text:
|Home | About | Journals | Submit | Contact Us | Français
The recent development of a cell culture model of hepatitis C virus (HCV) infection based on the JFH-1 molecular clone has enabled discovery of new antiviral agents. Using a cell-based colorimetric screening assay to interrogate a 1,200-compound chemical library for anti-HCV activity, we identified a family of 1,2-diamines derived from trans-stilbene oxide that prevent HCV infection at nontoxic, low micromolar concentrations in cell culture. Structure-activity relationship analysis of ~300 derivatives synthesized using click chemistry yielded compounds with greatly enhanced low nanomolar potency and a >1,000:1 therapeutic ratio. Using surrogate models of HCV infection, we showed that the compounds selectively block the initiation of replication of incoming HCV RNA but have no impact on viral entry, primary translation, or ongoing HCV RNA replication, nor do they suppress persistent HCV infection. Selection of an escape variant revealed that NS5A is directly or indirectly targeted by this compound. In summary, we have identified a family of HCV inhibitors that target a critical step in the establishment of HCV infection in which NS5A translated de novo from an incoming genomic HCV RNA template is required to initiate the replication of this important human pathogen.
Hepatitis C virus (HCV) is a hepatotropic, enveloped virus with a single-stranded RNA genome of positive polarity that causes acute and chronic hepatitis and hepatocellular carcinoma (36). More than 170 million people are chronically infected and are at risk of developing cirrhosis and hepatocellular carcinoma (25). There is currently no vaccine against HCV, and the only approved antiviral therapy, a combination of recombinant type I alpha interferon and ribavirin, is expensive, toxic, and only partially effective (50).
HCV is a member of the Flaviviridae family. Its 9.6-kb RNA genome carries a long open reading frame that is co- and posttranslationally cleaved by cellular and viral proteases (reviewed in reference 52) into structural proteins (core, E1, and E2) that constitute the major viral components of the viral particles and nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) that are required at multiple levels of the virus life cycle, including viral RNA replication (2) and infectious-particle assembly (46). The single open reading frame is located between two untranslated regions (UTRs), the 5′ UTR and the 3′ UTR, which contain RNA sequences essential for RNA translation and replication, respectively (15, 16, 24).
HCV infection is initiated when viral particles bind and enter the target cells in a process that involves multiple cellular receptors and clathrin-dependent endocytosis (7). Release of the viral genome into the cytoplasm is thought to occur after low-pH-induced membrane fusion, a process mediated by the viral envelope glycoproteins (60). Incoming viral genomes are translated into the viral polyprotein in a process that requires components of the autophagy machinery (12). Cleavage of the polyprotein into the individual viral proteins enables the establishment of replication complexes in endoplasmic reticulum (ER)-derived membranous compartments where viral RNA replication occurs via minus-strand synthesis (45). Progeny genomes are either translated to produce additional viral proteins or packaged to assemble progeny infectious virions. Virus particle assembly is thought to occur in an ER-related compartment in close proximity to cytosolic lipid droplets, where core and NS5A proteins colocalize (41, 44). HCV RNA-containing core particles acquire their envelope by budding through the ER membrane at sites where E1/E2 glycoprotein complexes are inserted (54). The HCV assembly process is dependent not only on structural and nonstructural proteins (p7, NS2, NS3, and NS5A) (1, 27, 29, 30, 39, 43, 47, 55, 58, 62) but also on cellular factors involved in lipoprotein biosynthesis (10, 18, 26, 28, 48). HCV assembly results in the production of high-density intracellular infectious precursors that undergo maturation during their passage through the secretory pathway, where they acquire their characteristic low-density extracellular configuration (21). Extracellular virions are spherical, pleomorphic particles that are heterogeneous in size (31, 35, 42, 53, 59), some of which are surrounded by a membrane bilayer, likely the viral envelope, that can be observed by cryoelectron microscopy (19).
The development of hepatoma-derived cell lines bearing autonomously replicating HCV RNA (replicons) (8, 38) permitted the development of potent antiviral molecules directed against the viral NS3 protease, the NS5B RNA-dependent RNA polymerase (49), and recently NS5A (17), a zinc metalloprotein that is required for viral replication and particle assembly (41, 58). The opportunity to develop compounds that target additional steps in the HCV life cycle awaited the development of cell culture infection models that recapitulated all aspects of the viral infection. We recently established a cell-based, unbiased screening procedure that permits interrogation of chemical libraries for antiviral activity against all steps in the HCV life cycle, including cellular and viral factors required for efficient viral spread (22). Using this technology, we have discovered a class of antiviral compounds that selectively blocks the initiation of HCV RNA replication in vitro without affecting ongoing HCV RNA replication, a unique and hitherto obscure step in HCV infection that can now be approached experimentally for basic and translational purposes.
The initial chemical library was composed of ~1,200 compounds with diverse skeletal architectures (i.e., acyclic, cyclic, fused/bridged polycyclic, heterocyclic, and aromatic). Their assembly relied on click chemistry, focusing primarily on carbon-heteroatom linkages (32). This synthetic strategy allowed for rapid generation of large quantities (grams) of material in high yields requiring relatively simple purification methods. Inherently incorporated into these structures were functional groups that immediately enabled further diversification for structure-activity relationship (SAR) analysis and hit compound optimization.
The syntheses of compounds 1 and 2 are illustrated elsewhere (http://www.scripps.edu/wieland/data/Gastaminza_2/Suppl.FIg1.pdf). Synthetic procedures for compound 1 have been described previously (20). A detailed preparation of compound 2 along with the SAR studies leading to compound 2 will be described in a future report (unpublished data). Dry powders of compounds 1 and 2 were dissolved in dimethyl sulfoxide (DMSO) to a final 10 mM concentration. The solution of compound 2 required heating at 65°C for 5 min before use, as this compound tends to precipitate in DMSO at a relatively high concentration (10 mM), although it is water soluble up to 50 μM.
The chemical library described above was screened using a colorimetric HCV infection readout as described previously (22). Briefly, compounds were dissolved in DMSO at a final concentration of 10 mM, diluted to a final concentration of 20 μM in 100 μl of culture medium (Dulbecco's modified Eagle's medium containing 10% fetal calf serum [DMEM-10% FCS]), and mixed (1:1) with 100 μl of a D183 virus (65) dilution (2 × 103 focus-forming units [FFU]/ml) in medium. One hundred microliters of this mixture was used to inoculate 104 Huh-7.5.1-clone 2 (Huh-7.5.1-c2) cells (51) per well (multiplicity of infection [MOI] of 0.01) in a 96-well format (Falcon flat-bottom 96-well cell culture microplate; BD Biosciences, La Jolla, CA) in duplicate, as previously described (20). The cells were incubated at 37°C for 72 h, after which the cells were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature. PFA-fixed cells were subsequently processed as described previously (22). Every test plate included control wells with uninfected cells that were used to subtract background values. Relative infection efficiency values were calculated from the colorimetric values using a standard curve generated by serial 2-fold virus dilutions starting at 200 FFU/well. Data were considered only if the standard curves displayed correlation coefficients (r2) above 0.97.
Compound toxicity was determined by evaluating the cell biomass remaining at 72 h postinoculation by crystal violet staining and colorimetry at 570 nm as described previously (6). Compounds resulting in a reduction of the biomass below ~70% of that of the controls were considered toxic and discarded for further analysis. Compounds reducing infection efficiency more than 10-fold in the absence of toxicity were considered hits and were further characterized.
Compound stock solutions (100 μM) were prepared by diluting a 10 mM solution in DMSO (1.5 μl) in complete medium (148 μl). Subsequent serial 3-fold dilutions of the compound were prepared in culture medium (DMEM-10% FCS). Compound dilutions (50 μl) were mixed with 50 μl of a virus dilution in complete medium containing 200 FFU mixtures containing virus, and compound dilutions ranging from 50 μM to 0.4 nM were added to Huh-7.5.1-c2 cells in 96-well plates and assayed as described above. Values of 50% effective concentration (EC50) and EC90 were obtained by graphic interpolation of the compound concentration resulting in 50% and 90% HCV infection inhibition in the linear portion of the curve. Values of 50% lethal dose (LD50) were determined by evaluating the cell biomass remaining at 72 h postinoculation as described above. LD50 values were interpolated graphically from the dose-response curves. Similar values were obtained using MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide]–formazan cellular viability assays performed in parallel (23).
Huh-7, Huh-7.5.1, and Huh-7.5.1-c2 cells were cultured in DMEM-10% FCS as previously described (51, 64). JFH-1 and D183 adapted virus stocks were produced as described previously (65). Establishment of persistent JFH-1 infections was previously described (18).
Infectivity titers and HCV RNA levels were measured by titration and reverse transcription-quantitative PCR (RT-qPCR) as previously described (21, 64).
HCV E1/E2-pseudotyped lentiviral particles (HCVpp) (or, as a control, vesicular stomatitis virus G-pseudotyped lentiviral particles [VSVpp]) bearing the luciferase (luc) reporter gene were generated as described previously (4). Comparable amounts of infectious HCVpp and VSVpp were used in every experiment. For the inhibition assays, particles were mixed 1:1 with medium containing DMSO or with compound dilutions to achieve a final 5 μM concentration. The mixture was used to inoculate Huh-7 cells at 37°C for another 72-h period, after which infection efficiency was evaluated by measuring reporter gene expression using a commercial kit (luciferase assay system; Promega, Madison, WI). Relative infection values were obtained using DMSO-treated cells as a control (100%). Background levels were established by measuring luciferase expression in cells transduced with HCVpp lacking envelope glycoproteins, as previously described (4).
Huh-7.5.1-c2 cells (5 × 105 cells, 6-well plate) were inoculated (MOI of 0.01) with D183 virus (65) in the presence of 5 μM doses and incubated for 6 days at 37°C. Cells were split (1:3) at days 2 and 4 postinoculation, at which times the cells were replenished with fresh medium containing the compounds. Samples of the cells and cell supernatants were tested for infectivity and HCV RNA at days 2, 4, and 6.
Huh-7 cells were seeded in 24-well plates (5 × 104 cells/well), inoculated with D183 virus 24 h later (MOI of 10) in the presence of 5 μM doses, and further incubated for 40 h in complete medium at 37°C. Samples of the cells and supernatants were collected at 40 h postinfection for HCV RNA and infectivity analyses.
Dilutions of compound 2 (10 μM) and D183 virus (106 FFU/ml) were prepared in DMEM-10% FCS. Huh-7 cells (2.5 × 104 cells/well, 48-well plate) were either inoculated at time zero with a 1:1 mixture of the compound (250 μl) and virus dilutions (250 μl) or pretreated with the compound (1:1 mixture with medium) or the virus (250 μl of virus). At the times indicated in Fig. 2C, pretreated cells were inoculated with the virus dilution (250 μl), and cells inoculated at time zero were treated with 250 μl of the compound dilution. Cells were further incubated at 37°C for 40 h and harvested to measure HCV RNA accumulation. The compounds remained present in the medium throughout the experiment, except as shown in Fig. 2 (Wash), where the pretreated cells were washed twice with phosphate-buffered saline (PBS) before virus inoculation. Infection efficiency was determined by measuring intracellular HCV RNA accumulation 40 h postinfection.
Single-cycle infections were performed as described above by incubating virus dilutions (MOI of 10) with target cells in the presence or absence of the compounds (5 μM) for 1 h on ice. Cells were then washed twice with cold PBS. Warm medium containing either the compounds (5 μM) or DMSO as a control was added to the cells, which were further incubated at 37°C. Infection efficiency was determined by measuring intracellular HCV RNA accumulation 40 h postinfection.
A genotype 2a (JFH-1) (12) or genotype 1b (Con1-Bart79ILuc [Con1]) (11) subgenomic replicon bearing a luciferase reporter gene was electroporated into Huh-7 or Huh-7.5.1 cells, respectively, as described previously (12). For primary-translation experiments, a replication-deficient mutant JFH-1 subgenomic replicon bearing a point mutation in the NS5B catalytic site (GND mutant) was transfected (12). Cells were then plated into replicate wells (5 × 104 cells/well) in the presence of the inhibitors (5 μM) or DMSO as a negative control. Unless otherwise stated, compounds were added immediately after transfection and replenished at 24 h. Samples were collected at 5, 10, 24, and 48 h postelectroporation and tested for luciferase activity using a commercially available kit (Renilla luciferase assay system; Promega, Madison, WI).
Persistent infections were established as described previously (18). Persistently infected cells were seeded (105 cells/well, 12-well plate) the day before treatment. Compounds were diluted in medium (5 μM) and added to the cell cultures. Cells were incubated for 24 h at 37°C, after which the medium was replenished with fresh compound. After 40 h of incubation at 37°C, samples of the cells and the supernatants were analyzed for infectivity and HCV RNA.
