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1.
Int J Mol Sci ; 20(19)2019 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-31561474

RESUMO

Small extracellular vesicles (sEVs) mediate cell-to-cell communication. We recently reported that circulating sEVs regulate systolic blood pressure in an animal model of human systemic hypertension. However, the underlying mechanisms still remain to be elucidated. As the first step for detailed analyses, we sought to increase the yield and purity of sEVs isolated from rat plasma. We compared the concentration and size distribution of sEVs as well as protein expression of the sEV marker and contaminants among plasma sEVs isolated by the ultracentrifugation (UC) method, the precipitation with polyethylene-glycol and ultracentrifugation (PEG-UC) method, or the precipitation with polyethylene-glycol (PEG) method. Effects of anticoagulants were also examined. The total concentration of plasma sEVs isolated by the PEG or PEG-UC method was much higher than that of the UC method. In the plasma sEVs isolated by the PEG-UC method, contaminating proteins were lower, while the protein expression of certain sEV markers was higher than that of the PEG method. There was no significant difference in total concentration or protein expression of sEV markers in sEVs isolated from rat plasma treated with three different anticoagulants (heparin, ethylenediaminetetraacetic acid, or acid citrate dextrose buffer) by the PEG-UC method. We, for the first time, determined that the PEG-UC method was optimal for sEV isolation from rat plasma.


Assuntos
Vesículas Extracelulares/metabolismo , Frações Subcelulares , Animais , Biomarcadores , Fracionamento Celular , Fracionamento Químico/métodos , Humanos , Masculino , Tamanho da Partícula , Plasma , Ratos
2.
Int J Mol Sci ; 20(19)2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31546622

RESUMO

A growing body of evidence emphasizes the important role exosomes in different physiological and pathological conditions. Exosomes, virus-size extracellular vesicles (EVs), carry a complex molecular cargo, which is actively processed in the endocytic compartment of parental cells. Exosomes carry and deliver this cargo to recipient cells, serving as an intercellular communication system. The methods for recovery of exosomes from supernatants of cell lines or body fluids are not uniformly established. Yet, studies of the quality and quantity of exosome cargos underlie the concept of "liquid biopsy." Exosomes are emerging as a potentially useful diagnostic tool and a predictor of disease progression, response to therapy and overall survival. Although many novel approaches to exosome isolation and analysis of their cargos have been introduced, the role of exosomes as diagnostic or prognostic biomarkers of disease remains unconfirmed. This review considers existing challenges to exosome validation as disease biomarkers. Focusing on advantages and limitations of methods for exosome isolation and characterization, approaches are proposed to facilitate further progress in the development of exosomes as biomarkers in human disease.


Assuntos
Biomarcadores/metabolismo , Exossomos/metabolismo , Comunicação Celular , Fracionamento Celular , Sistemas de Liberação de Medicamentos , Exossomos/genética , Humanos , Biópsia Líquida , Neoplasias/tratamento farmacológico
3.
Future Med Chem ; 11(10): 1225-1236, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31280675

RESUMO

Exosomes are secreted by mammalian cells and are widely distributed in cellular systems. They are a medium of information and material transmission. The complexity of exosome nature and function is not thoroughly understood. Nevertheless, they are being confirmed as mediators of intercellular communication and play significant roles in many physiological and pathological processes. Significant obstacles to the efficient and robust isolation of large quantities of pure and specific exosomes still exist. These include a lack of understanding of the relationship between exosome characteristics and function, and a shortage of scalable solutions to separate specific exosomes from other large entities remain. Hence, generic production platforms are desired. While solutions suitable for exosome manufacturing under GMP are available, most have been developed for other purposes.


