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Devices and methods for the production of particles |
The present invention provides methods and devices for producing particles with an average diameter less than about 15 μm using the precipitation with compressed fluid-antisolvent (PCA) process and the carbon-dioxide assisted nebulization with a bubble dryer (CAN-BD) process. In the methods and nozzles of the invention, at least one jet of supercritical or near-supercritical fluid and at least one jet of solution interact to mix the supercritical or near-supercritical fluid and the solution within a chamber. The solution contains at least one solvent and at least one solute. At least one of the jets is a swirling jet. To form particles, the solvent and supercritical or near-supercritical fluid are then injected into a PCA or a CAN-BD process chamber. The degree of mixing depends in part on the power input into the mixing chamber. Power inputs of about 6.5×109 W/m3 enhance the degree of mixing and allow production of nanoscale particles with the PCA process. The nanoscale particles have a size distribution so that polydispersity is less than about 1.75. |
1. A method for producing particles having a diameter less than about 15 microns comprising the steps of: a) mixing a first fluid and a second fluid in a mixing chamber, wherein mixing occurs through interaction of at least one first fluid jet and at least one second fluid jet in the mixing chamber and least one of the jets is a swirling jet; and b) spraying the mixed first and second fluids through an outlet into the chamber of an apparatus for forming particles wherein the first fluid is selected from the group comprising a supercritical fluid and a near-supercritical fluid and the second fluid is a solution comprising a solvent and a solute. 2. The method of claim 1, wherein the second fluid jet is a swirling jet. 3. The method of claim 1, wherein the particles have a size distribution so that the polydispersity is less than about 1.75. 4. The method of claim 1, wherein the particles have an average diameter less than about 1 micron. 5. The method of claim 4, wherein the particles have a size distribution so that the polydispersity is less than about 1.5. 6. The method of claim 1, wherein the power input into the nozzle is above about 6.5×109 W/m3. 7. The method of claim 1, wherein the particles are polymer particles. 8. The method of claim 7, wherein the solution further comprises a bioactive ingredient. 9. The method of claim 1, wherein the supercritical or near-supercritical fluid is CO2. 10. The method of claim 1, wherein the apparatus for forming particles is a PCA apparatus. 11. The method of claim 10, wherein the solvent is non-aqueous. 12. The method of claim 1, wherein the apparatus for forming particles is a CAN-BD apparatus. 13. The method of claim 12, wherein the solvent is aqueous. 14. An apparatus for forming particles with an average diameter less than about 15 microns comprising: a) a particle formation chamber capable of maintaining a selected temperature and pressure; and b) a nozzle for introduction into the particle formation chamber of a first fluid and a second fluid, the nozzle having an axis, wherein both the fluids flow through the nozzle so that each fluid has a component of flow parallel to the nozzle axis and wherein the nozzle comprises a mixing chamber having interior walls; at least a first and a second inlet to the mixing chamber; and at least one outlet from the mixing chamber, wherein at least the first inlet is a swirl inlet which is not parallel to the nozzle axis and the walls of the mixing chamber converge towards the outlet in the vicinity of the outlet and the first fluid is selected from the group consisting of a supercritical and a near-supercritical fluid and the second fluid is a solution comprising a solvent and at least one solute. 15. The apparatus of claim 14, wherein the second inlet is an axial inlet. 16. The apparatus of claim 15, wherein the outlet is axial. 17. The apparatus of claim 15, wherein the ratio of the diameters of the second inlet to the outlet is between about 1:10 and about 10:1. 18. The apparatus of claim 15, wherein the ratio of the diameters of the second inlet to the outlet is between about 1:2 and about 2:1. 19. An apparatus for forming particles with an average diameter less than about 15 microns comprising: a) a particle formation chamber capable of maintaining a selected temperature and pressure; and b) a nozzle for introduction into the particle formation chamber of a first fluid and a second fluid, the nozzle having an axis, wherein both the first fluid and the second fluid flow through the nozzle so that each fluid has a component of flow parallel to the nozzle axis and wherein the nozzle comprises a mixing chamber having interior walls, the walls having an angle with respect to the nozzle axis of less than 90° and the upper surface of the mixing chamber being formed by a swirl insert; at least a first and a second inlet to the mixing chamber, the first and second inlet being formed through the swirl insert; and at least one outlet from the mixing chamber, wherein at least the first inlet is a swirl inlet which is not parallel to the nozzle axis and the first fluid is selected from the group consisting of a supercritical and a near-supercritical fluid and the second fluid is a solution comprising a solvent and at least one solute. 20. The apparatus of claim 19, wherein the second inlet is an axial inlet. 21. The apparatus of claim 20, wherein the outlet is axial. 22. The apparatus of claim 20, wherein the ratio of the diameter of the second inlet to the diameter of the outlet is between about 1:10 and about 10:1. 23. The apparatus of claim 20, wherein the ratio of the diameter of the second inlet to the diameter of the outlet is between about 1:2 and about 2:1. 24. A nozzle for spraying a first fluid and a second fluid, the nozzle having an axis and comprising: a) a mixing chamber having interior walls; b) a first and second inlet to the mixing chamber; and c) at least one outlet from the mixing chamber, wherein both fluids flow through the nozzle so that each fluid has a component of flow parallel to the nozzle axis, at least the first inlet is a swirl inlet which is not parallel to the nozzle axis and the walls of the mixing chamber converge towards the outlet in the vicinity of the outlet and the first fluid is selected from the group consisting of a supercritical and a near-supercritical fluid and the second fluid is a solution comprising a solvent and at least one solute. |
<SOH> BACKGROUND OF THE INVENTION <EOH>This invention is in the field of particles having an average diameter less than about 15 microns, and in particular is directed to methods and devices for making particles using the precipitation with compressed-fluid antisolvent (PCA) process and the carbon-dioxide assisted nebulization with a bubble dryer (CAN-BD) process. Dry powder formulations with a controlled particle size and size distribution have many applications in the field of pharmaceutical drug delivery, especially in the areas of pulmonary delivery, controlled release, and needle-less powder injections. These formulations require precise control of particle size and size distribution. For example, pulmonary dosage forms of solid particles require particle diameters of about 1-3 μm in order to effectively target regions of the deep lung (J. Heyder et al. (1986) J. Aerosol Sci. 5:811). There has been considerable interest in developing polymeric nanoparticles (about 1-100 nm) that allow for targeted drug delivery to particular organs and tissues because they exhibit biodistribution profiles that are different from those of microparticles (greater than about 1 micron). For example, Desai et al. found the efficiency of uptake of 100 nm-sized poly (l-lactide-co-glycolide) particles by intestinal tissue was 15-250 fold higher compared to that for 1-10 μm sized particles (M. P. Desai et al (1996) Pharm. Res. 13:1838). Research in developing biodegradable polymeric nanoparticles has received attention because of their applications in controlled release formulations, as carriers for DNA in gene therapy, and their ability to deliver proteins, genes, and vaccines through a peroral route of administration (J. C Leurox, et al. (1996) J. Controlled Release 39:339; R. M. Kuntz, W. M. Saltzman, (1997) Trends Biotechnol, 15:364). Traditional techniques for the production of micrometer and sub-micrometer particles include mechanical comminution (e.g., grinding and milling), recrystallization of solutes from solution using liquid antisolvents, double emulsion/evaporation processes, freeze drying and spray drying (B. Subramaniam, R. A. et al (1997) J. Pharm. Sci. 86:885). However these techniques have limitations that include excessive solvent use, thermal and chemical degradation of the solute, large residual solvent concentrations, and difficulties in controlling particle size and size distribution during processing. These limitations may affect powder stability, flow properties, and delivery efficiency (P. H. Hirst, et al. (2002) Pharm. Res. 19:258). Overall, the production of monodisperse powders with a controlled particle size in the micro/nanoscale remains challenging. To address the shortcomings of traditional processing methods, supercritical fluid precipitation technologies that use supercritical carbon dioxide as an antisolvent have been investigated as a means for producing particulates. Supercritical fluids offer distinct advantages as antisolvents for precipitation. By adjusting both the temperature and pressure the physical properties of the supercritical fluid such as density, viscosity, and diffusivity can be readily varied. Favorable mass transfer characteristics, defined by a low viscosity and high diffusivity relative to liquids, offer the capability of producing monodisperse powders with low residual solvent concentrations. Supercritical carbon dioxide, by far the most common supercritical antisolvent, has a relatively low critical temperature (304.1 K) and pressure (7.38 MPa), low toxicity, and is inexpensive. Precipitation with a compressed-fluid antisolvent (PCA) is a method that is capable of producing micrometer and sub-micrometer sized powders in a single-stage, scalable process. Under the conditions typically used in PCA processing, a solute is dissolved in an organic solvent and the resulting solution is injected into, or mixed with, a supercritical fluid antisolvent. Typically, the method involves the use of a particle formation chamber containing compressed fluid antisolvent. The operating temperature and pressure are selected so the supercritical fluid is completely miscible with the organic solvent. Due to the very rapid two-way mass transfer in supercritical fluid mixtures (i.e. solvent diffusing into the supercritical fluid and the supercritical fluid diffusing into the solvent) high degrees of supersaturation of the solute occur and the solute precipitates. Past PCA research and development has focused on using jet hydrodynamics and liquid atomization theory as a guide for manipulating and controlling particle size during the PCA process. The Weber number, a ratio of inertial to surface tension forces, may be used to estimate droplet sizes in different atomization spray configurations (A. H. Lefebvre, Atomization and Sprays, Hemisphere Publishing Corp., Bristol, Pa., 1989). Liquid atomization theory predicts that larger Weber numbers will yield more intense atomization and hence smaller liquid droplet diameters. Smaller liquid droplets allow for an increase in the two-way mass transfer rates between the solvent/antisolvent, resulting in larger nucleation rates and smaller particles. With this theory as a guide, attempts have been made to control polymer particle size and size distribution (D. J. Dixon et al. (1993) AiChE J. 39:127; R. Bodemeier et al (1995) Pharm. Res. 12:1211; but only a weak variation in size with flow rate or Reynolds number was observed. In addition, various process parameters such as temperature, pressure, solute concentration, and solvent/solute flow rate have been manipulated to adjust particle size, but the results show there is a limited range in polymer size variation when these process parameters are varied. Overall, manipulation of particle size distributions has proved difficult using conventional PCA mixing configurations. More recent research suggests that gaseous mixing theory and mixing rates, or rather, mixing length scales for turbulent mixing, should be used as a guide for manipulating and controlling particle size during the PCA process. Lengsfeld et al. (2000) J. Phys. Chem B, 104:2725) recently explored the validity of the assumption that liquid atomization controls the size of particles produced in the PCA process. They demonstrated that surface tension for jets of miscible fluids injected into critical and near-critical solvents is reduced to negligible levels over timescales shorter than the characteristic jet breakup time, and that distinct droplets never form. Studies by Werling et al. (J. O. Werling, P. G. Debenedetti (2000) J. Supercrit. Fluids 18:11) and Bristow et al. (2001) J. Supercrit. Fluids 21:257) also show that for supercritical conditions, where the solvent and antisolvent are completely miscible, there is no well-defined droplet interface that is stabilized by surface tension. Based on these results, liquid atomization theory and Weber number based analysis are no longer considered the appropriate theory and parameter to characterize the process. Instead, gaseous mixing theory and mixing rates, or rather, mixing length scales for turbulent mixing, should be used to characterize sprays of miscible fluids (B. Y. Shekunov et al. (1999) J. Crystal Growth 198/199:1345). Various methods for mixing the solution and compressed antisolvent have been used. In one set of methods the solution is injected into a chamber of compressed antisolvent, and mixing between the two fluids occurs within the particle formation chamber. The solvent may be sprayed through a capillary tube (or capillary nozzle) directly into the chamber (Dixon et al. (1993), Materials, Interfaces, and Electrical Phenomena, 39(1), 127-139). Particle sizes obtained using this method are on the order of 0.1-5 microns. The solution may also be sprayed through a capillary tube onto a horn which is vibrated at ultrasonic frequencies. (Chattopadhyay and Gupta (2001), Ind. Eng. Chem. Res., 40, 3530-3539). In the later process, termed “SAS-EM”, mixing is carried out in the bulk of the particle formation chamber. The ultrasonic field generated by the horn surface is said to enhance turbulence and mixing within the supercritical phase, resulting in high mass transfer between the solution and the antisolvent and smaller particles (100-500 nm). The polydispersity of the particles reported was about 1.4 at a volume average particle size of 1100 nm and decreased to about 1.1 at a volume average particle size of 125 nm. However, the ultrasonic horns are expensive and have a limited life time at high pressure. Furthermore, heat is an unknown and uncontrolled variable. Heat may degrade molecules and affect the precipitation kinetics in the process as described. Furthermore, this process has not been demonstrated for polymer particles. The solution may also be co-introduced with a flow of antisolvent into the chamber of compressed antisolvent. The flow of antisolvent generates additional mixing of antisolvent and solvent. Typically, the solution and antisolvent are introduced coaxially through a coaxial nozzle. The velocities of the two flows can be manipulated independently and the antisolvent flow velocity is typically greater than that of the solvent. (Mawson et al. (1997) 64, 2105-2118). The solution can also be co-introduced with a flow of antisolvent through ultrasonic spray nozzles (Falk et al., (1997), J. Controlled Release, 44, 77-85; Randolph, T. W. et al., (1993), Biotechnology Progress, 9, 429-435). In some coaxial nozzle designs, mixing between the solution and the antisolvent does not occur within the nozzle. For example, Mawson et al. (1997, supra) show a coaxial nozzle having an inner nozzle tube which extends beyond an outer nozzle tube. The inner tube carries the solvent while the outer tube carries the antisolvent. Other coaxial nozzle designs do provide some mixing of the solution and the antisolvent within the nozzle. U.S. Pat. No. 5,851,453 to Hanna et al., issued Dec. 22, 1998 describes a coaxial nozzle in which the outer nozzle passage extends beyond the inner nozzle passage. The ends of both nozzle passages are tapered. In this configuration some mixing of the solvent and antisolvent can occur within the nozzle before the fluids pass into the main chamber of the PCA apparatus. The mean particle sizes demonstrated are greater than one micron. Yu et al. ((2001), Chem. Eng. Sci., 56, 2421-2433) describe a coaxial nozzle consisting of two tubes (for internal solution and external CO 2 flows) opening into a small premixing chamber. Although Yu et al. (2001) show a decrease in the mean particle diameter with an increase in the Reynolds number, the particles are not nanosized. Instead, the smallest mean particle diameter discussed appears to be about 5 microns in diameter. The size distribution of the particles does not appear to be reported, but the SEM photos of the smaller particles show clear size differences. The authors state that large-scale inhomogeneity may still exist in the flow through the nozzle. Another type of nozzle design for co-introduction of antisolvent and solution mixes two antisolvent flows and a solution within a chamber inside the nozzle (WO 98/35825, Hanna and York, published Aug. 27, 1998). In one embodiment, the nozzle provides a first fluid inlet means for the introduction into the chamber of a first supercritical fluid and a solution and second fluid inlet means for introducing simultaneously an impinging flow of the second supercritical fluid at an angle to, and directed at, the direction of flow of the first supercritical fluid. In a “cross-flow” nozzle, the two supercritical fluids were opposing (angle between the supercritical fluids of 180°). The first inlet means preferably has two or more concentric passages through which may be introduced a flow of the first supercritical fluid and a flow of the solution (i.e. the first inlet means preferably is a coaxial nozzle). Nozzle configurations are also described in which the solution is introduced at an angle to the flow of the first supercritical fluid, so long as it is then dispersed by the supercritical fluid(s) immediately as it comes into contact with them. In particular, a configuration is shown where the solution is introduced perpendicular to that of two countercurrent supercritical fluid flows. A cross-flow nozzle was demonstrated to produce particles in the size range 200-750 nm for a variety of materials. For nicotinic acid, it was claimed that the size distribution of (particles was narrowed compared to particles produced with a conventional coaxial nozzle. Carbon Dioxide-Assisted Nebulization with a Bubble Dryer (CAN-BD) is another particle formation method that is capable of producing powders less than about 6.5 microns in diameter. Under the conditions typically used in CAN-BD processing, a solute is dissolved in an aqueous solvent and the resulting solution is mixed with a supercritical fluid. In contrast to the PCA process, in the CAN-BD process the solvent is either insoluble or partially soluble in the supercritical fluid and the supercritical fluid is partially soluble in the aqueous solvent. The particles are formed through rapidly expanding the mixture into a region in which the temperature and pressure are below the critical temperature and pressure of the supercritical fluid. The rapid decompression of the supercritical fluid, coupled with the explosive release of dissolved supercritical fluid in the aqueous solution, acts to atomize the aqueous solution and produce an aerosol. This aerosol is directed into a stream of co-flowing heated gas (typically air or nitrogen), and particle formation results due to evaporation of the solvent. Solvent evaporation from droplets occurs in a plume that extends from the injection nozzle. The length of this plume is important for equipment design, as the plume must not impinge on any surfaces or vessel walls before evaporation is complete, or else severe agglomeration of wet particles may result. The size of this plume may be minimized by efficiently mixing the supercritical fluid with the aqueous solvent in a fashion so that the concentration of supercritical fluid dissolved in the aqueous phase immediately before expansion into the drying chamber is as high as possible. Sievers et al. (U.S. Pat. No. 5,639,441, issued Jun. 17, 1997) discloses a mixing tee with a dead volume less than about 10 μl for mixing the supercritical fluid and solvent. There remains a need in the art for improved methods and devices for producing particles with an average diameter less than 15 microns, preferably particles with an average diameter less than 1 micron. Furthermore, there remains a need in the art for particles with a controlled particle size. Preferably the particles have a narrow size distribution, with a polydispersity less than about 1.75. Although nanoscale particles have been made using the PCA and CAN-BD processes, the resulting particle size distributions have not always been optimal. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides methods and devices for producing particles with an average diameter of less than about 15 microns. The particles can be produced using the PCA process or by using the CAN-BD process. Particles produced using the PCA process can be made with an average diameter less than about 1 micron and with a polydispersity of less than about 1.75. The invention provides an apparatus for forming particles with an average diameter less than about 15 microns comprising: a particle formation chamber capable of maintaining a selected temperature and pressure; and a nozzle for introduction into the particle formation chamber of a first fluid and a second fluid, the nozzle having an axis, wherein both the fluids flow through the nozzle so that each fluid has a component of flow parallel to the nozzle axis and the first fluid is selected from the group consisting of a supercritical and a near-supercritical fluid, and the second fluid is a solution comprising a solvent and at least one solute; and wherein the nozzle comprises a mixing chamber having interior walls; at least a first and a second inlet to the mixing chamber; and at least one outlet from the mixing chamber, wherein at least the first inlet is a swirl inlet which is not parallel to the nozzle axis and the walls of the mixing chamber converge towards the outlet in the vicinity of the outlet. The nozzle creates at least two jets within the mixing chamber, wherein at least one of the jets is a swirling jet. Because of the swirling jet, swirling flow occurs within the mixing chamber which generates a rapidly expanding flow upon exit from the mixing chamber, which in turn acts to reduce particle growth by agglomeration. Either the solution or supercritical or near-supercritical fluid can swirl, or both fluids can swirl. In one configuration, the nozzle has at least one axial inlet to the mixing chamber which is parallel to the longitudinal axis of the nozzle and at least one swirl inlet which has an angle with respect to the longitudinal axis of less than 90°. The invention provides a method for producing particles comprising the steps of mixing a first fluid and a second fluid, wherein mixing occurs through interaction of at least one first fluid jet and at least one second fluid jet in the mixing chamber and least one of the jets is a swirling jet; and spraying the mixed fluids through an outlet into the chamber of an apparatus for forming particles, wherein the first fluid is selected from the group consisting of a supercritical and a near-supercritical fluid and the second fluid is a solution comprising a solvent and at least one solute. The apparatus for forming particles may be configured as a PCA-type apparatus or a CAN-BD-type apparatus. The interaction of the jets within the mixing chamber contributes to mixing of the solution and the supercritical or near-supercritical fluid within the chamber. The mixing within the chamber can be characterized by the energy for mixing inside the mixing chamber, or power input. In the methods of the invention, it is preferred that the power input into the nozzle is above about 6.5×10 9 W/m 3 . |
Lymphatic endothelial cells materials and methods |
The present invention is directed to methods and compositions for isolating lymphatic endothelial cells from a mixed population of cells. More particularly, the inventors have found that certain antibodies that recognize the extracellular domain of VEGFR-3 can be used to specifically isolated lymphatic endothelial cells substantially free of other contaminating non-lymphatic endothelial cells. Methods and compositions for producing such cells and using such cells are described. |
1. A method for isolating lymphatic endothelial cells from a biological sample comprising lymphatic endothelial cells, the method comprising: a) contacting said sample with an antibody that preferentially recognizes lymphatic endothelial cells as compared to other endothelial cells, under conditions where the antibody binds lymphatic endothelial cells, and b) isolating lymphatic endothelial cells bound to said antibody. 2. The method of claim 1, wherein said antibody is an antibody that is immunologically reactive with an epitope on the extracellular domain of VEGFR-3 that is specific for lymphatic endothelial cells. 3. The method of claim 1 wherein said biological sample is from a human patient. 4. The method of claim 1, wherein said antibody is immobilized on a solid support and said biological sample is contacted with said support to allow the lymphatic endothelial cells to become bound to said antibody. 5. The method of claim 1, wherein said antibody is labeled with a fluorescent label and said lymphatic endothelial cells are isolated using fluorescence activated cell sorting. 6. The method of claim 1, wherein said antibody is labeled with a magnetic label and lymphatic endothelial cells are isolated using magnetic activated cell sorting. 7. The method of claim 1, wherein said lymphatic endothelial cells are isolated using immunohistochemistry. 8. The method of claim 1, wherein said lymphatic endothelial cells are isolated using immunochromatography. 9. The method of claim 1, wherein the antibody is a polyclonal antibody. 10. The method of claim 1, wherein the antibody is a monoclonal antibody. 11. The method of claim 1, wherein the antibody is a binding reagent that comprises an antigen binding fragment of 2E11D11. 12. The method of claim 11, wherein the antibody recognizes the same epitope of VEGFR-3 protein that is recognized by 2E11D11. 13. The method of claim 1, wherein said antibody is 2E11D11. 14. The method of claim 1, wherein said antibody is an anti-podoplanin. 15. A method of isolating blood vascular endothelial cells from a sample of microvascular endothelial cells, the method comprising: a) contacting said cells with an antibody that preferentially binds to lymphatic endothelial cells as compared to other endothelial cells, under conditions where the antibody binds lymphatic endothelial cells, and b) removing said lymphatic endothelial cells that are bound by said antibody from microvascular cells that are not bound to said antibody, wherein said microvascular cells not bound to said antibody comprise a population of blood vascular endothelial cells substantially free of lymphatic endothelial cells. 16. The method of claim 15, wherein said antibody is an antibody that is immunologically reactive with the extracellular domain of VEGFR-3. 17. A lymphatic endothelial cell population isolated according to a method comprising: a) contacting a biological sample comprising lymphatic endothelial cells with an antibody that preferentially binds to lymphatic endothelial cells as compared to other endothelial cells, under conditions where the antibody binds lymphatic endothelial cells, and b) isolating lymphatic endothelial cells that are bound by said antibody. 18. The lymphatic endothelial cell population of claim 17, wherein said antibody is an antibody that is immunologically reactive with the extracellular domain of VEGFR-3. 19. The lymphatic endothelial cell population of claim 17, wherein said biological sample of cells comprises a heterogeneous population of endothelial cells. 20. The lymphatic endothelial cell population of claim 17, wherein said biological sample of cells is a microvascular endothelial cell population. 21. The lymphatic endothelial cell population of claim 17, wherein said lymphatic endothelial cell population is substantially free of contaminating blood vascular endothelial cells. 22. The method of claim 17, comprising expanding said lymphatic endothelial cells. 23. A blood vascular endothelial cell population isolated according to a method comprising: a) contacting a population of microvascular endothelial cells with an antibody that preferentially binds to lymphatic endothelial cells as compared to blood vascular endothelial cells, under conditions where the antibody binds to lymphatic endothelial cells, and b) removing said lymphatic endothelial cells that are bound by said antibody from microvascular cells that are not bound to said antibody, wherein said microvascular cells not bound to said antibody comprise a population of blood vascular endothelial cells substantially free of lymphatic endothelial cells. 24. The blood vascular cell population of claim 23, wherein said antibody is an antibody that is immunologically reactive with the extracellular domain of VEGFR-3. 25. The blood vascular cell population of claim 23, wherein the method further comprises expanding said blood vascular endothelial cell population. 26. A lymphatic endothelial cell population substantially free of other contaminating endothelial cells. 27. A blood vascular endothelial cell population substantially free of other contaminating endothelial cells. 28. A method of obtaining a composition substantially enriched in a subpopulation of lymphatic endothelial cells comprising: (a) obtaining, a source of cells comprising microvascular endothelial cells; (b) contacting the cells with a monoclonal antibody that preferentially binds to lymphatic endothelial cells as compared to other endothelial cells, under conditions to allow an antibody to bind lymphatic endothelial cells; (c) separating those cells that are specifically bound by the monoclonal antibody, thereby obtaining a composition substantially enriched in a subpopulation of lymphatic endothelial cells. 29. The method of claim 28, wherein said antibody is an anti-podoplanin antibody. 30. The method of claim 28, wherein said antibody is 2E11D11. 31. A composition comprising a substantially. enriched subpopulation of lymphatic endothelial cells obtained by the method according to claim 28. 32. A method of ameliorating a lymphatic endothelial cell disorder comprising targeting lymphatic endothelial cells with a therapeutic agent, wherein said therapeutic agent is targeted to said cells using an antibody that preferentially binds to lymphatic endothelial cells as compared to other endothelial cells, wherein said antibody is an antibody that is immunologically reactive with the extracellular domain of VEGFR-3. 33. The method of claim 30, wherein said disorder is selected from the group consisting of lymphoma, hereditary lymphedema, lymphedemas, lymphangiomas, lymphangiosarcomas, lymphangiomatosis, lymphangiectasis, and cystic hygroma. 34. A method of ameliorating a lymphatic disorder, wherein said method comprises ex vivo therapy comprising: a) obtaining microvascular endothelial cells of a patient in need of said therapy; b) contacting the microvascular endothelial cells with an antibody that preferentially binds to lymphatic endothelial cells as compared to other endothelial cells, under conditions that allow the binding of said antibody to lymphatic endothelial cells; c) isolating lymphatic endothelial cells that are bound by said antibody d) transfecting said lymphatic endothelial cells with an expression construct comprising a nucleic acid encoding a therapeutic protein operably linked to a promoter, in an amount effective to produce the expression of said protein in said cells and e) reintroducing said transfected cells to said patient. 35. The method of claim 34, wherein said antibody is an antibody that is immunologically reactive with the extracellular domain of VEGFR-3. 36. A method of promoting the growth of lymphatic endothelial cells in culture comprising: a) obtaining the lymphatic endothelial cells according to claim 1; b) stimulating said cells with a VEGFR-3 ligand; wherein stimulating the growth of said cells with said VEGFR-3 ligand promotes the survival of said cells in culture as compared to growth in the absence of said stimulation. 37. The method of claim 36, wherein said VEGFR-3 ligand is VEGF-C, VEGF-C156S or VEGF-D. 38. The method of claim 36, further comprising stimulating said cells with a VEGFR-2 ligand. 39. The method of claim 36, wherein said stimulation of said cells protects the cells from apoptosis. 40. The method of claim 36, wherein said protection of said cells is mediated through the activation of Akt or p42/MAPK signaling molecules. 41. The method of claim 36, wherein said stimulation allows said cells to maintain differentiated endothelial cell characteristics. 