Analysis of the subcellular distribution and trafficking of the insulin-responsive glucose transporter, GLUT4

The translocation of a facilitative glucose transporter, GLUT4, from an intracellular site to the cell surface accounts for the large insulin-dependent increase in glucose transport observed in muscle and adipose tissue. Recent studies have indicated that a large proportion of intracellular GLUT4 is segregated from the recycling endosomal system in a post-endocytic storage compartment in 3T3-L1 adipocytes. However, the nature of this GLUT4 storage compartment, and the mechanism by which GLUT4 reaches the cell surface upon insulin stimulation are poorly understood. Here, I have used Tf-HRP-mediated endosomal ablation analysis to examine the protein composition of the post-endocytic compartment with the aim of further defining the relationship of this compartment to other, well characterised, intracellular membrane structures. The results provide further evidence for the existence of a GLUT4 compartment segregated from the endosomal system, which is devoid of cell surface to endosome recycling markers including the TfR and SCAMPs. The observation that within this compartment, GLUT4 co-localises with TGN marker proteins, gamma-adaptin and the CD-M6PR, suggests that GLUT4 traffics through the TGN as part of its recycling itinerary. I further exploited this technique to examine the translocation of GLUT4 to the cell surface in response to insulin and GTPgammaS. The results indicate that insulin is still capable of stimulating GLUT4 translocation following endosomal ablation under conditions where GTPgammaS-stimulated GLUT4 translocation was effectively inhibited. In addition, both insulin- and GTPgammaS-stimulated GLUT1 translocation were blocked following ablation of the recycling endosomal system. On the basis of these and other data, I propose that insulin stimulates the exocytosis of two distinct compartments, one being the post-endocytic storage compartment and the other being the endosomal system, which may also be stimulated by GTPgammaS. Recent studies have implicated ARF proteins to function as regulators of regulated exocytosis. To investigate the role of ARF proteins in insulin-stimulated GLUT4 translocation, we examined the effect of myristoylated ARF peptides on insulin-stimulated GLUT4 translocation and glucose transport in permeabilised 3T3-L1 adipocytes. A myristoylated peptide corresponding to the N-terminus of ARF6 markedly inhibited insulin-stimulated GLUT4 translocation and 2-deoxy-D-glucose transport, whereas ARF5 and ARF1 peptides were without effect. In addition, an effective inhibitor of PLD, butan-1-ol was observed to have no effect on insulin-stimulated GLUT4 translocation and glucose transport. These results suggest that ARF6 plays a crucial role in insulin-stimulated GLUT4 translocation. However, PLD does not appear to function as a downstream effector of ARF in this event. Finally, I have shown that a chimeric protein comprising the signal sequence of the human growth hormone and HRP (ssHRP) may be targeted to the exocytic pathway and secreted from 3T3-L1 adipocytes. However, due to the time constraints of this study, I was unable to use this HRP probe to ablate the TGN, thus enabling me to investigate the role of this sorting compartment in GLUT4 trafficking

“I\u27m making a positive change in my life”: A mixed method evaluation of a well-being tertiary education unit

Issue Addressed: Mental health disorders (MHDs) are prevalent amongst university students with detrimental impacts on individual students, universities and the wider community. There is an urgent need for proactive and preventative strategies to address the mental health crisis in the university population. This study evaluated the efficacy of a 13-week unit developed to directly educate university students about ways to improve and maintain well-being. Methods: Fifty-eight university students from five disciplines participated in a 13-week elective undergraduate unit “Well-Being Fundamentals for Success” as part of their degree. The Act Belong Commit mental health promotion campaign framework formed the basis of teaching materials. Outcome well-being measures were self-assessed at weeks 1, 6 and 12 using four scales: (1) Warwick-Edinburgh Mental Well-being Scale (WEMWBS); (2) Perceived Stress Scale (PSS); (3) Brief Resilience Scale (BRS) and (4) Mindful Attention Awareness Scale (MAAS). Post-unit group interviews (n = 11) were analysed for key themes. Results: Linear mixed models demonstrated a significant improvement in BRS over the semester; well-being (WEMBS) and mindful attention (MAAS) did increase but not significantly. There was a significant increase in stress (PSS) over the semester. Key themes that emerged from the group interviews were that (1) University life contributes to well-being; (2) University life contributes to stress; (3) The well-being unit helped students see and do things differently; (4) An overall endorsement of the unit. Conclusion: University students’ resilience increased over the semester following participation in a curriculum focused on well-being which featured a combination of theoretical content and experiential workshops. So what? Incorporating mental well-being curriculum into tertiary education is proactive preventive health strategy which may assist with the increasing prevalence of MHD in Australia

VEGF isoforms have differential effects on permeability of human pulmonary microvascular endothelial cells

Abstract Background Alternative splicing of Vascular endothelial growth factor-A mRNA transcripts (commonly referred as VEGF) leads to the generation of functionally differing isoforms, the relative amounts of which have potentially significant physiological outcomes in conditions such as acute respiratory distress syndrome (ARDS). The effect of such isoforms on pulmonary vascular permeability is unknown. We hypothesised that VEGF165a and VEGF165b isoforms would have differing effects on pulmonary vascular permeability caused by differential activation of intercellular signal transduction pathways. Method To test this hypothesis we investigated the physiological effect of VEGF165a and VEGF165b on Human Pulmonary Microvascular Endothelial Cell (HPMEC) permeability using three different methods: trans-endothelial electrical resistance (TEER), Electric cell-substrate impedance sensing (ECIS) and FITC-BSA passage. In addition, potential downstream signalling pathways of the VEGF isoforms were investigated by Western blotting and the use of specific signalling inhibitors. Results VEGF165a increased HPMEC permeability using all three methods (paracellular and transcellular) and led to associated VE-cadherin and actin stress fibre changes. In contrast, VEGF165b decreased paracellular permeability and did not induce changes in VE-cadherin cell distribution. Furthermore, VEGF165a and VEGF165b had differing effects on both the phosphorylation of VEGF receptors and downstream signalling proteins pMEK, p42/44MAPK, p38 MAPK, pAKT and peNOS. Interestingly specific inhibition of the pMEK, p38 MAPK, PI3 kinase and eNOS pathways blocked the effects of both VEGF165a and VEGF165b on paracellular permeability and the effect of VEGF165a on proliferation/migration, suggesting that this difference in cellular response is mediated by an as yet unidentified signalling pathway(s). Conclusion This study demonstrates that the novel isoform VEGF165a and VEGF165b induce differing effects on permeability in pulmonary microvascular endothelial cells