A stock of D183 virus was used to inoculate Huh-7.5.1-c2 cells (1.5 × 106 cells, T75 flask) at a high MOI (MOI of 10) in the presence or absence of compound 2 (0.5 μM). Seventy-two hours postinfection, supernatants were collected and filtered through a 45-μm filter. Supernatants were diluted (1:10) in fresh medium and used to inoculate naïve Huh-7.5.1-c2 cells. After five blind passages in the presence of compound 2 (0.5 μM), supernatants were diluted 1:100, used to inoculate naïve cells at a low MOI in the presence of 2.5 μM compound 2, and subjected to five additional blind passages in the presence of compound 2 (2.5 μM). Diluted supernatants were used to inoculate naïve cells in the presence of 5 μM compound 2 for 10 additional blind passages in the presence of 5 μM compound. Samples of the supernatants were tested for their susceptibility to compound 2 by determining the infectivity titer in the presence or absence of 5 μM compound.
HCV RNA was isolated from the supernatants of passages 12, 14, and 18. Viral genome populations were bulk sequenced as previously described (65). All sequences were compared to the parental JFH-1 genotype, and mutations were considered only if they were present in all three samples.
Point mutants were generated by PCR-mediated mutagenesis using the following mutagenic primers (5′–3′): NsiI-RsrII fragment forward, CGCCACATGCATGCAAGCTGACCT; NsiI-RsrII fragment reverse (mutagenic E2371A), GCACGGACCGCCGGATgCGGCGG (mutations are indicated in bold, lowercase type); F2004L forward, CTGACCTCTAAATTGcTCCCCAAGCTGC; F2004L reverse, GCAGCTTGGGGAgCAATTTAGAGGTCAG; RsrII-EcoRV fragment forward, GAATCCGGCGGTCCGACGTCCCC; RsrII-EcoRV fragment reverse, GAAGAGATATCGGCCGCAAACGGCCG; K2493R forward, TCACAGAGGGCTAAAAgGGTAACTTTTGACAGG; K2493R reverse, CCTGTCAAAAGTTACCcTTTTAGCCCTCTGTGA; A2780T forward, GAGCGGAACCTGAGAaCCTTCACGGAGGCC; and A2780T reverse, GGCCTCCGTGAAGGtTCTCAGGTTCCGCTC. All PCR products were cloned into the pGEM-T vector (Promega, Madison, WI), and individual clones were sequenced to verify the presence of the mutation of interest and the absence of additional mutations. Mutant fragments were cloned into the parental vector using NsiI-RsrII sites for the F2004L and E2371A mutants and RsrII-EcoRV for the K2493R and A2780T mutants. The F2004L mutant was constructed by transferring the NsiI and BsshII fragments from the double mutant NsiI-RsrII fragment bearing the F2004L E2371A mutations into the parental plasmid. The K2493R A2780T double mutant was generated by site-directed mutagenesis of the replicon bearing the mutation A2780T.
We interrogated a small library of structurally diverse compounds by using an unbiased colorimetric cell-based screening assay for inhibitors of HCV infection that allows quantitation of JFH-1 spread in a 96-well format (20). Nontoxic compounds that strongly inhibited viral spread (>10-fold) were selected for further characterization. Analysis of the chemical structures of the antiviral compounds identified in the first round of screening led to the identification of a novel family of small molecules that significantly reduced viral spread at low concentrations (EC50 of <5 μM) in cell culture in the absence of measurable cytotoxicity (50% cytotoxic concentration [CC50] of >25 μM). The chemical structure of a representative member of the family (compound 1) is shown in Fig. 1A. Synthesis of over 300 compound 1 derivatives permitted extensive structure-activity relationship (SAR) analysis, resulting in the development of compound 2 (Fig. 1A), which displayed remarkably higher potency (EC50 of <50 nM) and comparable cytotoxicity (CC50 of >35 μM). A detailed description of the chemical library used for the screening, the SAR analysis conducted to obtain compound 2, and the structural elements required for optimal antiviral activity will be described elsewhere (unpublished data). In this report, we describe experiments that define the step of the viral life cycle that is targeted by these inhibitors and that provide insight into their mode of action against HCV in cell culture.
First, to independently confirm the ability of these compounds to interfere with HCV spread in cell culture, Huh-7 cells were inoculated at a low MOI (MOI of 0.01) with the cell culture-adapted variant of JFH-1, D183 virus (65), in the presence of compound 1 or 2 (5 μM). Extracellular-infectivity titers of supernatants collected at days 2, 4, and 6 postinfection indicated partial inhibition of HCV spread with compound 1, which caused a modest reduction in the titers found at every time point (Fig. 1B). As expected, compound 2 reduced viral titers to levels below detection (Fig. 1B), indicating that it efficiently prevented the initiation and spread of HCV infection. The antiviral activity of these compounds is underscored by the reduced intracellular HCV RNA content of compound 1-treated cultures and the complete abolition of HCV RNA replication in cells that had been pretreated with compound 2 (Fig. 1B). These results confirm the antiviral activity of these small molecules and the enhanced potency of the derivative compound 2.
Next, we performed single-cycle-infection experiments to determine the step in the viral life cycle that was targeted by the compounds. Huh-7 cells were inoculated at a high MOI (MOI of 10) in the presence of compounds 1 and 2 (5 μM). Intracellular HCV RNA levels were reduced 3-fold and >30-fold in compound 1- and compound 2-treated cells, respectively (Fig. 2A). This reduction resulted in the proportional decrease of intracellular- and extracellular-infectivity titers (Fig. 2A), indicating that compounds 1 and 2 interfere with an early step of the infection leading to the production of intracellular HCV RNA.
To examine the events preceding HCV RNA production in a single-cycle infection, we performed infections at 4°C to determine if the compounds were interfering with infection at the adsorption or postadsorption level. Huh-7 cells were inoculated at 4°C at a high MOI (MOI of 10) in the presence or absence of the antiviral compounds to permit particle adsorption without internalization, which is strongly inhibited at this temperature (33). After 1 h of incubation, the cells were washed extensively to remove free virus and compound, and infection was allowed to proceed by the addition of warm medium containing either the inhibitors or the vehicle control and by further incubation at 37°C. Analysis of intracellular HCV RNA at 40 h postinfection revealed that the compounds did not display any antiviral activity when added during particle adsorption (Fig. 2B) and that addition of the compounds postadsorption recapitulated the antiviral activity observed when the compounds were present throughout the experiment (Fig. 2B). These results suggest that the compounds target a postadsorption step in the infection and that they do not target the viral particles themselves.
In order to determine the optimal time of addition of these compounds, compound 2 was added to Huh-7 cells at different times relative to the time of inoculation (MOI of 10). Cells were pretreated (−6, −4, −2, or −1 h) with compound, or the compound was added at the time of inoculation (0 h) or after inoculation (1, 2, 4, or 6 h) and remained present throughout the experiment. Forty hours postinfection, intracellular HCV RNA levels were measured by RT-qPCR. Figure 2C shows the relative HCV RNA levels in cells treated at different times with compound 2. The curve indicates that the compound displays maximum efficacy when it is added before (Fig. 2C, no wash) or at the time of virus inoculation but is virtually inactive if it is added even shortly (<2 h) thereafter. In addition, the antiviral activity of the compound is severely attenuated if the compound is washed away before virus inoculation (Fig. 2C, wash), indicating that the compound must be present at the time of inoculation for optimal antiviral activity. This narrow time-of-addition window indicates that compound 2 targets a very early step in the viral life cycle.
Since the compounds fail to control infection if they are added shortly after inoculation, they should not have antiviral activity in persistently HCV-infected cells. To test this hypothesis, persistently infected cells were treated with compound 1 or 2 (5 μM) for 48 h. Parallel cultures were treated with a well-characterized polymerase inhibitor (2′-C-m-adenosine [2mAde]) as a positive control (9). As shown in Fig. 2D, compounds 1 and 2 had little or no effect on intracellular HCV RNA content or intracellular- or extracellular-infectivity titers. In contrast, 2mAde inhibited all of these parameters (Fig. 2D). These results demonstrate that compounds 1 and 2 have limited or no antiviral activity against established HCV infection. They also suggest that the compounds do not inhibit HCV RNA replication per se and that the ability of the compounds to suppress the accumulation of intracellular HCV RNA after inoculation at a high MOI (Fig. 2A) likely reflects their ability to inhibit a step preceding steady-state HCV RNA replication.
Similar single-cycle-infection experiments with HCV and other RNA viruses, such as lymphocytic choriomeningitis virus (LCMV) and Borna disease virus (BDV), were conducted in parallel using Huh-7 cells. Figure 3 shows that antiviral doses of compound 1 and 2 have little or no impact on LCMV or BDV infection efficiency, indicating that they display specific anti-HCV activity.
Since compounds 1 and 2 interfered with early events of HCV infection without affecting persistent HCV infection, we asked whether they blocked viral entry by using HCV-pseudotyped retroviral particles that recapitulate envelope glycoprotein-mediated receptor binding, particle internalization, and low-pH-mediated membrane fusion (3). Figure 4 shows that neither compound (5 μM) interfered significantly with either HCVpp or VSVpp infection, in contrast with the entry inhibitor fluphenazine (5 μM), which selectively reduced HCVpp infection by at least 1 order of magnitude without interfering with VSVpp infection, as previously described (22). These results suggest that the compounds interfere with a step downstream of glycoprotein-mediated fusion.
We examined the impact of compounds 1 and 2 on the ability of subgenomic HCV RNAs to initiate RNA replication after transfection into naïve target cells. This recapitulates the early steps of HCV infection after the viral genome is released into the cytoplasm. Huh-7 cells were transfected with a JFH-1 subgenomic replicon bearing a luciferase reporter gene in the absence or presence of compound 1 or 2 (5 μM) or the polymerase inhibitor 2mAde (5 μM), and luciferase activity was measured in total cell lysates at various times postinfection, as shown in Fig. 5A. Luciferase activities were comparable under all conditions at 5 and 12 h posttransfection, indicating that compounds 1 and 2 do not interfere with primary HCV internal ribosome entry site (IRES)-dependent translation of the incoming HCV RNA. This is underscored by the comparable luciferase activities observed in cells transfected with a replication-deficient mutant replicon and treated with compound 1 or 2 or 2mAde (Fig. 5B).
Luciferase activity increased in the transfected, DMSO-treated cells 24 and 48 h after transfection but not in the 2mAde-treated cells (Fig. 5A) or in the cells transfected with the replication-deficient replicon (Fig. 5B), indicating that luciferase activity reflected active HCV RNA replication. During the initial replication phase, from 12 to 24 h posttransfection, luciferase activity was slightly reduced (~3-fold) by compound 1 and more strongly reduced (~20-fold) in compound 2-treated cells (Fig. 5A). These results indicate that compounds 1 and 2 inhibit the initiation of HCV RNA replication, at a step downstream of primary translation, and that they do so proportionally to the antiviral activity they display in single-cycle infections. Interestingly, even though fresh compound was added to the cultures 24 h posttransfection, replicon replication between 24 and 48 h was inhibited only minimally in compound 1-treated cells and only moderately in compound 2-treated cells. This was in clear contrast with the profound antiviral activity of the polymerase inhibitor 2mAde (Fig. 5A). These results suggest that once HCV RNA replication is initiated, compound 1 and compound 2 are ineffective, implying that they address a unique step in the virus life cycle that is limited to the initiation phase of viral RNA replication.
To test this hypothesis, we treated the transfected cells with DMSO, the polymerase inhibitor 2mAde, or compound 1 or 2 at the time of transfection (0 h), 24 h later, or at both time points, and we measured luciferase activity 5, 24, and 48 h later (Fig. 5C, inset). As expected, administration of neither 2mAde nor compound 1 or 2 inhibited luciferase activity 5 h after transfection (prior to the onset of replication), but all agents inhibited luciferase activity 24 h after transfection (during which initiation of viral RNA replication occurs) (Fig. 5C). Inhibition was also observed at 48 h in cells that had been treated from the beginning of the experiment with compound 1 or 2, albeit slightly less than that observed at 24 h (Fig. 5C). However, when treatment was delayed until 24 h after transfection, neither compound 1 nor compound 2 displayed any antiviral activity, while the polymerase inhibitor was strongly suppressive (Fig. 5C), confirming that compounds 1 and 2 do not inhibit ongoing replication of HCV RNA. These results illustrate that compounds 1 and 2 inhibit the initiation of HCV RNA replication, thereby confirming the impact of these compounds in single-cycle-infection experiments and suggesting that they inhibit viral infection by interfering with the initiation of viral replication after translation of the incoming viral RNAs.
Similar transfection experiments were performed with a genotype 1b, Con1-based subgenomic replicon in Huh-7.5.1 cells. As expected based on this replicon's poor replicative capacity relative to that of JFH-1 (34), Con1-luc transfection induced luciferase activity that rapidly decreased after electroporation (Fig. 6A), while luciferase activity increased exponentially in JFH-1–luc–transfected cells (Fig. 6B). Nevertheless, Con1-luc replication was demonstrated by its sensitivity to the polymerase inhibitor 2mAde (Fig. 6A), similar to what has been reported using a replication-deficient mutant (34). However, because of the limited replication capacity of the Con1 replicon (Fig. 6A), the luciferase signal obtained at 24 h derived predominantly from translation of incoming genomes rather than from RNA replication, as shown by the similarity of the signals in the 2mAde-treated and untreated cells. Thus, using 2mAde-treated cells as a reference, we evaluated the antiviral activity of these compounds against establishment of Con1 RNA replication at 24 h, the earliest time point at which replication could be demonstrated (Fig. 6A). Figure 6C shows that compound 1 (5 μM) produced a small although significant (P < 0.05) reduction in luciferase accumulation at 24 h posttransfection and that compound 2 inhibited Con1 RNA replication with a magnitude comparable to that of the control inhibitor 2mAde (5 μM). These results suggest that this genotype 1b replicon is partially susceptible to the antiviral activity of these compounds, that compound 2 displays enhanced antiviral activity compared to that of compound 1, and that antiviral activity of this family of compounds is not limited to JFH-1.
