116 research outputs found

    Aryl hydrocarbon receptor activation in primary human keratinocytes and epidermal equivalents

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    The Aryl hydrocarbon Receptor (AhR) mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulting in the human specific toxicity, chloracne. To test whether the chloracnegenic potential of AhR-agonists depends upon binding affinity for the AhR, residency and/or down-regulation of the AhR, we investigated the effects of different AhR agonists in primary human keratinocytes and epidermal equivalents. The AhR agonists used were high-affinity, high-residency and high-potency TCDD, and two agonists not known to induce chloracne; low-affinity, low-residency and low-potency β-naphthoflavone (β-NF) and the low-affinity, low-residency and high-potency physiological agonist 2-(1‟H-indole-3‟-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). -NF, a partial agonist was used to test AhR dependency. The effects of these agonists on AhR activation, terminal differentiation, autophagy and expression of cathepsin D (CTSD) in primary human keratinocytes and epidermal equivalents were determined. All three agonists induced AhR activation by XRE-luciferase assay, which was inhibited by α-NF, demonstrating AhR dependence of the ligands. AhR degradation was induced by all ligands and CYP1A1 was induced strongly by TCDD but weakly by β-NF and ITE. CYP1A1 and XRE-luciferase induction correlated with ligand binding affinity; ranking levels of binding affinity as TCDD>β-NF>ITE. TCDD treatment induced a chloracne-like phenotype in epidermal equivalents, with a decrease in viable cell layer thickness and compacted stratum corneum. This was not induced by β-NF or ITE. To investigate the differential effects of AhR-ligands on epidermal equivalent phenotype, we studied differentiation markers filaggrin, involucrin and TGM-1. TGM-1 expression was induced specifically by TCDD while aberrant expression of involucrin and filaggrin were induced by TCDD, β-NF and ITE. AhR activation was not associated with increased apoptosis. Caspase-3 independent cell death has been implicated as a mechanism of decreased thickness of the viable cell layer, so we studied the effects of AhR-agonists on autophagy. Autophagy in keratinocytes and epidermal equivalents was characterised by induction of LC3 II, p62 degradation and transmission electron microscopy. TCDD robustly induced active autophagy, while ITE induced lower levels and β-NF blocked autophagy. TCDD- and ITE-induced autophagy in epidermal equivalents appeared to result in decreased numbers of lamellar bodies, which may account at least in part for the compacted stratum corneum phenotype shown by the TCDD-induced phenotype in epidermal equivalents and chloracne. As CTSD has been implicated in keratinocyte differentiation and an XRE domain has been identified upstream of CTSD, we studied the effects of ligand-dependent AhR activation on lysosomal aspartic protease CTSD expression. CTSD was increased by AhR activity in epidermal equivalents. Induction of CYP1A1 did not appear to be a specific biomarker of chloracnegenic potential of AhR agonists. The data presented have shown differential effects by TCDD, β-NF and ITE on autophagy that we hypothesise contributes to the chloracne phenotype. In this thesis, potential biomarkers specific to chloracne were identified in keratinocytes, TGM-1, CTSD, autophagy and decreased lamellar bodies, although further validation is required.EThOS - Electronic Theses Online ServiceBBSRCAstraZenecaGBUnited Kingdo

    WHITE PAPER: Environmental Scan for DataONE

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    This environmental scan (conducted by the U&AWG in fall 2018) features a multi-faceted analysis of projects/initiatives in the DataONE space. This report (1) provides context by identifying organizations in the data space; (2) analyzes those organizations most similar to DataONE regarding key services and products; and (3) explores the data training/education environment. As appropriate, the report offers key insights derived from the analysis

    ER exit in physiology and disease

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    The biosynthetic secretory pathway is comprised of multiple steps, modifications and interactions that form a highly precise pathway of protein trafficking and secretion, that is essential for eukaryotic life. The general outline of this pathway is understood, however the specific mechanisms are still unclear. In the last 15 years there have been vast advancements in technology that enable us to advance our understanding of this complex and subtle pathway. Therefore, based on the strong foundation of work performed over the last 40 years, we can now build another level of understanding, using the new technologies available. The biosynthetic secretory pathway is a high precision process, that involves a number of tightly regulated steps: Protein folding and quality control, cargo selection for Endoplasmic Reticulum (ER) exit, Golgi trafficking, sorting and secretion. When deregulated it causes severe diseases that here we categorise into three main groups of aberrant secretion: decreased, excess and altered secretion. Each of these categories disrupts organ homeostasis differently, effecting extracellular matrix composition, changing signalling events, or damaging the secretory cells due to aberrant intracellular accumulation of secretory proteins. Diseases of aberrant secretion are very common, but despite this, there are few effective therapies. Here we describe ER exit sites (ERES) as key hubs for regulation of the secretory pathway, protein quality control and an integratory hub for signalling within the cell. This review also describes the challenges that will be faced in developing effective therapies, due to the specificity required of potential drug candidates and the crucial need to respect the fine equilibrium of the pathway. The development of novel tools is moving forward, and we can also use these tools to build our understanding of the acute regulation of ERES and protein trafficking. Here we review ERES regulation in context as a therapeutic strategy

