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[PMID]: 28453955 [Au] Autor: Xue X; Hong X; Li Z; Deng CX; Fu J [Ad] Endereço: Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA. [Ti] Título: Acoustic tweezing cytometry enhances osteogenesis of human mesenchymal stem cells through cytoskeletal contractility and YAP activation. [So] Source: Biomaterials;134:22-30, 2017 Jul. [Is] ISSN: 1878-5905 [Cp] País de publicação: Netherlands [La] Idioma: eng [Ab] Resumo: Human mesenchymal stem cells (hMSCs) have great potential for cell-based therapies for treating degenerative bone diseases. It is known that mechanical cues in the cell microenvironment play an important role in regulating osteogenic (bone) differentiation of hMSCs. However, mechanoregulation of lineage commitment of hMSCs in conventional two-dimensional (2D) monocultures or bioengineered three-dimensional (3D) tissue constructs remains suboptimal due to complex biomaterial design criteria for hMSC culture. In this study, we demonstrate the feasibility of a novel cell mechanics and mechanobiology tool, acoustic tweezing cytometry (ATC), for mechanical stimulation of hMSCs. ATC utilizes ultrasound (US) pulses to actuate functionalized lipid microbubbles (MBs) which are covalently attached to hMSCs via integrin binding to exert forces to the cells. ATC stimulation increases cytoskeletal contractility of hMSCs regardless of the cell area. Furthermore, ATC application rescues osteogenic differentiation of hMSCs in culture conditions that are intrinsically repressive for hMSC osteogenesis (e.g., soft cell culture surfaces). ATC application activates transcriptional regulator YAP to enhance hMSC osteogenesis. Our data further show that F-actin, myosin II, and RhoA/ROCK signaling functions upstream of YAP activity in mediating ATC-stimulated hMSC osteogenesis. With the capability of applying controlled dynamic mechanical stimuli to cells, ATC provides a powerful tool for mechanoregulation of stem cell behaviors in tissue engineering and regenerative medicine applications. [Mh] Termos MeSH primário: Técnicas Citológicas/métodos Citoesqueleto/metabolismo Células Mesenquimais Estromais/citologia Osteogênese/fisiologia [Mh] Termos MeSH secundário: Western Blotting Diferenciação Celular/fisiologia Células Cultivadas Seres Humanos Células Mesenquimais Estromais/fisiologia Microbolhas Osteogênese/genética Reação em Cadeia da Polimerase em Tempo Real Transdução de Sinais/fisiologia [Pt] Tipo de publicação: JOURNAL ARTICLE [Em] Mês de entrada: 1802 [Cu] Atualização por classe: 180228 [Lr] Data última revisão: 180228 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 170429 [St] Status: MEDLINE

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[PMID]: 28448485 [Au] Autor: Radley AH; Schwab RM; Tan Y; Kim J; Lo EKW; Cahan P [Ad] Endereço: Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. [Ti] Título: Assessment of engineered cells using CellNet and RNA-seq. [So] Source: Nat Protoc;12(5):1089-1102, 2017 May. [Is] ISSN: 1750-2799 [Cp] País de publicação: England [La] Idioma: eng [Ab] Resumo: CellNet is a computational platform designed to assess cell populations engineered by either directed differentiation of pluripotent stem cells (PSCs) or direct conversion, and to suggest specific hypotheses to improve cell fate engineering protocols. CellNet takes as input gene expression data and compares them with large data sets of normal expression profiles compiled from public sources, in regard to the extent to which cell- and tissue-specific gene regulatory networks are established. CellNet was originally designed to work with human or mouse microarray expression data for 21 cell or tissue (C/T) types. Here we describe how to apply CellNet to RNA-seq data and how to build a completely new CellNet platform applicable to, for example, other species or additional cell and tissue types. Once the raw data have been preprocessed, running CellNet takes only several minutes, whereas the time required to create a completely new CellNet is several hours. [Mh] Termos MeSH primário: Diferenciação Celular Biologia Computacional/métodos Técnicas Citológicas/métodos Perfilação da Expressão Gênica Técnicas de Genotipagem/métodos Células-Tronco Pluripotentes/fisiologia Análise de Sequência de RNA [Mh] Termos MeSH secundário: Animais Camundongos [Pt] Tipo de publicação: JOURNAL ARTICLE [Em] Mês de entrada: 1706 [Cu] Atualização por classe: 180114 [Lr] Data última revisão: 180114 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 170428 [St] Status: MEDLINE [do] DOI: 10.1038/nprot.2017.022