In order to gain insight into the mode of action of compounds 1 and 2, we attempted to select JFH-1 variants that were resistant to the antiviral action of compound 2. We serially passaged D183 JFH-1 virus in the presence of sequentially increasing concentrations of compound 2. This led to the emergence of a compound 2-resistant (C2R) virus population; to reduce its infectivity by 50%, this virus required a concentration of compound 2 >10 fold higher than that for a control virus which was passaged in parallel in the absence of compound 2 (Fig. 7A). This reduced susceptibility was specific for compound 2, as the C2R virus displayed a sensitivity to 2mAde similar to that of the control virus (Fig. 7B). As shown in Fig. 7C, antiviral doses of compound 2 could not prevent intracellular HCV RNA accumulation in Huh-7 cells inoculated with the C2R variant at a high MOI, in contrast to the strong antiviral activity displayed against the control virus. These results confirmed that a resistant variant had been selected. In order to analyze the genetic determinants conferring resistance and therefore investigate which areas of the viral genome undergo selective pressure by compound 2, we sequenced the viral genomes present in the supernatants of three nonconsecutive passages of the virus under selection as well as control viruses passaged in the absence of compound. Common mutations were found in both control and C2R virus (Fig. 7D). Figure 7E summarizes the mutations found in all three passages of the C2R virus that were not found in the control supernatants and therefore might confer resistance to compound 2.
Nonsynonymous mutations were found in the coding sequences corresponding to E1 (V223D), E2 (N410D), p7 (H781Y), NS2 (Q931R), NS5A (F2004L and E2371A), and NS5B (K2493R and A2780T). Despite these genetic differences, control and C2R viruses display comparable growth curves (Fig. 8A). Since our results indicate that the antiviral activity could be recapitulated in the context of the subgenomic replicon (Fig. 5) that lacks the sequences encoding the structural proteins p7 and NS2, we focused our attention on the mutations in NS5A and NS5B. Individual, double, and quadruple mutations in NS5A and NS5B were engineered into a wild-type JFH-1 subgenomic replicon bearing a luciferase reporter gene. In vitro-transcribed RNAs (5 μg) were electroporated into Huh-7 cells in the presence of DMSO or antiviral doses (2.5 and 5 μM) of compound 2. Cell lysates were prepared at 5 and 24 h posttransfection and assayed for luciferase activity. All mutant replicons displayed comparable (<2-fold difference) replication capacities as measured by the luciferase signal obtained 24 h posttransfection in the absence of inhibitors (Fig. 8B). Figure 8C shows the expected dose-dependent reduction of luciferase signal for the wild-type replicon. The antiviral activity of compound 2 was clearly reduced against the replicons bearing all four mutations, the NS5A double mutation, and the single mutation F2004L, which were insensitive to 2.5 μM compound 2 and only partially inhibited at 5 μM (Fig. 7). In contrast, the NS5A E2371A mutant and both NS5B mutants displayed sensitivities similar to that of the wild type. These results indicate that the F2004L mutation (position F28 in NS5A), which is not conserved among different HCV genotypes (Fig. 9), is sufficient to confer partial resistance to the antiviral activity of compound 2 in the context of JFH-1 and suggest that NS5A might be directly or indirectly involved in the process targeted by these compounds.
Study of the HCV life cycle in surrogate models of infection, e.g., HCVpp and replicon cell lines, has permitted analysis of the cellular and molecular events that govern viral entry and steady-state HCV RNA replication. Aspects of the viral life cycle not recapitulated in these systems, such as those occurring downstream of endosomal internalization of the infectious particles, leading to primary translation of the viral genome and establishment of the replication complexes, are beginning to be characterized (5, 12) thanks to the development of cell culture infection systems that recapitulate every step of the HCV life cycle (37, 61, 64).
By adaptation of this cell culture system into an unbiased cell-based screening methodology (22), interrogation of chemical libraries for antiviral molecules against HCV enables discovery of antiviral compounds that interrupt aspects of the infection that were previously uncharacterized. Thus, this methodology has the potential not only to provide novel lead compounds for therapy against this important pathogen but also to identify novel chemical probes to study aspects of HCV infection that have been functionally and biochemically uncharacterized.
Using this methodology, we have identified and characterized a novel family of small molecules that efficiently inhibit viral spread at nanomolar concentrations. These compounds efficiently inhibited HCV infection without altering viral entry, as shown by the lack of antiviral activity against HCVpp (Fig. 4), or interfering with persistent HCV RNA replication and virus particle production in persistently infected cells (Fig. 2D).
Time-of-addition experiments indicate that the inhibition occurs at early steps of the infection, since addition of the compounds as early as 2 h after virus inoculation resulted in the virtual loss of antiviral activity (Fig. 2C). The results shown in Fig. 2C argue against a rapid loss of antiviral activity due to compound instability since the compounds retained full antiviral activity even when they were added to the cultures 6 h before virus inoculation. Since HCVpp entry was unaffected by these compounds, we postulated that they target steps downstream of viral entry. This hypothesis was confirmed by the antiviral activity of the compounds against the initiation of HCV RNA replication by HCV subgenomic replicons that were electroporated into naïve Huh-7 cells (Fig. 5A and B). This effect was dependent on the time of addition of the compounds and was not observed once HCV RNA replication had been initiated (Fig. 5C), recapitulating the lack of antiviral activity observed in persistently infected cells. Overall, these observations indicate that compounds 1 and 2 specifically target a very early step in HCV RNA replication without altering steady-state HCV RNA production. These results suggest that the compounds target a transient event that is rate limiting for the initiation of HCV RNA replication, rather than HCV RNA replication per se.
It has recently been shown that the cellular autophagy machinery is required for translation of incoming HCV genomes but not for translation of progeny genomes (12). Collectively, those observations and the results presented herein suggest that the cellular and viral factors required to initiate viral replication are different from those required to maintain it once replication complexes have formed. Another possible interpretation of our results derives from the fact that expression of HCV proteins, notably NS4B, causes a profound reorganization of cellular membranous compartments to promote the formation of replication complexes in modified ER membranes (5, 13). It is therefore formally possible that the compounds described above target HCV RNA replication per se but that virus-induced cellular changes (e.g., alteration of intracellular membranes) reduce the access of the compounds to their molecular target(s) sufficiently once replication complexes have been established to reduce their efficacy, resulting in an apparent lack of antiviral activity.
Selection of virus variants with reduced susceptibility to compound 2 (C2R virus) demonstrates the specific antiviral activity of this compound. We have shown that a point mutation in NS5A (F2004L) identified in the resistant virus variants is sufficient to confer the resistance phenotype to subgenomic replicons (Fig. 8), suggesting that the mechanisms by which these compounds inhibit HCV infection and establishment of the subgenomic-replicon replication are the same. Our results also indicate that mutation F2004L did not result in a significant fitness cost, as the C2R virus grows to titers comparable in magnitude (106 FFU/ml) and kinetics to those of the control virus (Fig. 8A). Moreover, introduction of the F2004L point mutation into the subgenomic replicon did not impair baseline replication (Fig. 8B), indicating that this mutation does not weaken viral replication even in a wild-type genetic background.
The NS5A residue F28 (F2004 in the polyprotein) is not conserved among all genotypes and strains, e.g., Con1 and J4L6 from genotype 1b display a leucine residue in that position (Fig. 9). However, a Con1-based replicon was susceptible to compound 2 (Fig. 6C) even at a concentration (2.5 μM) that was inactive against the JFH-1 F28L mutant, suggesting that a leucine residue in position 28 does not confer resistance to compound 2 in the context of genotype 1b. In any case, in the context of JFH-1 (genotype 2a), position F28 is located in a conserved class I proline-rich (PR) motif immediately adjacent to the membrane-anchoring amphipathic alpha helix located at the N terminus of NS5A (57). Although no specific function has been attributed to this conserved PR motif, PR motifs in NS5A have been proposed as structural elements that determine interaction of NS5A with cellular factors containing Src homology 3 (SH3) domains (57). This could be demonstrated for the C-terminal proline-rich motif, which mediates interaction with kinases like phosphatidylinositol 3-kinase (PI3K) (56), Lck, Hck, and Fyn (40), as well as adaptor proteins such as Grb2 (57) and amphiphysin II/BinI (63), but not for the N-terminal motif, in which the mutation conferring resistance to compound 2 (F28L) is included. It is therefore possible that compounds of the chemical family described here interfere with interactions of NS5A via its N-terminal PR motif and that mutation F28L restores such interactions that could be essential for the establishment of HCV RNA replication. Position F28 is located in close proximity to the N-terminal membrane-anchoring domain in NS5A and might contribute to the interaction of this protein with cellular membranes. It is possible that, during the initial phases of the infection, compounds 1 and 2 interfere with docking of NS5A into membranes devoid of viral replication complexes and that this does not occur once replication complexes have been formed. While these speculative scenarios would explain our observations, extensive biochemical studies are required to define the molecular events underlying both antiviral activity and resistance. Studies aimed at determining the composition of the molecular complexes that compounds 1 and 2 may bind in the cell will contribute to understanding the mechanisms underlying the establishment of HCV RNA replication complexes, a process in which NS5A could play a specific role.
NS5A has recently been revealed as a novel target for potent antiviral compounds that inhibit viral RNA replication (e.g., BMS-790052) (17). Genetic evidence indicates that compound 2 targets NS5A directly or indirectly (Fig. 6) but seems to do so by a different, unprecedented mechanism that targets initiation but not steady-state HCV RNA replication. Remarkably, despite this apparent differential mode of action, position F28 in JFH-1 is aligned with position 28 in genotypes 1a and 1b (Fig. 9), where mutations M28T and L28T, respectively, have been reported to confer partial resistance to BMS-790052 (14, 17). These genetic data indicate that both compounds impose similar selective pressures on the N terminus of NS5A, suggesting that they could target similar functions in NS5A but appear to do so by different molecular mechanisms.
Multiple functions involving different steps in the viral life cycle have been attributed to NS5A. However, no specific enzymatic activity has been ascribed to NS5A, and the molecular mechanisms by which it functions remain elusive. Thus, the inhibitors described herein constitute potentially powerful chemical tools that could facilitate our understanding of the functions that NS5A plays in the viral life cycle and may lead to the development of novel compounds with improved therapeutic potential.
We are grateful to Takaji Wakita (National Institute of Infectious Diseases, Tokyo, Japan) for providing the infectious JFH-1 molecular clone and replicon constructs, Charles Rice (Rockefeller University) for providing the Huh-7.5.1 cells from which the Huh-7.5.1-c2 cells were derived, Mansun Law and Dennis Burton (The Scripps Research Institute, La Jolla, CA) for providing the recombinant human IgG anti-E2, François-Loïc Cosset (Ecole Normale Superieure, INSERM U758, Lyon, France) for providing the vectors necessary for HCVpp production, Jeffrey Glenn from Stanford University (Palo Alto, CA) for providing the Con1-luc replicon, and Weidong Zhong from Gilead Sciences (Foster City, CA) for providing the 2′-C-m-adenosine. We thank our colleagues Urtzi Garaigorta and Stefan Wieland for their expert advice and useful discussions. We are grateful to Brian Boyd for excellent technical assistance.
This work was supported by NIH grants R01-CA108304 and R01-AI079043.
This is manuscript number 20959 from The Scripps Research Institute.
Published ahead of print on 23 March 2011.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
Michael got a good start in his career by studying at Lincoln Park High School, an institution well-known for its drama program. According to Michael, casting directors would come to his school in Chicago, allowing him and his schoolmates a chance to audition for movies, pilots and commercials.
User Score: 52
User Score: 21
User Score: 2

