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The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART.
RNA silencing (RNAi) is a new gene regulatory mechanism conserved from plants to humans. RNAi mediators are small non-coding RNAs (sncRNAs) that function through sequence specific mRNA targeting to either induce their degradation and/or inhibit translation [1,2]. In mammals, RNAi is mediated by different classes of small non-coding RNAs including piRNAs, microRNAs and siRNAs [3-5]. MicroRNAs are produced from a primary transcript (pri-miRNA) which is processed in the nucleus by the microprocessor complex containing RNase Drosha and DGCR8. The resulting product or pre-miRNA is exported to the cytoplasm through the exportin-5 pathway. Cytoplasmic pre-miRNA is processed by typeIII RNase Dicer to miRNA/miRNA* duplex of 19 to 25 nucleotides. miRNA/miRNA* is incorporated into the RNA-Induced Silencing Complex (RISC) where miRNA* is degraded while miRNA serves as a guide for mRNA targeting [2]. Key components of miRISC are proteins of the Argonaute family (Ago1 to Ago4) that are required for miRNA-mediated silencing [6,7]. To ensure mRNA translational inhibition and decay, miRISC, loaded with miRNA and its mRNA targets, associate with proteins involved in mRNA processing [2]. A key factor in this process is the GW182 protein that interacts directly with Argonaute1 (Ago1) [8], and the human homologs of GW182 that interact with Ago1–4 [9]. GW182 orchestrates both mRNA decapping, through the recruitment of p54/RCK that regulates the activity of the decapping enzymes DCP1/DCP2 [10], and mRNA deadenylation by recruiting the CCR4-NOT1 complex [11]. mRNA decapping and deadenylation leads to mRNA decay through the action of XRN1, a 5'-3' exonuclease [10]. Interestingly, RNAi effectors, including miRNAs and their target mRNAs, Ago proteins, GW182, RCK/p54, LSm-1 and DCP proteins co-localize in cytoplasmic structures called GW-bodies or P-bodies suggesting that miRNA-mediated silencing occurs at these sites [11-15]. Emerging evidence suggests that miRNA-mediated gene regulation serves as a defence mechanism against both RNA and DNA viruses in mammals [16-20]. The present study was designed to explore physical and functional interaction between effectors of miRNA-mediated silencing and HIV-1 replication.
Results and discussion
To investigate whether RNAi effectors regulate HIV-1 replication, we analyzed virus replication in cells where expression of RNAi effectors was reduced using specific siRNA. HeLa cells were transfected with siRNA specific to RCK/p54, GW182, LSm-1 or XRN1. As controls, HeLa cells were transfected with scrambled siRNA (Scr) or CDK9 specific siRNA and subsequently infected with HIV-1 (Figure 1a). Knockdown of RCK/p54, GW182, LSm-1 and XRN1 enhanced virus replication by up to 10 fold (Figure 1b). As we have previously shown, knockdown of Drosha [21] and DGCR8 (Figure 1b), the two subunits of the microprocessor complex, increased virus production while knockdown of the CDK9 subunit of the PTEFb complex that is required for viral gene expression, reduced HIV-1 production (Figure 1b). Interestingly, analysis of HIV-1 cytoplasmic mRNA distribution on glycerol gradient showed that knockdown of RCK/p54 shifted HIV-1 mRNA from the non-polysomal fraction to polysomes as compared to control siRNA transfected cells (Figure 2, upper panel). As control, we analyzed the distribution of endogenous mRNA expressed from a gene encoding Hdm2. Knockdown of RCK/p54 did not affect Hdm2 mRNA distribution (Figure 2, lower panel). These experiments show that GW182, RCK/p54, LSm-1 and XRN1, factors required for RNAi, are repressors of HIV-1 gene expression that act by preventing HIV-1 mRNA translation.
Figure 1. miRNA effectors are repressors of HIV-1 replication. HeLa cells were transfected with siRNA as indicated. 48 hours post transfection, cells were analyzed for RCK/p54, LSm-1, GW182, XRN1, DGCR8, DROSHA and CDK9 expression by Western blotting (a), or infected with a single round infectious virus (HIV-1-VSV-luc 200 ng/ml) and cell extracts were measured for luciferase activity 48 hours after infection (b). Results are presented as fold HIV production relative to Scr transfected cells, and data are representative of three independent experiments.
Figure 2. RCK/p54 restricts HIV-1 mRNA association with polysomes. Cytoplasmic extracts from HeLa cells that were transfected with the indicated siRNA and infected with HIV-1-VSVG-luc were run on glycerol gradient (7% to 47%). Fractions were collected and their RNA contents were monitored by measuring absorbance at 254 nm. HIV-1 mRNA (top panel) and Hdm2 mRNA (lower panel) were quantified in all the fractions by Q-RT-PCR using specific oligonucleotides.
We next investigated the physical interaction between RNAi effectors and HIV-1 mRNA. 293 cells were mock transfected or transfected with combinations of pNL4-3, Myc-Ago2, a central component of the RISC complex, or its RNA-binding mutant Myc-Ago2PAZ9 constructs as indicated in figures 3 and 4. First, we verified that Myc-Ago2 and Myc-Ago2PAZ9 were equally expressed (Figure 3a). Second, cytoplasmic extracts were prepared, and a fraction was used for total RNA extraction while the rest was subjected to immunoprecipitation using anti-Myc antibody to purify Myc-Ago2 associated mRNP. Both total RNA (Figure 3b, left panels) and Myc-Ago2 associated RNA (Figure 3b, right panels) were reverse transcribed and subjected to PCR amplification using oligonucleotides specific for HIV-1 TAR RNA (a structured motif associated with all HIV-1 mRNAs) or HIV-1 unspliced mRNA, Hdm2 mRNA or GAPDH mRNA. PCR analysis of total RNA showed that equal amounts of HIV-1, Hdm2 and GAPDH mRNAs were present in all samples (Figure 3b, left panels). HIV-1 mRNAs (both TAR and unspliced) were associated with Myc-Ago2, but not with Myc-Ago2PAZ9 mutant (Figure 3b, right panels). In agreement with the results shown in figure 2, Hdm2 mRNA was not detected in Myc-Ago2 mRNPs, suggesting that under these conditions Hdm2 is not regulated by RNAi. A similar experiment was performed to analyze the association of HIV-1 multispliced mRNA with Myc-Ago2 mRNPs. The RT-PCR reactions were performed in the presence of 32P-α ATP and were analyzed by autoradiography (Figure 3c). HIV-1 multispliced mRNAs associated with Myc-Ago2 (compare lane 3 to 2) and weakly with Myc-Ago2PAZ9 (compare lane 4 to lanes 3 and 2). Co-localization of HIV-1 mRNA and effectors of RNAi such as Ago2 and RCK/p54 within the P-bodies was also observed by immunofluorescence using HIV-1 containing MS2 binding sites and MS2-GFP constructs (Figure 4). Indeed, HIV-1 mRNAs visualized through their binding to MS2-GFP colocalized with endogenous RCK/p54 and ectopically expressed Myc-Ago2 (Figure 4). Our results show that HIV-1 mRNAs physically associate with Ago2, a central component of RISC, and co-localize with cellular proteins required for miRNA-mediated silencing such as RCK/p54 and Ago2 in P-bodies. We observed that all HIV-1 mRNA species associated with RISC. Accordingly, Huang et al. had identified 5 cellular miRNAs able to target the 3'UTR sequence present in all HIV-1 mRNAs [22]. Additionally, other cellular miRNAs able to target regions out side the 3'UTR may also participate [23].
