Increased expression of miR-187 in human islets from individuals with type 2 diabetes is associated with reduced glucose-stimulated insulin secretion

AIMS/HYPOTHESIS: Type 2 diabetes is characterised by progressive beta cell dysfunction, with changes in gene expression playing a crucial role in its development. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and therefore alterations in miRNA levels may be involved in the deterioration of beta cell function. METHODS: Global TaqMan arrays and individual TaqMan assays were used to measure islet miRNA expression in discovery (n = 20) and replication (n = 20) cohorts from individuals with and without type 2 diabetes. The role of specific dysregulated miRNAs in regulating insulin secretion, content and apoptosis was subsequently investigated in primary rat islets and INS-1 cells. Identification of miRNA targets was assessed using luciferase assays and by measuring mRNA levels. RESULTS: In the discovery and replication cohorts miR-187 expression was found to be significantly increased in islets from individuals with type 2 diabetes compared with matched controls. An inverse correlation between miR-187 levels and glucose-stimulated insulin secretion (GSIS) was observed in islets from normoglycaemic donors. This correlation paralleled findings in primary rat islets and INS-1 cells where overexpression of miR-187 markedly decreased GSIS without affecting insulin content or apoptotic index. Finally, the gene encoding homeodomain-interacting protein kinase-3 (HIPK3), a known regulator of insulin secretion, was identified as a direct target of miR-187 and displayed reduced expression in islets from individuals with type 2 diabetes. CONCLUSIONS/INTERPRETATION: Our findings suggest a role for miR-187 in the blunting of insulin secretion, potentially involving regulation of HIPK3, which occurs during the pathogenesis of type 2 diabetes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00125-013-3089-4) contains peer-reviewed but unedited supplementary material, which is available to authorised users

An alternative polyadenylation signal in TCF7L2 generates isoforms that inhibit T cell factor/lymphoid-enhancer factor (TCF/LEF)-dependent target genes.

Journal ArticleResearch Support, Non-U.S. Gov't© The Author(s) 2011. This article is published with open access at Springerlink.comAIMS/HYPOTHESIS: Intronic single nucleotide polymorphisms within the transcription factor 7-like 2 (TCF7L2) gene are associated with risk of type 2 diabetes. It is widely hypothesised that the predisposing variation is involved in cis-regulation of TCF7L2 activity. The aim of this study was to seek evidence for the existence of novel TCF7L2 isoforms encoded within the type 2 diabetes-associated genomic region. METHODS: We searched expressed sequence tag (EST) databases for novel TCF7L2 transcripts and sought to validate the function and integrity of any isoforms found using a combination of RT-PCR, western blotting and reporter gene techniques. RESULTS: Analysis of EST databases suggested the presence of an alternative polyadenylation site located in intron 4 of TCF7L2. We used 3' rapid amplification of cDNA ends and real-time PCR to validate the integrity of this polyadenylation signal and show its wide use across human tissues. Western blotting results are consistent with the use of this polyadenylation signal to generate novel protein isoforms. The alternative polyadenylation signal results in the production of isoforms that retain the β-catenin binding domain but do not possess the high-mobility group box DNA-binding domain. Promoter-reporter gene assays suggest that these isoforms inhibit TCF7L2-dependent target genes by sequestering β-catenin. CONCLUSIONS/INTERPRETATION: We have identified a novel polyadenylation signal within TCF7L2 that can result in the production of isoforms that act to repress TCF/LEF-dependent target genes. These findings may provide new insights into the association of TCF7L2 with susceptibility to type 2 diabetes.Wellcome TrustMRCEuropean Community’s Seventh Framework Programm

Increased expression of miR-187 in human islets from individuals with type 2 diabetes is associated with reduced glucose-stimulated insulin secretion

