RNA polymerase II stalling promotes nucleosome occlusion and pTEFb recruitment to drive immortalization by Epstein-Barr virus

Epstein-Barr virus (EBV) immortalizes resting B-cells and is a key etiologic agent in the development of numerous cancers. The essential EBV-encoded protein EBNA 2 activates the viral C promoter (Cp) producing a message of ~120 kb that is differentially spliced to encode all EBNAs required for immortalization. We have previously shown that EBNA 2-activated transcription is dependent on the activity of the RNA polymerase II (pol II) C-terminal domain (CTD) kinase pTEFb (CDK9/cyclin T1). We now demonstrate that Cp, in contrast to two shorter EBNA 2-activated viral genes (LMP 1 and 2A), displays high levels of promoter-proximally stalled pol II despite being constitutively active. Consistent with pol II stalling, we detect considerable pausing complex (NELF/DSIF) association with Cp. Significantly, we observe substantial Cp-specific pTEFb recruitment that stimulates high-level pol II CTD serine 2 phosphorylation at distal regions (up to +75 kb), promoting elongation. We reveal that Cp-specific pol II accumulation is directed by DNA sequences unfavourable for nucleosome assembly that increase TBP access and pol II recruitment. Stalled pol II then maintains Cp nucleosome depletion. Our data indicate that pTEFb is recruited to Cp by the bromodomain protein Brd4, with polymerase stalling facilitating stable association of pTEFb. The Brd4 inhibitor JQ1 and the pTEFb inhibitors DRB and Flavopiridol significantly reduce Cp, but not LMP1 transcript production indicating that Brd4 and pTEFb are required for Cp transcription. Taken together our data indicate that pol II stalling at Cp promotes transcription of essential immortalizing genes during EBV infection by (i) preventing promoter-proximal nucleosome assembly and ii) necessitating the recruitment of pTEFb thereby maintaining serine 2 CTD phosphorylation at distal regions

Ficolin-2 Levels and FCN2 Haplotypes Influence Hepatitis B Infection Outcome in Vietnamese Patients

Human Ficolin-2 (L-ficolins) encoded by FCN2 gene is a soluble serum protein that plays an important role in innate immunity and is mainly expressed in the liver. Ficolin-2 serum levels and FCN2 single nucleotide polymorphisms were associated to several infectious diseases. We initially screened the complete FCN2 gene in 48 healthy individuals of Vietnamese ethnicity. We genotyped a Vietnamese cohort comprising of 423 clinically classified hepatitis B virus patients and 303 controls for functional single nucleotide polymorphisms in the promoter region (-986G>A, -602G>A, -4A>G) and in exon 8 (+6424G>T) by real-time PCR and investigated the contribution of FCN2 genotypes and haplotypes to serum Ficolin-2 levels, viral load and liver enzyme levels. Haplotypes differed significantly between patients and controls (P = 0.002) and the haplotype AGGG was found frequently in controls in comparison to patients with hepatitis B virus and hepatocellular carcinoma (P = 0.0002 and P<0.0001) conferring a protective effect. Ficolin-2 levels differed significantly between patients and controls (p<0.0001). Patients with acute hepatitis B had higher serum Ficolin-2 levels compared to other patient groups and controls.The viral load was observed to be significantly distributed among the haplotypes (P = 0.04) and the AAAG haplotype contributed to higher Ficolin-2 levels and to viral load. Four novel single nucleotide polymorphisms in introns (-941G>T, -310G>A, +2363G>A, +4882G>A) and one synonymous mutation in exon 8 (+6485G>T) was observed. Strong linkage was found between the variant -986G>A and -4A>G. The very first study on Vietnamese cohort associates both Ficolin-2 serum levels and FCN2 haplotypes to hepatitis B virus infection and subsequent disease progression

Update on the Risk of Hepatocellular Carcinoma in Chronic Hepatitis B Virus Infection