Figure 3. HIV-1 mRNAs associate with Argonaute 2. 293 cells were transfected with HIV-1 molecular clone pNL4-3, Myc-Ago2 or Myc-AgoPAZ9 as indicated. 48 hours later cells were harvested and cytoplasmic extracts were prepared. Total RNA was purified from a fraction of harvested cells while the rest was subjected to immunoprecipitation using anti-Myc antibody. After washing, a fraction was used to analyze the amount of Myc-Ago2 and Myc-Ago2PAZ9 immunoprecipitated by Western blotting (a), and the rest of the Myc-IPs was used for RNA extraction. HIV-1 mRNAs (TAR and unspliced), Hdm2 and GAPDH mRNA were quantified from total RNA (b, left panel) or from Myc immunoprecipitated mRNPs (b, right panel) by RT-PCR using specific oligonucleotides. c) Experiment was performed as in fig 3 except that 293 cells were transfected with HIV-1 ΔPSP which contains a partial gag/pol deletion but retains all the mRNA splicing sites [66], and 32P-labelled nucleotides were used in the PCR reaction. PCR products were visualized by autoradiography.
Figure 4. HIV-1 mRNA co-localizes with RCK/p54 and Ago2. HeLa cells were transfected with Myc-Ago2 expression vector either alone (top panels) or co-transfected with HIV-1 vector containing 24 repeats of MS2 binding sites and MS2-GFP expression vectors [64,65] (lower panels). Endogenous RCK/p54 and transfected Myc-Ago2 were visualized using specific primary antibodies and appropriate secondary antibodies coupled with Cy5 (shown in blue) and Cy3 (red) respectively. HIV-1 RNA bound to MS2-GFP is shown in green. Green, blue and red merged images are shown.
Emerging evidence suggests the physical and functional interactions between P-bodies and the viral life cycles [24]. Viral mRNA trafficking through P-bodies may represent a pool of translationally repressed viral transcripts otherwise used for efficient packaging or formation of viral-replication complexes. Indeed, yeast retrotransposons Ty1 and Ty3 mRNA associate with P-bodies, and this association is required for efficient retrotransposition [25-27]. In the case of BMV (Brome Mosaic Virus), formation of the virus replication complex occurs in P-bodies [28]. In addition, P-bodies may also function in host defences against viruses and transposable elements. Indeed, the cellular factors APOBEC 3G (A3G) and 3F (A3F), which are viral restriction factors, are found to accumulate in P-bodies [29,30]. It has been suggested that A3G and A3F mediated HIV-1 restriction may involve viral mRNA targeting to P-bodies leading to their translational inhibition [30]. We, therefore, asked whether P-bodies are positive or negative regulators of HIV-1 replication. Thus, we analyzed HIV-1 replication in cells where P-bodies were disrupted by knocking down RCK/p54 or LSm-1 [31]. HeLa CD4+ cells were transfected with RCK/p54 or LSm-1 specific siRNA or control siRNA. Forty eight hours later, cells were infected with equal amounts of HIV-1 viral particles (as measured by p24 assay). HIV-1 p24 antigen was measured in cell culture supernatant 48 hours post-infection. As shown in figure 5b, knockdown of RCK/p54 or LSm-1 results in enhanced virus production as compared to infection of control siRNA transfected cells. To assess the infectivity of the produced viruses, an equal volume of supernatant from Scr, RCK/p54 and LSm-1 siRNAtransfected cells was used to infect HeLa CD4+ cells, and p24 release in the culture supernatant was measured 48 hours later (Figure 5c). Virus infectivity correlated with the amount of p24 produced (Figure 5b) showing that virions produced in RCK/p54 and LSm-1 knocked down cells are fully competent for replication and have no defect in steps such as RNA packaging. Since the knockdown of RCK/p54 and LSm-1 was shown to result in the disruption of P-bodies, we concluded from these experiments that accumulation of HIV-1 mRNA in P-bodies limits virus replication.
Figure 5. Disruption of P-bodies through knockdown of RCK/p54 and LSm-1 leads to enhanced production of infectious HIV-1 virions. HeLa CD4+ cells were transfected with siRNA as indicated. 48 hours post transfection cells were analyzed for RCK/p54 and LSm-1 expression by Western blotting (a) and infected with equal amounts of HIV-1 (200 ng/ml). b) Virus production was monitored 48 hours post infection by measuring p24 antigen in culture supernatant. c) To analyze the infectivity of new progeny virions, equal volumes of supernatant from siRNA transfected Hela CD4+cells were used to re-infect HeLa CD4+ cells. P24 antigen was measured in culture supernatant 48 hours post infection.
Next, we asked whether A3G-mediated HIV-1 restriction requires effectors of miRNA-mediated mRNA translational inhibition. Thus, we compared A3G-mediated HIV-1 restriction in cells where RCK/p54 or LSm-1 expression was reduced compared to control cells. HeLa cells were transfected with control siRNA or siRNA specific for RCK/p54 or LSm-1 (Figure 6, right panel). Forty-eight hours later, cells were transfected with an HIV-1 molecular clone lacking the vif gene (pNL4-3Δvif) either alone or with wild-type A3G or A3G mutant lacking antiviral activity (A3Gdm). HIV-1 p24 antigen was measured in culture supernatant 48 hours post-transfection. Interestingly, knock down of RCK/p54 or LSm-1 enhanced HIV-1 production regardless of A3G (Figure 6, left upper panel). Similarly, A3G but not A3Gdm reduced virus production regardless of RCK/p54 or LSm-1 expression (Figure 6, left upper panel). These results suggested that RCK/p54 or LSm-1 and A3G -mediated HIV-1 repression involves different mechanisms. We then analyzed the infectivity of HIV-1 produced from siRNA transfected cells. Equal amounts of p24 were used to infected HeLa CD4+ cells, and HIV-1 p24 antigen was measured in culture supernatant 48 hours post-infection. As shown in figure 6 (lower panel), virus produced in Scr siRNA transfected cells in the presence of A3G showed lower infectivity than those produced in its absence or in the presence of A3Gdm. Similar HIV-1-restriction activity of A3G was observed when the virus was produced in RCK/p54 or LSm-1 knocked down cells. This experiment showed that A3G-mediated HIV-1 restriction is independent of RNAi effectors RCK/p54 and LSm-1 and does not require P-bodies.
Figure 6. RNAi effectors and APOBEC 3G-mediated HIV-1 repression involve different pathways. HeLa CD4+ cells were transfected with the indicated siRNA. 48 hours later cells were analyzed for RCK/p54 and LSm-1 expression (right panel) or co-transfected with 1 μg of pNL4-3Δvif (lacking vif gene) and pcDNA or expression vectors for wild-type APOBEC3G or APOBEC3G double mutant lacking both deaminase and antiviral activity, A3G H65R/H257R [63]. HIV-1 production was measured 24 hours post-transfection in culture supernatant by quantifying p24 antigen (top left panel). Numbers on the top of the columns are fold increase relative to the respective Scr. Numbers on the top of Scr samples in A3Gwt and A3Gdm represent fold increase relative to Scr in pCDNA transfected cells. Infectivity assay was performed using equal amounts of p24 antigen to infect HeLa CD4+ cells. HIV-1 p24 antigen was measured 48 hours post infection (lower left panel). A representative experiment out of five is shown.
Taken together, our results show a physically repressive interaction between RNAi effectors and HIV-1 mRNA. Since cellular miRNAs were shown to play a role in HIV-1 latency [22], we asked whether RCK/p54, which is required for miRNA-mediated mRNA translational inhibition, contributes to HIV-1 silencing in vivo. Thus, PBMCs isolated from 3 HAART-treated HIV-1-infected patients with undetectable viremia were transfected with control siRNA or with siRNA specific for Drosha, DGCR8 or RCK/p54. Transfected cells were co-cultured with PHA/IL2-activated PBMCs isolated from healthy donors. Virus production was monitored every 3 days by measuring p24 antigen in the culture supernatant (Figure 7). As we have previously shown, knockdown of Drosha resulted in virus reactivation in PBMCs isolated from 3 HAART-treated HIV-1-infected patients [21]. Remarkably, viral replication from its natural reservoir resumed also when DGCR8 or RCK/p54 was silenced. No virus was isolated from control siRNA transfected PBMCs suggesting that virus production observed in Drosha, DGCR8 and RCK/p54 knock down was not due to actively infected PBMCs relieved from drug pressure. These results show that endogenous levels of Drosha, DGCR8 and RCK/p54 contribute to HIV-1 latency and/or its maintenance in infected patients.