Journal ArticleThis article is published with open access at Springerlink.com Electronic supplementary material. The online version of this article (doi:10.1007/s00125-013-3089-4) contains peer-reviewed but unedited supplementary material, which is available to authorised usersAims/hypothesis: Type 2 diabetes is characterised by progressive beta cell dysfunction, with changes in gene expression playing a crucial role in its development. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and therefore alterations in miRNA levels may be involved in the deterioration of beta cell function. Methods: Global TaqMan arrays and individual TaqMan assays were used to measure islet miRNA expression in discovery (n = 20) and replication (n = 20) cohorts from individuals with and without type 2 diabetes. The role of specific dysregulated miRNAs in regulating insulin secretion, content and apoptosis was subsequently investigated in primary rat islets and INS-1 cells. Identification of miRNA targets was assessed using luciferase assays and by measuring mRNA levels. Results: In the discovery and replication cohorts miR-187 expression was found to be significantly increased in islets from individuals with type 2 diabetes compared with matched controls. An inverse correlation between miR-187 levels and glucose-stimulated insulin secretion (GSIS) was observed in islets from normoglycaemic donors. This correlation paralleled findings in primary rat islets and INS-1 cells where overexpression of miR-187 markedly decreased GSIS without affecting insulin content or apoptotic index. Finally, the gene encoding homeodomain-interacting protein kinase-3 (HIPK3), a known regulator of insulin secretion, was identified as a direct target of miR-187 and displayed reduced expression in islets from individuals with type 2 diabetes. Conclusions/interpretation: Our findings suggest a role for miR-187 in the blunting of insulin secretion, potentially involving regulation of HIPK3, which occurs during the pathogenesis of type 2 diabetes. © 2013 The Author(s).This work was supported by the Wellcome Trust (project grant number 089845/Z/09/Z). GAR is the recipient of Royal Society Wolfson Research and Wellcome Trust Senior Investigator (WT098424AIA) Awards, and thanks the Medical Research Council (MRC) for Programme Grant MR/J0003042/1. GdSX and GAR were supported by a project grant from Diabetes UK (BDA 13/0004672) and HDR by MRC grant G1001644

Synthesis of novel methyl 3-(Hetero)arylthieno[3,2-b]pyridine-2-carboxylates and antitumor activity evaluation: Studies in vitro and in ovo grafts of chick chorioallantoic membrane (cam) with a triple negative breast cancer cell line

A series of novel functionalized methyl 3-(hetero)arylthieno[3,2-b]pyridine-2-carboxylates 2a–2h were synthesized by C-C Pd-catalyzed Suzuki-Miyaura cross-coupling of methyl 3-bromothie-no[3,2-b]pyridine-2-carboxylate with (hetero)aryl pinacol boranes, trifluoro potassium boronate salts or boronic acids. Their antitumoral potential was evaluated in two triple negative breast cancer (TNBC) cell lines—MDA-MB-231 and MDA-MB-468, by sulforhodamine B assay. Their effects on the non-tumorigenic MCF-12A cells were also evaluated. The results demonstrated that three compounds caused growth inhibition in both TNBC cell lines, with little or no effect against the non-tumorigenic cells. The most promising compound was further studied concerning possible effects on cell viability (by trypan blue exclusion assay), cell proliferation (by bromodeoxyuridine assay) and cell cycle profile (by flow cytometry). The results demonstrated that the GI50 concentration of compound 2e (13 µM) caused a decreased in MDA-MB-231 cell number, which was correlated with a decreased in the % of proliferating cells. Moreover, this compound increased G0/G1 phase and decreased S phases, when compared to control cells (although was not statistic significant). Interestingly, compound 2e also reduced tumor size using an in ovo CAM (chick chorioallantoic membrane) model. This work highlights the potential antitumor effect of a novel methyl 3-arylthieno[3,2-b]pyridine-2-carboxylate derivative.This research was funded by Fundação para a Ciência e Tecnologia (FCT)—Portugal that financially supports CQUM (UID/QUI/686/2019), also financed by European Regional Development Fund (ERDF), COMPETE2020 and Portugal2020, the PTNMR network also supported by Portugal2020. C.P.R.X. is supported through the post-doc grant SFRH/BPD/122871/2016 and J.M.R. through the doctoral grant SFRH/BD/115844/2016, by FCT, ESF (European Social Fund) and HCOP (Human Capital Operational Programme)

