Effects of insulin on intracellular GLUT4 vesicles in adipocytes: Evidence for a secretory mode of regulation

The facilitative glucose transporter, GLUT4 undergoes insulin-dependent movement to the cell surface in adipocytes, The magnitude of the insulin effect is much greater for CLUT4 than other recycling proteins such as the CD-MPR, In the present study we have studied the colocalisation of these proteins in adipocytes in an effort to explain this selective insulin-dependent recruitment of CLUT4, Using immunofluorescence microscopy or immuno-EM on 3T3-L1 adipocytes we find that there is considerable colocalisation between these proteins particularly within the area of the TGN, However, the distribution of CD-MPR was not significantly effected by insulin. The insulin-dependent recruitment of GLUT4 was concomitant with a selective decrease in GLUT4 labelling of cytoplasmic vesicles whereas the amount of CLUT4 in the TGN region (approx. 50% of total GLUT4) was relatively unaffected. To explore the possibility that the cytoplasmic GLUT4(+) vesicles represent an intracellular insulin-responsive storage compartment we performed quantitative immuno-EM on whole mounts of intracellular vesicles isolated from basal and insulin-stimulated adipocytes. These studies revealed that: (1) GLUT4 and CD-MPR were concentrated in small (30-200 nm) vesicles at a labelling density of 1-20+ gold particles/vesicle; (2) there was significant overlap between both proteins in that 70% of the total GLUT4 pool colocalised with CD-MPR; (3) a significant amount of GLUT4 (approx, 50% of total) was found in a subpopulation of vesicles that contained as little as 5% of the total CD-MPR pool; (4) the GLUT4(+)/CD-MPR(-) vesicles were highly insulin-responsive, and (5) the total number of GLUT4(+) vesicles, but not CD-MPR(+) vesicles, decreased by approx. 30% in response to insulin treatment. These data are consistent with a model in which GLUT4 is selectively sorted into a vesicular compartment in adipocytes that is recruited to the plasma membrane by insulin stimulation

Modulation of detoxification gene expression in human colon HT29 cells by glutathione-S-transferase inhibitors

SUMMAR

The Star Formation Reference Survey. I. Survey Description and Basic Data

Star formation is arguably the most important physical process in the cosmos. It is a fundamental driver of galaxy evolution and the ultimate source of most of the energy emitted by galaxies. A correct interpretation of star formation rate (SFR) measures is therefore essential to our understanding of galaxy formation and evolution. Unfortunately, however, no single SFR estimator is universally available or even applicable in all circumstances: the numerous galaxies found in deep surveys are often too faint (or too distant) to yield significant detections with most standard SFR measures, and until now there have been no global, multi-band observations of nearby galaxies that span all the conditions under which star-formation is taking place. To address this need in a systematic way, we have undertaken a multi-band survey of all types of star-forming galaxies in the local Universe. This project, the Star Formation Reference Survey (SFRS), is based on a statistically valid sample of 369 nearby galaxies that span all existing combinations of dust temperature, SFR, and specific SFR. Furthermore, because the SFRS is blind with respect to AGN fraction and environment it serves as a means to assess the influence of these factors on SFR. Our panchromatic global flux measurements (including GALEX FUV+NUV, SDSS ugriz, 2MASS JHKs, Spitzer 3-8{\mu}m, and others) furnish uniform SFR measures and the context in which their reliability can be assessed. This paper describes the SFRS survey strategy, defines the sample, and presents the multi-band photometry collected to date.Comment: 48 pages, 12 figures, 10 tables. Accepted by PASP. This version edited to correct references and typographical error

Seroprevalence of SARS-CoV-2 antibodies in children: a prospective multicentre cohort study.

BACKGROUND: Studies based on molecular testing of oral/nasal swabs underestimate SARS-CoV-2 infection due to issues with test sensitivity, test timing and selection bias. The objective of this study was to report the presence of SARS-CoV-2 antibodies, consistent with previous infection. DESIGN: This multicentre observational cohort study, conducted between 16 April to 3 July 2020 at 5 UK sites, recruited children of healthcare workers, aged 2-15 years. Participants provided blood samples for SARS-CoV-2 antibody testing and data were gathered regarding unwell contacts and symptoms. RESULTS: 1007 participants were enrolled, and 992 were included in the final analysis. The median age of participants was 10·1 years. There were 68 (6.9%) participants with positive SARS-CoV-2 antibody tests indicative of previous SARS-CoV-2 infection. Of these, 34/68 (50%) reported no symptoms prior to testing. The presence of antibodies and the mean antibody titre was not influenced by age. Following multivariable analysis four independent variables were identified as significantly associated with SARS-CoV-2 seropositivity: known infected household contact OR=10.9 (95% CI 6.1 to 19.6); fatigue OR=16.8 (95% CI 5.5 to 51.9); gastrointestinal symptoms OR=6.6 (95% CI 3.0 to 13.8); and changes in sense of smell or taste OR=10.0 (95% CI 2.4 to 11.4). DISCUSSION: Children demonstrated similar antibody titres in response to SARS-CoV-2 irrespective of age. Fatigue, gastrointestinal symptoms and changes in sense of smell or taste were the symptoms most strongly associated with SARS-CoV-2 antibody positivity. TRIAL REGISTRATION NUMBER: NCT0434740

