Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and GLUT4 translocation.

Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor-mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10. We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains. Although insulin activated the wild-type TC10 protein and a TC10/H-Ras chimera that were targeted to lipid raft microdomains, it was unable to activate a TC10/K-Ras chimera that was directed to the nonlipid raft domains. Similarly, only the lipid raft-localized TC10/ H-Ras chimera inhibited GLUT4 translocation, whereas the TC10/K-Ras chimera showed no significant inhibitory activity. Furthermore, disruption of lipid raft microdomains by expression of a dominant-interfering caveolin 3 mutant (Cav3/DGV) inhibited the insulin stimulation of GLUT4 translocation and TC10 lipid raft localization and activation without affecting PI-3 kinase signaling. These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways

Dynamin Is Functionally Coupled to Insulin Granule Exocytosis

The insulin granule integral membrane protein marker phogrin-green fluorescent protein was co-localized with insulin in Min6B1 beta-cell secretory granules but did not undergo plasma membrane translocation following glucose stimulation. Surprisingly, although expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis, it had no effect on phogringreen fluorescent protein localization in the basal or secretagogue-stimulated state. By contrast, co-expression of Dyn/K44A with human growth hormone as an insulin secretory marker resulted in a marked inhibition of human growth hormone release by glucose, KCl, and a combination of multiple secretagogues. Moreover, serial pulse depolarization stimulated an increase in cell surface capacitance that was also blocked in cells expressing Dyn/K44A. Similarly, small interference RNA-mediated knockdown of dynamin resulted in marked inhibition of glucose-stimulated insulin secretion. Together, these data suggest the presence of a selective kiss and run mechanism of insulin release. Moreover, these data indicate a coupling between endocytosis and exocytosis in the regulation of beta-cell insulin secretion

Study protocol for a multicentre longitudinal mixed methods study to explore the Outcomes of ChildrEn and fAmilies in the first year after paediatric Intensive Care: the OCEANIC study.

INTRODUCTION: Annually in the UK, 20 000 children become very ill or injured and need specialist care within a paediatric intensive care unit (PICU). Most children survive. However, some children and their families may experience problems after they have left the PICU including physical, functional and/or emotional problems. It is unknown which children and families experience such problems, when these occur or what causes them. The aim of this mixed-method longitudinal cohort study is to understand the physical, functional, emotional and social impact of children surviving PICU (aged: 1 month-17 years), their parents and siblings, during the first year after a PICU admission. METHODS AND ANALYSIS: A quantitative study involving 300 child survivors of PICU; 300 parents; and 150-300 siblings will collect data (using self-completion questionnaires) at baseline, PICU discharge, 1, 3, 6 and 12 months post-PICU discharge. Questionnaires will comprise validated and reliable instruments. Demographic data, PICU admission and treatment data, health-related quality of life, functional status, strengths and difficulties behaviour and post-traumatic stress symptoms will be collected from the child. Parent and sibling data will be collected on the impact of paediatric health conditions on the family's functioning capabilities, levels of anxiety and social impact of the child's PICU admission. Data will be analysed using descriptive and inferential statistics. Concurrently, an embedded qualitative study involving semistructured interviews with 24 enrolled families at 3 months and 9 months post-PICU discharge will be undertaken. Framework analysis will be used to analyse the qualitative data. ETHICS AND DISSEMINATION: The study has received ethical approval from the National Health Services Research Ethics Committee (Ref: 19/WM/0290) and full governance clearance. This will be the first UK study to comprehensively investigate physical, functional, emotional and social consequences of PICU survival in the first-year postdischarge.Clinical Trials Registration Number: ISRCTN28072812 [Pre-results]