Figure 7. Implication of RNAi in HIV-1 latency. PBMCs were isolated from three patients undergoing active HAART. Isolated PBMCs were transfected with the indicated siRNA and either analyzed for RCK/p54, DGCR8 and DROSHA expression by Western blotting 48 hours after transfection (right panel) or co-cultured with activated PBMCs obtained from healthy donors. Virus replication was monitored every 3 to 4 days post co-culture by measuring p24 antigen in culture supernatant. Shown is the amount of p24 antigen at day 15 post co-culture. No virus was isolated from Scr transfected-PBMCs for up to 27 days.
The outcome of HIV-1 infection results from complex interactions between viral components and host cell factors [32-35]. In most cases, HIV-1 successfully hijacks cellular pathways and bypasses restriction factors for optimal replication leading to continuous rounds of infection, replication, and cell death. Continuous viral replication causes the loss of CD4+T cells and progression to immunodeficiency in infected individuals. HAART treatment revealed the existence of a pool of resting memory CD4+ T cells harbouring integrated, but silent HIV-1 provirus [36,37]. This latent reservoir is believed to be the major obstacle for virus eradication by HAART. Therefore, it is critical to understand how HIV-1 latency is established and maintained [38]. Post-integration latency takes place at both transcriptional and post-transcriptional levels [39]. Transcriptional latency involves different mechanisms ranging from integration position effect [40-42], limitation in transcription factors [43-46], establishment of chromatin repressive marks and recruitment of chromatin silencers [47-51]. Post-transcriptional silencing involves defects in mRNA export and translation [52-54]. All together, these studies show that HIV-1 post-integration latency is a multi-factorial process. In the present study, we show that HIV-1 gene expression is additionally regulated by the miRNA pathway. HIV-1 mRNA associates with components of the RISC complex by a mechanism that does not involve APOBEC3G, but does need sncRNAs. Accordingly, it has been recently shown that the suppressor of RNAi P19 from tomato bushy stunt virus, known to bind and sequester sncRNAs including miRNA, enhances HIV-1 replication [55]. Additionally, the RNAi suppressor function of HIV-1 Tat [56] could be complemented by VP35 from Ebola virus [57] and the NS3 protein of rice hoja blanca virus through sequesteration of small non-coding RNAs [58]. HIV-1 mRNAs associated with RISC are sequestered in the non-polysomal fraction, thereby preventing translation. In agreement with two previous reports [19,21,22], we show that knockdown of RCK/p54, a protein required for miRNA-mediated silencing, led to virus reactivation from PBMCs isolated from HIV-1 infected patients who were undergoing suppressive HAART.
A challenge in AIDS treatment is the need to activate latent viral reservoirs in order to eradicate these viruses through HAART. In this respect, targeting the miRNA processing pathway could offer a strategy that could be exploited to activate latent viral reservoirs, for instance, during HAART. Several molecules have been used to reactivate viral reservoirs [59]. However, none of these approaches provides the sequence specific targeting that can be achieved using siRNA. Recent data suggest that siRNA can be used therapeutically in vivo in certain mouse disease models [60] and more recently in non-human primates [61,62]. It remains to be explored whether, as suggested here, the in vivo targeting of miRNA-effectors using siRNA can assist in activating latent HIV-1 reservoirs for eradication by HAART.
HIV-1 molecular clone pNL4-3Δvif and expression plasmids for APOBEC3G were gift from Olivier Schwartz (Pasteur, France). APOBEC3G H65R/H257R mutant was previously described [63]. HIV-1 vector containing MS2 binding sites and MS2-GFP expression plasmids [64,65] were gift from Alessandro Marcello (ICGEB. Trieste, Italy) and Edouard Bertrand (IGMM. Montpellier, France)
PBMCs were transfected with siRNA or miRNA using the Nucleofector II Device with the appropriate Nucleofection solution according to the manufacturer's instructions (Amaxa). siRNA corresponding to DGCR8 (5'-CAUCGGACAAGAGUGUGAU(dTdT)-3'), Drosha (5'-CGAGUAGGCUUCGUGACUU(dTdT)-3'), RCK/p54 (5'-GCAGAAACCCUAUGAGAUUUU(dTdT)-3'), LSm-1 (5'-GUGACAUCCUGCCACCUCACUU(dTdT)-3'), GW182 (5'-UAGCGGACCAGACAUUUCU(dTdT)-3'), XRN1 (5'-AGA UGA ACU UAC CGU AGA A(dTdT)-3') and CDK9 (5'-CCAAAGCUUCCCCCUAUAATT(dTdT)-3') were synthesized (MWG). Expression level of knock down proteins was analyzed by Western blotting 48 hours after transfection. Briefly, cell-extracts were resolved on SDS-PAGE gels. Proteins were transferred to PVDF membrane by semi-dry electroblotting and probed overnight at 4°C with the primary antibody (anti-Drosha, LSm1, GW182 (Abcam), DGCR8 (Proteintech Group), anti-RCK/p54 (Bethyl)or anti-CDK9 (Santa Cruz), washed and incubated with the appropriate secondary antibody (Amersham) for 1 hour. Proteins were visualized by chemiluminescence according to the manufacturer's protocol (Pierce).
PBMC isolation and co-culture assay for virus production
Peripheral blood mononuclear cells of HIV-1 infected patients were isolated by lymphocyte separation medium density centrifugation (Lonza). PBMCs from healthy donors were pre-activated using 5 μg/ml PHA (phytohemagglutinin-P, DIFCO)/10 U/ml IL-2 (interleukin-2, Roche) for 72 hours. They were then washed once with PBS and once with RPMI medium before co-culture assay. siRNA transfected HIV-infected PBMC (106 cells/ml) were co-cultured with pre-activated PBMC (106 cells/ml) from the same healthy donor in the presence of 10 U/ml IL-2. The culture medium was collected every 3 or 4 days. Fresh pre-activated healthy PBMCs were added to the culture every 7 days. Viral production was measured by quantifying the amounts of p24 in the culture medium using an ELISA kit (Ingen).
Pseudotyped virion production and single-round infections
The plasmid pNL4-3-env-Luc+ harboring a luciferase gene (obtained from the NIAID AIDS Reagent Program) was co-transfected with the envelope plasmid pMD.2G encoding the G protein of vesicular stomatitis virus (VSV.G) into human embryonic kidney cells-293T. The virions, named HIV-1VSV-Luc, were collected and filtered using 0.45 μm filters 48 hours post-transfection. HeLa or HeLa CD4+ cells were infected over-night at 37°C, washed and resuspended in DMEM containing 10% FCS. Virus production was monitored in culture supernatant by measuring p24 antigen (Ingen) and by following luciferase activity according to the manufacturer's instructions (Promega).
Cytoplasmic extracts analysis on sucrose gradients
To isolate cytoplasmic extracts, cells were lysed for 10 minutes in buffer B (5 mM Tris-HCl pH 7.4, 1.5 mM KCl, 2.5 mM MgCl2, 0.5% NP40 and protease inhibitor). Nuclei were pelleted by centrifugation for 10 minutes at 10,000 rpm. 2 mg of cytoplasmic extracts were loaded on a 7–47% sucrose gradient. Briefly, 5 layers of 7 to 47% sucrose were prepared in sucrose buffer (20 mM Tris-HCl pH7.4, 80 mM NaCl, 5 mM MgCl2, 1 mM DTT and protease inhibitors) and diffused at 4°C for 16 hours to obtain a linear sucrose gradient. 2 mg of cytoplasmic extracts were loaded on the top of the column, and centrifuged for 3 hours at 36,000 rpm in a SW41Ti rotor. After ultracentrifugation, 28 fractions were collected and OD at 254 nm was measured in each fraction using a Nanodrop apparatus (Labtech).
