text:
Security Trends & Vulnerabilities Review 2015
From 2013 to 2014, there was an increase in the vulnerability of the information systems of large enterprises. In about 60% of system attacks, the network perimeters were penetrated via web application vulnerabilities. Additionally in 2014, there was decreased awareness among employees regarding security issues.
Vulnerabilities of Mobile Internet (GPRS)
Modern mobile networks facilitate the most convenient access to the Internet without the need for static infrastructures. People can access email, messengers, social networks and online stores whenever and wherever they need it. A range of businesses use mobile Internet for remote administration, financial operations, e-commerce, M2M and some other purposes.
SS7 Vulnerabilities
Nowadays mobile networks are the most dynamic part of critical communication infrastructures and the key instrument used to perform daily activities ranging from voice and text messaging to providing signaling for emergency services and critical infrastructure.
Vulnerabilities in Corporate Systems 2014
This report includes statistics on penetration testing carried out by Positive Technologies in 2013 and compares them to a earlier study completed in 2012.
Web Application Vulnerability Statistics 2013
Web application security is becoming more essential by the day. Almost any organization, whether public or private, has its own website. New online services are introduced regularly. Personal data of clients and employees, and business and financial information of the organization, are stored in electronic form.
Modern civilization is largely dependent on ICS/SCADA industrial process automation systems. Computer technology now controls the operation of nuclear power plants, hydroelectricity plants, oil and gas pipelines as well as transport systems at national and global levels.
Vulnerability Statistics for E-Banking Systems 2011-2012
This report provides a summary of research carried out by Positive Research, the research division of Positive Technologies, into vulnerabilities in e-banking systems. For the purposes of this regional study, only e-banking systems used in Russia were included.
Web Application Vulnerability Statistics 2012
Web applications have many advantages including the ability to update and maintain them centrally, their cross-platform nature and the fact that they are available to anyone on the web, 24/7. But, of course, it's not just genuine users who have access to these applications.

subset: commoncrawl


text:
|16 april 1992
|27 sept 1991
|1 okt 1990
|Ook gepubliceerd als
|George Y Wu, Catherine H Wu
|Univ Connecticut
|Citatie exporteren
|BiBTeX, EndNote, RefMan
|Niet-patentcitaties (6), Verwijzingen naar dit patent (201), Classificaties (23), Juridische gebeurtenissen (7)
|Externe links: Patentscope, Espacenet
Background of the Invention
Viruses represent a natural and efficient means for the introduction of foreign genes into cells.
For this reason, they are useful tools for the study of genes, and gene regulation in vitro and for gene therapy. However, most viruses have broad cell specificity and can infect a wide variety of cell types. This can lead to foreign gene expression in many tissues, some of which may be undesirable, especially for clinical applications.
Generally, viral infection is mediated by interactions between viral envelopes and plasma membranes of target cells. In many cases, specific viral structures are recognized and bound by cellular receptors. For example, HIV employs envelope glycoproteins to bind to helper T lymphocytes via CD4 (T4) receptors. Dalgleich, A.G., et al. Nature 312:763-767 (1984). These interactions have been shown to be responsible for the observed species and organ specificity.
Some investigators have shown that virus specificity can be redirected by attaching antibodies to viruses. For example, Goud, B., et aL. Viroloσv 161:251-254 (1988) linked anti-transferrin receptor antibodies to obtain delivery of a retrovirus to human cells bearing the transferrin receptor. However, while binding and internalization occurred, infection and replication did not. A means for targeting viral or other types of nucleic acid vectors containing foreign genes to a target cell and obtaining infection and replication of the virus would be useful in gene therapy.
Summary of the Invention The invention pertains to a method of targeting a virus or a cell to a target cell for selective internalization in vivo (or i vitro) by the cell and to modified viruses and cells which are targeted for selective internalization by a target cell. A virus or cell is targeted to the target cell for internalization by introducing a receptor- specific molecule onto the surface of the virus or cell to produce a modified virus or cell which specifically binds to a receptor on the surface of the target cell. The modified virus or cell can be administered to an organism where it binds selectively to the receptor of the target cell. The receptor-binding results in internalization by the target cell. The cellular receptor can be a receptor which mediates endocytosis of a bound ligand such as the asialoglycoprotein receptor of hepatocytes and the receptor-specific molecule can be a natural or synthetic ligand for the receptor. The receptor-specific molecule can be introduced onto the surface of the virus or cell (e.g., onto a viral envelope or cellular membrane) by chemically coupling it, either directly or through bridging agents, to the surface or by treating the surface to expose the molecule for receptor recognition.
The method of this invention can be used to produce viral or cellular vectors for selective delivery of material such as nucleic acid (genes) to a target cell. For example, exogenous genes can be incorporated and expressed selectively in a target cell. These vectors can be used in gene therapy and in other applications which call for selective genetic alteration of cells. The method also provides a means for altering the natural tropism of an infective agent such as a virus or bacterium. An infective agent can be modified so that it will infect a cell which, in unmodified form, it would not normally infect. In this way, animal models of human diseases which do not have adequate experimental animal counterparts can be developed for study of the diseases. For example, an ecotropic human pathogen (such as the hepatitis or AIDS virus) can be modified to infect a non-human host to produce an experimental system for study of the pathogen and the disease. Brief Description of the Figures
Figure 1 shows in situ β-galactosidase expression in NIH 3T3, HepG2 and SK Hepl cells treated separately with unmodified or modified murine leukemia virus.
Figure 2 shows internalization of 35S-biolabeled modified Moloney murine leukemia virus.
Figure 3 shows a chromatogram of asialooro- mucoid-complexed Psi2 virus on Sephadex G150.
Figure 4 shows the β-galactosidase activity of various cells exposed to Psi2 virus-asialoglyco- protein conjugate.
Detailed Description of the Invention A virus or cell is targeted for selective internalization into a target cell by modifying the surface of the virus or cell to introduce a molecule which specifically binds to a surface receptor of the target cell. The cellular surface receptor is one which will mediate internalization of the targeted virus or cell. The modified virus or cell binds to the receptor of the target cell in vivo and is internalized by the cell.
According to the method of this invention, viruses can be modified to infect specific target cells. Such modified viruses can be used to selectively deliver exogenous, functional DNA to a target cell in order confer a new biological or biochemical property upon the cell or to abrogate an existing property. In addition, the tropism of a virus can be altered or redirected to target infectivity to a cell type or types not normally infected by the virus in natural (or unaltered) form. A variety of different enveloped viruses can be targeted by the method of this invention. The viruses can be RNA (retroviruses) or DNA viruses (e.g., hepatitis virus, adenovirus). The virus can be replication defective or otherwise defective in structure or function. For example, viral particles either essentially or completely devoid of genomic nucleic acid (e.g., "empty" viral envelope) can also be targeted. The present method also provides a means of targeting cells. These include cellular organisms such as bacteria, protozoa or trypanosomes whose tropism can be altered. In addition, mammalian cells can be targeted. The receptor-specific molecule can be a ligand for the surface receptor of the target cell. Preferably, the molecule is a ligand for a cellular surface receptor which mediates internalization of the ligand by the process of endocytosis, such as the asialoglycoprotein receptor of hepatocytes.
Glycoproteins having certain exposed terminal carbohydrate groups can be used as receptor-specific molecules. For specific targeting to hepatocytes, asialoglycoprotein (galactose-terminal) ligands are preferred. Examples of asialoglycoproteins include asialoorosomucoid or asialofetuin. Other useful galactose-terminal carbohydrates for hepatocyte targeting include carbohydrate trees obtained from natural glycoproteins, especially tri- and tetra-antennary structures that either contain terminal galactose residues or can be enzymatically treated to expose terminal galactose residues. In addition, naturally occurring plant carbohydrates, such as arabinogalactan can be used. For targeting other receptors, other types of carbohydrates can be used. For example, mannose and mannose-6 phosphate or carbohydrates having these terminal carbohydrate structures could used to target macrophages or endothelial cells.
Other receptor ligands such as peptide hormones could also be used to target viruses or cells to corresponding receptors. These include insulin, glucagon, gastrin polypeptides and their respective receptors.
Alternatively, the receptor-specific molecule can be a receptor or receptor-like molecule, such as an antibody, which binds a ligand (e.g., antigen) on the cell surface. Antibodies specific for cellular surface receptors can be produced by standard procedures.
The receptor-specific molecule is introduced onto the surface of the virus or cell so that it will be recognized by the cognate cellular surface receptor. For example, the receptor-specific molecule can be introduced onto the envelope of a virus or the membrane of a cell. In general, the molecule will be coupled to (or exposed on) a proteinaceous component of the surface but other components may be used.
The receptor-specific molecule can be introduced onto the surface of the virus or cell by different means. Preferably, the receptor-specific molecule is chemically coupled to the surface. For example, galactose moieties (ligand for the asialoglycoprotein receptor) can be covalently coupled to viral or cellular surface proteins by lactosamination, reductive amination, or via iminomethoxyethyl derivatives. In other embodiments, the receptor-specific molecule can be chemically coupled to components of the surface of the virus or cell through bridging agents such as biotin and avidin. For instance, a biotinylated receptor-specific molecule can be linked through avidin or streptavidin to a biotinylated surface component of the virus or cell.
Alternatively, the virus or cell can be chemically treated to expose a receptor-specific molecule on the surface. Surface polycarbohydrates can be enzymatically cleaved to expose desired carbohydrate residues (e.g., galactose residues) as terminal residues for specific receptor recognition and binding. For example, neurominidase treatment of certain polycarbohydrates leaves exposed terminal galactose residues in a tri- or tetra-antennary arrangement.
The modified virus or cell is administered in vivo, generally in an amount sufficient to saturate receptors of the target cell and thereby maximize uptake by the cell. They can be administered parenterally (typically intravenously) in a physiologically acceptable vehicle such as normal saline.
The method of this invention can be used to selectively deliver nucleic acid (DNA or RNA) to a target cell in vivo (or in vitro) so that it is expressed in the cell. The nucleic acid can be an exogenous gene, a genetic regulatory element or an antisense inhibitor of gene function. The nucleic acid is incorporated into a viral vector which has been modified, according to the method of this invention, to target it to the cell. Preferred viral vectors for delivery of foreign genes in vivo (or ex vivo) are retroviruses. The targeted viral vector is administered in vivo, as described, where i is selectively taken up by the target cell.
The method of this invention can be used to alter the natural tropism of an infectious agent. Ecotropic (species-restricted) agents can be made to infect species which they normally, in unmodified form, do not infect. The ability to target the infectivity of an infectious agent can be used to develop new experimental systems for the study of human infectious diseases to produce cells that can correct genetic defects in vivo, or target a corrective gene in vivo.