42. A method of selectively modulating lymphatic endothelial cells in a mammalian organism comprising: a) isolating lymphatic endothelial cells from said mammalian organism by the method of claim 1, b) contacting said isolated lymphatic endothelial cells with an agent to modulate the lymphatic endothelial cells; and c) reintroducing the lymphatic endothelial cells into said organism. 43. The method of claim 42, wherein the contacting step comprises introducing an exogenous polynucleotide into said cells. 44. The method of claim 42, wherein the organism has a disorder characterized by a genetic mutation in a gene expressed in lymphatic endothelial cells and the contacting comprises introducing an exogenous polynucleotide into the cells to overcome the effects of the genetic mutation in said gene. 45. The method of claim 44, wherein said disorder is hereditary lymphedema. 46. A method for imaging lymphatic endothelial cells in tissue from a vertebrate organism, comprising the steps of: (a) contacting vertebrate tissue suspected of containing a lymphatic endothelial cells with a composition comprising an antibody that preferentially binds to lymphatic endothelial cells as compared to other endothelial cells, under conditions that allow the binding of said antibody to lymphatic endothelial cells; (b) detecting said antibody bound to said lymphatic endothelial cells in said tissue; and (c) imaging lymphatic endothelial cells in the tissue by identifying lymphatic endothelial cells bound by said antibody, wherein said binding of the lymphatic endothelial cells to said antibody indicates the presence and location of lymphatic endothelial cells in the tissue. 47. The method of claim 46, wherein said tissue comprises human tissue. 48. The method of claim 46, further comprising the step of washing said tissue, after said contacting step and before said imaging step, under conditions that remove from said tissue antibody that is not bound to the lymphatic endothelial cells in said tissue. 49. The method of claim 46, wherein said antibody is an antibody that is immunologically reactive with the extracellular domain of VEGFR-3. 50. The method of claim 46, wherein said antibody is an anti-podoplanin antibody. 51. The method of claim 46, wherein said antibody further comprises a detectable label covalently bound thereto. 52. The method according to claim 46, further comprising steps of: contacting the tissue with a second compound that specifically binds to a lymphatic endothelial marker that is substantially absent in blood vascular endothelia; and detecting said second compound bound to cells in said tissue; wherein said imaging step comprises identifying lymphatic vessels labeled with both the antibody and the second compound, wherein lymphatic vessels labeled with both the antibody and the second compound correlate with the presence and location of lymphatic endothelial cells in the tissue. 53. The method of claim 52, wherein said antibody is an antibody that is immunologically reactive with the extracellular domain of VEGFR-3, and said second compound is an anti-podoplanin antibody. 54. A method of screening for a disease characterized by a change in lymphatic endothelial cells, comprising the steps of: (a) obtaining a tissue sample from a vertebrate organism suspected of being in a diseased state characterized by changes in lymphatic endothelial cells; (b) exposing said tissue sample to a composition comprising an antibody that preferentially binds to lymphatic endothelial cells as compared to other endothelial cells, under conditions that allow the binding of said antibody to lymphatic endothelial cells in said organism; (c) washing said tissue sample; and (d) screening for said disease by detecting the presence, quantity, or distribution of said bound antibody in said tissue sample. 55. A method for specifically detecting lymphatic endothelial cells in a mammal, comprising the steps of: (a) administering to said mammal a composition comprising an antibody that preferentially binds to lymphatic endothelial cells as compared to other endothelial cells, under conditions that allow the binding of said antibody to lymphatic endothelial cells, and (b) detecting said antibody bound to lymphatic endothelial cells, thereby detecting lymphatic endothelial cells in said organism. 56. The method of claim 55, further comprising administering to said mammal a second compound that specifically binds to a lymphatic endothelial cell marker; and wherein said detecting step comprises detection of said antibody and said second compound bound to lymphatic endothelial cells. 57. A method modifying lymphatic endothelial cells comprising: a) obtaining a microvascular endothelial cells; b) contacting the microvascular endothelial cells with an antibody that preferentially binds to lymphatic endothelial cells as compared to other endothelial cells, under conditions that allow the binding of said antibody to lymphatic endothelial cells; c) isolating lymphatic endothelial cells that are bound by said antibody; and d) transfecting said lymphatic endothelial cells with an expression construct comprising a nucleic acid encoding a therapeutic protein operably linked to a promoter, in an amount effective to produce the expression of said protein in said cells, wherein said transfecting produces modified lymphatic endothelial cells. 58. A lymphatic endothelial cell produced according to the method of claim 57. 59. A composition comprising a substantially enriched subpopulation of lymphatic endothelial cells obtained by the method according to claim 29. 60. A composition comprising a substantially enriched subpopulation of lymphatic endothelial cells obtained by the method according to claim 30. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The lymphatic system is a complex structure organized in parallel fashion to the circulatory system. In contrast to the circulatory system, which utilizes the heart to pump blood throughout the body, the lymphatic system pumps lymph fluid using the inherent contractility of the lymphatic vessels. The lymphatic vessels are not interconnected in the same manner as the blood vessels, but rather form a set of coordinated structures including the initial lymphatic sinuses [Jeltsch et al., Science, 276:1423-1425 (1997); and Castenholz, A., in Olszewski, W. L. (ed.), Lymph Stasis: Pathophysiology, Diagnosis, and Treatment. CRC Press: Boca Raton, Fla. (1991), pp. 15-42] which drain into the lymphatic capillaries and subsequently to the collecting lymphatics which drain into the lymphatic trunks and the thoracic duct which ultimately drains into the venous circulation. The composition of the channels through which lymph passes is varied [Olszewski, W. L., in Olszewski, W. L. (ed), Lymph Stasis: Pathophysiology, Diagnosis, and Treatment. CRC Press: Boca Raton, Fla. (1991), pp. 235-258; and Kinmonth, J. B., in Kinmonth, J. B. (ed), The Lymphatics: Diseases, Lymphography and Surgery. Edward Arnold Publishers: London, England (1972), pp. 82-86], including the single endothelial layers of the initial lymphatics, the multiple layers of the collecting lymphatics including endothelium, muscular and adventitial layers, and the complex organization of the lymph node. The various organs of the body such as skin, lung, and GI tract have components of the lymphatics with various unique features. [See Ohkuma, M., in Olszewski (1991), supra, at pp. 157-190; Uhley, H. and Leeds, S., in Olszewski (1991), supra, at pp. 191-210; and Barrowman, J. A., in Olszewski (1991), at pp. 221-234).] Vascular endothelial growth factor (VEGF) is a prime regulator of endothelial cell proliferation, angiogenesis, vasculogenesis and vascular permeability (Ferrara, J Mol Med 77:527-543, 1999). Besides VEGF, the VEGF family of growth factors currently contains five other known members, namely placenta growth factor (PIGF), VEGF-B, VEGF-C, VEGF-D and orf viral VEGF homologs (Eriksson and Alitalo, Curr Top Microbiol Immunol., 237:41-57; 1999). Additional novel VEGF-like heparin binding proteins were recently isolated from snake venom (Gasmi et al., Biochem Biophys Res Commun. 268(1):69-72, 2000; Gasmi et al., Biochim Biophys Acta 1481(1):209-12, 2000; Komori et al., 1999). Disruption of the genes encoding either VEGF or any of the three receptors of the VEGF family, VEGFR-1/Flt1, VEGFR-2/Flk1/KDR or VEGFR-3/Flt4 results in embryonic lethality because of failure of blood vessel development (Dumont et al., Science, 282:946-949, 1998; Fong et al., Nature, 376:66-70, 1995; Shalaby et al., Nature, 376:62-66, 1995). Detailed descriptions of these receptors and their ligands are presented in U.S. Pat. No. 5,776,755; U.S. Pat. No.5,607,918; U.S. Pat. No. 5,840,693; U.S. Pat. No. 5,928,939; U.S. Pat. No. 6,130,071; U.S. Pat. No. 6,221,839; U.S. Pat. No. 6,235,713; U.S. Pat. No. 6,245,530; U.S. patent application Ser. No. 09/427,657 filed Oct. 26, 1999; U.S. patent application Ser. No. 09/534,376 filed Mar. 24, 2000; U.S. Patent Application No. 60/262,476 filed Jan. 17, 2001; as well as PCT Application No. PCT/US98/01973, filed Feb. 2, 1998. Each of these documents is specifically incorporated herein by reference in its entirety. Additional disclosure relating to vascular endothelial growth factors and their receptors may be found in for example, U.S. Pat. No. 6,245,512; U.S. Pat. No. 6,168,778; U.S. Pat. No. 6,100,071; U.S. Pat. No. 6,051,698; U.S. Pat. No. 6,040,157; U.S. Pat. No. 6,020473; and U.S. Pat. No. 6,011,003, each incorporated herein by reference. VEGFR-2 is considered to be the main signal transducing VEGF receptor for angiogenesis and for mitogenesis of endothelial cells. VEGF induces endothelial cell proliferation, migration and survival via activation of VEGFR-2 and subsequent signal transduction pathways including the MAP (mitogen-activated protein) kinase/ERK (extracellular signal regulated kinase) and the phosphatidylinositol (PI) 3-kinase pathways (for a review, see Petrova et al., Exp. Cell. Res. 253:117-130, 1999; Shibuya et al., In Claesson-Welsh, L (ed.) Vascular growth factors and angiogenesis. Springer Verlag, GmbH&Co., KG, Heidelberg, 237:59-83, 1999). Activation of the p42/p44 MAPK (ERK1/ERK2) cascade is linked in many cells to a proliferation response. In addition, this pathway can lead to increased cell survival by stimulating the transcription of pro-survival genes and by posttranslational modification and inactivation of components of the cell death machinery (Bonni et al., Science, 286:1358-1362, 1999; Gupta et al., Exp. Cell. Res., 247:495-504, 1999). The PI3-kinase pathway was also initially linked to mitogenesis, but several studies have subsequently shown that this pathway has an important function in regulating cell survival by activation of the serine/threonine kinase Akt (protein kinase B) (Datta et al., Genes Dev. 13:2905-2927, 1999). Recent studies have also indicated some crosstalk between the MAPK and PI3-kinase signaling pathways: phosphorylation of Raf by Akt resulted in inhibition of the Raf-MEK (MAP kinase kinase)-ERK pathway (Rommel et al., Science 286:1738-1741, 1999; Zimmermann and Moelling, Science 286:1741-1744, 1999). Molecular biology has identified at least a few genes and proteins postulated to have roles mediating the growth and/or embryonic development of the lymphatic system. One such gene/protein is the receptor tyrosine kinase designated Flt4 (fmns-like tyrosine kinase 4; also referred to as vascular endothelial cell growth factor receptor 3 or VEGFR-3), cloned from human erythroleukaemia cell and placental cDNA libraries. [See U.S. Pat. No. 5,776,755 and U.S. Pat. no. 6,107,046; Aprelikova et al., Cancer Res., 52: 746-748 (1992); Galland et al., Genomics, 13: 475-478 (1992); Galland et al., Oncogene, 8: 1233-1240 (1993); and Pajusola et al., Cancer Res., 52:5738-5743 (1992), all incorporated herein by reference.] Studies showed that, in mouse embryos, a targeted disruption of the VEGFR-3 gene leads to a failure of the remodeling of the primary vascular network, and death after embryonic day 9.5 [umont et al., Science, 282: 946-949 (1998)]. Additional studies have indicated that certain mutations in VEGFR-3 have an apparent causal role in hereditary lymphedema (PCT Publication No. WO 00/58511). However, VEGFR-3 is not exclusive to lymphatic vessels. VEGFR-3 has an essential role in the development of die embryonic blood vasculature, before the emergence of the lymphatic vessels. However, additional studies indicated that, during further development, the expression of VEGFR-3 becomes restricted mainly to lymphatic vessels [Kaipainen, et al., Proc. Natl. Acad. Sci. USA, 92: 3566-3570 (1995)]. However, VEGFR-3 expression also is observed in neovascular blood vessels of at least some tumors (PCT Publication No. WO 00/21560). The expression of the VEGFR-3 gene starts during mouse embryonic day 8.5 in developing blood vessels, and VEGFR-3 deficient embryos die at midgestation due to defects in the remodeling of primary vascular networks (Dumont et al., Science, 282:946-949, 1998). However, in adult tissues VEGFR-3 expression occurs mainly in the lymphatic endothelia (Kaipainen, et al., Proc. Natl. Acad. Sci. USA, 92: 3566-3570, 1995; Partanen et al., FASEB J., 14:2087-2096, 2000), and VEGFR-3 ligands VEGF-C and VEGF-D can induce growth of the lymphatic vessels (Jeltsch et al., Science, 276:1423-1425, 1997; Veikkola et al., EMBO J. 20: 1223-1231, 2001). In contrast, blocking of VEGFR-3 signaling by use of a soluble VEGFR-3 protein caused regression of developing lymphatic vessels by inducing endothelial cell apoptosis (Mäkinen et al., Nature Med., 7:199-205, 2001). However, the biochemical signaling pathways activated via VEGFR-3 are less well characterized than those of VEGFR-2, making it difficult to ascertain the mechanism of action of these important regulators of lymphatic endothelial cells function. In the absence of such information, therapeutic and diagnostic implications of dysfunctions of these interactions remain elusive. Previously, a number of VEGFR-3 antibodies have been described, see for example, U.S. Pat. No. 6,107,046 (incorporated herein by reference). In addition, podoplanin has recently been identified as a specific marker for lymphatic endothelium (Breiteneder-Geleff et al., et al., Am. J. Path., 154(2) 385-394, 1999), and LYVE-1, a homolog of the CD44 glycoprotein is purported to be a lymph-specific receptor for hyaluron (Banerji et al., J. Biol. Chem., 144(4)789-801, 1999). However, despite the availability of these markers, at present, there are no adequate methods of obtaining isolated lymphatic endothelial cells. The study of therapeutic and diagnostic implications of various lymphatic cell disorders would be greatly facilitated if such isolated endothelial cells could be obtained and be made available for molecular studies. Moreover, the availability of such cells would provide useful information about the characteristic features of the lymphatic endothelial cells, thereby facilitating further identification of specific areas for therapeutic intervention. There are a number of disease states such as hereditary lymphedema, cancer metastases and post-surgical edema, which involve aberration in lymphatic endothelial cells and receptors thereon. The ability to isolate, grow and replace lymphatic endothelial cells would be in a useful palliative intervention, treatment or other ameliorative regimen against such disorders. Such interventions would be particularly useful against injury induced lymphedema, for example. Moreover, the availability of such cells would be particularly amenable to cell-specific treatment regimens thereby greatly reducing undesirable side effects as may be seen in e.g., non-cell specific gene therapy or chemotherapy protocols. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention, provides improvements in the ability to manipulate endothelial cells and lymphatic and vascular systems that have numerous practical uses in medicine and molecular biology. More particularly, the present invention provides a method for isolating lymphatic endothelial cells from a biological sample of comprising lymphatic endothelial cells, the method comprising contacting said biological sample with an antibody that preferentially recognizes lymphatic endothelial cells as compared to other endothelial cells, under conditions where the antibody binds lymphatic endothelial cells, and isolating lymphatic endothelial cells bound to said antibody. As used herein the term “antibody” is intended to refer to any antibody agent that specifically binds a target antigen (e.g., lymphatic endothelial cells) or any polypeptide that comprises an antigen binding fragment that specifically recognizes the antigen. More particularly, the antibody is one that is immunologically reactive with an epitope on the extracellular domain of VEGFR-3 that is specific for lymphatic endothelial cells. In the context of the present invention, “preferential” or “specific” means that the antibody binds the target antigen e.g., VEGFR-3 on lymphatic endothelial cells) with greater affinity or avidity than it binds similar antigens on other cells (e.g., VEGFR-3 on blood vascular endothelial cells). This differential binding permits the isolation of one cell type from another. It should be understood that the biological sample may be from any mammalian organism and may be any tissue or fluid sample that could be expected to contain lymphatic endothelial cells. Particularly preferred is a biological sample is from a human patient. In preferred embodiments, the antibody is immobilized on a solid support and said biological sample is contacted with said support to allow the lymphatic endothelial cells to become bound to said antibody and thereby to the support. In other preferred embodiments, the antibody is labeled with a fluorescent label and said lymphatic endothelial cells are isolated using fluorescence activated cell sorting. In alternative preferred embodiments, the antibody is labeled with a magnetic label and lymphatic endothelial cells are isolated using magnetic activated cell sorting. It is contemplated that the lymphatic endothelial cells in the biological sample may be isolated using immunohistochemistry. Other embodiments contemplate the use of immunochromatography to isolate the lymphatic endothelial cells. It should be understood that the antibody may be a polyclonal antibody or it may be a monoclonal antibody. In preferred embodiments, the antibody is a binding reagent that comprises an antigen binding fragment of 2E11D11. In other embodiments, the antibody is a derivative of 2E11D11. In still further embodiments, the antibody is a binding reagent that comprises an antigen binding fragment derived from the antigen binding fragment of 2E11D11 which has been mutated or altered to have greater binding specificity for a VEGFR-3 epitope that is specific for lymphatic endothelial cells. In other embodiments, the antibody recognizes the same epitope of VEGFR-3 protein that is recognized by 2E11D11. In particularly preferred embodiments, the antibody is 2E11D11. (deposited as accession 01083129 with European Collection of Cell Cultures, Center for Applied Microbiology and Research, Porton Down Salisbury, U.K.). The production of this antibody is described in U.S. Pat. No. 6,107,046 (incorporated herein by reference). In other preferred embodiments, antibody is an anti-podoplanin. In certain embodiments, antibody is mutant or derivative the anti-podoplanin antibody that has a binding specificity for lymphatic endothelial cells. Certain aspects of the present invention contemplate, a method of isolating blood vascular endothelial cells from a biological sample comprising microvascular endothelial cells, the method comprising contacting the biological sample with an antibody that preferentially recognizes lymphatic endothelial cells as compared to other endothelial cells, wherein the antibody is an antibody that is immunologically reactive with the extracellular domain of VEGFR-3, and removing the lymphatic endothelial cells that are bound by the antibody from microvascular cells that are not bound to the antibody, wherein the microvascular cells not bound to the antibody comprise a population of blood vascular endothelial cells substantially free of lymphatic endothelial cells. Anther aspect of the present invention contemplates a lymphatic endothelial cell population isolated according to a method comprising contacting a biological sample comprising lymphatic endothelial cells with an antibody that preferentially binds to lymphatic endothelial cells as compared to other endothelial cells, wherein the antibody is an antibody that is immunologically reactive with the extracellular domain of VEGFR-3, and isolating lymphatic endothelial cells that are bound by the antibody. In preferred embodiments, the biological sample comprises a heterogeneous population of endothelial cells. In other preferred embodiments, the sample of cells is a microvascular endothelial cell population. In particularly preferred embodiments, the lymphatic endothelial cell population is substantially free of contaminating blood vascular endothelial cells. In preferred aspects the method of isolating cells comprises expanding the lymphatic endothelial cells in culture. A further aspect of the present invention describes a blood vascular endothelial cell population isolated according to a method comprising: contacting a population of microvascular endothelial cells with an antibody that preferentially binds to lymphatic endothelial cells as compared to blood vascular endothelial cells, wherein the antibody is an antibody that is immunologically reactive with the extracellular domain of VEGFR-3, and; removing the lymphatic endothelial cells that are bound by the antibody from microvascular cells that are not bound to the antibody, wherein the microvascular cells not bound to the antibody comprise a population of blood vascular endothelial cells substantially free of lymphatic endothelial cells. In specific embodiments, the blood vascular cell population is produced by a method which further comprises expanding the blood vascular endothelial cell population. A preferred aspect of the present invention particularly contemplates a lymphatic endothelial cell population substantially free of other contaminating endothelial cells. Another preferred aspect of the invention describes a blood vascular endothelial cell population substantially free of other contaminating endothelial cells. Another preferred embodiment of the invention relates to a method of obtaining a composition substantially enriched in subpopulation of lymphatic endothelial cells comprising obtaining a source of cells comprising microvascular endothelial cells; contacting the cells with a monoclonal antibody that preferentially binds to lymphatic endothelial cells as compared to other endothelial cells, under conditions to allow the binding of the antibody to lymphatic endothelial cells; separating those cells that are specifically bound by the monoclonal antibody, thereby obtaining a composition substantially enriched in a subpopulation of lymphatic endothelial cells. In preferred aspects antibody is an antibody that is immunologically reactive with the extracellular domain of an antigen expressed on lymphatic endothelial cells. In further preferred embodiments, the antigen is VEGFR-3 The invention also encompasses, in preferred aspects, a composition comprising a substantially enriched subpopulation of lymphatic endothelial cells obtained by such a method. In preferred embodiments, the antibody is an anti-podoplanin. In other preferred embodiments, the antibody is 2E11D11, which preferentially recognizes VEGFR-3 expressed on lymphatic endothelial cells. Other embodiments contemplate a method of ameliorating a lymphatic endothelial cell disorder comprising targeting lymphatic endothelial cells with a therapeutic agent, wherein the therapeutic agent is targeted to the cells using an antibody that preferentially binds to lymphatic endothelial cells as compared to other endothelial cells, wherein the antibody is an antibody that is immunologically reactive with the extracellular domain of VEGFR-3. In specific embodiments, the disorder is selected from the group consisting of lymphoma, hereditary lymphedema, lymphedemas, lymphangiomas, lymphangiosarcomas, lymphangiomatosis, lymphangiectasis, and cystic hygroma. The present invention further provides a method of ameliorating a lymphatic disorder, wherein the method comprises ex vivo therapy comprising obtaining a biological sample from the patient in need of the therapy, wherein the biological sample comprises microvascular endothelial cells; contacting the microvascular endothelial cells with an antibody that preferentially binds to lymphatic endothelial cells as compared to other endothelial cells, wherein the antibody is an antibody that is immunologically reactive with the extracellular domain of VEGFR-3; isolating lymphatic endothelial cells that are bound by the antibody, transfecting the lymphatic endothelial cells with an expression construct comprising a nucleic acid encoding a protein operably linked to a promoter, in an amount effective to produce the expression of the protein in the cells; and reintroducing the transfected cells to the patient. The encoded protein can be any protein that one might wish to express in lymphatic endothelial cells (e.g., to treat a disease, palliate the symptoms of a disease, or to permit better diagnosis or imaging) The present invention also provides a method of promoting the growth of lymphatic endothelial cells in culture comprising obtaining the lymphatic endothelial cells according to a method of the present invention; and stimulating the cells with a VEGFR-3 ligand; wherein stimulating the growth of the cells with the VEGFR-3 ligand promotes the survival of the cells in culture as compared to growth in the absence of the stimulation. In particularly preferred embodiments, the VBGFR-3 ligand is VEGF-C, VEGF-C156S or VEGF-D. The method may further comprise stimulating the cells with a VEGFR-2 ligand. In specific embodiments, it is contemplated that the stimulation of the cells protects the cells from apoptosis. In preferred embodiments, the protection of the cells from apoptosis is mediated through the activation of Akt or p42/MAPK signaling molecules. In preferred embodiments, the stimulation of the cells allows the cells to maintain differentiated endothelial cell characteristics. Also encompassed by the present invention is a method of selectively modulating lymphatic endothelial cells in a mammalian organism comprising isolating lymphatic endothelial cells from the mammalian organism as described by the present invention, contacting the isolated lymphatic endothelial cells with an agent to modulate the lymphatic endothelial cells; and reintroducing the lymphatic endothelial cells into the organism. In preferred aspects, the contacting step comprises introducing an exogenous polynucleotide into the cells. In other preferred embodiments, the organism has a disorder characterized by a genetic mutation in a gene expressed in lymphatic endothelial cells and the contacting comprises introducing an exogenous polynucleotide into the cells to overcome the effects of the genetic mutation in the gene. In specific embodiments, the disorder is hereditary lymphedema For example, the disorder is hereditary lymphedema characterized by a VEGFR-3 mutation and the treatment comprises introducing a wild-type VEGFR-3 allele. Another aspect of the invention describes a method for imaging lymphatic endothelial cells in tissue from a vertebrate organism, comprising contacting vertebrate tissue suspected of containing a lymphatic endothelial cells with a composition comprising an antibody that preferentially binds to lymphatic endothelial cells as compared to other endothelial cells, under conditions that allow the binding of the antibody to lymphatic endothelial cells; detecting the antibody bound to the lymphatic endothelial cells in the tissue; and imaging lymphatic endothelial cells in the tissue by identifying lymphatic endothelial cells bound by the antibody, wherein the binding of the lymphatic endothelial cells to the antibody indicates the presence and location of lymphatic endothelial cells in the tissue. More particularly, the tissue comprises human tissue. In specific embodiments, the method further comprises the step of washing the tissue, after the contacting step and before the imaging step, under conditions that remove from the tissue antibody that is not bound to the lymphatic endothelial cells in the tissue. The antibody may be an antibody that is immunologically reactive with the extracellular domain of VEGFR-3. In other embodiments, the antibody is an anti-podoplanin antibody. In preferred embodiments, the antibody further comprises a detectable label covalently bound thereto. The method may be further defined as comprising contacting the tissue with a second compound that specifically binds to a lymphatic endothelial marker that is substantially absent in blood vascular endothelia; and detecting the second compound bound to cells in the tissue; wherein the imaging step comprises identifying lymphatic vessels labeled with both the antibody and the second compound, wherein lymphatic vessels labeled with both the antibody and the second compound correlate with the presence and location of lymphatic endothelial cells in the tissue. In preferred embodiments, the antibody is an antibody that is immunologically reactive with the extracellular domain of VEGFR-3, and the second compound is an anti-podoplanin antibody. Also contemplated herein is a method of screening for a disease characterized by a change in lymphatic endothelial cells, comprising obtaining a tissue sample from a vertebrate organism suspected of being in a diseased state characterized by changes in lymphatic endothelial cells; exposing the tissue sample to a composition comprising an antibody that preferentially binds to lymphatic endothelial cells as compared to other endothelial cells, under conditions that allow the binding of the antibody to lymphatic endothelial cells in the organism; washing the tissue sample; and screening for the disease by detecting the presence, quantity, or distribution of the bound antibody in the tissue sample. Another embodiment contemplates a method for specifically detecting lymphatic endothelial cells in a mammal, comprising administering to the mammal a composition comprising an antibody that preferentially binds to lymphatic endothelial cells as compared to other endothelial cells, under conditions that allow the binding of the antibody to lymphatic endothelial cells, and detecting the antibody bound to lymphatic endothelial cells, thereby detecting lymphatic endothelial cells in the organism. The method may further comprise administering to the mammal a second compound that specifically binds to a lymphatic endothelial cell marker; and wherein the detecting step comprises detection of the antibody and the second compound bound to lymphatic endothelial cells. In all the imaging methods of the invention, it is contemplated that the imaging methods may be used in disorders of lymphatic vessels in determining the presence of the disorder, as well as for monitoring the effects of treatment of the disorder. Such methods may be particularly useful in assessing lymphedema e.g., hereditary lymphedema or injury induced edema and other lymphatic vessel disorders. Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, and all such features are intended as aspects of the invention. Likewise, features of the invention described herein can be re-combined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specifically mentioned above as an aspect or embodiment of the invention. Also, only such limitations which are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention. Aspects of the invention may be summarized by genus, and it should be understood that every individual member of the genus is intended as an individual aspect of the invention. |
Methods of producing a package for semiconductor chips |
The inventive method is based on the a idea of releasing a mechanical connection between the semiconductor chip and the supporting substrate during the manufacturing of the packing. The mechanical connection required for producing the electrical contacts between the semiconductor chip and the supporting substrate ensues only temporarily. As a result, a critical interface in the packing is removed thereby resulting in distinctly reducing the thermomechanical stresses. |