subset: commoncrawl


text:
PDF to Speech Software
- Downloads:
- Views:
- Rating:
PDF Word to voice speech converter software turns words into audio format with natural speaking voices. PDF reader application program speaks read all type of text content like word, excel, html, plain text, WebPages, email, eBook, RTF, RSS feeds and save it to wav file format. Text speaker tool can read your PDF documents loudly, or you can convert them into WAV files for listening, anytime and anywhere just open a file and click speak button to hear it, understandable human sounding voice or audio sound just click save as button to convert and create a WAV file for future to listen. Listen to PDF document, instead of straining your eyes. Now you can hear text documents spoken in your native language since PDF speaker utility has word processing features, you can make generate produce and edit text files without the need of any other program. You can easily play this file in future using iPod, zune, PC audio players, mp3 player etc. Clipboard to talking program composer producer generator creator maker tools make build change composes converts all type of text content into wave audio sound file.
* PDF to speech software speaks your text files, eBooks, rich text files, emails, Word, Excel document and HTML files clearly and loudly.
* PDF to voice speak software converts your text content into spoken audio sound file.
* Listen to the PDF text data copied to the clipboard immediately.
* Product supports all computer voices.
* Text to speak software has simple, easy to use and user friendly GUI.
* Text reader utility works with Windows 7, 98, ME, NT, 2000, 2003, NT 3.x, NT 4.x, XP, Vista and Longhorn operating system.
Free download from Shareware Connection - PDF word to sound speech converter software makes your PC able to talk. Html to speak reader tool is used to read your eBooks Excel Word Email text documents and save it to wav file, used for future reference and it is play with any mp3 audio player
Version: 126.96.36.199 | Size: 307 KB | Platform: Win2000, Windows Server, Windows Vista Business, Windows Vista Business x64, Windows Vista Enterprise, Windows Vista Enterprise x64, Windows Vista Home Basic, Windows Vista Home Basic x64, Windows Vista Home Premium, Windows Vista Home Premium x64, Windows Vista Starter, Windows Vista Ultimate, Windows Vista Ultimate x64, WinOther
Released Date: 05-06-2009 | Rating: 0 | Title: PDF to Speech Software
Author Url: http://www.addpdffiles.com
Program Info Url: http://www.addpdffiles.com
Download Url: http://www.addpdffiles.com/text-speech-demo.exe
Screenshot Url: http://www.addpdffiles.com/text-to-speak-screen.jpg
Word Text to Speech - Word text to voice sound speech software makes your PC able to talk. Email to speak converter reader tool is used to read your eBooks Excel PDF documents and save it to wav format, used for future reference and it is play with any audio player.
Convert Picture to PDF - Pictures to PDF converter software convert photos to PDF, images to PDF, snapshot to PDF, JPG to PDF, BMP to PDF, TIFF to PDF, GIF to PDF, WMF to PDF. Image2PDF conversion creator utility create pdf from TIF PCD PSD TGA PNG PCX DCX JPEG EMF PIC files
TIFF to PDF Conversion - Tiff to PDF conversion program converting tif into pdf file. TIFF 2 PDF converter software support to convert images to PDF, photos to PDF and snapshot to PDF. TIFF2PDF creator utility create pdf document from single or multipage tif files formats.
Merging and Appending PDF Files Software - Add PDF Files has been released Adding PDF files pages together software adding pdfs, merge PDFs, deleting PDF pages, join PDFs, combining pdfs and split PDFs.
Convert image to PDF Software - Convert image to PDF Software convert photos to PDF, images to PDF, snapshot to PDF, JPG to PDF, BMP in PDF, TIFF in PDF, GIF in PDF, WMF to PDF. Image in PDF converter tool create PDF from TIFF PCD BMP JPEG JPG PSD TGA PNG PCX DCX EMF PIC files
MixMeister Express
Funny Voice
Karaoke Sound Tools
Blaze Audio Voice Cloak
Ringtone Convertor
New Musicmatch Jukebox
Music Recognition Pro
Text to Voice
Text to Speech
Text to Audio Software
Text to Voice Converter
Audio Encoders & Decoders
Audio File Players
Audio File Recorders
Authoring Tools
CD & DVD Tools
Desktop Publishing
Graphic Capture
Graphic Viewers
Image Editing
Tamil Pdf Speech Software
Mac Pdf Speech Software
Pdf Speech Software Mac
Pdf Speech Reader Software
Speech Pdf File Software
VeryDOC PDF Rendering SDK - VeryDOC PDF Rendering SDK (ActiveX) is a PDF file viewer component. PDF Rendering SDK provides a standalone embeddable PDF Reader for windows application developers. PDF Rendering SDK is usually used to quickly display PDF files.
PDF Suite NET - PDF Suite provides all the functionalities and features of PDF Converter and PDF
PDF to Multipage TIFF Converter - PDF to Multipage TIFF Converter convert PDF files into JPG, BMP, TIFF formats.
AEP Audio Studio - AEP Audio Studio is an all-in-one multi-track audio mixing, editing and recording utility. It lets you record, overdub edit and mix multiple tracks of .wav, .mp3, .wma and other major audio format files.
Mgosoft PDF Split Merge - Mgosoft PDF Split Merge is a simple, stand-alone application that lets you split any Acrobat pdf file into smaller pdf files or combine two or more Acrobat PDF files into a single PDF document.
Shareware Connection periodically updates pricing and software information of 'PDF to Speech Software' from company source 'Add PDF Files' , so some information may be slightly out-of-date. You should confirm all information before relying on it. Software piracy is theft, Using 'PDF to Speech Software' crack, password, serial numbers, registration codes, key generators is illegal and prevent future development of PDF to Speech Software.
Autodesk Smoke - Autodesk is the company well known for making some of the most feature rich and versatile tools for 3D modeling, animation and engineering design related work. Its Smoke is a Mac OS specific app that is aimed at professionals involved in ...
Freemake Video Converter - Video conversion can become a necessity for average PC users who find it increasingly hard to manage an ever growing collection of media and video files. The DVDs, videos obtained from web and clips shot with mobile devices keep growing. Over ...
WinKleaner Classic - Whether you use a laptop or desktop, with time it is likely to show deficit in performance and speed. This is owing to the inherent limitations of Windows OS and things can vary based on your usage pattern. Traces of removed programs existing in ...
TunnelBear - While internet access has become cheaper and faster over the years, certain downsides of the World Wide Web still looms and affects users. Even if you have the resources and monetary capacity to afford a blazing fast and unlimited internet ...
MacPilot - The Mac OS contains many advanced features that can make computing a different experience altogether. However, not all Mac users are aware of such deep rooted features of the Apple OS. The usual surface customizations are what most of them remain ...
Diver Reviver - It is not only malware and viruses that can affect performance of your computer. Malfunctioning and outdated drivers for hardware components can also affect speed and performance of a PC perceptibly. While seasoned users take the pain of ...
Undela - Computers users may lose data owing to a number of reasons. Data loss caused by hardware failure does not take place regularly. However, there are many instances when you may end up deleting files and folders by mistake. In shared computers, ...
Protector Plus 2014 (32-bit) - Without using suitable security software in PC, you cannot have peace of mind nowadays. Increasing instances of malware attacks and attacking exploits have prompted average PC users to resort to using antivirus and security suites for both home ...
Simpo PDF Creator Pro - Millions of PC users resort to using PDF format for exchanging and sharing documents for a number of needs. PDF is cross platform compatible, print friendly and can contain various objects like text, image and fields. While a majority of people ...
GhostSurf Platinum - While it is difficult to stay without resorting to the internet, you need to be aware of the risks that lurk in avenues of the Internet. Malicious users, hackers, data thieves are active online and they look for users with less secured ...