Certain pathogenic viruses such as hepatitis virus or human immunodeficiency virus infect only human cells. By the method of this invention, such viruses can be modified to enable them to infect experimental animals such as rodents. For example, the hepatitis virus which infects only human liver cells, can be modified so that it will infect non-human liver cells. To develop rodent models of hepatitis, for example, a ligand for rodent asialoglycoprotein receptor (e.g., galactose) can be introduced onto the surface of the hepatitis virus. This yields a modified hepatitis virus which will infect rodent liver cells. This modified hepatitis virus which can infect a rodent and the infected rodent or rodent cells, provides an experimental animal system for study of the hepatitis virus. The invention is illustrated further by the following examples.
Chemical Modification and Alteration of Host Cell Specificity of a Retrovirus
A model retroviral system was used. The virus, an ecotropic, replication-defective, Moloney murine leukemia virus containing the gene for bacterial β-galactosidase produced in a ψ ere cell line was kindly provided by Dr. James Wilson, University of Michigan. Wilson, J.M., e_Ł al. Proc. Natl. Acad. Sci. USA 87:439-443 (1990). Under normal circumstances, this virus infects only rodent cells. Wilson, J.M. , e_Ł al. Proc. Natl. Acad. Sci. USA Ł5_:3014-3018 (1988); Goud, B., et al. Virology JL__3.:251-254 (1988). The producer cells were grown in Dulbecco's modified Eagle's medium (GIBCO Laboratories, Grand Island, NY) supplemented with 10% heat-inactivated calf serum (GIBCO) . To prepare virus with as little contamination as possible from serum proteins, producer cells were cultured in serum-free Dulbecco's modified Eagle's medium for 3 days. Using this viral preparation, two strategies were developed for the modification of the surface of the harvested virus: A) chemical coupling of galactose residues to the virus and B) chemical coupling of an asialoglycoprotein to the virus. A. LACTOSAMINATION OF RETROVIRUSES
Virus was isolated from the culture medium according to the method of Goud, B., eŁ al. Virology 163:251-254 (1988), but modified to permit coupling of lactose during the isolation procedure. In brief, virus-containing medium was applied on a 10-20% sugar gradient in which α-lactose was substituted for sucrose (Sigma, St. Louis, MO) in 10 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, and was ultracentrifuged (LB-55, Beckman Instruments, San Ramon, CA) at 40,000 rpm in VTi 55 rotor (Beckman) at 4°C for 17 hours. Samples were adjusted to various pHs, 7.4-8.4, prior to centrifugation in order to determine optimal conditions for modification. After centrifugation, the bottom fraction of the lactose gradient containing 3.0 mg of protein (0.1 mg viral RNA) was reacted with sodium cyanoborohydride (Sigma) as described previously. Goud, B., e_Ł al. Virology 163:251-254 (1988). Following dialysis against minimum essential medium at 4°C for 24 hours, the samples were sterilized by passage through 0.45μ filters (Gelman Science Co., Ann Arbor, MI). Quantitation of the amount of virus present in samples prior to exposure to cells was determined by protein assay (Bio-Rad, Los Angeles, CA) according to the manufacturer's instructions, and confirmed by RNA assay (Nunro, H.N. and Fleck, A. Meth. Biochem. Anal. 14.:113-176 (1966)) after RNA extraction. ° Chomczynski, P. and Sacci, N. , Anal. Biochem.
162:156-159 (1987). Fetal bovine serum (GIBCO) was added subsequently to make a 10% solution. Except for stability experiments, all samples were used immediately after preparation. Viability of unmodified virus preparations was determined by transfection assays in NIH 3T3 mouse fibroblasts using limiting dilutions of viral stock (Danos, 0. and Mulligan, R.C. Proc. Natl. Acad. Sci. USA &_>:6460-6464 (1988)) and quantitated by determination of positive cells stained with X-gal. Sanes, J.R., et al. EMBO J. 5:3133-3142 (1986).
For uptake studies, virus was biosynthetically labeled by incubation of producer cells (5.0 x 106 cells) in 50% serum-free and 50% serum- and methionine-free Dulbecco's modified Eagle's medium containing 10 μCi/ml 35s_methionine (Amersham, Arlington Heights, IL) for 3 days. Virus was isolated from supernatants and modified as described above followed by dialysis against minimum essential medium.
Cells and Cell Culture
To evaluate the effects of chemical modification on viral infection specificity, several cell lines were employed: human hepatoma cell lines, HepG2, asialoglycoprotein receptor (+) (Schwartz, A.L., e± al. J. Biol. Chem. 25.6:8878-8881 (1981)) obtained from B.B. Knowles, Wistar Institute, Philadelphia, PA; and SK Hepl, receptor (-) from D.A. Shafritz, Albert Einstein College, of Medicine, Bronx, NY; a rat hepatoma cell line, Morris 7777, receptor (-) (Wu. G.Y., ej al. J. Biol. Chem. 263: 4719-4723 (1988)); and a murine fibroblast cell line NIH 3T3 (Goud, B., et aj___. Virology 163.:251-254 (1988)) which is also asialoglycoprotein receptor (-) . The latter two cell lines were purchased from American Type Culture Collection (Rockville, MD) . All were maintained in Eagle's minimum essential medium supplemented with 10% heat inactivated fetal bovine serum at 37°C under 5% Cθ2-
Assays for Viral Infection and Functional Gene Expression
In order to determine whether virus remained infectious and functional after chemical modification, the two human and two rodent cell lines were exposed to modified and unmodified virus followed by measurement of cellular β-galactosidase activity. Target cells were plated at a density of 0.5-2.0 x 105 cells/ml in 60 mm plastic dishes (Falcon Scientific Co, Lincoln Park, NJ) . Equal amounts, 16.7 μg RNA, (0.5 mg viral protein) of modified and unmodified virus, in Dulbecco's modified Eagle's medium were added to the culture medium and exposed to cells for 5 days at 37°C under 5% CO2. Cells were assayed for β-galactosidase activity as a measure of foreign gene expression according to the method of Gorman (Gorman, C. DNA Cloning, vol. 2 eds, Glover, D.M. IRL Press, Washington D.C. pp 157-158 (1986)). In brief, cell monolayers (approximately lxlO6 cells/60 mm dish) were washed with phosphate buffered saline, then lysed. The lysate, 0.1 ml, was reacted with o-nitrophenyl-galactopyranoside (ONPG, Sigma) and β-galactosidase activity quantitated by absorbance at 420 nm after addition of a Cθ3 to terminate the reaction. Results were expressed in U/mg of cellular protein according to the method by Norton, P.A. and Coffin, J.M. Mol. Cell. Biol. 1:281-290 (1985), using purified E___ coli β-galactosidase (Sigma) activity as a standard. Protein concentrations of the cellular samples were determined using a Bio-Rad Protein Assay Kit (Bio-Rad) following the manufacturer's instructions. For competition experiments, virus was added to the cell media together with a 100-fold molar excess of a natural asialoglycoprotein, asialoorosomucoid, prepared by desialylation (Oka, J.A., and Weigel, P.H. J. Biol. Chem. 258: 10253-10262 (1983)) of orosomucoid as previously described by Whitehead., D.H., and Sam ons, H.G. Biochim. Biophys. Acta 124:209-211 (1966). Background enzyme activity was determined in corresponding untreated cells and subtracted from the values of viral-treated samples. All assays were performed in triplicate and the results expressed as means + S.E. Table 1 shows that unmodified virus did not produce enzymatic activity in human HepG2 or SK Hepl cells as expected from the ecotropism of the virus. Also, modified virus did not produce β-galactosidase activity in SK Hepl, asialoglycoprotein receptor (-) cells. However, modified virus did produce high β-galactosidase activity, 71.2 ± 4.8U/mg of cellular protein, in human HepG2, asialoglycoprotein receptor (+) cells. Furthermore, this enzymatic activity was completely suppressed by addition of a large molar excess of asialoorosomucoid, supporting the notion that the transfection by modified virus was, in fact, mediated by asialoglycoprotein receptors. As expected from the ecotropism, β-galactosidase activity was high, 50.6 ± 5.2. in Morris 7777 rat cells after exposure to unmodified virus. Interestingly, β-galactosidase activity in these same cells was significantly lower when exposed to the same amount of modified virus. The same tendency was seen in originally susceptible murine NIH 3T3 cell as enzymatic activity after exposure to unmodified virus, 56.7 + 1.8, was more than double that following exposure to modified virus 27.0 ± 0.9. The coupling reaction linking lactose to protein has been shown to be enhanced under alkaline conditions. Schwartz, B.A. and Gray, G.R. Arch. Biochem. Biophys. 181:542-549 (1977). However, such conditions could be detrimental to the virus. To determine the optimal pH that results in modified, yet functional vectors, virus modified at different pHs were administered to HeρG2 cells, and β-galactosidase activity measured. Table 2 shows that enzymatic activity rose from 50.3 + 1.2, for virus modified at pH 7.4; to 71.2 + 4.8, for virus modified at pH 8.0. However, activity was significantly lower, 25.1 + 2.4, in cells treated with virus modified at pH 8.4.
Table 1
Cellular β-Galactosidase Activity Following Exposure to Viral Preparations-i-
β-Galactosidase Activity* Mean ± S.E. (U/mg)
AsG Unmodified Modified Modified Receptor Virus Virus Virus + Status ASOR**
Cell Line (Source) HepG2
(human) (+) 1.8 + 1.9 71.2 ± 4.8 2.9 + 1.1
SK Hepl
(human) (-) 1.7 ± 3.4 0.8 ± 4.6 1.6 ± 2.5
Morris 7777 (rat) (-) 50.6 ± 5.2 16.3 ± 4.4 15.7 ± 4.7
(mouse) (-) 52.1 + 4.9 15.4 + 1.1 16.3 + 3.9
+ virus was modified at pH 8.0 then incubated with cells for 5 days. * calculated as the difference in activity between treated and untreated cells.
** asialoorosomucoid (ASOR) in 100-fold molar excess.
AsG, Asialoglycoprotein Table 2
Effect of the pH During Modification of Viral Transfection in HepG2 Cells
Specific β-Galactosidase Activity (Mean ± S.E. U/mg protein)*
Modified Virus Modified Virus
+ Asialoorosomucoid** S ■
7.0 50.3 ± 1.2 6.4 ± 1.9
8.0 71.8 ± 4.1 4.9 ± 0.4 8.4 25.1 ± 2.4 0.0 ± 1.6
* after 5 days of exposure to modified virus.