Assuntos
Técnicas de Cultura de Células/métodos , Exossomos/metabolismo , Animais , Reatores Biológicos , Comunicação Celular , Técnicas de Cultura de Células/instrumentação , Fracionamento Celular/instrumentação , Fracionamento Celular/métodos , Desenho de Equipamento , Exossomos/química , Exossomos/patologia , Humanos
4.
PLoS One ; 14(7): e0220102, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31335892

RESUMO

The ability for safe and rapid pathogenic sample transportation and subsequent detection is an increasing challenge throughout the world. Herein, we describe and use bead-beating, vortex, sonication, 903 protein saver cards, and Lyse-It methods, aiming to inactivate Gram-positive and -negative bacteria with subsequent genome DNA (quantitative Polymerase Chain Reaction) qPCR detection. The basic concepts behind the four chosen technologies is their versatility, cost, and ease of use in developed and underdeveloped countries. The four methods target the testing of bacterial resilience, cellular extraction from general and complex media and subsequent DNA extraction for qPCR detection and amplification. These results demonstrate that conventional high temperature heating, 903 protein saver cards, and Lyse-It are all viable options for inactivating bacterial growth for safe shipping. Additionally, Lyse-It was found to be particularly useful as this technology can inactivate bacteria, extract cells from 903 protein saver cards, lyse bacterial cells, and additionally keep genomic DNA viable for qPCR detection.


Assuntos
Fracionamento Celular/métodos , DNA Bacteriano/normas , Técnicas de Diagnóstico Molecular/métodos , Fracionamento Celular/economia , Fracionamento Celular/normas , DNA Bacteriano/química , Bactérias Gram-Negativas/química , Bactérias Gram-Positivas/química , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/normas , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Kit de Reagentes para Diagnóstico/normas
5.
Cells ; 8(7)2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31311206

RESUMO

The use of extracellular vesicles, specifically exosomes, as carriers of biomarkers in extracellular spaces has been well demonstrated. Despite their promising potential, the use of exosomes in the clinical setting is restricted due to the lack of standardization in exosome isolation and analysis methods. The purpose of this review is to not only introduce the different types of extracellular vesicles but also to summarize their differences and similarities, and discuss different methods of exosome isolation and analysis currently used. A thorough understanding of the isolation and analysis methods currently being used could lead to some standardization in the field of exosomal research, allowing the use of exosomes in the clinical setting to become a reality.


Assuntos
Fracionamento Celular/métodos , Exossomos/química , Animais , Exossomos/metabolismo , Exossomos/ultraestrutura , Humanos , Proteômica/métodos
6.
Int J Mol Sci ; 20(12)2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31248089

RESUMO

Identification of novel proteins with changed expression in resistant cancer cells could be helpful in elucidation mechanisms involved in the development of acquired resistance to paclitaxel. In this study, we carried out a 2D-PAGE using the mitochondrial-enriched fraction from paclitaxel-resistant MCF7/PacR cells compared to original paclitaxel-sensitive MCF7 breast cancer cells. Differentially expressed proteins were identified employing mass spectrometry. We found that lysosomal cathepsin D and mitochondrial abhydrolase-domain containing protein 11 (ABHD11) had decreased expression in MCF7/PacR cells. On the other hand, mitochondrial carbamoyl-phosphate synthetase 1 (CPS1) and ATPase family AAA-domain containing protein 3A and 3B (ATAD3A, ATAD3B) were overexpressed in MCF7/PacR cells. Further, we showed that there was no difference in localization of CPS1 in MCF7 and MCF7/PacR cells. We demonstrated a significant increase in the number of CPS1 positive MCF7/PacR cells, using FACS analysis, compared to the number of CPS1 positive MCF7 cells. Silencing of CPS1 expression by specific siRNA had no significant effect on the resistance of MCF7/PacR cells to paclitaxel. To summarize, we identified several novel proteins of a mitochondrial fraction whose role in acquired resistance to paclitaxel in breast cancer cells should be further assessed.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Paclitaxel/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carbamoil-Fosfato Sintase (Amônia)/genética , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Fracionamento Celular , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Células MCF-7 , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteoma , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem
7.
Methods Mol Biol ; 1982: 75-101, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172467