CANFOR Portuguese version: validation study

This study is embedded in the larger project "Needs Assessment of the Mentally Disordered Prisoners" funded by Fundacao para a Ciencia e Tecnologia (PIC/IC/83257/2007). We acknowledge the General-Director for Prison Services for his authorization, the Prisons' Directors for their support, the medical and non-medical staff who collaborated in this study and detainees for their participation. Special thanks to Dr Ana Cabral for her support inside the prisons and Dr Jorge Pereira for his assistance in manuscript preparation. In memory of our late friend Dr Jose Morais, who collaborated on and supported this study from the beginning.BACKGROUND The increase in prisoner population is a troublesome reality in several regions of the world. Along with this growth there is increasing evidence that prisoners have a higher proportion of mental illnesses and suicide than the general population. In order to implement strategies that address criminal recidivism and the health and social status of prisoners, particularly in mental disordered offenders, it is necessary to assess their care needs in a comprehensive, but individual perspective. This assessment must include potential harmful areas like comorbid personality disorder, substance misuse and offending behaviours. The Camberwell Assessment of Need - Forensic Version (CANFOR) has proved to be a reliable tool designed to accomplish such aims. The present study aimed to validate the CANFOR Portuguese version. METHODS The translation, adaptation to the Portuguese context, back-translation and revision followed the usual procedures. The sample comprised all detainees receiving psychiatric care in four forensic facilities, over a one year period. A total of 143 subjects, and respective case manager, were selected. The forensic facilities were chosen by convenience: one prison hospital psychiatric ward (n=68; 47.6%), one male (n=24; 16.8%) and one female (n=22; 15.4%) psychiatric clinic and one civil security ward (n=29; 20.3%), all located nearby Lisbon. Basic descriptive statistics and Kappa weighted coefficients were calculated for the inter-rater and the test-retest reliability studies. The convergent validity was evaluated using the Global Assessment of Functioning and the Brief Psychiatric Rating Scale scores. RESULTS The majority of the participants were male and single, with short school attendance, and accused of a crime involving violence against persons. The most frequent diagnosis was major depression (56.1%) and almost half presented positive suicide risk. The reliability study showed average Kappa weighted coefficients of 0.884 and 0.445 for inter-rater and test-retest agreement, respectively. The convergent validity study presented highly significant correlations between unmet needs scores, GAF and BPRS scores. CONCLUSIONS The CANFOR Portuguese version revealed similar psychometric properties to the original English version. Moreover, the results of the reliability and validity studies indicate that the tool is appropriate for individual care needs assessment and as a guide for the mental health and social interventions in forensic psychiatric services.publishersversionpublishe

Different ability of multidrug-resistant and-sensitive counterpart cells to release and capture extracellular vesicles