** β-galactosidase activity of samples treated with modified virus plus a 100-fold molar excess of asialoorosomucoid.
Histochemical Staining to Demonstrate β-Galactosidase Activity
To confirm the colorimetric results, and to determine the fraction of cells that expressed the β-galactosidase gene after exposure to viral samples, histochemical staining of in situ β-galactosidase activity was performed according to the method of Sanes e_t al. EMBO J. 1:3133-3142 (1986). In brief, cultured cells in 35 mm dishes containing 0.5 -1 x 10*^ cells treated for 5 days with equal amounts, 8.4 μg of viral RNA (0.3 mg viral protein), of modified or unmodified virus. Cells were fixed in 0.5% glutaraldehyde (Sigma), phosphate buffered saline, then incubated with 1 mM MgCl2/ phosphate buffered saline, and overlaid with lmg/ml 4-Cl-5-Br-3-indoylyl-β-galactosidase (X-gal) (BRL, Washington, D.C.), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 2 mM MgCl2 in phosphate buffered saline. After incubation at 37°C for 1 hour, the dishes were washed in phosphate buffered saline to quench the reaction and evaluated by counting positive (blue) cells under a light microscope and the results expressed as the percent of positive/10 high power fields.
In situ staining for β-galactosidase activity in cells treated with various viral preparations is shown in Figure 1. In rodent NIH 3T3 cells treated with unmodified virus, panel B, 12.6% were positive for β-galactosidase activity using the X-gal stain. Background staining in untreated NIH 3T3 cells was not detectable, panel A. After exposure to modified virus, only 3.6% were positive under otherwise identical conditions, panel C. Human SK Hepl cells that were untreated, panel H, or exposed to either unmodified virus, panel I, or modified virus, panel J, failed to develop detectable staining. Similarly, HepG2, asialoglycoprotein receptor (+) cells treated with unmodified virus, panel E, did not develop evidence of significant β-galactosidase activity. However, HepG2, receptor (+) cells treated with modified virus, panel F, did develop substantial staining. Microscopic counting revealed that 36.4 % of the HepG2, cells possessed detectable marker enzyme. The observed color development was completely suppressed by addition of a 100-fold molar excess asialoorosomucoid to compete for uptake by asialoglycoprotein receptors, panel G, indicating involvement of asialoglycoprotein receptors in the transfection process.
Assays for Cellular Uptake of Virus
To determine whether the modified virus was actually taken up by cells and, if so, whether asialoglycoprotein receptors were involved, HepG2, SK Hepl and Morris 7777 cells, 5.0x10s cells/35 mm dish, were incubated at 37°C in serum-free Dulbecco's modified Eagle's medium containing 35s-biolabeled, modified virus, 3.3 μg viral RNA (98 μg viral protein) (Watanabe, N., eŁ al. Cancer Immunol. Immunother. 28:157-163 (1989)) with a specific activity of 6.1x10^ cpm/mg viral RNA. At various times, medium was removed, and cells were chilled to 4°C, washed with ice-cold minimum essential medium containing lmg/ml bovine serum albumin. Surface-bound radioactivity was stripped with cold 0.5 ml phosphate buffered saline, pH 7.2 containing 0.4% trypsin, 0.02% EDTA and separated from cells by centrifugation. The cell pellet was solubilized in 0.2 N NaOH and Poly-Fluor (Packard, Chicago, IL) , and trypsin-EDTA resistant
(internalized) radioactivity was measured by scintillation counting (TRI-CARB 4530, Packard). Schwartz, A.L., ej al. J. Biol. Chem. 256:8878-8881 (1981). Non-specific uptake was measured in the presence of a 100-fold molar excess of asialoorosomucoid, and specific uptake calculated as the difference between total and non-specific measurements. All assays were performed in triplicate and the results expressed as means ± S.E. in terms of ng viral RNA/10^ cells as a function of time.
Figure 2 shows that, of the two human and one rodent cell lines, only the human HepG2 asialoglycoprotein receptor (+) cells demonstrated significant specific uptake of labeled virus.
Counts resistant to EDTA and trypsin, increased as a function of time and continued to rise linearly through 120 minutes of incubation at a rate of approximately 800 ng viral protein/hr/105 cells. These data further support the notion that the observed expression of the galactosidase gene by modified virus was in fact due to internalization of the virus by asialoglycoprotein receptors.
Stability of Modified Virus To assess the stability of modified virus, samples of freshly prepared sterile, modified virus were incubated in serum-free Dulbecco's modified Eagle's medium at 4°C and 25°C. At various times, samples were added to the medium of HepG2 cells and incubated for 5 days. Cells were then assayed for β-galactosidase activity by colorimetric assay as described above. All assays were performed in triplicate and the results expressed as means ± S.E, in terms of U/mg cell protein normalized for the amount of virus added as a function of time of incubation. Table 3 shows that enzymatic activity at both 4 °C and 25°C, decreased with time to approximately 50% of original activity after 48 hours.
Table 3
S abilit of Mo ifi Vir
* after 5 days of exposure to modified virus
Specific β-galactosidase activity was calculated as the difference between samples treated with virus alone, and samples treated with modified virus plus a 100-fold molar excess of asialoorosomucoid. The coupling of lactose to proteins to target artificial asialoglycoproteins is based on the specificity of sodium cyanoborohydride to reduce Schiff's bases formed between aldehyde and amino groups to render the bonds irreversible. Treatment of viruses with aldehydes is not always similarly benign. For example formaldehyde has been used to inactivate viruses in the production of vaccines. Buynak, E.B., et al. J. Am. Med. Assoc. 235: 2832-2834 (1976). The data presented here indicate that under the conditions described, the modification process results not only in altered specificity of infection, but also results in preservation of viral gene expression. Furthermore, the data indicate that the production of modified yet functional virus increased with increasing pH of the modification reaction up to a limit of approximately 8.0, beyond which the function of the virus became compromised. Many retroviruses have been shown to enter cells normally via endocytosis and are thought to introduce their genetic material during an acidification step in the pathway. Andersen, K.B. and Nexo, P.A. Virology 125:85-98 (1983). Although the asialoglycoprotein endocytotic pathway is ultimately degradative with delivery of ligands to lysosomes (Tolleshaug, H., ei al. Biochim. Biophvs. Acta 585:71-84 (1979)), early in the internalization process, endosomal endocytotic compartments are acidified prior to fusion with lysosomes. Tycko, B. and Maxfield, R.F. Cell 28:643-651 (1982). This period of acid exposure may be analogous to the natural route of entry for some viruses (Nussbaum, 0., and Loyter, A. FEBS Lett. 221:61-67 (1987)) and may provide the requisite conditions for acid- mediated fusion of the viral envelope of endosomal membrane prior to destruction of the virus.
Helenius, A. Biol. Cell 51:181-186 (1984). The fact that modified virus is still able to introduce its genome into target cells suggests that the process of chemical modification did not abolish the function of those elements of the virus.
Crude preparations of virus obtained by low-speed centrifugation of medium from producer BAG cells followed by high-speed centrifugation through a discontinuous sucrose gradient as described previously was dialyzed against 0.9% saline, pH 7.5, at 4°C. After dialysis, NHS-LC-biotin (Pierce Chemical Co., Rockville, IL) was reacted with the virus (0.1 mg/ml of virus) at room temperature for four hours. The sample was then dialyzed against 0.9% saline, pH 7.5 at 4°C. Asialoorosomucoid (AsOR) was obtained by desialylation of serum orosomucoid originally derived from pooled human serum. Whitehead, D.H. and Sammons, H.P. Biochem. Biophvs. Acta 124:209 (1966). AsOR 0.1 mg was added to 1.0 mg of virus, thoroughly mixed and then 1.0 mg of avidin per mg of virus was added and allowed to incubate at room temperature for four hours. The complex was then dialyzed against Modified Eagle's Medium. Complexed virus was purified on a Sephadex G150 molecular sieve column. To determine conditions for purification, a viral complex was prepared in which asialoorosomucoid was labeled with 125I. Figure 3 shows that asialoorosomucoid alone was eluted from the column beginning at fraction number 32. Avidin, as detected by its optical density at 280 nm, eluted slightly later beginning at tube 33. However, unlabeled virus alone was much larger than either of the other two proteins and was eluted earlier with a peak at tube 29. The column was able to completely resolve virus from asialoorosomucoid and avidin. Figure 3 also shows that this virus complexed with l2^I-labeled AsOR mediated by biotin-avidin bonds, the radioactivity from the AsOR moved to the same position as expected for the intact virus, namely with a peak at tube 29. These data indicate that some labeled asialoorosomucoid was bound by the virus and migrated with it through the column.
In order to determine whether this complex could be used to target gene expression specifically to asialoglycoprotein receptor (+) cells, conjugated virus was incubated for 10 days with each of five cell lines: Hep G2, receptor (+); Huh-7, receptor (+); SK Hepl, human hepatoma receptor (-); Mahlavu, receptor (-) and Morris 7777, rat hepatoma receptor (-) cells. Figure 4, lane 1 shows that Hep G2 receptor (+) cells treated with conjugate had beta-galactosidase activity at a level of 2.3 units/mg of cell protein which is approximately 50% of the activity of the producer cell line, BAG shown in lane 11. Hep G2 cells without treatment were at a level of 1.81 units/mg. Huh-7 receptor (+) cells treated with conjugate had higher levels of beta-galactosidase, 3.8 units/mg as shown in lane 3 compared to those cells treated with biotinylated virus without asialoorosomucoid present in a complex shown in lane 4. This was similar to the levels obtained from these cells that were not treated at all as seen in lane 5. Lane 6 shows that Mahlavu receptor (-) cells treated with conjugate did not have any significant beta-galactosidase activity compared to those same cells that were untreated shown in lane 7. Similarly lanes 8 and 9 show that Morris 7777 cells treated with other conjugate or biotinylated virus without asialoorosomucoid, lanes 8 and 9 respectively, showed no significant beta-galactosidase activity compared to those same cells that were untreated shown in lane 10. SK HEPL cells responded similarly to the receptor (-) Morris 7777 cells.
In the staining procedure described in Example 1, Hep G2 cells treated with the conjugated virus produced a bluish coloration as did the Huh-7 cells treated similarly. However, cells that did not receive treatment had no staining.
Chemical Modification and Alteration of Host Cell Specificity of Hepatitis B Virus (HBV) Hepatitis B virus is a human pathogen that possesses very narrow host (species) and organ (liver) specificities, in vitro, the virus is also very fastidious as evidenced by the fact that human hepatocytes or hepatoma cells in culture cannot be infected by HBV without unusual and highly artificial conditions such as high concentrations of corticosteroids. Cells and Cell Culture
Hepatitis B virus (HBV) was obtained from Hep G2 producer cells chronically infected with HBV as described by Sells et. ai. Proc. Natl. Acad. Sci. :1005-1009 (1987), and maintained in Dulbecco's modified Eagle's medium (MEM) containing G418 as 380 mg/ml, supplemented with 10% heat inactivated fetal bovine serum. To test the infectivity and specificity of unmodified and modified HBV, two human cell lines were cultured. Huh7 human hepatoma cell line which possesses asialoglycoprotein receptors and IMR-90 fibroblasts which do not possess asialoglycoprotein receptors were maintained in Dulbecco's modified Eagle's minimum essential medium supplemented with 10% fetal bovine serum (FBS) .
