High density Huh7.5 cell hollow fiber bioreactor culture for high-yield production of hepatitis C virus and studies of antivirals

Abstract Chronic hepatitis C virus (HCV) infection poses a serious global public health burden. Despite the recent development of effective treatments there is a large unmet need for a prophylactic vaccine. Further, antiviral resistance might compromise treatment efficiency in the future. HCV cell culture systems are typically based on Huh7 and derived hepatoma cell lines cultured in monolayers. However, efficient high cell density culture systems for high-yield HCV production and studies of antivirals are lacking. We established a system based on Huh7.5 cells cultured in a hollow fiber bioreactor in the presence or absence of bovine serum. Using an adapted chimeric genotype 5a virus, we achieved peak HCV infectivity and RNA titers of 7.6 log10 FFU/mL and 10.4 log10 IU/mL, respectively. Bioreactor derived HCV showed high genetic stability, as well as buoyant density, sensitivity to neutralizing antibodies AR3A and AR4A, and dependency on HCV co-receptors CD81 and SR-BI comparable to that of HCV produced in monolayer cell cultures. Using the bioreactor platform, treatment with the NS5A inhibitor daclatasvir resulted in HCV escape mediated by the NS5A resistance substitution Y93H. In conclusion, we established an efficient high cell density HCV culture system with implications for studies of antivirals and vaccine development

HbA1c levels in non-diabetic older adults - No J-shaped associations with primary cardiovascular events, cardiovascular and all-cause mortality after adjustment for confounders in a meta-analysis of individual participant data from six cohort studies

Background:To determine the shape of the associations of HbA1c with mortality and cardiovascular outcomes in non-diabetic individuals and explore potential explanations. Methods: The associations of HbA1c with all-cause mortality, cardiovascular mortality and primary cardiovascular events (myocardial infarction or stroke) were assessed in non-diabetic subjects ≥50 years from six population-based cohort studies from Europe and the USA and meta-analyzed. Very low, low, intermediate and increased HbA1c were defined as <5.0, 5.0 to <5.5, 5.5 to <6.0 and 6.0 to <6.5 % (equals <31, 31 to <37, 37 to <42 and 42 to <48 mmol/mol), respectively, and low HbA1c was used as reference in Cox proportional hazards models. Results:Overall, 6,769 of 28,681 study participants died during a mean follow-up of 10.7 years, of whom 2,648 died of cardiovascular disease. Furthermore, 2,493 experienced a primary cardiovascular event. A linear association with primary cardiovascular events was observed. Adjustment for cardiovascular risk factors explained about 50 % of the excess risk and attenuated hazard ratios (95 % confidence interval) for increased HbA1c to 1.14 (1.03–1.27), 1.17 (1.00–1.37) and 1.19 (1.04–1.37) for all-cause mortality, cardiovascular mortality and cardiovascular events, respectively. The six cohorts yielded inconsistent results for the association of very low HbA1c levels with the mortality outcomes and the pooled effect estimates were not statistically significant. In one cohort with a pronounced J-shaped association of HbA1c levels with all-cause and cardiovascular mortality (NHANES), the following confounders of the association of very low HbA1c levels with mortality outcomes were identified: race/ethnicity; alcohol consumption; BMI; as well as biomarkers of iron deficiency anemia and liver function. Associations for very low HbA1c levels lost statistical significance in this cohort after adjusting for these confounders.Conclusions: A linear association of HbA1c levels with primary cardiovascular events was observed. For cardiovascular and all-cause mortality, the observed small effect sizes at both the lower and upper end of HbA1c distribution do not support the notion of a J-shaped association of HbA1c levels because a certain degree of residual confounding needs to be considered in the interpretation of the results. Keywords: Glycated hemoglobin, Cardiovascular disease, Myocardial infarction, Stroke, Mortality, Cohort study, Meta-analysi

HbA(1c) levels in non-diabetic older adults No J-shaped associations with primary cardiovascular events, cardiovascular and all-cause mortality after adjustment for confounders in a meta-analysis of individual participant data from six cohort studies

