Ribonomic analysis of human DZIP1 reveals its involvement in ribonucleoprotein complexes and stress granules

Submitted by Renata Fontoura ([email protected]) on 2014-11-26T12:39:22Z No. of bitstreams: 1 artigo 1.pdf: 1716335 bytes, checksum: 17480039c8f2b4b9a96d1131ed066e83 (MD5)Approved for entry into archive by Renata Fontoura ([email protected]) on 2014-11-26T13:11:26Z (GMT) No. of bitstreams: 1 artigo 1.pdf: 1716335 bytes, checksum: 17480039c8f2b4b9a96d1131ed066e83 (MD5)Made available in DSpace on 2014-11-26T13:11:26Z (GMT). No. of bitstreams: 1 artigo 1.pdf: 1716335 bytes, checksum: 17480039c8f2b4b9a96d1131ed066e83 (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Básica de Células Tronco. Curitiba, PR, Brasil.Instituto de Investigaciones Biológicas Clemente Estable. Genomics Department. Montevideo, Uruguay. / Universidad de la República Uruguay. Facultad de Ciencias. Departamento de Biología Celular y Molecular. Montevideo, Uruguay.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Básica de Células Tronco. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Básica de Células Tronco. Curitiba, PR, Brasil.Instituto Pasteur. Montevideo, Uruguay. / Universidad de la República Uruguay. Facultad de Ciencias. Departamento de Biología Celular y Molecular. Montevideo, Uruguay.Instituto Pasteur. Montevideo, Uruguay.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Básica de Células Tronco. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Básica de Células Tronco. Curitiba, PR, Brasil.National Laboratory for Cancer Research. Leidos Biomedical Research, Inc. Cancer Research Technology Program. Frederick, USA.Background: DZIP1 (DAZ-interacting protein 1) has been described as a component of the Hh signaling pathway with a putative regulatory role in ciliogenesis. DZIP1 interacts with DAZ RNA binding proteins in embryonic stem cells and human germ cells suggesting a role in mRNA regulation. Results: We investigated DZIP1 function in HeLa cells and its involvement in ribonucleoprotein complexes. DZIP1 was predominantly located in granules in the cytoplasm. Under oxidative stress conditions, DZIP1 re-localized to stress granules. DZIP appears to be important for the formation of stress granules during the stress response. We used immunoprecipitation assays with antibodies against DZIP1 and microarray hybridization to identify mRNAs associated with DZIP1. The genetic networks formed by the DZIP1-associated mRNAs were involved in cell cycle and gene expression regulation. DZIP1 is involved in the Hedgehog signaling pathway. We used cyclopamine, a specific inhibitor of this pathway, to analyze the expression of DZIP1 and its associated mRNAs. The abundance of DZIP1-associated mRNAs increased with treatment; however, the silencing or overexpression of DZIP1 in HeLa cells had no effect on the accumulation of the associated mRNAs. Polysomal profile analysis by sucrose gradient centrifugation demonstrated the presence of DZIP1 in the polysomal fraction. Conclusions: Our results suggest that DZIP1 is part of an RNP complex that occupies various subcellular locations. The diversity of the mRNAs associated with DZIP1 suggests that this protein is a component of different RNPs associated with translating polysomes and with RNA granules

Biological Synthesis of Low Cytotoxicity Silver Nanoparticles (AgNPs) by the Fungus <i>Chaetomium thermophilum</i>—Sustainable Nanotechnology

Fungal biotechnology research has rapidly increased as a result of the growing awareness of sustainable development and the pressing need to explore eco-friendly options. In the nanotechnology field, silver nanoparticles (AgNPs) are currently being studied for application in cancer therapy, tumour detection, drug delivery, and elsewhere. Therefore, synthesising nanoparticles (NPs) with low toxicity has become essential in the biomedical area. The fungus Chaetomium thermophilum (C. thermophilum) was here investigated—to the best of our knowledge, for the first time—for application in the production of AgNPs. Transmission electronic microscopy (TEM) images demonstrated a spherical AgNP shape, with an average size of 8.93 nm. Energy-dispersive X-ray spectrometry (EDX) confirmed the presence of elemental silver. A neutral red uptake (NRU) test evaluated the cytotoxicity of the AgNPs at different inhibitory concentrations (ICs). A half-maximal concentration (IC50 = 119.69 µg/mL) was used to predict a half-maximal lethal dose (LD50 = 624.31 mg/kg), indicating a Global Harmonized System of Classification and Labelling of Chemicals (GHS) acute toxicity estimate (ATE) classification category of 4. The fungus extract showed a non-toxic profile at the IC tested. Additionally, the interaction between the AgNPs and the Balb/c 3T3 NIH cells at an ultrastructural level resulted in preserved cells structures at non-toxic concentrations (IC20 = 91.77 µg/mL), demonstrating their potential as sustainable substitutes for physical and chemically made AgNPs. Nonetheless, at the IC50, the cytoplasm of the cells was damaged and mitochondrial morphological alteration was evident. This fact highlights the fact that dose-dependent phenomena are involved, as well as emphasising the importance of investigating NPs’ effects on mitochondria, as disruption to this organelle can impact health

Polysome profiling shows the identity of human adipose-derived stromal/stem cells in detail and clearly distinguishes them from dermal fibroblasts