Isolation of HBV
HeρG2 cells were cultured in serum free media for three days. The medium was centrifuged at 2000 rpm to remove debris and the supernatant applied on 10-20% lactose gradient, pH 7.4, 8.0 or 8.4, and ultracentrifuged at 40000 rpm in VTi55 rotor at 4°C for 16 hours to pellet and isolate the virus.
Chemical Modification of HBV HBV obtained (3.0 mg of protein) was lactosaminated in a similar fashion to that described in Example 1 using 10 mg of sodium cyanoborohydride for 3 hours at 25°C. The modified virus was sterilized by filtration through 0.45 μm membranes and then dialyzed against MEM through membranes with a 12-14000 molecular weight exclusion limit followed by dialysis against MEM plus 10% FBS. Infection of Cells with Unmodified and Modified HBV
Huh7 and IMR-90 cells were plated at 25-50% confluence in 35 or 100 mm diameter plastic dishes. Cell medium was removed and replaced with medium containing modified or unmodified virus and incubated at 37°C. Cells were washed and changed to fresh medium every three days and at regular intervals cells were studied for the presence of HBV DNA and medium analyzed for the presence of hepatitis B surface antigen (HBsAg) .
Detection of Targeted HBV DNA in Huh7 Cells Treated with Modified and Unmodified HBV
DNA was extracted from cells according to the method by Blin, N. and Stafford, D.W. Nucleic Acid Res. 2:2303-2312 (1976), in which the cells were washed twice with 10 ml of cold Tris-buffered saline (TBS), scraped off into TBS and centrifuged at 200 rpm. The cell pellet was resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, was added to the same buffer containing 20 mg/ml RNase, 0.5% SDS, and then treated with proteinase K. Cellular DNA was isolated by ethanol precipitation after phenol extraction. The DNA was analyzed by Southern blot using a γ32P-ATP labeled cDNA probe specific for HBV sequences (a Bam HI restriction fragment of plasmid adw HTD carrying the HBV genome, obtained from Dr. Jake Liang, Massachusetts General Hospital).
The Southern blot showed no hybridizable sequences when probed with our cDNA probe specific for HBV. This confirms the previous finding that Huh7 cells, even though of human origin, cannot be infected by unmodified HBV under the conditions of routine cell culture. In addition, the data indicate that the washing procedures eliminate any detectable non-specifically bound HBV DNA on these cells. However, treatment of the Huh7 cells with modified HBV for as little as one day of incubation resulted in a strong signal of hybridizable bands on the Southern blot corresponding to those expected for the plasmid sequences. IMR-90, asialoglycoprotein (-) cells did not produce hybridizable sequences under any conditions.
Detection of HBsAg in the Supernatant of Huh7 and IMR-90 Cells Exposed to Unmodified or Modified HBV
Medium from Huh7 and IMR-90 cells was incubated with modified or unmodified HBV as described above and at various intervals was assayed for HBsAg by an enzyme i munoassay kit (Auszyme Monoclonal) . The conditions were those recommended by the manufacturer, Abbott Labs.
As shown in Table 4, the background color absorbance was approximately 0.121 in untreated Huh7 cells and there was no significant difference between day 1 and day 7. Unmodified HBV did not result in significant production of HBsAg. Absorbance here was approximately 0.180. Similarly, the color absorbance reflecting HBV levels in IMR-90 cells did not exceed 0.110. However, Huh7 cells treated with modified HBV released HBsAg into their supernatants, with absorbance ranging from 0.760 to 0.865. Table 4
Levels of Hepatitis B Surface Antigen (HBsAg) in
Culture Medium as Determined by Auszyme Assay
(Absorbance Units)
IMR-90 Huh7
Day Modified Untreated Unmodified Modified HBV HBV HBV
1 .121 ± .054 .135 ± .017 .850 ± .010 3 .186 + .036 .700 ± .012 5 .171 ± .010 .865 ± .053 7 .110 + .023 .764 + .067
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.
|J. Gen. Virol., Vol. 68, issued 1987, GRUNDY, et al., "B2 Microglobulin Enhances the Infectivity of Cytomegalovirus and when Bound to the Virus Enables Class I HLA Molecules To Be Used as a Virus Receptor", pages 793-803, see figures 1-8, tables 1-2, pages 795-799.
|Proc. Natl. Acad. Sci. USA, Vol. 86, issued December 1989, ROUX et al., "A versatile and potentially general approach to the targeting of specific cell types by retroviruses: Application to the infection of human cells by means of major histocompatibility complex class I and class II antigens by mouse ecotropic murine leukemia virus-derived viruses", pages 979-982, see figures 1-2, pages 9080-9081.
|See also references of EP0553235A1
|The Journal of Biological Chemistry, Vol. 263, No. 29, issued 15 October 1988, WU et al., "Receptor - mediated Gene Delivery and Expression in Vivo", pages 14621-14624, see the entire document.
|Virology, Vol. 163, issued 1988, KOMAI et al., "The Vero Cell Receptor for the Hepatitis B Virus Small S Protein is a Sialoglycoprotein", pages 629-634, see figures 1-2, tables 1-3, pages 630-631 and 633.
|Virology, Vol. 172, issued 1989, PUGH et al., "Infection and Uptake of Duck Hepatitis B Virus by Duck Hepatocytes Maintained in the Presence of Dimethyl Sulfoxide", pages 564-572, see figures 1-8, pages 565-571.
|Citerend patent
|28 okt 1992
|13 mei 1993
|Thera Gene Hb
|Targeted delivery of virus vector to mammalian cells
|28 april 1993
|11 nov 1993
|Frank Andreas Harald Meyer
|Medicament for the gene-therapeutic treatment of human beings, animals and plants, especially to block virus multiplication and carcinogenes and process for producing the medicament
|25 juni 1993
|6 jan 1994
|British Tech Group
|Protein based delivery system
|23 sept 1993
|31 maart 1994
|Univ Connecticut
|Modification of a virus to redirect infectivity and enhance targeted delivery of polynucleotides to cells
|4 nov 1993
|11 mei 1994
|Imp Cancer Res Tech
|Virus with modified binding moiety specific for the target cells
|6 april 1994
|27 okt 1994
|Boehringer Ingelheim Int
|Adenovirus for the transfer of foreign dna into higher eucaryotic cells
|15 mei 1995
|23 nov 1995
|Viagene Inc
|Compositions and methods for targeting gene delivery vehicles
|7 april 1999
|14 okt 1999
|Corixa Corp
|Fusion proteins of mycobacterium tuberculosis antigens and their uses
|19 nov 1999
|25 mei 2000
|Univ Georgetown
|Systemic viral/ligand gene delivery system and gene therapy
|29 maart 2002
|10 okt 2002
|Avigenics Inc
|Avian lysozyme promoter
|2 april 2003
|16 okt 2003
|Arizeke Pharmaceuticals Inc
|Compositions and methods for targeted biological delivery of molecular carriers
|28 mei 2004
|6 jan 2005
|Svetlana Doronina
|Recombinant anti-cd30 antibodies and uses thereof
|4 okt 2004
|14 april 2005
|Vib Vzw
|Means and methods for the recruitment and identification of stem cells
|18 april 2005
|5 jan 2006
|Acuity Pharmaceuticals Inc
|Compositions and methods for inhibiting angiogenesis
|1 aug 2005
|4 mei 2006
|Sinai School Medicine
|Klf6 alternative splice forms and a germline klf6 dna polymorphism associated with increased cancer risk
|22 dec 2005
|29 juni 2006
|Auckland Uniservices Ltd
|Trefoil factors and methods of treating proliferation disorders using same
|25 jan 2006
|3 aug 2006
|Prolexys Pharmaceuticals Inc
|Quinoxaline derivatives as antitumor agents
|23 feb 2006
|31 aug 2006
|Halozyme Therapeutics Inc
|Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases
|17 maart 2006
|28 sept 2006
|Medimmune Inc
|Framework-shuffling of antibodies
|29 juni 2007
|4 sept 2008
|Squibb Bristol Myers Co
|Polynucleotides encoding novel pcsk9 variants
|25 juni 2008
|31 dec 2008
|Auckland Uniservices Ltd
|Polypeptides and polynucleotides for artemin and related ligands, and methods of use thereof
|25 maart 2009
|8 okt 2009
|Macrogenics, Inc.
|Her2/neu-specific antibodies and methods of using same
|25 maart 2009
|17 dec 2009
|Macrogenics, Inc.
|Bcr-complex-specific antibodies and methods of using same
|7 sept 2008
|11 maart 2010
|Glyconex Inc.
|Anti-extended type i glycosphingolipid antibody, derivatives thereof and use
|18 dec 2009
|1 juli 2010
|Universität Regensburg
|Norrin in the treatment of diseases associated with an increased tgf-beta activity
|16 feb 2010
|26 aug 2010
|Carnegie Mellon University
|Quenched dendrimeric dyes for bright detection
|28 mei 2010
|9 dec 2010
|Medimmune, Llc
|Molecules with extended half-lives and uses thereof
|13 aug 2010
|17 feb 2011
|Calmune Corporation
|Antibodies against human respiratory syncytial virus (rsv) and methods of use
|17 sept 2010
|24 maart 2011
|Calmune Corporation
|Antibodies against candida, collections thereof and methods of use
|8 nov 2010
|12 mei 2011
|Idexx Laboratories, Inc.
|Canine anti-cd20 antibodies
|25 mei 2011
|1 dec 2011
|Carnegie Mellon University
|Targeted probes of cellular physiology
|8 juli 2011
|12 jan 2012
|Calmune Corporation
|Anti-human respiratory syncytial virus (rsv) antibodies and methods of use
|16 mei 2012
|29 nov 2012
|Macrogenics, Inc.
|Deimmunized serum-binding domains and their use for extending serum half-life
|2 juli 2013
|9 jan 2014
|Medizinische Universität Wien
|Complement split product c4d for the treatment of inflammatory conditions
|18 nov 1993
|20 sept 1995
|University Of Medicine And Dentistry Of New Jersey
|Cell-type specific gene transfer using retroviral vectors containing antibody-envelope fusion proteins
|3 nov 1993
|11 dec 1996
|UNITED STATES GOVERNMENT as represented by THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES
|Targetable vector particles
|4 nov 1993
|27 sept 2000
|Transgene S.A.