text:
This profile was last updated on . .
Is this you? Claim your profile.
St Anne's College
Woodstock Road
Oxford, Oxfordshire OX2 6HS
United Kingdom
St Anne's is committed to keeping our world-wide community of 8,000 alumnae and friends in touch with each other, and with the College. The College is a vibrant, intellectual community, as demonstrated both by our busy events calendar - which is packed ... more
BPhil Course Director
Fixed-term Departmental Lecturer
Lecturer In the Philosophy Department
Doctoral Student At the Department of Philosophy
Examination Fellow
All Souls College
Prize Fellow
All Souls College
Research Fellow and Policy Analyst
Russell Group
Magdalen College , Oxford
Bachelor of Arts
physics and philosophy
University College London
All Souls College, Oxford
www.all-souls.ox.ac.uk [cached]
Dr Ian Phillips, Examination Fellow
New Arrivals - Trinity Term 2013 - Faculty of Philosophy
www.philosophy.ox.ac.uk [cached]
Ian Phillips has been appointed to a CUF Lecturership in association with the Gabrielle Taylor Tutorial Fellowship at St Anne's College.
Faculty Board Officers - Faculty of Philosophy
www.philosophy.ox.ac.uk [cached]
Faculty Members - Faculty of Philosophy
www.philosophy.ox.ac.uk [cached]
Dr Ian Phillips St Anne's College
www.russellgroup.ac.uk [cached]
Braden Phillips
California Conference for Equality and Justice
Gwen Phillips
Michael Phillips
Cisco Systems Inc
Tess Phillips
Outdoor Media Association Inc


text:
How to find "original" principal invested (ignoring rolling amounts due to gains from original investment in subsequent investments) ?I have a very important question which I could not figure out how Quicken can help answer regarding my investments. I will appreciate if someone can provide the answer / point to the right resource for same.
I need to find out as to How much of my own money (principal if I can use that word) is invested into my portfolio ? It appeared very simple initially as the Net amount invested report gives the same but here is the catch. I need only my "original principal" and not the net amount invested across all the stocks / mutual funds (i.e. bought - sold). This is because I might have purchased first stock, made a gain out of the same by selling it partially and bought second stock from the gains of the first stock. Hence, the amount invested in the second stock does not really come from my original principal but rather gains from the first stock. Now, I will have two stocks in the portfolio and the above scenario will repeat itself tens of times over the years as the stocks will keep on rolling through buys and sells. Hence, it becomes very difficult to find the original sum invested. At the end, if this sum is positive, that means my money is in stocks and if this sum is negative, it means that I have recovered all my principal and my stock holdings is entirely from the returns from the market or dividend income etc.
I also understand that as per IRS, the cost basis of each stock has to be separately calculated. However, I do not want the above mentioned report for tax purposes. It is only for my understanding of amounts invested irrespective of tax laws.


text:
- Sign in
- Join
- Find a rental
- Manage your booking
- Mineral rentals
- How to book
- Traveler Help Center
- Write a review
The Waters
From $500 / night
balance of due
Description from the manager
Description from the manager
Bedrooms: 6/
Bathrooms: 5/
Sleeps: 15/
Type: House
Book this 6 bedroom, 5 bathroom house in Mineral today! A total of 15 people can sleep here comfortably.
Guests can enjoy a pool, hot tub, and deck among other amenities offered by this rental.
Travelers are sharing their opinions on this property. It has an overall rating of 3/5 thanks to an average of 2 reviews.
The owner responds to 70% of inquiries, which is lower than average, and last updated the property's calendar yesterday.
About the manager
About the manager
Dockside Realty
Dockside Realty
- Response rate: 70%
- Response time: Within a day
- Calendar updated: yesterday
Overall rating:
3.6 / 5.0
More properties Call: +1-877-758-0098


text:
The Personal Philosophical Profile Test
Category 3
All possible test results
Category 1
Description 1 Read more
Category 2
Description 2 Read more
Category 3
Description 3 Read more
Category 4
Description 4 Read more
Category 5
Description 5 Read more
Category 6
Description 6 Read more
Category 7
Description 7 Read more
Category 8
Description 8 Read more
Category 9
Description 9 Read more
Category 10
Description 10 Read more
Category 11
Description 11 Read more
Category 12
Description 12 Read more
Category 13
Description 13 Read more
Category 14
Description 14 Read more
Category 15
Description 15 Read more
Category 16
Description 16 Read more
Tests others are taking
test of that you are falling in love to an older man
lutk427 / f / Bisexual
MoonLitGal56 / f / Gay
The Penis SiZe Preference For Girls Test
IamMrBrooks32 / m / Straight
subterfuge88927 / m / Straight
The Fucked Stream of Consciousness Test
last--exit20 / f / Bisexual
More tests we think you'll like
Taken 19768 times.
The Should You Teach High School Test
Find out if I think teaching high school is the right career for you! Keep in mind, it's only my personal opinion. This test will be based on me, basically.
Taken 6477 times.
The runescape Test
Hi! And welcome to my runescape Test. I'll be using advanced logic and knowledge to determine your true nature.
Taken 92183 times.
The Are you gay bi or straight? Test
This is the ultimate and most accurate test one could ever take in this area. This test is for boys, but if you're a girl and you feel you REALLY want...
Taken 9758 times.
Famous Movie Quotes Test
What is your knowledge of Famous Movie Quotes?
Taken 6736 times.
The Adventuresome Test
In my opinion, being adventurous is one of the best things you can do for yourself. If you try new things every now and then, sure you could run into...
Taken 1668 times.
O teste das facções
Find out witch of Divergent faction you belong.
Taken 42752 times.
The Which Wife are You Test
This test is to show which kind of wife you are, or will be (if that is your thing). Remember to answer honestly, otherwise any future spouses out th...
Taken 1180 times.
The Are You Lesbian Quiz Test
Find out if you are a lesbian, bi or straight
Taken 2086 times.
The Megaman NES Test
Hi! And welcome to my Megaman NES Test. I will ask you questions about the Megaman NES saga! Some questions will be easy, others will be hard! Good l...
Taken 6837 times.
The MeTest
now do you really know me?
Connect your existing OkCupid account
Sign in
Welcome Back!
Restore your account to continue meeting new people.
Reset your password
We'll email you a link to reset it.
Email sent!
An email is on its way to .
If you don't see it, try checking your Spam folder.
Enter Authentication Code
Sign in
Not so fast!
You need to complete your basic info on the mobile app before you can use the desktop site.