1. A method of producing a package for semiconductor chips, comprising: providing a carrier substrate; applying at least one adhesive layer, which loses its adhesive property when it is heated above a defined temperature, to the carrier substrate; applying a semiconductor chip to the carrier substrate and fixing it thereto by the adhesive layer; and applying a curable composition and carrying out at least one heat treatment, the temperature of the heat treatment lying at least temporarily above the temperature at which the adhesive layer loses its adhesive property, so that the adhesive layer loses its adhesive property and the curable composition cures. 2. The method as claimed in claim 1, wherein a carrier substrate with nubbins arranged on it is provided and the adhesive layer is applied to the nubbins. 3. The method as claimed in claim 1, wherein a curable composition is applied and a first heat treatment is carried out, so that the curable composition solidifies, the temperature of the first heat treatment lying below the temperature at which the adhesive layer loses its adhesive property, and subsequently a second heat treatment is carried out, the temperature of the second heat treatment lying above the temperature at which the adhesive layer loses its adhesive property, so that the adhesive layer loses its adhesive property and the curable compositions cures. 4. The method as claimed in claim 1, wherein the semiconductor chip is electrically connected to the carrier substrate by wire or film-carrier bonding, the bonding temperature lying below the temperature at which the adhesive layer loses its adhesive property. 5. The method as claimed in claim 1, wherein the adhesive layer is applied by a punching process. 6. The method as claimed in claim 1, wherein a first adhesive layer, a base layer and a second adhesive layer are applied, at least the second adhesive layer losing its adhesive property when it is heated above a defined temperature. 7. The method as claimed in claim 6, wherein the first adhesive layer, the base layer and the second adhesive layer are jointly applied in the form of a film. 8. The method as claimed in claim 1, wherein a polyimide, BT or FR4 substrate is used as the carrier substrate. 9. The method as claimed in claim 1, wherein the carrier substrate has at least one bonding channel. 10. The method as claimed in claim 1, wherein a mixture of silicone and epoxy resin or pure epoxy resin is used as the curable composition. 11. The method as claimed in claim 10, wherein the first heat treatment is carried out at a temperature between 130° and 150° C. and subsequently a second heat treatment is carried out at a temperature between 150° and 170° C. 12. The method as claimed in claim 1, wherein the curable compositions is applied by a printing process or a compression-molding process. 13. The method as claimed in claim 2, wherein the nubbins are applied to the carrier substrate by a printing process and a subsequent heat treatment. |
<SOH> BACKGROUND OF THE INVENTION <EOH>With the increasing integration in information and communication technology, the requirements imposed on the constructing and connecting techniques are becoming ever greater. Ever higher requirements are imposed in particular on the package of the semiconductor chips. The package is the main medium for connecting the integrated circuit on the semiconductor chip to the rest of the system. A further reduction in the size of the structures on the semiconductor chip would be largely senseless if there were not a corresponding development in the packaging of the semiconductor chips. To satisfy this high requirement on the packaging of the semiconductor chips, in recent years packaging methods for semiconductor chips have been proposed and developed under the designation “Chip Size Packaging” (CSP), in which the space requirement of the packaged semiconductor chip is not greater than approximately 1.2 times the unpackaged semiconductor chip. Accordingly, the integration density on the system board can be increased and parasitic effects of the package reduced, whereby ultimately the speed of the end product is significantly increased. In the production of miniature housings for semiconductor chips, generally a specially preprocessed carrier substrate is used. This carrier substrate may in this case be designed such that it is both relatively flexible and relatively rigid_ Generally serving for attaching the semiconductor chip on the carrier substrate are full-area chip bonding tapes or elastomer layers, which in addition to fixing the semiconductor chip also serve for minimizing mechanical stresses of the overall arrangement. In particular, in the case of these technologies, the semiconductor chip is permanently connected to the carrier substrate by means of the double-sided adhesive chip bonding tapes or elastomer layers. At the same time, packages of this type often have to satisfy extreme requirements with respect to stress absorption. In particular, it is necessary to compensate largely for thermally induced stresses, caused by different coefficients of expansion (CTE) of individual components of the package. In this respect it is generally attempted to reduce the thermomechanical stresses by means of the double-sided adhesive chip bonding tapes or elastomer layers, which to a certain extent have stress-absorbing properties. Unfortunately, so far there is no known method or material that satisfies this requirement satisfactorily. Therefore, instances of delamination in the package or damage to the semiconductor chip are repeatedly encountered. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides a method of producing a package for semiconductor chips which largely avoids the difficulties mentioned and which makes possible, in particular, a low-cost, virtually stress-free electrical bonding of the chip for miniature housings. anying drawings. According to one embodiment of the invention, there is a method of producing a package for semiconductor chips including a carrier substrate is provided, at least one adhesive layer, which loses its adhesive property when it is heated above a defined temperature, is applied to the carrier substrate, a semiconductor chip is applied to the carrier substrate and fixed by the adhesive layer, a curable composition is applied and at least one heat treatment is carried out, the temperature of the heat treatment lying at least temporarily above the temperature at which the adhesive layer loses its adhesive property, so that the adhesive layer loses its adhesive property and the curable composition cures. The invention has one advantage that, during the completion of the package, the mechanical connection between and the carrier substrate is released again. The mechanical connection between the semiconductor chip and the carrier substrate takes place temporarily. As a result, a critical interface in the package is removed, which has the consequence of a significant reduction in the thermomechanical stresses. Accordingly, the completed package is significantly more reliable, which is in turn reflected in a reduction in the costs. With the method according to the invention, miniature packages or miniature housings for semiconductor chips (CSP) can be produced in particular. According to a preferred embodiment of the invention, a curable first heat treatment is carried out, so that the curable composition solidifies, the temperature of the first heat treatment lying above the temperature at which the adhesive layer loses its adhesive property, so that the adhesive layer loses its adhesive property and the curable composition cures. This preferred embodiment of the invention has an advantages that, in the second heat treatment, the process parameters can be set exactly as required for a reliable and permanent release of the adhesive layer. At the same time, the second heat treatment acts like a post-curing process for the curable composition. According to a further preferred embodiment of the invention, before the curable heat treatment the semiconductor chip is electrically connected to the carrier substrate by wire or film-carrier bonding (lead bonding, TAB), the bonding temperature lying below the temperature at which the adhesive layer loses its adhesive property. According to a further preferred embodiment of the invention, the adhesive layer is applied by a punching process. Furthermore, it is preferable if a first adhesive layer, a base layer and a second adhesive layer are applied, at least the second adhesive layer property losing its adhesive property when it is heated above a defined temperature. In this case, it is preferred in particular if the first adhesive layer, the base layer and the second adhesive layer are jointly applied in the form of a film. According to a further preferred embodiment of the invention, a polyimide, BT or FR4 substrate is used as the carrier substrate. Furthermore, it is preferred if the carrier substrate has at least one bonding channel. According to a further preferred embodiment of the invention, a mixture of silicone and epoxy resin is used as the curable composition. The use of a mixture epoxy resin has the advantage over the use of pure epoxy resin that the encapsulating composition has a significantly higher flexibility even after curing. If, however, the production of a relatively rigid package is the aim, it is advantageous to use pure epoxy resin as the curable composition. According to a further preferred embodiment of the invention, the first heat treatment is carried out at a temperature between 1300 and 150° C. and subsequently a second heat treatment is carried out at a temperature between 150° and 170° C. According to a further preferred embodiment of the invention, the nubbins are applied to the carrier substrate by a printing process and a subsequent heat treatment. Furthermore, it is preferred if the curable composition is applied by a printing process or a compression-molding process. |
Method and device for optically detecting the open state of a vehicle door |
The invention relates to a method for detecting the position of an object within the interior or a vehicle equipped with a door (4). The inventive method is characterized by recording a sequence of images by means of a camera (2) and calculating the actual position of the object by electronically evaluating the images of the sequence of images, and generating a differential image by comparing an actual recorded image with a previously stored image. The position of the object is calculated on the basis of the differential image. To achieve this, the camera (2) records images of the door (4). The images of the door (4) are evaluated and the open state of the door (4) is established on the basis of the differential image between the closed state and the opened state. |
1. A method for detecting a position of an object within an interior of a vehicle equipped with a door (4), comprising: recording a sequence of images by a camera; and calculating an actual position of the object by electronically evaluating the images of the sequence of images, wherein a differential image is generated by comparing a currently recorded image with a previously stored image, and the position of the object is calculated on the basis of the differential image, and the camera records images of the door, the images of the door are evaluated and the open state of the door is established on the basis of the differential image between the closed state and the opened state. 2. The method according to claim 1, wherein an opening angle of the door is established from the differential image. 3. The method according to claim 1, wherein the camera generates a signal depending on the open state detected and feeds the signal to a monitoring system which, on the basis of the signal, switches on or off a security device for the protection of vehicle occupants. 4. A method for detecting a position of an object within an interior of a vehicle equipped with a door, comprising: recording a sequence of images by a camera; and calculating an actual position of the object by electronically evaluating the images of the sequence of images, wherein a differential image is generated by comparing an actually recorded image with a previously stored image, and the position of the object is calculated on the basis of the differential image, and the camera measures an open state of a window and the camera generates a signal depending on the open state detected and feeds the signal to a monitoring system. 5. The method according to claim 4, wherein the camera emits a signal to the monitoring system if it detects an object within a range of the opened windows, on the basis of which signal the monitoring system initializes a lockout which prevents the opened window from being closed. 6. The method according to claim 5, wherein the open state of the door and/or of the window is established from gray-scale images. 7. The method according to claim 6, wherein the gray-scale images virtual image areas are defined, in which image areas changes in the gray-scale values are expected if there is a change in the open state of the door and/or of the window. 8. The method according to claim 7, wherein, for each image established, a curve of gray-scale values as a function of a location is differentiated according to the location, in that the result of the differentiation is stored in each case and the open state is determined from comparison of the stored data. 9. The method according to claim 5, wherein the open state of the door and/or of the window is established from three-dimensional distance images of the camera. 10. The method according to claim 9, wherein after transformation into the vehicle system distance points are projected onto the vehicle floor or onto a plane parallel to the vehicle floor, such that the projected images are stored in each case and the open state is determined from comparison of the stored data. 11. The method according to claim 9, wherein point clouds of the door or of the window, are defined and the open state is determined from comparison of the stored data. 12. A device within an interior of a vehicle having a camera for implementing the method comprising: recording a sequence of images by a camera; and calculating an actual position of the object by electronically evaluating the images of the sequence of images, wherein a differential image is generated bv comparing a currently recorded image with a previously stored image, and the position of the object is calculated on the basis of the differential image, and the camera records images of the door, the images of the door are evaluated and the open state of the door is established on the basis of the differential image between the closed state and the opened state. 13. A device within an interior of a vehicle having a camera for implementing the method comprising: recording a sequence of images by a camera; and calculating an actual position of the object by electronically evaluating the images of the sequence of images, wherein a differential image is generated by comparing an actually recorded image with a previously stored image, and the position of the object is calculated on the basis of the differential image, and the camera measures an open state of a window and the camera generates a signal depending on the open state detected and feeds the signal to a monitoring system. |
<SOH> BACKGROUND OF THE INVENTION <EOH>WO 00 65538 discloses a differential image generated in which the contour of the object whose position is to be detected appears only if the object moves. In this method, only moving objects are automatically recorded in a scene, which makes it possible for images to be evaluated simply and reliably. A CCD or CMOS camera which is equipped with a wide-angle lens and mounted as high as possible within the interior of the vehicle, preferably in the roof lining of the vehicle, is used for recording images. The camera or a different optical sensor system can be arranged between the front seats; with its viewing angle it then covers the area between the dashboard and the seat backrest and between the roof lining and the seat cushion. The known method is used in order reliably to determine information about the position of the front passenger, in particular about the position of the front passenger's head, within the interior of the vehicle, and to use this information for releasing an airbag system. On the other hand, it is also known for a door contact of the vehicle to be read out in order for this information to be evaluated for monitoring the airbag system. A monitoring device classifies the occupancy of a vehicle seat, in particular of the passenger seat, into various possible occupancy classes (empty seat; child-seat facing backwards; child-seat facing forwards; adult person) and permits, by means of defined transitional probabilities, transitions between these occupancy classes. Provided that the vehicle door is open, the airbag system should not, however, be enabled. For that reason, a door contact, for example a mechanical door contact, of the vehicle generates a signal which is fed to the monitoring system. To achieve this, an electric line exists from the door contact to the camera or sensor system in the roof lining. A cable has, however, the disadvantage that laying it entails a cost. A long cable brings with it additional EMC problems (EMC=electromotive compatibility). The usually cheap door contact of the vehicle becomes a safety-relevant part because it affects the releasing of the airbag. |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention relates to a method for detecting the position of an object within the interior of a vehicle equipped with a door, in which method a sequence of images is recorded by means of a camera and the actual position of the object calculated by electronically evaluating the images of the sequence of images, a differential image being generated by comparing an actually recorded image with a previously stored image and the position of the object being calculated on the basis of the differential image. The invention is establishes a method for reliably detecting the vehicle door. According to one embodiment of the invention,a camera records images of the door, such that the images of the door are evaluated and in that, on the basis of the differential image between the closed state and the opened state, the open state of the door is established. In addition to ascertaining the open state, the method can in particular also be used for establishing from the differential image the specific opening angle of the door. This opening angle can be used in order to prevent, when the vehicle is parked in a car park, the vehicle door from being opened too widely, as a result of which these or the vehicle body parts of an adjacent vehicle or other adjacent objects could be damaged. The method for establishing a differential image is used in an advantageous manner in conjunction with a monitoring system. In this method, the camera generates, depending on the open state detected, a signal and feeds this signal to a monitoring system which, on the basis of the signal, switches on or off a security device for the protection of vehicle occupants. By this means, the disadvantages of a door contact connected with an electric line can be avoided. Additionally or alternatively, the method according to the invention can be used in order to measure the open state of a window. In this case, the camera generates, depending on the detected open state of a window, a signal and feeds this signal to a monitoring system. This can prevent the arm or head of a passenger who is leaning out of the window from being jammed or injured by a motorized window actuator or an object from being jammed or damaged by the rising window. A precondition for this, however, is that the endangered body parts of the passenger or the object are not concealed, for example by the torso of the passenger. When the camera detects an object within the range of the opened window, said camera supplies a signal to the monitoring system, based on which signal the monitoring system initializes a lockout which prevents the opened window from being closed. The evaluation of gray-scale images proves to be a particularly suitable method for evaluating open states of the vehicle door or of a window. In order, by means of a small number of gray-scale images, to reach a decision about the open state of the door and/or of a window, virtual image areas are defined for obtaining gray-scale images, in which virtual image areas changes in the gray-scale values are expected if there is a change in the open state of the door and/or window. In order, when evaluating gray-scale images, to have to use as little storage space as possible in a data memory, for each image obtained a gray-scale curve is differentiated as a function of the location according to the location. The result of the differentiation is stored in each case and the open state determined from comparison of the stored data. As an alternative to obtaining gray-scale images, three-dimensional distance images are recorded with a 3D camera, in order to establish from these the open state of the door and/or window. Measuring panels whose mean distance to the camera is measured are used here. In a further method, distance points are projected, after transformation into the vehicle system, onto a plane parallel to the floor of the vehicle. The projected images are then each stored and the open state determined from comparison of the stored data. Characteristic point clouds are determined by this means. When the door is opened, the position of the associated point cloud changes. The present invention also relates to a device for implementing a method as described above. |
Novel stress-associated genetic products from ashbya gossypii |
The present invention relates to novel polynucleotides from Ashbya gossypii; to oligonucleotides hybridizing therewith; to expression cassettes and vectors which comprise these polynucleotides; to microorganisms transformed therewith; to polypeptides encoded by these polynucleotides; and to the use of the novel polypeptides and polynucleotides as targets for improving stress resistance and, in particular, improving vitamin B2 production in microorganisms of the genus Ashbya. |
1. An isolated polynucleotide comprising a nucleic acid sequence that encodes a peptide sequence of a stress response protein that can be isolated from a microorganism of Ashbya gossypii, or a complementary sequence thereof, or another sequence or complementary sequence that can be derived therefrom by genetic degeneracy, or a fragment of any of the foregoing sequences. 2. The polynucleotide in of claim 1, wherein said stress-response protein is selected from the group of proteins consisting of proteins having a biological activity of a dioxygenase protein, a heat-shock protein, a methionine sulfoxide reductase protein, a DNA repair protein, a protein of the chaperonin-containing T complex and combinations thereof. 3. The polynucleotide of claim 1, comprising the nucleic acid sequence of SEQ ID NO: 1, 5, 8, 12 or 16, or a sequence complementary thereto; or another sequence or complementary sequence derived therefrom by genetic degeneracy, or a fragment of any of the foregoing sequences. 4. The polynucleotide of claim 1, which comprises the nucleic acid sequence of SEQ ID NO: 3, 10, 14 or 18, or a sequence complementary thereto; or a sequence or complementary sequence derived therefrom by genetic degeneracy, or a fragment of any of the foregoing sequences. 5. An isolated oligonucleotide that hybridizes to the polynucleotide of claim 1 under stringent hybridization conditions. 6. An isolated polynucleotide that hybridizes under stringent hybridization conditions to the oligonucleotide of claim 5, and codes for a gene product derived from said microorganism or a functional equivalent thereof. 7. An isolated polypeptide encoded by the polynucleotide of claim 1. 8. An expression cassette comprising the polynucleotide of claim 1 operatively linked to at least one regulatory nucleic acid sequence. 9. A recombinant vector comprising at least one expression cassette in of claim 8. 10. A prokaryotic or eukaryotic host cell transformed with at least one vector in of claim 9. 11. An isolated prokaryotic or eukaryotic host cell in which functional expression of at least one gene that codes for the polypeptide of claim 7 is modulated; or a biological activity of said polypeptide is reduced or increased. 12. The cell of claim 10, which is derived from the genus Ashbya. 13. A method for microbiological production of vitamin B2 or precursor or derivative thereof comprising expressing said vitamin B2 or precursors or derivatives thereof from the expression cassette of claim 8. 14. The method of claim 13, further comprising isolating said vitamin B2 or precursors or derivatives thereof. 15. A method for detecting an effector target for modulating microbiological production of vitamin B2 or precursor or derivative thereof, comprising: treating a microorganism capable of he microbiological production of vitamin B2 or precursor or derivative thereof with the effector target wherein said effector target interacts with the polypeptide of claim 7 or a nucleic acid sequence that encodes said polypeptide; and detecting an effect of the effector target by a change in an amount of vitamin B2 or precursor or derivative thereof that is produced by said microorganism. 16. A method for modulating microbiological production of vitamin B2 or precursors or derivatives thereof, comprising: treating a microorganism capable of microbiological production of vitamin B2 or precursors or derivatives thereof with an effector that interacts with the polypeptide of claim 7 or a nucleic acid sequence that encodes said polypeptide. 17. An isolated effector selected from the group consisting of antibodies or antigen-binding fragments thereof that bind to the polypeptide of claim 7; polypeptide ligands that are different from said antibodies or antigen-binding fragments and interact with the polypeptide low molecular weight effectors that can modulate biological activity of a said polypeptide and antisense nucleic acid sequences or ribozymes that can modulate biological activity of said polypeptide. 18. A method for microbiological production of vitamin B2 or precursors or derivatives thereof, comprising: culturing in a culture mixture the host cell of claim 10 under conditions favoring the production of vitamin B2 or precursors or derivatives thereof, and isolating a desired product from the culture mixture. 19. The method of claim 18, where the host is treated with an effector before or during culturing. 20. The method of claim 18, wherein the host a microorganism of the genus Ashbya. 21. The polypeptide of claim 7, which comprises at least 10 consecutive amino acid residues that correspond to the peptide sequence of SEQ ID NO: 2, 4, 6, 7, 9, 11, 13, 15, 17 or 19; or a functional equivalent thereof. 22. A method for microbiological production of vitamin B2 or precursors or derivatives thereof comprising: culturing a microorganism of the genus Ashby; and contacting the polynucleotide of claim 1 to said microorganism to modulate production of vitamin B2 or precursors or derivatives thereof of said microorganism. 23. A method for microbiological production of vitamin B2 or precursors or derivatives thereof, comprising: culturing a microorganism of the genus Ashby; and modulating expression of a stress response or a stress-induced subsequent state of said microorganism during culturing. 24. The host cell of claim 12, wherein said cell has an improved resistance to fermentive stress. 25. The cell of claim 11, which is derived from the genus Ashbya. 26. The method of claim 15, wherein the effector target binds to said polypeptide. 27. The method of claim 15, further comprising isolating the effector target. 28. The method of claim 16, wherein the effector is a ribozyme. 29. The method of claim 19, wherein the effector is selected from the group consisting of antibodies or antigen-binding fragments thereof that bind to a polypeptide encoded within a polynucleotide comprising: a nucleic acid sequence that encodes a peptide sequence of a stress response protein that can be isolated from a microorganism of Ashbya gossypii, or a complementary sequence thereof, or another sequence or complementary sequence that can be derived therefrom by genetic degeneracy, or a fragment of any of the foregoing sequences; polypeptide ligands that are different from said antibodies or antigen-binding fragments and interact with said polypeptide; low molecular weight effectors that can modulate a biological activity of said polypeptide; antisense nucleic acid sequences or ribozymes that can modulate biological activity of the polypeptide; and combinations thereof. 30. The polypeptide of claim 21, wherein said functional equivalent thereof possesses a biological activity of a dioxygenase protein, a heat-shock protein, a methionine sulfoxide reductase protein, a DNA repair protein, a protein of the chaperonin-containing T complex or a combination thereof. 31. The method of claim 22, further comprising isolating the vitamin B2 or precursors or derivatives thereof. 32. The method of claim 23, wherein modulating is performed with a polynucleotide comprising a nucleic acid sequence that encodes a peptide sequence of a stress response protein that can be isolated from a microorganism of Ashbya gossypii, or a complementary sequence thereof, or another nucleic acid sequence that can be derived from the nucleic acid or the complementary sequence by genetic degeneracy, or a fragment of any of the foregoing sequences. 33. The method of claim 23, wherein modulating is performed with a polypeptide encoded within a polynucleotide comprising a nucleic acid sequence that encodes a peptide sequence of a stress response protein that can be isolated from a microorganism of Ashbya gossypii, or a complementary sequence thereof, or another nucleic acid sequence that can be derived from the nucleic acid or the complementary sequence by genetic degeneracy, or a fragment of any of the foregoing sequences. 34. The method of claim 23, wherein modulating is performed with a ribozyme. 35. The method of claim 23, further comprising isolating the vitamin B2 or precursors or derivatives thereof. 36. An isolated effector that can modulate a biological activity of a stress response protein or a stress-induced state of a microorganism of Ashbya gossypii. |
Multichannel optical attenuator for multiplexed signal |
The invention relates to a multichannel optical attenuator for multiplexed signal. This optical attenuator includes at least one input optical fibre (1) intended to transport a set of light beams (2) centred on different wavelengths (λ1, . . . , λn) and at least one output optical fibre (3) intended to transport said set of light beams. The beams (2) are sent to a polarisation splitting assembly (4). This splitting assembly (4) includes first polarisation splitting means (5) generating two light beams (8-9) linearly polarised along orthogonal directions and a first lens (6). Controllable means (10) liable to change the polarisation of said beams (8-9) are inserted between the first lens (6) and a second lens (11). A recombination assembly (13) comprising the second lens (11) and second polarisation splitting means (14) receives the light beams linearly (8-9) polarised from said controllable means (10) to send them to the output optical fibres (3). |
1. A multichannel optical attenuator for wavelength multiplexed signal including: at least one input optical fibre (1) intended to transport a set of luminous fluxes (2) centred on different wavelengths (λ1, . . . , λn), at least one output optical fibre (3) intended to transport said set of luminous fluxes, characterised in that it comprises a polarisation splitting assembly (4) receiving the luminous flux (2) from the input optical fibres (1), said splitting assembly (4) including first polarisation splitting means (5) generating two light beams linearly polarised (8-9) along orthogonal directions and a first lens (6) having an optical axis (7), controllable means (10) liable to change the polarisation of said beams (8-9) being inserted at a common focus between the first lens (6) and a second lens (11) having an axis (12), a recombination assembly (13) comprising the second lens (11) and second polarisation splitting means, said second lens (11) sending the light beams linearly polarised (8-9) from said controllable means (10) to the second polarisation splitting means (14), and in that it comprises programmable electronic control means (28) of said means liable to change the polarisation. 2. A multichannel optical attenuator according to claim 1, characterised in that it comprises a mirror (21) situated after the controllable means (10) sending back the light beams linearly polarised (8-9), the splitting assembly (5) also forming a recombination assembly (14), the assembly including the first lens (6) and the mirror (21) forming a reflective system (22). 3. A multichannel optical attenuator according to claim 1, characterised in that a dispersive system (15) is inserted between the first polarisation splitting means (5) and the first lens (6). 4. A multichannel optical attenuator according to claim 3, characterised in that said dispersive system (15) is a diffraction grating dispersing at an angle the different wavelengths of the light beams linearly polarised (8-9) and generating separate luminous fluxes (16) centred on different wavelengths (λ1, . . . , λn). 5. A multichannel optical attenuator according to claim 4, characterised in that a first plate λ/2 (17) is positioned between the first polarisation splitting means (5) of polarisation and the grating (15) on the path of one of the two beams linearly polarised (8-9) and a second plate λ/2 (20) is positioned between the second lens and the second polarisation splitting means (14) on the path of the other beam. 6. A multichannel optical attenuator according to claim 5, characterised in that the plate λ/2 (17) is located so that the light beams linearly polarised (8-9) have a polarisation perpendicular to the lines (19) of the grating (15). 7. A multichannel optical attenuator according to one of the claims 4 to 6, characterised in that a prism (23) is placed between the diffraction grating (15) and the first lens (6), said prism linearising the spatial distribution of the separate luminous fluxes (16) as a function of the wavelength. 8. A multichannel optical attenuator according to claim 1, characterised in that each polarisation splitting means (5, 14) includes a polarisation splitter with parallel faces. 9. A multichannel optical attenuator according to claim 8, characterised in that said polarisation splitter with parallel faces is made of calcite (CaCO3). 10. A multichannel optical attenuator according to claim 1, characterised in that the axis (7) of the first lens (6) is positioned in the middle of the space separating the light beams linearly polarised (8-9) from the first polarisation splitting means (5). 11. A multichannel optical attenuator according to any one of the claims 4, 5, 6, 8, 9, or 10, characterised in that the first lens (6) is a lens whereof the digital aperture is such that no spatial overlapping of the separate fluxes (16) incident on the lens (6) occurs. 12. A multichannel optical attenuator according to claim 11, characterised in that the first lens (6) conjugates the grooves (19) of the grating (15) on the mirror (21). 13. A multichannel optical attenuator according to claim 12, characterised in that the object focus of the lens (6) is aligned with the centres (24′-24) of the spots created by the beams linearly polarised (8-9) from a single input fibre (1) on the dispersive system (15). 14. A multichannel optical attenuator according to claim 13, characterised in that a circulator (25) is placed in front of said input fibre (1) which is merged spatially with the output fibre (3). 15. A multichannel optical attenuator according to claim 1, characterised in that the controllable means (10) liable to change the polarisation of the beams (8-9) include a barrel-mounted birefringent plate. 16. A multichannel optical attenuator according to claim 1, characterised in that the controllable means (10) liable to change the polarisation of the beams (8-9) include a material with controllable bi-refringence. 17. A multichannel optical attenuator according to the claim 16, characterised in that the material with controllable bi-refringence comprises liquid crystals (26) distributed into pixels (27). 18. A multichannel optical attenuator according to 17, characterised in that each of the liquid crystals (26) receives a single separate flux (16) of wavelength λi(i=1 to n). 19. A multichannel optical attenuator according to any one of the claims 17 or 18, characterised in that the programmable electronic control means (28) of said liquid crystals (26) include a photo-conductive film deposited on the liquid crystals (26). |