|Virus with modified binding moiety specific for the target cells
|12 april 2001
|17 okt 2001
|Transgene S.A.
|Poxvirus with targeted infection specificity
|12 april 2001
|23 maart 2005
|Transgene S.A.
|Poxvirus with targeted infection specificity
|24 aug 2001
|10 sept 2008
|Pfizer Products Inc.
|Anti-IgE vaccines
|24 aug 2001
|10 sept 2008
|Pfizer Products Inc.
|Anti-IgE vaccines
|11 dec 2000
|8 okt 2008
|Invitrogen Corporation
|Use of multiple recombination sites with unique specificity in recombinational cloning
|26 nov 2002
|14 jan 2009
|Protein-protein interactions in human immunodeficiency virus
|28 nov 2001
|25 feb 2009
|Medimmune, Inc.
|Methods of administering/dosing anti-rsv antibodies for prophylaxis and treatment
|24 aug 2001
|3 juni 2009
|Pfizer Products Inc.
|Anti-IgE vaccines
|8 juli 1998
|7 okt 2009
|CANJI, Inc.
|Compositions and kits for enhancing delivery of therapeutic agents to cells
|9 aug 2004
|2 dec 2009
|Genenews Inc.
|Osteoarthritis biomarkers and uses thereof
|5 maart 2004
|17 maart 2010
|Halozyme, Inc.
|Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof
|5 maart 2004
|21 april 2010
|Halozyme, Inc.
|Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof
|11 dec 2000
|28 juli 2010
|Life Technologies Corporation
|Use of multiple recombination sites with unique specificity in recombinational cloning
|15 dec 2003
|18 aug 2010
|Halozyme, Inc.
|Human chondroitinase glycoprotein (chasegp), process for preparing the same, and pharmaceutical compositions comprising thereof
|12 april 2002
|15 sept 2010
|Human Genome Sciences, Inc.
|Antibodies against vascular endothelial growth factor 2
|18 aug 2004
|12 jan 2011
|MedImmune, LLC
|Humanisation of antibodies
|14 juni 2004
|23 maart 2011
|University Of Medicine And Dentistry Of New Jersey
|Recombinant protein production in the presence of mRNA interferase
|15 dec 2003
|23 maart 2011
|Halozyme, Inc.
|Human chondroitinase glycoprotein (CHASEGP), process for preparing the same, and pharmaceutical compositions comprising thereof
|14 juni 2004
|30 maart 2011
|University Of Medicine And Dentistry Of New Jersey
|Medical use of mRNA interferase
|13 feb 2003
|30 maart 2011
|American Diagnostica Inc.
|Methods for selecting treatment regimens and predicting outcomes in cancer patients
|29 maart 1999
|13 april 2011
|NorthWest Biotherapeutics, Inc.
|Therapeutic and diagnostic applications based on the role of the CXCR-4 and SDF-1 genes in tumourigenesis
|5 maart 2004
|20 april 2011
|Halozyme, Inc.
|Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof
|23 okt 2006
|11 mei 2011
|GeneNews Inc.
|Method and apparatus for correlating levels of biomarker products with disease
|5 maart 2004
|8 juni 2011
|Halozyme, Inc.
|Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof
|28 nov 2001
|29 juni 2011
|MedImmune, LLC
|Methods of administering/dosing anti-RSV antibodies for prophylaxis and treatment
|12 dec 2001
|6 juli 2011
|MedImmune, LLC
|Molecules with extended half-lives, compositions and uses thereof
|23 dec 2004
|3 aug 2011
|Genentech, Inc.
|Novel anti-IL 13 antibodies and uses thereof
|12 dec 2001
|10 aug 2011
|MedImmune, LLC
|Molecules with extended half-lives, compositions and uses thereof
|13 feb 2003
|24 aug 2011
|American Diagnostica Inc.
|Methods for selecting treatment regimens and predicting outcomes in cancer patients
|24 aug 2001
|31 aug 2011
|Pfizer Products Inc.
|Anti IgE vaccines
|9 jan 2004
|28 sept 2011
|Macrogenics, Inc.
|Identification and engineering of antibodies with variant Fc regions and methods of using same
|6 mei 2005
|2 nov 2011
|Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc.
|Methods of diagnosing or treating prostate cancer using the erg gene, alone or in combination with other over or under expressed genes in prostate cancer
|12 mei 2000
|9 nov 2011
|Arbor Vita Corporation
|Peptides or peptide analogues for modulating the binding of a PDZ protein and a PL protein
|29 maart 2007
|23 nov 2011
|PTC Therapeutics, Inc.
|Methods for the production of functional protein from DNA having a nonsense mutation and the treatment of disorders associated therewith
|28 nov 2001
|1 feb 2012
|MedImmune, LLC
|Methods of administering/dosing anti-RSV antibodies for prophylaxis and treatment
|21 juni 2005
|16 mei 2012
|The Board of Trustees of The Leland Stanford Junior University
|Genes and pathways differentially expressed in bipolar disorder and/or major depressive disorder
|24 aug 2007
|8 aug 2012
|Kyowa Hakko Kirin Co., Ltd.
|Antagonistic human light-specific human monoclonal antibodies
|4 nov 2006
|19 sept 2012
|Genentech, Inc.
|Use of complement pathway inhibitors to treat ocular diseases
|26 juni 2007
|3 okt 2012
|MacroGenics, Inc.
|Fcgamma-RIIB-specific antibodies and methods of the use thereof
|15 nov 2007
|24 okt 2012
|Functional Genetics, Inc.
|Anti-TSG101antibodies and their uses for treatment of viral infections
|9 okt 2007
|31 okt 2012
|The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc.
|Prostate cancer specific alterations in erg gene expression and detection and treatment methods based on those alterations
|6 feb 2006
|7 nov 2012
|GeneNews Inc.
|Mild osteoathritis biomarkers and uses thereof
|13 nov 2006
|6 maart 2013
|The Board of Trustees of the Leland
|FGF2-related methods for diagnosing and treating depression
|10 aug 2006
|27 maart 2013
|MacroGenics, Inc.
|Identification and engineering of antibodies with variant Fc regions and methods of using same
|17 dec 2007
|3 juli 2013
|Genentech, Inc.
|Antagonist anti-Notch3 antibodies and their use in the prevention and treatment of Notch3-related diseases
|24 jan 2008
|21 aug 2013
|Carnegie Mellon University
|Optical biosensors
|29 juni 2007
|18 sept 2013
|Bristol-Myers Squibb Company
|Polynucleotides encoding novel PCSK9 variants
|29 juni 2007
|11 dec 2013
|Bristol-Myers Squibb Company
|Polynucleotides encoding novel PCSK9 variants
|17 dec 2009
|8 okt 2014
|MacroGenics, Inc.
|Covalent diabodies and uses thereof
|15 dec 1993
|28 mei 1996
|Boehringer Ingelheim International, Gmbh
|Conjugates for introducing nucleic acid into higher eucaryotic cells
|23 sept 1992
|20 aug 1996
|Boehringer Ingelheim International Gmbh
|Composition for introducing nucleic acid complexes into higher eucaryotic cells
|18 juni 1993
|8 juli 1997
|Beth Israel Hospital Association
|Mesothelial cell gene therapy
|6 april 1994
|2 dec 1997
|Boehringer Ingelheim International Gmbh
|Adenovirus for delivering foreign DNA into higher eukaryotic cells
|28 okt 1992
|9 dec 1997
|Got-A-Gene Ab
|Targeted delivery of virus vector to mammalian cells
|7 juni 1995
|17 maart 1998
|University Of Conn.
|Use of a bacterial component to enhance targeted delivery of polynucleotides to cells
|28 sept 1994
|17 nov 1998
|American Home Products Corporation
|Complexes comprising a nucleic acid bound to a cationic polyamine having an endosome disruption agent
|28 aug 1997
|9 feb 1999
|University Of Medicine & Dentistry Of New Jersey
|Cell type specific gene transfer using retroviral vectors containing antibody-envelope fusion proteins and wild-type envelope fusion proteins
|25 mei 1994
|30 maart 1999
|Cold Spring Harbor Laboratory
|Cell cycle regulatory protein p16 gene
|1 feb 1992
|13 juli 1999
|Boehringer Ingelheim International Gmbh
|Complexes containing nucleic acid which can be taken-up by endocytosis into higher eukaryotic cells
|14 sept 1994
|5 okt 1999
|Cold Spring Harbor Laboratory
|Cell-cycle regulatory proteins, and uses related thereto
|15 juli 1997
|19 okt 1999
|Cold Spring Harbor Laboratories, Inc.
|Cell-cycle regulatory proteins, and uses related thereto
|25 mei 1995
|9 nov 1999
|Boehringer Ingelheim Int'l. Gmbh
|Composition comprising an endosomolytic agent for introducing nucleic acid complexes into higher eucaryotic cells
|7 juni 1995
|16 nov 1999
|The United States Of America As Represented By The Department Of Health And Human Sevices
|Targetable vector particles
|14 mei 1997
|21 dec 1999
|University Of Southern California
|Retroviral envelopes having modified hypervariable polyproline regions
|25 mei 1995
|8 feb 2000
|Boehringer Ingelheim International Gmbh
|Composition for introducing nucleic acid complexes into higher eucaryotic cells
|2 jan 1996
|28 maart 2000
|Cold Spring Harbor Laboratory
|Cell-cycle regulatory proteins, and uses related thereto
|6 aug 1998
|2 mei 2000
|Genvec, Inc.
|Targeting adenovirus with use of constrained peptide motifs
|27 sept 1996
|2 mei 2000
|Calydon, Inc.
|Tissue-specific enhancer active in prostate
|3 dec 1997
|30 mei 2000
|Beth Israel Hospital Association
|Mesothelial cell gene therapy
|14 feb 1997
|13 juni 2000
|Canji, Inc.
|Retinoblastoma fusion polypeptides
|28 sept 1995
|3 okt 2000
|American Home Products Corporation
|Multifunctional complexes for gene transfer into cells comprising a nucleic acid bound to a polyamine and having a endosome disruption agent
|11 juli 1997
|24 okt 2000
|Calydon, Inc.
|Prostate specific enhancer polynucleotides and methods of use thereof
|22 juli 1994
|14 nov 2000
|University Of Medicine And Dentistry Of New Jersey
|Cell-type specific gene transfer using retroviral vectors containing antibody-envelope fusion proteins
|17 juni 1999
|28 nov 2000
|Cornell Research Foundation, Inc.