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[PMID]: 28465336 [Au] Autor: Farrell DL; Weitz O; Magnasco MO; Zallen JA [Ad] Endereço: Howard Hughes Medical Institute and Developmental Biology Program, Sloan Kettering Institute, New York, NY 10065, USA. [Ti] Título: SEGGA: a toolset for rapid automated analysis of epithelial cell polarity and dynamics. [So] Source: Development;144(9):1725-1734, 2017 05 01. [Is] ISSN: 1477-9129 [Cp] País de publicação: England [La] Idioma: eng [Ab] Resumo: Epithelial remodeling determines the structure of many organs in the body through changes in cell shape, polarity and behavior and is a major area of study in developmental biology. Accurate and high-throughput methods are necessary to systematically analyze epithelial organization and dynamics at single-cell resolution. We developed SEGGA, an easy-to-use software for automated image segmentation, cell tracking and quantitative analysis of cell shape, polarity and behavior in epithelial tissues. SEGGA is free, open source, and provides a full suite of tools that allow users with no prior computational expertise to independently perform all steps of automated image segmentation, semi-automated user-guided error correction, and data analysis. Here we use SEGGA to analyze changes in cell shape, cell interactions and planar polarity during convergent extension in the embryo. These studies demonstrate that planar polarity is rapidly established in a spatiotemporally regulated pattern that is dynamically remodeled in response to changes in cell orientation. These findings reveal an unexpected plasticity that maintains coordinated planar polarity in actively moving populations through the continual realignment of cell polarity with the tissue axes. [Mh] Termos MeSH primário: Polaridade Celular Técnicas Citológicas/métodos Células Epiteliais/citologia Software [Mh] Termos MeSH secundário: Animais Automação Forma Celular Rastreamento de Células Drosophila melanogaster/citologia Drosophila melanogaster/embriologia Embrião não Mamífero/citologia Células Epiteliais/metabolismo Genótipo Processamento de Imagem Assistida por Computador [Pt] Tipo de publicação: JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL; RESEARCH SUPPORT, NON-U.S. GOV'T [Em] Mês de entrada: 1709 [Cu] Atualização por classe: 171126 [Lr] Data última revisão: 171126 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 170504 [St] Status: MEDLINE [do] DOI: 10.1242/dev.146837

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[PMID]: 28882542 [Au] Autor: Khamo JS; Krishnamurthy VV; Sharum SR; Mondal P; Zhang K [Ad] Endereço: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA. [Ti] Título: Applications of Optobiology in Intact Cells and Multicellular Organisms. [So] Source: J Mol Biol;429(20):2999-3017, 2017 Oct 13. [Is] ISSN: 1089-8638 [Cp] País de publicação: England [La] Idioma: eng [Ab] Resumo: Temporal kinetics and spatial coordination of signal transduction in cells are vital for cell fate determination. Tools that allow for precise modulation of spatiotemporal regulation of intracellular signaling in intact cells and multicellular organisms remain limited. The emerging optobiological approaches use light to control protein-protein interaction in live cells and multicellular organisms. Optobiology empowers light-mediated control of diverse cellular and organismal functions such as neuronal activity, intracellular signaling, gene expression, cell proliferation, differentiation, migration, and apoptosis. In this review, we highlight recent developments in optobiology, focusing on new features of second-generation optobiological tools. We cover applications of optobiological approaches in the study of cellular and organismal functions, discuss current challenges, and present our outlook. Taking advantage of the high spatial and temporal resolution of light control, optobiology promises to provide new insights into the coordination of signaling circuits in intact cells and multicellular organisms. [Mh] Termos MeSH primário: Técnicas Citológicas/métodos Regulação da Expressão Gênica Luz Transdução de Sinais [Mh] Termos MeSH secundário: Análise Espaço-Temporal [Pt] Tipo de publicação: JOURNAL ARTICLE; REVIEW [Em] Mês de entrada: 1710 [Cu] Atualização por classe: 171013 [Lr] Data última revisão: 171013 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 170909 [St] Status: MEDLINE