RNA immunoprecipitation
293 cells were grown in 60 mm dishes and transfected with the indicated plasmids using calcium-phosphate. Cells were harvested 48 hours after transfection, lysed for 15 minutes in RIP buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 2.5 mM MgCl2 × 6H2O, 250 mM sucrose, 0.05% NP40, 0.5% Triton X-100) containing RNASIN (Promega) and 1 mM DTT, and centrifuged to pellet debris. Supernatants were incubated overnight with mouse anti-Myc mAb 9E10 (Amersham) at 4°C followed by 2 hours incubation with protein G-Sepharose. Immunoprecipitates were washed with RIP buffer, and nucleic acids were extracted with phenol/chloroform/isoamyl alcohol, isopropanol-precipitated, ethanol-washed and resuspended in RNase-free water. Total RNA was DNase I treated and reverse-transcribed using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). RT products were PCR-amplified using either GAPDH (GAPDH forward: GTA TTG GGC GCC TGG TCA CC; reverse: CGC TCC TGG AAG ATG GTG ATG G), HIV-1 (HIV-1 forward: TAG TGT GTG CCC GTC TGT T; reverse: CTC TGG TTT CCC TTT CGC TTT C or Gag-reverse: GAT GGT TGT AGC TGT CCC AG for unspliced HIV RNA), or HDM2 specific oligonucleotides (HDM2 forward: GTA CCT GAG TCC GAT GAT TCC; reverse: ACC TAC TGA TGG TGC TGT AAC). PCR products were resolved on 1.5% agarose/TAE gels containing ethidium bromide. In vivo splicing assay and oligonucleotides BSS and SJ4.7A have been described [66]
Competing interests
The authors declare that they have no competing interest.
Authors' contributions
CBC, OL and LD carried out most experiments. TR initiated and performed the experiments shown in figures 3 and 4. AZ participated in experiments analyzing the involvement of APOBEC3. WA, JJM, RJ, LY, and SA participated in some of the experiments such as isolation of PBMCs from HIV-1 infected patients. BY and BM directed, supervised and wrote the manuscript.
We are grateful to Kiernan R, Jeang KT, Emiliani S, and Voinnet O for helpful discussions and for critically reading the manuscript. Work in MB's laboratory was supported by ANRS, SIDACTION, ANR and FRM. OM was supported by fellowship from "infectiopôle grand sud". DL was supported by ANRS scholarship. AZ was supported by SIDACTION fellowship.
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text:
Sleep Inn & Suites
2020 Franklin Terrace Ct.
Johnson City, TN 37604
Phone: (423) 915-0081
Fax: (423) 915-0029
Entertainment Venues
East Tennessee State University Memorial Center
The ETSU Memorial Center is a gleaming white domed structure on the East Tennessee University campus. The center, known ironically as the "Mini-Dome," is a 8,539-seat multi-purpose arena that changes its dimensions according to the sports seasons. Inside the center there is a basketball court floor, Gentry Field, two auxiliary basketball courts, a six-lane, 280-meter track, six racquetball and handball courts, six tennis courts, a rifle range, two gymnasiums, a strength training facility, several locker rooms and a physical education research laboratory. The arena hosted an NBA preseason game in 1994, when it stretched its already generous proportions to fit more than 14,000 spectators.
807 University Parkway
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+1 423 439 4161
Earn valuable points toward free nights.

subset: commoncrawl


text:
American BackroadsThursday, 9 to 10 p.m.
Program Host(s):
Greg Easterling
This program is available for sponsorship!
(Contact Ron Horan for more details.)
|Hosted by Greg Easterling, American Backroads combines folk, country, blues and roots-rock from the 50s through the 90s to create an aural portrait of American music.
Apr 2nd, 2015: 'New Beginnings'
Upcoming Programs
Including Jimmy Dale Gilmore, Shawn Colvin, Taj Mahal, the Jayhawks, Bonnie Raitt.Apr 9th, 2015: Encore Presentation with Johnny Cash, Dierks Bentley, Carolina Chocolate Drops, Cindy Bullens, and Indigenous
Apr 16th, 2015: Encore Presentation with Music by Mavis Staples, John Mellencamp, Marty Stuart, Robert Cray and John Prine
Apr 23rd, 2015: 'Live Shots'
Featuring concert recordings by Dave Alvin, Emmylou Harris, John Prine, Robert Earl Keen, Nanci Griffith, Gram Parsons.Apr 30th, 2015: 'Movin' On'
With Lucinda Williams, Roger McGuinn, Pure Prairie League, Wilco, John Hammond.
Previous Programsdisplays the previous month of programming
Mar 19th, 2015: Recent Releases from The Mavericks, Bob Seger, John Ballantyne's Crazy Heart, New Basement Tapes and Chris Smither
Mar 12th, 2015: Recent Releases from Elvin Bishop, Gary Clark, Jr., Westies, Lucinda Williams, and Eric Noden
Mar 5th, 2015: Encore Presentation Starring: Johnny Cash, Rosanne Cash, Heather Horton, Michael McDermott, Dolly Parton, and Patty Loveless


text:
Darnell Richards tries to sneak onto plane on Eastern Shore, gets arrested
WBOC reports that Darnell Richards, a 30-year-old Brooklyn man, was thrown out of the Wicomico Regional Airport on Sunday night after trying to find a way back to New York with no money.
Voice calls and airplanesComment
Kang interview December 23, 2013
British Airways jet wing hits building at South African airport
South African airport officials say four airport staff were slightly injured when the wing of a British Airways passenger aircraft clipped a building at Johannesburg's OR Tambo International Airport
Charlottesville plane crash kills pilot
The crash occurred Wednesday morning in a residential section near Route 29. No structures were struck by the plane.
Delta would continue to ban cell phone calls in-flight if FCC changes rules
Delta Airlines CEO Richard Anderson said in a memo to employees Wednesday that even if the FCC lifts the ban on in-flight telephone calls, his airline will continue to ban them.
NATO Afghanistan air crash kills six service members
NATO says six service members have died in an aircraft crash in southern Afghanistan, in an area where there was no fighting reported at the time.
Cell phone use in flight would still ban phone calls
The Federal Communications Commission may be close to allowing the use of cell phones in flight at all times, but with a catch - there would still be a ban on phone calls.
Storm air delays strand thousands at airports nationwide
As the pain from thousands of canceled or delayed flights from Sunday's storm lingered into Monday, the glut of cancellations stranded thousands of people and jammed ticket counters.
D.C.-area storm flight delays and cancellations
At Reagan Airport, all inbound flights are being held at their origin until 9 a.m.
Thanksgiving travel information: Forecast, flight information, cancellations
We're keeping an eye on travel conditions throughout the region. Bookmark this page and visit often for the very latest.
Boeing 747 lands at wrong airport in Kansas
A Boeing 747 that was mistakenly landed at a small Kansas airport took off again and successfully landed at McConnell Air Force Base in Wichita.
Cell phone use on planes expanded by FAA
Passengers will soon be allowed to use their mobile phones to do a variety of tasks on flights from gate-to-gate, according to new guidelines issued Thursday to airlines nationwide.
Narrower seats on airplanes a growing trend, executives say
Not only are airlines squeezing passengers for fees for a variety of things, including food and bags, the Wall Street Journal says that airlines both domestic and international are literally squeezing customers into smaller seats.
Spirit Airlines engine failure causes inspections, flight delays
Spirit spokeswoman Misty Pinson said today the Miramar-based airline is completing "a proactive and voluntary check" on aircraft as a precautionary measure following the engine failure on a flight last week.
FAA likely to revise electronics rules
An FAA advisory panel is likely to recommend new regulations for electronics on airplanes that would allow passengers to continue listening to podcasts and watching video during flights.
'Flugtag' takes flight in D.C.
Homemade flying vehicles will participate in a five-city national competition on Saturday.
Fire on 787 closes Heathrow Airport, sends Boeing stock tumblingComment
A fire aboard an empty 787 at Heathrow Airport shut down the runways and spooked Boeing investors Friday, as they feared the re-emergence of battery problems that grounded the plane for months earlier this year.
Solar-powered plane lands at DullesComment
A solar-powered plane nearing the close of a cross-continental journey landed at Dulles International Airport outside the nation's capital early Sunday.
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text:
The trial for the man accused of killing an 18-year-old man after an alleged argument in Dorchester County last year is underway.
