Mutations within the Primer Binding Site of the Human Immunodeficiency Virus Type 1 Define Sequence Requirements Essential for Reverse Transcription

AbstractThe primer binding site (PBS) is involved in two stages during the reverse transcription of the retroviral RNA genome. In the early stage, the PBS provides complementary sequences through which tRNALys,3binds the viral RNA genome to initiate minus-strand DNA synthesis; in the later stages, complementarity between the plus- and minus-strand copies of the PBS is required to facilitate the second template transfer needed to complete reverse transcription. We previously constructed a mutant HIV-1 proviral genome, designated as pHXB2PBS(pheC + 5) (now referred to as pheC + 5), which was used to identify regions of the PBS involved in the initiation and second template transfer steps of reverse transcription. To further define the sequence requirements of the PBS for the initiation of reverse transcription, we have made single nucleotide substitutions within the first six nucleotides of the pheC + 5 PBS. Our results demonstrate that mutations within the first five nucleotides of the PBS which disrupt base paring with tRNALys,3-PBS results in an noninfectious virus; a G-U base pair at position six of the tRNALys,3-PBS complex was tolerated. In contrast to the requirements for initiation, we found that complementary binding between only three base pairs of the plus- and minus-strand PBSs was required for the extension of plus-strand DNA during the second template transfer. Furthermore, regions of the minus-strand DNA of up to 24 nucleotides could be looped-out to facilitate the complementarity required for the completion of plus-strand DNA synthesis. Taken together, the results of our studies demonstrate that different features of the PBS with respect to RNA:RNA and DNA:DNA interactions are required for initiation of reverse transcription and the completion of plus-strand DNA synthesis, respectively

Comparative Analysis of the Glycosylation Profiles of Membrane-Anchored HIV-1 Envelope Glycoprotein Trimers and Soluble gp140

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer, which consists of the gp120 and gp41 subunits, is the focus of multiple strategies for vaccine development. Extensive Env glycosylation provides HIV-1 with protection from the immune system, yet the glycans are also essential components of binding epitopes for numerous broadly neutralizing antibodies. Recent studies have shown that when Env is isolated from virions, its glycosylation profile differs significantly from that of soluble forms of Env (gp120 or gp140) predominantly used in vaccine discovery research. Here we show that exogenous membrane-anchored Envs, which can be produced in large quantities in mammalian cells, also display a virion-like glycan profile, where the glycoprotein is extensively decorated with high-mannose glycans. Additionally, because we characterized the glycosylation with a high-fidelity profiling method, glycopeptide analysis, an unprecedented level of molecular detail regarding membrane Env glycosylation and its heterogeneity is presented. Each glycosylation site was characterized individually, with about 500 glycoforms characterized per Env protein. While many of the sites contain exclusively high-mannose glycans, others retain complex glycans, resulting in a glycan profile that cannot currently be mimicked on soluble gp120 or gp140 preparations. These site-level studies are important for understanding antibody-glycan interactions on native Env trimers. Additionally, we report a newly observed O-linked glycosylation site, T606, and we show that the full O-linked glycosylation profile of membrane-associated Env is similar to that of soluble gp140. These findings provide new insight into Env glycosylation and clarify key molecular-level differences between membrane-anchored Env and soluble gp140

