Clarifying mammalian RISC assembly in vitro

<p>Abstract</p> <p>Background</p> <p>Argonaute, the core component of the RNA induced silencing complex (RISC), binds to mature miRNAs and regulates gene expression at transcriptional or post-transcriptional level. We recently reported that Argonaute 2 (Ago2) also assembles into complexes with miRNA precursors (pre-miRNAs). These Ago2:pre-miRNA complexes are catalytically active <it>in vitro </it>and constitute non-canonical RISCs.</p> <p>Results</p> <p>The use of pre-miRNAs as guides by Ago2 bypasses Dicer activity and complicates <it>in vitro </it>RISC reconstitution. In this work, we characterized Ago2:pre-miRNA complexes and identified RNAs that are targeted by miRNAs but not their corresponding pre-miRNAs. Using these target RNAs we were able to recapitulate <it>in vitro </it>pre-miRNA processing and canonical RISC loading, and define the minimal factors required for these processes.</p> <p>Conclusions</p> <p>Our results indicate that Ago2 and Dicer are sufficient for processing and loading of miRNAs into RISC. Furthermore, our studies suggest that Ago2 binds primarily to the 5'- and alternatively, to the 3'-end of select pre-miRNAs.</p

Construção e validação de um instrumento de análise qualitativa do arremesso (lance-livre) do basquetebol Development and validation of an instrument to assess movement quality of the basketball free throw shooting

Quando o profissional da área de Educação Física está ensinando uma habilidade motora, ele procura avaliar o movimento realizado pelo aluno e reflete sobre quais intervenções poderiam ser feitas naquele momento. No entanto, muitas vezes, o professor não dispõe de recursos que possam auxiliá-lo no processo de avaliação do movimento a ser aprendido pelo aluno. O processo de aquisição habilidades motoras poderia ser facilitado se o professor dispusesse nesse momento de um instrumento que identificasse o erro(s) cometido(s), bem como, qual seria a informação prioritária a ser fornecida ao aprendiz. Portanto, como a literatura especializada apresenta escassez no que diz respeito a esse tipo de ferramenta, o objetivo do presente estudo foi construir, validar e testar a objetividade e fidedignidade de um instrumento (checklist) que avalia a qualidade do arremesso do basquetebol (lance livre). O checklist da habilidade foi desenvolvido e avaliado por especialistas da modalidade esportiva e, posteriormente, o checklist foi utilizado para analisar 10 arremessos (vídeo editado) de quatro indivíduos em diferentes fases de aprendizagem da habilidade. Os resultados foram organizados em função do erro crítico e somatória de erros verificados pelos especialistas em duas ocasiões com uma semana de intervalo. Contrastando as avaliações do mesmo avaliador e entre avaliadores, em ambas as medidas, observou-se um nível de erro médio de 16,9%. Concluiu-se, portanto que o checklist proposto no presente estudo é um instrumento de avaliação do lance livre consistente e, nesse sentido, pode auxiliar professores e técnicos na análise qualitativa do arremesso. Além disso, esse instrumento de avaliação permite fazer inferências sobre o processo de aprendizagem de indivíduos nessa habilidade.<br>When a physical activity professional is teaching a motor skill, he evaluates the movement's learner and considers which interventions could be done at the moment. However, many times the instructor does not have such resources which could help him/her to evaluate the learner movement. The skill acquisition process could be facilitated if instructors could have an instrument that identifies errors, prioritizing information to be given to the learner. Considering that the specialized literature presents a lack of information about such tool, the purpose of this study was to develop, and to determine the objectivity and reliability of an instrument to assess the movement quality of the basketball free throw shooting. The checklist was developed and evaluated by basketball experts. Additionally, the checklist was used to assess 10 trials (edited video) from four individuals in different learning stages. Data were organized by the critical error and the error sum appointed by the experts in two different occasions (one week interval). Contrasting both evaluations, and also, contrasting different experts assessments, in sum and critical error, it was observed an average error of 16.9%. It was concluded that the checklist to assess the basketball free throw is reliable, and could help instructors to make a qualitative analysis. Moreover, the checklist may allow instructors to make assumptions on the motor learning process

Gadolinium-induced fibrosis is counter-regulated by CCN3 in human dermal fibroblasts: a model for potential treatment of nephrogenic systemic fibrosis

We recently show that CCN3 is a counter-regulatory molecule for the pro-fibrotic protein CCN2, and a potentially novel fibrosis therapy. The goal of this study was to assess the role of CCN3 in fibroproliferative/fibrotic responses in human dermal fibroblasts exposed to Omniscan, one of the gadolinium-based contrast agents associated with development of nephrogenic systemic fibrosis (NSF) a rare but life-threatening disease thought to be complication of NMR diagnostics in renal impaired patients. Human dermal fibroblasts were exposed to Omniscan; or to platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) as controls. Proliferation was assessed along with matrix metalloproteinase-1, tissue inhibitor of metalloproteinases-1 and type 1 procollagen in the absence and presence of CCN3. In parallel, CCN3 production was assessed in control and Omniscan-treated cells. The results showed that PDGF stimulated fibroblast proliferation, production of Timp-1 and MMP-1 whereas exogenous CCN3 inhibited, in a dose response manner, cell proliferation (approx. 50 % max.) and production of MMP-1 (approx 35 % max.) but had little effect on TIMP-1. TGF-β stimulated type 1 procollagen production but not proliferation, Timp-1 or MMP-1 compared to non-TGF-ß treated control cells, and CCN3 treatment blocked (approx. 80 % max.) this up-regulation. Interestingly, untreated, control fibroblasts produced high constitutive levels of CCN3 and concentrations of Omniscan that induced fibroproliferative/fibrogenic changes in dermal fibroblasts correspondingly suppressed CCN3 production. The use of PDGF and TGF-β as positive controls, and the study of differential responses, including that to Omniscan itself, provide the first evidence for a role of fibroblast-derived CCN3 as an endogenous regulator of pro-fibrotic changes, elucidating possible mechanism(s). In conclusion, these data support our hypothesis of a role for fibroblast-derived CCN3 as an endogenous regulator of pro-fibrotic changes in these cells, and suggest that CCN3 may be an important regulatory molecule in NSF and a target for treatment in this and other fibrotic diseases

Epigenetics of Autoimmune Diseases

This chapter provides several examples of epigenetic deregulation in autoimmune diseases, a heterogeneous group of human conditions characterized by a deregulated immune response against the body own organs and tissues. Early studies based on the candidate gene approach have been flanked by genomewide screenings in the last few years, revealing global changes in DNA methylation or histone tail modifications, as well as deregulated methylation and/or expression of hundreds of genes and microRNAs in cells from patients affected by those disorders. This chapter will focus on epigenetic deregulations observed in systemic lupus erythematosus, rheumatoid arthritis, Sjögren’s syndrome, psoriasis, multiple sclerosis, systemic sclerosis, and autoimmune thyroid diseases, even though epigenetic modifications are increasingly being observed in many other autoimmune diseases. By contrast, only a few environmental factors have been shown or suspected to induce the observed epigenetic changes. Epigenetic drugs and RNA silencing experiments have often reversed autoimmune disease-like phenotypes in rodents or cell cultures, leading researchers to debate on their potential use in the treatment of these human conditions