Cancer multidrug resistance (MDR) is one of the main challenges for cancer treatment efficacy. MDR is a phenomenon by which tumor cells become resistant to several unrelated drugs. Some studies have previously described the important role of extracellular vesicles (EVs) in the dissemination of a MDR phenotype. EVs’ cargo may include different players of MDR, such as microRNAS and drug-efflux pumps, which may be transferred from donor MDR cells to recipient drug-sensitive counterparts. The present work aimed to: (i) compare the ability of drug-sensitive and their MDR counterpart cells to release and capture EVs and (ii) study and relate those differences with possible distinct fate of the endocytic pathway in these counterpart cells. Our results showed that MDR cells released more EVs than their drug-sensitive counterparts and also that the drug-sensitive cells captured more EVs than their MDR counterparts. This difference in the release and capture of EVs may be associated with differences in the endocytic pathway between drug-sensitive and MDR cells. Importantly, manipulation of the recycling pathway influenced the response of drug-sensitive cells to doxorubicin treatment.This article is a result of the project NORTE-01-0145-FEDER-000029, supported by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). We thank Spanish MINECO (SAF2015-66312 to JMF) and for the REDIEX (Spanish Excellence Network in Exosomes) and the Severo Ochoa Excellence Accreditation (SEV-2016-0644). The authors thank the Portuguese Foundation for Science and Technology (FCT) for the PhD grant of DS (SFRH/BD/98054/2013). Cristina P.R. Xavier is supported by FCT and Fundo Social Europeu (FSE), through the post-doc grant SFRH/BPD/122871/2016. The authors also acknowledge the European COST Action-European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD, BM1202) for short-term mission fellowship (ECOST-STSM-BM1202-150317-083396) and Grupo Español de investigacion en Vesiculas Extracelulares for GEIVEX mobility fellowship which allowed the work of DS in CICbioGUNE

Interviewer-driven Variability in Social Network Reporting: Results from Health and Aging in Africa: a Longitudinal Study of an INDEPTH community (HAALSI) in South Africa

Social network analysis depends on how social ties to others are elicited during interviews, a process easily affected by respondent and interviewer behaviors. We investigate how the number of self-reported important social contacts varied within a single data collection round. Our data come from Health and Aging in Africa: a Longitudinal Study of an INDEPTH community (HAALSI), a comprehensive population-based survey of individuals aged 40 years and older conducted over 13 months at the Agincourt health and demographic surveillance site in rural South Africa. As part of HAALSI, interviewers elicited detailed egocentric network data. The average number of contacts reported by the 5,059 respondents both varied significantly across interviewers and fell over time as the data collection progressed, even after adjusting for respondent, interviewer, and respondent–interviewer dyad characteristics. Contact numbers rose substantially after a targeted interviewer intervention. We conclude that checking (and adjusting) for interviewer effects, even within one data collection round, is critical to valid and reliable social network analysis. Measurements of social networks depend on the number and type of social ties to others (Berkman et al. 2000; Smith and Christakis 2008). These ties are typically elicited through interviews, a process easily affected by respondent or interviewer characteristics and behaviors. Understanding social network structure and composition requires substantial amounts of information from respondents (“egos”) about the people (“alters”) they have relationships with (Marsden 1990). Notably, the survey burden associated with network data collection depends heavily on the number of alters elicited through “name generator” questions: Each alter named leads to the repetition of all follow-up questions characterizing the ego–alter relationship (“name interpreters”; Burt 1984). Interviewers have been identified as a key source of variation in survey responses, particularly for questions that are attitudinal, ambiguous, or have complex skip patterns (West and Blom 2016). Several studies have previously identified interviewer effects on network size (Brüderl et al. 2013; Josten and Trappmann 2016; Marsden 2003; Paik and Sanchagrin 2013; van Tilburg 1998). These interviewer effects may arise from differential understanding of survey questions, and therefore how questions are presented to respondents. Interviewers can also affect which alters are elicited due to their own characteristics (e.g., sex, race, age, or experience), or the nature of the interviewer–respondent dyad (e.g., gender, race, or age homophily), leading to different lines of enquiry, levels of probing, or expectations of social acceptability (Collins 1980; Hox 1994; Marsden 2003; Phung et al. 2015). Furthermore, if respondents or interviewers are aware that naming more alters substantially increases survey length, then either group may consciously or unconsciously seek to minimize the number of alters named (Eagle and Proeschold-Bell 2015; van der Zouwen and van Tilburg 2001). In cross-sectional surveys, the opportunities for respondents to learn are limited, but those for interviewers will increase as the survey period progresses. Interviewers may try to reduce survey burden, either for themselves or for respondents, by favoring language or probes that decrease the number of alters elicited. Indeed, past studies in Europe have found evidence of interviewers intentionally filtering out questions by entering fewer responses that would trigger more questions. Such filtering behavior has been seen in Europe for interviewers who are being compensated by the interview rather than by the hour (Josten and Trappmann 2016; Kosyakova et al. 2014), for interviewers with prior experience using the relevant screening tool (Matschinger et al. 2005), and where interviewers are under substantial pressure to complete more interviews (Schnell and Kreuter 2000). We aim to extend this literature by assessing how the number of alters elicited systematically changed over the course of a cross-sectional social network survey of older adults in rural South Africa. We show a substantial drop in alter numbers over time, and a swift reversal following retraining, providing substantial evidence for interviewer effects