Anthony Willis Jr., 19, of Summerville is charged with the murder of 18-year-old Justin Smoot. Willis is accused of shooting and killing Smoot with a rifle on Muir Court on May 2, 2011, authorities said.
On Tuesday opening statements began at the Dorchester County courthouse in St. George. Prosecutors described Smoot as a high student who had his whole future and life in front of him.
Prosecutors told the jury that Smoot was at a bus stop at the Churchhill apartment complex on Old Trolley Road when Williams, referred to by witnesses as A.J., got into an argument with Smoot. Following the verbal and then physical altercation, Willis shot at Smoot, according to prosecutors.
After shooting at and missing Smoot, prosecutors claim Willis went up to Smoot and shot him.
Mr. Willis hunts him down,prosecutors told the jury. His life, terminated. His future, terminated.
Defense attorneys did not get into specifics about the series of events during opening statements but spoke to the jury about the burden of proof the state is required to present for a guilty verdict.
Attorneys for Willis, who is pleading not-guilty, told the jury they need to be skeptical of the evidence presented. Several witnesses were also called to the stand by the prosecution by mid-day on Monday including friends of Smoot, some claiming to see the shooting.
While the jury will hear no mention of it during the murder trial, in March authorities in Summerville charged Willis with a robbery and carjacking they said took place while he was out on bail for the murder charge.
The charges related to those cases remain pending, according to court records. Willis faces the possibility of a 30-year to life sentence, if convicted of murder this week.
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text:
Abstract available in
Claims available in
Description (OCR text may contain errors)
U A. ESCHLE April 14, 1970 DIVER'S WATCH' Filed April 10, 1968 INVENTOR. 910/: nmzt BY ,2 MN
Malay-r United States Patent O 3,505,808 DIVERS WATCH Urs Alois Eschle, Le Locle, Switzerland, assiguor to Manufacture des Montres Doxa SA, Le Locle, Switzerland, a body corporate of Switzerland Filed Apr. 10, 1968, Ser. No. 720,102 Claims priority, application Switzerland, Apr. 14, 1967,
5,295/ 67 Int. Cl. G04b 19/18 US. Cl. 58126 5 Claims ABSTRACT OF THE DISCLOSURE A divers watch having a rotatable bezel carrying twograduated scales having a common zero point. One of said scales forms a graduation dividing the circle of the bezel into minutes while the other scale comprises a graduation and indications referring to diving depths.
BACKGROUND OF THE INVENTION This invention relates to a divers watch of the type comprising a rotatable bezel carrying guide marks arranged so that the position of the minutes-hand of the watch with respect to the guide marks maygive to the diver an indication of time relative to a maximum duration of dive.
Divers Watches of this type are already known in the art. In one of these known watches the rotatable bezel is graduated in minutes. This graduation, however, obliges the diver to memorize a rule of correspondence between the reached depths and the limit durations of dive from which on it will be necessary for him to observe decompression stops on his way back to the surface. To remove this drawback, which may lead to a positive danger since at great depths the intellectual capacities of a diver may be substantially reduced, it has already been proposed to provide the bezel with a depth graduation so that when the zero point or origin of this graduation is placed opposite'the minutes-hand at the beginning of the dive, all the diver has to do. to know the limit duration is to know at which depth he is and to check the moment where the minutes-hand reaches the position opposite the corresponding indication of depth of the graduation.
It has also already been proposed, to the same end, to keep a bezel graduated'in minutes on the periphery of the watch case, and to make the minutes-hand move with a disc carrying a graduation indicating the depths. In order to use such a watch it is necessary to place the zero point of the bezel graduation opposite the indication of the reached depth at the moment of the diving. The limit duration of dive at this depth is reached when the minuteshand reaches the point at which it is opposite 'the zero point of the bezel. However, while the different solutions to the problem indicated above make unnecessary for the diver to memorize a decompression table or a rule, of correspondance between the limit durations of dive on the one hand and the depths on the other hand these solutions do not enable the diver to read directly on his watch -the time interval that has elapsed since he entered the water unless he had also memorized the exact time at which this event happened.
Now the limit duration of a dive may be calculated as a function of the depth according to different rules. On the other hand occasionally a diver may want to arbitrarily limit his dive to a period that is inferior to the maximum period givenv by a certain table, for example to half of the duration given by said table.
Thus the prime object of the present invention is to realize a divers watch which, while not being of a construction more complicated than the heretofore known watches, and while providing as well as these known Watches the indications of the limit durations of dive, in addition affords for the diver to read directly on this watch, the time interval in minutes which has elapsed since the beginning of the diving.
SUMMARY OF THE INVENTION To this end this invention has for its subject a divers watch, comprising a rotatable bezel carrying guide marks or indicating graduations arranged so that the position of the minute-hand with respect to the guide marks may give to the diver an indication of time referring to a maximum duration of dive, in which the guide marks form two concentrically arranged graduated scales comprising a common zero point, whereby the one of said scales divides the circle of the bezel into minutes, while the other scale comprises a graduation and indications referring to diving depths.
BRIEF DESCRIPTION OF THE DRAWINGS The single figure of the accompanying drawing illustrates by way of example in a plan view an embodiment of a divers watch according to the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENT In the drawing reference numeral 1 designates the casing of a divers watch to which are attached the two ends of a band or strap 2 and the windingbutton 3 of which is partially enclosed in a peripheral protecting collar 4. The dial of the watch is of the conventional type provided with horological marks and for this reason is not shown in detail. It is covered by the glass of the watch and between the glass and the dial there are arranged the usual hands, as the minute-hand 14, the hour-hand 15 and the second-hand 16.
The glass is surrounded by a rotatable bezel 5 which is arranged for rotation by hand in counterclockwise direction. This bezel has its periphery provided with a plurality of circumferentially spaced notches or cut-outs defining teeth 6. As clearly illustrated, the notches that define the circumferentially extending series of outwardly extending teeth are so formed that the extent of the opposite faces of the teeth are different. Thus, the faces 6 of successive teeth on the clockwise side of the axes of the teeth are substantially radially disposed while the faces 6" of successive teeth on the opposite side of the axes of said teeth are inclined toward the respective axes in the clockwise direction. Thus, the-faces 6 afford or offer a better grip when the bezel is turned in counterin clockwise direction than when it is tried to be turned mediately at the interior of the recessed rim with the cut-outs 6. The zeropoint indicating graduation or origin of this graduated scale is marked by a circular recess 8 to which corresponds a small projection 9 formed on i the periphery of the bezel. The indicating graduations 10 of the scale 7 are irregularly distributed over the circle of the bezel and are numerical indicia which decrease in the clockwise direction from 60 to 2.0. Concentrically arranged within this graduated scale extends a second scale marked by radial strokes 11 which are evenly distributed on the circumference of the bezel 5. A circular recess 12 arranged on the same radius on the same side of the axis of rotation of the minute hand 14 as in the recess 8 indicates the origin or zero point indicating graduation of this second graduated scale 11 which also carries indications 13 in form of numerical indicia from 5 to 55 and spaced 30 from one another. This inner scale thus provides indicating graduations denoting in minutes the time interval elapsed from the moment when the minutehand 14 has passed opposite the common zero point 12, 8.
As appears from the drawing the numerical indicia 5 of the inner graduated scale coincides with the numerical indicia 60 of the Outer graduated scale, while the numerical indicia 25 and 50 of the first scale coincide with the numerical indicia 30 and of the outer scale. The two scales thus bring about a relation between the durations and the vertical extent of depths, which relation indicates the limit duration for which a diver may remain at a given depth without having to observe decompressing stops when returning to the surface. The relation which results from the drawing is the so-called American relation.
If the indicating graduations of the bezel 5 would have been made in accordance with the so-called French scale, the coincidences would be dilferent.
The use of the described watch is exactly the same as that of a watch having a bezel 5 carrying only one scale.
Just before the dive the diver rotates the bezel 5 to position the radially aligned point indicating graduations O8 and 12 opposite or in radial alignment with the momentary position of the tip of the minute-hand. From this moment on all the diver has to do is to measure by means of a manometer the depth at which he is and to observe whether the minute-hand has already or has not yet reached the corresponding mark of scale 7 corresponding to this depth, to know whether he can return to the surface directly or not. But at the same time the position of the minute-hand with respect to the inner zero point indicating graduation 12 of the scale gives him directly the duration of his dive up to this moment without necessity for him to memorize the exact time at which the diving began.