Mexico City and the biogeochemistry of global urbanization

Mexico City is far advanced in its urban evolution, and cities in currently developing nations may soon follow a similar course. This paper investigates the strengths and weaknesses of infrastructures for the emerging megacities. The major driving force for infrastructure change in Mexico City is concern over air quality. Air chemistry data from recent field campaigns have been used to calculate fluxes in the atmosphere of the Valley of Mexico, for compounds that are important to biogeochemistry including methane (CH4), carbon monoxide (CO), nonmethane hydrocarbons (NMHCs), ammonia (NH3), sulfur dioxide (SO2), nitrogen oxides (NOx and NOy), soot, and dust. Leakage of liquified petroleum gas approached 10% during sampling periods, and automotive pollutant sources in Mexico City were found to match those in developed cities, despite a lower vehicle-to-person ratio of 0.1. Ammonia is released primarily from residential areas, at levels sufficient to titrate pollutant acids into particles across the entire basin. Enhancements of reduced nitrogen and hydrocarbons in the vapor phase skew the distribution of NOy species towards lower average deposition velocities. Partly as a result, downwind nutrient deposition occurs on a similar scale as nitrogen fixation across Central America, and augments marine nitrate upwelling. Dust suspension from unpaved roads and from the bed of Lake Texcoco was found to be comparable to that occurring on the periphery of the Sahara, Arabian, and Gobi deserts. In addition, sodium chloride (NaCl) in the dust may support heterogeneous chlorine oxide (ClOx) chemistry. The insights from our Mexico City analysis have been tentatively applied to the upcoming urbanization of Asia

A novel isolator-based system promotes viability of human embryos during laboratory processing

In vitro fertilisation (IVF) and related technologies are arguably the most challenging of all cell culture applications. The starting material is a single cell from which one aims to produce an embryo capable of establishing a pregnancy eventually leading to a live birth. Laboratory processing during IVF treatment requires open manipulations of gametes and embryos, which typically involves exposure to ambient conditions. To reduce the risk of cellular stress, we have developed a totally enclosed system of interlinked isolator-based workstations designed to maintain oocytes and embryos in a physiological environment throughout the IVF process. Comparison of clinical and laboratory data before and after the introduction of the new system revealed that significantly more embryos developed to the blastocyst stage in the enclosed isolator-based system compared with conventional open-fronted laminar flow hoods. Moreover, blastocysts produced in the isolator-based system contained significantly more cells and their development was accelerated. Consistent with this, the introduction of the enclosed system was accompanied by a significant increase in the clinical pregnancy rate and in the proportion of embryos implanting following transfer to the uterus. The data indicate that protection from ambient conditions promotes improved development of human embryos. Importantly, we found that it was entirely feasible to conduct all IVF-related procedures in the isolator-based workstations

Effect of nickel on the microstructure and mechanical property of die-cast Al–Mg–Si–Mn alloy

The effect of nickel on the microstructure and mechanical properties of a die-cast Al–Mg–Si–Mn alloy has been investigated. The results show that the presence of Ni in the alloy promotes the formation of Ni-rich intermetallics. These occur consistently during solidification in the die-cast Al–Mg–Si–Mn alloy across different levels of Ni content. The Ni-rich intermetallics exhibit dendritic morphology during the primary solidification and lamellar morphology during the eutectic solidification stage. Ni was found to be always associated with iron forming AlFeMnSiNi intermetallics, and no Al3Ni intermetallic was observed when Ni concentrations were up to 2.06 wt% in the alloy. Although with different morphologies, the Ni-rich intermetallics were identified as the same AlFeMnSiNi phase bearing a typical composition of Al[100–140](Fe,Mn)[2–7]SiNi[4–9]. With increasing Ni content, the spacing of the α-Al–Mg2Si eutectic phase was enlarged in the Al–Mg–Si–Mn alloy. The addition of Ni to the alloy resulted in a slight increase in the yield strength, but a significant decrease in the elongation. The ultimate tensile strength (UTS) increased slightly from 300 to 320 MPa when a small amount (e.g. 0.16 wt%) of Ni was added to the alloy, but further increase of the Ni content resulted in a decrease of the UTS.The Engineering and Physical Sciences Research Council (EPSRC), Technology Strategy Board (TSB) and Jaguar Land Rover (JLR) in the United Kingdom

Conversion From Calcineurin to Mammalian Target of Rapamycin Inhibitors in Liver Transplantation: A Meta-Analysis of Randomized Controlled Trials.

