprises a gear element selected from the group consisting of cam gears, screw drives, levers, crank shafts, pulleys and hydraulic gears. 18. A medicament dispenser according to claim 1, additionally comprising decoupling means adapted to enable the medicament container to return to a non-dispensing position independent of the drive means. 19. A medicament dispenser according to claim 18, wherein said decoupling means comprises a cam in contact either directly or indirectly with the medicament container and a coupling mechanism extending from the cam to a fixed point on the medicament dispenser. 20. A medicament dispenser according to claim 18, wherein the decoupling means comprising a beam, rotatably hinged at its centre point, said beam being movable between a first non-dispensing position through a dispensing position to a second non-dispensing position. 21. A medicament dispenser according to claim 18, wherein the decoupling means comprising a top crown and a push crown, the two crowns engaging with each other. 22. A medicament dispenser according to claim 1, additionally comprising drive-assisting means adapted to provide a mechanical advantage to the drive mechanism. 23. A medicament dispenser according to claim 22, wherein said drive-assisting means comprises one or more levers and one or more pivot points. 24. A medicament dispenser according to claim 22, wherein the drive-assisting means comprises a rotatable cylinder comprising a downwardly spiralling guide track and a guide arm fixed to the medicament container, wherein the guide arm is locatable within the guide track and the drive means connects to one end of the rotatable cylinder. 25. A medicament dispenser according to claim 22, wherein the drive-assisting means comprises a bowed beam connected at each end to a coupling mechanism, the beam having a notch removed in the lower surface thereof. 26. A medicament dispenser according to claim 22, wherein the drive-assisting means acts such as to change the work profile of the drive mechanism. 27. A medicament dispenser according to claim 1, wherein the medicament dispenser has a use configuration and a storage configuration such that the medicament dispenser in its storage configuration is smaller than when in the use configuration. 28. A medicament dispenser according to claim 27, wherein the housing comprises a telescopic arrangement. 29. A medicament dispenser according to claim 1, wherein the medicament container is an aerosol container. 30. A medicament dispenser according to claim 29, wherein, the aerosol container comprises a suspension of a medicament in a propellant. 31. A medicament dispenser according to claim 30, wherein the medicament is selected from the group consisting of albuterol, salmeterol, fluticasone propionate, fluticasone furoate, beclomethasone dipropionate, salts or solvates thereof and any mixtures thereof. 32. A medicament dispenser according claim 1, wherein actuation of the drive mechanism is responsive to a patient-actuable trigger comprising a sensor which senses the breath of a patient. 33. An actuator for a medicament container having a dispensing mechanism comprising a container seat for receipt of the medicament container; a dispenser seat for receipt of the dispensing mechanism; and drive means capable of moving the container seat relative to the dispenser seat to actuate the dispensing mechanism, said drive means being responsive to the application of non-mechanical energy thereto, wherein the medicament dispenser further comprises adjusting means adapted to enable medicament containers of varying sizes to be received by the housing. 34. An actuator for a medicament container having a dispensing mechanism comprising a housing; within said housing, a container seat for receipt of the medicament container; on the housing or connecting therewith, an anchor station; and drive means capable of moving the container seat relative to the anchor station to actuate the dispensing mechanism, said drive means being responsive to the application of non-mechanical energy thereto, wherein the medicament dispenser further comprises adjusting means adapted to enable medicament containers of varying sizes to be received by the housing. 35. An actuator for a medicament dispenser having a medicament container and separately an aerosol container having a dispensing mechanism comprising a housing, shaped for receipt of said medicament container for containing medicament for release; within said housing, a container seat for receipt of said aerosol container having a dispensing mechanism; an anchor station on the housing or connecting therewith; and drive means capable of moving the container seat relative to the anchor station to actuate the dispensing mechanism to energise released medicament, said drive means being responsive to the application of non-mechanical energy thereto, wherein the medicament dispenser further comprises adjusting means adapted to enable aerosol containers of varying sizes to be received by the housing. 36. An actuator for a medicament dispenser having a medicament container and separately an aerosol container having a dispensing mechanism comprising a housing, shaped for receipt of said medicament container for containing medicament for release; an aerosol container having a dispensing mechanism; a container seat for receipt of said aerosol container having a dispensing mechanism; a dispenser seat for receipt of the dispensing mechanism; and drive means capable of moving the container seat relative to the dispenser seat to actuate the dispensing mechanism to energise released medicament, said drive means being responsive to the application of non-mechanical energy thereto, wherein the medicament dispenser further comprises adjusting means adapted to enable aerosol containers of varying sizes to be received by the housing. 37. A laboratory test apparatus comprising at least one actuator according to any of claims 33 to 36 and a mounting for said at least one actuator. 38. A kit of parts comprising a medicament dispenser according to any of claims 1 to 32 in the form of a cartridge; and a housing shaped for receipt of said cartridge. 39. A kit of parts comprising an actuator according to any of claims 33 to 36 and, receivable by said actuator, a medicament container having a dispensing mechanism. |
Lignin-based polyurethane and process for producing the same |
Provided is a polyurethane which is inexpensive and excellent in physical properties and which contains a lignin sulfonic acid component incorporated into the molecular chain of the polyurethane. The polyurethane is obtainable by polycondensation of a polyol solution containing dissolved therein lignin sulfonic acid or a partially neutralized salt thereof with a polyisocyanate, wherein the content of said lignin sulfonic acid is 1-40% based on a total weight of said polyurethane. |
1. A polyurethane obtainable by polycondensation of a polyol solution containing dissolved therein lignin sulfonic acid or a partially neutralized salt thereof with a polyisocyanate, wherein the content of said lignin sulfonic acid or a partially neutralized salt thereof is 1 to 40% based on a total weight of said polyurethane. 2. A polyurethane as recited in claim 1, wherein said polycondensation is performed in the presence of water so that said polyurethane is in the form of a foam. 3. A process of producing a polyurethane, comprising subjecting a polyol solution containing dissolved therein lignin sulfonic acid or a partially neutralized salt thereof to polycondensation with a polyisocyanate, wherein the content of said lignin sulfonic acid or a partially neutralized salt thereof is 1 to 40% based on a total weight of said polyurethane. 4. A process as recited in claim 3, wherein said polycondensation is performed in the presence of water so that said polyurethane is in the form of a foam. |
<SOH> BACKGROUND ART <EOH>The present inventors found in the past that biodegradable polyurethanes produced from sugars such as monosaccharides and oligosaccharides or from lignins such as solvolysis lignin and craft lignin had excellent physical properties and that the physical properties were further improved when sugars and lignin were used conjointly. As a lignin-based material, known is a lignin sulfonate which is a by-product in a sulfite pulp manufacturing process. Because the lignin sulfonate is, however, insoluble in a polyol, it is extremely difficult to incorporate same into polyurethane molecules. The present inventors have found that lignin sulfonic acid in an acid form or a partially neutralized salt thereof is soluble in a polyol and can be incorporated into molecular chains of a polyurethane. It has also been found that the thus obtained polyurethane has excellent physical properties. It is an objective problem of the present invention to provide a polyurethane which is inexpensive and excellent in physical properties and which contains lignin sulfonic acid or a partially neutralized salt thereof incorporated into the molecular chain of the polyurethane. |
Animal model for allergy |
The invention relates to model systems for allergic conditions, and in particular to in vivo model systems in a large animal. The model systems of the invention are especially useful for providing large numbers of activated or non-activated eosinophils, for the discovery and evaluation of novel anti-inflammatory drug targets and for providing a model for the in vivo study of asthma and the effects of allergy treatments. In a preferred embodiment the animal is a sheep. In one embodiment, repeated infusion of house dust mite allergen (HDM) into the mammary gland is used to induce a specific allergic response, which is characterised by the recruitment of inflammatory cells, particularly eosinophils, into the mammary lumen; these cells can be harvested from peripheral blood and mammary lavage (MAL). In a second embodiment, the mammal is immunised with soluble antigen, for example by repeated subcutaneous immunisation, and then subjected to a single challenge with the same antigen administered directly to the lung. |
1-32. (canceled). 33. An in vivo model system for an allergic condition, comprising a mammal which has been subjected to sensitisation with an antigen or administration of a cytokine involved in response to allergen, in which a) the mammal is a female, and is sensitised by repeated administration of the antigen into the mammary gland; or b) the mammal is of either sex, and is sensitised by administration of the antigen, followed by administration directly to the lung; or c) the mammal is of either sex, and blood and tissue eosinophilia is induced by administration of a cytokine involved in response to allergen, in which the mammal is a member of the order Artiodactyla, and the antigen is not one derived from a helminth parasite. 34. A model according to claim 33, in which the mammal is of either sex, and is sensitised by administration of the antigen, followed by administration directly to the lung, and the allergic condition is one which is associated with eosinophilia and elevated levels of IgE. 35. A model according to claim 33, in which the antigen is selected from the group consisting of house dust mite, animal dander, feathers, plant antigens, moulds, and household or industrial chemicals. 36. A model according to claim 35, in which the antigen is house dust mite. 37. A model according to claim 35, in which the antigen is an extract of house dust mite. 38. A model according to claim 35, in which the animal dander is selected from the group consisting of cat dander, dog dander, bird dander and cockroach dander. 39. A model according to claim 35, in which the plant antigens are selected from the group consisting of grass pollens or tree pollens. 40. A model according to claim 39, in which the grass pollens are ryegrass pollen or Alternaria pollen. 41. A model according to claim 39, in which the tree pollens are birch or cedar pollens. 42. A model according to claim 33, in which the antigen is associated with asthma in humans. 43. A model according to claim 33, in which the cytokine involved in response to allergen is interleukin-5. 44. A model according to claim 33, in which the cytokine involved in response to allergen is eotaxin. 45. A lung model according to claim 33, in which the antigen or molecule involved in response to allergen is administered by intravenous, oral, subcutaneous, intradermal or intramuscular administration, followed by administration directly into the lung. 46. A model according to claim 45, in which the mammal is a ruminant or a pig. 47. A model according to claim 46, in which the mammal is a sheep, goat, or bovine. 48. A model according to claim 46, in which the mammal is a sheep or a goat. 49. A method of preparing a model according to claim 33, comprising the step of administration of antigen or of a cytokine involved in response to allergen to a mammal, thereby to induce a specific allergic response characterised by the recruitment of inflammatory cells into the blood of the mammal. 50. A method according to claim 49, comprising the step of repeated administration of antigen into the mammary gland of a mammal, thereby to induce a specific allergic response characterised by the recruitment of inflammatory cells into the mammary gland of the mammal. 51. A method according to claim 49, comprising the step of repeated administration of antigen into the lung of a mammal, thereby to induce a specific allergic response characterised by the recruitment of inflammatory cells into the lung of the mammal. 52. A method according to claim 49, further comprising the step of collection of the inflammatory cells. 53. A method according to claim 49, in which the administration is intravenous, oral, subcutaneous, intradermal, or intramuscular. 54. A method according to claim 50, in which the administration is subcutaneous. 55. A method according to claim 51, in which the administration to the lung is via a fibre-optic bronchoscope or nebulizer. 56. A method according to claim 51, in which the animal is a ruminant or a pig. 57. A method according to claim 56, in which the mammal is a sheep, goat, or bovine. 58. A method according to claim 57, in which the mammal is a sheep or a goat. 59. Use of a model according to claim 33 for: a) the study of asthma; b) the examination of the effects of chronic allergen exposure; c) in vivo testing of the efficacy of candidate drugs for the treatment of asthma; d) in vivo screening or testing of new anti-inflammatory drugs, therapies, and/or procedures; or e) in vitro screening assays for the development of new anti-inflammatory or anti-eosinophil degranulation drugs. 60. Use according to claim 59, in which candidate targets for anti-allergic drug targets are identified using molecular or biochemical techniques. 61. Use according to claim 60, in which the techniques are genomic, proteomic, or glycomic techniques. 62. Use according to claim 60, in which the techniques are differential display, representational difference analysis, microarrays, or 2-dimensional electrophoresis. 63. Inflammatory cells obtained by a method according to claim 52. 64. Use of inflammatory blood or MAL cells according to claim 63 for: a) the identification of processes or molecules differentially active or expressed in “activated” and “non-activated” eosinophils and/or other inflammatory cells; b) identification of processes and molecules involved in the recruitment of eosinophils and/or other inflammatory cells; c) identification of processes and molecules involved in degranulation of eosinophils and/or other inflammatory cells; d) in vivo testing of the efficacy of candidate drugs for the treatment of asthma; e) in vivo screening and testing of new anti-inflammatory drugs, therapies, and/or procedures; or f) in vitro screening assays for the development of new anti-inflammatory or anti-eosinophil degranulation drugs. |
<SOH> BACKGROUND OF THE INVENTION <EOH>All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country. The prevalence of allergic diseases, in particular asthma, has increased dramatically in the last 20 years, doubling in Westernised societies. The severity of asthma is a particularly serious health issue in Australia, as it has one of the highest incidences of asthma in the world, with 1 in 4 children suffering from this condition. Allergic asthma is an immunological disease associated with significant physiological changes in the lungs. The underlying immunological mechanisms directing the asthmatic response in the lungs are not clearly understood; however, a significant correlation between mast cells and eosinophils and the pathology of asthma has now been recognised. In particular, the pathophysiology of human asthma, including the development of airway hyperresponsiveness, is associated with the appearance of “activated” eosinophils and molecules released by these cells in bronchoalveolar lavage (BAL) fluid and in lung tissue (Walker et al, 1991; Desreumaux and Capron, 1996). Therefore there is a need in the art to investigate the processes involved in activation of eosinophils in an allergic response to a well-defined allergen, and to identify agents which can modulate this response. Eosinophils are produced in the bone marrow and released into circulation where they migrate to inflammatory or parasite-infected sites. Stimuli present within the tissue microenvironment can cause eosinophils to become “primed” or “activated”, a state in which the ability of the eosinophil to carry out its effector functions is fully developed (Jones, 1993). One manifestation of eosinophil activation is an enhanced capacity to mediate antibody-dependent killing of helminth larvae. Increased respiratory burst activity, resulting in the release of toxic oxygen metabolites, and increased release of lipid mediators, such as leukotriene C4 and platelet activating factor, are associated with eosinophil activation and parasite killing. A classic marker for the activation of eosinophils is the release of pre-formed granule proteins, both spontaneously and in response to exogenous stimuli (Butterworth and Thorne, 1993). These granule proteins are known to be toxic to helminths. In commonly used experimental systems in mice or humans it is very difficult to obtain large numbers of inflammatory cells, in particular eosinophils, because even in tissues where these cells are most prevalent they constitute only a small percentage of resident cells, and they can be isolated only with difficulty from these tissues. It is therefore not feasible to use normal eosinophils from these species for high through-put screening. Recently, an eosinophil cell line has been developed which could be used for screening, but since this is an immortalised cell line, it may react quite differently from normal cells, and does not provide an adequate model. Animal models of disease allow defined and controlled investigations of key issues in disease progression to be carried out, with the possibility of being able to relate findings to the human situation. Studies in mice in particular have used powerful tools such as genetic knock-outs, knock-ins, and neutralisation of specific molecules to demonstrate an important role for the cytokines interleukin-4 (IL-4) and interleukin-5 (IL-5) [Grunig et al, 1998], and more recently interleukin-13 (IL-13) [Grunig et al, 1998; Wills-Karp et al, 1998], in the pathophysiology of asthma. Unfortunately the smaller animal models, particularly those in mice, are limited, because they are not amenable to repeated sampling of cells, and/or because they yield only small numbers of cells for further studies. In addition, the development and physiology of the mouse lung is very different from that of human lung, and many of the pathological phenomena typical of human asthma are not adequately reproduced in the mouse models (Bice et al, 2000). Factors which may be responsible for the shortcomings of the mouse as a model for human asthmatic disease include poor development of smooth muscle structure associated with the lung airways, and poor responses to histamine in mice [Karol, 1994]. Sheep and other ruminants such as goats, and some non-ruminant animals such as pigs, have closer developmental and physiological similarities with humans than do mice, and are widely used as models for human physiological processes, including use of these animals in studies of immunological function. See for example “Handbook of Vertebrate Immunology” ed. P-P Pastoret et. al.,1998. In addition, large amounts of tissues and cells can be repeatedly harvested from a single such animal. It has previously been demonstrated that the allergic response in sheep lungs closely reproduces the development of the human asthmatic response, including a characteristic early- and late-phase asthmatic response, and bronchial hyperresponsiveness [Abraham et al, 1983; Fujimoto et al, 1996]. While sheep are now widely used to study the pharmacological effects of new anti-allergic compounds [Fujimoto et al, 1996; Fath et al, 1998; Abraham et al, 2000], so far none of the physiological studies in sheep have been combined with a detailed analysis of the associated immunological events. Although there have been reports of a model for allergic asthma using rhesus monkeys sensitised with house dust mite allergens (Schelegle et al, 2001) and dogs sensitised with Ascaris or ragweed