Variation in treatment of acute childhood wheeze in emergency departments of the United Kingdom and Ireland: An international survey of clinician practice

© 2015, BMJ Publishing Group. All rights reserved. Objective: National clinical guidelines for childhood wheeze exist, yet despite being one of the most common reasons for childhood emergency department (ED) attendance, signi ficant variation in practice occurs in other settings. We, therefore, evaluated practice variations of ED clinicians in the UK and Ireland. Design: Two-stage survey undertaken in March 2013. Stage one examined department practice and stage two assessed ED consultant practice in acute childhood wheeze. Questions interrogated pharmacological and other management strategies, including inhaled and intravenous therapies. Setting and participants: Member departments of Paediatric Emergency Research in the United Kingdom and Ireland and ED consultants treating children with acute wheeze. Results: 30 EDs and 183 (81%) clinicians responded. 29 (97%) EDs had wheeze guidelines and 12 (40%) had care pathways. Variation existed between clinicians in dose, timing and frequency of inhaled bronchodilators across severities. When escalating to intravenous bronchodilators, 99 (54%) preferred salbutamol first line, 52 (28%) magnesium sulfate (MgSO4) and 27 (15%) aminophylline. 87 (48%) administered intravenous bronchodilators sequentially and 30 (16%) concurrently, with others basing approach on case severity. 146 (80%) continued inhaled therapy after commencing intravenous bronchodilators. Of 170 who used intravenous salbutamol, 146 (86%) gave rapid boluses, 21 (12%) a longer loading dose and 164 (97%) an ongoing infusion, each with a range of doses and durations. Of 173 who used intravenous MgSO4, all used a bolus only. 41 (24%) used non-invasive ventilation. Conclusions: Signi ficant variation in ED consultant management of childhood wheeze exists despite the presence of national guidance. This reflects the lack of evidence in key areas of childhood wheeze and emphasises the need for further robust multicentre research studies

INSGFP/w human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells

AIMS/HYPOTHESIS: We aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS⁺) cells derived in vitro. METHODS: Homologous recombination was used to insert sequences encoding green fluorescent protein (GFP) into the INS locus, to create reporter cell lines enabling the prospective isolation of viable INS⁺ cells. RESULTS: Differentiation of INS(GFP/w) hESCs using published protocols demonstrated that all GFP⁺ cells co-produced insulin, confirming the fidelity of the reporter gene. INS-GFP⁺ cells often co-produced glucagon and somatostatin, confirming conclusions from previous studies that early hESC-derived insulin-producing cells were polyhormonal. INS(GFP/w) hESCs were used to develop a 96-well format spin embryoid body (EB) differentiation protocol that used the recombinant protein-based, fully defined medium, APEL. Like INS-GFP⁺ cells generated with other methods, those derived using the spin EB protocol expressed a suite of pancreatic-related transcription factor genes including ISL1, PAX6 and NKX2.2. However, in contrast with previous methods, the spin EB protocol yielded INS-GFP⁺ cells that also co-expressed the beta cell transcription factor gene, NKX6.1, and comprised a substantial proportion of monohormonal INS⁺ cells. CONCLUSIONS/INTERPRETATION: INS(GFP/w) hESCs are a valuable tool for investigating the nature of early INS⁺ progenitors in beta cell ontogeny and will facilitate the development of novel protocols for generating INS⁺ cells from differentiating hESCs

Phase 2 study of canfosfamide in combination with pegylated liposomal doxorubicin in platinum and paclitaxel refractory or resistant epithelial ovarian cancer

<p>Abstract</p> <p>Background</p> <p>Canfosfamide is a novel glutathione analog activated by glutathione S-transferase P1-1. This study evaluated the safety and efficacy of canfosfamide in combination with pegylated liposomal doxorubicin (PLD) in patients with platinum resistant ovarian cancer. Patients with platinum resistant ovarian carcinoma and measurable disease received canfosfamide at 960 mg/m²in combination with PLD at 50 mg/m², intravenously day 1 in every 28 day cycles until tumor progression or unacceptable toxicities. The primary endpoints were objective response rate (ORR) and progression-free survival (PFS).</p> <p>Results</p> <p>Canfosfamide plus PLD combination therapy was administered at 960/50 mg/m², respectively. Thirty-nine patients received a median number of 4 cycles (range 1.0-18.0). The ORR was 27.8% (95% CI, 14.2-45.2) with a disease stabilization rate of 80.6% (95% CI, 64.0-91.8) in the evaluable population. The CA-125 marker responses correlated with the radiological findings of complete response or partial response. The median PFS was 6.0 months (95% CI, 4.2-7.9) and median survival was 17.8 months. The combination was well tolerated. Myelosuppression was managed with dose reductions and growth factor support. Grade 3 febrile neutropenia was observed in 2 patients (5.1%). Non-hematologic adverse events occurred at the expected frequency and grade for each drug alone, with no unexpected or cumulative toxicities.</p> <p>Conclusions</p> <p>Canfosfamide in combination with PLD is well tolerated and active in platinum and paclitaxel refractory or resistant ovarian cancer. A randomized phase 3 study was conducted based on this supportive phase 2 study.</p> <p>Trial Registration</p> <p>This study was registered at www.clinicaltrials.gov: NCT00052065.</p

