Substance-tailored testing strategies in toxicology : an in silico methodology based on QSAR modeling of toxicological thresholds and Monte Carlo simulations of toxicological testing

International audienceThe design of toxicological testing strategies aimed at identifying the toxic effects of chemicals without (or with a minimal) recourse to animal experimentation is an important issue for toxicological regulations and for industrial decision-making. This article describes an original approach which enables the design of substance-tailored testing strategies with a specified performance in terms of false-positive and false-negative rates. The outcome of toxicological testing is simulated in a different way than previously published articles on the topic. Indeed, toxicological outcomes are simulated not only as a function of the performance of toxicological tests but also as a function of the physico-chemical Properties of chemicals. The required inputs for Our approach are QSAR predictions for the LOAELs of the toxicological effect of interest and statistical distributions describing the relationship existing between in vivo LOAEL values and results from in vitro tests. Our methodology is able to correctly predict the performance of testing strategies designed to analyze the teratogenic effects of two chemicals: di(2-ethylhexyl)phthalate and Indomethacin. The proposed decision-support methodology can be adapted to any toxicological context as long as a Statistical Comparison between in vitro and in Vivo results is possible and QSAR models for the toxicological effect of interest can be developed

Implanta??o e an?lise do framework scrum no desenvolvimento da plataforma aberta Nosso Exerc?cio

Este estudo prop?e a implementa??o e an?lise do Framework Scrum no desenvolvimento de novas funcionalidade para o website Nosso Exerc?cio. Esta aplica??o web consiste em um dos projetos do Programa de Educa??o Tutorial, PET-UFVJM/Campus do Mucuri situada na cidade de Te?filo Otoni-MG e tem como finalidade o compartilhamento aberto de exerc?cios did?ticos de diversas ?reas do conhecimento. Por solicita??o de seus idealizadores, novas funcionalidades foram demandadas para o Nosso Exerc?cio, por?m, n?o existiam para este projeto metas claras nem um plano de trabalho a ser seguido, os requisitos n?o estavam formalizados e a equipe dispon?vel n?o estava madura nas tecnologias utilizadas. Levando-se em considera??o o desafio enfrentado na gera??o de software de qualidade e o limite de tempo dispon?vel para a realiza??o desta pesquisa, foi proposto o uso de um processo da Engenharia de Software com intuito de se obter maior controle e qualidade do produto final a ser desenvolvido. O m?todo ?gil Scrum foi o escolhido para gerenciar as atividades de desenvolvimento para este software. Assim, o objetivo geral desta pesquisa consistiu em buscar uma resposta para a seguinte pergunta-problema: Quais benef?cios e/ou dificuldades podem ser obtidas atrav?s da aplica??o do Framework Scrum na evolu??o do desenvolvimento da plataforma aberta Nosso Exerc?cio? Seguindo o modelo sugerido por Coughlan e Coghlan (2002), o m?todo de pesquisa-a??o foi utilizado para descrever a din?mica conduzida durante este trabalho. A implanta??o do Scrum no Nosso Exerc?cio ocorreu em duas etapas, a primeira, visou realizar uma capacita??o sobre o Scrum e as ferramentas tecnol?gicas utilizadas no desenvolvimento do Nosso Exerc?cio. J? a segunda, tratou do desenvolvimento das funcionalidades para este website. A an?lise dos resultados mostrou v?rios benef?cios obtidos com a implanta??o do Scrum neste projeto, como: o foco e compromisso do Time durante o seu trabalho, o atendimento ?s reais necessidades do cliente (Product Owner), a flexibilidade do framework em se ajustar ?s condi??es de ambiente e trabalho de cada Time criado; o aprendizado cont?nuo do software e do processo resultante das discuss?es feitas nas Reuni?es de Planejamento, Revis?o e Retrospectiva. O ponto cr?tico do trabalho deu-se sobre a imaturidade com as tecnologias utilizadas para o desenvolvimento do website. As li??es aprendidas com esta pesquisa indicam que ? poss?vel obter benef?cios com a implanta??o do framework Scrum que superem as suas dificuldades, desde que sejam feitas as devidas an?lises do ambiente em que o mesmo for adotado.Disserta??o (Mestrado Profissional) ? Programa de P?s-Gradua??o em Tecnologia, Sa?de e Sociedade, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2018.This study proposes na implementation and analysis of Scrum Framework in the development of new functionalities to ?Nosso Exerc?cio? (Our Exercise) website. This web application consists in one of the Tutorial Education Program Projects, PET-UFVJM/Mucuri Campus, located in Te?filo Otoni city, Minas Gerais state and it has as its objective the open share of teaching exercises of several knowledge areas. At the request of its creators, new functionalities were demanded to ?Nosso Exerc?cio? (Our Exercise), however, there were not clear goals for this project nor a work plan to be followed, the requirements were not formalised and the available team was not mature enough on the tecnologies applied. Considering the challenge faced on the generation of a good quality software and the time limit available for taking this research, the use of a software engineering was proposed aiming to get bigger quality control of the final Product to be developed. The agile method Scrum was the one chosen to manage the developing activities to this software. So, the general goal of this research consisted in searching for an answer to the following question-problem: Which benefits and/or difficulties can be obtained through the apllication of Scrum Framework on the evolution of the development of ?Nosso Exerc?cio? open plataforma? According to the model suggested by Coughlan and Coghlan (2002), the research-action method was used to describe a dinamic conducted during this work. The Scrum implantation on ?Nosso Exerc?cio? occurred in two stages, the first one, aimed to do a training about Scrum and the technological tools used in the development of ?Nosso Exerc?cio?. The second one, dealt with the development of functionalities to this website. The analyses of the results showed many benefits gotten with the implatation of Scrum in this project, like focus and commitment of ?Time? during its work, the attendance to the costumer?s real needs (Product Owner), the framework flexibility in adjusting to environment and work conditions of each ?Time? created, the continuous learning of the software and of the process resulting of the discussions taken on planning, reviewing and retrospecto meetings. The critical work point was about the immaturity with the technologies used for the website development. The learned lessons with this research indicate that it?s possible to get benefits with the implantation of Scrum Framework which overcome the difficulties, as long as the needed analysis of the enviroment where it was adopted be done