Chronic hepatitis B virus infection is an important cause of liver-related morbidity and mortality, with hepatocellular carcinoma being the most life-threatening complication. Because of the highly variable clinical course of the disease, enormous research efforts have been made with the aim of revealing the factors in the natural history that are relevant to hepatocarcinogenesis. These include epidemiological studies of predisposing risk groups, viral studies of mutations within the hepatitis B viral genome, and clinical correlation of these risk factors in predicting the likelihood of development of hepatocellular cancer in susceptible hosts. This update addresses these risks, with emphasis on the latest research relevant to hepatocarcinogenesis

Smoke-free legislation and child health

In this paper, we aim to present an overview of the scientific literature on the link between smoke-free legislation and early-life health outcomes. Exposure to second-hand smoke is responsible for an estimated 166 ,000 child deaths each year worldwide. To protect people from tobacco smoke, the World Health Organization recommends the implementation of comprehensive smoke-free legislation that prohibits smoking in all public indoor spaces, including workplaces, bars and restaurants. The implementation of such legislation has been found to reduce tobacco smoke exposure, encourage people to quit smoking and improve adult health outcomes. There is an increasing body of evidence that shows that children also experience health benefits after implementation of smoke-free legislation. In addition to protecting children from tobacco smoke in public, the link between smoke-free legislation and improved child health is likely to be mediated via a decline in smoking during pregnancy and reduced exposure in the home environment. Recent studies have found that the implementation of smoke-free legislation is associated with a substantial decrease in the number of perinatal deaths, preterm births and hospital attendance for respiratory tract infections and asthma in children, although such benefits are not found in each study. With over 80% of the world’s population currently unprotected by comprehensive smoke-free laws, protecting (unborn) children from the adverse impact of tobacco smoking and SHS exposure holds great potential to benefit public health and should therefore be a key priority for policymakers and health workers alike

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Muslim Communities learning about second-hand smoke in Bangladesh (MCLASS II): study protocol for a cluster randomised controlled trial of a community-based smoke-free homes intervention, with or without Indoor Air Quality feedback

Background Second-hand smoke (SHS) is a serious health hazard costing 890,000 lives a year globally. Women and children in many economically developing countries are worst affected as smoke-free laws are only partially implemented and homes remain a major source of SHS exposure. There is limited evidence on interventions designed to reduce SHS exposure in homes, especially in community settings. Following a successful pilot, a community-based approach to promote smoke-free homes in Bangladesh, a country with a strong commitment to smoke-free environments but with high levels of SHS exposure, will be evaluated. The study aims to assess the effectiveness and cost-effectiveness of a community-based intervention, Muslims for better Health (M4bH), with or without Indoor Air Quality (IAQ) feedback, in reducing non-smokers’ exposure to SHS in the home. Methods/design Based on behaviour-change theories, M4bH and IAQ feedback are designed to discourage people from smoking indoors. M4bH consists of a set of messages couched within mainstream Islamic discourse, delivered weekly by faith leaders (imams and khatibs) in mosques over 12 weeks (one message each week). The messages address key determinants of current smoking behaviours including lack of knowledge and misconceptions on specific harms associated with SHS exposure. IAQ feedback consists of personalised information on IAQ measured by a particulate matter (PM2.5) monitor within the home. Following adaptation of M4bH and IAQ feedback for the Bangladeshi context, a three-arm cluster randomised controlled trial will be conducted in Dhaka. Forty-five mosques and 1800 households, with at least one smoker and one non-smoker, will be recruited. Mosques will be randomised to: M4bH and IAQ feedback; M4bH alone; or usual services only. The primary outcome is 24-h mean household concentration of indoor fine particulate matter (PM2.5) at 12 months post randomisation. Secondary outcomes are 24-h mean household PM2.5 at 3 months post randomisation, frequency and severity of respiratory symptoms, health care service use and quality of life. A cost-effectiveness analysis and process evaluation will also be conducted. Discussion The MCLASS II trial will test the potential of a community-based intervention to reduce second-hand smoke exposure at home and improve lung health among non-smokers in Bangladesh and beyond