Background To determine the shape of the associations of HbA1c with mortality and cardiovascular outcomes in non-diabetic individuals and explore potential explanations. Methods The associations of HbA1c with all-cause mortality, cardiovascular mortality and primary cardiovascular events (myocardial infarction or stroke) were assessed in non-diabetic subjects ≥50 years from six population-based cohort studies from Europe and the USA and meta-analyzed. Very low, low, intermediate and increased HbA1c were defined as <5.0, 5.0 to <5.5, 5.5 to <6.0 and 6.0 to <6.5 % (equals <31, 31 to <37, 37 to <42 and 42 to <48 mmol/mol), respectively, and low HbA1c was used as reference in Cox proportional hazards models. Results Overall, 6,769 of 28,681 study participants died during a mean follow-up of 10.7 years, of whom 2,648 died of cardiovascular disease. Furthermore, 2,493 experienced a primary cardiovascular event. A linear association with primary cardiovascular events was observed. Adjustment for cardiovascular risk factors explained about 50 % of the excess risk and attenuated hazard ratios (95 % confidence interval) for increased HbA1c to 1.14 (1.03–1.27), 1.17 (1.00–1.37) and 1.19 (1.04–1.37) for all-cause mortality, cardiovascular mortality and cardiovascular events, respectively. The six cohorts yielded inconsistent results for the association of very low HbA1c levels with the mortality outcomes and the pooled effect estimates were not statistically significant. In one cohort with a pronounced J-shaped association of HbA1c levels with all-cause and cardiovascular mortality (NHANES), the following confounders of the association of very low HbA1c levels with mortality outcomes were identified: race/ethnicity; alcohol consumption; BMI; as well as biomarkers of iron deficiency anemia and liver function. Associations for very low HbA1c levels lost statistical significance in this cohort after adjusting for these confounders. Conclusions A linear association of HbA1c levels with primary cardiovascular events was observed. For cardiovascular and all-cause mortality, the observed small effect sizes at both the lower and upper end of HbA1c distribution do not support the notion of a J-shaped association of HbA1c levels because a certain degree of residual confounding needs to be considered in the interpretation of the results

Placental transfer of the polybrominated diphenyl ethers BDE-47, BDE-99 and BDE-209 in a human placenta perfusion system: an experimental study

<p>Abstract</p> <p>Background</p> <p>Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants in consumer products. PBDEs may affect thyroid hormone homeostasis, which can result in irreversible damage of cognitive performance, motor skills and altered behaviour. Thus, in utero exposure is of very high concern due to critical windows in fetal development.</p> <p>Methods</p> <p>A human ex vivo placenta perfusion system was used to study the kinetics and extent of the placental transfer of BDE-47, BDE-99 and BDE-209 during four-hour perfusions. The PBDEs were added to the maternal circulation and monitored in the maternal and fetal compartments. In addition, the perfused cotyledon, the surrounding placental tissue as well as pre-perfusion placental tissue and umbilical cord plasma were also analysed. The PBDE analysis included Soxhlet extraction, clean-up by adsorption chromatography and GC-MS analysis.</p> <p>Results and Discussion</p> <p>Placental transfer of BDE-47 was faster and more extensive than for BDE-99. The fetal-maternal ratios (FM-ratio) after four hours of perfusion were 0.47 and 0.25 for BDE-47 and BDE-99, respectively, while the indicative permeability coefficient (IPC) measured after 60 minutes of perfusion was 0.26 h^-1and 0.10 h^-1, respectively. The transport of BDE-209 seemed to be limited. These differences between the congeners may be related to the degree of bromination. Significant accumulation was observed for all congeners in the perfused cotyledon as well as in the surrounding placental tissue.</p> <p>Conclusion</p> <p>The transport of BDE-47 and BDE-99 indicates in utero exposure to these congeners. Although the transport of BDE-209 was limited, however, possible metabolic debromination may lead to products which are both more toxic and transportable. Our study demonstrates fetal exposure to PBDEs, which should be included in risk assessment of PBDE exposure of women of child-bearing age.</p

Team Reasoning and Collective Intentionality

Different versions of the idea that individualism about agency is the root of standard game theoretical puzzles have been defended by Regan 1980, Bacharach (Research in Economics 53: 117–147, 1999), Hurley (Behavioral and Brain Sciences 26: 264–265, 2003), Sugden (Philosophical Explorations 6(3):165–181, 2003), and Tuomela 2013, among others. While collectivistic game theorists like Michael Bacharach provide formal frameworks designed to avert some of the standard dilemmas, philosophers of collective action like Raimo Tuomela aim at substantive accounts of collective action that may explain how agents overcoming such social dilemmas would be motivated. This paper focuses on the conditions on collective action and intention that need to be fulfilled for Bacharach’s “team reasoning” to occur. Two influential approaches to collective action are related to the idea of team reasoning: Michael Bratman’s theory of shared intention and Raimo Tuomela’s theory of a we-mode of intending. I argue that neither captures the “agency transformation” that team reasoning requires. That might be an acceptable conclusion for Bratman but more problematic for Tuomela, who claims that Bacharach’s results support his theory. I sketch an alternative framework in which the perspectival element that is required for team reasoning - the ‘we-perspective’ - can be understood and functionally characterized in relation to the traditional distinction between mode and content of intentional states. I claim that the latter understanding of a collective perspective provides the right kind of philosophical background for team reasoning, and I discuss some implications in relation to Tuomela’s assumption that switching between individual and collective perspectives can be a matter of rational choice