text:
Trending Now
Top Definition
Pig fucking asshole who deserves nothing short of sodomy with razor wire
Nothing sucks worse than being Flirmy.
by PROPAIN October 27, 2003
A stupid lazy whore mother fucking bitch piece of shit.
Don't insult Saddam by calling him a flirmy, thats going WAY too far.
by PumpMasterCunty April 08, 2003
Keeps the ancient Roman tradition of sodomizing boys alive.
Look at flirmy fucking that little boy in the asshole!
by PumpMasterCunty April 08, 2003


text:
TOPIC : Culture, integration and European public space
|Topic identifier:
|Publication date:
|14 October 2015
|Types of action:
|ERA-NET-Cofund ERA-NET Cofund
|DeadlineModel: Planned opening date:
|single-stage 04 October 2016
|02 February 2017 17:00:00
|Time Zone : (Brussels time)
Topic DescriptionSpecific Challenge:
"Europe" has existed as a cultural, political and economic identity for centuries, although the nature and coherence of that identity has been contested greatly over time. One of the major challenges for Europe since the mid twentieth century has been the challenge of linking European identity to forms of integration that produce mutual benefit through tolerance, respect and creative interaction, and avoid the devastating conflicts of the past. Culture plays an essential role in this process, since the realm of culture is where values, belief-systems, memories, languages, educational systems, artistic practices and social lives operate and evolve. Culture is where forms of creativity and diversity can be modelled or practiced; it is also a place where separateness, difference and specificity can be asserted and maintained in productive ways that enhance our quality of life. The cultural, political and economic spheres exist in dynamic relation to each other, and the coherence of Europe at the political and economic levels is closely related to and impacted by events and practices at the level of culture.
The attempt to create European integration (which is to be distinguished from simple "homogenisation") at political, economic and cultural levels has been incomplete; this is evident in the phenomena of xenophobia, extremism, ethnic conflict, Euro-scepticism and the revival of the North-South and East-West divides, as well as electoral apathy or disenchantment. At the same time, there is strong awareness among citizens and immigrants of living in a distinct "European" space, both real and imagined. It is made real in geographical focal points like cities, particular regions and landscapes, and in other forms of cultural heritage. Arts and creative industries have also proved to be vehicles of European integration: a growing number of vibrant urban spaces, populated by international communities of creative practitioners, are now acknowledged as engines of cultural and economic development and innovation. The challenge for research is to identify how the relations between culture and integration have been modelled and how they can be better understood for the immediate future.
Specifically, there is an urgent need to understand the role of culture in defining "public space", and role of culture in enabling or problematizing cultural integration whilst respecting diversity. "Public spaces" are the arenas in which key cultural interactions and societal dynamics take place and can be observed. This notion of "public space" can be interpreted widely: it includes physical and built environments, landscapes and material culture. But it also includes cultural zones, public spheres and "virtual" spaces defined by media, language, ethnicity, shared values, political allegiances, religious identities, creative practices, and communication technologies. These spaces are populated not just by people but by material objects, texts, art works, performances and institutions, all of which contribute to the creation of a sense of place. Recent research is acknowledging the crucial importance of physical things, objects and material traces of culture, and the value of material culture for providing new ways of looking at multiple histories and identities within a diversified Europe.Scope:
Activities under this topic should explore the dynamics through which European "public spaces" both shape, and are shaped, by cultural activity, and the dynamics through which integration can be practised and understood. This will involve investigating a variety of perspectives: for example, historical models for European public space, and the relevance of such models for today; the impact of migration on culture and the creation of public space; the role of material culture in shaping public spaces; the role of creativity in defining European identity; and the role of the cultural sphere in either enabling or challenging integration at political and economic levels. Activities may also include researching questions such as: How meaningful is it to speak of a common "European" culture or history or public space? How should such a concept be modelled, and how has it been modelled in the past? How has the circulation of knowledge (through media, research, publishing practices, intellectual and education networks) contributed to or challenged European integration? How do various European spaces and identities make use of the media and systems of representation? Are representations of Europe mainly created and captured by Northern/continental Europe and how do Southern and Central and Eastern Europe citizens contribute and participate to a European public space? What are the relations between material objects and cultural and political practices? How can multiple histories and cultures be integrated into a notion of national or "European" identities?
There is a strong need for humanities-oriented research in this topic, as a well as a need for multi-disciplinary engagement representing also a large variety of European countries well balanced geographically, including from Southern and Central and Eastern Europe. There is significant potential also to facilitate new collaborations between researchers and partners in the media, heritage, creative and other sectors, thus adding value to research-led knowledge exchange.
The proposed ERA-NET Cofund will have a wide geographical coverage, including Southern and Central and Eastern Europe. It aims at coordinating the research efforts of the participating Member States, Associated States and Regions in the field described. Proposals should pool the necessary financial resources from the participating national (or regional) research programmes and implement a joint transnational call for proposals with EU co-funding (resulting in grants to third parties) to fund multinational innovative research initiatives in this domain. Proposers are encouraged to implement other joint activities, including additional joint calls without EU co-funding.
Participation of legal entities from international partner countries is encouraged in the joint call as well as in other joint activities. Participants from countries which are not automatically eligible for funding[1] may nonetheless request a Union contribution on to cover the coordination costs of additional activities on the basis of the ERA-NET unit cost.
The Commission considers that proposals requesting a contribution from the EU of a maximum of EUR 5 million would allow this specific challenge to be addressed appropriately.Expected Impact:
The ERA-NET Co-fund Action will mobilise the wide range of transnational and multi-disciplinary perspectives necessary to understanding the relationships of culture, European integration and "public space", including in Southern and Central and Eastern Europe. The research is expected to lead to a richer, more complex and comprehensive understanding of the cultural dimensions of European integration; to discover and analyse historical models and precedents for integration in Europe; to give new insights that promote the full potential of citizens' engagement with European public and cultural spaces; to stimulate public, political and scholarly debate about the future prospects of European integration; to provide a platform for changes to existing structures, policies and practices, such as the development of new modes of interactive and reciprocal engagement between users, academics and those working in the media, creative industries, and heritage sectors.Delegation Exception Footnote:
This activity directly aimed at supporting the development and implementation of evidence base for R&I policies and supporting various groups of stakeholders. It is excluded from the delegation to Research Executive Agency and will be implemented by the Commission services.
Topic conditions and documents
Please read carefully all provisions below before the preparation of your application.
- List of countries and applicable rules for funding: described in part A of the General Annexes of the General Work Programme.
Note also that a number of non-EU/non-Associated Countries that are not automatically eligible for funding have made specific provisions for making funding available for their participants in Horizon 2020 projects (follow the links to Australia, Brazil, Canada, China, Hong Kong & Macau, India, Japan, Republic of Korea, Mexico, Russia, Taiwan).
- Eligibility and admissibility conditions: described in part B and C of the General Annexes of the General Work Programme
Proposal page limits and layout: Please refer to Part B of the standard proposal template.
- Evaluation
3.1 Evaluation criteria and procedure, scoring and threshold: described in part H of the General Annexes of the General Work Programme
3.2 Specific evaluation procedure: At least 1 proposal per topic will be selected for funding provided it passes all evaluation thresholds.
3.3 Submission and evaluation process: Guide to the submission and evaluation process
- Indicative timetable for evaluation and grant agreement:
Information on the outcome of single-stage evaluation: maximum 5 months from the deadline for submission.
Signature of grant agreements: maximum 8 months from the deadline for submission.
Members of consortium are required to conclude a consortium agreement, in principle prior to the signature of the grant agreement.
- Provisions, proposal templates and evaluation forms for the type(s) of action(s) under this topic:
Research and Innovation Action:
Specific provisions and funding rates
Standard proposal template
Standard evaluation form
H2020 General MGA -Multi-Beneficiary
Annotated Grant Agreement
- Additional provisions:
Horizon 2020 budget flexibility
Classified information
Technology readiness levels (TRL) – where a topic description refers to TRL, these definitions apply.
Financial support to Third Parties – where a topic description foresees financial support to Third Parties, these provisions apply.
- Open access must be granted to all scientific publications resulting from Horizon 2020 actions, and proposals must refer to measures envisaged. Where relevant, proposals should also provide information on how the participants will manage the research data generated and/or collected during the project, such as details on what types of data the project will generate, whether and how this data will be exploited or made accessible for verification and re-use, and how it will be curated and preserved.
This topic participates per default in the open access to research data pilot which aims to improve and maximise access to and re-use of research data generated by projects:
• The pilot applies to the data needed to validate the results presented in scientific publications. Additionally, projects can choose to make other data available for open access and need to describe their approach in a Data Management Plan (to be provided within six months after the project start).
• Note that the evaluation phase proposals will not be evaluated more favourably because they are part of the Pilot, and will not be penalised for opting out of the Pilot.
• Projects can at any stage opt-out of the pilot.
The legal requirements for projects participating in this pilot are in the article 29.3 of the Model Grant Agreement.
Further information on the Open Research Data Pilot is made available in the H2020 Online Manual.
- Additional documents
H2020 Work Programme 2016-17: Introduction
H2020 Work Programme 2016-17: Europe in a changing world - inclusive, innovative and reflective societies
H2020 Work Programme 2017-17: Dissemination, Exploitation and Evaluation
H2020 Work Programme 2016-17: General Annexes
Legal basis - Framework Programme
Legal basis - Rules for Participation
Legal basis - Specific Programme
Submission Service
The submission system is planned to be opened on the date stated on the topic header.
Get support
H2020 Online Manual your online guide on the procedures from proposal submission to managing your grant.
Participant Portal FAQ – Submission of proposals.
National Contact Points (NCP) - contact your NCP for further assistance.
Research Enquiry Service – ask questions about any aspect of European research in general and the EU Research Framework Programmes in particular.
Enterprise Europe Network – contact your EEN national contact for advice to businesses with special focus on SMEs. The support includes guidance on the EU research funding.
IT Helpdesk- contact the Participant Portal IT helpdesk for questions such as forgotten passwords, access rights and roles, technical aspects of submission of proposals, etc.
Ethics – for compliance with ethical issues, see the Participant Portal and Science and Society Portal
European IPR Helpdesk assists you on intellectual property issues
CEN and CENELEC, the European Standards Organisations, advise you how to tackle standardisation in your project proposal. Contact CEN-CENELEC Research Helpdesk at email@example.com.
The European Charter for Researchers and the Code of Conduct for their recruitment
Partner Search Services help you find a partner organisation for your proposal


text:
The Big 12 agreed to a $2.5 billion college football rights deal with ESPN and FOX that runs through 2025.
The deal is worth approximately $20 million per school per year and was initially reported by CBS Sports nearly six months ago.
ESPN announced the deal which is similar to network broadcast partnerships with the Pac-12 -- a joint deal with ESPN and Fox -- that brings stability to a conference that experienced great upheaval in recent years.
The now 10-team league subtracted Nebraska (Big Ten), Colorado (Pac-12), Missouri (SEC) and Texas A&M (SEC) since 2011. The exodus helped contribute to a change in conference leadership and brought in two new schools -- TCU and
The broadcast contract will include at least six primetime games on FOX and shared "TV everywhere" rights with ESPN covering tablets, smart phones and unique applications.
Copyright (C) 2012 The Sports Xchange. All Rights Reserved.