|Nucleic acid that encodes a chimeric adenoviral coat protein
|25 juni 1993
|12 dec 2000
|Btg International Limited
|RNA bacteriophage-based delivery system
|5 juni 1998
|19 dec 2000
|The Regents Of The University Of Michigan
|Neuregulin response element and uses therefor
|29 nov 1994
|3 april 2001
|Cold Spring Harbor
|Cell-cycle regulatory proteins, and uses related thereto
|16 dec 1999
|14 aug 2001
|Boehringer Ingelheim International Gmbh
|Composition for introducing nucleic acid complexes into higher eucaryotic cells
|6 dec 1999
|11 dec 2001
|Genvec, Inc.
|Targetting adenovirus with use of constrained peptide motifs
|30 juni 1995
|18 dec 2001
|Cold Spring Harbor Laboratory
|Cell-cycle regulatory proteins, and uses related thereto
|19 mei 1999
|30 april 2002
|Canji, Inc.
|Retinoblastoma fusion proteins
|22 okt 1999
|30 april 2002
|American Home Products Corporation
|Multifunctional complexes for gene transfer into cells comprising a nucleic acid bound to a polyamine and having an endosome disruption agent
|8 juli 1998
|21 mei 2002
|Canji, Inc.
|Compositions for enhancing delivery of nucleic acids to cells
|27 nov 1996
|15 okt 2002
|Genvec, Inc.
|Vectors and methods for gene transfer to cells
|30 jan 1998
|26 nov 2002
|Cold Spring Harbor Laboratory
|Cell-cycle regulatory proteins, and uses related thereto
|13 aug 1999
|7 jan 2003
|W. French Anderson
|Targetable vector particles
|17 aug 1998
|18 maart 2003
|University Of Medicine And Dentistry Of New Jersey
|Cell type specific gene transfer using retroviral vectors containing antibody-envelope fusion proteins and wild-type envelope fusion proteins
|4 juni 1999
|10 juni 2003
|Cornell Research Foundation, Inc.
|Chimeric adenovirus fiber protein
|1 okt 2001
|18 nov 2003
|Genvec, Inc.
|Targeting adenovirus with use of constrained peptide motifs
|10 april 2000
|9 dec 2003
|Health Protection Agency
|Treatment of intracellular infection
|20 nov 2001
|2 maart 2004
|University Of Maryland Biotechnology Institute
|Treatment and prevention of HIV infection by administration of derivatives of human chorionic gonadotropin
|27 aug 1997
|1 feb 2005
|Bio-Rad Laboratories, Inc.
|Methods and compositions for diagnosis and treatment of iron misregulation diseases
|13 juli 2001
|8 maart 2005
|University Of Southern California
|Modified viral surface proteins for binding to extracellular matrix components
|30 nov 2001
|5 april 2005
|Avigenics, Inc.
|Ovomucoid promoter and methods of use
|19 mei 1999
|7 juni 2005
|Canji, Inc.
|Methods of treating hyperproliferative disorders using retinoblastoma fusion proteins
|5 juni 2001
|12 juli 2005
|Cell Genesys, Inc.
|Human glandular kallikrein enhancer, vectors comprising the enhancer and methods of use thereof
|24 okt 2001
|4 okt 2005
|Genvec, Inc.
|Vectors and methods for gene transfer
|8 juli 1997
|21 feb 2006
|Canji, Inc.
|Compositions and methods for therapeutic use
|7 mei 1997
|11 april 2006
|Bio-Rad Laboratories, Inc.
|Polymorphisms in the region of the human hemochromatosis gene
|20 nov 2002
|30 mei 2006
|Bio-Rad Laboratories, Inc.
|Polymorphisms in the region of the human hemochromatosis gene
|2 mei 2002
|27 juni 2006
|Bio-Rad Laboratories, Inc.
|Hereditary hemochromatosis gene
|19 aug 2002
|18 juli 2006
|University Of Southern California
|Retroviral vectors including modified envelope escort proteins
|14 maart 2002
|14 nov 2006
|University Of Cincinnati
|Avian iFABP gene expression controlling region
|3 aug 2001
|13 feb 2007
|Avigenics, Inc.
|Avian lysozyme promoter
|1 april 2002
|3 april 2007
|Avigenics, Inc.
|Recombinant promoters in avian cells
|13 nov 2001
|10 april 2007
|Multifunctional molecular complexes for gene transfer to cells
|20 nov 2000
|2 okt 2007
|Dendreon Corporation
|Nucleic acids encoding endotheliases, endotheliases and uses thereof
|28 mei 2004
|13 nov 2007
|Avigenics, Inc.
|Ovomucoid promoters and methods of use
|31 jan 2005
|26 feb 2008
|Avigenics, Inc.
|Avian gene expression controlling regions
|15 dec 2004
|25 maart 2008
|University Of Southern California
|Method of delivering therapeutic agents to site of tissue injury
|7 sept 2004
|25 maart 2008
|Transgene, S.A.
|Poxvirus with targeted infection specificity
|12 april 2001
|8 april 2008
|Transgene S.A.
|Poxvirus with targeted infection specificity
|2 dec 2002
|20 mei 2008
|Avigenics, Inc.
|Transgenic avians with an ovomucoid gene expression control region linked to a nucleotide sequence encoding a heterologous polypeptide
|15 juli 2004
|1 juli 2008
|Barros Research Institute
|Compositions and methods for immunotherapy of cancer and infectious diseases
|30 jan 1998
|16 sept 2008
|Cold Spring Harbor Laboratory
|Antibodies to the cell cycle regulatory protein p16
|28 juni 2002
|11 nov 2008
|Novartis Ag
|PERV screening method and use thereof
|3 mei 2002
|3 maart 2009
|Fit Biotech Oyj Plc
|Expression vectors and uses thereof
|4 jan 2007
|24 maart 2009
|Avigenics, Inc.
|Transgenic avians containing recombinant ovomucoid promoters
|3 mei 2002
|31 maart 2009
|Fit Biotech Oyj Plc
|Expression vectors and uses thereof
|22 jan 2002
|19 mei 2009
|Canji, Inc.
|Compositions and methods for enhancing delivery of therapeutic agents to cells
|26 jan 2007
|2 juni 2009
|Synageva Biopharma Corp.
|Avians containing a lysozyme promoter transgene
|14 april 1994
|23 juni 2009
|Cold Spring Harbor Laboratory
|p16INK4 polypeptides
|2 okt 2003
|23 juni 2009
|Synageva Biopharma Corp.
|Production of a transgenic avian by cytoplasmic injection
|4 mei 1999
|28 juli 2009
|New York University
|Cancer treatment with endothelin receptor antagonists
|5 jan 2006
|25 aug 2009
|Bio-Rad Laboratories, Inc.
|Hereditary hemochromatosis gene
|20 juni 2005
|29 sept 2009
|Bio-Rad Laboratories, Inc.
|Polymorphisms in the region of the human hemochromatosis gene
|20 aug 2008
|6 april 2010
|Cold Spring Harbor Laboratory
|Kit for detecting the level of cyclin-dependent kinase inhibitor P16 gene expression
|2 feb 2001
|20 april 2010
|Dendreon Corporation
|Nucleic acid molecules encoding transmembrane serine proteases, the encoded proteins and methods based thereon
|17 nov 2008
|12 okt 2010
|Synageva Biopharma Corp.
|Methods and protein production using ovomucoid promoters
|18 jan 2008
|26 okt 2010
|University Of Southern California
|Transgene delivering retrovirus targeting collagen exposed at site of tissue injury
|4 aug 2005
|1 maart 2011
|Pharmacia & Upjohn Company, Llc
|Anti-IgE vaccines
|5 mei 2008
|19 april 2011
|Ottawa Health Research Institute
|PCSK9 polypeptide fragment
|15 nov 2007
|21 juni 2011
|Limin Li
|Anti-TSG101 antibodies and their uses for treatment of viral infections
|25 maart 2005
|5 juli 2011
|Human Genome Sciences, Inc.
|Antibodies against nogo receptor
|19 feb 2009
|16 aug 2011
|Bio-Rad Laboratories, Inc.
|Determining genotype of a polymorphic site in the hereditary hemochromatosis gene
|28 maart 2006
|23 aug 2011
|Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw
|Means and methods for the recruitment and identification of stem cells
|12 feb 2004
|20 sept 2011
|Functional Genetics, Inc.
|Method for reducing HIV viral budding by administering a VPS28-specfic antibody that disrupts Gag-TSG101-VPS28 binding interactions
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|10 sept 2010
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|7 juli 2011
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|Hereditary hemochromatosis gene
|24 nov 2009
|25 sept 2012
|Pfizer Inc.
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|29 maart 2012
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|Human chondroitinase glycoprotein (CHASEGP), process for preparing the same, and pharmaceutical compositions comprising thereof
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SOCHI SCENE: Bronze Headstand
KRASNAYA POLYANA, Russia (AP) – After his silver in downhill, Christof Innerhofer couldn't stop screaming in delight.
When he added a bronze in super-combined Friday, the colorful Italian skier performed a headstand on the podium.
Innerhofer suffers from so much back pain, he doesn't even train during the summer anymore.
Somehow, though, Innerhofer tied with silver medalist Ivica Kostelic for the third-fastest slalom run in the super-combi. Not bad for a racer who hasn't entered a World Cup slalom in more than two years.
"I didn't believe in myself," said Innerhofer, who stood eighth after the downhill leg. "I didn't think I could race that well in slalom. It was more or less a miracle."
If Innerhofer wins Sunday's super-G, he'll match his haul from the 2011 world championships in Garmisch-Partenkirchen, Germany, when he won a medal of each color.
_ By Andrew Dampf _ Twitter
Associated Press reporters are filing dispatches about happenings in and around Sochi during the 2014 Winter Games. Follow AP journalists covering the Olympics on Twitter:
(Copyright 2014 The Associated Press. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.)


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So a month or so ago I was asked by the New York Post to review Marty Appel's new Thurman Munson biography. They sent me the book. I read it. Liked it. I wrote a review. Then I traded the book to Jason at IIATMS for a Chipper Jones jersey he didn't want anymore (query: […]
Cooperstown Confidential: Opening a new book on Munson
A new baseball biography is spurring conversation, especially as it climbs the New York Times best-seller list. Bruce Markusen interviews the author.
Meet Selena Roberts (and an actually worthy person too)
Here's an event for you, at least if you're in Brooklyn this evening: Gelf's Varsity Letters sports reading series returns on Thursday, July 2, at 7:30 p.m., with a night dedicated to baseball. At this free monthly event in DUMBO, Brooklyn, hosted by Gelf and Jan Larsen Art, Scott Price, Selena Roberts, and members of […]
Looking back at Satchel Paige
Reviewing a new, and long overdue, book about the extraordinary Satchel Paige.