allergens (Bice et al, 2000), there is still a need in the art for an IgE-specific large animal model of asthma. In particular, the monkey model requires repeated intranasal challenge following initial subcutaneous sensitisation, full anaesthesia of animals for measuring airway responsiveness, and is too expensive for large scale and detailed drug evaluation. All of the previously-available sheep models of asthma have utilised acute allergic responses against an allergen derived from a nematode parasite, Ascaris suum , which is not an antigen relevant to asthma in humans. The use of Ascaris suum as the allergen in sheep asthma models was described about 20 years ago; no other allergens have been investigated in such a system, and no detailed immunological studies of the inflammatory response induced by the Ascaris antigen have been reported. Ascaris -sensitised sheep are an inefficient physiological model for asthma, as only a small proportion of the sensitised sheep respond with the desired late-phase asthmatic response, which must be measured using complicated lung-function test equipment, and responders must be identified by trial and error. Different breeds of sheep may also react differently to Ascaris sensitisation; for example, only a small proportion of Australian merino sheep seem to respond. The expectation in the art was that sheep would only react to very strongly allergenic allergens such as Ascaris , and that therefore this approach is very strictly limited in its applicability to human allergies. A sheep mammary infusion model has been described previously for the collection of large numbers of eosinophils for parasite killing assays (Rainbird et al, 1998; Duffus and Franks, 1980) and for the study of the cellular kinetics of an allergic-type response (Greenhalgh et al, 1996; Bischof and Meeusen, 2002). In these studies, parasite larvae or parasite extracts were infused through the teat canal into the mammary gland, and leukocytes thus induced to migrate into the mammary lumen were collected by infusion of sterile saline, followed by “milking” of the glands. While the basic technique has been known for some time, this method was mainly used for performing parasite killing assays, and more recently for basic studies of inflammation (Greenhalgh et al, 1996; Rainbird et al, 1998; Bischof and Meeusen, 2002). Its use for identifying novel target molecules or for high through-put in vitro screening assays has not previously been suggested, and is not a logical extension from the prior art. It is now realised that long-term structural and functional changes to lung tissues, usually referred to as airway remodelling, in patients suffering from chronic asthma lead to significant increases in morbidity. The underlying biological processes involved in airway remodelling are poorly understood, and scientific progress in this area has been severely restricted by the lack of a suitable experimental system. Various mouse models of asthma exhibit some, but not all, of the morphological and functional lesions of the chronic human disease. A recently-described mouse model involving inhalation of ovalbumin aerosols shows subepithelial fibrosis, mucous cell hyperplasia, chronic inflammation of the lamina propria, and accumulation of intraepithelial eosinophils, but does not exhibit mast cell recruitment into the airway wall, or increase in smooth muscle mass (Kumar and Foster, 2001). Clearly, better animal models reflecting the human situation are required. We have developed two novel approaches for the study of allergic responses in sheep, other ruminants, and pigs, which have distinct advantages over existing models for the discovery of novel therapeutic molecules and processes: (a) a mammary infusion model for the collection of large numbers of eosinophils at different stages of activation, and (b) an asthma model based on sensitisation with allergens which affect humans, such as an extract of the house dust mite, Dermatophagoides pteronyssinus (HDM),ragweed pollen, or food allergens. |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention generally provides an in vivo model system for an allergic condition, comprising a mammal of the order Artiodactyla, a non-human primate, or a member of the family Canidae, which has been subjected to allergic sensitisation with an antigen, with the proviso that the antigen is not one derived from Ascaris suum. In a first aspect, the invention provides an in vivo model system for an allergic condition, comprising a mammal which has been subjected to sensitisation with an antigen or administration of a molecule involved in response to allergen, in which a) the mammal is a female, and is sensitised by repeated administration of the antigen into the mammary gland; or b) the mammal is of either sex, and is sensitised by administration of the antigen, followed by administration directly to the lung; or c) the mammal is of either sex, and blood and tissue eosinophilia is induced by administration of a molecule involved in response to allergen, in which the mammal is not a rodent, and the antigen is not one derived from Ascaris suum. The antigen may be any antigen which is capable of inducing allergic sensitisation. Allergens contemplated to be suitable for use in the invention include those from house dust mite, animal danders such as cat, dog or bird dander, feathers, cockroach, grass pollens such as those from ryegrass or alternaria, tree pollens such as those from birch or cedar, other plant allergens, moulds, and household or industrial chemicals. Preferably the antigen is one which is associated with asthma in humans. In a particularly preferred embodiment the antigen is an extract of the house dust mite, Dermatophagoides pteronyssinus (HDM). The order Artiodactyla includes sheep, goats, cattle, pigs, deer and antelope. Preferably the animal of this order is a ruminant, such as a sheep, goat, or cow, or is a pig. More preferably the mammal is a sheep or a goat. The order Primates includes apes, Old World and New World monkeys, lemurs and tarsiers. Preferably the non-human primate is an ape or a monkey, more preferably a rhesus monkey ( Macaca mulatta ). The family Canidae includes dogs, wolves, jackals, and the like. Preferably the animal of this family is a dog. In one embodiment of this method, repeated infusion of house dust mite allergen (HDM) into the mammary gland is used to induce a specific allergic response, which is characterised by the recruitment of inflammatory cells, particularly eosinophils, into the mammary lumen; these cells can be harvested from peripheral blood and mammary lavage (MAL). The development of eosinophilia in blood and tissues after allergen challenge is due to the induction of host regulatory molecules (e.g. cytokines) which drive the increased production of eosinophils from the bone marrow and their recruitment via the blood to the allergen-challenged tissue. Mammary and/or peripheral blood eosinophilia can therefore also be induced directly by administering host molecules involved in the response to allergens(e.g. cytokines such as interleukin-5 and eotaxin) (Foster et al, 2001). The large numbers of inflammatory blood and MAL cells collected by these procedures can be used for the following applications: (a) Identification of processes and molecules differentially active or expressed in “activated” and “non-activated” eosinophils and other inflammatory cells; (b) Identification of processes and molecules involved in the recruitment of eosinophils and other inflammatory cells; (c) Identification of processes and molecules involved in degranulation of eosinophils and other inflammatory cells; (d) In vivo screening and testing of new anti-inflammatory drugs and therapies; and (e) Use of inflammatory blood and MAL cells, including but not limited to eosinophils, for in vitro screening assays for the development of new anti-inflammatory or anti-degranulation/activation drugs. In a second embodiment, the mammal is immunised with soluble antigen, for example by repeated subcutaneous immunisation, and then subjected to a single challenge with the same antigen administered directly to the lung. Preferably the lung challenge is administered using a fibre-optic bronchoscope; this permits localised delivery of the antigen challenge deep into the caudal lobe of the lung. For repeated sensitisation and evaluation of airway mechanics, the antigen is preferably administered as an aerosol. This embodiment of the model of the invention provides a direct model system for the study of asthma, in which broncho-constriction can be measured in un-anaesthesised animals. The effects of chronic allergen exposure, including tissue remodelling, can be examined. Airway remodelling is also characteristic of chronic asthma. This model is also suitable for in vivo testing of the efficacy of candidate drugs or drug delivery methods for the treatment of asthma, including the testing of long-term therapeutic procedures. This model is also suitable for studies of airway remodelling. The model of the invention provides a convenient system in which a reproducible inflammatory response can be induced, and can be studied with significantly greater ease than has hitherto been possible. The present application describes for the first time: (a) the use of a major human allergen, house dust mite extract (HDM), in a sheep asthma model, (b) a correlation between high IgE responder (atopic) sheep and the induction of a sustained allergic response (eosinophil recruitment) in the lung after challenge, consistent with the human situation, and (c) the chronic stimulation of sheep lungs with HDM to induce tissue remodelling changes of the kind which are typical of chronic asthma in human patients. As a result of the well-known physiological similarity between sheep and human respiratory systems, and between humans and other primates, we expect that the sheep models can readily be extended to non-human primates. Similarly, dogs have widely used in studies of allergy and asthma; see for example Bice, et al. (2000). We therefore also expect that the sheep models can be extended to dogs. The allergens used in the model according to the invention may be administered by any suitable route, and the person skilled in the art will readily be able to determine the most suitable route and dose for the condition to be induced For example, in the mammary infusion model antigen is infused directly into the teat canal. For the lung model, initial sensitization may be effected by a variety of routes; however, preferably the antigen is administered by oral, subcutaneous, intradermal or intramuscular injection, more preferably by subcutaneous injection with alum as adjuvant. Optionally other adjuvants or immunomodulators such as Freund's adjuvant, iscoms or cytokines may be used. Many alternative adjuvants are known in the art. It is known that interleukin-5 (IL-5) induces eosinophilia and eotaxin recruits eosinophils into tissues; for example IL-5 gives a high eosinophil response in a variety of animal models (Foster et al, 2001). We therefore expect that the model of the invention can be reproduced by treatment of animals with IL-5 or eotaxin. Preferably this modification is used with the mammary model of the invention. The nature of the carrier or diluent, and other excipients, which are used for the allergen will depend on the allergen and the route of administration, and again the person skilled in the art will readily be able to determine the most suitable formulation for each particular case. For example, methods and pharmaceutical carriers for preparation of pharmaceutical compositions are well known in the art, as set out in textbooks such as Remington's Pharmaceutical Sciences, 20th Edition, Williams & Wilkins, Pennsylvania, USA. For the purposes of this specification it will be clearly understood that the word “comprising” means “including but not limited to”, and that the word “comprises” has a corresponding meaning. |
Use of a peptide which activates guanylate-cyclase c for the treatment of respiratory airway problems via the airways, medicament, inhaltion devices and method of diagnosis |
The invention relates to the use of a guanylate cyclase C activated peptide for the treatment of respiratory airway problems and problems associated with ventilation disorder and/or mucous secretion disorders via the airways, in addition to a medicament which is fed via the airways. The invention also relates to an inhalation device which contains the medicament and a method for diagnosing the illnesses associated with inhalation disorders and mucous secretion disorders in the airways, by detecting a gualylate cyclase C activated peptide. The peptides which are used are guanylin, uroguanylin and lymphoguanylin or a heat resistant enterotoxin. |
1. The use of a peptide which activates guanylate cyclase C for producing a medicament for the treatment of respiratory tract disorders and disorders associated with impairments of ventilation and/or impairments of mucus secretion via the airways, the medicament being formulated in such a way that the peptide is delivered on the air side of the respiratory tract, namely to the apical membrane of the mucosal epithelial cells. 2. The use as claimed in claim 1, characterized in that the peptide is a natural or recombinant guanylin, uroguanylin, lymphoguanylin or heat-resistant enterotoxin, or is a peptide which is homologous thereto and has substantially the same function, in particular such a peptide variant having a sequence modification linked through deletion, insertion or exchange of one and/or more amino acids, sequence-extending attachment of one and/or more amino acids and/or chemical derivatization, in particular of the terminal amino acids). 3. The use as claimed in claim 1, characterized in that the peptide includes one of the sequences Seq. ID 1 to Seq. ID 7. 4. The use of a peptide as indicated in claim 1 for producing a diagnostic aid for respiratory tract disorders and disorders associated with impairments of ventilation and/or impairments of mucus secretion. 5. A medicament in a preparation which is delivered via the airways to the apical membrane, characterized in that it comprises at least one peptide which activates guanylate cyclase C. 6. The medicament as claimed in claim 5, characterized in that the peptide is guanylin, uroguanylin, lymphoguanylin or a heat-resistant enterotoxin, or a peptide which is homologous to the latter and has substantially the same function, in particular such a peptide variant having a sequence modification linked through deletion, insertion or exchange of one and/or more amino acids, sequence-extending attachment of one and/or more amino acids and/or chemical derivatization, in particular of the terminal amino acids, or a peptide mixture comprising at least one of these peptides. 7. The medicament as claimed in claim 5, characterized in that at least one of the peptides comprises one of the sequences Seq. ID 1 to Seq. ID 7. 8. The medicament as claimed in claim 5, characterized in that, besides the at least one peptide, the medicament comprises as active ingredient at least one further active ingredient and, where appropriate, excipients and additives. 9. The medicament as claimed in claim 5, characterized in that the medicament is in the form of an inhalation means and comprises at least one propellant, at least one aerosol former or at least one smoke former. 10. An inhalation device comprising the medicament as claimed in claim 5. 11. The inhalation device as claimed in claim 10, characterized in that it comprises a spray device, in particular a metering spray device or a metering inhaler. 12. A method for diagnosing disorders which are associated with impairments of ventilation and impairments of mucus secretion in the respiratory tract through detection of at least one peptide which activates guanylate cyclase C. 13. The method as claimed in claim 12, characterized in that the detection is directed at at least one of the sequences Seq. ID 1 to Seq. ID 7. 14. The method as claimed in claim 12, characterized in that the peptide is detected in exudate, bronchial mucus or lavage. 15. The method as claimed in claim 12, characterized in that a concentration of at least one of the peptides which activate guanylate cyclase C differing from comparison samples from healthy subjects is regarded as a positive test result for the detection of an impairment. |
Article of footwear, binding assembly and article of footwear-binding assembly combination |
The present invention relates to an article of footwear which consists of a forward portion and a rear portion which are articulated to one another about an axis that is almost coincident with the metatarsophalangeal joint in the hallux when the foot is located in the article of footwear, a binding assembly for releasable attachment of the article of footwear, in particular a ski boot, to a sports device, in particular a ski, under the toe portion of the article of footwear about an essentially horizontal axis almost perpendicular to the longitudinal direction of the article of footwear which, after fastening, permits a rotational movement about the axis where none of the elements in the unit have faces which rotate directly in contact with elements in the article of footwear, and an article of footwear-binding assembly combination which comprises an article of footwear and a binding assembly. |
1. An article of footwear, whereby it consists of a forward portion and a rear portion which are articulated to one another about an axis which is almost coincident with the metatarsophalangeal joint in the hallux when the foot is located in the article of footwear, and that the toe portion under the forward portion of the article footwear is designed for articulation with a sports device about an essentially horizontal axis almost perpendicular to the longitudinal direction of the article of footwear. 2. An article of footwear according to claim 1, wherein the article of footwear is a ski boot and the sports device is a ski. 3. An article of footwear according to claim 1, wherein the article of footwear is made of a torsion-proof material that is not deformed during use. 4. An article of footwear according to claim 2, wherein the elements in the articulation with the sports device, during use, rotate about faces which are both located in the binding. 5. An article of footwear according to claim 2, wherein the elements in the articulation with the sports device, during use, rotate about faces which are both located in the article of footwear. 6. An article of footwear according to claim 2, wherein the horizontal axis is located in the area between the front edge of the article of footwear and the axis. |
Method to genetate non virulent microorganisms from pathogenic ones through permanent genetic modification of their biological membrane for vaccine production |
Here we describe a methodology to generate non virulent microorganisms from pathogenic ones through permanent genetic modification of the physical state of their membrane (MPS). Thus, at the onset of infection, in these transformed organisms, as they infect a host (e.g. a target cell of a higher eukaryote, particularly mammals, and more specifically human cells, or injecting them in a model of animal infection, the expression of heat shock (stress) genes and the accumulation of the coded proteins (stress proteins or HSPs) and that of other species-specific gene products, the regulation is altered as a consequence of the coded modification of MPS. Among others, we refer also to genes whose regulation is mediated by signaling transduction pathways. Therefore, as a result of this procedure, pathogens became non-virulent (attenuated, live microorganisms) that can be used for vaccine production. |