RESUMO

The NADPH oxidase NOX2 complex consists of assembled cytosolic and redox membrane proteins. In mammalian cells, natural arachidonic acid (cis-AA), released by activated phospholipase-A2, plays an important role in the activation of the NADPH oxidase, but the mechanism of action of cis-AA is still a matter of debate. In cell-free systems, cis-AA is commonly used for activation although its structural effects are still unclear. Undoubtedly cis-AA participates in the synergistic multi-partner assembly that can be hardly studied at the molecular level in vivo due to cellular complexity. The capacity of this anionic amphiphilic fatty acid to activate the oxidase is mainly explained by its ability to disrupt intramolecular bonds, mimicking phosphorylation events in cell signaling and therefore allowing protein-protein interactions. Interestingly the geometric isomerism of the fatty acid and its purity are crucial for optimal superoxide production in cell-free assays. Indeed, optimal NADPH oxidase assembly was hampered by the substitution of the cis form by the trans forms of AA isomers (Souabni et al., BBA-Biomembranes 1818:2314-2324, 2012). Structural analysis of the changes induced by these two compounds, by circular dichroism and by biochemical methods, revealed differences in the interaction between subunits. We describe how the specific geometry of AA plays an important role in the activation of the NOX2 complex.


Assuntos
Ácido Araquidônico/metabolismo , NADPH Oxidases/metabolismo , Fagócitos/enzimologia , Ácido Araquidônico/química , Fracionamento Celular , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Sistema Livre de Células , Colorimetria , Ativação Enzimática , Isomerismo , Estrutura Molecular , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/química , NADPH Oxidases/isolamento & purificação , Neutrófilos/enzimologia , Fagócitos/imunologia , Proteínas Recombinantes de Fusão , Análise Espectral
8.
Methods Mol Biol ; 1982: 103-111, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172468

RESUMO

NADPH oxidases (NOX) are a family of transmembrane enzymes, which catalyze the formation of O2 ˙- and H2O2. Membrane fractions of leukocytes are highly enriched in the phagocyte NOX isoform (NOX2). This feat has allowed the development of a complex NOX2 cell-free assay, which has been a key tool for the understanding of the mode of action of NOX2, its biochemistry, pharmacology, and identification of NOX2-specific inhibitors. In addition to NOX2, there are six other NOX isoforms in humans, but cell-free assays of non-phagocytic oxidases are infrequently used, and their specificity has recently been debated. Here we describe a NOX5 cell-free assay. We present a method to purify the membranous component of cells stably transduced with NOX5 and to measure O2 ˙- in a high-throughput format (96-w or 384-w plates). The experimental description allows high-throughput screening of small molecules with limited cost.


Assuntos
Sistema Livre de Células , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , NADPH Oxidase 5/antagonistas & inibidores , Fracionamento Celular , Membrana Celular/enzimologia , Sistema Livre de Células/enzimologia , Descoberta de Drogas , Inibidores Enzimáticos/química , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , NADPH Oxidase 5/química , NADPH Oxidase 5/genética , NADPH Oxidase 5/metabolismo , Oxirredução , Espécies Reativas de Oxigênio , Bibliotecas de Moléculas Pequenas , Análise Espectral
9.
Methods Mol Biol ; 1982: 461-472, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172489

RESUMO

Reactive oxygen species (ROS) convey signals essential for proliferation, maintenance, and senescence of a growing list of cell types. Compartmentalization of these signals is integral to cell viability as well as the signaling pathways ROS direct. Redox-active endosomes (redoxosomes) are formed downstream of several ligand-activated receptors. NADPH oxidase (NOX) is a main component of redoxosomes, which recruits multiple proteins (Rac1, NOX2, p67phox, SOD1). Isolation of redoxosomes and evaluation of how superoxide (O2˙-) production directs receptor signaling at the level of the endosome have enabled a better understanding of biologic processes controlled by ROS. In this chapter, we will first review the major signaling pathways that utilize redoxosomes and components that control its redox-dependent functions. We will then outline biochemical and biophysical methods for the isolation and characterization of redoxosome properties.