Predicting in vivo gene expression in macrophages after exposure to benzo(a)pyrene based on in vitro assays and toxicokinetic/toxicodynamic models

International audiencePredictive toxicology aims at developing methodologies to relate the results obtained from in vitro experiments to in vivo exposure. In the case of polycyclic aromatic hydrocarbons (PAHs), a substantial amount of knowledge on effects and modes of action has been recently obtained from in vitro studies of gene expression. In the current study, we built a physiologically based toxicokinetic (PBTK) model to relate in vivo and in vitro gene expression in case of exposure to benzo(a)pyrene (BaP), a referent PAH. This model was calibrated with two toxicokinetic datasets obtained on rats exposed either through intratracheal instillation or through intravenous administration and on an in vitro degradation study. A good agreement was obtained between the model's predictions and the concentrations measured in target organs, such as liver and lungs. Our model was able to relate correctly the gene expression for two genes targeted by PAHs, measured in vitro on primary human macrophages and in vivo in rat macrophages after exposure to BaP. Combining in vitro studies and PBTK modeling is promising for PAH risk assessment, especially for mixtures which are more efficiently studied in vitro than in vivo

Roles of heat shock factor 1 and 2 in response to proteasome inhibition: consequence on p53 stability.

International audienceA single heat shock factor (HSF), mediating the heat shock response, exists from yeast to Drosophila, whereas several related HSFs have been found in mammals. This raises the question of the specific or redundant functions of the different members of the HSF family and in particular of HSF1 and HSF2, which are both ubiquitously expressed. Using immortalized mouse embryonic fibroblasts (iMEFs) derived from wild-type, Hsf1(-/-), Hsf2(-/-) or double-mutant mice, we observed the distinctive behaviors of these mutants with respect to proteasome inhibition. This proteotoxic stress reduces to the same extent the viability of Hsf1(-/-)- and Hsf2(-/-)-deficient cells, but through different underlying mechanisms. Contrary to Hsf2(-/-) cells, Hsf1(-/-) cells are unable to induce pro-survival heat shock protein expression. Conversely, proteasome activity is lower in Hsf2(-/-) cells and the expression of some proteasome subunits, such as Psmb5 and gankyrin, is decreased. As gankyrin is an oncoprotein involved in p53 degradation, we analyzed the status of p53 in HSF-deficient iMEFs and observed that it was strongly stabilized in Hsf2(-/-) cells. This study points a new role for HSF2 in the regulation of protein degradation and suggests that pan-HSF inhibitors could be valuable tools to reduce chemoresistance to proteasome inhibition observed in cancer therapy

BAG-6 is essential for selective elimination of defective proteasomal substrates

The ubiquitin-like protein BAG-6 protects cells from newly synthesized misfolded proteins by tethering them to the proteasome

Expressions of glutathione S-transferase alpha, mu, and pi in brains of medically intractable epileptic patients