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[PMID]: 28796234 [Au] Autor: Martell JD; Deerinck TJ; Lam SS; Ellisman MH; Ting AY [Ad] Endereço: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [Ti] Título: Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells. [So] Source: Nat Protoc;12(9):1792-1816, 2017 Sep. [Is] ISSN: 1750-2799 [Cp] País de publicação: England [La] Idioma: eng [Ab] Resumo: Electron microscopy (EM) is the premiere technique for high-resolution imaging of cellular ultrastructure. Unambiguous identification of specific proteins or cellular compartments in electron micrographs, however, remains challenging because of difficulties in delivering electron-dense contrast agents to specific subcellular targets within intact cells. We recently reported enhanced ascorbate peroxidase 2 (APEX2) as a broadly applicable genetic tag that generates EM contrast on a specific protein or subcellular compartment of interest. This protocol provides guidelines for designing and validating APEX2 fusion constructs, along with detailed instructions for cell culture, transfection, fixation, heavy-metal staining, embedding in resin, and EM imaging. Although this protocol focuses on EM in cultured mammalian cells, APEX2 is applicable to many cell types and contexts, including intact tissues and organisms, and is useful for numerous applications beyond EM, including live-cell proteomic mapping. This protocol, which describes procedures for sample preparation from cell monolayers and cell pellets, can be completed in 10 d, including time for APEX2 fusion construct validation, cell growth, and solidification of embedding resins. Notably, the only additional steps required relative to a standard EM sample preparation are cell transfection and a 2- to 45-min staining period with 3,3-diaminobenzidine (DAB) and hydrogen peroxide (H O ). [Mh] Termos MeSH primário: Ascorbato Peroxidases/genética Técnicas Citológicas/métodos Técnicas Genéticas Microscopia Eletrônica/métodos Imagem Molecular/métodos [Mh] Termos MeSH secundário: Animais Células COS Células Cultivadas Estruturas Celulares/ultraestrutura Cercopithecus aethiops Células HEK293 Hipocampo/citologia Seres Humanos Ratos Proteínas Recombinantes de Fusão/genética [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (Recombinant Fusion Proteins); EC 1.11.1.11 (Ascorbate Peroxidases) [Em] Mês de entrada: 1708 [Cu] Atualização por classe: 170821 [Lr] Data última revisão: 170821 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 170811 [St] Status: MEDLINE [do] DOI: 10.1038/nprot.2017.065

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[PMID]: 28739858 [Au] Autor: Bellevicine C; Malapelle U; Vigliar E; Pisapia P; Vita G; Troncone G [Ad] Endereço: Department of Public Health, Anatomic Pathology Unit, University of Napoli Federico II School of Medicine, Napoli, Italy. [Ti] Título: How to prepare cytological samples for molecular testing. [So] Source: J Clin Pathol;70(10):819-826, 2017 Oct. [Is] ISSN: 1472-4146 [Cp] País de publicação: England [La] Idioma: eng [Ab] Resumo: This review is focused on the challenges in standardising and optimising molecular testing workflow in cytopathology. Although cytological samples yield optimal quality DNA, whose minimal amounts in most cases suffice even for multigene mutational profiling, the success of molecular testing is strongly dependent on standardised preanalytical protocols for maximising DNA yield and quality. Sample cytopreparation influences, even more, the quality of RNA and consequently the potential success of reverse transcription-PCR. Here, the educational and technical involvement of the cytopathologist as a relevant component of a multidisciplinary team, in the issues related to test request, specimen collection, fixation, processing, staining, tumour fraction enrichment, DNA quality/quantity assessment and storage conditions is discussed. In addition, the specific sample requirements related to more recent technological developments are examined, underlining the modern role of the cytopathologist, whose continuous education is crucial to meet the opportunities of molecular medicine. [Mh] Termos MeSH primário: Técnicas Citológicas Análise Mutacional de DNA/métodos Sequenciamento de Nucleotídeos em Larga Escala/métodos [Mh] Termos MeSH secundário: Seres Humanos [Pt] Tipo de publicação: JOURNAL ARTICLE; REVIEW [Em] Mês de entrada: 1710 [Cu] Atualização por classe: 171002 [Lr] Data última revisão: 171002 [Sb] Subgrupo de revista: AIM; IM [Da] Data de entrada para processamento: 170726 [St] Status: MEDLINE [do] DOI: 10.1136/jclinpath-2017-204561