Assuntos
Fracionamento Celular , Endossomos/metabolismo , NADPH Oxidases/metabolismo , Oxirredução , Fracionamento Celular/métodos , Linhagem Celular , Centrifugação com Gradiente de Concentração , Cromatografia de Afinidade , Ativação Enzimática , Humanos , Espécies Reativas de Oxigênio/metabolismo
10.
BMC Bioinformatics ; 20(1): 360, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253078

RESUMO

BACKGROUND: Because of its non-destructive nature, label-free imaging is an important strategy for studying biological processes. However, routine microscopic techniques like phase contrast or DIC suffer from shadow-cast artifacts making automatic segmentation challenging. The aim of this study was to compare the segmentation efficacy of published steps of segmentation work-flow (image reconstruction, foreground segmentation, cell detection (seed-point extraction) and cell (instance) segmentation) on a dataset of the same cells from multiple contrast microscopic modalities. RESULTS: We built a collection of routines aimed at image segmentation of viable adherent cells grown on the culture dish acquired by phase contrast, differential interference contrast, Hoffman modulation contrast and quantitative phase imaging, and we performed a comprehensive comparison of available segmentation methods applicable for label-free data. We demonstrated that it is crucial to perform the image reconstruction step, enabling the use of segmentation methods originally not applicable on label-free images. Further we compared foreground segmentation methods (thresholding, feature-extraction, level-set, graph-cut, learning-based), seed-point extraction methods (Laplacian of Gaussians, radial symmetry and distance transform, iterative radial voting, maximally stable extremal region and learning-based) and single cell segmentation methods. We validated suitable set of methods for each microscopy modality and published them online. CONCLUSIONS: We demonstrate that image reconstruction step allows the use of segmentation methods not originally intended for label-free imaging. In addition to the comprehensive comparison of methods, raw and reconstructed annotated data and Matlab codes are provided.


Assuntos
Fracionamento Celular/métodos , Microscopia/métodos , Algoritmos , Humanos , Processamento de Imagem Assistida por Computador
12.
Methods Mol Biol ; 1965: 351-374, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069686

RESUMO

Known for its tumor suppressor activity in breast and ovarian cancers, the breast cancer 1 susceptibility gene (Brca1) is involved in a variety of cellular pathways including DNA repair, antioxidant signaling, apoptosis, and cell cycle regulation. BRCA1 can translocate between the cytoplasm and nucleus to perform its various roles. Herein is a procedure for measuring BRCA1 protein levels in the whole cell lysate (WCL), as well as in the nuclear (N) and cytoplasmic (C) fractions of mouse tissues at different gestational ages. The method employs multiple loading controls to ensure proper separation of fractions and a total protein stain for more consistent comparisons of dissimilar samples. This method is useful for identifying BRCA1 deficiencies and localization in a variety of research fields, including development, neurodegeneration, and cancer.


Assuntos
Proteína BRCA1/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Animais , Western Blotting , Fracionamento Celular , Feminino , Idade Gestacional , Camundongos , Gravidez , Transporte Proteico
13.
Methods Mol Biol ; 1983: 79-106, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31087294

RESUMO

Posttranslational modifications of proteins control many complex biological processes, including genome expression, chromatin dynamics, metabolism, and cell division through a language of chemical modifications. Improvements in mass spectrometry-based proteomics have demonstrated protein acetylation is a widespread and dynamic modification in the cell; however, many questions remain on the regulation and downstream effects, and an assessment of the overall acetylation stoichiometry is needed. In this chapter, we describe the determination of acetylation stoichiometry using data-independent acquisition mass spectrometry to expand the number of acetylation sites quantified. However, the increased depth of data-independent acquisition is limited by the spectral library used to deconvolute fragmentation spectra. We describe a powerful approach of subcellular fractionation in conjunction with offline prefractionation to increase the depth of the spectral library. This deep interrogation of subcellular compartments provides essential insights into the compartment-specific regulation and downstream functions of protein acetylation.


Assuntos
Lisina/metabolismo , Proteínas/metabolismo , Acetilação , Técnicas de Cultura de Células , Fracionamento Celular , Cromatografia Líquida , Cromatografia de Fase Reversa , Interpretação Estatística de Dados , Concentração de Íons de Hidrogênio , Lisina/química , Espectrometria de Massas/métodos , Mitocôndrias/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/química , Proteômica/métodos , Espectrometria de Massas em Tandem
14.
Anal Bioanal Chem ; 411(19): 4963-4971, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31093701