Device and method for bio-membrane filtration |
The present invention has an object to provide a biomembrane filtration apparatus which can treat a nitrate nitrogen and nitrite nitrogen in an anaerobic state to stably remove them together with SS with efficiency and, simultaneously, can biologically remove the residual hydrogen donor as well remaining in the water to be treated in one and the same biomembrane filtration apparatus by a fixed bed biomembrane filtration method and a method therefor. As the means to achieve such an object, one embodiment of the present invention provides a biomembrane filtration apparatus having a packed layer packed with a particulate packing material to which microorganisms are to attach; an inlet for a liquid to be treated arranged above the packed layer to feed the liquid to be treated; and a gas feed opening arranged under the packed layer to feed a substantially oxygen-free gas or a gas containing trace amounts of oxygen. |
1. A biomembrane filtration apparatus having a packed layer packed with a particulate packing material to which microorganisms are to attach; an inlet for a liquid to be treated arranged above the packed layer to feed the liquid to be treated; a gas feed opening arranged under the packed layer to feed a substantially oxygen-free gas or a gas containing trace amounts of oxygen gas. 2. The biomembrane filtration apparatus of claim 1 having piping which receives a discharge gas discharged from the packed layer and is connected to said gas feed opening. 3. A method of biomembrane filtration of a liquid to be treated comprising allowing a liquid to be treated to pass through a microorganism-attached packed layer packed with a packing material in which microorganisms are attached on to the surface of a particulate packing material as a downward flow while allowing a substantially oxygen-free gas or a gas containing trace amounts of oxygen to pass through the packed layer from the bottom of the packed layer as an upward flow. 4. The method of biomembrane filtration of a liquid to be treated according to claim 3, wherein the gas discharged from the packed layer is recycled as the gas to be fed from the bottom of the packed layer. 5. The method of biomembrane filtration of a liquid to be treated according to claim 3, wherein the gas containing trace amounts of oxygen contains 0.1 to 4% by volume of oxygen. 6. The method of biomembrane filtration of a liquid to be treated according to claim 3, wherein the gas is allowed to pass through the packed layer at a superficial velocity of 0.02 to 0.1 m3/m2·min (NTP). |
<SOH> BACKGROUND ART <EOH>The method for purifying water with the use of a microorganism-attached packing material obtained by allowing microorganisms to attach on to the surface of a particulate packing material such as sand, anthracite, active carbon and a plastic filter medium to form a biomembrane is a so-called “biomembrane filtration method”, and various types of methods have been quite lately investigated. Among them, a method comprising sprinkling water to be treated over a packed layer in which microorganisms are attached on to a particulate packing material in a fixed bed state from above, and simultaneously flowing air or oxygen from the bottom of the packed layer to form a gas-liquid counter current state, thereby bringing the water to be treated into contact with air or oxygen to effect purification of the water to be treated, and intermittently washing the packed layer and discharging a slurry built up in the packed layer, has many advantages such that a slurry can be treated without circulation; bulking is not caused; clogging of the packed layer by the slurry is prevented; a settling and separation zone is unnecessary since the method also has a physical filtration action; the efficiency of using oxygen is high; and the like, and is popularized. However, although the biomembrane filtration method as described above is very efficient as to the removal of BOD and SS (suspended solids) and the nitrification of ammonia, the denitrification which is an anaerobic reaction could not be effectively conducted by this method. For example, in the above described fix bed biomembrane filtration method, it is possible to remove a nitrate nitrogen and a nitrite nitrogen in the water to be treated by stopping the flow of air or oxygen into the packed layer to render the inside of the packed layer anaerobic. However, in this case, a nitrogen gas formed is built up within the packed layer, which prevents formation of a uniform downward flow of the water-to be treated, and thus there is a problem of the difficulty of obtaining stabilized removal performance. In addition, in order to remove the nitrate nitrogen and nitrite nitrogen, it is necessary to add a hydrogen donor (for example, an organic substance such as methanol) necessary for the nitrogen removal reaction to the packed layer, and this hydrogen donor sometimes remains in the water to be treated. Accordingly, in order to remove the hydrogen donor such as methanol from the water to be treated, a reaeration tank is typically provided. However, to provide the reaeration tank unfavorably means to add a step and it is clearly preferred to remove the residual hydrogen donor within the biomembrane filtration apparatus. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a flow block diagram of a biomembrane filtration apparatus relating to one embodiment of the present invention. FIG. 2 is a schematic view of a gas-liquid counter current state in a packed layer. FIG. 3 is a graph showing change of properties of the treated water and the filtration resistance with the progression of time after back wash in one example of the present invention. FIG. 4 is a graph showing the relationship between the superficial velocity of gas flow and the removal ratio of NO x —N in one example of the present invention. detailed-description description="Detailed Description" end="lead"? |
Chemical compounds |
Compounds of formula (I) in which R1, R2 and X4 have the meanings given in the specification are Factor Xa inhibitors useful in the treatment of thrombotic disorders. |
1. A compound of formula (I) in which R1 represents (1-4C)alkyl, (2-4C)alkenyl or (2-4C)alkynyl; and R2 is selected from in which X1 represents a hydrogen atom or a halogen atom; X2 represents a hydrogen atom, a methyl group, a chlorine atom or a bromine atom; X3 represents a hydrogen atom, a methyl group or a halogen atom; X5 represents chloro, methoxy or methyl; X6 represents a hydrogen atom, a halogen atom or a methyl group; and X4 represents CH or N; or a pharmaceutically acceptable salt thereof. 2. A compound as claimed in claim 1, in which R2 is selected from 3. A compound as claimed in claim 2, in which R2 is selected from 4. A compound as claimed in claim 1, in which R2 is selected from 5. A compound as claimed in claim 1, in which R1 is a (1-4C)alkyl group. 6. A compound as claimed in claim 3, in which R1 is methyl, ethyl, propyl, 2-propyl, butyl, t-butyl, 1-methylpropyl or 2-methylpropyl. 7. A compound as claimed in claim 6, in which R1 is 2-propyl. 8. A compound as claimed in claim 1, in which R1 is methyl, ethyl, propyl, 2-propyl, butyl, t-butyl, 1-methylpropyl, 2-methylpropyl, prop-2-enyl or prop-2-ynyl. 9. A compound as claimed in claim 1, in which X1 represents a hydrogen atom or a fluorine atom; X2 represents a hydrogen atom or a chlorine atom; X3 represents a fluorine or chlorine atom; and X6 represents a chlorine atom. 10. A compound as claimed in claim 9, in which R2 is 4-methoxyphenyl, indol-6-yl, 3-methylindol-6-yl, 3-chloroindol-6-yl, 5-chloroindol-2-yl, 3-fluoro-4-methoxyphenyl, 5-fluoroindol-2-yl or 6-chlorobenzo[b]thiophen-2-yl. 11. A compound as claimed in claim 3, in which X1 represents a hydrogen atom or a fluorine atom; X2 represents a hydrogen atom or a chlorine atom; and X3 represents a chlorine atom. 12. A compound as claimed in claim 11, in which R2 is 4-methoxyphenyl, indol-6-yl, 3-methylindol-6-yl, 3-chloroindol-6-yl, or 5-chloroindol-2-yl. 13. A compound as claimed in claim 11, in which R2 is 4-methoxyphenyl, indol-6-yl or 5-chloroindol-2-yl. 14. A compound as claimed in claim 1, in which X4represents CH. 15. A compound as claimed in claim 1, which is selected from: 1-(4-methoxybenzoyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperidine and 1-(indole-6-carbonyl-D-valinyl)-4-(1-methylpiperidin-4-yl)piperidine; and pharmaceutically acceptable salts thereof. 16. A compound as claimed in claim 1, in which X4 represents N. 17. A compound as claimed in claim 1, which is selected from: 1-(5-chloroindole-2-carbonyl-D-valinyl)-4-(1-methyl-piperidin-4-yl)piperazine, and pharmaceutically acceptable salts thereof. 18. A pharmaceutical composition, which comprises a compound as claimed in claim 1, together with a pharmaceutically acceptable diluent or carrier. 19. A process for preparing a compound as claimed in claim 1, which comprises (a) reacting a compound of formula (II) or a salt thereof, with a compound of formula (III) or a reactive derivative thereof; or (b) reacting a compound of formula (IV) or a salt thereof, with a compound of formula (V) HOOC—R2 (V) or a reactive derivative thereof; followed, if a pharmaceutically acceptable salt is desired, by forming a pharmaceutically acceptable salt. 20. A compound of formula (III) or a salt thereof, in which R1 and R2 are as defined in claim 19. 21. A compound of formula (IV) or a salt thereof, in which R1 and X4 are as defined in claim 19. 22. (canceled): 23. (canceled): 24. A method of treating a thrombotic disorder in a subject requiring treatment, which comprises administering an effective amount of a compound as claimed in claim 1. |
Napthoquinone derivatives as inhibitors of tau aggregation for the treatment of alzheimer's and related neurodegenerative disorders |
Provided are napthoquinone-type compounds which can be used to modulate the aggregation of protein (e.g. tau) associated with neurodegenerative disease (e.g. Alzheimer's disease). Structure-function characteristics for oxidised and reduced napthoquinone-type compounds, such as menadione-related compounds, are disclosed. The invention further provides methods of treatment or prophylaxis of neurodegenerative diseases and/or clinical dementias based on the compounds. |
1-39. (canceled) 40. A method of treatment or prophylaxis of a neurodegenerative disease and/or clinical dementia associated with protein aggregation, which method comprises administering to a subject a prophylactically or therapeutically effective amount of a compound having the following formula: wherein: J1 and J2 are both ═O; the covalent bonds marked β and δ are single bonds, and the covalent bond marked γ is a double bond with R1B and R2B both absent, or the covalent bond marked γ is a single bond, or: J1 is —OR7 and J2 is —OR8; the covalent bonds marked β and δ are double bonds, and the covalent bond marked γ is a single bond with R1B and R2B both absent, wherein: R1A is selected from —H, unsubstituted C1-7alkyl, C1-7haloalkyl, C1-7hydroxyalkyl, C1-7aminoalkyl, C1-7carboxyalkyl, —OH, C1-7alkoxy, acyloxy, —COOH, ester, —SO3H, —SO3M, sulfonate, C1-7alkylsulfonate, or a short chain alkyl group; R2A is selected from —H, unsubstituted C1-7alkyl, C1-7haloalkyl, C1-7hydroxyalkyl, C1-7aminoalkyl, C1-7carboxyalkyl, —OH, C1-7alkoxy, acyloxy, —COOH, ester, —SO3H, —SO3M, sulfonate, C1-7alkylsulfonate, or a short chain alkyl group; R1B if present is selected from the same groups as R1A, and may be the same as or different to R1A; R2B if present is selected from the same groups as R2A, and may be the same as or different to R2A; M denotes a cation or cations of charge or cumulative charge to counter the charge on the —SO3− group; R3, R4, R5, and R6 is independently —H, —OH, C1-7alkyl, C1-7alkoxy, or acyloxy; and, R7 and R8 is independently —H, C1-7alkyl, acyl, —SO3H, —SO3M, or sulfonate; or a pharmaceutically acceptable salt, solvate, amide, ester, or ether thereof, or a therapeutic composition comprising the same, such as to inhibit the aggregation of the protein associated with said disease or dementia. 41. A method as claimed in claim 40 wherein the compound is used in combination with another treatment for said disease or dementia. 42. A method as claimed in claim 40 wherein the protein is tau protein. 43. A method as claimed in claim 40 wherein the disease is selected from the list consisting of Familial Multiple System Tauopathy, Corticobasal Degeneration, Familial Gerstmann-Straussler-Scheinker Disease, Motor Neurone Disease; Lewy body disease; Pick's disease; Progressive Supranuclear Palsy; Alzheimer's disease. 44. A method as claimed in claim 40 wherein J1 is —OR7 and J2 is —OR8; the covalent bonds marked β and δ are double bonds, and the covalent bond marked γ is a single bond with R1B and R2B both absent. 45. A method as claimed in claim 44 wherein each of R7 and R8 is independently —H, -Me, -Et, —C(═O)Me, —C(═O)Et, —SO3H, —SO3M, —SO3Me, or —SO3Et. 46. A method as claimed in claim 45 wherein each of R7 and R8 is independently —H, -Me, —C(═O)Me, —SO3H, —SO3M, or —SO3Me. 47. A method as claimed in claim 45 wherein each of R7 and R8 is —H. 48. A method as claimed in claim 40 wherein J1 and J2 are both =O; the covalent bonds marked β and δ are single bonds, and the covalent bond marked γ is a double bond with R1B and R2B both absent, and the compound has the formula: 49. A method as claimed in claim 40 wherein J1 and J2 are both =O; the covalent bonds marked β and δ are single bonds, and the covalent bond marked γ is a single bond, and the compound has the formula: 50. A method as claimed in claim 49 wherein R1B and R2B are both —H: 51. A method as claimed in claim 49 wherein R2A and R2B are both —H: 52. A method as claimed in claim 40 wherein the short chain alkyl group in R2A is one of the following groups, where n is 0, 1, or 2: 53. A method as claimed in claim 40 wherein each of R1A, R2A, and R1B and R2B if present, is independently —H; -Me, -Et, -nPr, -iPr, -nBu, -sBu, -iBu, -tBu; —OH; —OMe, —OEt, —O(nPr), —O(iPr), —O(nBu), —O(sBu), —O(iBu), —O(tBu); —OC(═O)Me, —OC(═O)Et, —OC(═O)(nPr), —OC(═O)(iPr), —OC(═O)(nBu), —OC(═O)(sBu), —OC(═O)(iBu), —O(C═O)(tBu); —C(═O)OMe, —C(═O)OEt, —C═O)O(nPr), —C(═O)O(iPr), —C(═O)O(nBu), —C(═O)O(sBu), —C(═O)O(iBu), —C(═O)O(tBu); —SO3H, —SO3M, —SO3Me, —SO3Et, —SO3(nPr), —SO3(iPr), —SO3(nBu), —SO3(sBu), —SO3(iBu), or —SO3(tBu). 54. A method as claimed in claim 53 wherein each of R1A, R2A, and R1B and R2B if present, is independently —H, -Me, -Et, —OMe, —OEt, —OH, —OMe, —OEt, —OC(═O)Me, —OC(═O)Et, —COOH, —COOMe, —COOEt, —SO3H, —SO3M, —SO3Me, —SO3Et, or —CH2CH═C(CH3)2. 55. A method as claimed in claim 54 wherein each of R1A, R2A, and R1B and R2B if present, is independently —H, -Me, —OMe, —OH, —OMe, —OC(═O)Me, —COOH, —COOMe, —SO3H, —SO3M, —SO3Me, or —CH2CH═C(CH3)2. 56. A method as claimed in claim 40 wherein each of R3, R4, R5, and R6 is independently: —H, —OH, -Me, -Et, —OMe, —OEt, —OC(═O)Me, or —OC(═O)Et. 57. A method as claimed in claim 40 wherein each of R3, R4, R5, and R6 is independently: —H, —OH, -Me, —OMe, or —OC(═O)Me. 58. A method as claimed in claim 57 wherein each of R3, R4, R5, and R6 is independently: —H or —OH. 59. A method as claimed in claim 58 wherein each of R4, R5, and R6 is —H. 60. A method as claimed in claim 40 wherein R3 is —H. 61. A method as claimed in claim 40 wherein R3 is —OH. 62. A method as claimed in claim 48 wherein R1A is methyl, and each of R2A, R3, R4, R5, and R6 is —H. 63. A method as claimed in claim 48 wherein R1A is —SO3H or —SO3M, and each of R2A, R3, R4, R5, and R6 is —H. 64. A method as claimed in claim 48 wherein R1A and R2A are methyl, and each of R3, R4, R5, and R6 is —H. 65. A method as claimed in claim 48 wherein R1A is methyl and R3is —OH, and each of R2A, R4, R5, and R6 is —H. 66. A method as claimed in claim 48 wherein R1A is —OMe, and each of R2A, R3, R4, R5, and R6 is —H. 67. A method as claimed in claim 48 wherein R1A is methyl and R2A —OMe, and each of R3, R4, R5, and R6 is —H. 68. A method as claimed in claim 48 wherein R1A is —COOH; each of R2A, R3, R4, R5, R6 is —H; and R7 and R8 are —OH. 69. A method as claimed in claim 48 wherein R1A is methyl; each of R2A, R3, R4, R5, R6 is —H; and R7 and R8 are —C(═O)Me. 70. A method as claimed in claim 48 wherein R1A is methyl; each of R2A, R3, R4, R5, R6 is —H; and R7 and R8 are both either —SO3H or —SO3M. 71. A method as claimed in claim 48 wherein R1A and R2A are methyl; each of R3, R4, R5, R6 is —H; and R7 and R8 are —C(═O)Me. 72. A method as claimed in claim 40 wherein M is selected from the group consisting of: Na+ and K+. 73. A method as claimed in claim 40 which has an B50 value obtained as determined with reference to the Examples herein of <20. 74. A method as claimed in claim 40 which has an Rxindx value obtained as determined with reference to Example 4 herein of greater than 4. |
<SOH> BACKGROUND TO INVENTION <EOH>Conditions of dementia such as AD are frequently characterised by a progressive accumulation of intracellular and/or extracellular deposits of proteinaceous structures such as β-amyloid plaques and neurofibrillary tangles (NFTs) in the brains of affected patients. The appearance of these lesions largely correlates with pathological neurofibrillary degeneration and brain atrophy, as well as with cognitive impairment (Mukaetova-Ladinska, E. B. et al. (2000) Am. J. Pathol. Vol. 157, No. 2, 623-636). In AD, both neuritic plaques and NFTs contain paired helical filaments (PHFs), of which a major constituent is the microtubule-associated protein tau (Wischik et al. (1988a) PNAS USA 85, 4506-4510). Plaques also contain extracellular β-amyloid fibrils derived from the abnormal processing of amyloid precursor protein (APP; Kang et al. (1987) Nature 325, 733). An article by Wischik et al. (in ‘Neurobiology of Alzheimer's Disease’, 2nd Edition (2000) Eds. Dawbarn, D. and Allen, S. J., The Molecular and Cellular Neurobiology Series, Bios Scientific Publishers, Oxford) discusses in detail the putative role of tau protein in the pathogenesis of neurodegenerative dementias. Loss of the normal form of tau, accumulation of pathological PHFs and loss of synapses in the mid-frontal cortex all correlate with associated cognitive impairment. Furthermore, loss of synapses and loss of pyramidal cells both correlate with morphometric measures of tau-reactive neurofibrillary pathology, which parallels, at a molecular level, an almost total redistribution of the tau protein pool from a soluble to a polymerised form (i.e. PHFs) in Alzheimer's disease. Tau exists in alternatively-spliced isoforms, which contain three or four copies of a repeat sequence corresponding to the microtubule-binding domain (Goedert, M., et al. (1989) EMBO J. 8, 393-399; Goedert, M., et al. (1989) Neuron 3, 519-526). Tau in PHFs is proteolytically processed to a core domain (Wischik, C. M., et al. (1988b) PNAS. USA 85, 4884-4888; Wischik et al. (1988a) Loc cit .); Novak, M., et al. (1993) EMBO J. 12, 365-370) which is composed of a phase-shifted version of the repeat domain; only three repeats are involved in the stable tau-tau interaction (Jakes, R., et al. (1991) EMBO J. 10, 2725-2729). Once formed, PHF-like tau aggregates act as seeds for the further capture and provide a template for proteolytic processing of full-length tau protein (Wischik et al. 1996 Proc Natl Acad Sci USA 93, 11213-11218). The phase shift which is observed in the repeat domain of tau incorporated into PHFs suggests that the repeat domain undergoes an induced conformational change during incorporation into the filament. During the onset of AD, it is envisaged that this conformational change could be initiated by the binding of tau to a pathological substrate, such as damaged or mutated membrane proteins (see Wischik, C. M., et al. (1997) in “ Microtubule - associated proteins: modifications in disease ”, eds. Avila, J., Brandt, R. and Kosik, K. S. (Harwood Academic Publishers, Amsterdam) pp.185-241). In the course of their formation and accumulation, PHFs first assemble to form amorphous aggregates within the cytoplasm, probably from early tau oligomers which become truncated prior to, or in the course of, PHF assembly (Mena, R., et al. (1995) Acta Neuropathol. 89, 50-56; Mena, R., et al. (1996) Acta Neuropathol. 91, 633-641). These filaments then go on to form classical intracellular NFTs. In this state, the PHFs consist of a core of truncated tau and a fuzzy outer coat containing full-length tau (Wischik., C. M., et al, (1996) loc. cit.). The assembly process is exponential, consuming the cellular pool of normal functional tau and inducing new tau synthesis to make up the deficit (Lai, R. Y. K., et al., (1995), Neurobiology of Ageing , Vol. 16, No. 3, 433-445). Eventually, functional impairment of the neurone progresses to the point of cell death, leaving behind an extracellular NFT. Cell death is highly correlated with the number of extracellular NFTs (Wischik et al. 2000, loc.cit). As tangles are extruded into the extracellular space, there is progressive loss of the fuzzy outer coat of the neurone with corresponding loss of N-terminal tau immunoreactivity, but preservation of tau immunoreactivity associated with the PHF core (Bondareff, W. et al., (1994) J. Neuropath. Exper. Neurol ., Vol. 53, No. 2, 158-164). Clearly the identification of compounds that could modulate the aggregation of disease-associated proteins such as tau is of great interest. WO 96/30766 (F Hoffman-La Roche) discloses assays for the inhibition of tau-tau association, and certain inhibitors identified using the assays. FIGS. 23 and 24 therein rank certain compounds according to their inhibitory properties. Vitamin K (=K2) has a value of 0.674 and menadione (also known as Vitamin K3) is denoted as having a value of 1.042. In the ranking a value of 1 represents binding equivalent to that observed in the absence of compound. Of course, vitamin K is well known, per se, as a therapeutic. A brief overview of Vitamin K is given in “Goodman and Gilman's The Pharmacological Basis of Therapeutics”, 9th edition, pp 1582-1585, 1998. More comprehensive reviews are provided in William Friedrich, “Vitamins”, pp 285-338, 1988; Thorp et al (1995), Drugs 49, 376-387; Vermeer and Schurgers (2000), Blood Stasis and Thrombosis, 14, 339-353. The reduced form of vitamin K acts as a cofactor for the enzyme gamma-glutamyl carboxylase. This enzyme is responsible for the conversion of glutamic acid residues to gamma-carboxyglutamate on the vitamin K—dependent clotting factors (factors II, VII, IX, X and the anticoagulation proteins, protein C and protein S). Other gamma-carboxyglutamic acid containing proteins (so called Gla-proteins) have been found in plasma (protein Z), bone (osteocalcin), kidney, lung and testicular tissue. The functions of non-haematological Gla-proteins are outlined in Vermeer and Schurgers (2000, loc cit.). However such proteins are not found in the brain (Vermeer (1990), Biochem J, 266, 625-636). Traditional therapeutic uses of Vitamin K analogues include hypoprothrominaemia in adults and the newborn, inadequate absorption of lipid-soluble substances, and intestinal malabsorbtion syndromes such as cystic fibrosis, sprue, Crohn's disease and enterocolitis. In addition to the therapeutic uses described above, vitamin K3 is also known to have anti-tumour activity in vitro against a broad range of rodent and human tumour cell lines (Hu et al., 1996). The mechanism of this activity is not known. It has been shown that vitamin K3 has complex effects on several second messenger kinase cascades (Markovits et al., 1998; Wu and Sun 1999), and it has been proposed specifically that vitamin K3 forms a covalent bond with kinases/phsophatases containing the peptide sequence (I/V)HCXXXXXR(S/T)G inducing cell-cycle arrest and cell death by inhibitng Cdc25 phosphatase. However the consensus sequence [HCXXXXXR(S/T)G] is not found in the repeat domain of tau. One study (Nakajima et al., 1993) examined the effects of vitamin K derivatives on cultured CNS neurones and found that vitamins K1 and K2 had prominent survival promoting effects in the range 10 nM—1 μM. By contrast, vitamin K3 (menadione) had only ˜10% of this survival promoting activity, and this only at 1 μM. Whatever the mechanism of this effect, it was not dependent on the vitamin K cycle, since coumarin anticoagulant which interferes with epoxide reductase step had no effect on the survival promotion assay. Using cultured human neuroblastoma cells, Ko et al. (1997) showed that menadione at high doses (200 μM) caused both prominent dephosphorylation of tau protein, and oxidation of a broad range of proteins. Interestingly, for the reasons discussed in detail in Wischik et al. (2000), tau protein dephosphorylation might be expected to enhance tau protein aggregation. More recently, Ko et al. (2000) discusses the role of pathogenic mutations in alpha-synuclein in sensitising neuronal cells to oxidative stress induced by high dose menadione. In this paper, the authors argue that thiol-depletion induced by compounds which generate oxidative-stress is a general mechanism responsible for toxicity of mutant alpha-synuclein in hereditary Parkinson's disease, with the implication that rational approaches to therapy would be based on counteracting the oxidant damage produced by substances such as menadione. However, apart from the isolated data given in WO 96/30766 (F Hoffman-La Roche), no investigation has been carried out to demonstrate and optimise a role for napthoquinone-type compounds in the inhibition of aggregation of protein associated with neurodegenerative disease. |
Specific proliferation in tumour cell which can express antioncogene with high efficiency and the use of it |
The present invention provides a recombinant virus, which highly expresses a cancer therapeutic gene and specifically proliferates in tumor cells, and a method for the proliferation of the same. In said recombinant virus, the transcription of at least one VPEG is under the control of a hTERT promoter, whereby the virus selectively proliferates in telomerase activity positive tumor cells, but substantially not in normal cells negative in the telomerase activity. Said recombinant virus further contains a cancer therapeutic gene inserted in its genome, whereby the cancer therapeutic gene is highly expressed in tumor cells. The tumor cells are killed by the synergistic effects of the virus and the cancer therapeutic gene. Such recombinant viruses can be used to treat various tumors. |
1. A recombinant virus, wherein the transcription of at least one virus proliferating essential gene (VPEG) of said virus is under the control of a telomerase promoter, and the genome of said virus comprises a nucleotide sequence of a cancer therapeutic gene. 2. The recombinant virus according to claim 1, wherein said telomerase promoter is selected from the group being consisted of a hTERT promoter and a telomerase RNA component promoter. 3. The recombinant virus according to claim 2, wherein said telomerase promoter is a hTERT promoter. 4. The recombinant virus according to claim 3, wherein multiple copies of E-box (SEQ ID NO:27) are introduced downstream of the transcription initiation site of the hTERT promoter. 5. The recombinant virus according to claim 1, wherein said recombinant virus is an adenovirus, and at least one of said VPEGs is selected from the following adenoviral early genes: E1A, E1 B, E2 and E4. 6. The recombinant virus according to claim 1, wherein said recombinant virus is an adenovirus, and at least one of said VPEGs is an adenoviral late gene. 7. The recombinant virus according to claim 1, wherein said cancer therapeutic gene is selected from the group being consisted of a cancer suppressor gene; a antiangiogenic gene; a cytokine gene; a prodrug convertase gene; and an apoptosis gene. 8. The recombinant virus according to claim 7, wherein said cancer suppressor gene is selected from the group being consisted of P53, P21, Rb. NF1, VHL and APC; said antiangiogenic gene is selected from the group being consisted of endostatin gene, angiogenesis inhibitor gene, kringle 1-4 structure, kringle 1-5 structure, kringle 1-3 structure and kringle 1-5 structure plus kringle 1-3 structure of plasminogen, Interferon-α gene, interferon-β gene, interferon-v gene, thrombospondin gene, platelet cofactor 4 gene, plasminogen activator inhibitor gene, interleukin 12 and fibronectin gene; said cytokine gene is selected from the group being consisted of interleukin 2, interleukin 12, granulocyte-monocyte colony stimulating factor, tumor necrosis factor, interferon α, interferon β, interferon v, Light and F1t3 ligand; said prodrug convertase gene is selected from the group being consisted of herpes simplex virus thymidine kinase, varicella-zoster virus thymidine kinase and E. coli cytosine deaminase; said apoptosin gene is selected from the group being consisted of ICE, capase-3, capase-8 and capase-9. 9. The recombinant virus according to claim 8, wherein said antiangiogenic gene comprises a sequence which encodes a secretory signal peptide. 10. The recombinant virus according to claim 9, wherein said secretory signal peptide is selected from the group being consisted of the signal peptide natively associated with angiogenesis-inhibitor, the signal peptide from Oncostatln-M and the signal peptide from immunoglobulin K chain. 11. The recombinant virus according to claim 1, wherein said recombinant virus has the following features: 1) transcription of an other VPEG is under the control of a cis-acting element specifically activated in tumor cells; and/or 2) at least one VPEG is deficient in functions, while the virus can still specifically propagate in tumor cells. 12. The recombinant virus according to claim 11, wherein the transcription of another VPEG of said virus is under the control of at least one cis-acting element specifically activated in tumor cells, wherein said cis-acting element is selected from the group being consisted of hypoxia response element, S phase specific promoter, α-fetoprotein enhancer and promoter, carcino-embryonic antigen enhancer and promoter, tyrosinase enhancer and promoter, urokinase-type plasminogen activator enhancer and promoter, ErbB2 enhancer and promoter, ErbB3 enhancer and promoter, ErbB4 enhancer and promoter, DF3 breast cancer-associated antigen enhancer, prostaglandin specific antigen enhancer and promoter, vasodilatin enhancer and promoter, Orip from EB virus, FR enhancer of Orip from EB virus, and Bam HI C-promoter of EB virus. 13. The recombinant virus according to claim 11, wherein said virus is a recombinant adenovirus, wherein the proteins encoded by E1A, E1B55 Kda and/or E1B19Kda are deficient in functions in said adenovirus. 14. The recombinant virus according to claim 11, wherein said recombinant virus is a herpes simplex virus, the proteins encoded by ICP6 and two-copied ICP34.5 are deficient in functions in said herpes simplex virus. 15. The method for the proliferation of the recombinant virus according to claim 1, comprising in vitro injecting the telomerase positive tumor cells positive with said recombinant virus, whereby causing cyto toxicity to said tumor cells. 16. Use of the recombinant virus according to claim 1, comprising in vitro infecting telomerase positive tumor cells with said recombinant virus, whereby causing cyto toxicity to said tumor cells. 17. A method of treating tumors in mammalian, especially in human, with the recombinant virus according to claim 1, which method comprises: 1) infecting tumor cells in vitro or in vivo with said virus; and 2) restricting the replication and proliferation of the virus essentially in tumor cells, causing increased copies of the nucleotide sequences encoding the cancer therapeutic gene as well as increased expression of the cancer therapeutic gene in tumor cells, thus specifically killing the tumor cells and inhibiting the formation, growth and metastasis of tumors. 18. The method according to claim 17, further comprising the step of administering a chemical antineoplastic agent before, during and/or after the infection of tumor cells with the recombinant virus according to claim 1. 19. Use of the recombinant virus according to claim 1, in the inhibition of tumor cell growth. 20. Use of the recombinant virus according claim 1 in the preparation of a medicine for the treatment of tumors. 21. A pharmaceutical composition, comprising the recombinant virus according to claim 1 and a pharmaceutically acceptable carrier. |