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[PMID]: 28726849 [Au] Autor: Lewis DM; Blatchley MR; Park KM; Gerecht S [Ad] Endereço: Department of Chemical and Biomolecular Engineering, Johns Hopkins Physical Sciences-Oncology Center and Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, Maryland, USA. [Ti] Título: O -controllable hydrogels for studying cellular responses to hypoxic gradients in three dimensions in vitro and in vivo. [So] Source: Nat Protoc;12(8):1620-1638, 2017 Aug. [Is] ISSN: 1750-2799 [Cp] País de publicação: England [La] Idioma: eng [Ab] Resumo: Oxygen (O ) acts as a potent upstream regulator of cell function. In both physiological and pathophysiological microenvironments, the O concentration is not uniformly distributed but instead follows a gradient that depends on distance from oxygen-carrying blood vessels. Such gradients have a particularly important role in development, tissue regeneration, and tumor growth. In this protocol, we describe how to use our previously reported gelatin-based O -controllable hydrogels that can provide hypoxic microenvironments in vitro. The hydrogel polymeric network is formed via a laccase-mediated cross-linking reaction. In this reaction, laccase catalyzes diferulic acid (diFA) formation to form hydrogels with an O -consuming reaction. Cells, such as cancer or endothelial cells, as well as tumor/tissue grafts, can be encapsulated in the hydrogels during hydrogel formation and then analyzed for cellular responses to 3D hypoxic gradients and to elucidate the underlying mechanisms governing these responses. Importantly, oxygen gradients can be precisely controlled in standard cell/tissue culture conditions and in vivo. This platform has been applied to study vascular morphogenesis in response to hypoxia and to understand how oxygen gradients mediate cancer cell behavior. Herein, we describe the means to validate the assay from polymer synthesis and characterization-which take 1-2 weeks and include verification of ferulic acid (FA) conjugation, rheological measurements, and O monitoring-to the study of cellular responses and use in rodent models. Time courses for biological experiments using this hydrogel are variable, and thus they may range from hours to weeks, depending on the application and user end goal. [Mh] Termos MeSH primário: Técnicas de Cultura de Células/métodos Técnicas Citológicas/métodos Hidrogéis Hipóxia/metabolismo Oxigênio/metabolismo Estresse Fisiológico [Mh] Termos MeSH secundário: Animais Células Cultivadas Ácidos Cumáricos/metabolismo Gelatina Lacase/metabolismo Camundongos Endogâmicos C57BL [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (Coumaric Acids); 0 (Hydrogels); 0 (diferulic acid); 9000-70-8 (Gelatin); EC 1.10.3.2 (Laccase); S88TT14065 (Oxygen) [Em] Mês de entrada: 1707 [Cu] Atualização por classe: 170731 [Lr] Data última revisão: 170731 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 170721 [St] Status: MEDLINE [do] DOI: 10.1038/nprot.2017.059

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[PMID]: 28686585 [Au] Autor: Lowndes M; Junyent S; Habib SJ [Ad] Endereço: Centre for Stem Cells and Regenerative Medicine, King's College London, London, UK. [Ti] Título: Constructing cellular niche properties by localized presentation of Wnt proteins on synthetic surfaces. [So] Source: Nat Protoc;12(7):1498-1512, 2017 Jul. [Is] ISSN: 1750-2799 [Cp] País de publicação: England [La] Idioma: eng [Ab] Resumo: Wnt signaling is crucial during embryonic development and for the maintenance of adult tissues. Depending on the tissue type, the Wnt pathway can promote stem cell self-renewal and/or direct lineage commitment. Wnt proteins are subject to lipid modification, often restricting them to act in a localized manner on responsive cells. Most methods for inducing Wnt signaling in stem cell cultures do not control the spatial presentation of the protein. To recreate the local presentation of Wnt proteins often seen in vivo, we previously developed a method to immobilize the protein onto synthetic surfaces. Here we describe a detailed protocol based on covalent binding of nucleophilic groups on Wnt proteins to activated carboxylic acid (COOH) or glutaraldehyde (COH) groups functionalized on synthetic surfaces. As an example, we describe how this method can be used to covalently immobilize Wnt3a proteins on microbeads or a glass surface. This procedure requires ∼3 h and allows for the hydrophobic protein to be stored in the absence of detergent. The immobilization efficiency of active Wnt proteins can be assessed using different T-cell factor (TCF) reporter assays as a readout for Wnt/ß-catenin-dependent transcription. Immobilization efficiency can be measured 12-18 h after seeding the cells and takes 2-4 h. The covalent immobilization of Wnt proteins can also be used for single-cell analysis using Wnt-coated microbeads (12-18 h of live imaging) and to create a Wnt platform on a glass surface for stem cell maintenance and cell population analysis (3 d). The simple chemistry used for Wnt immobilization allows for adaptation to new materials and other developmental signals. Therefore, this method can also be incorporated into tissue engineering platforms in which depletion of the stem cell pool restricts the complexity and maturity of the tissue developed. [Mh] Termos MeSH primário: Técnicas Citológicas/métodos Proteínas Imobilizadas/metabolismo Proteínas Wnt/metabolismo [Mh] Termos MeSH secundário: Vidro Microesferas Ligação Proteica Análise de Célula Única/métodos Fatores de Tempo [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (Immobilized Proteins); 0 (Wnt Proteins) [Em] Mês de entrada: 1707 [Cu] Atualização por classe: 170726 [Lr] Data última revisão: 170726 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 170708 [St] Status: MEDLINE [do] DOI: 10.1038/nprot.2017.061