In order to clearly differentiate the two graduated scales, the numerical indicia of one of these scales may be marked with a vivid or bright colour, for example orange, while the indicating graduations of the other scale could be made in black, for example. If the two above-said colours are selected it will be preferable to mark in orange the numerical indicia and the zero point indicating graduation 8 of the outer scale corresponding to the depths.
What is claimed is:
1. In a divers watch for indicating conditions to a diver, a minute hand, a rotatable bezel radially outwards of the path of movement of the tip of said minute hand, said bezel having two series of circumferentially spaced condition indicating graduations defining two concentrically arranged graduated scales, each scale having a zero point indicating graduation, the zero point indicating graduations of the respective scales being in radial alignment on the same side of the axis of rotation of said minute hand, the indicating graduations of the first of said scales dividing the circular extent of said bezel into minutes with progressive minute indicating graduations increasing in the clockwise direction from said zero point indicating graduation of said first of said scales, the indicating graduations of the second of said scales dividing the circular extent of said bezel into vertical extent of depth indicating graduations with said last mentioned indicating graduations decreasing in the clockwise direction from the zero point indicating graduation of the second of said scales, each indicating graduation of each scale forming an angle with its associated zero point indicating graduation and the axis of rotation of said minute hand, whereby upon rotation of said bezel to position said radially aligned zero point indicating graduations in at least approximate alignment with the radial position of the tip of said minute hand substantially at the moment of the start of a dive, observance during a dive of the extent of movement of and the radial position of said minute hand in relation to said positioned radially aligned zero point indicating graduations provides a direct indication'of the maximum period of time that a diver may remain at the depth then indicated by the radial position of the minute hand with relation to the then radially aligned "vertical extent of depth indicating graduation on the second of said scales without the diver having to observe decompression stops on the way back to the surface and a direct indication on said first scale of the duration of dive up to the moment of observance in accordance with the ex-' tent of movement of said minute hand.
,2. A divers watch as claimed in claim 1 and the periphery of said bezel having circumferentially spaced notches therein defining a circumferentially extending series of outwardly extending teeth each having opposite faces, the faces of successive teeth on the clockwise side of the axes of said teeth being substantially radially disposed and the faces of successive teeth on the opposite side of the axes of said teeth being inclined toward the respective axes in the clockwise direction so as to afiord a better grip for turning the bezel in a counter-clockwise direction.
, v3. A divers watch as claimed in claim 1 and the indicating graduations on each of said scales including colored numerical indicia and the color of the numerical indicia on one scale being different than the color of the numerical indicia on the other scale.
4. A divers watch as claimed in claim 1 and the indicating graduations on the first of said scales including circumferentially spaced minute indicating numerical indicia progressively increasing in the clockwise direction and radially extending marks between successive numerical indicia and the indicating graduations on the second of said scales comprising vertical extent of depth indicating numerical indicia.
' 5. A divers watch as claimed in claim 4 and the numerical indicia on both scales being colored, and the color of the numerical indicia on one scale being different than the color of the numerical indicia on the other scale.
References Cited UNITED STATES PATENTS 3,124,928 3/1964 Brunet 5 8126 X FOREIGN PATENTS 365,027 11/1962 Switzerland. 436,140 1/1967 Switzerland.
RICHARD B. WILLIAMS, Primary Examiner STANLEY A. WAL, Assistant Examiner US. Cl. X.R.,
Patent Citations
Referenced by
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Got inspired to try a photo setup with candles to experiment with lighting/shadow/depth of field. A trip to wal-mart and 32 candles later here we go! I might upload the color version of this too...it's much warmer feeling whereas the black and white is more..moody.
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SOURCE: The Evening Sun
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American Civil War Wax Museum up for saletags: museums, Gettysburg, American Civil War Wax Museum
After 51 years educating and entertaining Gettysburg visitors, the American Civil War Wax Museum is up for sale.
"The owners are looking to retire," said Tammy Myers, general manager of the museum. It has always been a family business and the owners' children are not interested in operating it, she added, so they are ready to sell.
The 12,450-square-foot property located at 297 Steinwehr Av. is currently on the market for $1,695,000, acording to a listing by Prudential Bob Yost-Sites Homesale. As to whether or not the museum will remain open once it is sold, that will depend on the buyers, Myers said....
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- Joan Baez, Sly Stone, Steve Martin, Ben E. King -- all honored by the Library of Congress
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20th October 2008
Abortion dominates debate as lesbian fertility rights come before Commons
2:58 PM — The Human Fertilisation and Embryology Bill is back before MPs this week. If it passes the Commons without amendments on Wednesday it will become law.
Experts predict sharp rise in HIV infections among Hong Kong gays
1:41 PM — One third of men who have sex with men could be infected with HIV unless prevention measures work, according to experts in Hong Kong.
18th October 2008
Preview: Stephen Fry In America – The Deep South 22
12:10 PM — "I'd rather be curled up in a snowy cabin with a hot whisky or, quite frankly, a Horlicks than I would spend half an hour in this rotting place." Stephen Fry exclaims after visiting the USA's southern states in his six part BBC series.
17th October 2008
Dutch court hears evidence from victims of 'HIV gang'
2:18 PM — The trial of three men accused of deliberately injecting others with HIV-infected blood has heard testimony from some of their alleged victims.
15th October 2008
GMFA launches 'Know your HIV status' campaign
5:39 PM — Gay men's health charity GMFA have launched a new campaign highlighting the benefits of knowing you HIV status following new figures released by the British Association for Sexual Health and HIV (BASHH).
Four in ten gay HIV+ men are undiagnosed, say experts
1:58 PM — The British HIV Association (BHIVA) Conference last week head that almost half gay men in the UK with HIV are unaware of their condition.
13th October 2008
Three men stand trial accused of deliberate HIV infection
4:09 PM — Three gay men go on trial today in Holland charged with picking up more than a dozen men on the Internet, drugging them and injecting them with HIV positive blood.
10th October 2008
Children of lesbians 'at risk' due to prolific sperm donors 2
12:12 AM — A health official in South Australia has warned that the state's rules on sperm donation could put the children of some lesbians at risk.
9th October 2008
MPs and peers celebrate National AIDS Trust's 21st birthday
6:14 PM — Lord Fowler was among the guests at a Houses of Parliament reception for the National AIDS Trust yesterday.
8th October 2008
Circumcision not "significant" in HIV rates among men who have gay sex
2:16 PM — Circumcised men who have sex with men do not have significantly lower rates of HIV transmission, a US Centres for Disease Control report has concluded.
7th October 2008
Health minister took Mandelson to hospital after emergency call to Downing St 3
2:57 PM — Business Secretary Peter Mandelson called the Prime Minister's office to seek help after he fell ill on Sunday night, it has emerged.
6th October 2008
Mandelson to undergo surgery just days after taking office 2
4:17 PM — The new Secretary of State for Business, Enterprise and Regulatory Reform is to have surgery later today to remove a kidney stone.
1st October 2008
Health minister asks Indian PM to intervene over decriminalisation
4:10 PM — An Indian Cabinet minister has said that he will ask the country's Prime Minister to intervene in the row over the decriminalisation of homosexual acts.
US accused of not overturning ban on HIV+ visitors 1
12:55 PM — A New York-based LGBT immigration pressure group has accused the US government of failing to follow a new policy on HIV+ people visiting the country.
26th September 2008
Indian government argues to retain ban on gay sex 5
3:13 PM — A row between two government departments in India over the decriminalisation of "un-natural" sexual acts has resulted in a victory for those who oppose the change.
25th September 2008
London professor condemns fundamentalists for misinterpreting his research 5
2:09 PM — A leading British academic has spoken out after his work into homosexuality and depression was presented as proof that gay people are mentally disordered.