RESUMO

Cu isotope fractionation was investigated in the human neuroblastoma SH-SY5Y cell line, in a proliferating/tumor phase (undifferentiated cells), and in a differentiated state (neuron-like cells), induced using retinoic acid (RA). The SH-SY5Y cell line displays genetic aberrations due to its cancerous origin, but differentiation drives the cell line towards phenotypes suitable for the research of neurological diseases (e.g., Alzheimer's disease or Parkinson's disease). Cellular Cu distribution was first explored by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) imaging and, subsequently, Cu isotopic analysis was performed at cellular and sub-cellular levels via multi-collector inductively coupled plasma-mass spectrometry (MC-ICP-MS). The SH-SY5Y cells showed a re-distribution of intracellular Cu upon RA differentiation. Both undifferentiated and differentiated cells became systematically enriched in the light 63Cu isotope with increasing intracellular Cu content. Differentiated neuron-like SH-SY5Y cells showed a heavier Cu isotopic composition (+ 0.3‰) than did the undifferentiated proliferating cells when exposed to Cu for 24 h. However, after a longer exposure time (72 h), no difference was observed between both cellular phenotypes. Mitochondrial fractions were enriched in the light 63Cu isotope, compared to whole cells, for both undifferentiated and differentiated cells (no significant difference). The Cu isotopic composition of the remaining cell lysates was heavier than that of the whole cells and + 0.2‰ heavier in the differentiated cells than in the undifferentiated cells. These results indicate that neuronal differentiation affects the Cu isotope fractionation accompanying Cu uptake in the cells, but this effect does not seem to be associated with the mitochondrial Cu pathway. Cu isotope fractionation can be an interesting tool for studying Cu metabolism at a (sub)-cellular level in functional neurons. Graphical abstract.


Assuntos
Fracionamento Celular , Cobre/isolamento & purificação , Isótopos/isolamento & purificação , Neuroblastoma/metabolismo , Neurônios/metabolismo , Frações Subcelulares/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Mitocôndrias/metabolismo , Neuroblastoma/patologia , Neurônios/citologia
15.
Methods Mol Biol ; 1979: 9-21, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028629

RESUMO

The starting material for all single-cell protocols is a cell suspension. The particular functions and spatial distribution of immune cells generally make them easy to isolate them from the tissues where they dwell. Here we describe tissue dissociation protocols that have been used to obtain human immune cells from lymphoid and nonlymphoid tissues to be then used as input to single-cell methods. We highlight the main factors that can influence the final quality of single-cell data, namely the stress signatures that can bias its interpretation.


Assuntos
Perfilação da Expressão Gênica/métodos , RNA/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Manejo de Espécimes/métodos , Fracionamento Celular/métodos , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Linfonodos/citologia , Linfonodos/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismo , RNA/sangue , RNA/isolamento & purificação , Pele/citologia , Pele/metabolismo , Baço/citologia , Baço/metabolismo
16.
Anaerobe ; 57: 75-81, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30935994

RESUMO

Seven protocols were tested to prepare cell wall extracts from live Cutibacterium acnes. Different parameters were modified: thawing/freezing and sonication/freezing cycles, to impact on mechanical degradation of the bacteria. Finally, the immunogenic potential of the extracts generated was evaluated by measuring IL-8 releases using an in vitro skin explants system. The aim of this article was to compare the existing protocols from the scientific literature, and also propose a standardized method developed in our facilities.


Assuntos
Extratos Celulares/imunologia , Extratos Celulares/isolamento & purificação , Parede Celular/imunologia , Propionibacterium acnes/imunologia , Fracionamento Celular/métodos , Humanos , Imunidade Inata , Pele/imunologia
17.
PLoS One ; 14(4): e0215324, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30973950

