A yeast three-hybrid system that reconstitutes mammalian hypoxia inducible factor regulatory machinery

Background: Several human pathologies, including neoplasia and ischemic cardiovascular diseases, course with an unbalance between oxygen supply and demand ( hypoxia). Cells within hypoxic regions respond with the induction of a specific genetic program, under the control of the Hypoxia Inducible Factor (HIF), that mediates their adaptation to the lack of oxygen. The activity of HIF is mainly regulated by the EGL-nine homolog (EGLN) enzymes that hydroxylate the alpha subunit of this transcription factor in an oxygen-dependent reaction. Hydroxylated HIF is then recognized and ubiquitinilated by the product of the tumor suppressor gene, pVHL, leading to its proteosomal degradation. Under hypoxia, the hydroxylation of HIF by the EGLNs is compromised due to the lack of oxygen, which is a reaction cosubstrate. Thus, HIF escapes degradation and drives the transcription of its target genes. Since the progression of the aforementioned pathologies might be influenced by activation of HIF-target genes, development of small molecules with the ability to interfere with the HIF-regulatory machinery is of great interest.Results: Herein we describe a yeast three-hybrid system that reconstitutes mammalian HIF regulation by the EGLNs and VHL. In this system, yeast growth, under specific nutrient restrictions, is driven by the interaction between the beta domain of VHL and a hydroxyproline-containing HIF alpha peptide. In turn, this interaction is strictly dependent on EGLN activity that hydroxylates the HIFa peptide. Importantly, this system accurately preserves the specificity of the hydroxylation reaction toward specific substrates. We propose that this system, in combination with a matched control, can be used as a simple and inexpensive assay to identify molecules that specifically modulate EGLN activity. As a proof of principle we show that two known EGLN inhibitors, dimethyloxaloylglycine (DMOG) and 6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamide (S956711), have a profound and specific effect on the yeast HIF/EGLN/VHL system.Conclusion: The system described in this work accurately reconstitutes HIF regulation while preserving EGLN substrate specificity. Thus, it is a valuable tool to study HIF regulation, and particularly EGLN biochemistry, in a cellular context. In addition, we demonstrate that this system can be used to identify specific inhibitors of the EGLN enzymes

Cytotoxicity of the mycotoxins deoxynivalenol and ochratoxin A on Caco-2 cell line in presence of resveratrol

Exposure to mycotoxins through dietary food intake involves a highly complex scenario where co-contamination of different mycotoxins has been frequently demonstrated. On the other hand, the effect of the interaction of mycotoxins with other generally considered beneficial food components, as the antioxidants, has been scarcely studied. The main goal of the present work was to assess the cytotoxic effects on Caco-2 cells of the mycotoxins deoxynivalenol (DON) and ochratoxin A (OTA), alone or combined, and to explore potential protective effects of resveratrol (RES), an antioxidant frequently found in wine. In parallel, reactive oxygen species (ROS) production has also been studied as a first approach to understand the underlying mechanism of cytotoxicity. Results indicate a higher toxic effect of the mycotoxins when they are co-exposed. This increase in cytotoxicity was not accompanied by an increase in ROS production. The co-exposure of OTA or DON with RES did not result in a decrease in cytotoxicity; on the contrary, it resulted in increased cytotoxicity not associated with an increase in ROS production.The authors are grateful to the CYTED Action 109AC0371, to the INIA project (RTA 2012-00053-00-00) as well as the Spanish (Project AGL2011-24862) and Catalonian (XaRTA-Reference Network on Food Technology) Governments for their financial support. C.A. González-Arias thanks the Secretaria de Universitats i Recerca del Departament de Economia i Coneixement of the Generalitat de Catalunya for the pre-doctoral grant

Mean-Payoff Optimization in Continuous-Time Markov Chains with Parametric Alarms

Continuous-time Markov chains with alarms (ACTMCs) allow for alarm events that can be non-exponentially distributed. Within parametric ACTMCs, the parameters of alarm-event distributions are not given explicitly and can be subject of parameter synthesis. An algorithm solving the $\varepsilon$-optimal parameter synthesis problem for parametric ACTMCs with long-run average optimization objectives is presented. Our approach is based on reduction of the problem to finding long-run average optimal strategies in semi-Markov decision processes (semi-MDPs) and sufficient discretization of parameter (i.e., action) space. Since the set of actions in the discretized semi-MDP can be very large, a straightforward approach based on explicit action-space construction fails to solve even simple instances of the problem. The presented algorithm uses an enhanced policy iteration on symbolic representations of the action space. The soundness of the algorithm is established for parametric ACTMCs with alarm-event distributions satisfying four mild assumptions that are shown to hold for uniform, Dirac and Weibull distributions in particular, but are satisfied for many other distributions as well. An experimental implementation shows that the symbolic technique substantially improves the efficiency of the synthesis algorithm and allows to solve instances of realistic size.Comment: This article is a full version of a paper accepted to the Conference on Quantitative Evaluation of SysTems (QEST) 201

Do Interventions Designed to Support Shared Decision-Making Reduce Health Inequalities? : A Systematic Review and Meta-Analysis