1-26. (canceled) 27. Method to transform pathogenic micro-organisms into non virulent ones comprising the step of modifying the physical and/or dynamic state of their biological membrane (MPS) by genetic modification of a gene whose product modulates the membrane saturated fatty acid/unsaturated fatty acid (SFA/UFA) or the protein/lipid ratios. 28. Method according to claim 27 wherein the modified physical and/or dynamic state of biological membranes alters the capacity of the pathogens to accumulate at the onset of infection an appropriate amount of stress proteins. 29. Method according to claim 27 wherein said genetic modification is an overexpression. 30. Method according to claim 27 wherein the genetic modification is obtained according to the following main steps: construction of a vector containing a gene under the control of a promoter, that regulates the expression of a gene whose protein product is able to modify the physical and/or dynamic state of the membranes of the microbial pathogens in which such vector is inserted; genetic transformation of pathogens with such a vector; expression of the protein product with such vector. 31. Method according to claim 30 wherein the gene whose protein product is able to modify the physical and/or dynamic state of the membranes of the microbial pathogens is selected in the group consisting of: desaturase genes, genes whose product is a membrane bound protein. 32. Method according to claim 31 wherein said desaturase gene is selected from: Δ12-desaturase gene, Δ9-desaturase gene, Δ6-desaturase gene, other desaturase genes, genes coding for integral membrane proteins. 33. Method according to claim 30 wherein said protein product is an enzyme that modifies the level of unsaturation of membrane fatty acids and phospholipids. 34. Method according to claim 30 wherein said membrane physical state modification consists in changes of membrane permeability. 35. Method according to claim 30 wherein said membrane physical state modification consists in changes of thermal phase transition profile of membrane. 36. Method according to claim 27 wherein the pathogenic micro-organisms are selected in the group of: strictly intracellular bacteria, facultative intracellular bacteria, fungi and parasites and non-intracellular pathogens. 37. Method according to claim 27 wherein the pathogenic microorganisms are selected in the group: Chlamydia species, such as pneumoniae and trachomatis, Coxiella burnetii, Ehrlichia chaffeensis, Rickettsiae; Legionella pneumophila, Mycobacteria, such as M. tuberculosis, M. marinum, M. leprae, Nocardia species (mycetoma), Bartonella species, Brucella species, Francisella tularensis, Listeria monocytogenes, Salmonella species, Shigella species; Borrelia burgdorferi (Lyme disease), Treponemapallidum, Campylobacter, Haemophilus influenzae Klebsiella pneumoniae, Leptospira interrogans, Neisseriae species, Staphylococci, Streptococcus pyrogenes, S. agalactiae, S. pneumoniae, Yersiniae, Bacillus anthracis; Aspergillus fumigatus, Candida species, Cryptococcus neoformans, Histoplasma capsulatum, Pneumocystis carinii; Entamoeba histolytica, Leishmania species, Plasmodium falciparum and vivax, Toxoplasma gondii, Trypanosoma cruzi. 38. Method for the production of attenuated non virulent pathogenic micro-organisms, selected among S. typhimurium, M marinum, and H. capsulatum, comprising the following steps: construction of a vector; transformation of the pathogen with such a vector that expresses, under the control of a promoter regulating the expression of a downstream gene during infection of the pathogen, one of the following genes: Cyanobacterium Synechocystis PCC6803 Δ12-desaturase gene or Δ9-desaturase of the virulent H. capsulatum G217B strain, S. cerevisiae or H. capsulatum Δ9-desaturase gene, or other desaturase genes of prokaryotic or eukaryotic organisms or other genes coding for integral membrane proteins that cause a perturbation of the physical and/or dynamic state of said biological membranes; over-expression of the inserted gene. 39. Method according to claim 38 wherein the promoter is an up-regulated promoter of the Downs strain. 40. Salmonella obtained with a method according to claim 38 characterized by a protein/lipid ratio of isolated outer membrane that is about 100 in the virulent strain and 170 in the genetically modified strain. 41. M. marinum obtained with method according claim 38 characterized by protein/lipid ratio of the isolated outer membrane that is increased 40% in the genetically modified strain. 42. Pathogenic micro-organisms obtained with the method according to claim 27 and characterized for being attenuated in their mechanism of virulence but immuno-competent. 43. Modified micro-organisms obtained with the method according to claim 27 to be used in medical applications. 44. Modified micro-organisms obtained with the method according to claim 27 for the manufacture of a vaccine. 45. Use of modified micro-organisms according to claim 43 to produce vaccines. 46. Vaccines comprising as active ingredient an effective amount of modified microorganisms according to claim 44 in combination with suitable excipients and additives. 47. Vaccines according to claim 46 in which the microorganisms are selected in the group comprising: Chiamydia species, such as pneumoniae e trachomatis, Coxiella burnetii, Ehrlichia chaffeensis, Rickettsiae; Legionella pneumophila, Mycobacteria, such as M. tuberculosis, M. marinum, M. leprae, Nocardia species (mycetoma), Bartonella species, Brucella species, Francisella tularensis, Listeria monocytogenes, Salmonella species, Shigella species; Borrelia burgdorferi (Lyme disease), Treponema pallidum, Campylobacter, Haemophilus influenzae Klebsiella pneumoniae, Leptospira interrogans, Neisseriae species, Staphylococci, Streptococcus pyrogenes, S. agalactiae, S. pneumoniae, Yersiniae, Bacillus anthracis; Aspergillus fumigatus, Candida species, Cryptococcus neoformans, Histoplasma capsulatum, Pneumocystis carinii; Entamoeba histolytica, Leishmania species, Plasmodium falciparum, vivax, Toxoplasma gondii, Trypanosoma cruzi, and relative combinations. 48. Vaccines according to claim 46 formulated to be administered intradermically, intramuscularly, intravenously, in the mucosa, nasally, vaginally, orally and rectally. |
<SOH> BACKGROUND ART <EOH>The Heat Shock Response, or stress response, is one of the better studied homeostatic cell responses, mainly involved in the maintenance of cell functionality in response to diverse environmental stresses and/or in pathologic states. This response is mediated by a rapid increase of the transcription of those genes that codify for the stress proteins (Lindquist. 1986). It has been largely demonstrated that such increase in mRNA synthesis of stress genes, and the relative intracellular accumulation of HSPs, is associated with the acquisition of thermotolerance, with protection to subsequent exposure to other forms of stresses or in pathological conditions, etc. (Singer & Lindquist 1998; van Eden & Young 1996). It has been demonstrated that the primary sensor(s) of temperature variations, and in general to other forms of stresses, is (are) localized in the membrane (Carratu et al 1996; Horvath et al 1998, Vigh & Maresca, 1998; Suzuki et al 2000, Piper et al 2000; Torok et al 2001; Vigh & Maresca, 1998). Further, recent studies have shown that an abrupt temperature change or exposure to other forms of stress, determine a physical re-organization of lipid and protein membrane components (Slater et al 1994), that is followed by a specific gene response aimed to compensate variations in MPS. Thus, a cross-talk between changes in MPS and regulation of gene expression exists, particularly for heat shock genes. We have focused our attention on the crucial role of membranes as primary targets of heat stress and have attempted to understand how proper lipid/protein interactions within the membrane determines the transcriptional regulation of HS genes. Such molecular interactions have been shown to be critically involved in the conversion of physical and chemical signals from the environment into sequential processes culminating, in a specific manner, in the transcriptional activation of stress regulated genes. In turn, the interactions between certain HSPs and specific regions (domains) of membranes remodel the status of membrane physical state (overall phase state, order, permeability, etc.). We have shown that the specificity of gene expression is obtained by the uneven distribution of these membrane domains that precisely sense biological and physical environmental regulating signals and different forms of stresses. These studies have strongly modified our vision of the functions of biological membranes. We proposed that the composition, organization and physical state of membranes play central and determining roles in the cellular responses during acute heat stress and pathological states (Vigh & Maresca, 1998). Among the agents responsible for an appropriate MPS, we mention the desaturase enzymes, that through their enzymatic activities, control membrane phospholipid composition. Desaturases are enzymes that introduce double bonds in SFA to form UFA. The ratio SFA/UFA is one of the critical factors that determine an appropriate MPS in all cells (Cossins, 1994). Recently, it has been shown that synthesis of inducible HSPs is controlled by abrupt and local variations of a number of factors that include: Membrane lipid composition Membrane lipid/protein interactions Membrane lipid dynamic (MPS modification) (Vigh et al 1998, Vigh & Maresca, unpublished, 1998) Thus, change of the MPS under stress condition re-determines the threshold value at which HSPs are normally synthesized. Intracellular pathogens, such as Salmonella typhimurium, Mycobacterium tuberculosis, Mycobacterium marinum Histoplasma capsulatum , trypanosomes, etc., at the onset and during infection of macrophages and of other cells, induce a genetic response through transcriptional activation of stress genes and other sets of species-specific genes, here defined, in general terms, as virulence genes. The gene products are directly involved in the mechanisms of invasion/adaptation, operate in a coordinate fashion, and are responsible of the capacity for the pathogen to invade, replicate and induce disease (virulence genes) in the host (Groisman & Ochman 1997). This vast, coordinated and generalized genetic response allows intracellular pathogens such as S. typhimurium, M. marinum to induce the disease avoiding the immune response of the host. All bacteria, fungi and parasites in general, agents responsible for deadly diseases not yet eradicated, such as salmonellosis, typhoid fever, tuberculosis, histoplasmosis, candidosis, malaria, trypanosomiasis, etc, induce HSPs as an essential part of their response to the conditions encountered in the host at the onset of infection (Groisman & Saier 1990). Hsp70s of eukaryotic pathogens and bacterial Hsp60 (GroEL) are primary antigens, that constitute up to 15% of the cell's dry weight (Feige and van Eden, 1996). Thus, stress genes and those involved in virulence are strictly interconnected and genetically coordinated (Groisman & Ochman 1997). While the details of the regulation of these virulence genes have not elucidated yet, it is known that the proper expression of these sequences is also under the control of an appropriate amount of HSPs that are synthesized at the onset of infection. The traditional methods to fight pathogens are believed to be scarcely effective since they are based on the use of vaccines that stimulate an immune response against one or few antigens. For example, some strains of Mycobacterium tuberculosis , the etiologic agent of tuberculosis, have become progressively resistant to the available antibiotics. Since the identification of new effective non toxic antibiotics requires many years of experimental work and huge investments, it is urgent to produce new types of vaccines against the new resistant strains. Regarding Salmonella , the present vaccines are: Vaccines with attenuated strains injectable parenterally; Vaccines made of purified bacterial capsules that can be injected parenterally; A Ty21 vaccine composed of attenuated strains of Salmonella administered orally. Such vaccines, though give a permanent immunity, have several disadvantages. For example, it is not determined that vaccines made of killed bacteria are virtually free of live cells, and that the attenuated ones do not revert to the virulent form. It is also possible that vaccines obtained with killed organisms contain substances, cells or traces of medium that may be toxic for humans. Further, vaccines with killed organisms often have high incidence of side effects, that combine with a low level of protection. Vaccines made of infective attenuated particles, obtained by a rapid thermal or chemical treatment, may contain antigens whose physiological protein folding is modified (denatured) thus altering the natural immunological response. Vaccines not involving the use of attenuated strains are generally composed of a single or a few antigens that give only a partial and incomplete protection compared to the natural immunological response. In general, this has a drawback that results in the low efficacy of today's vaccines, since, in man, as in mammals in general, and more in general in higher eukaryotes, the natural mechanisms of protections against infective agents comprise a complex immunological response against the entire microorganism and all its combined antigens. Authors have now hypothesized, and experimentally proved, that, altering the physiological MPS and the stress response of pathogens, it is possible to attenuate strains to be utilized for vaccine production, voided of the above mentioned side affects. |
<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the present invention to provide a method for the permanent genetic modification of microorganism's MPS, in particular intracellular pathogens (e.g. bacteria, fungi, parasites) such as S. typhimurium, M. marinum, H. capsulatum , trypanosomes, etc. with the aim of altering the synthesis and accumulation of HSPs in such pathogens at the onset of infection and of those species-specific gene products, whose regulation is modified as consequence of MPS, including genes whose activation is mediated by signaling transduction pathways (e.g. c-fos, fos B, junB, junD, MAP kinase, genes), where the host can be a target cell such as a macrophage, cells of higher organisms in general, or of mammals or of humans. Therefore, as a result of this modification, pathogens become non-virulent (attenuated). Another object is the use of these attenuated strains to produce vaccines. A further object is the development of a new class of vaccines for human use, and for animals in general, comprising the modified pathogens. A further object is the method for producing attenuated non-virulent strains of pathogenic microorganisms, such as S. typhimurium, M. marinum, H. capsulatum , etc. through the transformation with a vector carrying and express a gene coding for Δ 12 -desaturase of Synechocystis PCC6803 or for other desaturase genes of Salmonella or for other prokaryotic and eukaryotic microorganisms (e.g. Δ 9 -desaturase of S. cerevisiae or of H. capsulatum ) or for genes coding for integral membrane proteins that cause a perturbation of MPS (lipid phase transitions, permeability). With the genetic modification produced according to the invention it is possible to obtain, for example in Salmonella , an increases in the ratio protein/lipid of total membrane (mixture of outer and inner or cytoplasmic membranes) from about 100 (virulent strain) to about 170 (strain genetically modified) ( FIG. 4 ) or, for example in M. marinum a decrease of at least 4° C. in the mutant strain of the major lipid phase transition of its outer cell membrane. The method can be extended. to other intracellular pathogens listed herein below as a non-limitative example. Further objects will be evident form the following detailed description of the invention. |
High efficiency silicon light emitting device and modulator |
The present invention provides a high performance silicon light emitting device. A method and device providing both emission and modulation from a single device is provided, with modulation of the emission characteristics being achieved by application of an electric field across the device, so as to induce quantum confined Stark effects, Franz-Keldysh effects or the like. |
1. A light emitting device with alterable emission characteristics, the device comprising: a light emitting portion comprising a pn junction for emission of light by application of current to flow through the pn junction; and means for applying an electric field across the light emitting portion by applying a voltage to one or more contacts that are electrically isolated from the pn junction in order to alter a light emission characteristic of the light emitting portion. 2 The light emitting device as claimed in claim 1 wherein the light emitting portion and pn junction comprise silicon. 3. The light emitting device as claimed in claim 2 wherein the light emitting portion and pn junction comprise crystalline silicon. 4. The light emitting device as claimed in claim 1 wherein the light emitting portion and pn junction comprise SiGe. 5. The light emitting device as claimed in claim 1, wherein the light emitting portion of the device comprises a thin layer which has a sufficiently small thickness to allow quantum confinement effects to occur. 6. The light emitting device as claimed in claim 5 wherein the thin layer comprises a quantum well. 7. The light emitting device as claimed in claim 5, wherein the layer has a thickness less than 20 nm. 8. The light emitting device as claimed in claim 7 wherein the layer has a thickness of substantially 2 nm. 9. The light emitting device as claimed in claim 1, wherein the light emitting portion of the device comprises a layer which has a thickness greater than a maximum thickness which can achieve quantum confined Stark effects. 10. The light emitting device as claimed in claim 1, wherein the means for applying an electric field across the light emitting portion is arranged such that the electric field is applied across the layer substantially normal to a plane defined by the layer. 11. The light emitting device as claimed in claim 1, wherein the device is integrated in an integrated circuit. 12. The light emitting device as claimed in claim 1, wherein the light emitting portion of the device is formed of a material having high excited carrier lifetimes. 13. The light emitting device as claimed in claim 1, wherein the means for applying the electric field across the light emitting portion is operable to repeatedly apply the electric field at a switching rate greater than substantially ten GHz. 14. The light emitting device as claimed in claim 1, wherein the light emitting device is a waveguide edge emitting device. 15. The light emitting device as claimed in claim 14 wherein the waveguide is formed by layers having a lower refractive index than the light emitting portion positioned either side of the light emitting portion, such that light generated within the light emitting portion travels within the light emitting portion to emerge laterally from the light emitting portion. 16. The light emitting device as claimed in claim 15, wherein the light emitting portion comprises silicon, and the layers having a lower refractive index comprise silicon oxide. 17. The light emitting device as claimed in claim 14, wherein the dimensions of the light emitting portion are smaller than a typical diffusion length of the material of the light emitting portion. 18. The light emitting device as claimed in claim 14, wherein a thickness of the light emitting portion is chosen in order to optimise the performance of the waveguide at a desired frequency of emission. 19. The light emitting device as claimed in claim 1, wherein the light emitting device is a surface emitting device. 20. The light emitting device as claimed in claim 19 wherein the surface emitting device is in bulk form. 21. The light emitting device as claimed in claim 19 wherein the surface emitting device is in integrated form. 22. The light emitting device as claimed in claim 19, further comprising transparent layers between the light emitting portion of the device and the region into which the light is to be emitted. 23. The light emitting device as claimed in claim 22, wherein the means for applying the electric field comprises a transparent electrode positioned over the light emitting portion, and wherein a transparent dielectric is positioned between the transparent electrode and the light emitting portion. 24. The light emitting device as claimed in claim 19, wherein the means for applying the electric field comprises an electrode positioned over the light emitting portion in a grid pattern for limiting shading, and wherein a transparent dielectric is positioned between the electrode and the light emitting portion. 25. The light emitting device as claimed in claim 1, wherein the light emitting portion comprises a layer, wherein contacts are provided for biasing the p and n regions of the pn junction, and wherein said contacts are positioned at opposed lateral edges of the layer. 26. The light emitting device as claimed in claim 25, wherein a heavily doped p+ region is provided close to the p contact. 27. The light emitting device as claimed in claim 25, wherein a region of p+ or p+/intrinsic amorphous silicon is provided close to the p contact. 28. The light emitting device as claimed in claim 25, wherein a heavily doped n+ region is provided close to the n contact. 29. The light emitting device as claimed in claim 25, wherein a region of n+ or n+/intrinsic amorphous silicon is provided close to the n contact. 30. The light emitting device as claimed in claim 25, wherein said contacts contact the layer only at discrete spaced positions along the lateral edges of the layer. 31. The light emitting device as claimed in claim 25, wherein the surfaces of the layer of the light emitting portion are passivated in regions where the contacts do not contact the layer. 32. The light emitting device as claimed in claim 31, wherein the surfaces of the layer of the light emitting portion are passivated by a silicon oxide layer formed over the surfaces. 33. The light emitting device as claimed in claim 31, wherein the surfaces of the layer of the light emitting portion are passivated by a polycrystalline layer. 34. The light emitting device as claimed in claim 31, wherein the surfaces of the layer of the light emitting portion are passivated by a microcrystalline layer. 35. The light emitting device as claimed in claim 31, wherein the surfaces of the layer of the light emitting portion are passivated by an amorphous silicon layer. 