The stress of starvation: glucocorticoid restraint of beta cell development

Developmental insults during gestation, such as under-nutrition, are known to restrict the number of beta cells that form in the fetal pancreas and are maintained in adulthood, leading to increased risk of type 2 diabetes. There are now substantial data indicating that glucocorticoids mediate this effect of under-nutrition on beta cell mass and that even at physiological levels they restrain fetal beta cell development in utero. There are emerging clues that this occurs downstream of endocrine commitment by neurogenin 3 but prior to terminal beta cell differentiation. Deciphering the precise mechanism will be important as it might unveil new pathways by which to manipulate beta cell mass that could be exploited as novel therapies for patients with diabetes

Presentations of children to emergency departments across Europe and the COVID-19 pandemic: A multinational observational study

BACKGROUND: During the initial phase of the Coronavirus Disease 2019 (COVID-19) pandemic, reduced numbers of acutely ill or injured children presented to emergency departments (EDs). Concerns were raised about the potential for delayed and more severe presentations and an increase in diagnoses such as diabetic ketoacidosis and mental health issues. This multinational observational study aimed to study the number of children presenting to EDs across Europe during the early COVID-19 pandemic and factors influencing this and to investigate changes in severity of illness and diagnoses. METHODS AND FINDINGS: Routine health data were extracted retrospectively from electronic patient records of children aged 18 years and under, presenting to 38 EDs in 16 European countries for the period January 2018 to May 2020, using predefined and standardized data domains. Observed and predicted numbers of ED attendances were calculated for the period February 2020 to May 2020. Poisson models and incidence rate ratios (IRRs), using predicted counts for each site as offset to adjust for case-mix differences, were used to compare age groups, diagnoses, and outcomes. Reductions in pediatric ED attendances, hospital admissions, and high triage urgencies were seen in all participating sites. ED attendances were relatively higher in countries with lower SARS-CoV-2 prevalence (IRR 2.26, 95% CI 1.90 to 2.70, p < 0.001) and in children aged <12 months (12 to <24 months IRR 0.86, 95% CI 0.84 to 0.89; 2 to <5 years IRR 0.80, 95% CI 0.78 to 0.82; 5 to <12 years IRR 0.68, 95% CI 0.67 to 0.70; 12 to 18 years IRR 0.72, 95% CI 0.70 to 0.74; versus age <12 months as reference group, p < 0.001). The lowering of pediatric intensive care admissions was not as great as that of general admissions (IRR 1.30, 95% CI 1.16 to 1.45, p < 0.001). Lower triage urgencies were reduced more than higher triage urgencies (urgent triage IRR 1.10, 95% CI 1.08 to 1.12; emergent and very urgent triage IRR 1.53, 95% CI 1.49 to 1.57; versus nonurgent triage category, p < 0.001). Reductions were highest and sustained throughout the study period for children with communicable infectious diseases. The main limitation was the retrospective nature of the study, using routine clinical data from a wide range of European hospitals and health systems. CONCLUSIONS: Reductions in ED attendances were seen across Europe during the first COVID-19 lockdown period. More severely ill children continued to attend hospital more frequently compared to those with minor injuries and illnesses, although absolute numbers fell. TRIAL REGISTRATION: ISRCTN91495258 https://www.isrctn.com/ISRCTN91495258

Research priorities for European paediatric emergency medicine

Objective Research in European Paediatric Emergency Medicine (REPEM) network is a collaborative group of 69 paediatric emergency medicine (PEM) physicians from 20 countries in Europe, initiated in 2006. To further improve paediatric emergency care in Europe, the aim of this study was to define research priorities for PEM in Europe to guide the development of future research projects. Design and Setting We carried out an online survey in a modified three-stage Delphi study. Eligible participants were members of the REPEM network. In stage 1, the REPEM steering committee prepared a list of research topics. In stage 2, REPEM members rated on a 6-point scale research topics and they could add research topics and comment on the list for further refinement. Stage 3 included further prioritisation using the Hanlon Process of Prioritisation (HPP) to give more emphasis to the feasibility of a research topic. Results Based on 52 respondents (response rates per stage varying from 41% to 57%), we identified the conditions 'fever', 'sepsis' and 'respiratory infections', and the processes/interventions 'biomarkers', 'risk stratification' and 'practice variation' as common themes of research interest. The HPP identified highest priority for 4 of the 5 highest prioritised items by the Delphi process, incorporating prevalence and severity of each condition and feasibility of undertaking such research. Conclusions While the high diversity in emergency department (ED) populations, cultures, healthcare systems and healthcare delivery in European PEM prompts to focus on practice variation of ED conditions, our defined research priority list will help guide further collaborative research efforts within the REPEM network to improve PEM care in Europe.publishersversionPeer reviewe