Variable pulmonary manifestations in Chitayat syndrome: Six additional affected individuals

Hand hyperphalangism leading to shortened index fingers with ulnar deviation, hallux valgus, mild facial dysmorphism and respiratory compromise requiring assisted ventilation are the key features of Chitayat syndrome. This condition results from the recurrent heterozygous missense variant NM_006494.2:c.266A>G; p.(Tyr89Cys) in ERF on chromosome 19q13.2, encoding the ETS2 repressor factor (ERF) protein. The pathomechanism of Chitayat syndrome is unknown. To date, seven individuals with Chitayat syndrome and the recurrent pathogenic ERF variant have been reported in the literature. Here, we describe six additional individuals, among them only one presenting with a history of assisted ventilation, and the remaining presenting with variable pulmonary phenotypes, including one individual without any obvious pulmonary manifestations. Our findings widen the phenotype spectrum caused by the recurrent pathogenic variant in ERF, underline Chitayat syndrome as a cause of isolated skeletal malformations and therefore contribute to the improvement of diagnostic strategies in individuals with hand hyperphalangism

Should patients with abnormal liver function tests in primary care be tested for chronic viral hepatitis: cost minimisation analysis based on a comprehensively tested cohort

Background Liver function tests (LFTs) are ordered in large numbers in primary care, and the Birmingham and Lambeth Liver Evaluation Testing Strategies (BALLETS) study was set up to assess their usefulness in patients with no pre-existing or self-evident liver disease. All patients were tested for chronic viral hepatitis thereby providing an opportunity to compare various strategies for detection of this serious treatable disease. Methods This study uses data from the BALLETS cohort to compare various testing strategies for viral hepatitis in patients who had received an abnormal LFT result. The aim was to inform a strategy for identification of patients with chronic viral hepatitis. We used a cost-minimisation analysis to define a base case and then calculated the incremental cost per case detected to inform a strategy that could guide testing for chronic viral hepatitis. Results Of the 1,236 study patients with an abnormal LFT, 13 had chronic viral hepatitis (nine hepatitis B and four hepatitis C). The strategy advocated by the current guidelines (repeating the LFT with a view to testing for specific disease if it remained abnormal) was less efficient (more expensive per case detected) than a simple policy of testing all patients for viral hepatitis without repeating LFTs. A more selective strategy of viral testing all patients for viral hepatitis if they were born in countries where viral hepatitis was prevalent provided high efficiency with little loss of sensitivity. A notably high alanine aminotransferase (ALT) level (greater than twice the upper limit of normal) on the initial ALT test had high predictive value, but was insensitive, missing half the cases of viral infection. Conclusions Based on this analysis and on widely accepted clinical principles, a "fast and frugal" heuristic was produced to guide general practitioners with respect to diagnosing cases of viral hepatitis in asymptomatic patients with abnormal LFTs. It recommends testing all patients where a clear clinical indication of infection is present (e.g. evidence of intravenous drug use), followed by testing all patients who originated from countries where viral hepatitis is prevalent, and finally testing those who have a notably raised ALT level (more than twice the upper limit of normal). Patients not picked up by this efficient algorithm had a risk of chronic viral hepatitis that is lower than the general population

Simulation techniques for cosmological simulations

Modern cosmological observations allow us to study in great detail the evolution and history of the large scale structure hierarchy. The fundamental problem of accurate constraints on the cosmological parameters, within a given cosmological model, requires precise modelling of the observed structure. In this paper we briefly review the current most effective techniques of large scale structure simulations, emphasising both their advantages and shortcomings. Starting with basics of the direct N-body simulations appropriate to modelling cold dark matter evolution, we then discuss the direct-sum technique GRAPE, particle-mesh (PM) and hybrid methods, combining the PM and the tree algorithms. Simulations of baryonic matter in the Universe often use hydrodynamic codes based on both particle methods that discretise mass, and grid-based methods. We briefly describe Eulerian grid methods, and also some variants of Lagrangian smoothed particle hydrodynamics (SPH) methods.Comment: 42 pages, 16 figures, accepted for publication in Space Science Reviews, special issue "Clusters of galaxies: beyond the thermal view", Editor J.S. Kaastra, Chapter 12; work done by an international team at the International Space Science Institute (ISSI), Bern, organised by J.S. Kaastra, A.M. Bykov, S. Schindler & J.A.M. Bleeke