36. The light emitting device as claimed in claim 1, wherein a layer below the light emitting portion is formed of a material having a lower refractive index than the light emitting portion, so as to internally reflect light. 37. The light emitting device as claimed in claim 36, wherein the layer below the light emitting portion comprises a dielectric/metal reflecting layer combination. 38. The light emitting device as claimed in claim 1, wherein an anti-reflective coating is provided over a surface from which the device emits light. 39. The light emitting device as claimed in claim 1, wherein at least one dimension of the light emitting portion is a significant fraction of a diffusion length of the material of the light emitting portion. 40. The light emitting device as claimed in claim 1, comprising a plurality of light emitting portions. 41. The light emitting device of claim 40 wherein the light emitting device comprises alternating layers of dielectrics and light emitting portions. 42. The light emitting device as claimed in claim 1, wherein the light emitting portion comprises a bulk region being only relatively lightly doped, and comprises only relatively small amounts of relatively heavily doped silicon. 43. The light emitting device as claimed in claim 1, wherein the light emitting portion comprises a bulk region with introduced defects for enhancing sub-bandgap absorption. 44. The light emitting device as claimed in claim 43, wherein the introduced defects comprise Ge. 45. The light emitting device as claimed in claim 43, wherein the introduced defects comprise dopants from Groups III or V of the periodic table. 46. The light emitting device as claimed in claim 43, wherein the introduced defects give rise to states relatively deeply within the bandgap. 47. The light emitting device as claimed in claim 46, wherein the introduced defects comprise indium dopants. 48. The light emitting device as claimed in claim 46, wherein the introduced defects comprise thallium dopants. 49. The light emitting device as claimed in claim 43, wherein the introduced defects more tightly bind electrons. 50. The light emitting device as claimed in claim 49, wherein the introduced defects comprise rare earth metals. 51. The light emitting device as claimed in claim 50, wherein the introduced defects comprise Er or Yb. 52. The light emitting device as claimed in claim 43, wherein the introduced defects comprise dislocations. 53. The light emitting device as claimed in claim 1, wherein an emitting surface of the device is of a geometry which directs emissions in a predetermined direction. 54. The light emitting surface as claimed in claim 53, wherein the emitting surface of the device is bevelled. 55. The light emitting device as claimed in claim 53, wherein the device comprises a periodic structure to produce a diffraction effect. 56. The light emitting device as claimed in claim 1, wherein the emitting surface comprises a textured geometry comprising (111) equivalent crystallographic planes present in a (100) oriented wafer surface plane. 57. The light emitting device as claimed in claim 53, wherein the emitting surface is textured by anisotropic etching. 58. A method of altering light emissions from a light emitting diode, the method comprising the step of: applying a current across a pn junction of the light emitting diode to cause the light emitting diode to emit light; and applying an electric field across the light emitting diode portion by applying a voltage to one or more contacts that are electrically isolated from the pn junction so as to alter light emission characteristics of the light emitting diode. 59. The method as claimed in claim 58 wherein the light emitting diode comprises silicon. 60. The method as claimed in claim 59 wherein the light emitting diode comprises crystalline silicon. 61. The method as claimed in claim 58 wherein the light emitting diode comprises SiGe. 62. The method as claimed in claim 58, wherein the light emitting diode comprises a thin layer which has a sufficiently small thickness to allow quantum confinement effects to occur, such that application of the electric field causes the quantum confined Stark effect to occur. 63. The method as claimed in claim 62 wherein the thin layer comprises a quantum well. 64. The method as claimed in claim 58, wherein the step of applying the electric field across the light emitting diode comprises applying the electric field across the diode substantially normal to a plane defined by a layer of the diode. 65. The method as claimed in claim 58, wherein the diode is integrated in an integrated circuit. 66. The method as claimed in claim 58, wherein the step of applying the electric field across the light emitting diode comprises repeatedly applying the electric field at a switching rate greater than substantially ten GHz. 67. The method as claimed in claim 58, wherein the light emitting diode is a waveguide edge emitting device. 68. The method as claimed in claim 58, wherein the step of applying the electric field serves to encode data on a light signal produced by the diode, for transmission through an optical transmission system. 69. A silicon light emitting device comprising an emitting surface having geometry for directing emissions in a predetermined direction. 70. The silicon light emitting device as claimed in claim 69, wherein the emitting surface geometry is implemented by bevelling of the emitting surface. 71. The silicon light emitting device as claimed in claim 69, wherein the emitting surface geometry is implemented by a periodic structure which produces a diffraction effect. 72. A silicon light emitting device with surface geometry adapted to control the passage of light in the device so as to improve absorption. 73. The silicon light emitting device as claimed in claim 72 wherein the surface geometry is created by texturing one or more surfaces of the device. 74. The silicon light emitting device as claimed in claim 73 wherein the textured surface geometry comprises (111) equivalent crystallographic planes present in a (100) orientated wafer surface plane, exposed by anisotropic etching. 75. A method of design of a silicon light emitting diode, comprising the step of designing a silicon device having high light absorption, for reverse operation as a light emitting diode. 76. A light modulator comprising: a layer of silicon, to be positioned in the path of light to be modulated; and means to apply an electric field across the silicon layer so as to alter the bandgap of the silicon layer, thus controlling whether light is absorbed by the silicon layer or transmitted through the silicon layer. 77. The modulator of claim 76, wherein the silicon layer is of a sufficiently small thickness to exhibit quantum confinement effects, such that application of the electric field induces bandgap alterations by way of the quantum confined Stark effect. 78. The modulator of claim 76 wherein the silicon layer is of a thickness to exhibit Franz-Keldysh effects upon application of the electric field. 79. The modulator of claim 76 wherein the modulator is in integrated form on a silicon integrated circuit. 80. The modulator of claim 76 wherein the modulator is in bulk form on a silicon integrated circuit. 81. A method of modulating light, the method comprising the steps of: positioning a layer of silicon in the path of the light; and applying an electric field across the thin silicon layer so as to alter the bandgap of the silicon layer, thus controlling whether light is absorbed by the silicon layer or transmitted through the silicon layer. 82. The method of claim 81, wherein the silicon layer is of a sufficiently small thickness to exhibit quantum confinement effects, such that application of the electric field induces bandgap alterations by way of the quantum confined Stark effect. 83. The method of claim 81 wherein the silicon layer is of a thickness to exhibit Franz-Keldysh effects upon application of the electric field. 84. The method of claim 81, wherein the step of applying the electric field comprises repeatedly applying the electric field at a switching rate greater than substantially 10 GHz. 85. The method of claim 81, wherein the step of applying the electric field causes modulation of data onto a light signal for transmission in an optical transmission system. |
<SOH> BACKGROUND ART <EOH>Light emitting diodes (LEDs) are an extremely common electronic device with a wide range of applications, including displays, short distance communications over fibres, opto-couplers, indicators and infrared wireless communication (including television remote controls and free space data links). LEDs typically comprise a light-emitting gallium arsenide pn junction (which emits based on direct band-to-band transitions) or may comprise GaAs 1-x P x , with an isoelectronic dopant such as nitrogen (which emits based on impurity states in the bandgap). GaAs LEDs have been found to offer acceptable quantum efficiencies, approaching 1%, while the use of nitrogen as an isoelectronic dopant in a GaAs 1-x P x LED can improve the quantum efficiency of the LED to over 1%. The materials of a LED may also be chosen in order to produce light of a desirable frequency. For example, GaAs LEDs produce around 900 nm (infrared) light, GaP:ZnO LEDs produce around 650 nm (red) light, GaAs 1-x P x LEDs produce light around the 580 nm (yellow) to 620 nm (orange) range, depending on the value of x, GaP:N LEDs produce green light, and GaN LEDs have recently been introduced to provide emission at around 400 nm (violet). However, the use of such materials prevents integration of LEDs into silicon devices. While attempts have been made in the past to manufacture silicon LEDs, the conversion efficiency of silicon LEDs has, for most applications, been unacceptably low. Conversion efficiencies of silicon devices are usually around 0.001%, while the highest performance silicon junction devices have achieved efficiencies around 0.01%. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. |
<SOH> SUMMARY OF THE INVENTION <EOH>According to a first aspect the present invention provides a light emitting device with alterable emission characteristics, the device comprising: a light emitting portion comprising a pn junction for emission of light by application of current to the pn junction; and means for applying an electric field across the light emitting portion in order to alter a light emission characteristic of the light-emitting portion. By providing a light emitting device with alterable emission characteristics, the present invention enables both generation and modulation of a light signal by a single device. This may be important in devices where physical size is a consideration, for example when integrating such devices in an integrated circuit. Furthermore, by alleviating physical size concerns in this manner, the present invention may allow the light emitting portion of such a device to be formed of materials having lesser quantum efficiency while forming the device of larger size so as to maintain a sufficient intensity output. For instance, the light emitting portion and pn junction of such devices may be formed of silicon. While Si is of lower quantum efficiency than GaAs, a larger light emitting Si device may provide similar light intensity as a relatively smaller GaAs device. Further, Si is a particularly suitable material for integration into silicon integrated circuits. The light emitting portion of the device may alternatively be formed of SiGe or other materials. The light emitting portion of the device of the first aspect of the present invention preferably comprises a thin layer which is thin enough for quantum confinement effects to occur. For example the thin layer may comprise a quantum well. In such embodiments, the means for applying the electric field is preferably arranged such that the electric field is applied across the thin layer substantially normal to a plane defined by the layer. In such an orientation the electric field enables significant quantum confined Stark effect modulation of absorption and emission properties of the thin silicon layer, thus enabling modulation of peak emission wavelength and emission intensity at a given wavelength. However, modulation of the light emission characteristics of the light emitting portion may also be achieved by application of an electric field across the light emitting portion in a direction other than substantially normal to the plane. The thin layer may comprise a crystalline silicon layer comprising the quantum well. Alternatively, the light emitting portion may comprise a layer which has a thickness greater than a maximum thickness which can achieve quantum confined Stark effects. In such embodiments, the application of the electric field may nevertheless cause modulation of the emission characteristics of the light emitting portion due to the Franz-Keldysh effect. According to a second aspect the present invention provides a method of altering light emissions from a light emitting diode, the method comprising the step of: applying an electric field across the light emitting diode so as to alter light emission characteristics of the diode. To date, light modulation schemes have often been comprised of two separate components, namely a light source and a modulator. Light is continuously generated by the light source, such as a light emitting diode, and then passed through a modulator which is controlled so as to either allow the light to continue or to prevent further passage of the light. Such systems require a separate physical area for each of the two components, for example increasing chip space demands. Alternatively, an LED may be used as a modulator by simply turning the pn junction on and off, however the recombination time of the LED material limits application of such modulation to low frequencies, for example less than MHz. However, by applying an electric field across an LED in order to exploit quantum confined Stark effects, Franz-Keldysh effects or the like so as to alter emission characteristics of the diode, the present invention avoids the need for a separate modulator and hence the need for separate chip space, and also uses a modulation technique which permits very high switching rates for example tens or hundreds of GHz. Such a technique may be particularly useful in silicon diode applications, in which high quality silicon (with a relatively large recombination time) is desired to be used, and in which a relatively large physical bulk of silicon may be required in order to produce a given power output. By alleviating the chip-space issues involved with having a separate modulator, and by avoiding the trade-off of recombination time against switching rate, the present invention may thus provide for silicon LEDs with high quality (and high recombination time) material providing appropriate output intensity levels, yet allowing for high speed switching. Accordingly, in preferred embodiments of the present invention, the light emitting portion of the device is formed of a material having high excited carrier lifetimes, in order to assist in reducing bulk recombination. Preferably, the light emitting diode comprises a thin layer quantum well, wherein application of an electric field to the quantum well results in an alteration of the emission characteristics of the well due to the quantum confined Stark effect. In such embodiments, the electric field is preferably applied in a direction substantially normal to a plane defined by the thin layer. Alternatively, even where the light emitting diode is thicker than required for quantum confinement, application of an appropriate electric field across the diode may generate sufficient alteration in the emission characteristics of the diode due to the Franz-Keldysh effect, thus providing measurable modulation of the light emissions of the device. The light emitting diode may be formed of crystalline silicon, GaAs or other materials appropriate for operation as a light emitting diode and exhibiting electroabsorption, that is, absorption and emission characteristics which vary with applied electric field. In embodiments in which the light emitting portion of the device is formed of a thin layer of crystalline silicon exhibiting quantum confinement, the device is preferably a waveguide edge emitting device. In such devices, the waveguide is preferably formed by lower refractive index layers such as SiO 2 positioned either side of the thin silicon layer such that light generated within the thin silicon layer travels within the silicon layer to emerge laterally from the silicon layer. Such embodiments allow for summation of the light intensity along the waveguide, and may thus produce an output light intensity at an appropriate level despite the relatively low intrinsic quantum efficiency of silicon. In such embodiments, the largest dimensions of the device are preferably kept smaller than a typical diffusion length of the semiconductor material. Of course, where the silicon layer is thin enough to exhibit quantum confinement effects, the silicon layer thickness will be significantly less than the diffusion length. For example, to exhibit quantum confinement effects, the silicon layer is likely to be 20 nm thick at most, and in some embodiments may be around 2 nm. The thickness of the silicon layer may be chosen in order to optimise the performance of the waveguide at the desired frequency of emission. It has been realised that both bulk and integrated edge emitting silicon light emitting devices may be formed, as the increase in dark saturation current density associated with edge emitting structures is to some extent offset by shorter emission wavelengths and higher probability of occupation of photon states at such energies. Embodiments of the present invention may comprise selected oxide and/or doping regions for controlling surface contributions to the dark saturation current density. Further, embodiments of the invention may be particularly advantageous in integrating silicon light emitting devices into integrated circuits. Alternatively the device may be a surface emitting device in bulk or integrated form, which may comprise transparent layers between the light emitting portion of the device and the region into which the light is to be emitted. For example, a surface emitting device may comprise a transparent silicon oxide layer over the light emitting portion, and a transparent electrode over the silicon oxide layer, for applying the electric field. Alternatively a non-transparent electrode for applying the electric field may be positioned sparsely over the light emitting portion; such as in a grid pattern, so as to limit the shading of the light emitting portion by the electrode. Preferably, contacts provided for biasing the p and n regions of the pn junction are situated at opposed lateral edges of the emission layer. This feature is preferable both where the layer is very thin such as in integrated form, and in cases where the layer is relatively thick so as to act as a bulk region emitting device. In such embodiments, the p and n contacts do not shade the light emitting region of the device, and thus will provide improved emission efficiency over arrangements where the p or n contact is positioned over the emitting region of the device. Preferably, a heavily doped p+ region is provided close to the metal p contact, and a heavily doped n+ region is provided close to the metal n contact. Further, a region of n+ or n+/intrinsic amorphous silicon may be provided instead of the n+ region, and a p+ or p+/intrinsic amorphous silicon region may be provided instead of the p+ region. Such regions of amorphous silicon can maintain good conduction into the metal conductor and also provide a good surface interface. The layer below the silicon quantum well layer may be formed of a reflective material such that light emitted away from the intended direction of emission is reflected towards the intended direction of emission. For instance, a dielectric/metal reflecting layer combination may be provided in order to internally reflect light from a surface from which emission is not desired. Such a reflector preferably has a high reflectance in order to reduce metal absorption losses. Embodiments of the present invention which exploit the quantum confined Stark effect (QCSE) and field controlled modulation or switching, have the potential to be very useful because the QCSE has application at very high frequencies as there is no intrinsic limitations at least up to picosecond switching intervals (tens of GHz). In fact, any switching limitation is likely to be in the capacitance and resistance of the external voltage supply circuitry. To the contrary, the switching rate of normal LEDs is limited by the recombination time of the bulk material, which limits the switching applications of high performance silicon LEDs to a few hundred kHz. High speed (but lower efficiency) LEDs with introduced impurities have a reduced recombination time and can provide switching rates of up to many Mb/s, however this is still significantly slower than may be offered by quantum confined Stark effect switching. In preferred embodiments of the first and second aspects of the invention, an anti-reflective coating over a surface from which the device emits light is provided in order to increase emissivity at desired frequencies. According to a third aspect the present invention resides in a