O artigo encontra-se disponível em acesso aberto no site do Editor.Submitted by Michel Batista ([email protected]) on 2014-12-11T23:53:17Z No. of bitstreams: 1 zych2014 (1).pdf: 669092 bytes, checksum: c4215d46bb3118c318f1160caf288d86 (MD5)Approved for entry into archive by Michel Batista ([email protected]) on 2014-12-12T00:08:23Z (GMT) No. of bitstreams: 1 zych2014 (1).pdf: 669092 bytes, checksum: c4215d46bb3118c318f1160caf288d86 (MD5)Made available in DSpace on 2014-12-12T00:08:23Z (GMT). No. of bitstreams: 1 zych2014 (1).pdf: 669092 bytes, checksum: c4215d46bb3118c318f1160caf288d86 (MD5) Previous issue date: 2014This work was supported by grants from Ministério da Saúde and Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq, FIOCRUZ-Pasteur Research Program, and Fundação Araucária. L.S. received fellowship from ANII (Agencia Nacional de Investigación e Innovación, Uruguay); S.G., J.Z., and B.D. from CNPq; P.S. from FIOCRUZ; and A.C. from Fundação Araucária.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Instituto Pasteur. Unidad de Bioinformática. Montevideo, Uruguay.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Pontifícia Universidade Católica do Paraná. Núcleo de Tecnologia Celular. Curitiba, PR, Brasil.Pontifícia Universidade Católica do Paraná. Núcleo de Tecnologia Celular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Instituto Pasteur. Unidad de Bioinformática. Montevideo, Uruguay.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Although fibroblasts and multipotent stromal/stem cells, including adipose-derived stromal cells (ADSCs), have been extensively studied, they cannot be clearly distinguished from each other. We, therefore, investigated the cellular and molecular characteristics of ADSCs and fibroblasts. ADSCs and fibroblasts share several morphological similarities and surface markers, but were clearly found to be different types of cells. Contrary to previous reports, fibroblasts were not able to differentiate into adipocytes, osteoblasts, or chondrocytes. Polysome-bound mRNA profiling revealed that*1,547 genes were differentially expressed (DE) in the two cell types; the genes were related to cell adhesion, the extracellular matrix, differentiation, and proliferation. These findings were confirmed by functional analyses showing that ADSCs had a greater adhesion capacity than fibroblasts; the proliferation rate of fibroblasts was also higher than that of ADSCs. Importantly, 185 DE genes were integral to the plasma membrane and, thus, candidate markers for ADSC isolation and manipulation. We also observed that an established marker of fibroblasts and ADSCs, CD105, was overexpressed in ADSCs at both mRNA and protein levels. CD105 expression seemed to be related to differentiation capacity, at least for adipogenesis. This study shows that ADSCs and fibroblasts are distinct cell types. These findings should be taken into account when using these two cell types in basic and therapeutic studies

Polysome profiling shows extensive posttranscriptional regulation during human adipocyte stem cell differentiation into adipocytes

Submitted by Manoel Barata ([email protected]) on 2017-08-18T12:45:14Z No. of bitstreams: 1 Spangenbergpoly.pdf: 1003715 bytes, checksum: 4e042c381ca6191b3ccac780cc2def23 (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2017-08-18T14:28:45Z (GMT) No. of bitstreams: 1 Spangenbergpoly.pdf: 1003715 bytes, checksum: 4e042c381ca6191b3ccac780cc2def23 (MD5)Made available in DSpace on 2017-08-18T14:28:45Z (GMT). No. of bitstreams: 1 Spangenbergpoly.pdf: 1003715 bytes, checksum: 4e042c381ca6191b3ccac780cc2def23 (MD5) Previous issue date: 2013Institut Pasteur Montevideo. Unidad de Bioinformática. Montevideo, Uruguay.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Pontifícia Universidade Católica do Paraná. Núcleo de Tecnologia Celular. Curitiba, PR, Brasil.Pontifícia Universidade Católica do Paraná. Núcleo de Tecnologia Celular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Institut Pasteur Montevideo. Unidad de Bioinformática. Montevideo, Uruguay.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Adipocyte stem cells (hASCs) can proliferate and self-renew and, due to their multipotent nature, they can differentiate into several tissue-specific lineages, making them ideal candidates for use in cell therapy. Most attempts to determine the mRNA profile of self-renewing or differentiating stem cells have made use of total RNA for gene expression analysis. Several lines of evidence suggest that self-renewal and differentiation are also dependent on the control of protein synthesis by posttranscriptional mechanisms. We used adipogenic differentiation as a model, to investigate the extent to which posttranscriptional regulation controlled gene expression in hASCs. We focused on the initial steps of differentiation and isolated both the total mRNA fraction and the subpopulation of mRNAs associated with translating ribosomes. We observed that adipogenesis is committed in the first days of induction and three days appears as the minimum time of induction necessary for efficient differentiation. RNA-seq analysis showed that a significant percentage of regulated mRNAs were posttranscriptionally controlled. Part of this regulation involves massive changes in transcript untranslated regions (UTR) length, with differential extension/reduction of the 3'UTR after induction. A slight correlation can be observed between the expression levels of differentially expressed genes and the 3'UTR length. When we considered association to polysomes, this correlation values increased. Changes in the half lives were related to the extension of the 3'UTR, with longer UTRs mainly stabilizing the transcripts. Thus, changes in the length of these extensions may be associated with changes in the ability to associate with polysomes or in half-life