Website for gay men newly diagnosed with HIV launched 3
1:26 PM — A new interactive website aims to tackle the isolation many gay men feel when they are first diagnosed as HIV+.
18th September 2008
New guidelines suggest gay men should have annual HIV test 1
12:43 PM — Sexually active gay men should be tested every 12 months, new guidelines on HIV testing have recommended.
17th September 2008
Muslim leader denies issuing progressive fatwa on gender surgery
5:52 PM — A Sunni Muslim cleric in Kuwait has denied media reports that he had become the first religious authority in the country to approve gender reassignment surgery.
16th September 2008
Gays more prone to depression, psychiatrists claim 1
3:45 PM — A new study published by the journal BMC Psychiatry claims that lesbian, gay and bisexual people are 50% more likely than straight people to have suffered depression or anxiety disorders.


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Home > Graphic Apps::CAD > PDF to DXF Converter 9.6.2 9.6
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Our special coverage of the closest race for years
Explore the personal stories of Syrian refugees in Lebanon
Listening Post | 05 Jul 2014 18:58 GMT | Media, Latin America, Brazil, World Cup
In 2007, when Brazil won the right to host the World Cup, fans everywhere were excited at the prospect of feast of football in a country with such a rich tradition in the world's favourite sport.
For the government there, the fringe benefit was showing Brazil off to the world and reaping the PR benefits. But as the cost of the tournament grew, so did the size of anti-World Cup protests on the streets. And the demonstrators had a bone to pick with the mainstream media and their coverage of the demos.
Either the media failed to understand what was unfolding - or there was a reluctance in newsrooms to be critical of an event the government wanted to showcase.
The Listening Post's Gouri Sharma now, on some of the new media startups filling the gaps in the coverage of news stories, that can sometimes get in the way of a big sports story.
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In religiously conservative Brazil, women risk jail time and their lives to undergo the procedure that is illegal but commonplace.
Health, Human Rights, Latin America
Trash is a fast-growing import in the Scandinavian country, which turns it into heat for people's homes.
Environment, Europe, Sweden
Critics say the UK's home secretary is using Muslims as a 'political punchbag' in a bid to shore up right-wing support.
Human Rights, Politics, Racism
2014 was the worst year in more than a decade for polio infections, with at least 306 new cases reported.
Polio, Asia, Pakistan
It is a source of sustenance but also of tension and new political realities are now increasing uncertainty.
Egypt, Sudan, Middle East
A media post-mortem into the UK's surveillance report. Plus, why five years on in Argentina the media looks so familiar.
Media, Edward Snowden, Security
The untold story of how the Lebanese community overcame the odds and found their place in multicultural Australia.
Asia Pacific, Australia, Lebanon
How to keep a telenovela profitable and demanding sponsors happy while inspiring the production team to stay passionate.
Arts & Culture, Mexico, Latin America


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A good year for cricket with some brilliant action! India was declared the world's No. 1 cricket team. And the icing on the cake was the recognition for Master Blaster Sachin Tendulkar as he crossed 30,000 runs in international cricket.
Read about the year that was — the good and the not so good, as we bid goodbye to 2009.
More In: YOUNG WORLD | FEATURES


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Oct 27, 2011 ~ Love for your member. Your one band member.
by Lissa
posted Oct 27 2011 10:42AM
Sometimes I close my eyes and imagine I'm on a tropical beach somewhere, sipping mai tais and using the word "fabulous" to describe everything. In this fantasy, I have a gold-encrusted bikini and a killer tan. And I'm surrounded by people who laugh hysterically at all my jokes. In real life, I'm in Wilkes Barre PA, my co-workers seldom get my sarcastic humor, I wear SPF 50, and a gold-encrusted bikini would probably burn my hoo-ha. Guh....Itchy. But it's fun to imagine, isn't it?
I also like to imagine the original lineup of Guns n' Roses getting back together. Another fantasy that will never come true. Guns n' Roses is my all-time FAVORITE band. I got a rose tattoo on my stomach the day I turned eighteen….. It's an "Axl Rose". I know. Stupid….played. But hey, I was eighteen. Could've been a locomotive. Could've been a November raindrop. Could've been a rocket queen. My point: could've been sooooooo much dumber. So leave me alone.
I got my GnR tickets for the big show at Mohegan Sun Arena November 20 and I am officially PSYCHED! I've been getting a little flak from other diehard Guns fans because I honestly don't give a crap that Axl's the only ORIGINAL member on this tour. Big whoop. He was the main songwriter – we all know this. And yes, it sucks ass that they can't reconcile, but much like we are never again gonna see Meg Ryan's original face, we must come to terms with these new developments and move on.
Sure, it's not the same. But it's still AWESOME.
I've been in a rock band myself, and I know first hand how things can go south between band members. Of course, this is different in my case because we made little to no money and had little to no success. The only way we were getting booked at the state fair is if we joined 4-H. But I digress. I just mean to say I have equal sympathy for BOTH sides.
I would LOVE to see Slash, Duff, Izzy, and Steven onstage with Axl again, but let's be real. GnR is gonna kick ass no matter what! Because those songs are timeless. And Axl's bigger than life (which is also why he's a complete f%#king whackadoo). But I love him. I put Kanye in the same category: you're crazy, but you're a genius and I love you. Crazy/talented/genius. I'm lucky if I get weird/slow-witted/follower.
So I hope to see you at Mohegan Sun Arena November 20! I'll bring my 4-H pig. Name's "Wiggly". You can pet him. And if you see me down front, I'll be the girl spazzing out to every song, trying to use my radio station laminates to get backstage (and by "radio station laminates", of course I mean boobs).
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1963 1/2 FORD GALAXIE Vintage Racecar. IOLA Poster car. See at the 2015 show. Circle Track, Road Race or Show. 2011 parade car at Brickyard 400 and... More
2005 350 Mercedes SLK offered for sale. Low mileage two owner car with warranty replacement engine 39,000 miles ago. Only 30,000 on the motor.... More
1949 Hillegass, This is a very rare opportunity to acquire one of the very last unrestored, rail frame "Big Car" Sprint Cars designed and... More
1964 Chrysler 300K offered for sale in the San Francisco Bay area. 440CID bored MOPAR engine complete with cross-ram intake manifold, MOPAR accessories and... More
***1967 DODGE DART*** *4 LINK* DANA 60* CONE COIL OVER ON REAR* FULL CAGE (TIG WELDED)* BACK HALF* AFTERMARKET UPPER LOWER CONTROL ARMS* TUBULAR COIL... More
Body: New urethane paint with vinyl stripes and number balls Bonnet opens forward for easy access to engine Drive shaft tunnel modified with tubular space frame for... More
Vehicle Type : Midget auto racer, rear engine. One of a kind. Museum piece Year built : 1975. Suspension : Cross torsion bar front, one cross... More
2004 Ford Mustang Mach 1 Cobra Jet with Shaker Hood and 40th Anniversary badge. Azure Blue with Black Racing Stripe down center of hood.... More
1977 OLDSMOBILE/BUICK REGAL. Built by Hutchinson-Pagan and campaigned in the former NASCAR Winston Cup Series by Tighe Scott with crew chief Harry Hyde. Finished... More
Rebuilt 327 motor Bulletproof Delco Heavy Duty 350 transmission Lakewood Ladder Bars 411 Ratio differential New ring and pinion posi traction rear end Stock suspension MSP ignition and distributor, push... More
1968 Ford Cortina restored from bare chassis to be a track-day car. It is fully stripped, weighs about 1800 lb, and is powered by... More
2007 Chrysler 300 SRT 8 ONLY 44,762 MILES!!!!!! 6.1L HEMI V8 425 HP and 420 pound feet of torque 0-60 in 4.7 seconds 5 speed shiftable... More
1980 Chevy Malibu, Seattle, Washington. Selling for $15,500 Engine Specs - 434ci "World Engine" Rocker Arms comp 1.16 Hydraulic Roller Lifters Comp 850 Holly Double Pumper Carb Edelbrock Manifold 2 piece... More
3.5 L EGJ V6 engined 2001 Chrysler Prowler offered for sale in Oakland, CA. Low mileage great example of a car that saw Chrysler let... More
2003 Audi S8 in Immaculate Condition. Original Paint, New Trans, Complete service history! Audi Space frame aluminum Supercar! Well documented miles currently at- 113,830 Clean Carfax and well documented Classic Avus Silver Clear... More


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cityscapes greeting cards, cityscape greeting cards, cincinnati greeting cards, cincinnati ohio greeting cards, ohio greeting cards, sidewalk shadows greeting cards, shadow greeting cards, montgomery ohio greeting cards, downtown greeting cards, urban greeting cards, small town america greeting cards
( Copyright Mel Steinhauer all rights reserved ) Just like most major metropolitan areas, Cincinnati has many smaller communities which appear to blend in with the official city limits. Although each one is a separate entity with their own unique characteristics, they are individual contributors to the sum total of this beautiful All American City. As you depart one, you immediately enter the next and frequently there is only a sign to inform you so. But if you slow down, look a little harder, you will notice some differences and see some beauty. Like I did one day when I arrived in the colorful and quaint downtown area of Montgomery. An early morning sun cast a golden glow just so I could really enjoy these ... Sidewalk Shadows. I...