silicon light emitting device formed using a silicon wafer substrate and comprising metal contacts, wherein the metal contacts are isolated from the substrate by an insulating dielectric apart from small local contact areas. Isolation of metal contacts from the substrate assists in reducing metal-semiconductor recombination. For instance, in embodiments of the first and second aspects of the invention, the metal contacts are preferably formed after a layer of silicon oxide has been formed over the substrate, thereby isolating the metal contacts from the substrate. According to a fourth aspect the present invention resides in a silicon light emitting device comprising means for controlling carrier concentrations in surface regions to minimise surface recombination. The means for controlling carrier concentrations may comprise regions of the silicon device proximal to the surfaces having controlled doping levels. Alternatively or additionally the means for controlling carrier concentrations may comprise electrostatic means such as overlying heterostructures. Preferred embodiments of the fourth aspect of the invention may have a device thickness which is a significant fraction of a diffusion length. Increasing the device thickness results in an approximately linear increase in emitted light output, while causing only small increases in the diode dark saturation current density, which is largely dependent on surface recombination rather than on device thickness. At thicknesses greater than a diffusion length, performance will start to degrade. However, embodiments of the invention may have multiple layers forming a multiple junction device, providing an overall device thickness of greater than a diffusion length. Of course, in embodiments in which the device is of a thickness to support quantum confinement effects, the device thickness will be significantly less than a diffusion length. According to a fifth aspect the present invention resides in a light emitting silicon device comprising a bulk region being only relatively lightly doped, and wherein the device comprises only relatively small amounts of relatively heavily doped silicon. According to a sixth aspect, the present invention resides in a method of fabricating a silicon light emitting device comprising metal contacts, the method comprising the step of minimising the area of the metal contacts. Minimising the area of the metal contacts minimises surface recombination in the vicinity of the metal contacts. For example, in embodiments of the first and second aspects of the invention in which the light emitting portion of the device acts as a waveguide, the p and n metal contacts may comprise small metal contacts positioned only at a limited number of locations along the lateral edges of the light emitting portion. Thus, as carriers pass along the waveguide, surface recombination will be limited due to the limited number of positions and limited total surface area of the respective contacts. According to a seventh aspect, the present invention resides in a method of fabricating a silicon light emitting device comprising the step of passivating non-contacted surface areas of the device. The step of passivating may comprise forming a heterostructure over the surface, such as a surface oxide layer. Alternatively or additionally, the step of passivating may comprise deposition of a polycrystalline layer, a microcrystalline layer and/or an amorphous silicon layer, or the like. Preferred embodiments of the seventh aspect of the invention provide a sufficiently high level of surface passivation to allow quasi-fermi potentials to be approximately constant throughout the device. Thus, in embodiments of the first and second aspects of the invention in which the light emitting portion acts as a waveguide with n and p contacts only at a small number of spaced locations along the lateral edges of the waveguide, the edge portion of the waveguide between each contact is preferably passivated by providing a transparent dielectric layer such as silicon oxide over the light emitting portion. Similarly, the light emitting portion is preferably formed over a passivation layer, and a passivation layer is preferably formed over the light emitting portion. According to an eighth aspect, the present invention resides in a method of fabricating a silicon light emitting device comprising the step of introducing silicon defects to enhance sub-bandgap absorption. Of course, enhancement of sub-bandgap absorption corresponds to enhanced sub-bandgap emission. The step of introducing silicon defects may comprise introducing defects to produce energy states in the bandgap corresponding to a desired emission wavelength. For example, emissions at 1.55 micron may be desirable in optical communication systems, and the introduction of appropriate silicon defects may provide a silicon device having appropriate emission wavelengths, and importantly, relatively high emission efficiencies at such wavelengths. Preferably, the introduced silicon defects of embodiments of the eighth aspect of the invention are such that carriers from the band that is closest in energy to the defect level energy make rapid transitions to and from the defect energy, while transitions to and from the other band are relatively slow and radiatively efficient. Such embodiments preferably ensure that occupancy of the defect is governed by the occupancy of the nearer band and that radiative emission to the other band will increase exponentially with voltage, as for band-to-band emission. Introduced defects may comprise Ge from Group IV of the periodic table, or dopants from Groups III or V. In particular, the introduced defects may comprise dopants such as indium and thallium so as to give rise to states relatively deeply within the bandgap, or the introduced defects may comprise defects that more tightly bind electrons such as rare earth metals, for example Er or Yb. Er may be particularly appropriate in optical communications applications, as it offers an absorption peak close to 1.55 microns. The introduced defects may comprise combinations of such dopants, and further, combinations of rare earth dopants may increase the efficiency of absorption processes. Additionally or alternatively, the introduced defects may comprise defects such as dislocations. According to a ninth aspect the present invention resides in a silicon light emitting device comprising an emitting surface having geometry for directing emissions in a predetermined direction. Embodiments of the ninth aspect of the invention may be particularly advantageous in optical communications systems. The emitting surface geometry may be implemented by bevelling of the emitting surface or by using a periodic structure to produce a diffraction effect. In preferred embodiments of the ninth aspect, the surface geometry will also serve to enhance emissions from the device. According to a tenth aspect the present invention resides in a silicon light emitting device with surface geometry adapted to control the passage of light in the device so as to improve absorption. The surface geometry is preferably adapted so as to improve the passage of long wavelength light, for instance light of wavelength longer than 1000 nm. Preferred embodiments of the tenth aspect of the invention have a surface geometry created by texturing one or more surfaces of the device. The textured surface geometry may comprise (111) equivalent crystallographic planes present in a (100) orientated wafer surface plane, exposed for example by anisotropic etching. Further embodiments of the tenth aspect of the invention may comprise surface geometry having crystallographic features in distinct locations. Such locations may be controlled by photolithography, or may arise through random nucleation. In embodiments of the tenth aspect of the invention where the light emitting device is required to have emissions concentrated at a particular range of energies or emission angles, the device may have surface geometry adapted to maximise absorptance (and hence emittance) at those particular energies or emission angles. According to an eleventh aspect the present invention resides in a method of design of a silicon light emitting diode, comprising the step of: designing a silicon device having high light absorption, for reverse operation as a light emitting diode. It has now been realised that techniques and features which have been developed in relation to silicon devices having high light absorption, such as silicon solar cells, are often applicable in design of a relatively high efficiency silicon light emitting device, such as a silicon LED. Accordingly, it is envisaged that significant improvements in silicon LED efficiency may be achieved by use of absorption enhancement techniques in emission optimisation. According to a twelfth aspect, the present invention provides a light modulator comprising: a thin layer of silicon exhibiting quantum confinement effects, to be positioned in the path of light to be modulated; and means to apply an electric field across the thin silicon layer so as to alter the bandgap of the silicon layer by way of the quantum confined Stark effect, thus controlling whether light is absorbed by the silicon layer or transmitted through the silicon layer. According to a thirteenth aspect, the present invention provides a method of modulating light, the method comprising the steps of: positioning a thin layer of silicon in the path of the light, the silicon layer exhibiting quantum confinement effects; and applying an electric field across the thin silicon layer so as to alter the bandgap of the silicon layer by way of the quantum confined Stark effect, thus controlling whether light is absorbed by the silicon layer or transmitted through the silicon layer. It has now been realised that quantum confined structures may be formed of silicon. Thus, the twelfth and thirteenth aspects of the invention provide for modulation devices which utilise quantum confinement effects in modulating light passing through the thin silicon layer. As the quantum confined Stark effect causes an alteration of the bandgap of the silicon upon application of an electric field, such devices will be of application when modulating light of particular frequencies. Such frequencies will be those which are either absorbed by the silicon or transmitted through the silicon, depending on whether the electric field is applied. Further, the twelfth and thirteenth aspects of the invention may be particularly advantageous in providing for convenient integration of a silicon quantum confined Stark effect light modulating device onto a silicon integrated circuit, whether in bulk or integrated form. Preferred embodiments of the invention may comprise features of two or more of the first to thirteenth aspects of the invention as previously described. The silicon light emitting device of any of the first to thirteenth aspects may be a silicon light emitting diode, an optical coupler or the like. |
Sequences for detection and identification of methicillin-resistant staphyloccocus |
The present invention describes novel SCCmec right extremity junction sequences for the detection of methicillin-resistant Staphyloccocus aureus (MRSA). It relates to the use of these DNA sequences for diagnostic purposes. |
1. A method to detect the presence of a methicillin-resistant Staphylococcus aureus (MRSA) strain in a sample, said MRSA strain being resistant because of the presence of an SCCmec insert containing a mecA gene, said SCCmec being inserted in bacterial nucleic acids thereby generating a polymorphic right extremity junction (MREJ), said method comprising the step of annealing the nucleic acids of the sample with a plurality of probes and/or primers, characterized by: (i) said primers and/or probes are specific for MRSA strains and capable of annealing with polymorphic MREJ nucleic acids, said polymorphic MREJ comprising MREJ types i to x; and (ii) said primers and/or probes altogether can anneal with at least four MREJ types selected from MREJ types i to x. 2. The method of claim 1, wherein the primers and/or probes are all chosen to anneal under common annealing conditions. 3. The method of claim 2, wherein the primer and/or probes are placed altogether in the same physical enclosure. 4. The method of claim 1, wherein the primers and/or probes have at least 10 nucleotides in length and are capable of annealing with MREJ types i to iii, defined in any one of SEQ ID NOs: 1, 20, 21, 22, 23, 24, 25, 41, 199; 2, 17, 18, 19, 26, 40, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 185, 186, 197; 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 104, 184, 198 and with one or more of MREJ types iv to ix, having SEQ ID NOs: 42, 43, 44, 45, 46, 51, 47, 48, 49, 50; 171; 165, 166; 167; 168. 5. The method of claim 1, wherein the primers and/or probes altogether can anneal with said SEQ ID NOs of MREJ types i to ix. 6. The method of claim 1, wherein said primers and/or probes have the following sequences SEQ ID NOs: 66, 100, 101, 105, 52, 53, 54, 55, for the detection of MREJ type i 56, 57, 64, 71, 72, 73, 74, 75, 76, 70, 103, 130, 132, 158, 159, 59, 62, 126, 127, 128, 129, 131, 200, 201, 60, 61, 63 32, 83, 84, 160, 161, 162, 163, 164 85, 86, 87, 88, 89 66, 97, 99, 100, 101, 106, 117, for the detection of MREJ type ii 118, 124, 125, 52, 53, 54, 55, 56, 57 64, 71, 72, 73, 74, 75, 76, 70, 103, 130, 132, 158, 159 59, 62 126, 127 128, 129, 131, 200, 201 60, 61, 63 32, 83, 84, 160, 161, 162, 163, 164 85, 86, 87, 88, 89 67, 98, 102, 107, 108 for the detection of MREJ type iii 64, 71, 72, 73, 74, 75, 76, 70, 103, 130, 132, 158, 159 58, 59, 62 126, 127 128, 129, 131, 200, 201 60, 61, 63 32, 83, 84, 160, 161, 162, 163, 164 85, 86, 87, 88, 89 79, 77, 145, 147 for the detection of MREJ type iv 64, 71, 72, 73, 74, 75, 76, 70, 103, 130, 132, 158, 159 59, 62 126, 127 128, 129, 131, 200, 201 60, 61, 63 68 32, 83, 84, 160, 161, 162, 163, 164 85, 86, 87, 88, 89 65, 80, 146, 154, 155 for the detection of MREJ type v 64, 71, 72, 73, 74, 75, 76, 70, 103, 130, 132, 158, 159 59, 62 126, 127 128, 129, 131, 200, 201 60, 61, 63 32, 83, 84, 160, 161, 162, 163, 164 85, 86, 87, 88, 89 202, 203, 204 for the detection of MREJ type vi 64, 71, 72, 73, 74, 75, 76, 70, 103, 130, 132, 158, 159 59, 62 126, 127 128, 129, 131, 200, 201 60, 61, 63 32, 83, 84, 160, 161, 162, 163, 164 85, 86, 87, 88, 89 112, 113, 114, 119, 120, 121, 122, for the detection of MREJ type vii 123, 150, 151, 153 64, 71, 72, 73, 74, 75, 76, 70, 103, 130, 132, 158, 159 59, 62 126, 127 128, 129, 131, 200, 201 60, 61, 63 32, 83, 84, 160, 161, 162, 163, 164 85, 86, 87, 88, 89 115, 116, 187, 188, 207, 208 for the detection of MREJ type viii 64, 71, 72, 73, 74, 75, 76, 70, 103, 130, 132, 158, 159 59, 62 126, 127 128, 129, 131, 200, 201 60, 61, 63 32, 83, 84, 160, 161, 162, 163, 164 85, 86, 87, 88, 89 109, 148, 149, 205, 206 for the detection of MREJ type ix. 64, 71, 72, 73, 74, 75, 76 70, 103, 130, 132, 158, 159 59, 62 126, 127 128, 129, 131, 200, 201 60, 61, 63 32, 83, 84, 160, 161, 162, 163, 164 85, 86, 87, 88, 89 7. The method of claim 6, wherein primer pairs have the nucleotide sequence which are defined in SEQ ID NOs: 64/66, 64/100, 64/101; 59/52, for the detection of type i MREJ 59/53, 59/54, 59/55, 59/56, 59/57, 60/52, 60/53, 60/54, 60/55, 60/56 60/57, 61/52, 61/53, 61/54, 61/55 61/56, 61/57, 62/52, 62/53, 62/54 62/55, 62/56, 62/57, 63/52, 63/53 63/54, 63/55, 63/56, 63/57 64/66, 64/97, 64/99, 64/100, 64/101 for the detection of type ii MREJ 59/52, 59/53, 59/54, 59/55, 59/56, 59/57, 60/52, 60/53, 60/54, 60/55, 60/56, 60/57, 61/52, 61/53, 61/54, 61/55, 61/56, 61/57, 62/52, 62/53, 62/54, 62/55, 62/56, 62/57, 63/52 63/53, 63/54, 63/55, 63/56, 63/57 64/67, 64/98, 64/102; 59/58, for the detection of type iii MREJ 60/58, 61/58, 62/58, 63/58 64/79 for the detection of type iv MREJ 64/80 for the detection of type v MREJ 64/204 for the detection of type vi MREJ 64/112, 64/113 for the detection of type vii MREJ 64/115, 64/116 for the detection of type viii MREJ 64/109 for the detection of type ix MREJ 8. The method of claim 7, further comprising probes having the following sequences: SEQ ID NOs: 32, 83, 84, 160, 161, 162, 163, 164 for the detection of MREJ types i to ix. 9. The method of claim 6, wherein said primers and probes have the following nucleotide sequences: i) SEQ ID NOs: 64, 66, 84, 163, 164 for the detection of MREJ type i ii) SEQ ID NOs: 64, 66, 84, 163, 164 for the detection of MREJ type ii iii) SEQ ID NOs: 64, 67, 84, 163, 164 for the detection of MREJ type iii iv) SEQ ID NOs: 64, 79, 84, 163, 164 for the detection of MREJ type iv v) SEQ ID NOs: 64, 80, 84, 163,164 for the detection of MREJ type v vi) SEQ ID NOs: 64, 112, 84, 163, 164 for the detection of MREJ type vii. 10. The method of claim 1, wherein said probes and primers are used together. 11. The method of claim 9, wherein said probes and/or primers are used together in the same physical enclosure. 12. A method for typing a MREJ of a MRSA strain, which comprises the steps of: reproducing the method of claim 1 with primers and/or probes specific for a determined MREJ type, and detecting an annealed probe and/or primer as an indication of the presence of a determined MREJ type. 13. A nucleic acid selected from: i) SEQ ID NOs: 42, 43, 44, 45, 46, 51 for sequence of MREJ type iv; ii) SEQ ID NOs: 47, 48, 49, 50 for sequence of MREJ type v; iii) SEQ ID NOs: 171 for sequence of MREJ type vi; iv) SEQ ID NOs: 165, 166 for sequence of MREJ type vii; v) SEQ ID NOs: 167 for sequence of MREJ type viii; vi) SEQ ID NOs: 168 for sequence of MREJ type ix. 14. An oligonucleotide of at least 10 nucleotides in length which hybridizes with the nucleic acid of claim 13 and which hybridizes with one or more MREJ of types selected from iv to ix. 15. An oligonucleotide pair which has the nucleotide sequences defined in any one of SEQ ID NOs: 64/66, 64/100, 64/101; 59/52, for the detection of type i MREJ 59/53, 59/54, 59/55, 59/56, 59/57, 60/52, 60/53, 60/54, 60/55, 60/56 60/57, 61/52, 61/53, 61/54, 61/55 61/56, 61/57, 62/52, 62/53, 62/54 62/55, 62/56, 62/57, 63/52, 63/53 63/54, 63/55, 63/56, 63/57 64/66, 64/97, 64/99, 64/100, 64/101 for the detection of type ii MREJ 59/52, 59/53, 59/54, 59/55, 59/56, 59/57, 60/52, 60/53, 60/54, 60/55, 60/56, 60/57, 61/52, 61/53, 61/54, 61/55, 61/56, 61/57, 62/52, 62/53, 62/54, 62/55, 62/56, 62/57, 63/52 63/53, 63/54, 63/55, 63/56, 63/57 64/67, 64/98, 64/102; 59/58, for the detection of type iii MREJ 60/58, 61/58, 62/58, 63/58 64/79 for the detection of type iv MREJ 64/80 for the detection of type v MREJ 64/204 for the detection of type vi MREJ 64/112, 64/113 for the detection of type vii MREJ 64/115, 64/116 for the detection of type viii MREJ 64/109 for the detection of type ix MREJ 16. An oligonucleotide which has the nucleotide sequence defined in any one of SEQ ID NOs: 32, 83, 84, 160, 161, 162, 163, 164. 17. A composition of matter comprising primers and/or probes, the nucleotide sequences of which have at least 10 nucleotides in length which hybridize with any nucleic acid defined in claim 13, and which hybridize with one or more MREJ of types selected from iv to ix. 18. The composition of claim 17, which further comprises primers and/or probes, which hybridize with one or more MREJ of types selected from i to iii. 19. The composition of claim 18, wherein the primers pairs have the nucleotide sequences defined in SEQ ID NOs: 64/66, 64/100, 64/101; 59/52, for the detection of type i MREJ 59/53, 59/54, 59/55, 59/56, 59/57, 60/52, 60/53, 60/54, 60/55, 60/56 60/57, 61/52, 61/53, 61/54, 61/55 61/56, 61/57, 62/52, 62/53, 62/54 62/55, 62/56, 62/57, 63/52, 63/53 63/54, 63/55, 63/56, 63/57 64/66, 64/97, 64/99, 64/100, 64/101 for the detection of type ii MREJ 59/52, 59/53, 59/54, 59/55, 59/56, 59/57, 60/52, 60/53, 60/54, 60/55, 60/56, 60/57, 61/52, 61/53, 61/54, 61/55, 61/56, 61/57, 62/52, 62/53, 62/54, 62/55, 62/56, 62/57, 63/52 63/53, 63/54, 63/55, 63/56, 63/57 64/67, 64/98, 64/102; 59/58, for the detection of type iii MREJ 60/58, 61/58, 62/58, 63/58 64/79 for the detection of type iv MREJ 64/80 for the detection of type v MREJ 64/204 for the detection of type vi MREJ 64/112, 64/113 for the detection of type vii MREJ 64/115, 64/116 for the detection of type viii MREJ 64/109 for the detection of type ix MREJ 20. The composition of claim 18, which further comprises probes, which SEQ ID NOs are: 32, 83, 84, 160, 161, 162, 163, 164. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Clinical Significance of Staphylococcus aureus The coagulase-positive species Staphylococcus aureus is well documented as a human opportunistic pathogen. Nosocomial infections caused by S. aureus are a major cause of morbidity and mortality. Some of the most common infections caused by S. aureus involve the skin, and they include furuncles or boils, cellulitis, impetigo, and postoperative wound infections at various sites. Some of the more serious infections produced by S. aureus are bacteremia, pneumonia, osteomyelitis, acute endocarditis, myocarditis, pericarditis, cerebritis, meningitis, scalded skin syndrome, and various abcesses. Food poisoning mediated by staphylococcal enterotoxins is another important syndrome associated with S. aureus. Toxic shock syndrome, a community-acquired disease, has also been attributed to infection or colonization with toxigenic S. aureus (Murray et al. Eds, 1999, Manual of Clinical Microbiology, 7 th Ed., ASM Press, Washington, D.C.). Methicillin-resistant S. aureus (MRSA) emerged in the 1980s as a major clinical and epidemiologic problem in hospitals. MRSA are resistant to all β-lactams including penicillins, cephalosporins, carbapenems, and monobactams, which are the most commonly used antibiotics to cure S. aureus infections. MRSA infections can only be treated with more toxic and more costly antibiotics, which are normally used as the last line of defence. Since MRSA can spread easily from patient to patient via personnel, hospitals over the world are confronted with the problem to control MRSA. Consequently, there is a need to develop rapid and simple screening or diagnostic tests for detection and/or identification of MRSA to reduce its dissemination and improve the diagnosis and treatment of infected patients. Methicillin resistance in S. aureus is unique in that it is due to acquisition of DNA from other coagulase-negative staphylococci (CNS), coding for a surnumerary β-lactam-resistant penicillin-binding protein (PBP), which takes over the biosynthetic functions of the normal PBPs when the cell is exposed to β-lactam antibiotics. S. aureus normally contains four PBPs, of which PBPs 1, 2 and 3 are essential. The low-affinity PBP in MRSA, termed PBP 2a (or PBP2′), is encoded by the choromosomal mecA gene and functions as a β-lactam-resistant transpeptidase. The mecA gene is absent from methicillin-sensitive S. aureus but is widely distributed among other species of staphylococci and is highly conserved (Ubukata et al., 1990, Antimicrob. Agents Chemother. 34:170-172). By nucleotide sequence determination of the DNA region surrounding the mecA gene from S. aureus strain N315 (isolated in Japan in 1982), Hiramatsu et al. have found that the mecA gene is carried by a novel genetic element, designated staphylococcal cassette chromosome mec (SCCmec), inserted into the chromosome. SCCmec is a mobile genetic element characterized by the presence of terminal inverted and direct repeats, a set of site-specific recombinase genes (ccrA and ccrB), and the mecA gene complex (Ito et al., 1999, Antimicrob. Agents Chemother. 43:1449-1458; Katayama et al., 2000, Antimicrob. Agents Chemother. 44:1549-1555). The element is precisely excised from the chromosome of S. aureus strain N315 and integrates into a specific S. aureus chromosomal site in the same orientation through the function of a unique set of recombinase genes comprising ccrA and ccrB. Two novel genetic elements that shared similar structural features of SCCmec were found by cloning and sequencing the DNA region surrounding the mecA gene from MRSA strains NCTC 10442 (the first MRSA strain isolated in England in 1961) and 85/2082 (a strain from New Zealand isolated in 1985). The three SCCmec have been designated type I (NCTC 10442), type II (N315) and type III (85/2082) based on the year of isolation of the strains (Ito et al., 2001, Antimicrob. Agents. Chemother. 45:1323-1336) ( FIG. 1 ). Hiramatsu et al. have found that the SCCmec DNAs are integrated at a specific site in the methicillin-sensitive S. aureus (MSSA) chromosome. They characterized the nucleotide sequences of the regions around the left and right boundaries of SCCmec DNA (i.e. attL and attR, respectively) as well as those of the regions around the SCCmec DNA integration site (i.e. attBscc which is the bacterial chromosome attachment site for SCCmec DNA). The attBscc site was located at the 3′ end of a novel open reading frame (ORF), orfX. The orfX potentially encodes a 159-amino acid polypeptide sharing identity with some previously identified polypeptides, but of unknown function (Ito et al., 1999, Antimicrob. Agents Chemother. 43:1449-1458). Recently, a new type of SCCmec (type IV) has been described by both Hiramatsu et al. (Ma et al, 2002, Antimicrob. Agents Chemother. 46:1147-1152) and Oliveira et al. (Oliveira et al, 2001, Microb. Drug Resist. 7:349-360). The sequences of the right extremity of the new type IV SCCmec from S. aureus strains CA05 and 8/6-3P published by Hiramatsu et al. (Ma et al., 2002, Antimicrob. Agents Chemother. 46:1147-1152) were nearly identical over 2000 nucleotides to that of type II SCCmec of S. aureus strain N315 (Ito et al., 2001, Antimicrob. Agents Chemother. 45:1323-1336). No sequence at the right extremity of the SCCmec type IV is available from the S. aureus strains HDE288 and PL72 described by Oliveira et al. (Oliveira et al., 2001, Microb. Drug Resist. 7:349-360). Previous methods used to detect and identify MRSA (Saito et al., 1995, J. Clin. Microbiol. 33:2498-2500; Ubukata et al., 1992, J. Clin. Microbiol. 30:1728-1733; Murakami et al., 1991, J. Clin. Microbiol. 29:2240-2244; Hiramatsu et al., 1992, Microbiol. Immunol. 36:445-453), which are based on the detection of the mecA gene and S. aureus -specific chromosomal sequences, encountered difficulty in discriminating MRSA from methicillin-resistant coagulase-negative staphylococci (CNS) because the mecA gene is widely distributed in both S. aureus and CNS species (Suzuki et al., 1992, Antimicrob. Agents. Chemother. 36:429-434). Hiramatsu et al. (U.S. Pat. No. 6,156,507) have described a PCR assay specific for MRSA by using primers that can specifically hybridize to the right extremities of the 3 types of SCCmec DNAs in combination with a primer specific to the S. aureus chromosome, which corresponds to the nucleotide sequence on the right side of the SCCmec integration site. Since nucleotide sequences surrounding the SCCmec integration site in other staphylococcal species (such as S. epidermidis and S. haemolyticus ) are different from those found in S. aureus, this PCR assay was specific for the detection of MRSA. This PCR assay also supplied information for MREP typing (standing for <<mec right extremity polymorphism>>) of SCCmec DNA (Ito et al., 2001, Antimicrob. Agents Chemother. 45:1323-1336; Hiramatsu et al., 1996, J. Infect. Chemother. 2:117-129). This typing method takes advantage of the polymorphism at the right extremity of SCCmec DNAs adjacent to the integration site among the three types of SCCmec. Type III has a unique nucleotide sequence while type II has an insertion of 102 nucleotides to the right terminus of SCCmec type II. The MREP typing method described by Hiramatsu et al. (Ito et al., 2001, Antimicrob. Agents Chemother. 45:1323-1336; Hiramatsu et al., 1996, J. Infect. Chemother. 2:117-129) defines the SCCmec type I as MREP type i, SCCmec type II as MREP type ii and SCCmec type III as MREP type iii. It should be noted that the MREP typing method cannot differentiate the new SCCmec type IV described by Hiramatsu et al. (Ma et al., 2002, Antimicrob. Agents Chemother. 46:1147-1152) from SCCmec type II because these two SCCmec types exhibit the same nucleotide sequence to the right extremity. The set of primers described by Hiramatsu et al. as being the optimal primer combination (SEQ ID NOs.: 22, 24, 28 in U.S. Pat. No. 6,156,507 corresponding to SEQ ID NOs.: 56, 58 and 60, respectively, in the present invention) have been used in the present invention to test by PCR a variety of MRSA and MSSA strains ( FIG. 1 and Table 1). Twenty of the 39 MRSA strains tested were not amplified by the Hiramatsu et al. multiplex PCR assay (Tables 2 and 3). Hiramitsu's method indeed was successful in detecting less than 50% of the tested 39 MRSA strains. This finding demonstrates that some MRSA strains have sequences at the right extremity of SCCmec-chromosome right extremity junction different from those identified by Hiramatsu et al. Consequently, the system developed by Hiramatsu et al. does not allow the detection of all MRSA. The present invention relates to the generation of SCCmec-chromosome right extremity junction sequence data required to detect more MRSA strains in order to improve the Hiramatsu et al. assay. There is a need for developing more ubiquitous primers and probes for the detection of most MRSA strains around the world. |
<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the present invention to provide a specific, ubiquitous and sensitive method using probes and/or amplification primers for determining the presence and/or amount of nucleic acids from all MRSA strains. Ubiquity of at least 50% amongst the strains representing MRSA strains types IV to X is an objective of this invention. Therefore, in accordance with the present invention is provided a method to detect the presence of a methicillin-resistant Staphylococcus aureus (MRSA) strain in a sample, the MRSA strain being resistant because of the presence of an SCCmec insert containing a mecA gene, said SCCmec being inserted in bacterial nucleic acids thereby generating a polymorphic right extremity junction (MREJ), the method comprising the step of annealing the nucleic acids of the sample with a plurality of probes and/or primers, characterized by: (i) the primers and/or probes are specific for MRSA strains and capable of annealing with polymorphic MREJ nucleic acids, the polymorphic MREJ comprising MREJ types i to x; and (ii) the primers and/or probes altogether can anneal with at least four MREJ types selected from MREJ types i to x. In a specific embodiment, the primers and/or probes are all chosen to anneal under common annealing conditions, and even more specifically, they are placed altogether in the same physical enclosure. A specific method has been developed using primers and/or probes having at least 10 nucleotides in length and capable of annealing with MREJ types i to iii, defined in any one of SEQ ID NOs: 1, 20, 21, 22,23, 24, 25, 41, 199; 2, 17, 18, 19, 26, 40, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 185, 186, 197; 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 104, 184, 198 and with one or more of MREJ types iv to ix, having SEQ ID NOs: 42, 43, 44, 45, 46, 51; 47, 48, 49, 50; 171; 165, 166; 167; 168. To be perfectly ubiquitous with the all the sequenced MREJs, the primers and/or probes altogether can anneal with said. SEQ ID NOs of MREJ types i to ix. The following specific primers and/or probes having the following sequences have been designed: 66, 100, 101, 105, 52, 53, 54, 55, for the detection of MREJ type i 56, 57, 64, 71, 72, 73, 74, 75, 76, 70, 103, 130, 132, 158, 159, 59, 62, 126, 127, 128, 129, 131, 200, 201, 60, 61, 63 32, 83, 84, 160, 161, 162, 163, 164 85, 86, 87, 88, 89 66, 97, 99, 100, 101, 106, 117, for the detection of MREJ type ii 118, 124, 125, 52, 53, 54, 55, 56, 57 64, 71, 72, 73, 74, 75, 76, 70, 103, 130, 132, 158, 159 59, 62 126, 127 128, 129, 131, 200, 201 60, 61, 63 32, 83, 84, 160, 161, 162, 163, 164 85, 86, 87, 88, 89 67, 98, 102, 107, 108 for the detection of MREJ type iii 64, 71, 72, 73, 74, 75, 76, 70, 103, 130, 132, 158, 159 58, 59, 62 126, 127 128, 129, 131, 200, 201 60, 61, 63 32, 83, 84, 160, 161, 162, 163, 164 85, 86, 87, 88, 89 79, 77, 145, 147 for the detection of MREJ type iv 64, 71, 72, 73, 74, 75, 76, 70, 103, 130, 132, 158, 159 59, 62 126, 127 128, 129, 131, 200, 201 60, 61, 63 68 32, 83, 84, 160, 161, 162, 163, 164 85, 86, 87, 88, 89 65, 80, 146, 154, 155 for the detection of MREJ type v 64, 71, 72, 73, 74, 75, 76, 70, 103, 130, 132, 158, 159 59, 62 126, 127 128, 129, 131, 200, 201 60, 61, 63 32, 83, 84, 160, 161, 162, 163, 164 85, 86, 87, 88, 89 202, 203, 204 for the detection of MREJ type vi 64, 71, 72, 73, 74, 75, 76, 70, 103, 130, 132, 158, 159 59, 62 126, 127 128, 129, 131, 200, 201 60, 61, 63 32, 83, 84, 160, 161, 162, 163, 164 85, 86, 87, 88, 89 112, 113, 114, 119, 120, 121, 122 for the detection of MREJ type vii, 123, 150, 151, 153 64, 71, 72, 73, 74, 75, 76, 70, 103, 130, 132, 158, 159 59, 62 126, 127 128, 129, 131, 200, 201 60, 61, 63 32, 83, 84, 160, 161, 162, 163, 164 85, 86, 87, 88, 89 115, 116, 187, 188, 207, 208 for the detection of MREJ type viii 64, 71, 72, 73, 74, 75, 76, 70, 103, 130, 132, 158, 159 59, 62 126, 127 128, 129, 131, 200, 201 60, 61, 63 32, 83, 84, 160, 161, 162, 163, 164 85, 86, 87, 88, 89 109, 148, 149, 205, 206 for the detection of MREJ type ix. 64, 71, 72, 73, 74, 75, 76 70, 103, 130, 132, 158, 159 59, 62 126, 127 128, 129, 131, 200, 201 60, 61, 63 32, 83, 84, 160, 161, 162, 163, 164 85, 86, 87, 88, 89 Amongst these, the following primer pairs having the following sequences are used: 64/66, 64/100, 64/101; 59/52, for the detection of type i MREJ 59/53, 59/54, 59/55, 59/56, 59/57, 60/52, 60/53, 60/54, 60/55, 60/56 60/57, 61/52, 61/53, 61/54, 61/55 61/56, 61/57, 62/52, 62/53, 62/54 62/55, 62/56, 62/57, 63/52, 63/53 63/54, 63/55, 63/56, 63/57 64/66, 64/97, 64/99, 64/100, 64/101 for the detection of type ii MREJ 59/52, 59/53, 59/54, 59/55, 59/56, 59/57, 60/52, 60/53, 60/54, 60/55, 60/56, 60/57, 61/52, 61/53, 61/54, 61/55, 61/56, 61/57, 62/52, 62/53, 62/54, 62/55, 62/56, 62/57, 63/52 63/53, 63/54, 63/55, 63/56, 63/57 64/67, 64/98, 64/102; 59/58, for the detection of type iii MREJ 60/58, 61/58, 62/58, 63/58 64/79 for the detection of type iv MREJ 64/80 for the detection of type v MREJ 64/204 for the detection of type vi MREJ 64/112, 64/113 for the detection of type vii MREJ 64/115, 64/116 for the detection of type viii MREJ 64/109 for the detection of type ix MREJ As well, amongst these, the following probes having the following sequences are used: SEQ ID NOs: 32, 83, 84, 160, 161, 162, 163, 164 for the detection of MREJ types i to ix. In the most preferred embodied method, the following primers and/or probes having the following nucleotide sequences are used together. The preferred combinations make use of: i) SEQ ID NOs: 64, 66, 84, 163, 164 for the detection of MREJ type i ii) SEQ ID NOs: 64, 66, 84, 163, 164 for the detection of MREJ type ii iii) SEQ ID NOs: 64, 67, 84, 163, 164 for the detection of MREJ type iii iv) SEQ ID NOs: 64, 79, 84, 163, 164 for the detection of MREJ type iv v) SEQ ID NOs: 64, 80, 84, 163, 164 for the detection of MREJ type v vi) SEQ ID NOs: 64, 112, 84, 163, 164 for the detection of MREJ type vii. All these probes and primers can even be used together in the same physical enclosure. It is another object of this invention to provide a method for typing a MRJE of a MRSA strain, which comprises the steps of: reproducing the above method with primers and/or probes specific for a determined MREJ type, and detecting an annealed probe or primer as an indication of the presence of a determined MREJ type. It is further another object of this invention to provide a nucleic acid selected from SEQ ID NOs: i) SEQ ID NOs: 42, 43, 44, 45, 46, 51 for sequence of MREJ type iv; ii) SEQ ID NOs: 47, 48, 49, 50 for sequence of MREJ type v; iii) SEQ ID NOs: 171 for sequence of MREJ type vi; iv) SEQ ID NOs: 165, 166 for sequence of MREJ type vii; v) SEQ ID NOs: 167 for sequence of MREJ type viii; vi) SEQ ID NOs: 168 for sequence of MREJ type ix. Oligonucleotides of at least 10 nucleotides in length which hybridize with any of these nucleic acids and which hybridize with one or more MREJ of types selected from iv to ix are also objects of this invention. Amongst these, primer pairs (or probes) having the following SEQ ID NOs: 64/66, 64/100, 64/101; 59/52, for the detection of type i MREJ 59/53, 59/54, 59/55, 59/56, 59/57, 60/52, 60/53, 60/54, 60/55, 60/56 60/57, 61/52, 61/53, 61/54, 61/55 61/56, 61/57, 62/52, 62/53, 62/54 62/55, 62/56, 62/57, 63/52, 63/53 63/54, 63/55, 63/56, 63/57 64/66, 64/97, 64/99, 64/100, 64/101 for the detection of type ii MREJ 59/52, 59/53, 59/54, 59/55, 59/56, 59/57, 60/52, 60/53, 60/54, 60/55, 60/56, 60/57, 61/52, 61/53, 61/54, 61/55, 61/56, 61/57, 62/52, 62/53, 62/54, 62/55, 62/56, 62/57, 63/52 63/53, 63/54, 63/55, 63/56, 63/57 64/67, 64/98, 64/102; 59/58, for the detection of type iii MREJ 60/58, 61/58, 62/58, 63/58 64/79 for the detection of type iv MREJ 64/80 for the detection of type v MREJ 64/204 for the detection of type vi MREJ 64/112, 64/113 for the detection of type vii MREJ 64/115, 64/116 for the detection of type viii MREJ 64/109 for the detection of type ix MREJ, are also within the scope of this invention. Further, internal probes having nucleotide sequences defined in any one of SEQ ID NOs: 32, 83, 84, 160, 161, 162, 163, 164, are also within the scope of this invention. Compositions of matter comprising the primers and/or probes annealing or hybridizing with one or more MREJ of types selected from iv to ix as well as with the above nucleic acids, comprising or not primers and/or probes, which hybridize with one or more MREJ of types selected from i to iii, are further objects of this invention. The preferred compositions would comprise the primers having the nucleotide sequences defined in SEQ ID NOs: 64/66, 64/100, 64/101; 59/52, for the detection of type i MREJ 59/53, 59/54, 59/55, 59/56, 59/57, 60/52, 60/53, 60/54, 60/55, 60/56 60/57, 61/52, 61/53, 61/54, 61/55 61/56, 61/57, 62/52, 62/53, 62/54 62/55, 62/56, 62/57, 63/52, 63/53 63/54, 63/55, 63/56, 63/57 64/66, 64/97, 64/99, 64/100, 64/101 for the detection of type ii MREJ 59/52, 59/53, 59/54, 59/55, 59/56, 59/57, 60/52, 60/53, 60/54, 60/55, 60/56, 60/57, 61/52, 61/53, 61/54, 61/55, 61/56, 61/57, 62/52, 62/53, 62/54, 62/55, 62/56, 62/57, 63/52 63/53, 63/54, 63/55, 63/56, 63/57 64/67, 64/98, 64/102; 59/58, for the detection of type iii MREJ 60/58, 61/58, 62/58, 63/58 64/79 for the detection of type iv MREJ 64/80 for the detection of type v MREJ 64/204 for the detection of type vi MREJ 64/112, 64/113 for the detection of type vii MREJ 64/115, 64/116 for the detection of type viii MREJ 64/109 for the detection of type ix MREJ, or probes, which SEQ ID NOs are: 32, 83, 84, 160, 161, 162, 163, 164, or both. |
Blood sugar tester and data uploading method |
The present invention relates to a blood sugar test device and a method for transmitting a measured blood sugar level to a blood sugar level administration server on the Internet. In one embodiment, blood sugar test function is integrated in a mobile terminal. In another embodiment, a blood sugar test adaptor is connected to a connection terminal of a mobile terminal, the adaptor being equipped with minimal functions to perform blood sugar tests. In addition, both the mobile terminal with integrated blood sugar test function and the mobile terminal connected to the above blood sugar test adaptor can transmit measured blood sugar level to the blood sugar level administration server. A strip case is provided for at an outer surface of the mobile terminal itself in the former, while a strip case is located at an outer surface of the adaptor in the latter. |
1. A mobile communication terminal with integrated glucose test function comprising: a strip connector into which a strip applied with blood sample can be inserted, a sensor part for measuring electric current existent in blood sample on said strip, a temperature sensor part for measuring temperature at the time of a glucose test, a signal conversion part for converting said measured electric current transmitted from said sensor part into a glucose value referring to said temperature transmitted from said temperature sensor part, a radio part that transmits said glucose value measured to a glucose level administration server on the Internet through radio communication with a mobile communication base station, a display part for displaying measured glucose value, an input part for user interface, and a central processing part which controls function parts of said mobile communication terminal and transmits said glucose value received from said signal conversion part to said display part as well as to said radio part. 2. The mobile communication with integrated glucose test function as set forth in claim 1, further comprising a strip case for storage of strips at an outer surface thereof, said strips being strips to be applied with blood sample. 3. A glucose test adaptor for use in connection with a mobile communication terminal comprising: a strip connector into which a strip applied with blood sample can be inserted, a sensor part for measuring electric current existent in blood sample on said strip, a temperature sensor part for measuring temperature at the time of a glucose test, a signal conversion part for converting said measured electric current transmitted from said sensor part into a glucose value referring to said temperature transmitted from said temperature sensor part, a serial port for serial communication with a mobile communication terminal capable of transmitting measured glucose value to said mobile communication terminal, and a central processing part which controls function parts of said glucose test adaptor and transmits said glucose value received from said signal conversion part to said serial port. 4. The glucose test adaptor for use in connection with a mobile communication terminal as set forth in claim 3, comprising a strip case for storage of strips at an outer surface thereof, said strips being strips to be applied with blood sample. 5. A method for transmitting measured glucose value to a glucose level administration server on the Internet via a radio network, comprising the steps of: automatic connecting to and logging in a predetermined glucose level administration server on the Internet by a mobile communication terminal after a glucose test has been performed by said mobile communication terminal with integrated glucose test function or by a glucose test adaptor connected to said mobile communication terminal, and transmitting measured glucose value to a glucose level administration server on the Internet by said mobile communication terminal via a radio network. |
