42 research outputs found
Small molecule and RNAi induced phenotype transition of expanded and primary colonic epithelial cells
Recent progress in mammalian intestinal epithelial cell culture led to novel
concepts of tissue modeling. Especially the development of phenotypically
stable cell lines from individual animals enables an investigation of distinct
intestinal loci and disease states. We here report primary and prolonged
culture of normal porcine epithelial cells from colon for cell line
development. In addition, a novel primary three-dimensional intestinal culture
system is presented, which generated organoids composed of a highly polarized
epithelial layer lining a core of subepithelial tissue. Cellular
characterization of monolayer cell lines revealed epithelial identity and
pointed to a proliferative crypt cell phenotype. We evaluated both RNAi and
chemical approaches to induce epithelial differentiation in generated cell
lines by targeting promoters of epithelial to mesenchymal transition (EMT). By
in silico prediction and ectopic expression, miR-147b was proven to be a
potent trigger of intestinal epithelial cell differentiation. Our results
outline an approach to generate phenotypically stable cell lines expanded from
primary colonic epithelial cultures and demonstrate the relevance of miR-147b
and chemical inhibitors for promoting epithelial differentiation features
Ninety-day oral toxicity studies on two genetically modified maize MON810 varieties in Wistar Han RCC rats (EU 7th Framework Programme project GRACE)
The GMO Risk Assessment and Communication of Evidence (GRACE; www.grace-fp7.eu) project is funded by the European Commission within the 7th Framework Programme. A key objective of GRACE is to conduct 90-day animal feeding trials, animal studies with an extended time frame as well as analytical, in vitro and in silico studies on genetically modified (GM) maize in order to comparatively evaluate their use in GM plant risk assessment. In the present study, the results of two 90-day feeding trials with two different GM maize MON810 varieties, their near-isogenic non-GM varieties and four additional conventional maize varieties are presented. The feeding trials were performed by taking into account the guidance for such studies published by the EFSA Scientific Committee in 2011 and the OECD Test Guideline 408. The results obtained show that the MON810 maize at a level of up to 33 % in the diet did not induce adverse effects in male and female Wistar Han RCC rats after subchronic exposure, independently of the two different genetic backgrounds of the event
Down regulated lncRNA MEG3 eliminates mycobacteria in macrophages via autophagy
Small non-coding RNA play a major part in host response to bacterial agents.
However, the role of long non-coding RNA (lncRNA) in this context remains
unknown. LncRNA regulate gene expression by acting e.g. as transcriptional
coactivators, RNA decoys or microRNA sponges. They control development,
differentiation and cellular processes such as autophagy in disease
conditions. Here, we provide an insight into the role of lncRNA in
mycobacterial infections. Human macrophages were infected with Mycobacterium
bovis BCG and lncRNA expression was studied early post infection. For this
purpose, lncRNA with known immune related functions were preselected and a
lncRNA specific RT-qPCR protocol was established. In addition to expression-
based prediction of lncRNA function, we assessed strategies for thorough
normalisation of lncRNA. Arrayed quantification showed infection-dependent
repression of several lncRNA including MEG3. Pathway analysis linked MEG3 to
mTOR and PI3K-AKT signalling pointing to regulation of autophagy. Accordingly,
IFN-γ induced autophagy in infected macrophages resulted in sustained MEG3
down regulation and lack of IFN-γ allowed for counter regulation of MEG3 by
viable M. bovis BCG. Knockdown of MEG3 in macrophages resulted in induction of
autophagy and enhanced eradication of intracellular M. bovis BCG
CRIAÇÃO DE UM SISTEMA WEB PARA O SISAE
O Serviço Integrado de Suporte e Acompanhamento Educacional (SISAE) é o setorresponsável por dar suporte ao aluno, promovendo sucesso acadêmico, bem-estar, saúdee inclusão. Apesar disso, o setor lida com desafios constantes como o agendamento dehorários satisfatórios de atendimento para todas as partes interessadas, o acessocentralizado a informações pertinentes e a realização de registros uniformemente. Dessaforma, o presente trabalho, que faz parte de um projeto da matéria de Pesquisa eExtensão do curso de Ciência da Computação, tem como objetivo melhorar a qualidadedos serviços do SISAE para todos que consultam o setor, bem como facilitar parte deseus processos. Assim, propõe-se a criação de um Sistema Web que segue boas práticasde acessibilidade, usabilidade e segurança da informação a fim de melhorar osprocessos do setor do SISAE e resolver os desafios relatados. Para tanto, o projeto temcomo metodologia a pesquisa bibliográfica de caráter exploratório, a fim de familiarizaros autores com os temas propostos. Por outro lado, a implementação do projeto