Perturbing HIV-1 Ribosomal Frameshifting Frequency Reveals a cis Preference for Gag-Pol Incorporation into Assembling Virions

HIV-1 virion production is driven by Gag and Gag-Pol (GP) proteins, with Gag forming the bulk of the capsid and driving budding, while GP binds Gag to deliver the essential virion enzymes protease, reverse transcriptase, and integrase. Virion GP levels are traditionally thought to reflect the relative abundances of GP and Gag in cells (;1:20), dictated by the frequency of a 21 programmed ribosomal frameshifting (PRF) event occurring in gag-pol mRNAs. Here, we exploited a panel of PRF mutant viruses to show that mechanisms in addition to PRF regulate GP incorporation into virions. First, we show that GP is enriched ;3-fold in virions relative to cells, with viral infectivity being better maintained at subphysiological levels of GP than when GP levels are too high. Second, we report that GP is more efficiently incorporated into virions when Gag and GP are synthesized in cis (i.e., from the same gag-pol mRNA) than in trans, suggesting that Gag/GP translation and assembly are spatially coupled processes. Third, we show that, surprisingly, virions exhibit a strong upper limit to trans-delivered GP incorporation; an adaptation that appears to allow the virus to temper defects to GP/Gag cleavage that may negatively impact reverse transcription. Taking these results together, we propose a "weighted Goldilocks"scenario for HIV-1 GP incorporation, wherein combined mechanisms of GP enrichment and exclusion buffer virion infectivity over a broad range of local GP concentrations. These results provide new insights into the HIV-1 virion assembly pathway relevant to the anticipated efficacy of PRF-targeted antiviral strategies.National Institutes of Health R01AI110221, P01CA022332, R35GM118131, T32GM00834

The Parmodulin NRD-21 is an Allosteric Inhibitor of PAR1 Gq Signaling with Improved Anti-Inflammatory Activity and Stability

Novel analogs of the allosteric, biased PAR1 ligand ML161 (parmodulin 2, PM2) were prepared in order to identify potential anti-thrombotic and anti-inflammatory compounds of the parmodulin class with improved properties. Investigations of structure-activity relationships of the western portion of the 1,3-diaminobenzene scaffold were performed using an intracellular calcium mobilization assay with endothelial cells, and several heterocycles were identified that inhibited PAR1 at sub-micromolar concentrations. The oxazole NRD-21 was profiled in additional detail, and it was confirmed to act as a selective, reversible, negative allosteric modulator of PAR1. In addition to inhibiting human platelet aggregation, it showed superior anti-inflammatory activity to ML161 in a qPCR assay measuring the expression of tissue factor in response to the cytokine TNF-alpha in endothelial cells. Additionally, NRD-21 is much more plasma stable than ML161, and is a promising lead compound for the parmodulin class for anti-thrombotic and anti-inflammatory indications

Academic well-being and smoking among 14-to 17-year-old schoolchildren in six European cities

It is well established that poor academic performance is related to smoking, but the association between academic well-being and smoking is less known. We measured academic well-being by school burnout and schoolwork engagement and studied their associations with smoking among 14- to 17-year-old schoolchildren in Belgium, Germany, Finland, Italy, the Netherlands, and Portugal. A classroom survey (2013 SILNE survey, N = 11,015) was conducted using the Short School Burnout Inventory and the Schoolwork Engagement Inventory. Logistic regression, generalized linear mixed models, and ANOVA were used. Low schoolwork engagement and high school burnout increased the odds for daily smoking in all countries. Academic performance was correlated with school burnout and schoolwork engagement, and adjusting for it slightly decreased the odds for smoking. Adjusting for socioeconomic factors and school level had little effect. Although high school burnout and low schoolwork engagement correlate with low academic performance, they are mutually independent risk factors for smoking. (C) 2016 The Authors. Published by Elsevier Ltd on behalf of The Foundation for Professionals in Services for Adolescents.Peer reviewe

Using breath carbon monoxide to validate self-reported tobacco smoking in remote Australian Indigenous communities

Background: This paper examines the specificity and sensitivity of a breath carbon monoxide (BCO) test and\ud optimum BCO cutoff level for validating self-reported tobacco smoking in Indigenous Australians in Arnhem Land,\ud Northern Territory (NT).\ud \ud Methods: In a sample of 400 people (≥16 years) interviewed about tobacco use in three communities, both selfreported\ud smoking and BCO data were recorded for 309 study participants. Of these, 249 reported smoking tobacco\ud within the preceding 24 hours, and 60 reported they had never smoked or had not smoked tobacco for ≥6\ud months. The sample was opportunistically recruited using quotas to reflect age and gender balances in the\ud communities where the combined Indigenous populations comprised 1,104 males and 1,215 females (≥16 years).\ud Local Indigenous research workers assisted researchers in interviewing participants and facilitating BCO tests using\ud a portable hand-held analyzer.\ud \ud Results: A BCO cutoff of ≥7 parts per million (ppm) provided good agreement between self-report and BCO\ud (96.0% sensitivity, 93.3% specificity). An alternative cutoff of ≥5 ppm increased sensitivity from 96.0% to 99.6% with no change in specificity (93.3%). With data for two self-reported nonsmokers who also reported that they smoked\ud cannabis removed from the analysis, specificity increased to 96.6%.\ud \ud Conclusion: In these disadvantaged Indigenous populations, where data describing smoking are few, testing for\ud BCO provides a practical, noninvasive, and immediate method to validate self-reported smoking. In further studies\ud of tobacco smoking in these populations, cannabis use should be considered where self-reported nonsmokers\ud show high BCO

Predisposition to Childhood Otitis Media and Genetic Polymorphisms within the Toll-Like Receptor 4 (TLR4) Locus

Background Predisposition to childhood otitis media (OM) has a strong genetic component, with polymorphisms in innate immunity genes suspected to contribute to risk. Studies on several genes have been conducted, but most associations have failed to replicate in independent cohorts. Methods We investigated 53 gene polymorphisms in a Finnish cohort of 624 cases and 778 controls. A positive association signal was followed up in a tagging approach and tested in an independent Finnish cohort of 205 cases, in a British cohort of 1269 trios, as well as in two cohorts from the United States (US); one with 403 families and the other with 100 cases and 104 controls. Results In the initial Finnish cohort, the SNP rs5030717 in the TLR4 gene region showed significant association (OR 1.33, P=.003) to OM. Tagging SNP analysis of the gene found rs1329060 (OR 1.33, P=.002) and rs1329057 (OR 1.29, P=.003) also to be associated. In the more severe phenotype the association was stronger. This finding was supported by an independent Finnish case cohort, but the associations failed to replicate in the British and US cohorts. In studies on TLR4 signaling in 20 study subjects, the three-marker risk haplotype correlated with a decreased TNF alpha secretion in myeloid dendritic cells. Conclusions The TLR4 gene locus, regulating the innate immune response, influences the genetic predisposition to childhood OM in a subpopulation of patients. Environmental factors likely modulate the genetic components contributing to the risk of OM.Peer reviewe