<SOH> BACKGROUND ART <EOH>A blood sugar test, being measurement of glucose level in blood, shall be conducted everyday to a patient with fluctuating glucose level. Repeated blood sugar test even within one single day shall be made for a patient diagnosed as a heavy diabetic or other specific disease. Although a blood sugar test is frequently conducted in a clinic or other medical institution, various portable blood sugar test devices are currently in supply to enable a patient to test his glucose level personally in his everyday life. For a self-test of glucose level, a patient normally requires a blood sugar test device capable of measuring a glucose level, a needle (lancet) for taking blood sample, a strip which is a test paper, and a diabetic diary for recording the test results. FIG. 1 shows a conventional portable blood sugar test device 100 . This blood sugar test device functions as follows: If a test strip 130 is applied with a blood sample and inserted into connector 120 of the blood sugar test device, glucose level of the blood sample is automatically measured and the measured glucose level is displayed on the display window 110 . A glucose level thus measured and recorded in a diabetic diary can serve as a guideline for controlling and monitoring glucose fluctuation by diet, exercise, stress, etc. as well as for administration of a diabetic. A well administration of glucose level can prevent complications in the eyes, the kidneys, the nerval systems, the blood vessels, etc. caused by high glucose level, and contribute to a rapid monitoring of a high or low blood pressure as well as curing thereof. However, for self-test of glucose level, a patient must carry a blood sugar test device, a strip case, and a diabetic diary for recording the test results, which is very inconvenient in daily life. After a test, he shall record the test result and take the records to a physician for further diagnostic and treatment. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a plane view showing a conventional portable blood sugar test device. FIG. 2 shows an internal block diagram of a mobile communication terminal with integrated blood sugar test function. FIG. 3 is a perspective view of a mobile communication terminal with integrated blood sugar test function. FIG. 4 is a perspective view showing how a mobile communication terminal is connected to a blood sugar test adaptor. FIG. 5 shows an internal block diagram of a blood sugar test adaptor. detailed-description description="Detailed Description" end="lead"? |
Nucleic acid amplication utilizing intermediate duplexes |
This invention provides for a novel amplification procedure for nucleic acid. The method uses a wild type or mutant RNA polymerase designed to transcribe both deoxyribonucleotides and ribonucleotides (see FIGS. 1-7). |
1. An oligonucleotide primer comprising in the following order from 5′ to 3′: a phage-encoded RNA polymerase recognition sequence, a spacer sequence comprising a sequence of from 12 to 21 nucleotides, and a target complementary sequence which can bind a segment of a target nucleic acid. 2. The primer of claim 1, wherein said spacer sequence comprises a nucleotide sequence having the formula (XY)n, wherein n is from 6 to 10, wherein X and Y are independently selected from the group consisting of an adenine nucleotide, a guanine nucleotide, a cytosine nucleotide, and a thymidine nucleotide, wherein X and Y are not the same. 3. The primer of claim 2, wherein X is an adenine nucleotide and Y is a guanine nucleotide. 4. The primer of claim 3, wherein n is 9. 5. The primer of claim 1, wherein said spacer sequence comprises adenine nucleotides, guanine nucleotides, and either cytosine nucleotides, or thymidine nucleotides. 6. The primer of claim 5, wherein said spacer sequence comprises a nucleotide sequence having the formula (XYZ)n, wherein n is from 4 to 7, and wherein X is an adenine nucleotide, Y is a guanine nucleotide, and Z is either a cytosine nucleotide or a thymidine nucleotide. 7. The primer of claim 1, wherein said spacer sequence comprises a nucleotide sequence having the formula (X)n, wherein n is from 12 to 20, wherein X is selected from the group consisting of an adenine nucleotide, a guanine nucleotide, a cytosine nucleotide, and a thymidine nucleotide. 8. The primer of claim 7, wherein n is 18. 9. A method of amplifying a target nucleic acid in an aqueous solution with a first and a second primer, said method comprising: i.) transcribing an intermediate duplex with a phage-encoded RNA polymerase to form a sense transcription product having a 5′ end and a 3′ end, wherein said intermediate duplex comprises a double-stranded molecule, wherein said double-stranded DNA molecule comprises a first and a second strand, wherein said first strand comprises in the following order from 5′ to 3: a phage-encoded RNA polymerase recognition sequence, a first spacer sequence comprising a sequence of from 12 to 20 nucleotides, and a first target complementary sequence which can bind to a segment of said target nucleic acid, wherein said second strand comprises in the following order from 5′ to 3′: a second target complementary sequence which can bind to a segment of said target nucleic acid, a second spacer sequence comprising a sequence of from 12 to 20 nucleotides, and a phage-encoded RNA polymerase recognition sequence, wherein said transcribing takes place in the presence of Mn++, of all four dNTPs, and of those rNTPs represented in said first spacer sequence; ii.) hybridizing said second primer to said sense transcription product to form a second primer-sense transcription product complex, wherein said second primer comprises in the following order from 5′ to 3′: a phage-encoded RNA polymerase recognition sequence, said second spacer sequence, and said second target complementary sequence which can bind to a 3′ segment of said target nucleic acid; iii.) extending said second primer-sense transcription product complex with a Reverse Transcriptase that lacks RNAseH activity to form a first amplification duplex; iv.) transcribing said first amplification duplex with a phage-encoded RNA polymerase, in the presence of Mn++, of all four dNTPs, and of those rNTPs represented in said second spacer sequence, to form an antisense transcription product; v.) hybridizing said first primer to said antisense transcription product to form a first primer-antisense transcription product complex, wherein said first primer comprises in the following order from 5′ to 3′: a phage-encoded RNA polymerase recognition sequence, said first spacer sequence, and said first target complementary sequence which can bind to a 5′ segment of said target nucleic acid; vi.) extending said second primer-antisense transcription product complex with a Reverse Transcriptase that lacks RNAseH activity to form a second amplification duplex; and vii.) transcribing said second amplification duplex with a phage-encoded RNA polymerase, in the presence of Mn++, of all four dNTPs, and of those rNTPs represented in said first spacer sequence to form said sense transcription product. 10. The method of claim 9, wherein the method further comprises repetitively carrying out steps i to vii. 11. The method of claim 9, wherein said first or said second spacer sequence comprises a nucleotide sequence having the formula (XY)n, wherein n is from 6 to 10, wherein X and Y are independently selected from the group consisting of an adenine nucleotide, a guanine nucleotide, a cytosine nucleotide, and a thyrmidine nucleotide, wherein X and Y are not the same. 12. The method of claim 11, wherein X is an adenine nucleotide and Y is a guanine nucleotide. 13. The method of claim 12, wherein n is 9. 14. The method of claim 9, wherein said spacer sequence comprises adenine nucleotides, guanine nucleotides, and either cytosine nucleotides, or thymidine nucleotides. 15. The method of claim 14, wherein said spacer sequence comprises a nucleotide sequence having the formula (XYZ)n, wherein n is from 4 to 7, and wherein X is an adenine nucleotide, Y is a guanine nucleotide, and Z is either a cytosine nucleotide or a thymidine nucleotide. 16. The method of claim 9, wherein said first or said second spacer sequence comprises a nucleotide sequence having the formula (X)n, wherein n is from 12 to 20, wherein X is selected from the group consisting of an adenine nucleotide, a guanine nucleotide, a cytosine nucleotide, and a thymidine nucleotide. 17. The method of claim 16, wherein n is 18. 18. The method of claim 9, wherein said sense and antisense transcription products comprise a nucleic acid strand comprising both ribonucleotides and deoxyribonucleotides. 19. The method of claim 9, wherein said first and said second amplification duplexes consist of deoxyribonucleotides and ribonucleotides. 20. The method of claim 9, wherein said method is carried out at a single temperature. 21. The method of claim 9, wherein said method is carried out at a single temperature of between 25° C. and 55° C. 22. The method of claim 9, wherein the method is carried out at a single temperature of greater than 50° C. 23. The method of claim 9, wherein said intermediate duplex comprises a double-stranded DNA comprising one complete primer sequence followed by the entire sequence that is to amplified. 24. The method of claim 9, wherein said intermediate duplex is formed from double-stranded DNA, single-stranded DNA, or RNA. 25. The method of claim 9, wherein said intermediate duplex is formed by the process comprising the following steps of: denaturing a double-stranded DNA target to form an upper strand and a lower strand; hybridizing said first primer to said lower strand to form a first primer-lower strand complex; extending said first primer-lower strand complex with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to form a first long sense strand product-lower strand complex; denaturing said first long sense strand product-lower strand complex into a first long sense strand product and said lower strand; hybridizing said second primer to said first long sense strand product to form a second primer-first long sense strand product; and extending said first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield said intermediate duplex. 26. The method of claim 9, wherein said intermediate duplex is formed by the process comprising the following steps of: denaturing a double-stranded DNA target to form an upper strand and a lower strand; hybridizing said first primer to said lower strand to form a first primer-lower strand complex; extending said first primer-lower strand complex with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to form a first long sense strand product-lower strand complex, wherein said first long sense strand product has a 5′ and a 3′ end; displacing said first sense strand product from said lower strand by: hybridizing a bumper oligonucleotide to a subsequence on said lower strand adjacent to said 5′ end of said first sense strand product on the first sense strand product-lower strand complex; extending said bumper oligonucleotide with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase, thereby displacing said first sense strand product; hybridizing said second primer to said first long sense strand product to form a second primer-first long sense strand product; and extending said first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield said intermediate duplex. 27. The method of claim 9, wherein said intermediate duplex is formed by the process comprising the following steps of: hybridizing said second primer to a target RNA molecule to form a second primer-RNA template complex; extending said second primer-target RNA molecule complex with a Reverse Transcriptase that lacks RNAseH activity or a DNA Polymerase to form a first long antisense strand product-template complex, wherein said first long antisense strand product has a 5′ and a 3′ end; displacing said first long antisense strand product from said target RNA molecule by: hybridizing a bumper oligonucleotide to a subsequence on said target RNA molecule adjacent to said 5′ end of said first sense strand product on the first sense strand product-lower strand complex; extending said bumper oligonucleotide with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase, thereby displacing said first long antisense strand product; hybridizing said first primer to said first long antisense strand product to form a first primer-first long antisense strand product complex; and extending said first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield said intermediate duplex. 28. The method of claim 9, wherein said intermediate duplex is formed by the process comprising the following steps of: hybridizing said second primer to a single-stranded target RNA molecule to form a second primer-RNA template complex; extending said second primer-RNA template complex with a Reverse Transcriptase that lacks RNAseH activity or a DNA Polymerase to form a first long antisense strand product-template complex; denaturing said first long antisense strand product-RNA template complex into a first long antisense strand product and said single-stranded RNA molecule; hybridizing said first primer to said first long antisense strand product to form a first primer-first long antisense strand product complex; and extending said first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield said intermediate duplex. 29. The method of claim 9, wherein said phage-encoded RNA polymerase is polymerase selected from the group consisting of: a T7 RNA polymerase, a T4 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase and a K11 RNA polymerase. 30. The method of claim 29, wherein said phage-encoded RNA polymerase is a mutant phage-encoded RNA polymerase that is competent to incorporate dNTPs into a template nucleic acid. 31. The method of claim 30, wherein said phage-encoded RNA polymerase is a T7 RNA polymerase. 32. The method of claim 31, wherein said T7 RNA polymerase contains a Y639F mutation. 33. The method of claim 31, wherein said T7 RNA polymerase contains a S641A mutation. 34. The method of claim 31, wherein said T7 RNA polymerase contains at least two mutations. 35. The method of claim 9, wherein said Mn++ is present in a concentration of between 10 μM to 20 mM. 36. The method of claim 35, wherein said concentration is 10 mM. 37. The method of claim 9, wherein said target nucleic acid is single-stranded DNA. 38. The method of claim 9, wherein the target nucleic acid is comprised of RNA. 39. The method of claim 9, further detecting said sense transcription product, said antisense transcription product, said first amplification duplex, or said second amplification duplex, wherein said detecting comprises hybridizing a detection oligonucleotide comprising a detectable moiety, wherein said detection oligonucleotide is complementary to a subsequence of said sense transcription product, said antisense transcription product, said first amplification duplex, or said second amplification duplex. 40. A kit for copying a target nucleic acid comprising: a container containing: a first nucleotide primer comprising in the following order from 5′ to 3′: a phage-encoded RNA polymerase recognition sequence, a first spacer sequence comprising a sequence of from 12 to 20 nucleotides, a first target complementary sequence which can bind to a segment of said target nucleic acid; and a second primer comprising in the following order from 5′ to 3′: a phage-encoded RNA polymerase recognition sequence, a second spacer sequence comprising a sequence of from 12 to 20 nucleotides, and a second target complementary sequence which can bind to a segment of said target nucleic acid. 41. The kit of claim 40, wherein said phage-encoded RNA polymerase is polymerase selected from the group consisting of: a T7 RNA polymerase, a T4 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase and a K11 RNA polymerase. 42. The kit of claim 41, wherein said phage-encoded RNA polymerase is a mutant phage-encoded RNA polymerase that is competent to incorporate dNTPs into a template nucleic acid. 43. The kit of claim 41, wherein said phage-encoded RNA polymerase is a T7 RNA polymerase. 44. The kit of claim 43, wherein said T7 RNA polymerase contains a Y639F mutation. 45. The kit of claim 43, wherein said T7 RNA polymerase contains a S641 A mutation. 46. The kit of claim 43, wherein said T7 RNA polymerase contains at least two mutations. 47. The kit of claim 40, further comprising a member selected from the group consisting of: a DNA polymerase; a Reverse Transcriptase that lacks RNAseH activity; a phage-encoded RNA polymerase; all four dNTPs; those rNTPs represented in said first and second spacer sequences; reaction buffer containing manganese in a concentration from 10 μM to 20 mM and magnesium in a concentration from 5 mM to 20 mM; a positive control target nucleic acid; and instructions for carrying out a method of copying a nucleic acid using said first primer and said second primer. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Many forms of nucleic acid amplification reactions have been developed in recent years. The first method was the Polymerase Chain Reaction (PCR) which involved repeated cycles/ of heating to separate the DNA strands, primer annealing to the strands, and primer extension by a DNA polymerase. Product accumulation from the PCR reaction is exponential; that is, the amount of product doubles for every cycle of amplification. Therefore, the expected amount of product may be calculated by the formula (Eff*2)n where Eff is the efficiency of the primer annealing and primer extension reaction, and n is the number of cycles. An alternative method for target amplification was developed called NASBA (Nucleic Acid Sequence Based Amplification) (see e.g., Compton (1991) Nature 350: 91-92). This method relies on the concerted action of three enzymatic activities, Reverse transcriptase, RNaseH, and RNA Polymerase, to amplify an RNA target. Reverse transcriptases generally possess an endogenous RNase H activity, which can under the correct conditions, substitute for exogenously added RNase H activity. Primers are first designed which have an RNA polymerase site together with a target recognition sequence. Then, the primers are added to the target nucleic acid together with the three enzyme activities. First, primer binds followed by primer extension across the sequence of interest. The result is a double-stranded RNA-DNA hybrid. The RNA portion of the hybrid is digested by the RNase H activity allowing binding of the other primer. The reverse transcriptase activity then extends this primer back across the sequence of interest finishing at the RNA polymerase binding sequence. The RNA polymerase activity then transcribes the sequence of interest making multiple single-stranded RNA copies. These RNAs may bind more primers and the cycle continues. Because each transcription step yields 10-100 copies of RNA per copy of template, product accumulates rapidly and logarithmically. Still, another method has been developed which is called SDA or Strand Displacement Amplification (see e.g., Walker (1993) PCR Meth. Appl. 3: 1-6. This method utilizes four primer sequences with two primers binding on either end of the sequence of interest. It also requires a DNA polymerase and a restriction endonuclease (A restriction endonuclease binds to a specific sequence called its recognition site, and then cleaves the DNA a specific sequence). In the first step, nucleic acid strands are heat separated allowing the binding of the first primer pair. The inner primer contains a restriction enzyme site which is non-complementary to the target sequence, while the outer primer binds just upstream of the inner primer. DNA polymerase extends both primers, but extension from the outer primer displaces the newly synthesized inner strand yielding a single strand template for primer binding. Extension reactions are done in the presence of a nucleotide analog (alpha-thio-dATP such that the newly synthesized strands are fully substituted making them immune to cleavage by the restriction endonuclease. However, since the inner primers are not substituted, and the complement of the inner primer is substituted, the restriction enzyme will create a nick within the inner primer sequence by cutting only within the unsubstituted sequence. The nick can act as a priming site for DNA polymerase. In the process of extending the nick, the DNA strands are separated or displaced by the DNA polymerase creating single strand primers which can then bind inner primers for the next round of amplification. Accumulation of product for SDA is therefore exponential since every priming event doubles the amount of product. Other amplification schemes have been devised, but they all require generating a single strand intermediate that allows primer binding for continued rounds of amplification (see e.g., Fahy et al. (1991) PCR Meth. Appl. 1: 25-33; Guatelli et al. (1990) Proc. Nat. Acad. Sci., U.S.A. 87: 1874-1878. While the methods described above have been shown to work well, they do have some drawbacks. PCR requires the use of a thermocycler to obtain rounds of strand separation and primer extension. Furthermore, the process of heating and cooling can be slow resulting in a PCR reaction requiring a few hours to complete from start to finish. NASBA circumvents this issue by being run isothermally, that is at a single temperature. The products are single-stranded RNA which can be relatively unstable especially if an RNase activity, which are ubiquitous, is inadvertently introduced. RNA products are also generally chemically less stable. Furthermore, the length of the expected product dictates the efficiency of the amplification reaction. This is in part due to the RNAaseH activity which can inactivate RNA transcripts with bound primers. NASBA reactions also require the addition of high concentrations of both ribonucleotides and deoxyribonucleotides increasing the cost of running a reaction. NASBA reactions are also run at lower temperatures leading to the production of spurious amplification products. In SDA, while the amplification products are DNA, the products are modified by the presence of the alpha-thio-dATP used to inhibit strand cleavage by the restriction endonuclease which may make further manipulation of the product difficult, especially in research applications. There is a need for improved methods of nucleic amplification. This invention meets those needs. |