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[PMID]: 28686583 [Au] Autor: Zhang Y; Wei F; Poh YC; Jia Q; Chen J; Chen J; Luo J; Yao W; Zhou W; Huang W; Yang F; Zhang Y; Wang N [Ad] Endereço: Laboratory for Cellular Biomechanics and Regenerative Medicine, School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China. [Ti] Título: Interfacing 3D magnetic twisting cytometry with confocal fluorescence microscopy to image force responses in living cells. [So] Source: Nat Protoc;12(7):1437-1450, 2017 Jul. [Is] ISSN: 1750-2799 [Cp] País de publicação: England [La] Idioma: eng [Ab] Resumo: Cells and tissues can undergo a variety of biological and structural changes in response to mechanical forces. Only a few existing techniques are available for quantification of structural changes at high resolution in response to forces applied along different directions. 3D-magnetic twisting cytometry (3D-MTC) is a technique for applying local mechanical stresses to living cells. Here we describe a protocol for interfacing 3D-MTC with confocal fluorescence microscopy. In 3D-MTC, ferromagnetic beads are bound to the cell surface via surface receptors, followed by their magnetization in any desired direction. A magnetic twisting field in a different direction is then applied to generate rotational shear stresses in any desired direction. This protocol describes how to combine magnetic-field-induced mechanical stimulation with confocal fluorescence microscopy and provides an optional extension for super-resolution imaging using stimulated emission depletion (STED) nanoscopy. This technology allows for rapid real-time acquisition of a living cell's mechanical responses to forces via specific receptors and for quantifying structural and biochemical changes in the same cell using confocal fluorescence microscopy or STED. The integrated 3D-MTC-microscopy platform takes ∼20 d to construct, and the experimental procedures require ∼4 d when carried out by a life sciences graduate student. [Mh] Termos MeSH primário: Fenômenos Fisiológicos Celulares Técnicas Citológicas/métodos Processamento de Imagem Assistida por Computador/métodos Microscopia Confocal/métodos Microscopia de Fluorescência/métodos Estresse Mecânico [Mh] Termos MeSH secundário: Animais Seres Humanos Magnetismo Microesferas [Pt] Tipo de publicação: JOURNAL ARTICLE [Em] Mês de entrada: 1707 [Cu] Atualização por classe: 170815 [Lr] Data última revisão: 170815 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 170708 [St] Status: MEDLINE [do] DOI: 10.1038/nprot.2017.042

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[PMID]: 28686582 [Au] Autor: Vinegoni C; Fumene Feruglio P; Brand C; Lee S; Nibbs AE; Stapleton S; Shah S; Gryczynski I; Reiner T; Mazitschek R; Weissleder R [Ad] Endereço: Center for System Biology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA. [Ti] Título: Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging. [So] Source: Nat Protoc;12(7):1472-1497, 2017 Jul. [Is] ISSN: 1750-2799 [Cp] País de publicação: England [La] Idioma: eng [Ab] Resumo: The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon fluorescence anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPE microscope, the protocol can be extended to other commercial or custom-built microscopes. [Mh] Termos MeSH primário: Técnicas Citológicas/métodos Polarização de Fluorescência/métodos Imagem Óptica/métodos Preparações Farmacêuticas/análise Farmacologia/métodos [Mh] Termos MeSH secundário: Processamento de Imagem Assistida por Computador/métodos [Pt] Tipo de publicação: JOURNAL ARTICLE [Nm] Nome de substância: 0 (Pharmaceutical Preparations) [Em] Mês de entrada: 1707 [Cu] Atualização por classe: 170726 [Lr] Data última revisão: 170726 [Sb] Subgrupo de revista: IM [Da] Data de entrada para processamento: 170708 [St] Status: MEDLINE [do] DOI: 10.1038/nprot.2017.043

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