<SOH> BACKGROUND ART <EOH>Malignant tumors severely threat the human life. At present, the conventional means for the treatment of malignant tumors are surgery, radiotherapy and chemotherapy, which, however, cannot achieve satisfactory effect on most tumors. The therapeutic index of most chemotherapeutics is very low, that is, their therapeutic dose and toxic dose are almost the same. Thus the aforementioned therapeutic means are accompanied with substantive toxic effect, including the life-threatening suppression of bone marrow. Therefore, it is very important for tumor treatment to develop methods which selectively kill the tumor cells but not affect the normal cells. These methods mainly rely on the characteristic marker of tumor cells, and their therapeutic effects depend on whether said tumor marker is strictly restricted to tumor cells. Telomere is a structure of eukaryotic cells, which protects the end of chromosomes. The telomere of normal cells shortens by 50-200 nucleotides after each cell division. When the length of the telomere decreases to a certain degree, the cells will die. While in malignant cells, the telomerase is activated, whereby elongating the shortened telomere and maintaining the stability of the chromosome. Then the cells escape from death and become immortal. It means that the tumor cells can propagate continuously. Therefore, the activation of telomerase plays an essential role in the onset and development of tumor. The telomerase activity is positive in about 90% tumor cells, while it is negative in most of the normal cells. Based on this fact, the telomerase can be used as a characteristic marker of tumor cells. At present, the telomerase in human is known to be consisted of the following three components: (1) RNA component (hTERC), which acts as an endogenous template for repeated synthesis of telomere; (2) telomerase binding protein (TEP1); and (3) telomerase catalytic subunit (abbreviated as hTERT), also named as telomerase reverse transcriptase. RNA component and hTERT are essential for the activity of telomerase. Recent study further shows that hTERT plays a critical role in the telomerase activity, and is highly expressed in most tumor cells or tumor cell lines, while is expresses at a lower level or even not expressed in normal cells. Thus it is believed that the promoter of hTERT is in an activated state in most tumor cells. Various researches have been carried out on tumor therapy, which aims at using telomerase as the target. These studies include the following: (1) tumor therapy, which aims to inhibit telomerase activity; (2) gene therapy, which uses telomerase promoter (i.e. hTERT promoter and/or telomerase RNA component promoter) to drive an apoptosis gene or suicide gene; and (3) virotherapy, which employs a tumor-specific propagating virus controlled by the hTERT promoter. However, the aforementioned therapy still has significant defects. Firstly, the telomerase activity is negative in about 10% of the human tumors. In these tumor cells, the length of the telomere is maintained by the mechanism termed as Alternative Lengthening of Telomere (ALT). Furthermore, due to the heterogenicity of tumor cells, the fact that the telomerase activity is positive in certain tumor cells of a patient does not means that the telomerase activity is positive in all of the tumor cells in said patient. ALT mechanism may also exist in said patient. Thus antitumor strategy only aiming at the telomerase cannot kill all the tumor cells. Studies have shown that, telomerase inhibitor can inhibit the telomerase activity, but also enhance ALT mechanism in tumor cells, whereby not being efficient to kill the tumor cells. Secondly, in human body, the telomerase activity is also positive in germ celler, hemopoietic stem cells and diverticulum cells in the gastrointestinal tract. Thus the tumor therapy aiming at the telomerase may cause toxicity to said cells. In the last decade, gene therapy was developed in clinical research. Clinical practice shows that, gene therapy is very safe, but its efficiency in treating tumor is also very low, and in some cases, it even has no therapeutic effect. Although there are various reasons for said defect, the main reasons are lower efficiency of in vivo transfection of the vector system, lower expression of the cancer therapeutic gene and being unable to target tumor cells. Thus the cancer therapeutic gene cannot be transfected to sufficient tumor cells and be highly expressed in said cells, which adversely affects the therapeutic effect of gene therapy in tumor clinical practice. It is a key point in tumor gene therapy that how to increase the transfection efficiency of the vector system, how to increase the expression of the cancer therapeutic gene and the specificity to tumor cells. In recent years, due to the rapid development in virology, molecular biology and oncology, the genomes of various virus have been sequenced, and their gene structures and functions have been studied in detail, thus virus genes can be effectively modified. The modified virus has enhanced ability to transfect tumor cells, replicate in cells as well as increased lysis effect. Whereas, in normal cells, said ability is decreased or even lost. Thus such virus can replicate selectively in tumor cells, and propagate to thousands or even millions folds, causing tumor cell lysis. Upon cell lysis, new virus is released, and again transfects and propagates in tumor cells, until all the tumor cells are killed. Since such virus cannot propagate in normal cells, it has little effect on them. Such virus, which specifically propagates in tumor cells, is termed as tumor-specific propagating virus. As yet, about ten types of tumor-specific propagating virus are in clinical test. However, virotherapy, which only employs a tumor-specific replicating virus, has its own limits. Firstly, due to the complicated mechanism of tumor formation, and The heterogenicity of tumors, there is obvious difference among patients, tumors or even cells in a tumor, With regard to the telomerase, the fact that the telomerase activity is positive in certain tumor cells of a patient does not mean that the telomerase is positive in all the tumor cells of said patient. Thus the virus employing a certain tumor mechanism to propagate cannot kill all the tumor cells. Secondly, the diffusion of virus may be inhibited by various factors in tumors, such as fibrosis, existence of normal cells and necrosis region. Thirdly, in certain tumors, the insufficient expression of the receptor of the virus (e.g. Coxsackie virus receptor) inhibits the infection of said virus. Fourthly, the immune response of the patient to the viruses also inhibits the proliferation and diffusion of virus. ONYX Pharmaceutical Company (USA) applied only E1b 55 kDa protein deleted virus (ONYX-015) to treat tumor, but only achieved 15-20% efficiency in clinics (Nemunaitis, J. et al, Cancer Res., 2000, 60(22):6359; U.S. Pat. No. 5,677,178; and U.S. Pat. No. 5,801,029). Therefore, more effective and specific tumor therapy with minimized side-effect is urgently desired. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides a recombinant virus, wherein the transcription of at least one of the virus proliferation essential gene (VPEG) is under the control of a telomerase promoter, and wherein the genome of said virus comprises a nucleotide sequence of an antitumor gene. In one embodiment, said telomerase promoter is selected from the group consisting of hTERT promoter and the telomerase RNA component promoter. Where hTERT promoter is used, several copies of E-box sequence (5′cacgtg 3′(SEQ ID NO:27)) are added downstream of the transcription initiation site. The recombinant virus of this invention may additionally have the following features: 1) transcription of other VPEG is under the control of a cis-acting element specifically activated in tumor cells; and/or 2) at least one VPEG is inactivated, while the virus can still specifically propagate in tumor cells. The present invention also provides a method, by using said recombinant virus, to treat tumors in mammalian, especially in human, which method comprises: 1) infecting tumor cells in vitro or in vivo using said virus; and 2) the virus selectively replicates and propagates essentially in tumor cells, resulting in the increase of the copies of the nucleotide sequences of a cancer therapeutic gene and the increase of the expression of the cancer therapeutic gene in tumor cells, thereby inhibiting the formation, growth and metastasis of the tumors. Appropriately, said method may also comprise administering chemical antitumor drugs before, during and/or after the infection of tumor cells. The present invention also relates to the use of said recombinant virus to inhibit the growth of tumor cells. Furthermore, the present invention relates to the use of said recombinant virus in the preparation of drugs for treating tumor. |
Degassing of flowable masses in a multiple-screw extruder |
The invention relates to a multiple-screw extruder that comprises a first process chamber (1) and a second process chamber (2). In the degassing zones of said extruder, at least the first process chamber is provided with at least one degassing opening. The radius R of the conveyor element in the degassing zones, in at least one conveyor element (7; 9) of at least one screw (5, 7; 5, 9) configured as a passage screw, in a subsection configured as a passage subsection (71a, 71e; 91a, 91e), along the peripheral direction of the at least one conveyor element (7; 9) is by ΔR smaller than the full radius Rv of the conveyor element (6; 8) required for the mutual stripping of adhering product in a closely intermeshing screw operation with die adjacent conveyor elements (6; 8) of adjacent conventional screws (5, 6; 5, 8). The invention also relates to a method for degassing viscous or elasticoviscous flowable masses in such a multi-screw extruder having several process chambers, the respective process chambers communicating by means of a connecting opening. |
1. A multiple-screw extruder, particularly a ring extruder, having multiple screws positioned parallel to one another, in particular like a collar, having processing elements which are implemented along the axial conveyance direction of the extruder in at least some sections as closely intermeshing, at least double-threaded conveyor elements having one land per thread, the process chamber of the extruder being subdivided in these sections into a first process chamber (1) and a second process chamber (2) by the screws having the closely intermeshing conveyor elements, and at least the first process chamber being provided with at least one degassing opening in the degassing zones, characterized in that for at least one conveyor element (7; 9) of at least one screw (5, 7; 5, 9), which is implemented as a passage screw, in a region around the circumference of the at least one conveyor element (7; 9) which is implemented as a passage region (71a, . . . , 71e; 91a, . . . , 91e), the radius R of the conveyor element is smaller by ΔR than the complete radius Rv of the conveyor element (6; 8), which is necessary for mutual stripping of adhering product during closely intermeshing operation with the neighboring conveyor elements (6; 8) of neighboring typical screws (5, 6; 5, 8). 2. The multiple-screw extruder according to claim 1, characterized in that the radius R of the conveyor element is smaller than the complete radius Rv by ΔR in the degassing zones of the extruder. 3. The multiple-screw extruder according to claim 1 or 2, characterized in that the region around the circumference of the at least one conveyor element (7; 9) which is implemented as a passage region (71a, . . . , 71e; 91a, . . . , 91e) is a subsection around the circumference of this conveyor element. 4. The multiple-screw extruder according to one of the preceding claims, characterized in that the differential radius ΔR of the conveyor element is a function of the peripheral angle φ around the circumference of the conveyor element and a function of the axial location x along the axial lengthwise direction of the conveyor element. 5. The multiple-screw extruder according to claim 4, characterized in that the differential radius ΔR of the conveyor element is a symmetrical function of the peripheral angle φ over a complete circumference (0<φ<360°) of the conveyor element. 6. The multiple-screw extruder according to one of claims 3 to 5, characterized in that the subsection is the surface of at least one land of the lands (71, 72; 91, 92, 93) of the conveyor element. 7. The multiple-screw extruder according to one of claims 3 to 6, characterized in that the subsection is the surface of at least one side of the sides of the conveyor element. 8. The multiple-screw extruder according to one of claims 3 to 7, characterized in that the subsection is the surface of at least one core region of the core regions of the conveyor element. 9. The multiple-screw extruder according to one of the preceding claims, characterized in that the multiple screws, which are parallel to one another, are alternately passage screws (5, 7; 5, 9) and typical screws (5, 6; 5, 8). 10. The multiple-screw extruder according to one of the preceding claims, characterized in that the subsection of the conveyor element of a passage screw (5, 7; 5, 9) which has a radius R smaller than the complete radius Rv is a land (71; 91) of the conveyor element (7; 9). 11. The multiple-screw extruder according to one of the preceding claims, characterized in that the subsections of the conveyor elements (6; 8) of a typical screw (5, 6; 5, 8) which may not be stripped using the passage subsections (71a, 71e; 91a, . . . , 91e) of a conveyor element (7; 9) of a passage screw (5, 7; 5, 9) may be stripped by other complete radius circumference subsections of the same conveyor element of the passage screw. 12. The multiple-screw extruder according to one of the preceding claims, characterized in that the multiple-screw extruder is a ring extruder having parallel screws (5) positioned like a collar, particularly circularly, the first process chamber being the outer chamber (1) and the second process chamber being the inner chamber (2) of the ring extruder process chamber (1, 2). 13. The multiple-screw extruder according to one of the preceding claims, characterized in that the conveyor element (7; 9) having the passage subsection (71a, . . . , 71e; 91a, . . . , 91e) is an n-threaded conveyor element (7; 9), in which the radius of at most n-1 lands is a smaller radius than the complete radius Rv and the radius of at least one land is the complete radius Rv. 14. The multiple-screw extruder according to claim 13, characterized in that the conveyor element (7; 9) having the passage subsection (71a, . . . , 71e; 91a, . . . , 91e) is a double-threaded conveyor element, in which the radius of the first land is a smaller radius than the complete radius Rv and the radius of the second land is the complete radius Rv. 15. The multiple-screw extruder according to claim 14, characterized in that sequential passage screws (5, 7; 5, 9), which are positioned on both sides of a typical screw (5, 6; 5, 8) positioned between them, are laid out in such a way that as they rotate around their lengthwise axis A, the particular passage subsection (71a, . . . , 71e; 91a, . . . , 91e) of a conveyor element is angularly offset around the circumference by approximately 180° to the corresponding passage subsection (71a, . . . , 71e; 91a, . . . , 91e) of the following passage screw. 16. The multiple-screw extruder according to one of the preceding claims, characterized in that no passage conveyor elements (7; 9), i.e., only closely intermeshing conveyor elements (6; 8), are positioned in a degassing zone, at least in the region of the degassing opening of the extruder housing. 17. The multiple-screw extruder according to one of the preceding claims, characterized in that the passage conveyor element (7; 9) having the passage subsection (71a, . . . , 71e; 91a, . . . , 91e) is implemented around its circumference in such a way that viscous or viscoelastic mass adhering to the passage conveyor element (7; 9) may be stretched by the rotating passage conveyor element into a thin film in a cyclic way. 18. A method of degassing viscous or viscoelastic flow masses in a multiple-screw extruder having multiple process chambers, which are each separated from one another by multiple screws, positioned parallel to one another, having essentially closely intermeshing conveyor elements, at least one of the process chambers having at least one degassing zone having a degassing opening and each of the process chambers being partially filled with the mass to be degassed, which is processed in each process chamber and conveyed through these process chambers using the conveyor elements of the screws delimiting the particular process chamber, characterized in that there is at least one connection opening between the particular process chambers. 19. The method according to claim 18, characterized in that the multiple-screw extruder has a first process chamber and a second process chamber, which is separated from the first process chamber by multiple screws positioned parallel to one another having essentially closely intermeshing conveyor elements. 20. The method according to claim 18 or 19, characterized in that the connection opening continuously changes during the operation of the multiple-screw extruder. 21. The method according to claim 20, characterized in that the change of the connection opening is that the connection opening moves back and forth cyclically in the region of the degassing zone along the lengthwise direction A. 22. The method according to claim 20 or 21, characterized in that the change of the connection opening is that the extent of the connection opening cyclically increases and reduces. 23. The method according to one of claims 18 to 22, characterized in that viscous or viscoelastic mass adhering to the passage conveyor element is so greatly and rapidly opened cyclically by the rotating conveyor element, having its cyclically and/or periodically expanding and reducing connection opening, that the gap is only partially filled with product. 24. The method according to claim 22 or 23, characterized in that the cyclic and/or periodic enlargement and reduction of the connection opening occurs in such a way that the mass in the region of the connection opening is stretched into a thin film and pulled over at least a subsection of the connection opening and subsequently destroyed. 25. The method according to one of claims 22 to 24, characterized in that the cyclic and/or periodic enlargement and reduction of the connection opening occurs in such a way that the mass in the region of the connection opening changes over from one process chamber into the other process chamber, the surface, of the material being cyclically enlarged and reduced. 26. The method according to claim 24 or 25, characterized in that the destruction of the thin film occurs through film rupture due to the sufficiently rapid stretching of the film. 27. The method according to one of claims 24, 25 or 26, characterized in that the destruction of the thin film occurs through film rupture due to the pressure differential between the process chambers on both sides of the connection opening. 28. The method according to one of claims 18 to 27, characterized in that the connection opening is a slot-like opening and the cyclic enlargement and/or reduction of the connection opening is generated in that the diametrically opposite longer edge regions of the slot are cyclically moved away from one another and/or toward one another. 29. The method according to one of claims 18 to 28, characterized in that it is performed on a multiple-screw extruder according to one of claims 1 to 12, the cyclic movement of the edge regions of the at least one connection opening toward and away from one another being produced through the rotation of the at least one conveyor element (7; 9) having its passage subsection (71a, . . . , 71e; 91a, . . . , 91e). |
Monkey visual field measurement system and visual field measurement method |
The present invention relates to a system and a method for determining a field of a vision of a monkey. Its object is to provide a system and a method which can cause a monkey to effect accurate visual field determination like a human subject. To achieve the object, according to the invention, there is provided a system for determining visual field of a monkey, comprising: a head fixing unit (A) for fixing a chair (1) to be seated by the monkey and the monkey's head; an eye target display unit (B) for displaying a gazing point (2) at which the monkey is caused to fix its eye and displaying also an eye target (3) at a predetermined position within the monkey's visual field with a predetermined brightness; a gazing monitor unit (C) for monitoring the direction of the monkey's gazing; and a response lever (4) by which the monkey shows that it has recognized the eye target; wherein the head fixing unit (A) includes an attachment member (5) fixable to the top of the monkey's head, a support member (6) connectable to the attachment member (5), and a base member (7) for fixing the support member (6). There is also provided a method for determining a visual field of a monkey using this system. |
1. A system for determining visual field of a monkey, comprising: a head fixing unit (A) for fixing a chair (1) to be seated by the monkey and the monkey's head; an eye target display unit (B) for displaying a gazing point (2) at which the monkey is caused to fix its eye and displaying also an eye target (3) at a predetermined position within the monkey's visual field with a predetermined brightness; a gazing monitor unit (C) for monitoring the direction of the monkey's gazing; a calculating unit (12) for analyzing data; and a response lever (4) by which the monkey shows that it has recognized the eye target; wherein the head fixing unit (A) includes an attachment member (5) fixable to the top of the monkey's head, a support member (6) connectable to the attachment member, and a base member (7) for fixing the support member. 2. The visual field determining system for a monkey according to claim 1, wherein the head fixing unit (A) is configured to be out of the monkey's visual field. 3. The visual field determining system for a monkey according to claim 1, wherein the support member (6) is adapted to be speedily connectable with the attachment member (5) and adapted also to be length-adjustable. 4. The visual field determining system for a monkey according to claim 1, wherein the gazing monitor unit (C) includes an infrared LED array (10) for irradiating infrared beam to the pupil of the monkey's eye, and a CCD camera (11); the shape of the monkey's pupil imaged by the CCD camera is approximated to an oval with the center of the oval representing the center of the pupil, and a comparison calculation is made between the gazing point and the position coordinates of the pupil obtained in advance by causing the monkey to fix its eye on a desired position adjacent the gazing point, thereby to continuously determine the direction of the monkey's gazing in realtime. 5. The visual field determining system for a monkey according to claim 1, wherein said calculating unit (12) comprises a general-purpose personal computer. 6. A method for fixing a head of a test subject animal, wherein after an attachment member (5) is fixedly attached to a top of the head of a monkey, a portion of a support member (6) is connected to this attachment member and then the support member (6) is fixed to a base member (7). 7. A method of determining a field of a vision of a monkey comprising the steps of: seating the monkey at a chair (1) with an attachment member (5) being fixed to the top of the monkey's head; connecting a portion of a support member (6) to the attachment member; fixing the support member to a base member (7); and then illuminating a gazing point (2) in an eye target display screen placed in front of the monkey in response to the monkey's pressing a response lever (4); illuminating an eye target (3) at a predetermined position in the monkey's visual field with predetermined brightness while the monkey is gazing at the gazing point and pressing the response lever; and releasing the pressing of the response lever when the eye target is illuminated. 8. The method of determining a field of a vision of a monkey according to claim 7, wherein for confirming whether the monkey is gazing at the gazing point (2) or not, an infrared LED array (10) is used for irradiating infrared beam to the pupil of the monkey's eye, and a CCD camera (11) is used for monitoring the monkey's pupil, the shape of the monkey's pupil being approximated to an oval, with the center of the oval representing the center of the pupil, and a comparison calculation is made between the gazing point and the position coordinates of the pupil obtained in advance by causing the monkey to gaze at a desired position adjacent the gazing point, thereby to continuously determine the direction of the monkey's gazing in realtime. 9. A method of determining efficacy of a therapeutic agent for glaucoma using the visual field determining system as defined in claim 1. |
<SOH> BACKGROUND ART <EOH>Glaucoma, a disease generally caused by rise in blood pressure in the eye, gives damage to the visual nerve system, leading to a symptom of loss of vision. Various medical products have been developed and put into use as therapeutical agents for this glaucoma. In research and development of medical products in general, not limited to those of the therapeutical agents for glaucoma, an efficacy test of the agent using animals as subjects is essential before it is put for use in a clinical test. Since glaucoma is a disease leading to loss of vision as described above, it is preferred that such efficacy test be effected in the form of determination of visual field. However, it is extremely difficult to conduct determination of visual field with high accuracy with using an animal subject. Therefore, in actuality, the efficacy test has been conducted not in the form of determination of visual field, but in the form of determination of ophathalmotonometry (eye blood pressure). On the other hand, as an instrument for determining visual field, a Humphrey field analyzer is usually employed. This field analyzer was developed for determining visual field of human. In its use, a subject is seated in front of an eye target display screen and instructed to fix his/her eye on a gazing point illuminated at a substantially center position in the eye target display screen. Under this condition, an eye target is illuminated at a predetermined position in the eye target display screen, and when the subject recognizes this illumination, the subject shows it by e.g. pressing a response button. During this determination, the subject assumes a posture with his/her chin placed on a fixing platform for fixing the subject's head. As described above, in the research and development of a therapeutic agent for glaucoma, the determination of visual field has an important roll. Hence, there has been a need to develop a technique which allows an efficacy test to be conducted by way of visual field determination using an animal subject. It is known that there exists close correlation of central cavity visual function between the human and the monkey. Then, if the determination of visual field with using a monkey is made possible, this will help deduce the efficacy of the agent in a human subject with higher accuracy, thus being very useful for the research and development of therapeutic agent for glaucoma. However, the above-described Humphrey field analyzer was developed for determination of visual field of humans. So, although this instrument can be appropriately used when smooth communication is possible between the operator and the subject, it is difficult to use this for determination of visual field of a money with which communication is not easy. Further, in the determination of visual field, it is determined to which level of illumination the subject can recognize each position in his/her field of vision. The greater the number of points determined and the greater the number of levels of brightness determined at the respective points, the higher the accuracy of the measurement result. However, in the case of the Humphrey field analyzer, considering the time period when a human subject can keep his/her concentration in the determination, the instrument is designed so that the determination is completed in a short period of time. Therefore, it is not possible to determine a threshold value with varying the brightness in small increments or to conform the reproducibility of the determined values. In this respect, the instrument is not satisfactory in terms of the accuracy of the visual field determination. For this reason, there has been a need for developing a system and a method which allow accurate determination of visual field of a monkey. In addition, since the Humphrey field analyzer is expensive, there has also been a need for a less costly determining system utilizing a general-purpose instrument. An object of the present invention is to provide a system and a method for determining visual field of a monkey, which allow high accuracy determination of visual field of a monkey just like that of a human subject. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is an explanatory view showing a construction of a system for determining a field of a vision of a monkey relating to the present invention, FIG. 2 is an explanatory view showing a procedure for fixing a monkey's head, and FIG. 3 is an explanatory view showing result of visual field determination of a monkey. detailed-description description="Detailed Description" end="lead"? |
Method and system for producing formatted information related to defects of appliances |
A method and a system for producing formatted information related to defects of appliances of a chain of appliances. To produce the formatted information related to the defects of an image-capture appliance of the chain, the method includes a first calculation algorithm with which there can be chosen, within a set of parameterizable transformation models, within a set of parameterizable reverse transformation models, within a set of synthesis images, within a set of reference scenes, and within a set of transformed images: a reference scene, and/or a transformed image, and/or a parameterizable transformation model, with which a reference image of the reference scene can be transformed to the transformed image, and/or a parameterizable reverse transformation model, with which the transformed image can be transformed to the reference image, and/or a synthesis image obtained from the reference scene and/or obtained from the reference image. The formatted information is at least partly composed of the parameters of the chosen parameterizable transformation model and/or of the parameters of the chosen parameterizable reverse transformation model. |