BACKGROUND: Conversion to mammalian target of rapamycin inhibitors (mTORi) is often used in liver transplantation to overcome calcineurin inhibitor (CNI) nephrotoxicity but the evidence base for this approach is not well defined. To summarize the evidence, from randomized clinical trials (RCTs), for conversion from CNI to mTORi-based immunosuppression after liver transplantation. METHODS: Databases and conference abstracts were searched up to August 2015. The RCTs evaluating conversion from CNI to mTORi-based maintenance immunosuppression after adult liver transplantation. Descriptive and quantitative information was extracted; summary mean difference and risk ratio (RR) estimates were synthesized under a random-effects model. Heterogeneity was assessed using the Q statistic and I. RESULTS: Ten RCTs, with a total of 1927 patients, met the final inclusion criteria. Patients converted to mTORi had significantly better renal function at 1 year after randomization compared with patients remaining on CNI (mean difference, 7.48 mL/min per 1.73 m; 95% confidence interval [95% CI], 3.18-11.8). The risks of graft loss (RR, 0.77; 95% CI, 0.29-2.09; I, 31%) and patient death (RR, 1.05; 95% CI, 0.63-1.73; I, 0%) were similar for patients converted to mTORi and patients remaining on CNI. However, conversion to mTORi was associated with a higher risk of acute rejection (RR, 1.76; 95% CI, 1.33-2.34; I, 0%) and study discontinuation due to adverse events (RR, 2.17; 95% CI, 1.38-3.44; I, 63%) up to 1 year after randomization. CONCLUSIONS: Conversion from CNI to mTORi after liver transplantation is associated with improved renal function after 1 year but increases the risk of acute rejection and may be poorly tolerated.The study was funded in part by the NIHR Cambridge Biomedical Research Centre.This is the author accepted manuscript. The final version is available from Lippincott Williams & Wilkins via http://dx.doi.org/10.1097/TP.000000000000100

Robust diagnostic genetic testing using solution capture enrichment and a novel variant-filtering interface.

Targeted hybridization enrichment prior to next-generation sequencing is a widespread method for characterizing sequence variation in a research setting, and is being adopted by diagnostic laboratories. However, the number of variants identified can overwhelm clinical laboratories with strict time constraints, the final interpretation of likely pathogenicity being a particular bottleneck. To address this, we have developed an approach in which, after automatic variant calling on a standard unix pipeline, subsequent variant filtering is performed interactively, using AgileExomeFilter and AgilePindelFilter (http://dna.leeds.ac.uk/agile), tools designed for clinical scientists with standard desktop computers. To demonstrate the method's diagnostic efficacy, we tested 128 patients using (1) a targeted capture of 36 cancer-predisposing genes or (2) whole-exome capture for diagnosis of the genetically heterogeneous disorder primary ciliary dyskinesia (PCD). In the cancer cohort, complete concordance with previous diagnostic data was achieved across 793 variant genotypes. A high yield (42%) was also achieved for exome-based PCD diagnosis, underscoring the scalability of our method. Simple adjustments to the variant filtering parameters further allowed the identification of a homozygous truncating mutation in a presumptive new PCD gene, DNAH8. These tools should allow diagnostic laboratories to expand their testing portfolios flexibly, using a standard set of reagents and techniques

Significance of herpesvirus immediate early gene expression in cellular immunity to cytomegalovirus infection

Interstitial pneumonia linked with reactivation of latent human cytomegalovirus due to iatrogenic immunosuppression can be a serious complication of bone marrow transplantation therapy of aplastic anaemia and acute leukaemia1. Cellular immunity plays a critical role in the immune surveillance of inapparent cytomegalovirus infections in man and the mouse1−7. The molecular basis of latency, however, and the interaction between latently or recurrently infected cells and the immune system of the host are poorfy understood. We have detected a so far unknown antigen in the mouse model. This antigen is found in infected cells in association with the expression of the herpesvirus 'immediate early' genes and is recognized by cytolytic T lymphocytes (CTL)8. We now demonstrate that an unexpectedly high proportion of the CTL precursors generated in vivo during acute murine cytomegalovirus infection are specific for cells that selectively synthesize immediate early proteins, indicating an immunodominant role of viral non-structural proteins