Decreased venous thrombosis with an oral inhibitor of P selectin

BackgroundP-selectin inhibition with protein therapeutics such as antibodies or soluble ligands given intravenously can decrease thrombosis in a mouse ligation model of venous thrombosis. In this study, we hypothesized that oral inhibition of P selectin with a novel oral nonprotein inhibitor (PSI-697) would decrease thrombosis and circulating microparticle populations. This study evaluated the effects on thrombosis and circulating microparticle populations in this murine venous thrombosis model.MethodsMice underwent inferior vena cava ligation to induce thrombosis. Mice with high circulating level of P selectin, Delta Cytoplasmic Tail (^CT), mice gene-deleted for both E- and P-selectin knockout (EPKO), and wild-type C57BL/6 mice (WT) were studied without and with administration of PSI-697 in food (100 mg/kg daily) from 2 days before thrombosis until the end of the study. Animals were killed 2 and 6 days later. Evaluations included thrombus weight (TW), vein wall morphometrics, microparticle quantification by using fluorescence-activated cell sorter analysis, and vein wall enzyme-linked immunosorbent assays for interleukin (IL)-10, P selectin, and monocyte chemotactic protein 1.ResultsPSI-697 significantly decreased TW in WT and ^CT mice, with a treated vs nontreated TW of 132 ± 24 vs 228 ± 29 × 10−4 g (P = .014) and 166 ± 19 vs 281 ± 16 × 10−4 g (P = .001), respectively. At day 6, the effect was significant only in the ^CT group (P < .05). Drug therapy at day 2 significantly increased vein wall monocytes in WT mice and increased monocytes and total inflammatory cells in ^CT animals. A significant decrease in neutrophils and total inflammatory cells was seen in EPKO mice at day 2 with therapy. Therapy significantly increased platelet-derived microparticles and total microparticles in ^CT mice on day 2. Changes in treated WT and treated EPKO animals were not significant compared with respective vehicle treatments at day 2. On day 6, therapy significantly decreased total microparticles in EPKO animals. Vein wall expression of IL-10 increased in all groups with therapy at day 2 (n = 18) and was significantly increased in WT (2687.5 ± 903 pg/mL vs 636 ± 108 pg/mL total protein; P = .038) and ^CT (2078 ± 295 pg/mL vs 432 ± 62 pg/mL total protein; P = .001) mice. Therapy significantly decreased vein wall P selectin, monocyte chemotactic protein 1, and IL-10 levels at day 6.ConclusionsPSI-697 decreased thrombosis. P-selectin inhibition allowed vein wall inflammatory cell extravasation in this model of complete ligation. Circulating microparticles (platelet-derived microparticles and total microparticles) increased with P-selectin inhibition, possibly because of decreased consumption into the thrombus. In summary, the oral administration of an inhibitor to P selectin provides significant TW reduction.Clinical RelevanceDeep venous thrombosis is a significant national health problem in the general population. The average annual incidence of deep venous thrombosis is approximately 250,000 cases per year. The selectin family of adhesion molecules is thought to be largely responsible for the initial attachment and rolling of leukocytes on stimulated vascular endothelium. Recent studies have explored the possible therapeutic implications of P-selectin inhibition to modulate venous thrombosis. For example, prophylactic dosing of a recombinant P-selectin ligand decreases venous thrombosis in a dose-dependent fashion in both feline and nonhuman primate animal models. Additionally, treatment of 2-day iliac thrombi with a recombinant protein, P-selectin inhibitor, significantly improves vein reopening in nonhuman primates. It is interesting to note that P-selectin inhibition decreases thrombosis without adverse anticoagulation. On the basis of the results from these previous studies, the use of P-selectin antagonism is a logical therapeutic approach to treat venous thrombosis. All inhibitors developed to date are either proteins or small molecules with low oral bioavailability that require intravenous or subcutaneous injection. This study evaluates, for the first time, a novel orally bioavailable inhibitor of P-selectin (PSI-697)

Movies and TV Influence Tobacco Use in India: Findings from a National Survey

Background: Exposure to mass media may impact the use of tobacco, a major source of illness and death in India. The objective is to test the association of self-reported tobacco smoking and chewing with frequency of use of four types of mass media: newspapers, radio, television, and movies. Methodology/Principal Findings: We analyzed data from a sex-stratified nationally-representative cross-sectional survey of 123,768 women and 74,068 men in India. All models controlled for wealth, education, caste, occupation, urbanicity, religion, marital status, and age. In fully-adjusted models, monthly cinema attendance is associated with increased smoking among women (relative risk [RR]: 1·55; 95% confidence interval [CI]: 1·04–2·31) and men (RR: 1·17; 95% CI: 1·12–1·23) and increased tobacco chewing among men (RR: 1·15; 95% CI: 1·11–1·20). Daily television and radio use is associated with higher likelihood of tobacco chewing among men and women, while daily newspaper use is related to lower likelihood of tobacco chewing among women. Conclusion/Significance: In India, exposure to visual mass media may contribute to increased tobacco consumption in men and women, while newspaper use may suppress the use of tobacco chewing in women. Future studies should investigate the role that different types of media content and media play in influencing other health behaviors