<p>Abstract</p> <p>Background</p> <p>Glutathione S-transferases (GSTs) play an important role in metabolizing anti-epileptic drugs (AEDs) in liver. Expressions of GSTs in brain, which may result in poor efficacy of AEDs, have not been well studied. Using clinical cortex specimen from 32 intractable epileptic subjects and 8 non-epileptic controls, the present study investigated the correlation between GSTs and intractable epilepsy.</p> <p>Results</p> <p>Three different GST isoforms (α, μ, and π) were detected with immunohistochemistry. GST-α expression was not seen in any cortex specimens. Sixty three percent (63%) of control and 53% of intractible epileptic specimens showed GST-μ immunoreactivity. No significant difference in intensity of GST-μ staining was observed between these two groups. GST-π expression was found in endothelial cells and glial cells/astrocytes. Fifty percent (50%) of the control patients and 66% of the epileptic patients were GST-π positive. The grading of epileptic patients was significantly higher than that of control patients (<it>p </it>< 0.01).</p> <p>Conclusion</p> <p>High levels of GST-π in endothelial cells and glial cells/astrocyte correlate to medical intractable epilepsy, suggesting that GST-π contributes to resistance to AED treatment.</p

A Novel, Non-Apoptotic Role for Scythe/BAT3: A Functional Switch between the Pro- and Anti-Proliferative Roles of p21 during the Cell Cycle

BACKGROUND: Scythe/BAT3 is a member of the BAG protein family whose role in apoptosis has been extensively studied. However, since the developmental defects observed in Bat3-null mouse embryos cannot be explained solely by defects in apoptosis, we investigated whether BAT3 is also involved in cell-cycle progression. METHODS/PRINCIPAL FINDINGS: Using a stable-inducible Bat3-knockdown cellular system, we demonstrated that reduced BAT3 protein level causes a delay in both G1/S transition and G2/M progression. Concurrent with these changes in cell-cycle progression, we observed a reduction in the turnover and phosphorylation of the CDK inhibitor p21, which is best known as an inhibitor of DNA replication; however, phosphorylated p21 has also been shown to promote G2/M progression. Our findings indicate that in Bat3-knockdown cells, p21 continues to be synthesized during cell-cycle phases that do not normally require p21, resulting in p21 protein accumulation and a subsequent delay in cell-cycle progression. Finally, we showed that BAT3 co-localizes with p21 during the cell cycle and is required for the translocation of p21 from the cytoplasm to the nucleus during the G1/S transition and G2/M progression. CONCLUSION: Our study reveals a novel, non-apoptotic role for BAT3 in cell-cycle regulation. By maintaining a low p21 protein level during the G1/S transition, BAT3 counteracts the inhibitory effect of p21 on DNA replication and thus enables the cells to progress from G1 to S phase. Conversely, during G2/M progression, BAT3 facilitates p21 phosphorylation by cyclin A/Cdk2, an event required for G2/M progression. BAT3 modulates these pro- and anti-proliferative roles of p21 at least in part by regulating cyclin A abundance, as well as p21 translocation between the cytoplasm and the nucleus to ensure that it functions in the appropriate intracellular compartment during each phase of the cell cycle.Dissertatio

Identification of Kinases Regulating Prostate Cancer Cell Growth Using an RNAi Phenotypic Screen

As prostate cancer progresses to castration-resistant disease, there is an increase in signal transduction activity. Most castration-resistant prostate tumors continue to express the androgen receptor (AR) as well as androgen-responsive genes, despite the near absence of circulating androgen in these patients. The AR is regulated not only by its cognate steroid hormone, but also by interactions with a constellation of co-regulatory and signaling molecules. Thus, the elevated signaling activity that occurs during progression to castration resistance can affect prostate cancer cell growth either through the AR or independent of the AR. In order to identify signaling pathways that regulate prostate cancer cell growth, we screened a panel of shRNAs targeting 673 human kinases against LNCaP prostate cancer cells grown in the presence and absence of hormone. The screen identified multiple shRNA clones against known and novel gene targets that regulate prostate cancer cell growth. Based on the magnitude of effect on growth, we selected six kinases for further study: MAP3K11, DGKD, ICK, CIT, GALK2, and PSKH1. Knockdown of these kinases decreased cell growth in both androgen-dependent and castration-resistant prostate cancer cells. However, these kinases had different effects on basal or androgen-induced transcriptional activity of AR target genes. MAP3K11 knockdown most consistently altered transcription of AR target genes, suggesting that MAP3K11 affected its growth inhibitory effect by modulating the AR transcriptional program. Consistent with MAP3K11 acting on the AR, knockdown of MAP3K11 inhibited AR Ser 650 phosphorylation, further supporting stress kinase regulation of AR phosphorylation. This study demonstrates the applicability of lentiviral-based shRNA for conducting phenotypic screens and identifies MAP3K11, DGKD, ICK, CIT, GALK2, and PSKH1 as regulators of prostate cancer cell growth. The thorough evaluation of these kinase targets will pave the way for developing more effective treatments for castration-resistant prostate cancer