Welcome to a collection of my favorite "Scenes From Far and Near" ! Just select a gallery and click on any image for better viewing and for an easy selection of print sizes, mats, frames, canvas wraps, metal or acrylic prints, cards, pricing and shipping information. My love for photography began as a boy when I was taught the basics by my father. During a tour of duty in Vietnam, I purchased an expensive Nikon SLR camera and dreamed of becoming a photographer when I returned home. A family of my own and a new career put that dream on hold, however I always enjoyed taking photographs. Several years ago, after surviving another kind of war with...
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2 hours lecture, 2 hours laboratory; 3 credits
Physiological factors involved in bodily movement and performance of work. Such phenomena as responsive changes in circulation and respiration coincident with exercise, and adaptation to environmental conditions. Efficiency, physical fitness, coordination, and fatigue.
Prerequisite: Physical Education/Kinesiolgy 3271 [22.71] and 3275 [22.75] or the equivalent.
Take advantage of our two five-week summer sessions for both undergraduate and graduate students.
If you can't be here in person, take our virtual tour and discover the beauty and vitality of our campus.
Our faculty not only share their discoveries and knowledge with you, they invite you to be active members of their research teams.
A privately owned residence hall is located just blocks from the college.
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AMA Affiliate Program
- The Missouri Mudders participates in the AMA affiliate program. Join the AMA or renew your membership by clicking here or in the footer of our web pages.
Event Shelter and Construction Progress
Sunday, May 17th, 2015 National Enduro Championship Series RD-5
- Staging from the Mudder's shelter at St. Joe State Park
- Here is the flier
Sunday, July 19th, 2015 Leadbelt XC UFD Mountain Bike Event
- Staging from the Pim Day Use Area at St. Joe State Park
Sunday, October 25th, 2015 Leadbelt MHSC Hare Scrambles
- Staging from the Mudder's shelter at St. Joe State Park


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HELP!! Ideas needed for Backsplash with Madre Perla Countertops
This is my first post on this forum and hope to get some feedback from all you creative types...
We need to choose a backsplash to compliment our beautiful madre perla quartzite countertops. I have been looking at polished white (pearl) onyx staggered tile to maybe glass tile. Any ideas or feedback is appreciated!
Absolutely gorgeous choices so far. The colors are subtle and incredibly beautiful. I love the wall color, cabinet color, stone, floor - home run.
How do you feel about mother-of-pearl tile? It is available in almost any imaginable format like the 2" x 2" mosaic below, and can have touches of pink to cream, to brown.
One thing to look for is how the tile looks with the lights on and lights off. I tried the MOP shell tile in my current kitchen. It was gorgeous with the lights off and only daylight in the room.
As soon as I turned on the ucl or the other lights, it went flat. Someone else mentioned this. I was surprised that it happened.
I think Ann Sachs might have an Onyx tile available.
Did you find a bs tile? I'd love to see the result. Yours is one of my favorite neutral kitchens!


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TUG 2015 will take place at the Loews Royal Pacific Resort in Orlando, Florida on May 4-6, 2015.
Register today to join us as we explore the universe of test by generating ideas and sharing solutions. TUG is the longest running ATE Users Group and is a great opportunity for users of Teradyne products to collaborate with peers in the industry, as well as the experts from Teradyne. View the most up-to-date program, including abstracts, panel discussion topics and current list of participants in the vendor fair.
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Olmscheid -- Baklund
Niki Olmscheid and Joshua Baklund were united in marriage Dec. 19 at Bingham Hall Bed and Breakfast in New Ulm.
The bride is the daughter of Al and Arlis Feser of Hutchinson. The groom is the son of Greg and Ruth Baklund of Atwater.
Maid of honor was Stacey Bern.
Best man was Mike Meyer. Ring bearer was Javan Olmscheid.
The couple lives in Hutchinson.
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Fresh Air Weekend: The Coen Brothers And 'Anchorman 2'
Originally published on Sat December 21, 2013 11:50 am
Fresh Air Weekend highlights some of the best interviews and reviews from past weeks, and new program elements specially paced for weekends. Our weekend show emphasizes interviews with writers, filmmakers, actors, and musicians, and often includes excerpts from live in-studio concerts. This week:
The Coen Bros. On Writing, 'Lebowski' And Literally Herding Cats: Inside Llewyn Davis — starring Oscar Isaac and a disobedient cat — is the latest from the filmmaking duo. The brothers talk with Fresh Air's Terry Gross about their writing process ("It's mostly napping") and the cult status of their 1998 film The Big Lebowski ("How do you explain that? I have no idea.")
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AT&T, Microsoft reveal AT&T Mobile Office Suite for SMBs
The new mobile suite aims to make voice, video and web collaboration easier.
The new mobile suite aims to make voice, video and web collaboration easier.
AusCERT's annual conference has wrapped up for 2013, and this time, it was personal.
Quickflix is asking its customers to buy up to AU$1 million in new shares after selling only AU$650,000 worth of the AU$5.7 million share offer that it hoped would bolster its presence prior to the Australian launch of rival Netflix next year.
Robotics and automation could put well over half a million jobs in professions such as bookkeeping, accounting, and stenography at risk for the year ending 2014, according to the Department of Industry's latest report.
A 44-year-old man has been charged with seven counts of corruptly receiving payments from a US-based IT company totalling almost AU$1.9 million.
Apple acquired a UK Cassandra-based analytics firm before Foundation DB, around the time it was buying tech to improve Siri.
Central to the new features is an extra layer of privacy that BlackBerry said allows users to take more control over their shared messages and content.
BYOD is no longer a fad, and has for many companies become part of the IT strategy. But what happens to a device once it reaches end of life?
The government has laid down €6bn to give Italy better broadband, and it's hoping local telcos will do the same.
The company says the buyout will bolster Akamai's DNS product portfolio offerings.


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Want to meet us at the Market this year? We're moving to the Waterfront Plaza, along the Riverwalk in downtown Mount Vernon!
Regular Market hours: Saturdays at the Plaza, 9am-2pm, starting May 23, 2015 and running through Oct 17, 2015 – for a total of 22 markets (dates listed below).
Wednesdays at the Hospital, 11am-4pm through during summer months. For questions concerning the Market, please email co-managers JillQ and Jodie B at firstname.lastname@example.com, or you can reach the Market's Board of Directors, at email@example.com.


text: Stayed here for 1 night while on recent trip to the area. Easy to find and free car parking on site which is always a BONUS !! The rooms are nothing special but adequate. Did not eat in as there are plenty of places to dine out but the breakfast was OK. Good for anyone on tight budget My only... More


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Stars arrive at the 2013 Film Independent Spirit Awards in Santa Monica, California, on February 23, 2013.
2013 Independent Spirit Awards Red Carpet Arrivals
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Melanie Lynskey
Melanie Lynskey attends the 2013 Film Independent Spirit Awards at Santa Monica Beach on February 23, 2013 in Santa Monica, California.
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