Copyright: © 2014 Durand et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background: Increasing patient engagement in healthcare has become a health policy priority. However, there has been concern that promoting supported shared decision-making could increase health inequalities. Objective: To evaluate the impact of SDM interventions on disadvantaged groups and health inequalities. Design: Systematic review and meta-analysis of randomised controlled trials and observational studies.Peer reviewe

Mineral analysis of complete dog and cat foods in the UK and compliance with European guidelines

Mineral content of complete pet food is regulated to ensure health of the companion animal population. Analysis of adherence to these regulatory guidelines has not been conducted. Here, mineral composition of complete wet (n = 97) and dry (n = 80) canine and feline pet food sold in the UK was measured to assess compliance with EU guidelines. A majority of foods complied with ≥8 of 11 guidelines (99% and 83% for dry and wet food, respectively), but many failed to provide nutritional minimum (e.g. Cu, 20% of wet food) or exceeded nutritional maximum (e.g. Se, 76% of wet food). Only 6% (6/97) of wet and 38% (30/80) of dry food were fully compliant. Some foods (20–30% of all analysed) had mineral imbalance, such as not having the recommended ratio of Ca:P (between 1:1 to 2:1). Foods with high fish content had high levels of undesirable metal elements such as arsenic. This study highlights broad non-compliance of a range of popular pet foods sold in the UK with EU guidelines (94% and 61% of wet and dry foods, respectively). If fed exclusively and over an extended period, a number of these pet foods could impact the general health of companion animals

Vitamin D supplementation and breast cancer prevention : a systematic review and meta-analysis of randomized clinical trials

In recent years, the scientific evidence linking vitamin D status or supplementation to breast cancer has grown notably. To investigate the role of vitamin D supplementation on breast cancer incidence, we conducted a systematic review and meta-analysis of randomized controlled trials comparing vitamin D with placebo or no treatment. We used OVID to search MEDLINE (R), EMBASE and CENTRAL until April 2012. We screened the reference lists of included studies and used the “Related Article” feature in PubMed to identify additional articles. No language restrictions were applied. Two reviewers independently extracted data on methodological quality, participants, intervention, comparison and outcomes. Risk Ratios and 95% Confident Intervals for breast cancer were pooled using a random-effects model. Heterogeneity was assessed using the I2 test. In sensitivity analysis, we assessed the impact of vitamin D dosage and mode of administration on treatment effects. Only two randomized controlled trials fulfilled the pre-set inclusion criteria. The pooled analysis included 5372 postmenopausal women. Overall, Risk Ratios and 95% Confident Intervals were 1.11 and 0.74–1.68. We found no evidence of heterogeneity. Neither vitamin D dosage nor mode of administration significantly affected breast cancer risk. However, treatment efficacy was somewhat greater when vitamin D was administered at the highest dosage and in combination with calcium (Risk Ratio 0.58, 95% Confident Interval 0.23–1.47 and Risk Ratio 0.93, 95% Confident Interval 0.54–1.60, respectively). In conclusions, vitamin D use seems not to be associated with a reduced risk of breast cancer development in postmenopausal women. However, the available evidence is still limited and inadequate to draw firm conclusions. Study protocol code: FARM8L2B5L

Norwegian Physicians' Knowledge of and Opinions about Evidence-Based Medicine: Cross-Sectional Study

Objective: To answer five research questions: Do Norwegian physicians know about the three important aspects of EBM? Do they use EBM methods in their clinical practice? What are their attitudes towards EBM? Has EBM in their opinion changed medical practice during the last 10 years? Do they use EBM based information sources? Design: Cross sectional survey in 2006. Setting: Norway. Participants: 966 doctors who responded to a questionnaire (70% response rate). Results: In total 87% of the physicians mentioned the use of randomised clinical trials as a key aspect of EBM, while 53% of them mentioned use of clinical expertise and only 19% patients' values. 40% of the respondents reported that their practice had always been evidence-based. Many respondents experienced difficulties in using EBM principles in their clinical practice because of lack of time and difficulties in searching EBM based literature. 80% agreed that EBM helps physicians towards better practice and 52% that it improves patients' health. As reasons for changes in medical practice 86% of respondents mentioned medical progress, but only 39% EBM. Conclusions: The results of the study indicate that Norwegian physicians have a limited knowledge of the key aspects of EBM but a positive attitude towards the concept. They had limited experience in the practice of EBM and were rather indifferent to the impact of EBM on medical practice. For solving a patient problem, physicians would rather consult a colleague than searching evidence based resources such as the Cochrane Library

Coastal risk mitigation by green infrastructure in Latin America

This paper aims to highlight the prevailing experiences of Latin America and to clarify what ‘green infrastructure’ entails in addition to describing seven case studies from a range of coastal ecosystems (wetlands, coastal dunes, beaches and coral reefs) at scales varying from local to regional. The case studies are categorised according to their degree of naturalness (nature-based, engineered ecosystems, soft engineering, ecologically enhanced hard infrastructure and de-engineering). Generally, the implementation of green infrastructure projects aims to increase resilience, enhance the provision of ecosystem services, recover biodiversity, reduce the negative effects of hard infrastructure and implement corrective measures. The greatest benefits of these projects relate to the creation of multi-functional spaces, which often combine the above advantages with improved opportunities for recreation and/or economic activities. It is hoped that this paper will disseminate the experience in green infrastructure among academics and practitioners and stimulate wider adoption of green infrastructure projects and good practices