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD(369)↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed

A giant comet-like cloud of hydrogen escaping the warm Neptune-mass exoplanet GJ 436b

Exoplanets orbiting close to their parent stars could lose some fraction of their atmospheres because of the extreme irradiation. Atmospheric mass loss primarily affects low-mass exoplanets, leading to suggest that hot rocky planets might have begun as Neptune-like, but subsequently lost all of their atmospheres; however, no confident measurements have hitherto been available. The signature of this loss could be observed in the ultraviolet spectrum, when the planet and its escaping atmosphere transit the star, giving rise to deeper and longer transit signatures than in the optical spectrum. Here we report that in the ultraviolet the Neptune-mass exoplanet GJ 436b (also known as Gliese 436b) has transit depths of 56.3 +/- 3.5% (1 sigma), far beyond the 0.69% optical transit depth. The ultraviolet transits repeatedly start ~2 h before, and end >3 h after the ~1 h optical transit, which is substantially different from one previous claim (based on an inaccurate ephemeris). We infer from this that the planet is surrounded and trailed by a large exospheric cloud composed mainly of hydrogen atoms. We estimate a mass-loss rate in the range of ~10^8-10^9 g/s, which today is far too small to deplete the atmosphere of a Neptune-like planet in the lifetime of the parent star, but would have been much greater in the past.Comment: Published in Nature on 25 June 2015. Preprint is 28 pages, 12 figures, 2 table

TRESK channel contribution to nociceptive sensory neurons excitability: modulation by nerve injury

<p>Abstract</p> <p>Background</p> <p>Neuronal hyperexcitability is a crucial phenomenon underlying spontaneous and evoked pain. In invertebrate nociceptors, the S-type leak K⁺channel (analogous to TREK-1 in mammals) plays a critical role of in determining neuronal excitability following nerve injury. Few data are available on the role of leak K_2Pchannels after peripheral axotomy in mammals.</p> <p>Results</p> <p>Here we describe that rat sciatic nerve axotomy induces hyperexcitability of L4-L5 DRG sensory neurons and decreases TRESK (K2P18.1) expression, a channel with a major contribution to total leak current in DRGs. While the expression of other channels from the same family did not significantly change, injury markers ATF3 and Cacna2d1 were highly upregulated. Similarly, acute sensory neuron dissociation (<it>in vitro </it>axotomy) produced marked hyperexcitability and similar total background currents compared with neurons injured <it>in vivo</it>. In addition, the sanshool derivative IBA, which blocked TRESK currents in transfected HEK293 cells and DRGs, increased intracellular calcium in 49% of DRG neurons in culture. Most IBA-responding neurons (71%) also responded to the TRPV1 agonist capsaicin, indicating that they were nociceptors. Additional evidence of a biological role of TRESK channels was provided by behavioral evidence of pain (flinching and licking), in vivo electrophysiological evidence of C-nociceptor activation following IBA injection in the rat hindpaw, and increased sensitivity to painful pressure after TRESK knockdown in vivo.</p> <p>Conclusions</p> <p>In summary, our results clearly support an important role of TRESK channels in determining neuronal excitability in specific DRG neurons subpopulations, and show that axonal injury down-regulates TRESK channels, therefore contributing to neuronal hyperexcitability.</p