Book Review: Cooperstown Confidential
Not to be confused with Bruce Markusen's feature here at THT, this Cooperstown Confidential is a new book by Zev Chafets.
The new Satchel Paige biography sounds really, really good: How fast could Satchel Paige throw a baseball? It's hard to know because there were no radar guns to measure ball velocity when Leroy Paige, better known as Satchel, became a pitching star of the Negro Leagues in the 1930s. In his discerning, empathetic and hype-free […]
Selena Roberts < James Frey
Couldn't happen to a nicer book: Remember that tell-all book about A-Rod? Just a month after making headlines with its allegations that the New York Yankees star likely used steroids as far back as high school, Selena Roberts' "A-Rod: The Many Lives of Alex Rodriguez" has vanished from best seller lists. Published in early May […]
Book Review: "Miracle Ball"
I'm not going to say that I've stretched myself a bit thin recently, but I had forgotten that I wrote a book review for the New York Post a week or two ago. In fact, I hadn't even realized that it had been published this past Sunday until I happened to run into it while […]
Book Review: 100 Things Dodgers Fans Should Know & Do Before They Die
A review of a definitive Dodgers guide
Heart of the Game
The Bats blog's Ihsan Taylor has an interview with S.L. Price, author of a new book about the late Mike Coolbaugh and Tino Sanchez, the man whose foul ball ended Coolbaugh's life. I'll admit that when I first heard about this book I was skeptical that it would be something of a ghoulish cash-in on […]
Quote of the Day
Mark Purdy of the San Jose Mercury-News, lamenting the fact that the Selena Roberts book isn't selling all that well: It does confirm one of my beliefs, however. There's a perception that fans love to read dirt about famous athletes. Maybe they do, on salacious Web sites full of half-truths — but not in well-reported […]
Statistics tell more truths than Selena Roberts
The New York Times, with some help from Tom Tango and Leverage Index, blows up the pitch-tipping allegations: If a tipping conspiracy were in place, one would expect that Rodriguez and rival middle infielders in games he played to have hit better in low-leverage situations than in high-leverage ones. Using a fairly loose definition of […]
"On the Road" was really about splits
The guy who wrote this: They danced down the streets like dingledodies, and I shambled after as I've been doing all my life after people who interest me, because the only people for me are the mad ones, the ones who are mad to live, mad to talk, mad to be saved, desirous of everything […]
BOB Book Review: Forever Blue
A biography of former Dodgers owner Walter O'Malley
The Selena Roberts book is about what you'd expect
After a brief detour to deal with Manny-mania, I finally finished the Selena Roberts/A-Rod book over the weekend. I originally had thoughts of dissecting the sucker, but upon its completion I realized that, despite all of the hype (to which I helped contribute, I'll admit), writing a highly-detailed takedown would represent a massively disproportionate response […]
Book Review: '78 by Bill Reynolds
Chris' takes on the new book '78: The Boston Red Sox, A Historic Game and a Divided City
Great Moments in Scouts' Notes
For all of the things I've written, reading the A-Rod book is not without its enjoyable moments. I may later assemble some random ones in a post, but I want to share this one while it's fresh. From the field notes of Mariners' scout Roger Jongewaard after surveying Alex Rodriguez, high school senior: Premium prospect […]
The Fisking of Roberts has begun
A little over 48 hours since the book's release, and people are starting to pick the nits. I'm not sure I care about the "was he in Milwaukee or was he in Detroit" kind of nits that hell of a lot, but if there are tons and tons of them, it suggests that the commercial […]
Quote of the Day
Via Whitlock, who continues to lay waste to Selena Roberts, comes a quote from Roberts herself from last night's Jim Rome show which pretty much explains why I've been writing what I've been writing since last Thursday: "You give people a litmus test, Jim . . . You say to them, you go back to […]
Doug Glanville questions the pitch tipping allegations
My daily spewing about the Roberts-A-Rod book is up over at NBC.
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All quotes tagged Futurity
(6612 quotes found)
"It is the experience of learning and not the record of perfection that ultimately define ourselves."
"To all those who look towards the pessimistic side that the future will be bleak, I have one thing to say. Without hope you are already dead."
"Tomorrow is not even the possibility of a broken promise. It is not anything but a hope, a dream, an illusion."
"They booked credits from multiple suppliers, with cash tied to future business. It might be perceived as another piece of bad news. But it's not a big issue."
"Life Begins but Life does End. Life can win, But life can Bend. Life can give and life can Take. Life can Love and life can Hate. So ladies and gents, i ask you this. Think about the words, which pass through my lips. Tomorrow has come, Yesterday has passed. Look to the future, forget about the past. Think about next week, next month and then after. Fill your life with joy, good friends and laughter. Life can be cruel and life can be fast. So we must cherish every moment, As if it's our last."


text:
Lalwani Ferro Alloys Ltd.View mapKolkata, West Bengal
Company Profile
The company is promoted by Sri Kamal Kishore Lalwani to produce Noble Alloys , Master Alloys and Bulk Alloys. The first Group Company was Lalwani Ferro Alloys (P) Ltd. which was established on 27.11.1986 to manufacture & trade in Ferro Alloys.
Lalwani Ferro Alloys Ltd.
Lalwani Metallics (P) Ltd....view all
Fact Sheet
|Year of Establishment
|Nature of Business
Manufacturer, Trader
Contact UsLalwani Ferro Alloys Ltd.Sundeep LalwaniView Contact Number
View map32 Jawaharlal Nehru Road,
Om Towers , Suit No 205
Kolkata - 700 017
West Bengal, India
Om Towers , Suit No 205
Kolkata - 700 017
West Bengal, India
Send your enquiry to this supplier


text:
Mitt Romney returns to CPAC, tells crowd thank you and shares optimistic vision of America (+video)
Jacquelyn Martin, Associated Press
Former Republican presidential candidate Mitt Romney took to the stage at the Conservative Political Action Conference in Washington, D.C., telling the audience that although he's sorry he can't be their president, he'll gladly work shoulder-to-shoulder alongside them as their co-worker.
Romney, who was greeted at the conference with a standing ovation, has maintained a relatively low profile since he conceded the presidential race to President Barack Obama in November 2012, being spotted at Disneyland, joining the board of Marriott International and emerging for a recent interview with Chris Wallace.
His message at CPAC this year, he said, was one of thanks and optimism.
"It's fashionable in some circles to be pessimistic about America, about conservative solutions, about the Republican Party," Romney said. "I utterly reject pessimism."
While Republicans may have lost on Nov. 7, they haven't lost the country, and they haven't lost their way, he said.
Highlighting stories of individuals he's encountered in his experiences, Romney talked about meeting determined Americans, men of God, heroes in the armed forces and heroes in the home.
Gov. Nikki Haley, R-S.C., introduced Romney, saying that he and his wife Ann had a love for the country that the election only strengthened. Romney returned the compliment, saying Americans and Republicans needed to learn from leaders like her.
"I would urge us all to learn the lessons that come from some of our greatest success stories, and that's the 30 Republican governors across the country," Romney said. "They're winning elections, but more importantly, they're solving problems — big problems. Important problems."
In his speech, Romney also emphasized the importance of a strong U.S. military and discussed the role America should play in the world. He highlighted U.S. efforts to fight AIDS in Africa, the philanthropic efforts of the country and its citizens, and its military interventions in places like Europe, Korea and Iraq.
"Whenever you think of these — whatever you think of these interventions — the impulse behind every single one of them was liberation, not conquest," Romney said. "What nation but ours would've enjoyed hegemonic military power for 25 years and never used it to seek revenge against its foes or to seize resources from the weak?"
The challenges facing the country today are not small, Romney said, and each individual has been summoned by history to step up and meet his or her responsibilities.
"In the end we'll win," Romney said. "We'll win for the same reason we've won before — because our cause is just and it is right. I want to thank you again for your support and your help along the journey."
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The rooms are comfortable and well appointed. All rooms are equipped with private bathroom, direct telephone, radio, TV, minibar and safe. The restaurant serves simple and traditional cooking prepared with fresh ingredients. Their cuisine ranges from International dishes to the more traditional typical of Aosta Valley. A typical mountain resort hotel The lobby is spacious with a wooden welcome desk situated at the entrance. Some comfortable sofas are placed in the lounge for guests to relax. This is a good quality hotel, with an excellent location for skiing or for exploring the mountains.
Situated in a panoramic position at the foot of the Mount Cervino and the Grandes Murailles chain.
Just 30mt away from the sky slopes and a few steps away from the pedestrian area of downtown. 1 kms to city centre 185 kms to the nearest airport (malpensa international airport) 29 km to the nearest station (chatinon train) 5 minute walk to the nearest bus stop


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Is this exam room ready?
Jan 01, 2013
After an exam, we turn off the lights, close the back door and leave the front door open. If the front door is open and the lights are off, the front staff knows the room is available for use.
If the back door is open and the lights are on, the back staff knows the room needs to be cleaned. It also saves on electric bills.
—Ryan Cook, DVM
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Actually, he says FUCHING but carry on
Album Rating: 5.0bloc knows what's up
Album Rating: 4.5This album is still hands down their best LP.
Seeing these guys at the Roundhouse tomorrow, looking forward to them Follow The Water riffs
Digging: Knocked Loose - Pop Culture
Album Rating: 5.0Fuck man i really wanted to go that gig but just forgot they were touring haha
Digging: Palm Reader - Beside The Ones We Love
When I saw that Every Time I Die were supporting I basically had to go, two of the best live bands I've ever seen tbh
Album Rating: 5.0Saw enter shikari at the roundhouse couple weeks ago, great venue. Im sure youll have a blast
seriously sounds like i shoulda called emergency services
Got to sing into the microphone during Every Time I Die, splitting my lip open has never been more worth it.
Album Rating: 5.0cool man. did they play anything off this?
Early Grave and Follow The Water, it was mostly the new album with bits from Daybreaker
Album Rating: 4.5From what I've read in interviews I don't think they're massive on their eariler sound these days.
Album Rating: 5.0Especially the Here and Now.
Yeah they only played one song from The Here And Now (Day In Day Out). I think they've pretty much settled into metalcore now.
Album Rating: 4.5They've regressed imo, but, hey, whatever does it for them.
Album Rating: 4.0They've always been metalcore and yeah band sucks now. The only reason they probably don't like their earlier material is cause they can't replicate it and that's what their fans want/keep asking for. Next they'll be saying they wanted to go for a more mature sound
Album Rating: 5.0Theyve all said that the here and now is just weak in all ways except for one or 2 songs, i dont know their thoughts on not playing Nightmares and Ruin
Album Rating: 3.5Shame since Ruin and Nightmares are their best 2 albums.