faz usoda metodologia ágil de desenvolvimento, que proporciona flexibilidade e dinamismo.Nesse sentido, pretende-se realizar ciclos curtos de desenvolvimento e validação comcerca de 14 a 21 dias e acompanhamentos frequentes com o setor, a fim de validar asentregas por meio de formulários e entrevistas, consideradas nos ciclos seguintes. Até omomento, foi realizada uma conversa inicial com o SISAE para definição de escopo elevantamento de requisitos, onde os principais pontos de dificuldade foram notados. Emseguida, estipulou-se a criação de módulos do sistema que resolvam essas dificuldades:agendamento de atendimentos, cadastro de anotações, registro de ocorrências e perfilacadêmico do aluno. Em seguida, estipulou-se um cronograma que considera cada partedo sistema, dividindo-se nos ciclos curtos. Por fim, foi criado o esboço das telas e daidentidade visual do sistema, a ser seguido a partir do primeiro ciclo dedesenvolvimento. Dessa forma, espera-se que o projeto auxilie toda comunidade do IFC, incluindo alunos, pais, assim como pessoas com necessidades especiais e quepossa ser mantido pelos próximos alunos da instituição
Prevention of methamphetamine-induced microglial cell death by TNF-α and IL-6 through activation of the JAK-STAT pathway
<p><b>Abstract</b></p> <p><b>Background</b></p> <p>It is well known that methamphetamine (METH) is neurotoxic and recent studies have suggested the involvement of neuroinflammatory processes in brain dysfunction induced by misuse of this drug. Indeed, glial cells seem to be activated in response to METH, but its effects on microglial cells are not fully understood. Moreover, it has been shown that cytokines, which are normally released by activated microglia, may have a dual role in response to brain injury. This led us to study the toxic effect of METH on microglial cells by looking to cell death and alterations of tumor necrosis factor-alpha (TNF-α) and interleukine-6 (IL-6) systems, as well as the role played by these cytokines.</p> <p><b>Methods</b></p> <p>We used the N9 microglial cell line, and cell death and proliferation were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay and incorporation of bromodeoxyuridine, respectively. The TNF-α and IL-6 content was quantified by enzyme-linked immunosorbent assay, and changes in TNF receptor 1, IL-6 receptor-alpha, Bax and Bcl-2 protein levels by western blotting. Immunocytochemistry analysis was also performed to evaluate alterations in microglial morphology and in the protein expression of phospho-signal transducer and activator of transcription 3 (pSTAT3).</p> <p><b>Results</b></p> <p>METH induced microglial cell death in a concentration-dependent manner (EC<sub>50</sub> = 1 mM), and also led to significant morphological changes and decreased cell proliferation. Additionally, this drug increased TNF-α extracellular and intracellular levels, as well as its receptor protein levels at 1 h, whereas IL-6 and its receptor levels were increased at 24 h post-exposure. However, the endogenous proinflammatory cytokines did not contribute to METH-induced microglial cell death. On the other hand, exogenous low concentrations of TNF-α or IL-6 had a protective effect. Interestingly, we also verified that the anti-apoptotic role of TNF-α was mediated by activation of IL-6 signaling, specifically the janus kinase (JAK)-STAT3 pathway, which in turn induced down-regulation of the Bax/Bcl-2 ratio.</p> <p><b>Conclusions</b></p> <p>These findings show that TNF-α and IL-6 have a protective role against METH-induced microglial cell death via the IL-6 receptor, specifically through activation of the JAK-STAT3 pathway, with consequent changes in pro- and anti-apoptotic proteins.</p
Genetic Evidence Implicates the Immune System and Cholesterol Metabolism in the Aetiology of Alzheimer's Disease
Background
1Late Onset Alzheimer's disease (LOAD) is the leading cause of dementia. Recent large genome-wide association studies (GWAS) identified the first strongly supported LOAD susceptibility genes since the discovery of the involvement of APOE in the early 1990s. We have now exploited these GWAS datasets to uncover key LOAD pathophysiological processes.
Methodology
We applied a recently developed tool for mining GWAS data for biologically meaningful information to a LOAD GWAS dataset. The principal findings were then tested in an independent GWAS dataset.
Principal Findings
We found a significant overrepresentation of association signals in pathways related to cholesterol metabolism and the immune response in both of the two largest genome-wide association studies for LOAD.
Significance
Processes related to cholesterol metabolism and the innate immune response have previously been implicated by pathological and epidemiological studies of Alzheimer's disease, but it has been unclear whether those findings reflected primary aetiological events or consequences of the disease process. Our independent evidence from two large studies now demonstrates that these processes are aetiologically relevant, and suggests that they may be suitable targets for novel and existing therapeutic approaches