1-44. (Canceled). 45. A method for producing formatted information related to defects of appliances of an appliance chain, addressed to an image-processor for modifying quality of images processed by the image-processor the appliance chain including at least one image-capture appliance and/or at least one image-restitution means and/or at least one observer, the method comprising: producing data of the formatted information characterizing the defects of the appliances of the appliance chain. 46. A method according to claim 45, wherein the appliance chain includes at least one image-capture appliance, the method configured to produce the formatted information related to the defects of the image-capture appliance, the method further comprising: producing data characterizing the defects of the image-capture appliance, the formatted information further including the data characterizing the defects of the image-capture appliance. 47. A method according to claim 46, wherein the appliance chain includes at least one image-capture appliance, to produce the formatted information related to the defects of the image-capture appliance, the method includes, for calculating the formatted information, a first algorithm by which it is possible to choose, within a set of parameterizable transformation models, within a set of parameterizable reverse transformation models, within a set of synthetic images, within a set of reference scenes, and within a set of transformed images: a reference scene, and/or a transformed image, and/or a parameterizable transformation model with which the reference image obtained by capturing the reference scene by the image-capture appliance can be transformed to the transformed image, and/or a parameterizable reverse transformation model with which the transformed image can be transformed to the reference image, and/or a synthetic image obtained from the reference scene and/or obtained from the reference image, such that the transformed image is close to the synthetic image; the transformed image exhibiting a deviation compared with the synthetic image; the formatted information being at least partly composed of parameters of the chosen parameterizable transformation model and/or of parameters of the chosen parameterizable reverse transformation model. 48. A method according to claim 47, wherein the first calculation algorithm makes it possible to choose, within a set of mathematical projections, one mathematical projection with which the synthetic image can be constructed from the reference scene. 49. A method according to claim 46, wherein the appliance chain includes at least one image-capture appliance, to produce the formatted information related to the defects of the image-capture appliance, the method includes, for calculating the formatted information, a second algorithm that includes: choosing at least one reference scene, capturing at least one reference image of each reference scene by the image-capture appliance, choosing, within a set of parameterizable transformation models and within a set of synthetic images: a parameterizable transformation model with which the reference image can be transformed to a transformed image, and/or a synthetic image obtained from the reference scene and/or obtained from the reference image, such that the synthetic image is close to the transformed image; the transformed image exhibiting a deviation compared with the synthetic image; the formatted information being at least partly composed of parameters of the chosen parameterizable transformation model and/or of parameters of the chosen parameterizable reverse transformation model, the parameterizable reverse transformation model permitting the transformed image to be transformed to the reference image. 50. A method according to claim 49, wherein the second calculation algorithm makes it possible to choose, within a set of mathematical projections, one mathematical projection with which the synthetic image can be constructed from the reference scene. 51. A method according to claim 47, the method including, for calculating the deviations, a third algorithm that includes: calculating the deviations between the transformed image and the synthetic image, associating the deviations with the formatted information. 52. A method according to claim 46, wherein the appliance chain includes at least one image-capture appliance, the image or images being images of scenes captured by a user by the image-capture appliance; to produce the formatted information related to the defects of the image-capture appliance, wherein the method further comprises, for calculating the formatted information, a fourth algorithm that includes: constructing a synthetic-image class by specified mathematical projections of at least one reference scene onto a surface, capturing at least one reference image of each reference scene by the image-capture appliance, choosing, within a set of parameterizable transformation models, that with which the reference image can be transformed to a transformed image close to the synthetic-image class of the reference scene; the transformed image exhibiting a deviation compared with the synthetic-image class, the formatted information being at least partly composed of the parameters of the chosen parameterizable transformation models. 53. A method according to claim 52, the method including, for calculating the deviations, a fifth algorithm that includes: calculating the deviations between the transformed image and the synthetic-image class, associating the deviations with the formatted information. 54. A method according to claim 47, wherein in the method the image-capture appliance of the appliance chain is provided with at least one variable characteristic depending on the image, a fraction of the formatted information being related to the defects of the image-capture appliance provided with the variable characteristic or characteristics, each variable characteristic being configured to be associated with a value to form a combination composed of the set of the variable characteristics and of the values; the method further comprising: producing a fraction of the formatted information for a predetermined selection of the combinations, the obtained formatted information in the fraction of the formatted information being entered in a field of the formatted information as measured formatted information. 55. A method according to claim 59, the method further comprising: obtaining the fraction of the formatted information related to an arbitrary combination from measured formatted information, as extended formatted information, the formatted information including the extended formatted information instead of and in place of the measured formatted information. 56. A method according to claim 55, wherein the measured formatted information and the extended formatted information exhibit an interpolation deviation, the method further comprising: selecting zero or one or more of the variable characteristics, such that the interpolation deviation for the extended formatted information obtained for the selected variable characteristics is below a predetermined interpolation threshold. 57. A method according to claim 45, wherein the appliance chain includes at least one image-restitution means, the method further configured to provide to the image-processor formatted information related to the defects of the image-restitution means; the method further comprising: producing data characterizing the defects of the image-restitution means, the formatted information also including the data characterizing the defects of the image-restitution means. 58. A method according to claim 57, wherein the appliance chain includes at least one image-restitution means to produce the formatted information related to the defects of the image-restitution means, the method including, for calculating the formatted information, a sixth algorithm by which it is possible to choose, within a set of parameterizable restitution transformation models, within a set of parameterizable reverse restitution transformation models, within a set of mathematical restitution projections, within a set of restitution references, and within a set of corrected reference restitution images: a restitution reference, and/or a corrected reference restitution image, and/or a parameterizable restitution transformation model with which the restitution reference can be transformed to the corrected reference restitution image, and/or a parameterizable reverse restitution transformation model with which the corrected reference restitution image can be transformed to the restitution reference, and/or a mathematical restitution projection with which a synthetic restitution image can be constructed from the corrected reference restitution image, such that the synthetic restitution image is close to the restituted reference obtained by restitution of the restitution reference by the image-restitution means; the restituted reference exhibiting a restitution deviation compared with the synthetic restitution image; the formatted information being at least partly composed of the parameters of the chosen parameterizable restitution transformation model and/or of the parameters of the chosen parameterizable reverse restitution transformation model. 59. A method according to claim 57, wherein the appliance chain includes at least one image-restitution means to produce the formatted information related to the defects of the image-restitution means, the method including, for calculating the formatted information, a seventh algorithm that includes: choosing at least one restitution reference, restituting the restitution reference to a restituted reference by the image-restitution means, choosing, within a set of parameterizable restitution transformation models and within a set of mathematical restitution projections: a parameterizable restitution transformation model with which the restitution reference can be transformed to a corrected reference restitution image, and a mathematical restitution projection with which a synthetic restitution image can be constructed from the corrected reference restitution image; such that the synthetic restitution image is close to the restituted reference; the restituted reference exhibiting a restitution deviation compared with the synthetic restitution image; the formatted information being at least partly composed of the parameters of the chosen parameterizable restitution transformation model and/or of the parameters of the parameterizable reverse restitution transformation model, the parameterizable reverse restitution transformation model permitting the corrected reference restitution image to be transformed to the restitution reference. 60. A method according to claim 57, wherein the appliance chain includes at least one image-restitution means, to produce the formatted information related to the defects of the image-restitution means, the method including, for calculating the formatted information, an eighth algorithm that includes: choosing a corrected reference restitution image, choosing, within a set of parameterizable restitution transformation models, within a set of mathematical restitution projections, and within a set of restitution references: a restitution reference, and/or a parameterizable restitution transformation model with which the restitution reference can be transformed to the corrected reference restitution image, and/or a parameterizable reverse restitution transformation model with which the corrected reference restitution image can be transformed to the restitution reference, and/or a mathematical restitution projection with which a synthetic restitution image can be constructed from the corrected reference restitution image, such that the synthetic restitution image is close to the restituted reference obtained by restitution of the restitution reference by the image-restitution means; the restituted reference exhibiting a restitution deviation compared with the synthetic restitution image; the formatted information being at least partly composed of the parameters of the chosen parameterizable restitution transformation model and/or of the parameters of the chosen parameterizable reverse restitution transformation model. 61. A method according to claim 58, the method including, for calculating the restitution deviations, a ninth algorithm that includes: calculating the restitution deviations between the restituted reference and the synthetic restitution image, associating the restitution deviations with the formatted information. 62. A method according to claim 58, wherein in the method the image-restitution means of the appliance chain is provided with at least one variable restitution characteristic depending on the image, a fraction of the formatted information being related to the defects of the image-restitution means provided with the variable restitution characteristic or characteristics, each variable restitution characteristic configured to be associated with a value to form a restitution combination composed of the set of the variable restitution characteristics and of the values; the method further comprising: producing the fraction of the formatted information for a predetermined selection of the restitution combinations produced entered in the field of the formatted information as measured formatted restitution information. 63. A method according to claim 62, the method further comprising: obtaining the fraction of the formatted information related to an arbitrary combination from measured formatted restitution information, as extended formatted restitution information, the formatted information including the extended formatted restitution information instead of and in place of the measured formatted restitution information. 64. A method according to claim 63, wherein the measured formatted restitution information and the extended formatted restitution information exhibit a restitution interpolation deviation, the method further comprising: selecting zero or one or more of variable restitution characteristics, such that the restitution interpolation deviation for the extended formatted restitution information obtained for the selected variable restitution characteristics is below a predetermined restitution interpolation threshold. 65. A method according to claim 58, wherein the image-restitution means is associated with an image-capture appliance to restitute, in digital form, the restituted reference from the restitution reference, wherein in the method to produce the formatted information related to the defects of the said image-restitution means, the formatted information related to the image-capture appliance associated with the image-restitution means is used. 66. A method according to claim 45, wherein the appliance chain includes at least two appliances, to produce formatted information related to the defects of the appliances of the appliance chain, the method further comprising: producing formatted information related to each of the appliances of the appliance chain, combining the formatted information related to each of the appliances of the appliance chain, to obtain the formatted information related to the appliance chain. 67. A system for producing formatted information related to defects of appliances of an appliance chain, addressed to an image-processor, for modifying quality of images processed by the image-processor, the appliance chain including at least one image-capture appliance and/or at least one image-restitution means and/or at least one observer, the system comprising: data-processing means for producing data of formatted information characterizing the defects of the appliances of the appliance chain. 68. A system according to claim 67, wherein the appliance chain includes at least one image-capture appliance, the system configured to produce the formatted information related to defects of the image-capture appliance, the system further comprising: data-processing means for producing data characterizing the defects of the image-capture appliance, the formatted information also including the data characterizing the defects of the image-capture appliance. 69. A system according to claim 68, wherein the appliance chain includes at least one image-capture appliance, to produce the formatted information related to the defects of the image-capture appliance, the data-processing means configured to employ a first algorithm for calculating the formatted information, the first calculation algorithm making it possible to choose, within a set of parameterizable transformation models, within a set of parameterizable reverse transformation models, within a set of synthetic images, within a set of reference scenes, and within a set of transformed images: a reference scene, and/or a transformed image, and/or a parameterizable transformation model with which the reference image obtained by capturing the reference scene by the image-capture appliance can be transformed to the transformed image, and/or a parameterizable reverse transformation model with which the transformed image can be transformed to the reference image, and/or a synthetic image obtained from the reference scene and/or obtained from the reference image, such that the transformed image is close to the synthetic image; the transformed image exhibiting a deviation compared with the synthetic image; the formatted information being at least partly composed of parameters of the chosen parameterizable transformation model and/or of parameters of the chosen parameterizable reverse transformation model. 70. A system according to claim 69, wherein the data-processing means employs the first calculation algorithm including calculating means for choosing, within a set of mathematical projections, one mathematical projection with which the synthetic image can be constructed from the reference scene. 71. A system according to claim 68, wherein the appliance chain includes at least one image-capture appliance, to produce the formatted information related to the defects of the image-capture appliance, the data-processing means employing a second algorithm for calculating the formatted information, the data-processing means employing the second calculation algorithm including calculating means for choosing at least one reference scene, the image-capture appliance making it possible to capture at least one reference image of each reference scene, the calculating means additionally making it possible to make a choice, within a set of parameterizable transformation models and within a set of synthetic images: of a parameterizable transformation model with which the reference image can be transformed to a transformed image, and/or of a synthetic image obtained from the reference scene and/or obtained from the reference image, the choice being made such that the synthetic image is close to the transformed image; the transformed image exhibiting a deviation compared with the synthetic image; the formatted information being at least partly composed of the parameters of the chosen parameterizable transformation model and/or of the parameters of the chosen parameterizable reverse transformation model, the parameterizable reverse transformation model permitting the transformed image to be transformed to the said reference image. 72. A system according to claim 71, wherein the calculating means with which the second calculation algorithm can be employed includes calculating means for choosing, within a set of mathematical projections, one mathematical projection with which the synthetic image can be constructed from the reference scene. 73. A system according to claim 69, wherein the data-processing means is configured to employ a third algorithm for calculating the deviations including calculating means for: calculating the deviations between the transformed image and the synthetic image, associating the deviations with the formatted information. 74. A system according to claim 68, wherein in the system, to calculate the formatted information related to an image-capture appliance of the appliance chain, the system further comprises at least one reference scene, the image-capture appliance constructing a reference image for each reference scene, to calculate the formatted information related to the image-capture appliance, the system further comprising calculating and processing means for: constructing a synthetic-image class by specified mathematical projections of at least one reference scene onto a surface, choosing, within a set of parameterizable transformation models, that with which the reference image can be transformed to a transformed image close to the synthetic-image class of the reference scene; the transformed image exhibiting a deviation compared with the synthetic-image class, the formatted information being at least partly composed of the parameters of the chosen parameterizable transformation models. 75. A system according to claim 74, wherein the system includes calculating means for: calculating the deviations between the transformed image and the synthetic-image class, associating the deviations with the formatted information. 76. A system according to claim 69, wherein in the system the image-capture appliance of the appliance chain is provided with at least one variable characteristic depending on the image, a fraction of the formatted information being related to the defects of the image-capture appliance provided with the variable characteristic or characteristics, each variable characteristic configured to be associated with a value to form a combination composed of the set of the variable characteristics and of the values; the system further comprising: data-processing means for producing the fraction of the formatted information for a predetermined selection of the combinations, the produced formatted information in the fraction of the formatted information being entered in a field of the formatted information as measured formatted information. 77. A system according to claim 76, wherein the data-processing means is configured to obtain the fraction of the formatted information related to an arbitrary combination from measured formatted information as extended formatted information, the formatted information including the extended formatted information instead of and in place of the measured formatted information. 78. A system according to claim 77, wherein the measured formatted information and the extended formatted information exhibit an interpolation deviation, the system further comprising: selection means for selecting zero or one or more of the variable characteristics, such that the interpolation deviation for the extended formatted information obtained for the variable characteristics selected is below a predetermined interpolation threshold. 79. A system according to claim 67, wherein the appliance chain includes at least one image-restitution means, the system further configured to provide, to the image-processor, formatted information related to the defects of the image-restitution means; the system further comprising: data-processing means for producing data characterizing the defects of the image-restitution means, the formatted information also including the data characterizing the defects of the image-restitution means. 80. A system according to claim 79, wherein the appliance chain includes at least one image-restitution means to produce the formatted information related to the defects of the image-restitution means, the system further including calculating means for making a choice, within a set of parameterizable restitution transformation models, within a set of parameterizable reverse restitution transformation models, within a set of mathematical restitution projections, within a set of restitution references, and within a set of corrected reference restitution images: a restitution reference, and/or a corrected reference restitution image, and/or a parameterizable restitution transformation model with which the restitution reference can be transformed to the corrected reference restitution image, and/or a parameterizable reverse restitution transformation model with which the corrected reference restitution image can be transformed to the restitution reference, and/or a mathematical restitution projection with which a synthetic restitution image can be constructed from the corrected reference restitution image, the choice being made such that the synthetic restitution image is close to the restituted reference obtained by restitution of the restitution reference by the image-restitution means, the restituted reference exhibiting a restitution deviation compared with the synthetic restitution image, the formatted information being at least partly composed of the parameters of the chosen parameterizable restitution transformation model and/or of the parameters of the chosen parameterizable reverse restitution transformation model. 81. A system according to claim 79, wherein the appliance chain includes at least one image-restitution means to produce the formatted information related to the defects of the image-restitution means, the system further including calculating means for choosing at least one restitution reference, the image-restitution means making it possible to restitute the restitution reference to a restituted reference, the calculating means further making it possible to make a choice within a set of parameterizable restitution transformation models and within a set of mathematical restitution projections: of a parameterizable restitution transformation model with which the restitution reference can be transformed to a corrected reference restitution image, and of a mathematical restitution projection with which a synthetic restitution image can be constructed from the corrected reference restitution image; the choice being made such that the synthetic restitution image is close to the restituted reference, the restituted reference exhibiting a restitution deviation compared with the synthetic restitution image, the formatted information being at least partly composed of the parameters of the chosen parameterizable restitution transformation model and/or of the parameters of the parameterizable reverse restitution transformation model, the parameterizable reverse restitution transformation model permitting the corrected reference restitution image to be transformed to the said restitution reference. 82. A system according to claim 79, wherein the appliance chain includes at least one image-restitution means to produce the formatted information related to the defects of the image-restitution means, the system including calculating means for choosing a corrected reference restitution image, the calculating means further making it possible to make a choice within a set of parameterizable restitution transformation models, within a set of mathematical restitution projections, and within a set of restitution references: of a parameterizable restitution transformation model with which a specified restitution reference can be transformed to the corrected reference restitution image, and/or of a parameterizable reverse restitution transformation model with which the corrected reference restitution image can be transformed to the restitution reference, and/or of a mathematical restitution projection with which a synthetic restitution image can be constructed from the corrected reference restitution image, the choice being made such that the synthetic restitution image is close to the restituted reference obtained by restitution of the restitution reference by the image-restitution means, the restituted reference exhibiting a restitution deviation compared with the synthetic restitution image, the formatted information being at least partly composed of the parameters of the chosen parameterizable restitution transformation model and/or of the parameters of the chosen parameterizable reverse restitution transformation model. 83. A system according to claim 80, the system further comprising calculating means for: calculating the restitution deviations between the restituted reference and the synthetic restitution image, associating the restitution deviations with the formatted information. 84. A system according to claim 80, wherein in the system the image-restitution means of the appliance chain is provided with at least one variable restitution characteristic depending on the image, a fraction of the formatted information being related to the defects of the image-restitution means provided with the variable restitution characteristic or characteristics, each variable restitution characteristic configured to be associated with a value to form a restitution combination composed of the set of the variable restitution characteristics and of the values, the system further comprising: data-processing means for producing the fraction of the formatted information for a predetermined selection of the restitution combinations, the produced formatted information in the fraction and entered in a field of the formatted information as the measured formatted restitution information. 85. A system according to claim 84, the system further comprising: data-processing means for obtaining the fraction of the formatted information related to an arbitrary combination from measured formatted restitution information, the fraction of the formatted information related to an arbitrary restitution combination as extended formatted restitution information, the formatted information including the extended formatted restitution information instead of and in place of the measured formatted restitution information. 86. A system according to claim 85, wherein the measured formatted restitution information and the extended formatted restitution information exhibit a restitution interpolation deviation, the system further comprising: selection means for selecting zero or one or more variable restitution characteristics, such that the restitution interpolation deviation for the extended formatted restitution information obtained for the variable restitution characteristics selected is below a predetermined restitution interpolation threshold. 87. A system according to claim 80, the restitution means being associated with an image-capture appliance to restitute, in digital form, the restituted reference from the restitution reference, the system further comprising: data-processing means for producing the formatted information related to the defects of the restitution means, by using the formatted information related to the image-capture appliance associated with the restitution means. 88. A system according to claim 67, wherein the appliance chain includes at least two appliances to produce formatted information related to the defects of the appliances of the appliance chain, the system further comprising: data-processing means for producing the formatted information related to each of the appliances of the appliance chain and for combining the formatted information related to each of the appliances of the appliance chain, to obtain the formatted information related to the appliance chain. |
<SOH> FIELD IN QUESTION, PROBLEM POSED <EOH>The present invention relates to a method and a system for producing formatted information related to defects of appliances of an appliance chain. The present invention also relates to the formatted information obtained in this way and addressed to image-processing means. |
Allene oxide synthase and divinyl ether synthase from the cyp74-enzyme family isolated out of physcomitrella patens, the nucleotide sequences that code these synthases, and method for producing pathogen-resistant plants |
The present invention relates to enzymes from the cytochrome P450 family and to the nucleotide sequences encoding them, and to their use in a method for the generation of pathogen-resistant plants. |
1. An allene oxide synthase with the amino acid sequence of SEQ ID No. 2. 2. A divinyl ether synthase with the amino acid sequence of SEQ ID No. 4. 3. The synthase of claim 1, which converts both 9-HPOD/TE and 13-HPOD/TE as substrates. 4. An isolated nucleotide sequence encoding an allene oxide synthase, which is involved in the biosynthesis of polyunsaturated fatty acids, and is selected from the group of sequences consisting of: a) the nucleotide sequence of SEQ ID No.1; b) a nucleotide sequence with at least 70% identity to the nucleotide sequence of SEQ ID No.1; and c) a nucleotide sequence which is complementary to a) or b). 5. An isolated nucleotide sequence encoding a divinyl ether synthase, which is involved in the biosynthesis of polyunsaturated fatty acids, and is selected from the group of sequences consisting of: a) the nucleotide sequence of SEQ ID No.3; b) a nucleotide sequence with at least 70% identity to the nucleotide sequence of SEQ ID No.3; and c) a nucleotide sequence which is complementary to a) or b). 6. The sequence of claim 4, which is derived from moss or higher plants. 7. The sequence of claim 4, which is derived from Physcomitrella patens. 8. A gene construct comprising the sequence of claim 4, or an allele, derivative or part thereof and a regulatory nucleotide sequence operatively linked thereto. 9. A vector comprising the gene construct of claim 8 and an additional nucleotide sequence for selection or replication in a host cell or integration into the genome of a host cell. 10. A transgenic plant cell, intact plant or progeny therefrom, comprising, in replicable form, the gene construct of claim 8, which shows increased expression of the nucleotide sequence encoding said allene oxide synthase in comparison with the endogenous gene expression, which brings about an increased resistance of plants to pathogens. 11. The transgenic plant cell, intact plant or progeny therefrom of claim 10, wherein the nucleotide sequence is present in extrachromosomal form or integrated stably into the plant genome. 12. The transgenic plant cell, intact plant or progeny therefrom of claim 10 wherein the allene oxide synthase has an increased specific activity in comparison with corresponding endogenous specific enzyme activity in a plant cell. 13. The transgenic plant cell, intact plant or progeny therefrom of claim 10, which is derived from the Solanaceae family or the cereal family. 14. A method for increasing the resistance of plants to pathogens, which comprises transferring into plant cells, the nucleotide sequence of claim 4 in replicable form and regenerating intact plants from the transformed plant cells. 15. The method of claim 14, wherein the plant cells are derived from the Solanaceae family or from cereals. 16. A method for increasing resistance of plant cells, intact plants, or progeny therefrom to pathogens comprising transferring, into plant cells, the nucleotide sequence of claim 4 in replicable form. 17. A method for increasing resistance of plant cells, intact plants or progeny therefrom to pathogens comprising administering the synthase of claim 1 thereto. 18. The method of claim 17, wherein an increased specific activity of said synthase brings about an increase in resistance to pathogens by 20-90%, in comparison with corresponding endogenous specific enzyme activity. 19. The method of claim 17, wherein the synthase brings about an increased resistance to mildew. 20. The synthase of claim 2, which converts both 9-HPOD/TE and 13-HPOD/TE as substrates. 21. The synthase of claim 2, which is derived from moss or higher plants. 22. The synthase of claim 2, which is derived from Physcomitrella patens. 23. The sequence of claim 5, which is derived from moss or higher plants. 24. The sequence of claim 5, which is derived from Physcomitrella patens. 25. A gene construct comprising the sequence of claim 5, or an allele, derivative or part thereof and a regulatory nucleotide sequence operatively linked thereto. 26. A vector comprising the gene construct of claim 25 and an additional nucleotide sequence for selection or replication in a host cell or integration into the genome of a host cell. 27. A transgenic plant cell, intact plant or progeny therefrom, comprising, in replicable form, the gene construct of claim 25, which shows increased expression of the nucleotide sequence encoding said divinyl ether synthase in comparison with the endogenous gene expression, which brings about an increased resistance of plants to pathogens. 28. The transgenic plant cell, intact plant or progeny therefrom of claim 27, wherein the nucleotide sequence is present in extrachromosomal form or integrated stably into the plant genome. 29. The transgenic plant cell, intact plant or progeny therefrom of claim 27, wherein the divinyl ether synthase has an increased specific activity in comparison with corresponding endogenous specific enzyme activity in a plant cell 30. The transgenic plant cell, intact plant or progeny therefrom of claim 27, which is derived from the Solanaceae family or the cereal family. 31. The transgenic plant cell, intact plant or progeny therefrom of claim 13, which is derived from potato, barley or wheat. 32. The transgenic plant cell, intact plant or progeny therefrom of claim 30, which is derived from potato, barley or wheat. 33. A method for increasing the resistance of plants to pathogens, which comprises transferring, into plant cells, the nucleotide sequence of claim 5 in replicable form and regenerating intact plants from the transformed plant cells. 34. The method of claim 33, wherein the plant cells are derived from the Solanaceae family or from cereals. 35. The transgenic plant cell, intact plant or progeny therefrom of claim 15, wherein the plant cells are derived from potato, barley or wheat. 36. The transgenic plant cell, intact plant or progeny therefrom of claim 34, wherein the plant cells are derived from potato, barley or wheat. 37. A method for increasing resistance of plant cells, intact plants, or progeny therefrom to pathogens comprising transferring into plant cells, the nucleotide sequence of claim 5 in replicable form and regenerating intact plants from the transformed plant cells. 38. A method for increasing resistance of plant cells, intact plants or progeny therefrom to pathogens comprising administering the synthase of claim 2 thereto. 39. The method of claim 38, wherein an increased specific activity of said synthase brings about an increase in resistance to pathogens by 20-90%, in comparison with corresponding endogenous specific enzyme activity. 40. The method of claim 18, wherein the increased resistance to pathogens is 30-80%. 41. The method of claim 39, wherein the increased resistance to pathogens is 30-80%. 42. The method of claim 18, wherein the increased resistance to pathogens is 40-70%. 43. The method of claim 39, wherein the increased resistance to pathogens is 40-70%. 44. The method of claim 38, wherein the synthase brings about an increased resistance to mildew. 45. The method of claim 19, wherein the mildew is from a strain of Blumeria graminis, Blumeria hordei, Blumeria tritici or Phytophtora infestans. 46. The method of claim 44, wherein the mildew is from a strain of Blumeria graminis, Blumeria hordei, Blumeria tritici or Phytophtora infestans. |
