Engineering supported membranes for cell biology

Cell membranes exhibit multiple layers of complexity, ranging from their specific molecular content to their emergent mechanical properties and dynamic spatial organization. Both compositional and geometrical organizations of membrane components are known to play important roles in life processes, including signal transduction. Supported membranes, comprised of a bilayer assembly of phospholipids on the solid substrate, have been productively served as model systems to study wide range problems in cell biology. Because lateral mobility of membrane components is readily preserved, supported lipid membranes with signaling molecules can be utilized to effectively trigger various intercellular reactions. The spatial organization and mechanical deformation of supported membranes can also be manipulated by patterning underlying substrates with modern micro- and nano-fabrication techniques. This article focuses on various applications and methods to spatially patterned biomembranes by means of curvature modulations and spatial reorganizations, and utilizing them to interface with live cells. The integration of biological components into synthetic devices provides a unique approach to investigate molecular mechanisms in cell biology

Myosin IIA Modulates T Cell Receptor Transport and CasL Phosphorylation during Early Immunological Synapse Formation

Activation of T cell receptor (TCR) by antigens occurs in concert with an elaborate multi-scale spatial reorganization of proteins at the immunological synapse, the junction between a T cell and an antigen-presenting cell (APC). The directed movement of molecules, which intrinsically requires physical forces, is known to modulate biochemical signaling. It remains unclear, however, if mechanical forces exert any direct influence on the signaling cascades. We use T cells from AND transgenic mice expressing TCRs specific to the moth cytochrome c 88–103 peptide, and replace the APC with a synthetic supported lipid membrane. Through a series of high spatiotemporal molecular tracking studies in live T cells, we demonstrate that the molecular motor, non-muscle myosin IIA, transiently drives TCR transport during the first one to two minutes of immunological synapse formation. Myosin inhibition reduces calcium influx and colocalization of active ZAP-70 (zeta-chain associated protein kinase 70) with TCR, revealing an influence on signaling activity. More tellingly, its inhibition also significantly reduces phosphorylation of the mechanosensing protein CasL (Crk-associated substrate the lymphocyte type), raising the possibility of a direct mechanical mechanism of signal modulation involving CasL

Concordance between vocal and genetic diversity in crested gibbons

<p>Abstract</p> <p>Background</p> <p>Gibbons or small apes are, next to great apes, our closest living relatives, and form the most diverse group of contemporary hominoids. A characteristic trait of gibbons is their species-specific song structure, which, however, exhibits a certain amount of inter- and intra-individual variation. Although differences in gibbon song structure are routinely applied as taxonomic tool to identify subspecies and species, it remains unclear to which degree acoustic and phylogenetic differences are correlated. To trace this issue, we comparatively analyse song recordings and mitochondrial cytochrome b gene sequence data from 22 gibbon populations representing six of the seven crested gibbon species (genus <it>Nomascus</it>). In addition, we address whether song similarity and geographic distribution can support a recent hypothesis about the biogeographic history of crested gibbons.</p> <p>Results</p> <p>The acoustic analysis of 92 gibbon duets confirms the hypothesised concordance between song structure and phylogeny. Based on features of male and female songs, we can not only distinguish between <it>N. nasutus</it>, <it>N. concolor </it>and the four southern species (<it>N. leucogenys, N. siki, N. annamensis</it>, <it>N. gabriellae</it>), but also between the latter by applying more detailed analysis. In addition to the significant correlation between song structure and genetic similarity, we find a similar high correlation between song similarity and geographic distance.</p> <p>Conclusions</p> <p>The results show that the structure of crested gibbon songs is not only a reliable tool to verify phylogenetic relatedness, but also to unravel geographic origins. As vocal production in other nonhuman primate species appears to be evolutionarily based, it is likely that loud calls produced by other species can serve as characters to elucidate phylogenetic relationships.</p

Assessing transmissibility of SARS-CoV-2 lineage B.1.1.7 in England

The SARS-CoV-2 lineage B.1.1.7, designated variant of concern (VOC) 202012/01 by Public Health England1, was first identified in the UK in late summer to early autumn 20202. Whole-genome SARS-CoV-2 sequence data collected from community-based diagnostic testing for COVID-19 show an extremely rapid expansion of the B.1.1.7 lineage during autumn 2020, suggesting that it has a selective advantage. Here we show that changes in VOC frequency inferred from genetic data correspond closely to changes inferred by S gene target failures (SGTF) in community-based diagnostic PCR testing. Analysis of trends in SGTF and non-SGTF case numbers in local areas across England shows that B.1.1.7 has higher transmissibility than non-VOC lineages, even if it has a different latent period or generation time. The SGTF data indicate a transient shift in the age composition of reported cases, with cases of B.1.1.7 including a larger share of under 20-year-olds than non-VOC cases. We estimated time-varying reproduction numbers for B.1.1.7 and co-circulating lineages using SGTF and genomic data. The best-supported models did not indicate a substantial difference in VOC transmissibility among different age groups, but all analyses agreed that B.1.1.7 has a substantial transmission advantage over other lineages, with a 50% to 100% higher reproduction number