text:
Key Content
- UNICEF: Philippines Humanitarian Situation Report – July 2016
- OCHA Philippines Humanitarian Bulletin Issue 6, 1 to 30 Jun 2016
- ECHO Factsheet – Philippines - Jun 2016
Appeals & Funding
Useful Links
The cessation of hostilities between the Government and the Moro Islamic Liberation Front (MILF) continued to be respected in the first half of the year, furthering the return and resettlement of the internally displaced people. However, the conflict-affected populations' high level of vulnerability and the perseverance of violence connected to other armed groups and to clan violence (locally referred to as rido) have contributed to the persistence of humanitarian needs.
I always sort of had an idea that women and girls living in extreme poverty suffered a bit more than men from disasters like war, a flood, an earthquake or a drought -- it just seems logical, if you think about it. But I had no idea how much more acutely they feel pain from these events until I started to research it.


text:
Hello, Hello. Sign In
Please enter a valid city, state or zip code
48 reviews | 4.1 out of 5
the watchface is small and feminine as is the leather watch band. The full size black numbers on white background make it easy to read.The Walmart price is less than the timex company retail price!
I like the watch easy to read the white back ground makes the date easier to see its just a great watch I would buy it again or even get another one for back up....
This is an excellent inexpensive watch. It keeps accurate time and it easy to read. When the winding stem is pushed in, the face lights up, so it is helpful even int he dark. The only problem is the strap, which comes apart after about a year and can't be replaced. But at this price, you can afford to throw it away.
This is a workhorse of a watch, not a lot of bells and whistles or rhinestones, nothing fancy, just a good, solid, easy to read watch. Like everything else, the price has gone up, but it's still a good time-keeper and has the Indiglo feature so you can see how long until the movie's over.
the watch works great but buckle strap broke first time i put the watch on. otherwise it is an ok product.
Great watch, the only watch I wear. I love number instead of digital or roman numberal. I have to have a leather band and not metal and I have to have a lighted watch and this one is. I've had it in gold and in silver and love both.
I purchased this watch online because the store did not have it in stock. It was even cheaper online. Numbers are very easy to read and I love the light.
Nice watch would give 5 stars if I could get the strap to bend more so it stays in the right spot. It constantly twists so the dial is on the side of my wrist, but I cannot tighten it up without cutting off the circulation.
I've always like Timex watches -- inexpensive, yet dependable.
I got two of these since June- the first one fogged up - I couldn't dry it out but I liked a lot so I ordered another a few weeks ago- the strap is cracked and will break soon. too bad - so cute
i got this watch for my mother...it is terrific! adjustable band for her little wrist, large numbers so she can read the time. she loves it!
I can read the time when I'm in bed with my glasses off.
I bought this for my sister for Christmas. She was having trouble seeing the hands and numbers on her wristwatch and was thrilled to receive this. I fully expect it to serve her many years as Timex watches always just "keep on ticking."
My last Timex lasted so long that I decided to buy another one with a large face. Actually, I think this is a man's watch but it is suitable for a casual watch for a woman. The only thing I'm not to crazy about is that the band is rather stiff. I hope it gets more pliable as I wear it. Other than that I like the look of this watch.
I have bought this watch multiple times in the past. However, the crystal face has been changed to a cheap material. It scratches so easily that I have only had the watch a couple of months, and I can barely read the numbers. I will not buy it again.
This is my 2nd or 3rd Timex watch. It's not fancy. However, I really like it's easy to read features; especially the Indiglo. Enjoy!
This is the second watch that I've purchased of this exact same model. It's nice without being fancy and the first one lasted for years before it stopped working correctly. I highly recommend this as an everyday watch.
Love the big numbers. Timex still does it right.
I've owned Timex watches all of my life, and I knew I wanted the next one to work as hard as all the rest. My last one is still working perfectly after 15 years, but it was looking a bit beat up. My husband said I could have any watch that I wanted, but I didn't want to mess with perfection. Order a Timex; it's always been a good choice for me.
This watch works very well. I like the watch because it's very easy to see the numbers and it also can be lit up when nessesary. I wear it all the time. Timex has been around for years and I hope they stay around for many more years.


text:
Select the store
nearest you to check available inventory*
3 Review(s)
Too hot to handle? Skillet handles won't be with Arden Benhar's duraSERVE® Silicone Hot Handle Holders. Made with non-stick silicone exterior with a non-melting 100% cotton fill and terry cotton liner, this handle holder provides excellent cold and heat protection. With an insulation value of 450°F, it will keep hands safe. Easily wipes clean and is machine washable.
This is one of the greatest kitchen aids I've ever bought - and I buy a lot! Not only do these do away with having to "test" the handle to make sure it's not too hot, it helps with gripping the handle very well and that's important when you have wrist problems, believe me. Greatest little invention ever!!! (Posted on 3/1/2010)
These are great helpers in the kitchen. They are handy for pots that have handles that get hot on the stove. And they are super great to stick on pot/pan handles after the pot/pan comes out of the oven. No more forgetting the pot/pan just came out of the oven and grabbing a scalding hot handle. (Posted on 9/8/2009)
Pot handles get hot on a 4 burner stove, and the handle holder is just right for the kitchen. (Posted on 8/20/2009)
Use spaces to separate tags. Use single quotes (') for phrases.
There are no product questions yet.
Ask a question, and give an answer. Help your fellow customer.
* Required Fields
Select the number of stars that reflect your overall item rating
share your opinion with others by writing a detailed review
when writing your review, try to avoid:
What do you like about this item?
What do you dislike about this item?
Would you recommend this product to a friend?
pictures speak a thousand words!
post your youtube video link to show how you style this item
Back to top


text:
The Preliminary Training of Railway Employés.
—In view of the very large number of accidents, resulting either fatally, or in the maiming of the unfortunate ones for life, it is proposed to establish schools of training at favorable railway points. In the aggregate the number—2,000 killed, and 20,000 injured in 1890— of sufferers is great, and the utmost means of prevention should be taken. It is true that carelessness is responsible for a goodly proportion of the accidents, and it is this very factor which will be largely overcome by a proper drilling, and a due presentation of consequences.
A New Disease.
—Two English physicians— Dr. Hale White and Mr. Golding-Bird—have recently described an affection to which they give the name " Idioglossia." It appears that the patients hear well, and express themselves in articulate sounds, but such sounds are unlike those of any known language. The patients really have a
Some tools below are only available to our subscribers or users with an online account.
Download citation file:
Customize your page view by dragging & repositioning the boxes below.
More Listings atJAMACareerCenter.com >
Enter your username and email address. We'll send you a link to reset your password.
Enter your username and email address. We'll send instructions on how to reset your password to the email address we have on record.
Need assistance?
Athens and Shibboleth are access management services that provide single sign-on to protected resources. They replace the multiple user names and passwords necessary to access subscription-based content with a single user name and password that can be entered once per session. It operates independently of a user's location or IP address. If your institution uses Athens or Shibboleth authentication, please contact your site administrator to receive your user name and password.


text:
Hi. I have a very annoying problem..
I have embedded a media player on my site showing a video stream which is working well..
The only problem is, when you scroll down the page, the video scrolls down with you, covering information and generally giving me a pain in the back side..
Is there any way to resolve this problem??
Please HEEEEELP


text:
Silver Springs is a 2nd generation campground nestled in 170 acres of beautiful countryside. We have 300 sites and offer many amenities for the entire family to enjoy.
Our Recreational Vehicle sites are right on the open water of Florida Bay and located adjacent to the Marina for easy access to your boat. The waterfront location provides spectacular views.
Saskatchewan Accommodation: Campgrounds/RV Parks
Waterside RV park close to swimming beaches
Nova Scotia Accommodation: Campgrounds/RV Parks Located at the beginning of the Historic Cabot Trail, Joyful Journey's is a must visit campground. It has the breathtaking view of the mountains coupled with the amazing views of the ocean.
Located on beautiful Lake Hamilton, LLRVP is the perfect base for your Hot Springs National Park adventures!
Crazy Horse is a family campground located along the banks of the beautiful Sugar River in Brodhead, Wisconsin. You can enjoy tubing, canoeing kayaking, or fishing on the river. Our heated pool mini golf jumping pillow. Something for everyone.
Saskatchewan Accommodation: Campgrounds/RV Parks, ( hole golf, mini golf, showers/laundry, trout pond, concession, canoe rentals, hiking trails, fishing, boating.
130 unit campground w/14 cabins, tents & RV sites. Storybook Land and Land of Oz theme parks are located within park area. Mini-golf, go-karts, train, carousel, balloon, & roller coaster rides, volleyball, basketball, softball, & zoo. No admission.
We are a beaitfull counrty campground with a beach area from Lake Eufaula. We have a catch and release pound and canoe, kayak you can rent from us to take out on the lake and watch either the sun set ot sun rise they are beaitful or do some fishing.
Browse thousands of listings for North America.


text:
August 18, 2015
What is a compiler? Ask an average engineer and you will get an answer something like: "A software... Read more
May 16, 2016
A common compiler optimization is the inclusion of a function's code at the location(s) from where the function... Read more
Electronics & Electrical EngineeringReal-Time Embedded Systems
August 28, 2015
An embedded system is an electronic system that are designed to perform a dedicated function within a larger... Read more
August 18, 2015
What is a compiler? Ask an average engineer and you will get an answer something like: "A software... Read more
July 30, 2015
I often get emails from students asking me how to get started in a career in embedded software.... Read more
Computer Science; Electronics & Electrical EngineeringVintage Multi-core – the IPC
March 17, 2015
Last time, I wrote about a "multi-core" project that I was working on 30 years ago. To be... Read more
December 15, 2014
The idea of inlining code – placing the actual code of a small function at each call site... Read more
Popular on SciTechConnect
November 24, 2014
The computer world is often accused of being mired in jargon and I think that is a fair... Read more
Electronics & Electrical EngineeringIntroducing the iBrush
February 18, 2014
Today, for a change, instead of discussing some embedded software technology, I would like to put forward a... Read more