Swithcing assembly and method |
A method of switching electrical equipment between power supplies external to the equipment and a switching assembly is disclosed which includes: connecting first electrical connector means to the electrical equipment; connecting second electrical connector means to a first electrical power supply; providing third electrical connector means for electrical connection to a second electrical power supply, and operating switching means to electrically disconnect the first electrical connector means and the second electrical connector means and to electrically connect the fust electrical connector means and the third electrical connector means; the first, second and third electrical connector means and the switching means being included in the switching assembly. |
1. A switching assembly for switching electrical equipment between a first electrical power supply external to the equipment and switching assembly and to which the equipment is normally connected and a second electrical power supply external to the equipment and switching assembly and to which the equipment is not normally connected, the switching assembly including: first electrical connector means for electrical connection to electrical equipment; second electrical connector means for electrical connection to the first electrical power supply; third electrical connector means for electrical connection to the second electrical power supply, and switching means for switching an electrical connection between the first electrical connector means and the second electrical connector means to and from an electrical connection between the first electrical connector means and the third electrical connector means, the switching means effecting the switching substantially instantaneously. 2. A switching assembly as claimed in claim 1, wherein the first electrical connector means is a plug receivable in a socket on the electrical equipment. 3. A switching assembly as claimed in claim 2, wherein the second and third electrical connector means are sockets for receiving the plug of a power lead for electrically connecting the switching assembly to an electrical power supply. 4. A switching assembly as claimed in claim 3, wherein the switching means is manually operable. 5. A switching assembly as claimed in claim 1, wherein the first and second electrical connector means are the plug and socket respectively of a power lead. 6. A switching assembly as claimed in claim 5, wherein the third electrical connector means is a socket for receiving the plug of a power lead. 7. A switching assembly as claimed in claim 6, wherein the switching means is automatically operable upon insertion and withdrawal of a plug into the socket comprising the third electrical connector means. 8. A switching assembly as claimed in claim 1, wherein the first electrical connector means is a socket for receiving the plug of a power lead. 9. A switching assembly as claimed in claim 8, wherein the third electrical connector means is a plug for receiving the socket of a power lead. 10. A switching assembly as claimed in claim 9, wherein the switching means is manually operable. 11. A switching assembly as claimed in claim 1, wherein the switching assembly is not fixedly wired to the equipment or the power supplies. 12. A method of switching electrical equipment between a first electrical power supply external to the equipment and to which the equipment is normally connected and a second electrical power supply external to the equipment and to which the equipment is not normally connected, the method including: connecting first electrical connector means to the electrical equipment; connecting second electrical connector means to the first electrical power supply; providing third electrical connector means for electrical connection to the second electrical power supply, and operating switching means to electrically disconnect the first electrical connector means and the second electrical connector means and to electrically connect the first electrical connector means and the third electrical connector means, the switching means effecting the switching substantially instantaneously. 13. Electrical equipment including: an internal power supply point; first electrical connector means on the exterior of the equipment for electrical connection to a first electrical power supply to which the equipment is normally connected; second electrical connector means on the exterior of the equipment for electrical connection to a second electrical power supply to which the equipment is not normally connected, and switching means for switching an electrical connection between the internal power supply point and the first electrical connector means to and from an electrical connection between the internal power supply point and the second electrical connector means, the switching means effecting the switching substantially instantaneously; whereby the electrical equipment may be switched substantially instantaneously from the first to the second electrical power supply. 14. Electrical equipment as claimed in claim 13, wherein the switching means includes a manual switch accessible from the exterior of the equipment. 15. Electrical equipment as claimed in claim 13, wherein the switching means includes an electronic switch actuated electronically by a command signal generated by the equipment. 16. Electrical equipment as claimed in claim 13, wherein the switching means is automatically actuated upon failure of the first electrical power supply. 17. Electrical equipment as claimed in claim 13, wherein the switching means includes a preferential automatic switch for providing power from a more preferred power source upon activation thereof. 18. An electrical connector assembly for alternately connecting electrical equipment between a first electrical power supply external to both the equipment and to the connector assembly and to which the equipment is normally connected, and to a second electrical power supply external to both the equipment and to the connector assembly and to which the equipment is not normally connected, such that a substantially continuous supply of power is provided to the electrical equipment, the connector assembly being separate to the electrical equipment and the power sources and being connectable thereto and disconnectable therefrom, and including: first electrical conductor means having ari electrical output means for connection to the electrical equipment; second electrical conductor means having a first electrical input means for connection to the first electrical power supply; third electrical conductor means having a second electrical input means for connection to the second electrical power supply; the electrical output means outputting electrical power from the connector assembly to the electrical equipment when connected thereto, the first electrical input means inputting electrical power to the connector assembly from the first electrical power supply when connected thereto, and the second electrical input means inputting electrical power to the connector assembly from the second electrical power supply when connected thereto, and switching means connected to the first, second and third electrical conductor means and being operable to change the power supplied to the electrical equipment from one power supply to the other while maintaining an operationally effective power supply to the equipment. 19. Electrical equipment alternately connectable between a first electrical power supply external to the equipment and to which the equipment is normally connected, and to a second electrical power supply external to the equipment and to which the equipment is not normally connected, such that a substantially continuous supply of power is provided to the electrical equipment, the electrical equipment including: an internal power supply point, and an electrical connector assembly including: first electrical conductor means having an electrical output means for connection to the power supply point; second electrical conductor means having a first electrical input means for connection to the first electrical power supply; third electrical conductor means having a second electrical input means for connection to the second electrical power supply; the electrical output means outputting electrical power from the connector assembly to the power supply point when connected thereto, the first electrical input means inputting electrical power to the connector assembly from the first electrical power supply when connected thereto, and the second electrical input means inputting electrical power to the connector assembly from the second electrical power supply when connected thereto, and switching means connected to the first, second and third electrical conductor means and being operable to change the power supplied to the power supply point from one power supply to the other while maintaining an operationally effective power supply to the equipment. |
<SOH> BACKGROUND OF INVENTION <EOH>Currently only a very few computing or network devices are available with multiple power supplies and multiple power connectors. For continuous equipment operation, managing the physical environment and power supply becomes important. In most installations, equipment is being frequently added, moved and removed. All these equipment changes require planning and co-ordination of facilities—space, access, cooling, network connections and power outlets. Major installation changes, such as installing or adding a UPS for additional reliability, requires reconnecting the power supply of most or all computing and network devices in an installation. For those few devices fitted with multiple power supplies, this is a trivial matter performed without affecting normal operation. Otherwise, equipment must be shutdown, causing service interruptions requiring careful planning, scheduling, co-ordination, and considerable extra effort. FIG. 1 schematically illustrates the conventional manner of connecting electrical equipment to a power supply wherein a power lead 40 connects a power outlet 50 to a socket 10 in the equipment. |
<SOH> SUMMARY OF INVENTION <EOH>The present invention aims to provide an alternative to known switching assemblies and methods. In one aspect this invention resides broadly in a switching assembly for switching electrical equipment between a first electrical power supply external to the equipment and switching assembly and to which the equipment is normally connected and a second electrical power supply external to the equipment and switching assembly and to which the equipment is not normally connected, the switching assembly including: first electrical connector means for electrical connection to electrical equipment; second electrical connector means for electrical connection to the first electrical power supply; third electrical connector means for electrical connection to the second electrical power supply; switching means for switching an electrical connection between the first electrical connector means and the second electrical connector means to and from an electrical connection between the first electrical connector means and the third electrical connector means, the switching means effecting the switching substantially instantaneously. In one preferred embodiment the first electrical connector means is a plug receivable in a socket on the electrical equipment. In this embodiment it is preferred that the second and third electrical connector means are sockets for receiving the plug of a power lead for electrically connecting the switching assembly to an electrical power supply, and that the switching means is manually operable. In another preferred embodiment the first and second electrical connector means are the plug and socket respectively of a power lead. In this embodiment it is preferred that the third electrical connector means is a socket for receiving the plug of a power lead, and that the switching means is automatically operable upon insertion and withdrawal of a plug into the socket comprising the third electrical connector means. In another preferred embodiment the first electrical connector means is a socket for receiving the plug of a power lead. In this embodiment the third electrical connector means is a plug for receiving the socket of a power lead, and the switching means is manually operable. It is preferred that the switching assembly is not fixedly wired to the equipment or the power supplies. In another aspect this invention resides broadly in a method of switching electrical equipment between a first electrical power supply external to the equipment and to which the equipment is normally connected and a second electrical power supply external to the equipment and to which the equipment is not normally connected, the method including: connecting first electrical connector means to the electrical equipment; connecting second electrical connector means to the first electrical power supply; providing third electrical connector means for electrical connection to the second electrical power supply, and operating switching means to electrically disconnect the first electrical connector means and the second electrical connector means and to electrically connect the first electrical connector means and the third electrical connector means, the switching means effecting the switching substantially instantaneously. It will be appreciated that the switching means need not itself be external to the electrical equipment. Thus the second and third electrical connector means themselves may be located, for example with respect to the illustration in FIGS. 2 and 3 , on the back face of equipment 12 , with the first electrical connector means being internal and constituting the connection between an internally located switching means and the initial point of power delivery within equipment 12 , such as for example an AC-DC power supply (not shown). In this aspect the invention may be regarded as residing broadly in electrical equipment including: an internal power supply point; first electrical connector means on the exterior of the equipment for electrical connection to a first electrical power supply to which the equipment is normally connected; second electrical connector means on the exterior of the equipment for electrical connection to a second electrical power supply to which the equipment is not normally connected, and switching means for switching an electrical connection between the internal power supply point and the first electrical connector means to and from an electrical connection between the internal power supply point and the second electrical connector means, the switching means effecting the switching substantially instantaneously; whereby the electrical equipment may be switched substantially instantaneously from the first to the second electrical power supply. In one preferred embodiment of this aspect of the invention the switching means can include a manual switch accessible from the exterior of the equipment. Alternatively in another embodiment of this aspect of the invention, the switching means may be an electronic switch actuated electronically by a command signal generated by the equipment. Alternatively in another embodiment of this aspect of the invention, the switching means may be automatically actuated upon failure of the first electrical power supply. The switching means may also include a preferential automatic switch for providing power from a more preferred power source upon activation thereof. Thus it is to be understood that references herein to the switching being substantially instantaneous, and references to the switching assembly per se, extend to embrace what may be termed automatic switching means wherein designated power sources are used preferentially. In the simplest case of two power sources, there is a primary source or electrical connection and a secondary. A simple embodiment of this arrangement is a ‘latch-up’ relay in which only if power is not available from the primary source will the alternate (and powered) source be connected. It will be realised that there has to be some ‘hysteresis’ or short settling period allowed by the switch logic when an unused but more preferred, or primary, power source becomes active. The switching is still substantially instantaneous. The automatic switching means can switch over to power sources either (a) in a simple ‘next available active supply’ manner only on the loss of the current supply, or (b), in preferential manner between power sources, with switching initiated not only on loss of the current supply but also when more preferred supplies become active, as described above with reference to the latch-up relay. The extension of preferential automatic switch logic to multiple power sources will be obvious to those skilled in the art. In accordance with an alternative definition of the invention, as will be subsequently described and claimed, in one aspect the invention can be seen as residing broadly in an electrical connector assembly for alternately connecting electrical equipment between a first electrical power supply external to both the equipment and to the connector assembly and to which the equipment is normally connected, and to a second electrical power supply external to both the equipment and to the connector assembly and to which the equipment is not normally connected, such that a substantially continuous supply of power is provided to the electrical equipment, the connector assembly being separate to the electrical equipment and the power sources and being connectable thereto and disconnectable therefrom, and including: first electrical conductor means having an electrical output means for connection to the electrical equipment; second electrical conductor means having a first electrical input means for connection to the first electrical power supply; third electrical conductor means having a second electrical input means for connection to the second electrical power supply; the electrical output means outputting electrical power from the connector assembly to the electrical equipment when connected thereto, the first electrical input means inputting electrical power to the connector assembly from the first electrical power supply when connected thereto, and the second electrical input means inputting electrical power to the connector assembly from the second electrical power supply when connected thereto, and switching means connected to the first, second and third electrical conductor means and being operable to change the power supplied to the electrical equipment from one power supply to the other while maintaining an operationally effective power supply to the equipment. In accordance with this alternative definition of the invention, in another aspect the invention can also be seen as residing broadly in electrical equipment alternately connectable between a first electrical power supply external to the equipment and to which the equipment is normally connected, and to a second electrical power supply external to the equipment and to which the equipment is not normally connected, such that a substantially continuous supply of power is provided to the electrical equipment, the electrical equipment including: an internal power supply point, and an electrical connector assembly including: first electrical conductor means having an electrical output means for connection to the power supply point; second electrical conductor means having a first electrical input means for connection to the first electrical power supply; third electrical conductor means having a second electrical input means for connection to the second electrical power supply; the electrical output means outputting electrical power from the connector assembly to the power supply point when connected thereto, the first electrical input means inputting electrical power to the connector assembly from the first electrical power supply when connected thereto, and the second electrical input means inputting electrical power to the connector assembly from the second electrical power supply when connected thereto, and switching means connected to the first, second and third electrical conductor means and being operable to change the power supplied to the power supply point from one power supply to the other while maintaining an operationally effective power supply to the equipment. |
Heap bioleaching process for the extraction of zinc |
A method of extracting zinc from a sulphidic ore is provided which comprises bioleaching the ore in a heap with acidophilic microorganisms to produce a pregnant leach solution which is recovered from the bottom of the heap. An integrated process which comprises subjecting the pregnant leach solution to neutralization and solvent extraction to produce a concentrated zinc solution is also provided. Zinc may be recovered from the concentrated solution by means of electrowinning, either in the absence or presence of manganese. Alternatively zinc may be recovered in the form of a zinc compound. |
1. A method of extracting zinc from a sulphidic ore, comprising the steps of: selecting a sulphidic ore having a maximum particle size of about 50 mm; forming the ore into a heap and bio-oxidizing the ore in the heap with acidophilic microorganisms by providing air to the bottom of the heap at a rate of at least 5 L/m2·min and irrigating the top of the heap with an acidic solution containing up to about 30 g/L sulphuric acid at a rate to produce a pregnant leach solution with a predetermined acid and zinc content; and recovering zinc from the pregnant leach solution. 2. The method according to claim 1, wherein air is provided to the bottom of the heap at a rate of at least 30 L/m2·min. 3. The method according to claim 1, wherein air is provided to the bottom of the heap at a rate in the range of 10 to 100 L/m2·min. 4. The method according to claim 1, wherein air is provided to the bottom of the heap at a rate in the range of 30 to 60 L/m2·min. 5. The method according to claim 1, wherein air is provided to the bottom of the heap at a rate in excess of 100 L/m2·min. 6. The method according to claim 1, wherein said acidic solution is added to the top of the heap at a rate of at least 0.05 L/m2·min. 7. The method according to claim 1, wherein the heap has a predetermined flooding limit and said acidic solution is added to the top of the heap at a rate which is below the flooding limit of the heap. 8. The method according to claim 1, wherein the heap has a predetermined flooding limit and said acidic solution is added to the top of the heap at a rate in the range of from 0.01 L/m2·min up to the flooding limit of the heap. 9. The method according to claim 1, wherein said acidic solution is added to the top of the heap at a rate in the range of from 0.05 L/m2·min to 0.27 L/m2·min. 10. The method according to claim 1, wherein said acidic solution is added to the top of the hear at a rate of about 0.15 L/2·min. 11. The method according to claim 1, wherein zinc is extracted from the ore in the heap at an average rate of about 2.5 kg/m2·d, whereby the average concentration of zinc in the acidic solution is increased during passage through the heap to produce the pregnant leach solution. 12. The method according to claim 11, wherein the average concentration of zinc in the acidic solution is increased by up to about 20 g/L. 13. The method according to claim 11, wherein the average concentration of zinc in the acidic solution is increased by at least about 5 g/L. 14. The method according to claim 1, wherein the acidic solution contains about 15 g/L sulphuric acid. 15. The method according to claim 1, wherein the acidic solution contains about 15 g/L to 30 g/L sulphuric acid. 16. The method according to claim 1, wherein the acidic solution has a sufficient acid content to counteract iron precipitation in the heap. 17. The method according to claim 1, wherein the pregnant leach solution has a pH≦4. 18. The method according to claim 1, wherein the pregnant leach solution has a pH≦3.0. 19. The method according to claim 1, wherein the pregnant leach solution has a pH≦2.5. 20. The method according to claim 1, wherein the pregnant leach solution has a pH≦2.0. 21. The method according to claim 1, wherein the acidic solution contains iron. 22. The method according to claim 21, wherein the acidic solution contains at least about 0.04 g/L iron. 23. The method according to claim 21, wherein the acidic solution contains at least about 0.5 g/L iron. 24. The method according to claim 1, wherein the bio-oxidizing of the ore is carried out at an average temperature of at least about 30° C. 25. The method according to claim 1, wherein the bio-oxidizing of the ore is carried out at an average temperature of at least about 35° C. 26. The method according to claim 1, wherein the bio-oxidizing of the ore is carried out at an average temperature of about 30° C. to 85° C. 27. The method according to claim 1, wherein the bio-oxidizing of the ore is carried out at an average temperature of about 35° C. to 70° C. 28. The method according to claim 1, further comprising the step of recovering the pregnant leach solution at a temperature of at least 30° C. at the bottom of the heap. 29. The method according to claim 1, further comprising the step of recovering the pregnant leach solution at a temperature of at least 35° C. at the bottom of the heap. 30. The method according to claim 1, wherein said maximum particle size is about 25 mm. 31. The method according to claim 1, wherein said maximum particle size is about 12 mm. 32. The method according to claim 1, wherein the ore having said maximum particle size is produced by subjecting a larger particle size ore to crushing to reduce the ore to said maximum particle size. 33. The method according to claim 32, wherein after said crushing, the ore is subjected to agglomeration prior to the step of forming the ore into a heap. 34. The method according to claim 33, wherein said agglomeration is effected with water. 35. The method according to claim 33, wherein said agglomeration is effected with a solution containing iron. 36. The method according to claim 33, wherein said agglomeration is effected with an acidic solution. 37. The method according to claim 33, wherein said agglomeration is effected with a concentrated acid. 38. The method according to claim 37, wherein the acid is sulphuric acid. 39. The method according to claim 33, wherein said agglomeration is effected with acid mine drainage. 40. The method according to claim 1, wherein the microorganisms are indigenous to the ore. 41. The method according to claim 1, wherein the ore is inoculated directly or indirectly with said microorganisms. 42. The method according to claim 41, wherein the ore is inoculated with the microorganisms by adding to said ore an acid mine drainage solution containing indigenous microorganisms. 43. The method according to claim 1, wherein the heap has a height of at least 2 meters. 44. The method according to claim 1, wherein the heap has a height of at least 3 meters. 45. The method according to claim 1, wherein the heap has a height of at least 5 meters. 46. The method according to claim 1, wherein the heap has a height of about 2 to 10 meters. 47. The method according to claim 1, wherein the heap has a height of about 4 to 8 meters. 48. The method according to claim 1, wherein the heap has a height of about 6 meters. 49. The method according to claim 1, wherein the ore is derived from a sedimentary exhalative type deposit. 50. The method according to claim 1, wherein the ore is derived from a volcanogenic massive sulphide type deposit. 51. The method according to claim 1, wherein the ore is derived from a carbonate replacement deposit. 52. The method according to claim 1, wherein the ore contains zinc in the form of a zinc sulphide mineral. 53. The method according to claim 52, wherein the zinc sulphide mineral is sphalerite. 54. The method according to claim 52, wherein the zinc sulphide mineral is marmatite. 55. The method according to claim 52, wherein the zinc sulphide mineral is wurtzite. 56. The method according to claim 1, wherein the ore is a zinc-copper ore and the pregnant leach solution contains both zinc and copper in solution, further comprising the steps of removing zinc and copper from the pregnant leach solution by separate solvent extraction steps. 57. The method according to claim 56, wherein the copper is removed from the pregnant leach solution by solvent extraction prior to the removal of zinc. 58. The method according to claim 1, wherein the ore is a complex zinc-containing ore. 59. The method according to claim 1, wherein the ore is a predominantly sulphidic ore. 60. The method according to claim 1, wherein the ore is selected from the group consisting of a complex ore, mixed ore and an iron-containing ore. 61. The method according to claim 1, wherein the ore is selected from the group consisting of a weathered ore, partially oxidized ore, oxidic ore and siliceous ore, which ore still contains some sulphidic minerals. 62. The method according to claim 1, wherein the ore contains up to about 30% iron. 63. The method according to claim 1, wherein the ore contains at least 3% zinc. 64. The method according to claim 1, wherein the ore contains at least 5% zinc. 65. The method according to claim 1, wherein the ore contains at least 10% zinc. 66. The method according to claim 1, wherein said microorganisms are selected from the group consisting of mesophiles, thermophiles and extreme thermophiles. 67. The method according to claim 1, wherein said microorganisms are selected from the group consisting of Acidithiobacillus spp. (Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Acidithiobacillus caldus); Leptospirillum ssp. (Leptospirillum ferrooxidans); Acidiphilium spp. (Acidiphilium cryptum); Ferromicrobium acidophilus; Ferroplasma acidiphilum; Sulfobacillus spp. (Sulfobacillus thermosulfidooxidans, Sulfobacillus acidophilus); Alicyclobacillus spp. (Alicyclobacillus acidocaldrius); Acidimicrobium ferrooxidans; Sulfolobus spp. (Sulfolobus metallicus); Acidianus spp. (Acidianus infernus); Metallosphaera spp. (Metallosphaera sedula); and Thermoplasma spp. (Thermoplasma acidophilum). 