Studi Volume Lalu Lintas di Jalan Raya Narogong Cileungsi, Kabupaten Bogor, Periode Agustus 2011

Survey of traffic volume is one of the simplest methods to obtain traffic data in order to better understand optimalisation andefficiency so that it can minimize vehicle traffic congestion problems on the highway. The method used is based on descriptive andanalytical methods, which is done is to classify the vehicles in classes manually by counting the number of vehicles per time unitbased on class - class. The purpose of the volume of traffic surveys carried out in the classified Narogong Cullinan Road, Bogorregency during the period August, 2011, results that can be found is the degree of saturation of the highway is still in an acceptablelevel. Analysis of traffic flow at the study site is still under the limit congestion. It is suggested that to reduce the traffic density, thetype of heavy vehicles such as out of the factory operates around the study site between the hours of 10:00 pm to 3:00 am

Treatment outcomes in 1p19q co-deleted/partially deleted gliomasa

AbstractBackground:Radiotherapy with procarbazine, lomustine, and vincristine improves overall survival (OS) in patients with 1p19q co-deleted anaplastic oligodendroglioma/anaplastic oligoastrocytoma.Methods:This retrospective analysis investigated outcomes in patients with 1p19q co-deleted/partially deleted oligodendroglioma, oligoastrocytoma, anaplastic oligodendroglioma, or anaplastic oligoastrocytoma. OS and progression-free survival (PFS) were analyzed using the Kaplan-Meier method and prognostic factors using the Cox proportional hazard model.Results:A total of 106 patients (between December 1997 and December 2013) were included. Median age was 40 years (19-66), 58 were male (55%), Eastern Cooperative Oncology Group performance status was 0 in 80 patients (75%). 1p19q status was co-deleted in 66 (62%), incompletely co-deleted in 27 (25%), and 1p or 19q loss alone in four (4%) and nine (8%) patients, respectively. Isocitrate dehydrogenase-1 R132H mutation was found in 67 of 85 patients with sufficient material. Upfront treatment was given in 72 (68%) patients and temozolomide alone in 52 (49%). Median time to radiotherapy in 47 patients (44%) was 34.7 months and 41.2 months in 9 patients with co-deleted/incompletely co-deleted anaplastic oligodendroglioma/anaplastic oligoastrocytoma who received upfront temozolomide alone. Median OS was not reached and 5-year OS was 91% for all groups (median follow-up, 5.1 years). On multivariable analysis for all patients, receipt of therapy upfront versus none (p=0.04), PS 1 versus 0 (p&lt;0.001) and 1p19q co-deletion/incomplete deletion versus 1p or 19q loss alone (p=0.005) were prognostic for PFS. Isocitrate dehydrogenase-1 status was not prognostic for PFS.Conclusions:With similar survival patterns in low-grade/anaplastic gliomas, molecular characteristics may be more important than histological grade. Longer follow-up and results of prospective trials are needed for definitive guidance on treatment of these patients.</jats:p

Differential expression of VEGF-Axxx isoforms is critical for development of pulmonary fibrosis

RATIONALE Fibrosis after lung injury is related to poor outcome, and idiopathic pulmonary fibrosis (IPF) can be regarded as an exemplar. Vascular endothelial growth factor (VEGF)-A has been implicated in this context, but there are conflicting reports as to whether it is a contributory or protective factor. Differential splicing of the VEGF-A gene produces multiple functional isoforms including VEGF-Aa and VEGF-Ab, a member of the inhibitory family. To date there is no clear information on the role of VEGF-A in IPF. OBJECTIVES To establish VEGF-A isoform expression and functional effects in IPF. METHODS We used tissue sections, plasma, and lung fibroblasts from patients with IPF and control subjects. In a bleomycin-induced lung fibrosis model we used wild-type MMTV mice and a triple transgenic mouse SPC-rtTATetoCreLoxP-VEGF-Ato conditionally induce VEGF-A isoform deletion specifically in the alveolar type II (ATII) cells of adult mice. MEASUREMENTS AND MAIN RESULTS IPF and normal lung fibroblasts differentially expressed and responded to VEGF-Aa and VEGF-Ab in terms of proliferation and matrix expression. Increased VEGF-Ab was detected in plasma of progressing patients with IPF. In a mouse model of pulmonary fibrosis, ATII-specific deficiency of VEGF-A or constitutive overexpression of VEGF-Ab inhibited the development of pulmonary fibrosis, as did treatment with intraperitoneal delivery of VEGF-Ab to wild-type mice. CONCLUSIONS These results indicate that changes in the bioavailability of VEGF-A sourced from ATII cells, namely the ratio of VEGF-Aa to VEGF-Ab, are critical in development of pulmonary fibrosis and may be a paradigm for the regulation of tissue repair

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children