Retail Price and Point of Sale Display of Tobacco in the UK: A Descriptive Study of Small Retailers

Background: Since the implementation of the 2002 Tobacco Advertising and Promotion Act, point-of-sale (PoS) tobacco displays are one of few remaining means of communication between the tobacco industry and customers in the UK. This study aimed to explore the characteristics of tobacco displays in a UK city, and particularly to assess the tobacco prices and promotional offers, types and pack sizes on display. Methods: Digital pictures of PoS displays were taken in 117 small retail shops in Nottingham in mid 2010. Data were analysed using Windows Photo Gallery software and SPSS version 16. Results: Just over half (52%) of cigarette packs on display were packs of 20, and 43 % packs of 10. Cigarette prices differed substantially between brands, ranging from £4.19 to £6.85 for 20-packs, and from £2.12 to £3.59 for 10-packs. Forty four percent of cigarette packs and 40 % of RYO (Roll-Your-Own) tobacco pouches, almost exclusively lower priced brands, were displayed with a pricemark, implying a promotional price offer. Eighty percent of 20-pack cigarette brand or brand variants on sale were priced below the EU-defined Most Popular Price Category (MPPC) for the UK in 2010; 45 % were priced below the Weighted Average Price (WAP), which replaced the MPPC in 2011. Conclusion: PoS displays communicate value by displaying a high proportion of lower cost brands, and smaller and hence lower-cost packs, and by displaying price discounts on packs. The MPPC substantially overestimated the prices at whic

Pathways to a cancer-free future: a protocol for modelled evaluations to minimise the future burden of colorectal cancer in Australia.

INTRODUCTION:With almost 50% of cases preventable and the Australian National Bowel Cancer Screening Program in place, colorectal cancer (CRC) is a prime candidate for investment to reduce the cancer burden. The challenge is determining effective ways to reduce morbidity and mortality and their implementation through policy and practice. Pathways-Bowel is a multistage programme that aims to identify best-value investment in CRC control by integrating expert and end-user engagement; relevant evidence; modelled interventions to guide future investment; and policy-driven implementation of interventions using evidence-based methods. METHODS AND ANALYSIS: Pathways-Bowel is an iterative work programme incorporating a calibrated and validated CRC natural history model for Australia (Policy1-Bowel) and assessing the health and cost outcomes and resource use of targeted interventions. Experts help identify and prioritise modelled evaluations of changing trends and interventions and critically assess results to advise on their real-world applicability. Where appropriate the results are used to support public policy change and make the case for optimal investment in specific CRC control interventions. Fourteen high-priority evaluations have been modelled or planned, including evaluations of CRC outcomes from the changing prevalence of modifiable exposures, including smoking and body fatness; potential benefits of daily aspirin intake as chemoprevention; increasing CRC incidence in people aged <50 years; increasing screening participation in the general and Aboriginal and Torres Strait Islander populations; alternative screening technologies and modalities; and changes to follow-up surveillance protocols. Pathways-Bowel is a unique, comprehensive approach to evaluating CRC control; no prior body of work has assessed the relative benefits of a variety of interventions across CRC development and progression to produce a list of best-value investments. ETHICS AND DISSEMINATION:Ethics approval was not required as human participants were not involved. Findings are reported in a series of papers in peer-reviewed journals and presented at fora to engage the community and policymakers