68. The method according to claim 1, further comprising the step of recycling a portion of the pregnant leach solution to the heap. 69. The method according to claim 68, wherein the pregnant leach solution being recycled contains at least about 0.04 g/L iron. 70. The method according to claim 68, wherein the pregnant leach solution being recycled contains at least about 0.5 g/L iron. 71. The method according to claim 1, further comprising the step of subjecting the pregnant leach solution to solvent extraction to obtain a concentrated zinc solution and a raffinate. 72. The method according to claim 71, wherein zinc is only partially extracted during said solvent extraction. 73. The method according to claim 72, wherein zinc is extracted in an amount of about 30-50% of zinc in the pregnant leach solution. 74. The method according to claim 71, wherein the ore is subjected to crushing to produce said maximum particle size and after said crushing, the ore is subjected to agglomeration, and further comprising the step of recycling the raffinate for use in effecting said agglomeration. 75. The method according to claim 71, a further comprising the step of subjecting the pregnant leach solution to neutralization to a pH of about 4 to 4.5 prior to said solvent extraction. 76. The method according to claim 75, wherein the neutralization is carried out in the absence of forced aeration. 77. The method according to claim 71, further comprising the step of recycling at least part of the raffinate for use as said acidic solution for irrigating the heap. 78. The method according to claim 77, wherein the raffinate being recycled contains at least about 0.04 g/L iron. 79. The method according to claim 77, wherein the raffinate being recycled contains at least about 0.5 g/L iron. 80. The method according to claim 71, wherein zinc is recovered from the concentrated zinc solution by means of precipitation. 81. The method according to claim 71, wherein zinc is recovered from the concentrated zinc solution in the form of a zinc compound. 82. The method according to claim 81, wherein the zinc compound is selected from the group consisting of zinc hydroxide, zinc sulphate, zinc oxide, zinc carbonate and zinc oxalate. 83. The method according to claim 71, further comprising the step of subjecting the concentrated zinc solution to electrowinning to recover zinc therefrom. 84. The method according to claim 83, wherein the electrowinning is carried out in the presence of manganese. 85. The method according to claim 83, wherein the electrowinning is carried out in the presence of manganese using an anode comprising 0.5 to 1.0% by weight of a silver-lead alloy. 86. The method according to claim 83, wherein the electrowinning is carried out in the absence of manganese in the concentrated zinc solution. 87. The method according to claim 86, wherein the electrowinning is carried out using an anode of a silver-lead alloy comprising at least 1% silver by weight. 88. The method according to claim 87, wherein the electrowinning is carried out using an anode of a silver-lead alloy comprising about 2% silver by weight. 89. The method according to claim 86, wherein the electrowinning is carried out using an anode comprising a bismuth-silver-lead alloy. 90. The method according to claim 89, wherein the alloy comprises about 0.7 to 0.8% by weight of silver and about 1.7 to 1.9% by weight of bismuth. 91. The method according claim 83, wherein the electrowinning is carried out using an aluminum cathode and wherein zinc metal is deposited on said cathode during the electrowinning to produce cathode zinc. 92. The method according to claim 91, further comprising the step of melting the cathode zinc to produce molten zinc and casting the molten zinc into ingots. 93. The method according to claim 1, further comprising the step of providing a nutrient to the microorganisms. 94. The method according to claim 93, wherein the nutrient comprises nitrogen in the form of an ammonium salt and a source of potassium and phosphorous. 95. The method according to claim 1, wherein air is provided to the bottom of the heap at a rate of at least 10 L/m2·min. 96. A method of extracting zinc from a sulphidic ore, comprising the steps of: selecting a sulphidic ore derived from the grout consisting of a sedimentary exhalative type deposit and a volcanogenic massive sulphide type deposit; forming the ore into a heap and bio-oxidizinc the ore in the heap with acidophilic microorganisms by providing air to the bottom of the heap at a rate of at least 5 L/m2·min and irrigating the top of the heap with an acidic solution containing up to about 30 g/L sulphuric acid at a rate to produce a pregnant zinc solution with a predetermined acid and zinc content; recycling a first portion of the pregnant leacb solution to the heap; and recovering zinc from a second portion of the pregnant leach solution. 97. The method according to claim 96, wherein the second portion of the pregnant leach solution is subjected to solvent extraction to obtain a concentrated zinc solution and a raffinate. 98. The method according to claim 97, further comprising the step of subjecting the pregnant leach solution to neutralization to a pH of about 4 to 4.5 prior to said solvent extraction. 99. The method according to claim 98, wherein said neutralization is carried out in the absence of forced aeration. 100. The method according to claim 97, further comprising the step of recycling at least part of the raffinate for use as said acidic solution for irrigating the heap. 101. The method according to claim 97, wherein zinc is only partially extracted during said solvent extraction. 102. The method according to claim 101, wherein the zinc is extracted in an amount of about 30-50% of zinc in the pregnant solution. 103. A method of extracting zinc from a sulphidic ore, comprising the steps of: heap leaching the ore with an acidic leach solution in the presence of acidophilic microorganisms to produce a pregnant leach solution containing zinc in solution; subjecting the pregnant leach solution to zinc solvent extraction to obtain a manganese-free concentrated zinc solution and a raffinate; and subjecting the concentrated zinc solution to electrowinning to recover zinc therefrom, wherein the electrowinning is carried out in the absence of manganese in the concentrated zinc solution. 104. The method according to claim 103, wherein zinc is only partially extracted during said solvent extraction. 105. The method according to claim 104, wherein the zinc is extracted in an amount of about 30-50% of zinc in the pregnant leach solution. 106. The method according to claim 103, wherein the electrowinning is carried out using an anode of a silver-lead alloy comprising at least 1% silver by weight. 107. The method according to claim 106, wherein the electrowinning is carried out using an anode of a silver-lead alloy comprising about 2% silver by weight. 108. The method according to claim 103, wherein the electrowining is carried out using an anode comprising a bismuth-silver-lead alloy. 109. The method according to claim 108, wherein the alloy comprises about 0.7 to 0.8% weight of silver and about 1.7 to 1.9% by weight of bismuth. 110. The method according to claim 103, further comprising the step of subjecting the pregnant zinc solution to neutralization to a pH of about 4 to 4.5 prior to said solvent extraction. 111. The method according to claim 110, wherein the neutralization is carried out in the absence of forced aeration. 112. The method according to claim 103, further comprising the step of recycling the raffinate for use as said acidic leach solution. 113. The method according to claim 103, wherein the heap leaching of the ore is carried out at an average temperature of from about 30° C. to 85° C. in the heap. 114. The method according to claim 103, wherein the heap leaching is carried out at an average temperature of about 35° C. to 70° C. 115. The method according to claim 103, wherein the ore contains at least 5% zinc. 116. The method according to claim 103, further comprising the step of taking a bleed stream from the pregnant leach solution, thereby to remove impurities. 117. The method according to claim 116, further comprising the step of treating the bleed stream for the removal of impurities therefrom. 118. The method according to claim 116, further comprising the step of subjecting the bleed stream to solvent extraction to produce a zinc solution and a raffinate which is subjected to said treatment for the removal of impurities therefrom. 119. The method according to claim 116, wherein the bleed stream is preheated to at least about 50° C. and treated in a first stage of treatment for the removal of cadmium therefrom by cementation with zinc dust to produce a zinc-cadmium cementation product and a liquor. 120. The method according to claim 119, wherein the liquor from the first stage of treatment is heated to at least 80° C. and then subjected to a second stage of treatment wherein the liquor is subjected to neutralization to a pH of about 6 to produce a basic zinc sulphate precipitate and a liquor. 121. The method according to claim 120, wherein the basic zinc sulphate is recycled to said neutralization of the pregnant leach solution to recover zinc therefrom. 122. The method according to claim 120, wherein the liquor from the second stage of treatment is subjected to a third stage of treatment with lime slurry to a pH of about 10 for the removal of heavy metals therefrom and then discarded. 123. The method according to claim 103, further comprising the step of taking a bleed stream from the raffinate, thereby to remove impurities from the raffinate. 124. The method according to claim 123, further comprising the step of treating the bleed stream for the removal of impurities therefrom. 125. A method of extracting zinc from a sulphidic ore also containing iron, comprising the steps of: subjecting the ore to a heap leach with an acidic solution in the presence of acidophilic microorganisms to produce a pregnant leach solution containing zinc and ferrous and ferric iron; subjecting the leach solution to neutralization without the benefit of forced air flow to the leach solution, thereby maintaining the presence of the ferrous iron in the neutralized solution; subjecting the neutralized solution to zinc solvent extraction with an organic extractant to produce a loaded extractant and a raffinate containing ferrous iron in solution; stripping the loaded organic with a solution to produce a concentrated zinc solution; and recycling at least part of the raffinate to the heap leach. 126. The method according to claim 125, further comprising the step of treating the concentrated zinc solution to recover a zinc compound therefrom. 127. The method according to claim 125, further comprising the step of subjecting the concentrated zinc solution to electrowinning to recover zinc therefrom. 128. The method according to claim 125, further comprising the step of subjecting the organic to an iron removal step to remove any ferric iron contained in said organic, therefrom. 129. A method of extracting zinc from a sulphidic ore, comprising the steps of: selecting an ore having a maximum particle size of about 50 mm; forming the ore into a heap and bio-oxidizing the ore in the heap with acidophilic microorganisms; further comprising the steps of: providing air to the bottom of the heap at a rate of at least 5 L/m2·min; and irrigating the top of the heap with an acidic solution containing up to about 30 g/L sulphuric acid at a predetermined rate to extract zinc from the ore in the heap at an average rate of about 2.5 kglm2·d, whereby the average concentration of zinc in the acid solution is increased by about up to 20 g/L during passage through the heap to produce a pregnant leach solution containing zinc in solution. 130. A method of extracting zinc from a sulphidic ore, comprising the steps of: heap leaching the ore with an acidic leach solution in the presence of acidophilic microorganisms to produce a pregnant leach solution containing zinc in solution; subjecting the pregnant leach solution to zinc solvent extraction to obtain a manganese-free concentrated zinc solution and a raffinate; and treating at least a portion of the concentrated zinc solution to recover a zinc compound therefrom. 131. The method according to claim 130, wherein the zinc compound is selected from the group consisting of zinc hydroxide, zinc sulphate, zinc oxide, zinc carbonate and zinc oxalate. 132. A method of extracting zinc from a sulphidic ore, comprising the steps of: heap leaching the ore with an acidic leach solution in the presence of acidophilic microorganisms to produce a pregnant leach solution containing zinc in solution; subjecting the pregnant leach solution to zinc solvent extraction to obtain a manganese-free concentrated zinc solution and a raffinate, wherein zinc is only partially extracted during said solvent extraction, the solvent extraction being carried out in the absence of neutralization; and recycling at least part of the raffinate to the heap leaching. 133. A method of extracting zinc from a zinc solution, comprising the steps of: subjecting the zinc solution to zinc solvent extraction to obtain a manganese free concentrated zinc solution and a raffinate; and subjecting the concentrated zinc solution to electrowinning to recover zinc therefrom, wherein the electrowinning is carried out in the absence of manganese in the concentrated zinc solution. 134. The method according to claim 133, wherein the zinc solution is obtained by leaching a zinc ore or concentrate. 135. The method according to claim 133, wherein the zinc solution is obtained by leaching an electric arc furnace dust. 136. The method according to claim 133, wherein the zinc solution is obtained by leaching a recyclable zinc containing material. 137. The method according to claim 133, wherein the electrowinning is carried out using an anode of a silver-lead alloy comprising at least 1% silver by weight. 138. The method according to claim 137, wherein the electrowinning is carried out using an anode of a silver-lead alloy comprising about 2% silver by weight. 139. The method according to claim 134, wherein the ore is an oxidic ore. 140. The method according to claim 134, wherein the ore or concentrate is a sulphidic ore or concentrate. 141. The method according to claim 134, a further comprising the step of subjecting the zinc solution to neutralization to a pH of about 4 to 4.5 prior to said solvent extraction. 142. The method according to claim 141, wherein the neutralization is carried out in the absence of forced aeration. 143. The method according to claim 137, wherein the electrowinning is carried out using an anode comprising a bismuth-silver-lead alloy. 144. The method according to claim 143, wherein the alloy comprises about 0.7 to 0.8% by weight of silver and about 1.7 to 1.9% by weight of bismuth. 145. A method of extracting zinc from a sulphidic ore, comprising the steps of: selecting a sulphidic ore having a maximum particle size of about 50 mm; forming the ore into a heap and bio-oxidizing the ore in the heap with acidophilic microorganisms by providing air to the bottom of the heap at a rate of at least 5 L/m2·min and irrigating the top of the heap with an acidic solution containing at least about 30 g/I sulphuric acid at a rate to produce a pregnant leach solution with a predetermined acid and zinc content; and recovering zinc from the pregnant leach solution. |
<SOH> BACKGROUND TO THE INVENTION <EOH>Conventional processes for the extraction of zinc mostly involve crushing, grinding and concentration of the ore and then roasting of the resulting concentrate followed by leaching, purification and electrowinning of the zinc. Metals such as copper, nickel and gold have been extracted using heap leaching. See for example U.S. Pat. Nos. 6,168,766; 6,110,253; 4,017,309; 5,196,052; and 4,721,526 where several copper and gold extraction processes are described. Heap leaching of nickel is described in Canadian Patent Application No. 2,155,050. Typically the heap leaching is applied to low grade ores, e.g. <5 g/t for gold and <1% for copper. Therefore, the part of the heap representing the metal being extracted is relatively small compared to the total amount of material in the heap. Nevertheless, the value of the metal being extracted renders the application of heap leaching for these metals economically feasible. Since small amounts of material are leached from such heaps, problems related to decrepitation, slumping and compaction are relatively minor concerns. Zinc has a lower market value than copper or gold and particularly where the higher grade zinc ores are concerned, the tried and tested methods of concentrate roasting, leaching and electrowinning have been employed for the extraction of the ore. To the applicant's knowledge there are no commercial heap bioleach processes in operation for the extraction of zinc. Therefore, despite the fact that heap leaching has changed the economics in so far as the recovery of gold and copper is concerned, this has not been applied to the recovery of zinc. A reason for this may be the expectation that leaching of zinc presents problems unique to the heap leaching of zinc ores, such as the precipitation of iron oxides within the heap. Australian Patent Application No. 654322 states that a particular problem of treatment of transition ores in-situ or in heap is the tendency for iron present in the ore or in the treating liquor to precipitate as an insoluble precipitate leading to percolation problems. While problems relating to decrepitation, slumping and compaction are relatively minor concerns in the heap leaching of copper and gold, these problems threaten to become major concerns with zinc ore leaching where considerable physical changes of the ore, especially with run-of-mine ores of good grade, can be encountered. These changes might be expected to result in percolation and irrigation problems, such as flooding, channelling and cold spots. In addition, permeability problems might be expected in view of the larger amounts of material that need to be leached from the heap in order to render the process economically feasible. A bioleaching process for the recovery of zinc is described in WO01/18266 but this process is carried out in a reactor tank or vessel and employs oxygen enrichment in order to render the process feasible. U.S. Pat. No. 6,096,113 describes a tank/heap biooxidation process for recovering a metal from a refractory sulphide ore by splitting the ore in two portions. The first portion is partially biodigested in a reactor to acclimatize the sulphide-digestion microorganism. The partially digested ore is then combined with the second portion. The resulting material is dewatered, biooxidized and subjected to lixiviation. U.S. Pat. No. 5,429,659 describes a process for recovering precious or base metals from particulate refractory sulphide material comprising contacting the material with an aqueous solution containing a thermotolerant bacteria culture. Hearne et al (1) propose a process for the recovery of zinc from its sulphide ores or concentrates by an entirely hydrometallurgical route. It consists of bacteria-assisted heap-leaching of sphalerite ore, or leaching zinc concentrate at elevated temperatures with ferric sulphate and re-oxidising the formed ferrous iron with the aid of bacteria in a ferric ion generator. The results of column leaching of different sized ores are reported. The economic feasibility of a moderate-scale heap leach operation is assessed but the authors conclude that for the purely hydrometallurgical recovery route for zinc, industrial acceptance is still some time away until the technology is fully developed and demonstrated on a large scale. The authors further conclude that heap leaching may be beneficial to recover zinc from marginal ore and foresee a process development stage in which a small heap leach/solvent extraction/electrowinning plant is incorporated as an “add on” to another process, i.e. there is no teaching of heap leaching being operated as a stand alone process. The authors also state that zinc solvent extraction, crucial to both ore and concentrate leaching, is not yet satisfactorily solved. Konishi et al (2) have reported on the kinetics of the bioleaching of ZnS concentrate by Thiobacillus ferrooxidans in a well-mixed batch reactor. Experimental studies were done at 30° C. and pH 2.2 on adsorption of the bacteria to the mineral, ferric iron leaching and bacterial leaching. A mathematical model for bioleaching is presented for quantitatively examining the effects of certain operating variables with the object of selecting optimum bioleaching conditions for zinc concentrates. Sandström et al (3) have performed bioleaching with moderate thermophilic bacteria at 45° C. and with extreme thermophilic archaea at 60° C. on a complex zinc sulphide ore. The ore was fine grained and contained refractory gold as an additional value. The bioleaching was carried out in continuous stirred tank reactors and although the authors conclude that biooxidation of a complex zinc sulphide ore at 45° C. and 65° C. has proven to be a viable process, especially at the higher temperature, the ore must be finely ground (20 to 28 microns) in order to obtain high zinc recoveries at modest pyrite oxidation. U.S. Pat. No. 4,401,531 describes a process for the recovery of zinc from secondary zinc raw materials by leaching followed by solvent extraction and electrowinning. However, again the leaching is carried out in stirred tank reactors and in this case no bio-leaching is involved. Steemson et al (4) describe a process for zinc metal production from zinc concentrates by integrating zinc bioleaching with zinc solvent extraction and electrowinning. The bioleaching again was carried out in reactor tanks. The temperature was controlled at 40° C. to 45° C. In carrying out the process, the concentrate was slurried in water to produce a 6.5% w/w slurry which is then fed to the reactor tanks. See Australian Patent No. 673929 where the Steemson et al process is more fully described. Krafft et al (5) report on the leaching of two Swedish zinc sulphide ores in columns. Five grain sizes ranging from 4 mm to 128 mm were used. The authors state that, despite intermittent sulphuric acid additions, pH values varied between 2.5 and 3.5 most of the time and it was impossible to avoid precipitation of iron compounds in the columns with the result that for the two smallest grain size fractions of the one ore, the column was clogged by precipitates of iron to such an extent that the leachate could not penetrate the ore mass. Dutrizac (6) reports on ferric sulphate perolation leaching of a pyritic Zn—Pb—Cu ore and states that zinc recovery from acidic iron-bearing solutions is difficult and that much work still needs to be done in this regard. It is further stated that the problem is especially severe for low zinc concentrations. Problems were also encountered when attempts were made to use higher iron concentrations since part of the iron precipitated. In light of the above it can be seen that heap bioleaching of ore on its own, or in combination with solvent extraction and electrowinning, has not been established or proven as a viable process for the extraction of zinc on a commercial scale vis-à-vis the conventional processes involving roasting of the concentrate or the bioleaching of finely-ground zinc concentrates in reaction tanks where temperature, pH and bacteria concentration would be expected to be more even than with heap leaching. It is an object of the present invention to provide a process for the extraction of zinc from an ore by means of heap bioleaching the ore and also to provide an integrated zinc extraction process which includes solvent extraction to produce a pure concentrated zinc solution for the production of zinc metal by electrowinning or for the production of zinc compounds. The invention is the basis of the Cominco HydroZinc™ process. |
<SOH> SUMMARY OF THE INVENTION <EOH>According to the invention there is provided a method of extracting zinc from a sulphidic ore, comprising the steps of selecting a sulphidic ore having a maximum particle size of about 50 mm; forming the ore into a heap and bio-oxidizing the ore in the heap with acidophilic microorganisms by providing air to the bottom of the heap at a rate of at least 5 L/m 2 ·min and irrigating the top of the heap with an acidic solution containing up to about 30 g/L sulphuric acid at a rate to produce a pregnant leach solution with a predetermined acid and zinc content; and recovering zinc from the pregnant leach solution. Air may be provided to the bottom of the heap at a rate of from 10 to 100 L/m 2 ·min. The air may be provided at a rate of at least 30 L/m 2 ·min, such as in the range of 30 to 60 L/m 2 ·min. According to another aspect of the process, air may be provided to the bottom of the heap at a rate in excess of 100 L/m 2 ·min. Acidic solution may be added to the top of the heap at a rate of at least 0.05 L/m 2 ·min. The acidic solution may be added at a rate which is below the flooding limit of the heap, or in the range of 0.01 L/m 2 ·min up the flooding limit of the heap. The rate may be in range of 0.05 L/m 2 ·min to 0.27 L/m 2 ·min, such as 0.15 L/m 2 ·min. The units L/m 2 ·min represent a volumetric flow rate (L/min) per unit area (m 2 ) of the heap taken in plan view (i.e. in horizontal cross-section). Flooding is understood by those skilled in the art to mean the condition in which elevated liquid flow rates result in liquid accumulation within the heap which affects flow patterns, preventing even distribution of gas and liquid phases and rendering the heap inoperable. The flooding limit can vary with certain characteristics and parameters such as ore size, bulk density and heap height. The acidic solution may contain from about 15 g/L to about 30 g/L sulphuric acid. The pregnant leach solution may be subjected to solvent extraction to obtain a concentrated zinc solution and a raffinate. Preferably, zinc is only partially extracted during the solvent extraction. The amount of zinc extracted may be typically about 30% to 50% of the zinc in the pregnant leach solution. Thus, because less acid is generated in the solvent extraction, the solvent extraction may be carried out in stages without requiring neutralization between stages. The concentrated zinc solution may be subjected to electrowinning to produce zinc metal. Alternatively, zinc may be recovered from the concentrated zinc solution in the form of a compound, such as zinc hydroxide, zinc sulphate, zinc oxide, zinc carbonate or zinc oxalate. The ore may be derived from a sedimentary exhalative type deposit or a volcanogenic massive sulphide type deposit or, less preferably, from a carbonate replacement deposit. The ore may be a complex ore, a mixed ore or an iron-containing ore. The ore may also be any one of the following ores that still contains some sulphidic minerals: a weathered ore, a partially oxidized ore, an oxidic ore and a siliceous ore. The ore may be a good grade run-of-mine ore containing at least 5% zinc or at least 10% and even more than 20% zinc but it may also contain a lesser amount, such as at least 3% zinc, if economically feasible. The ore may contain significant quantities of iron, for example, up to 30% iron. The process may also be applicable to low grade dump material of various grades. The bio-oxidizing of the ore may be carried out at an average temperature of about 30° C. to 85° C., preferably about 35° C. to 70° C. The autogenous heating of the heap, from the heat of reaction, assists the leaching process. The microorganisms may be indigenous to the ore or the ore may be inoculated with the microorganisms, e.g. by adding a culture or a solution containing the microorganisms, such as mine drainage solution with indigenous microorganisms, to the ore. Given the wide temperature range, the nature of the microorganisms can vary in the heap. The process may further comprise the step of providing nutrient to the microorganisms. The nutrient may comprise nitrogen in the form of an ammonium salt and a source of potassium and phosphorous. The ore may be a complex zinc-containing ore. It may be a zinc-copper ore in which case the pregnant leach solution contains both zinc and copper in solution. The zinc and copper may be recovered in separate solvent extraction circuits, e.g. copper may be extracted prior to removal of the zinc. The ore may be derived from a sedimentary exhalative type deposit or a volcanogenic massive sulphide type deposit. The ore may contain zinc in the form of a zinc sulphide mineral, such as sphalerite, marmatite or wurtzite. The heap may have a height of at least 2 meters, for practical reasons. The maximum height of the heap may also be limited due to certain physical constraints. It may have a height of from about 2 to 10 meters, preferably about 4 to 8 meters. Different configurations of heaps are possible from an engineering point of view. The ore may be agglomerated before forming into the heap. The agglomeration may be effected with solutions at different acid strengths, including concentrated acid, depending on ore type and neutralization characteristics of the ore. The solution used for agglomeration may contain iron. The agglomeration may also be effected with pregnant leach solution, raffinate from the zinc solvent extraction or acid mine drainage. The microorganisms may be mesophiles, thermophiles or extreme thermophiles, which are categories according to temperature ranges for growth. In this specification, mesophiles are those microorganisms that grow in the moderate temperature range up to about 45° C. Thermophiles are heat-loving organisms having an optimum growth temperature in the range of 45° C. to 60° C. Extreme thermophiles have an optimum growth temperature above 60° C. As mentioned above, the microorganisms may vary according to the temperatures in the heap. These microorganisms may be selected from the following non-limiting examples of genus groups and species: Acidithiobacillus spp. ( Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Acidithiobacillus caldus ); Leptospirillum ssp. ( Leptospirillum ferrooxidans ); Acidiphilium spp. ( Acidiphilium cryptum ); Ferromicrobium acidophilus; Ferroplasma acidiphilum; Sulfobacillus spp. ( Sulfobacillus thermosulfidooxidans, Sulfobacillus acidophilus ); Alicyclobacillus spp. ( Alicyclobacillus acidocaldrius ); Acidimicrobium ferrooxidans; Sulfolobus spp. ( Sulfolobus metallicus ); Acidianus spp. ( Acidianus infernus ); Metallosphaera spp. ( Metallosphaera sedula ); Thermoplasma spp. ( Thermoplasma acidophilum ). According to another aspect of the invention the heap leaching may be effected with an acidic solution containing at least 30 g/L sulphuric acid. Further according to the invention there is provided a method of extracting zinc from a sulphidic ore, comprising the steps of heap leaching the ore with an acidic leach solution in the presence of acidophilic microorganisms to produce a pregnant zinc solution; subjecting the pregnant zinc solution to zinc solvent extraction to obtain a manganese-free concentrated zinc solution and a raffinate; and subjecting the concentrated zinc solution to electrowinning to recover zinc therefrom, wherein the electrowinning is carried out in the absence of manganese in the concentrated zinc solution. Also according to the invention there is provided a method of extracting zinc from a sulphidic ore also containing iron, comprising the steps of subjecting the ore to a heap leach with an acidic solution in the presence of acidophilic microorganisms to produce a pregnant leach solution containing zinc and iron; subjecting the leach solution to neutralization without the benefit of forced air flow to the leach solution, thereby maintaining the presence of the ferrous iron in the neutralized solution; subjecting the neutralized solution to zinc solvent extraction with an organic extractant to produce a loaded extractant and a raffinate containing ferrous iron in solution; stripping the loaded organic with an aqueous solution to produce a concentrated zinc solution; and recycling at least part of the raffinate to the heap leach. Furthermore, a portion of the pregnant leach solution, which contains iron, may be recycled to the heap so that the iron may assist in the leaching process. The acidic solution preferably has sufficient acid content such that iron precipitation in the heap is avoided. The pregnant leach solution may have a pH≦4. It may have a pH≦3.0 but preferably pH≦2.5. Further according to the invention there is provided a method of extracting zinc from a zinc solution, comprising the steps of subjecting the zinc solution to zinc solvent extraction to obtain a manganese free concentrated zinc solution and a raffinate; and subjecting the concentrated zinc solution to electrowinning to recover zinc therefrom, wherein the electrowinning is carried out in the absence of manganese in the concentrated zinc solution. The zinc solution may be obtained by leaching a zinc ore or concentrate or by the leaching of an electric arc furnace dust or a recyclable zinc containing material. Further objects and advantages of the invention will become apparent from the description of preferred embodiments of the invention below. |
Method and transparent patterning of a material web and device for carrying out said method |
The invention relates to a method for generating three-dimensional transparent patterns on a non-woven fabric or similar, whereby, for example, hot air is sucked through openings which form the pattern in the circumferential surface of a drum. The openings are straight or curved gaps instead of conventional holes and together with other openings form the desired pattern. The air or similar, flowing through the gaps, presses the fibres into the gaps and the vacuum on the inner side of the drum sucks said fibres onto the patterning. |
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