Spatio-Temporal Dependence of the Signaling Response in Immune-Receptor Trafficking Networks Regulated by Cell Density: A Theoretical Model

Cell signaling processes involve receptor trafficking through highly connected networks of interacting components. The binding of surface receptors to their specific ligands is a key factor for the control and triggering of signaling pathways. In most experimental systems, ligand concentration and cell density vary within a wide range of values. Dependence of the signal response on cell density is related with the extracellular volume available per cell. This dependence has previously been studied using non-spatial models which assume that signaling components are well mixed and uniformly distributed in a single compartment. In this paper, a mathematical model that shows the influence exerted by cell density on the spatio-temporal evolution of ligands, cell surface receptors, and intracellular signaling molecules is developed. To this end, partial differential equations were used to model ligand and receptor trafficking dynamics through the different domains of the whole system. This enabled us to analyze several interesting features involved with these systems, namely: a) how the perturbation caused by the signaling response propagates through the system; b) receptor internalization dynamics and how cell density affects the robustness of dose-response curves upon variation of the binding affinity; and c) that enhanced correlations between ligand input and system response are obtained under conditions that result in larger perturbations of the equilibrium . Finally, the results are compared with those obtained by considering that the above components are well mixed in a single compartment

Towards the clinical implementation of pharmacogenetics in bipolar disorder.

BackgroundBipolar disorder (BD) is a psychiatric illness defined by pathological alterations between the mood states of mania and depression, causing disability, imposing healthcare costs and elevating the risk of suicide. Although effective treatments for BD exist, variability in outcomes leads to a large number of treatment failures, typically followed by a trial and error process of medication switches that can take years. Pharmacogenetic testing (PGT), by tailoring drug choice to an individual, may personalize and expedite treatment so as to identify more rapidly medications well suited to individual BD patients.DiscussionA number of associations have been made in BD between medication response phenotypes and specific genetic markers. However, to date clinical adoption of PGT has been limited, often citing questions that must be answered before it can be widely utilized. These include: What are the requirements of supporting evidence? How large is a clinically relevant effect? What degree of specificity and sensitivity are required? Does a given marker influence decision making and have clinical utility? In many cases, the answers to these questions remain unknown, and ultimately, the question of whether PGT is valid and useful must be determined empirically. Towards this aim, we have reviewed the literature and selected drug-genotype associations with the strongest evidence for utility in BD.SummaryBased upon these findings, we propose a preliminary panel for use in PGT, and a method by which the results of a PGT panel can be integrated for clinical interpretation. Finally, we argue that based on the sufficiency of accumulated evidence, PGT implementation studies are now warranted. We propose and discuss the design for a randomized clinical trial to test the use of PGT in the treatment of BD

The common rs9939609 variant of the fat mass and obesity-associated gene is associated with obesity risk in children and adolescents of Beijing, China

<p>Abstract</p> <p>Background</p> <p>Previous genome-wide association studies for type 2 diabetes susceptibility genes have confirmed that a common variant, rs9939609, in the fat mass and obesity associated (<it>FTO</it>) gene region is associated with body mass index (BMI) in European children and adults. A significant association of the same risk allele has been described in Asian adult populations, but the results are conflicting. In addition, no replication studies have been conducted in children and adolescents of Asian ancestry.</p> <p>Methods</p> <p>A population-based survey was carried out among 3503 children and adolescents (6-18 years of age) in Beijing, China, including 1229 obese and 2274 non-obese subjects. We investigated the association of rs9939609 with BMI and the risk of obesity. In addition, we tested the association of rs9939609 with weight, height, waist circumference, waist-to-height ratio, fat mass percentage, birth weight, blood pressure and related metabolic traits.</p> <p>Results</p> <p>We found significant associations of rs9939609 variant with weight, BMI, BMI standard deviation score (BMI-SDS), waist circumference, waist-to-height ratio, and fat mass percentage in children and adolescents (<it>p </it>for trend = 3.29 × 10^-5, 1.39 × 10^-6, 3.76 × 10^-6, 2.26 × 10^-5, 1.94 × 10^-5, and 9.75 × 10^-5, respectively). No significant associations were detected with height, birth weight, systolic and diastolic blood pressure and related metabolic traits such as total cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol and fasting plasma glucose (all <it>p </it>> 0.05). Each additional copy of the rs9939609 A allele was associated with a BMI increase of 0.79 [95% Confidence interval (CI) 0.47 to 1.10] kg/m², equivalent to 0.25 (95%CI 0.14 to 0.35) BMI-SDS units. This rs9939609 variant is significantly associated with the risk of obesity under an additive model [Odds ratio (OR) = 1.29, 95% CI 1.11 to 1.50] after adjusting for age and gender. Moreover, an interaction between the <it>FTO</it> rs9939609 genotype and physical activity (<it>p </it>< 0.001) was detected on BMI levels, the effect of rs9939609-A allele on BMI being (0.95 ± 0.10), (0.77 ± 0.08) and (0.67 ± 0.05) kg/m², for subjects who performed low, moderate and severe intensity physical activity.</p> <p>Conclusion</p> <p>The <it>FTO </it>rs9939609 variant is strongly associated with BMI and the risk of obesity in a population of children and adolescents in Beijing, China.</p