text:
|Publication number
|Publication type
|Publication date
|Jul 8, 1919
|Filing date
|Jul 23, 1918
|Publication number
|Wtllaxd J. Nobbis
|Export Citation
|BiBTeX, EndNote, RefMan
|Referenced by (2), Classifications (3)
|External Links: USPTO, USPTO Assignment, Espacenet
W. J. NORRiSV HEADUGHT. "mamon mso JULY 2a. unaL 1,308,858. Patented July a, 1919.
' 2 SHEETS-SHEET l.
f Z/ fia. K
MJ.' /VRR/s" @f MMM.
W. i. NORRIS.
Armcmou msn mv za. ma.
1,308,858. Patented July 8, 1919.`
@mm1 a r ramyfr mm J'.` NOEIBIS, 0F CHARLESTON, WEST VIRGINIA.
Heumann Specication' of Letters Patent.
Application fued my as, 191s. semi no. 248,341.
To all whom it may concern:
Be it known that I', WiLLAno J. Noluus, a citizen of,the United States, residing at Charleston, in the county of Kanawha and Stute of West Virginia, have invented certain new and useful Improvements in eadlights, of which the following is a specifica tion.
My invention relates to improvements in lamps, adapted for use upon automobiles, railway vehicles, ships or the like.
An important object of the invention is to provide a lamp of the above 'mentioned character, having a. lens or lenses which are adjustable for directing or distributing the light over an increased area, when desired.
A further object of the invention is to vprovide means to shift the source of light,
with the angular adjustment of the lens or lenses, .so that the source of light will occupy a proper position to cause the rays of light therefrom to pass generally 4through the lenses, at a right angle thereto.
A further object of the invention is to provide a. lamp of the above mentioned character, paratively inexpensive to manufacture, and convenient in use.
Other objects and advantages of the inf' vention will vbe aparent during the course of the following escrlption.
In the accompanyin drawings forming a,
i part of this specificatlon and inwhich like numerals are employed to designate like parts throughout the same,'
Figure 1 is a plan view of a' lamp em-A bofplying my invention, l,
1g. 2 is a front end elevation of the In the drawings, wherein for .the purpose vof illustration is shown a preferred embodi ment of my invention, the numeral 10 designatos a longitudinal casing, which is preferably horizontally arranged. This longitudinalcasing is provided at its forward end -with a pair of lateral extensions 11, which which is simple in'construction, com- 'upper en are angularly arranged, and diverge forwardly. These lateral extensions have outer portions 12; which are longitudinally curved and are struck upon arcs having their oenters at 13, this numeral also designating pivot elements or pins. y
Theforward end 14 of the lon itudinal casing 10 is formed open, and is a apted to be covered by a `main lens 15, carried in a swinging frame 16. This frame is equipped' with ears or knuckles 17, which are free to turn upon the pin 13. A suitable latch 18, is
employed to retain the; frame 16 ixi the' closed position.
The numeral 19 designates auxiliary casing sections, arranged to swing within the extensions 12, and being longitudinally curved, to conform to the curvature of the same. The casing sections 19 are rigidly s'el cured to frames 20, carrying auxiliary lenses 21, as shown. The frames 20 have apertured ears or knuckles 22, receiving the pins 13, and rigidly clamped thereto. It is thus apparent that by turning the pins 13, the auxiliary lenses 21 may be swung or angularl ,ad]usted in a horizontal plane.
eens' are provided to turn the pins 13, comprisin cranks 23, rigidly secured to the s of' the pins. These cranks' extend inwardly and have overlapping slotted rod 27.
Rigidly secured to a suitable support, ini
convenient reach of the operator, 1s a tubular housing 28, receivin therein a reciprocatory rod- 29, Vhaving t e rod 27 connected therewith. Mounted upon the tubular housing 28 is a reciprocating sleeve 30. A head or button 31 is rigidly attached to the rod 29 and sleeve 30. The sleeve 30 has a lon- 32, receiving a. transversely block 33, provided with teeth teeth 35, which are formed upon the base 28. The block 33 is pivote to a thumb-lever 37. as shown at 38, and this thumb-lever is pivoted at 39. The rear end of the thumb-lever is forced upwardly gitudinal slot reciprocatory 34, to engage with by a spring 40.
The numeral 41 designates a source of light, 'which maybe an electric lamp, gas lamp, or oil lamp. This source of lightis suitably mounted upon a carriage 42, which is. movable longitudinally of the casing 10, in a manner to be described. Arranged rearwardly of the source of light 4 1 and rigidly Patented July 8,1919.`
en 1,aos,`ets
secured to the carri 42,iee releter 43,
which4 is longitudine y curved in a Ahorizonk tel plane, yes shown. 4This reflector ig `sulliciently long and is suitabl curved whereb 6 portions thereof serve to r ect li ht throug 10 horizontal arm 45, to the outer end of which eomprisi all of the lenses. The carriage is mount:-
Od' `to slide within or wpon "longitudinal track :14 secured within the easing 10.`
Bigidly secured to the reflector 48'is a enerated by the -souce of li ht 41. It is?? to be taken as a preferred exemple' of the same, and that various changes in the shape.
size, and arrangement of parts muy be resorted to without departing from the spirit of the invention or the scope of the sul,- j'oined claims.
Having thus described my invention, 4l claim:
1. A lamp of the character described, u swinging lens, n source of light, an means to simultaneously swing the loris and move the source of light toward. or away from the lens.
2. lamp 0f the character described, comprising a lens adapted to he shifted in a horizontal plane to assume rlierent :ingut us seen that the lon itudinu movement of ler positions, a source/of light adopted to the 27 is utiliz to shift the carriage 42 longitudinally. I
The, operation of the lamp is as follows; When it is desired to have the rays -of li htpassing through the three lenses 15 and 2 me or form in 'eiect a si le light, the lenses l area so that t eir axes are substantial] para el with the 4exis of the .lens 15, or t e lenses 21 may have their axes Aslightly forward] converging. When the lenses 21 are in t s position, the buttonor lcnob 31 mayassume the roer position as mdicated in dotted lines in 4.
To swing the lenses 21 rearward! so that their axes diverge forwardly, the utton 31 is pushed forwardly, after the rod is released by manipulation of the thumb-lever be moved horizontally toward and uwuy from the lens, and common means to shift the lens and move the light.
3. A; lamp of the character described. comprising a relatively stationary lens, n source ofrlight arranged rearwardly of the lens and in substantial alinement with the longitudinal axis `thereof, means whereby the sour of light may be moved toward and away from the lens nt substantially :i riight angle thereto, and or. nngninl'ly a: justable lens arranged upon conside of the relatively stationary lens and adapted to be shifted to different angular positions with respect to the longitudinal axis of the relatively stationery lens.
4.. A lamp of the character described, com
37. This forward movement of the rod 29 prising a casing, a main lens connected with moves the rod 27 forwardly,l which in turn 'swi the bell'crank levers 23 forwardly, causing the casing sections 19 and frames 20 to move rearwardlv. Upon the forward movement of the ro 27, the carriage 42 'moves forwardly, thereby advancing the source .of light 41 and reflector 43, with relation to the singularly arranged 'lenses 21.
As illustrated diagrammatically in Fig. 6, the rays of light from the source of light 41,1111: retuinedmore or less perpendicular to the lenses 21, when these lenses occupy the several adjusted positions which is effected by -shifting the source of light toward or away from these lenses.
From the foregoing description it 1s obvios that rays of light pass through the mein lens 15 and are projected forwardly in a streitgg'ht` line,4while the rays of light lineassing through the auxiliary lenses 21, may
rejected forwardly in parallel relation to t e rays of light passin through the lens 15, or may assume angu arpositlons, conr verging or diver "ng with relntion thereto. YHy this Ineens t e rays of. llglilt may be spread over en area of varying sues.
It is to be understood that the form of my linvention herewith shown and described is the forward end thereof, angularly adjust able lensesconnected with the forward end of the casing and disposed upon opposite sides of the main lens, means to move the anulnrly ad'ustable lenses, n source of li t arrange within the casing rearwnrdly o the lenses, l,and mea-ns connecting the source of light and singularly adjustable lenses to move them.
5. A lamp of the character described, comprising a. casing, a main lens connected with the forward end of the casing, angulnrly adjustable 'lenses connected with the forward end of the casing and disposed upon opposite sides of the main lens, a carriage longitudinally `movubly mounted within the casing, a source of light connected with the carriage, a reflector secured to the carriage i rearwardly ofthe source of light and 'ceneting with all of the lenses,and connecting the carriage and singularly adjustable lenses to move then in umson. 'L
In testimony whereof I ailing, my signature in presence of two witnesses..
S. E. mits C. W. Tmc.
|Citing Patent
|Filing date
|Publication date
|May 8, 1947
|Dec 19, 1950
|Tucker Corp
|Automobile dome light
|Apr 17, 1995
|Jan 19, 1999
|Musco Corporation
|Light fixture with controllable light block
|U.S. Classification
|362/284, 362/285
|Cooperative Classification


text:
A-DATA X Series Ver 2 PC3-12800 6GB DDR3 Kit
SiSoft Sandra
Version and / or Patch Used: Professional Home
Developer Homepage: http://www.sisoftware.co.uk
Product Homepage: http://sisoftware.jaggedonline.com/index.php?location=home&a=TTA&lang=en
SiSoft Sandra (System ANalyser, Diagnostic and Reporting Assistant) is a synthetic Windows benchmark that features different tests used to evaluate different PC subsystems.
Like EVEREST we can see that with the modules at 2000MHz we see some serious performance. We can also see that compared to the Corsair modules which are down clocked we get a jump on them thanks to the lower latencies on offer.
PRICING: You can find products similar to this one for sale below.
United States: Find other tech and computer products like this over at Amazon's website.
United Kingdom: Find other tech and computer products like this over at Amazon UK's website.
Canada: Find other tech and computer products like this over at Amazon Canada's website.
- Page 1 [Introduction]
- Page 2 [The Package and Modules]
- Page 3 [Overclocking]
- Page 4 [Test System Setup and wPrime]
- Page 5 [Benchmarks - EVEREST Ultimate Edition]
- Page 6 [Benchmarks - SiSoft Sandra]
- Page 7 [Benchmarks - Sciencemark]
- Page 8 [Benchmarks - Far Cry 2]
- Page 9 [Final Thoughts]
Recommended for You
- We at TweakTown openly invite the companies who provide us with review samples / who are mentioned or discussed to express their opinion of our content. If any company representative wishes to respond, we will publish the response here.
Related Tags
Latest News Posts
- Apple sold less iPhones this quarter, revenues are also down
- Life is Strange getting a live-action episodic series
- Nintendo financials plunge, even with Pokemon GO's immense success
- Crucial expands its affordable MX300 SSD range with 3 new drives
- AMD Radeon Pro graphics card powers 16K display, or 15,360 x 8640
Forum Activity
- PW-IE20AH51T0 compatible with PC-G70 ?
- D-Link DIR-879 EXO AC1900 Wireless Router Review
- Asrock z77 Extreme 6 Ram Overclocking Stuck at 2000mhz
- MDD BP5e 480GB M.2 SATA III SSD Review
- ASRock Beebox-S (Intel Core i5-6200U) Mini PC Review
Press Releases
- Polaris Nitro-charged - introducing the SAPPHIRE NITRO+ Radeon RX 480
- AccelStor debuting NeoSapphire 3706-ES1 at Flash Memory Summit 2016
- Elitegroup ceremoniously launches ECS H110S-2P mini-STX motherboard and the all-new LIVA Pro Mini PC
- MSI announces custom GeForce GTX 1060 graphics cards
- Seagate unveils the industry's broadest 10TB portfolio