    Assessment, Usability, and Sociocultural Impacts of DataONE

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    DataONE, funded from 2009-2019 by the U.S. National Science Foundation, is an early example of a large-scale project that built both a cyberinfrastructure and culture of data discovery, sharing, and reuse. DataONE used a Working Group model, where a diverse group of participants collaborated on targeted research and development activities to achieve broader project goals. This article summarizes the work carried out by two of DataONE’s working groups: Usability & Assessment (2009-2019) and Sociocultural Issues (2009-2014). The activities of these working groups provide a unique longitudinal look at how scientists, librarians, and other key stakeholders engaged in convergence research to identify and analyze practices around research data management through the development of boundary objects, an iterative assessment program, and reflection. Members of the working groups disseminated their findings widely in papers, presentations, and datasets, reaching international audiences through publications in 25 different journals and presentations to over 5,000 people at interdisciplinary venues. The working groups helped inform the DataONE cyberinfrastructure and influenced the evolving data management landscape. By studying working groups over time, the paper also presents lessons learned about the working group model for global large-scale projects that bring together participants from multiple disciplines and communities in convergence research

    Developmental contributions to macronutrient selection: A randomized controlled trial in adult survivors of malnutrition

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    Background and objectives: Birthweight differences between kwashiorkor and marasmus suggest that intrauterine factors influence the development of these syndromes of malnutrition and may modulate risk of obesity through dietary intake. We tested the hypotheses that the target protein intake in adulthood is associated with birthweight, and that protein leveraging to maintain this target protein intake would influence energy intake (EI) and body weight in adult survivors of malnutrition.Methodology: Sixty-three adult survivors of marasmus and kwashiorkor could freely compose a diet from foods containing 10, 15 and 25 percentage energy from protein (percentage of energy derived from protein (PEP); Phase 1) for 3 days. Participants were then randomized in Phase 2 (5 days) to diets with PEP fixed at 10%, 15% or 25%.Results: Self-selected PEP was similar in both groups. In the groups combined, selected PEP was 14.7, which differed significantly (P < 0.0001) from the null expectation (16.7%) of no selection. Self-selected PEP was inversely related to birthweight, the effect disappearing after adjusting for sex and current body weight. In Phase 2, PEP correlated inversely with EI (P = 0.002) and weight change from Phase 1 to 2 (P = 0.002). Protein intake increased with increasing PEP, but to a lesser extent than energy increased with decreasing PEP.Conclusions and implications: Macronutrient intakes were not independently related to birthweight or diagnosis. In a free-choice situation (Phase 1), subjects selected a dietary PEP significantly lower than random. Lower PEP diets induce increased energy and decreased protein intake, and are associated with weight gain

    Intensive family preservaton services to prevent out-of-home placement of children: a systematic review and meta-analysis

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    Background Intensive Family Preservation Services (IFPS) are in-home crisis intervention services designed to help families with children at imminent risk of out-of-home placement. Objectives To assess the evidence of the effectiveness and cost-effectiveness of IFPS in reducing the need for children to enter out-of-home care. Participants and setting Children <18 years and their families in the home setting. Methods A systematic review and meta-analysis was carried out by searching 12 databases and 16 websites for publications up to January 2019. Results 1948 potentially relevant papers were identified, of which 37 papers, relating to 33 studies, met our inclusion criteria. Studies reported outcomes at child or family level. There were significant reductions in relative risk (RR) of out-of-home placements in children who received IFPS compared with controls at child level at three, six, 12 and 24 months’ follow-up (RR 0.57, 95 % CI 0.35 to 0.93, RR 0.51, 95 % CI 0.27 to 0.96, RR 0.60, 95 % CI 0.48 to 0.76, RR 0.51, 95 % CI 0.30 to 0.87 respectively). At family level, there was not a significant reduction in RR of placement. Economic evidence was limited to cost analyses or cost-cost offset analyses. Conclusion The available evidence, at child level, suggests that IFPS are effective in preventing children from entering care up to 24 months after the intervention. Placement outcomes reported at family level did not demonstrate a significant reduction in out-of-home placements. The economic analyses suggest that IFPS could be cost-saving; however, evidence of cost-effectiveness generated by full economic evaluations is needed
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