Methodological issues associated with collecting sensitive information over the telephone - experience from an Australian non-suicidal self-injury (NSSI) prevalence study

<p>Abstract</p> <p>Background</p> <p>Collecting population data on sensitive issues such as non-suicidal self-injury (NSSI) is problematic. Case note audits or hospital/clinic based presentations only record severe cases and do not distinguish between suicidal and non-suicidal intent. Community surveys have largely been limited to school and university students, resulting in little much needed population-based data on NSSI. Collecting these data via a large scale population survey presents challenges to survey methodologists. This paper addresses the methodological issues associated with collecting this type of data via CATI.</p> <p>Methods</p> <p>An Australia-wide population survey was funded by the Australian Government to determine prevalence estimates of NSSI and associations, predictors, relationships to suicide attempts and suicide ideation, and outcomes. Computer assisted telephone interviewing (CATI) on a random sample of the Australian population aged 10+ years of age from randomly selected households, was undertaken.</p> <p>Results</p> <p>Overall, from 31,216 eligible households, 12,006 interviews were undertaken (response rate 38.5%). The 4-week prevalence of NSSI was 1.1% (95% ci 0.9-1.3%) and lifetime prevalence was 8.1% (95% ci 7.6-8.6).</p> <p>Methodological concerns and challenges in regard to collection of these data included extensive interviewer training and post interview counselling. Ethical considerations, especially with children as young as 10 years of age being asked sensitive questions, were addressed prior to data collection. The solution required a large amount of information to be sent to each selected household prior to the telephone interview which contributed to a lower than expected response rate. Non-coverage error caused by the population of interest being highly mobile, homeless or institutionalised was also a suspected issue in this low prevalence condition. In many circumstances the numbers missing from the sampling frame are small enough to not cause worry, especially when compared with the population as a whole, but within the population of interest to us, we believe that the most likely direction of bias is towards an underestimation of our prevalence estimates.</p> <p>Conclusion</p> <p>Collecting valid and reliable data is a paramount concern of health researchers and survey research methodologists. The challenge is to design cost-effective studies especially those associated with low-prevalence issues, and to balance time and convenience against validity, reliability, sampling, coverage, non-response and measurement error issues.</p

Hypoxia regulates human lung fibroblast proliferation via p53-dependent and -independent pathways

<p>Abstract</p> <p>Background</p> <p>Hypoxia induces the proliferation of lung fibroblasts in vivo and in vitro. However, the subcellular interactions between hypoxia and expression of tumor suppressor p53 and cyclin-dependent kinase inhibitors p21 and p27 remain unclear.</p> <p>Methods</p> <p>Normal human lung fibroblasts (NHLF) were cultured in a hypoxic chamber or exposed to desferroxamine (DFX). DNA synthesis was measured using bromodeoxyuridine incorporation, and expression of p53, p21 and p27 was measured using real-time RT-PCR and Western blot analysis.</p> <p>Results</p> <p>DNA synthesis was increased by moderate hypoxia (2% oxygen) but was decreased by severe hypoxia (0.1% oxygen) and DFX. Moderate hypoxia decreased p21 synthesis without affecting p53 synthesis, whereas severe hypoxia and DFX increased synthesis of both p21 and p53. p27 protein expression was decreased by severe hypoxia and DFX. Gene silencing of p21 and p27 promoted DNA synthesis at ambient oxygen concentrations. p21 and p53 gene silencing lessened the decrease in DNA synthesis due to severe hypoxia or DFX exposure. p21 gene silencing prevented increased DNA synthesis in moderate hypoxia. p27 protein expression was significantly increased by p53 gene silencing, and was decreased by wild-type p53 gene transfection.</p> <p>Conclusion</p> <p>These results indicate that in NHLF, severe hypoxia leads to cell cycle arrest via the p53-p21 pathway, but that moderate hypoxia enhances cell proliferation via the p21 pathway in a p53-independent manner. In addition, our results suggest that p27 may be involved in compensating for p53 in cultured NHLF proliferation.</p