<SOH> SUMMARY OF THE INVENTION <EOH>In one aspect, the present invention provides for oligonucleotide primers that comprise in the following order from 5′ to 3′: a phage-encoded RNA polymerase recognition sequence, a spacer sequence comprising a sequence of from 12 to 20 nucleotides that consists of one nucleotide type or two different nucleotide types, and a target complementary sequence which can bind a segment of a target nucleic acid. In certain embodiments, the space sequence comprises a nucleotide sequence having the formula (XY) n , wherein n is from 6 to 10, wherein X and Y are independently selected from the group consisting of an adenine nucleotide, a guanine nucleotide, a cytosine nucleotide, and a thymidine nucleotide, wherein X and Y are not the same. In certain preferred embodiments, X is an adenine nucleotide and Y is a guanine nucleotide. In other embodiments, the spacer sequence comprises a nucleotide sequence having the formula (X) n , wherein n is from 12 to 20, wherein X is selected from the group consisting of an adenine nucleotide, a guanine nucleotide, a cytosine nucleotide, and a thymidine nucleotide. In some embodiments, all four nucleotides are present in the spacer. In another aspect, the present invention provides for methods of amplifying a target nucleic acid in an aqueous solution with a first and a second primer, said method comprising: i.) transcribing an intermediate duplex with a phage-encoded RNA polymerase to form a sense transcription product having a 5′ end and a 3′ end, wherein said intermediate duplex comprises a double-stranded molecule, wherein said double-stranded DNA molecule comprises a first and a second strand, wherein said first strand comprises in the following order from 5′ to 3: a phage-encoded RNA polymerase recognition sequence, a first spacer sequence comprising a sequence of from 12 to 20 nucleotides, and a first target complementary sequence which can bind to a segment of said target nucleic acid, wherein said second strand comprises in the following order from 5′ to 3′: a second target complementary sequence which can bind to a segment of said target nucleic acid, a second spacer sequence comprising a sequence of from 12 to 20 nucleotides, and a phage-encoded RNA polymerase recognition sequence, wherein said transcribing takes place in the presence of Mn ++ , with all four dNTPs, and with those rNTPs represented in said first spacer sequence; ii.) hybridizing said second primer to said sense transcription product to form a second primer-sense transcription product complex, wherein said second primer comprises in the following order from 5′ to 3′: a phage-encoded RNA polymerase recognition sequence, said second spacer sequence, and said second target complementary sequence which can bind to a 3′ segment of said target nucleic acid; iii.) extending said second primer-sense transcription product complex with a Reverse Transcriptase that lacks RNAseH activity to form a first amplification duplex; iv.) transcribing said first amplification duplex with a phage-encoded RNA polymerase, in the presence of Mn ++ , with all four dNTPs, and with those rNTPs represented in said second spacer sequence, to form an antisense transcription product; v.) hybridizing said first primer to said antisense transcription product to form a first primer-antisense transcription product complex,wherein said first primer comprises in the following order from 5′ to 3′: a phage-encoded RNA polymerase recognition sequence, said first spacer sequence, and said first target complementary sequence which can bind to a 5′ segment of said target nucleic acid; vi.) extending said second primer-antisense transcription product complex with a Reverse Transcriptase that lacks RNAseH activity to form a second amplification duplex; and vii.) transcribing said second amplification duplex with a phage-encoded RNA polymerase, in the presence of Mn ++ , with all four dNTPs, and with those rNTPs represented in said first spacer sequence to form said sense transcription product. Typically, these methods further involve repetitively carrying out steps i to vii. For example, steps i to vii can be carried out one, two, etc. The rNTPs represented in the spacer sequences can be present in the reaction mixture. For example if a spacer sequence is (AG) 12-20 then rATP and RGTP can be present in the reaction mixture. Similarly, the first or said second spacer sequence can comprise a nucleotide sequence having the formula (X) 12-20 , where X is a dNTP. Then, the corresponding rNTP should be present in the reaction mixture. The sense and antisense transcription products can comprise a nucleic acid strand comprising both ribonucleotides and deoxyribonucleotides. In addition, the first and said second amplification duplexes can consist of deoxyribonucleotides and ribonucleotides. In certain embodiments, the intermediate duplex comprises a double-stranded DNA comprising one complete primer sequence followed by the entire sequence that is to be amplified. The intermediate duplex can be formed from a variety of sources including, without limitation, double-stranded DNA, single-stranded DNA, or RNA. For example, in certain embodiments, the intermediate duplex is formed by the process comprising the following steps of: denaturing a double-stranded DNA target to form an upper strand and a lower strand; hybridizing the first primer to the lower strand to form a first primer-lower strand complex; extending the first primer-lower strand complex with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to form a first long sense strand product-lower strand complex; denaturing the first long sense strand product-lower strand complex into a first long sense strand product and the lower strand; hybridizing the second primer to the first long sense strand product to form a second primer-first long sense strand product; and extending the first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield the intermediate duplex (see e.g., FIG. 4 ). In other embodiments, the intermediate duplex is formed from DNA by the process comprising the following steps of: denaturing a double-stranded DNA target to form an upper strand and a lower strand; hybridizing the first primer to the lower strand to form a first primer-lower strand complex; extending the first primer-lower strand complex with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to form a first long sense strand product-lower strand complex, wherein the first long sense strand product has a 5′ and a 3′ end; displacing the first sense strand product from the lower strand by: hybridizing a bumper oligonucleotide to a subsequence on the lower strand adjacent to the 5′ end of the first sense strand product on the first sense strand product-lower strand complex; extending the bumper oligonucleotide with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase, thereby displacing the first sense strand product; hybridizing the second primer to the first long sense strand product to form a second primer-first long sense strand product; and extending the first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield the intermediate duplex (see e.g., FIG. 5 ). In still other embodiments, the intermediate duplex is formed from RNA. For example, the intermediate duplex can be formed by the process comprising the following steps of: hybridizing the second primer to a target RNA molecule to form a second primer-RNA template complex; extending the second primer-target RNA molecule complex with a Reverse Transcriptase that lacks RNAseH activity or a DNA Polymerase to form a first long antisense strand product-template complex, wherein the first long antisense strand product has a 5′ and a 3′ end; displacing the first long antisense strand product from the target RNA molecule by: hybridizing a bumper oligonucleotide to a subsequence on the target RNA molecule adjacent to the 5′ end of the first sense strand product on the first sense strand product-lower strand complex; extending the bumper oligonucleotide with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase, thereby displacing the first long antisense strand product; hybridizing the first primer to the first long antisense strand product to form a first primer-first long antisense strand product complex; and extending the first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield the intermediate duplex (see e.g., FIG. 6 ). In still other embodiments, the intermediate duplex can be formed by the process comprising the following steps of: hybridizing the second primer to a single-stranded target RNA molecule to form a second primer-RNA template complex; extending the second primer-RNA template complex with a Reverse Transcriptase that lacks RNAseH activity or a DNA Polymerase to form a first long antisense strand product-template complex; denaturing the first long antisense strand product-RNA template complex into a first long antisense strand product and the single-stranded RNA molecule; hybridizing the first primer to the first long antisense strand product to form a first primer-first long antisense strand product complex; and extending the first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield the intermediate duplex (see e.g., FIG. 7 ). The temperature of the reaction mixture can vary over the range at which the enzymes in the mixture are active and products are produced. However, the methods can be carried out at a single temperature, e.g., isothermally. For example, the methods can be carried out at a single temperature of between 25° C. and 55° C., or at a single temperature of greater than 50° C. The methods described herein can comprise a reaction mixture further containing a bumper oligonucleotide which is: (i) able to hybridize to a DNA sequence about or adjacent to the 5′ end of the first long strand and (ii) able to serve as polymerase primer which displaces the first long strand when extended towards the 3′ end of the target nucleic acid. This invention further comprises a novel composition comprising a double-stranded DNA having a first and second end comprising a phage-encoded RNA polymerase recognition sequences on both the first and second ends wherein at least one end has a complementary sequence that forms a phage polymerase recognition site. This is termed an intermediate duplex. The composition may also be a double-stranded DNA having phage-encoded RNA polymerase recognition sequences on both the first and second ends wherein the sites may be the same or different. The composition may also have a single functional RNA polymerase binding site on one end and a blunt end on the other end in which the blunt end has the target complementary sequence with the RNA polymerase binding site. The composition may optionally comprise a signature sequence for a specific genus or species of organism. This invention also provides for a novel aqueous reaction mixture comprising: i. a target nucleic acid for amplification; ii. a first and second amplification primer each having a phage-encoded RNA polymerase recognition sequence, a spacer unit of 12 to 20 bases or more, and a target binding sequence wherein the first target binding sequence is complementary to the 5′ end of the target sequence and the second target binding sequence is complementary to the antisense sequence of the 3′ end of the target sequence; iii. phage-encoded RNA polymerase either wild type or an RNA polymerase mutated to recognize and polymerize dNTP and rNTP; and, iv. an excess of dNTPs and appropriate rNTPs. The reaction mixture may also comprise target nucleic acid which is a double-stranded DNA having a first 5′ end which bears a phage-encoded RNA polymerase recognition site and a second 5′ end which bears a phage-encoded RNA polymerase recognition sequence. Essentially any RNA polymerase that is active under the method conditions and can transcribe rNTPs and dNTPs into the products in the method can be used. An especially preferred class of RNAPs are the phage-encoded polymerases which include, without limitation, a T7 RNA polymerase, a T4 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase and a K11 RNA polymerase. In certain embodiments, the phage-encoded RNA polymerase is a mutant phage-encoded RNA polymerase (e.g., a mutated T7 RNAP) that is competent to incorporate dNTPs into a template nucleic acid. For example, T7 RNAPs with a Y639F mutation, or a S641A mutation, or Y639F/S641A mutations can be used. In certain embodiments, the T7 RNAP contains at least two mutations. Manganese ions are critical in the method for the incorporation of dNTPs into the transcription products. Preferably, the Mn ++ is present in a concentration of between 10 μM to 20 mM. In an especially preferred embodiment, the concentration is 10 mM. The target nucleic acid can be single-stranded DNA or comprised of RNA. In some embodiments, one or more of the products of the method can be detected using a labeled reagent. For example, the methods can further comprise the sense transcription product, the antisense transcription product, the first amplification duplex, or the second amplification duplex, wherein the detecting comprises hybridizing a detection oligonucleotide comprising a detectable moiety, wherein the detection oligonucleotide is complementary to a subsequence of the sense transcription product, the antisense transcription product, the first amplification duplex, or the second amplification duplex. This invention further provides for a kit for amplifying a target nucleic acid comprising a container containing a first primer having a sequence complementary to a 5′ end of the target nucleic acid and a phage polymerase recognition sequence and a spacer sequence of 12 to 20 bases or more, and a container containing a second primer having a sequence which is the anti-complement to the 3′ end of the target nucleic acid and a phage polymerase recognition sequence and a second spacer of 12 to 20 bases or more. The kit may also have a wild type RNA polymerase or a mutant phage polymerase competent to incorporate dNTP and rNTP simultaneously into a template nucleic acid. The kit may also have a bumper oligonucleotide which is able to hybridize to a template DNA sequence where that sequence is about or immediately adjacent to the 3′ base of the sequence to which one of the amplification primer binds. |
Wheel, in particular a spoked wheel for motor vehicles constructed from sheet-steel |
The invention relates to a wheel rim, in particular a spoked wheel rim for motor vehicles consisting of sheet-steel. Said rim is assembled from various parts: an inner screw-on flange (1b), a central rim ring (1a) and an external rim well (3, 4, 5). The rim ring (1a) and the screw-on flange (1b) form an inner bearing shell (1) for the main bearing load of the wheel rim. Said bearing shell (1) is covered by an external design shell (2) consisting of sheet-steel, forming a hollow chamber (7). Said design shell is connected in a fixed manner to the screw-on flange (1b) and the rim flange (3) and is configured to support an auxiliary bearing load. |
1. Wheel for motor vehicles, in particular passenger cars or motorcycles, built up in several parts, with a rim ring, with a design shell located in the opening surrounded by the rim ring and capable of being attached to a wheel hub of the motor vehicle concerned, and with a covering shell located on the outside of the wheel, whereby the design shell and the covering shell support the rim ring jointly and the force absorbed by the covering shell when the rim ring is under load is essentially equal to the force absorbed by the design shell. 2. Wheel as in claim 1, wherein the covering shell is connected non-positively with the rim ring. 3. Wheel as in claim 1, wherein the covering shell is connected positively with the rim ring. 4. Wheel as in claim 1, wherein the covering shell is connected material-interlockingly with the rim ring, in particular by welding, soldering or bonding. 5. Wheel as in claim 1, wherein the design shell is provided with screw-down openings through which the attachment to the corresponding hub of the motor vehicle is effected. 6. Wheel as in claim 5, wherein the screw-down openings are formed in a screw-on flange of the design shell. 7. Wheel as in claim 5, wherein the covering shell and the design shell can be screwed together to the corresponding hub of the motor vehicle by means of at least one screw connection. 8. Wheel as in claim 1, wherein the covering shell is connected firmly to the design shell in the area of the screw-down openings. 9. Wheel as in claim 6, wherein the design shell is provided with at least one spoke via which the rim ring is connected to the screw-on flange. 10. Wheel as in claim 1, wherein the covering shell and the design shell are held at a distance from each other, at least in some sections. 11. Wheel as in claim 7, wherein the spoke is made with a U-shaped profile at least in some sections. 12. Wheel as in claim 1, wherein it is made of steel. 13. Wheel as in claim 1, wherein the rim ring, the design shell and/or the covering shell are made of special steel. 14. Wheel as in claim 1, wherein the rim ring is provided with a rim flange on its side associated with the covering shell. 15. Wheel as in claim 14, wherein the covering shell is connected firmly by its border assigned to the rim ring at least in sections to the rim flange. 16. Wheel as in claim 15, wherein the border of the covering shell assigned to the rim ring is bent around the rim flange and/or is material-interlockingly connected to the rim flange, being in particular welded, bonded or soldered. 17. Wheel as in claim 1, wherein the rim flange is formed from a border section of the covering shell assigned to the rim ring. 18. Wheel as in claim 1, wherein the rim ring is formed of several ring elements with different thicknesses and/or firmness. 19. Wheel as in claim 1, wherein a ring element is located on the inside of the design shell away from the covering shell, said ring element holding the wheel at a distance from the corresponding hub of the motor vehicle in its mounted state on the motor vehicle. 20. Wheel as in claim 19, wherein the ring element possesses low heat conductivity. 21. Wheel as in claim 19, wherein the ring element attenuates sound. 22. Wheel as in claim 1, wherein the covering shell is provided with a colored coating on its visible outside. 23. Wheel as in claim 1, wherein the design shell covers a border area of the rim with a border area formed on its outer circumference and in that the design shell is material-interlockingly connected, in particular welded, to the rim ring in the area of the channel formed between the bottom of the rim ring as well as on the circumferential border of the design shell. 24. Wheel as in claim 1, wherein it is intended to be mounted on a passenger car or truck and in that the covering shell is mounted on the outside of the wheel on its side away from the body of the motor vehicle when mounted. |
Multipurpose hydraulic shock absorber for vehicle |
The invention concerns a shock absorber wherein all the adjustable means (70, 71) determining compression phase damping are arranged in the head (61) of the reservoir (R) and are externally accessible without disassembling, and those (71) adjusting low-speed compression, comprise a tubular body (75) for preload adjustment by being screwed in the head (61) of the reservoir, and overlapping outside thereof through an external manoeuvring head (75a), and, inside the reservoir, through an active end configured as a seat (82), said tubular body (75): a) communicating with the shock absorber wide cross-section chamber (G), b) being axially traversed by the adjusting screw (70) bearing, at its end inside the reservoir (R), the valve (72) with its spring-type setting means, and c) including, in its part configured as a seat (82) for the valve (72), at least a radial groove (81) forming an outlet channel determining the preload, the passage cross-section of said channel being determined by the distance between the terminal side (82) of the tubular body (75) and the inner side (61a) of the head (61). |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.