Mapping the sex determination locus in the hāpuku (Polyprion oxygeneios) using ddRAD sequencing

Background&nbsp; Hāpuku (Polyprion oxygeneios) is a member of the wreckfish family (Polyprionidae) and is highly regarded as a food fish. Although adults grow relatively slowly, juveniles exhibit low feed conversion ratios and can reach market size in 1&ndash;2 years, makingP. oxygeneiosa strong candidate for aquaculture. However, they can take over 5years to reach sexual maturity in captivity and are not externally sexually dimorphic, complicating many aspects of broodstock management. Understanding the sex determination system ofP. oxygeneiosand developing accurate assays to assign genetic sex will contribute significantly towards its full-scale commercialisation.&nbsp; Results&nbsp; DNA from parents and sexed offspring (n = 57) from a single family of captive bredP. oxygeneioswas used as a template for double digestion Restriction-site Associated DNA (ddRAD) sequencing. Two libraries were constructed usingSbfI&ndash;SphI andSbfI &ndash;NcoI restriction enzyme combinations, respectively. Two runs on an Illumina MiSeq platform generated 70,266,464 raw reads, identifying 19,669 RAD loci. A combined sex linkage map (1367cM) was constructed based on 1575 Single Nucleotide Polymorphism (SNP) markers that resolved into 35 linkage groups. Sex-specific linkage maps were of similar size (1132 and 1168cM for male and female maps respectively). A single major sex-determining locus, found to be heterogametic in males, was mapped to linkage group 14. Several markers were found to be in strong linkage disequilibrium with the sex-determining locus. Allele-specific PCR assays were developed for two of these markers, SphI6331 and SphI8298, and demonstrated to accurately differentiate sex in progeny within the same pedigree. Comparative genomic analyses indicated that many of the linkage groups within theP. oxygeneiosmap share a relatively high degree of homology with those published for the European seabass (Dicentrarchus labrax).&nbsp; Conclusion&nbsp; P. oxygeneioshas an XX/XY sex determination system. Evaluation of allele-specific PCR assays, based on the two SNP markers most closely associated with phenotypic sex, indicates that a simple molecular assay for sexingP. oxygeneiosshould be readily attainable. The high degree of synteny observed withD. labraxshould aid further molecular genetic study and exploitation of hāpuku as a food fish

Search for the standard model Higgs boson in the H to ZZ to 2l 2nu channel in pp collisions at sqrt(s) = 7 TeV

A search for the standard model Higgs boson in the H to ZZ to 2l 2nu decay channel, where l = e or mu, in pp collisions at a center-of-mass energy of 7 TeV is presented. The data were collected at the LHC, with the CMS detector, and correspond to an integrated luminosity of 4.6 inverse femtobarns. No significant excess is observed above the background expectation, and upper limits are set on the Higgs boson production cross section. The presence of the standard model Higgs boson with a mass in the 270-440 GeV range is excluded at 95% confidence level.Comment: Submitted to JHE

Search for anomalous t t-bar production in the highly-boosted all-hadronic final state

A search is presented for a massive particle, generically referred to as a Z', decaying into a t t-bar pair. The search focuses on Z' resonances that are sufficiently massive to produce highly Lorentz-boosted top quarks, which yield collimated decay products that are partially or fully merged into single jets. The analysis uses new methods to analyze jet substructure, providing suppression of the non-top multijet backgrounds. The analysis is based on a data sample of proton-proton collisions at a center-of-mass energy of 7 TeV, corresponding to an integrated luminosity of 5 inverse femtobarns. Upper limits in the range of 1 pb are set on the product of the production cross section and branching fraction for a topcolor Z' modeled for several widths, as well as for a Randall--Sundrum Kaluza--Klein gluon. In addition, the results constrain any enhancement in t t-bar production beyond expectations of the standard model for t t-bar invariant masses larger than 1 TeV.Comment: Submitted to the Journal of High Energy Physics; this version includes a minor typo correction that will be submitted as an erratu