Digging: Bryson Tiller - T R A P S O U L
Album Rating: 5.0they probs dont do nightmares because only 2 of them were on it and its a pretty different vocal style to what sam does
Bands: A B C D E F G H I J K L M N O P Q R S T U V W X Y Z


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Funds lead the way
Relevant offers
The University of Otago has committed more than $2.5 million to support 14 research centres during the next five years.
Deputy Vice Chancellor of Research and Development, Professor Richard Blaikie, says the centres are an important facet of the university.
"It's something we are committed to supporting, even in times when budgets are constrained, " he says.
"We have increased the level of support for research centres by about 50 per cent from university resources in this round compared to the round just completed."
Two of the centres, which have been formally recognised as University of Otago Research Centres for the next five years, are new. They are the Centre for Research on Colonial Culture and the International Centre for Governance, Science, and Society.
Among the existing 12 are the Brain Health Research Centre, the Centre for Neuroendocrinology, the Jack Dodd Centre for Quantum Technology, and the New Zealand Centre for Sustainable Cities.
The funding from the University of Otago supplements grants from academic divisions.
"The funding is to support some sustaining activities, not to support all the research activities, " Blaikie says.
"We'll support some student activities, some collaborative activities, and workshops and seminars. But these groups have to be very strongly committed to going out and receiving money from grants and awards for their research, as well."
The 14 centres went through a rigorous application process in order to receive funding from the university. Blaikie says the University Research Committee encourages research groups which embrace collaboration and multidisciplinary work.
"Some of the really strong applications were from two or more groups who had established themselves with strong international reputations, " he says. "So by combining the relatively modest amounts from the University Research Committee we can make the whole greater than the sum of the parts."
Some of the research centres are involved in internationally cutting edge research work.
But Blaikie says it is important to the University of Otago that the research activities can be translated into real-world benefits where possible.
"You will see, in this collection of centres, people with very strong evidence of that translational activity, " he says.
"The Centre for Translational Cancer Research is very strongly connected to Pacific Edge Biotechnology, which is developing commercial cancer diagnostics, and the Edgar National Centre for Diabetes and Obesity Research are one of the leading lights in a very important area for New Zealand.
"As these problems grow, the research effort needs to grow."
- The Southland Times
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CPD courses for practitioners
Our therapy service offers personal development, while our programme of CPD courses provides professional development for therapists.
Contact us now to discuss the kind of training you may be considering for yourself or your organisation, or email us on: firstname.lastname@example.com.
Diploma in integrative supervision: starting March 2016
This training offers counsellors, psychotherapists, psychologists and those in the helping professions the opportunity to develop therapeutic supervisory skills. The course is run on a multi-modal, multi-disciplinary basis. We recognise that nowadays supervisors may be supervising practitioners from different theoretical backgrounds from their own, particularly in organisational contexts. So our approach in this training is integrative – including a range of models and supervision concepts – while upholding the existing framework and background of each course participant.
Click here to learn more about the supervision course.
Certificate in Cognitive Behavioural Therapy (CBT) skills
This is a post-qualification training in Cognitive Behavioural Therapy skills. It is a part-time structured programme of 6 days, taught over three weekends across a three-month period, providing an opportunity for practitioners to integrate CBT concepts and techniques with their existing theoretical background and way of working.
Click here to read more about the CBT course.
Introduction to psychosexual therapy
This 2-day weekend workshop is facilitated by Cabby Laffy and is designed as an initial training to equip therapists with knowledge-based skills to work with common psychosexual problems that are presented in counselling. The dates are Saturday 25th & Sunday 26th October 2015.
Click here to read more about the psychosexual therapy 2-day workshop.
Certificate in couple therapy: starting March 2016
Cabby Laffy facilitates this 8-day CPD course as an introduction to couple counselling for therapists who are already practising with individual clients. Cabby brings to this workshop her experience of teaching a 2-year diploma course is psychosexual and relationship therapy, as well as her knowledge as a published writer on sex and relationships. The course is taught in 4 x 2-day blocks, each on a Friday and Saturday.
Click here to read more about this CPD course.
2-day CPD workshop: Cognitive Behavioural Couple Therapy
This 2 day experiential workshop provides an introduction to and a foundational training in CBCT (Coginitive Behavioural Couple Therapy). It will be of interest to therapists who have training in other modalities of couple therapy (who will find that the approach complements and extends their practice) as well as therapists who do not have experience working with couples.
The workshop is facilitated by Dr Michael Worrell on Saturday 19th and Sunday 20th September 2015.
Click here to read more about this CPD workshop.
Bespoke in-house courses
Sometimes organisations approach The Grove to run a tailored programme in-house at their own premises for their own teams. We are happy to discuss your requirements and tailor a programme according to your brief. Please email Sarah Briggs, Training Director, at firstname.lastname@example.com or call her on 07973 36851207973 368512.
If you'd like to stay informed when dates are announced or would like to suggest a topic to be included in the series, email us on: email@example.com.
Alternatively, if you are a trainer or facilitator who'd like to run a workshop with The Grove, please get in touch with Sarah Briggs at email@example.com to discuss your idea.


text:
BPL Community Profile
Kevin Babione (KBabione) - BPL Lifetime Member
You cannot send this user a personal message (PM) because they have not specified a PM email address yet.
All Forum Posts by Kevin Babione
Montbell fit for big guys
I'm six feet even and tip the scales at around 260. I have a Montbell Ex Light jacket in XXL that fits well, but I've only ever had to put a Patagonia R1 & R2 under it. Montbell ret...
Thermarest Lite Seat = Awesome Pillow
Travis, I too love the Lite Seat as my pillow but use a cheap polyester book cover that I "borrowed" from my high school daughter as a pillowcase. It's one of those super-stretchy...
Platy Hoser vs Big Zip
I'm the proud owner of both a 2L Hoser and a 2L Big Zip (along with every possible size Camelbak bladder). There is a weight difference between the two. On my scale: - 2L Hoser = 98 ...
Travel Bikes
Although not a folding bike, Freeman Transport handcrafts "transportable" bikes in the US that disassemble and reassemble with one simple tool. Just another option, but another one t...
Susquehannock Trail
Chuck Dillon at Pine Creek Outfitters also has a guide to the Susquehannock Trail that's pretty reasonable at $11.50. Both trails are on my list to do...
Someone had posted these a year or so ago... http://www.sprintaquatics.com/prodinfo.asp?number=901 Apologies for not imbedding it... They're cheap ($4.00) and light (53 grams on my sca...
Gatewood Cape Video
Chris, Nice video. You may want to consider using your second trekking pole as a lift for one of the additional guyouts (especially in the snow). Here's a photo: I use it at my head...
Inov-8 Roclite 390 GTX
I have a pair of size 11.5 that I'd like to sell for $60 delivered. They have 30 miles on them (three 10-mile trips...I REALLY wanted to like them). They were just too narrow for my feet. ...
Solo Shelter Options
Aaron, Just because you're carrying a poncho-tarp doesn't necessarily mean that you have to use it as your raingear. If carrying the Gatewood Cape is lighter than carrying a tarp/bivy c...
Smaller Tarp
Look, if you haven't already, at the SMD Gatewood Cape. There are lots of posts around if you search. I don't have a bivy and haven't had any problems with getting wet - I simply pitc...
All Reader Reviews by Kevin Babione
Showing 1 - 2 of 2 | Page: 1
Montbell Extremely Light Down Jacket
I'm a big guy and have the Ex Light in XXL - weighs 7.8 ounces. In addition to being a great piece of insulation to wear I've found that I can use it in my hammock as an extra top quilt by op...
Lighten Up! by Don Ladigin
I just finished reading the book - it's an enjoyable and quick read. I've got two weekend trips planned this spring with guys who have never backpacked in their lives. I'm passing the bo...


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January 08, 2015
Jessica Dipre - Civil Litigation Issues
What the civil litigation cases for Brown and Garner might look likePolice training and proper documentation is more important than ever in order to prevent or defend Section 1983 actions - Full Story
January 05, 2015
Force Science Institute - Destroying Myths & Discovering Cold Facts
What appeals court says cops should know about prone positioningBolick v. City of East Grand Rapids (MI), is a civil rights action against police, alleging the use of excessive force in the excited delirium death of a mentally ill and highly combative 30-year-old who had been "freaking out" and threatening to kill his father - Full Story
December 18, 2014
P1 Community - The Question
Why cops don't 'shoot to injure'A question posted recently on Quora asked, "Why aren't U.S. cops trained to "shoot to... - Full Story
December 04, 2014
Chief Joel F. Shults, Ed.D. - Passion for the Job
5 things police leaders can do to better report violence against officersThere is a vast under-reporting on assaults against our officers, and it is not the fault of the FBI — it is the fault of police leadership - Full Story
November 24, 2014
Doug Wyllie, PoliceOne Editor in Chief - Be Advised...
Why Officer Darren Wilson wasn't indictedA cop may use lethal force when he or she "reasonably believes that the action is in defense of human life, including the officer's own life" and in the Ferguson incident, that's precisely what happened - Full Story
November 19, 2014
Force Science Institute - Destroying Myths & Discovering Cold Facts
Amid shooting furors, politicians get a special "training day"The St. Louis Police Officers Association invited about 60 elected officials to a day of immersion training in hopes of opening their eyes and minds to the realities behind the badge - Full Story
November 17, 2014
Duane Wolfe - The Warrior's Path
5 common use-of-force myths debunkedToo many administrators, chiefs, mayors, city council members, community activists, and members of the media are unaware of Graham v. Connor, let alone the four-pronged test of 'reasonableness' - Full Story
November 04, 2014
Lt. Dan Marcou - Blue Knights
Dealing with dogs on SWAT operations: 10 tactical optionsNo matter how you feel about dogs, do not put your life — or the lives of your team — in jeopardy because of an aggressive dog - Full Story
October 24, 2014
Matt Stiehm - Campus Safety
3 keys to improve public's perception of forceWhen used lawfully, reasonably, and within the totality of the circumstances, force is appropriate; we need to work hard to educate the press and the public that this is the case - Full Story
October 20, 2014
Force Science Institute - Destroying Myths & Discovering Cold Facts
Success story: Cops 2, Plaintiffs 0 in excessive force lawsuits"These were the first major cases I've been involved in where I applied material from Force Science since attorneys began calling on me for expert witness work 13 years ago," Sgt. Thomas Ovens told Force Science News. - Full Story


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Capital Markets
Company Overview of iShares Trust - iShares Agency Bond ETF
Company Overview
400 Howard Street
San Francisco, CA 94105
United States
Founded in 2008
Key Executives for iShares Trust - iShares Agency Bond ETF
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