RESUMO

Ultracentrifugation (UC) is recognized as a robust approach for the isolation of extracellular vesicles (EVs). However, recent studies have highlighted limitations of UC including low recovery efficiencies and aggregation of EVs that could impact downstream functional analyses. We tested the benefit of using a liquid cushion of iodixanol during UC to address such shortcomings. In this study, we compared the yield and purity of EVs isolated from J774A.1 macrophage conditioned media by conventional UC and cushioned-UC (C-UC). We extended our study to include two other common EV isolation approaches: ultrafiltration (UF) and polyethylene glycol (PEG) sedimentation. After concentrating EVs using these four methods, the concentrates underwent further purification by using OptiPrep density gradient ultracentrifugation (DGUC). Our data show that C-DGUC provides a two-fold improvement in EV recovery over conventional UC-DGUC. We also found that UF-DGUC retained ten-fold more protein while PEG-DGUC achieved similar performance in nanoparticle and protein recovery compared to C-DGUC. Regarding purity as assessed by nanoparticle to protein ratio, our data show that EVs isolated by UC-DGUC achieved the highest purity while C-DGUC and PEG-DGUC led to similarly pure preparations. Collectively, we demonstrate that the use of a high-density iodixanol cushion during the initial concentration step improves the yield of EVs derived from cell culture media compared to conventional UC. This enhanced yield without substantial retention of protein contaminants and without exposure to forces causing aggregation offers new opportunities for the isolation of EVs that can subsequently be used for functional studies.


Assuntos
Fracionamento Celular/métodos , Centrifugação com Gradiente de Concentração/métodos , Vesículas Extracelulares/ultraestrutura , Animais , Linhagem Celular , Meios de Cultivo Condicionados , Vesículas Extracelulares/metabolismo , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão , Nanopartículas/metabolismo , Nanopartículas/ultraestrutura , Polietilenoglicóis , Proteínas/metabolismo , RNA/metabolismo , Ácidos Tri-Iodobenzoicos
18.
Biotechniques ; 66(4): 171-178, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987443

RESUMO

Fractionation in Gram-negative bacteria is used to identify the subcellular localization of proteins, in particular the localization of exported recombinant proteins. The process of cell fractionation can be fraught with cross-contamination issues and often lacks supporting data for fraction purity. Here, we compare three periplasm extraction and two cell disruption techniques in different combinations to investigate which process gives uncontaminated compartments from Escherichia coli. From these data, a robust method named PureFrac was compiled that gives pure periplasmic fractions and a superior recovery of soluble cytoplasmic proteins. The process extracts periplasm using cold osmotic shock with magnesium, prior to sonication and ultracentrifugation to separate the cytoplasm from insoluble material. This method handles cells cultivated in various conditions and allows preparation of active proteins in their respective compartments.


Assuntos
Fracionamento Celular/métodos , Proteínas de Escherichia coli/análise , Escherichia coli/citologia , Periplasma/química , Proteínas Recombinantes/análise , Western Blotting/métodos , Temperatura Baixa , Escherichia coli/química , Pressão Osmótica
19.
Front Immunol ; 10: 202, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30873152

RESUMO

Exosomes and microvesicles are two major categories of extracellular vesicles (EVs) released by almost all cell types and are highly abundant in biological fluids. Both the molecular composition of EVs and their release are thought to be strictly regulated by external stimuli. Multiple studies have consistently demonstrated that EVs transfer proteins, lipids and RNA between various cell types, thus mediating intercellular communication, and signaling. Importantly, small non-coding RNAs within EVs are thought to be major contributors to the molecular events occurring in the recipient cell. Furthermore, RNA cargo in exosomes and microvesicles could hold tremendous potential as non-invasive biomarkers for multiple disorders, including pathologies of the immune system. This mini-review is aimed to provide the state-of-the-art in the EVs-associated RNA transcriptome field, as well as the comprehensive analysis of previous studies characterizing RNA content within EVs released by various cells using next-generation sequencing. Finally, we highlight the technical challenges associated with obtaining pure EVs and deep sequencing of the EV-associated RNAs.


Assuntos
Vesículas Extracelulares/metabolismo , Transcriptoma , Animais , Transporte Biológico , Biomarcadores , Comunicação Celular , Fracionamento Celular/métodos , Micropartículas Derivadas de Células/metabolismo , Exossomos/metabolismo , Humanos , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , RNA/metabolismo
20.
Nat Commun ; 10(1): 1287, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894536

RESUMO

Close proximities between organelles have been described for decades. However, only recently a specific field dealing with organelle communication at membrane contact sites has gained wide acceptance, attracting scientists from multiple areas of cell biology. The diversity of approaches warrants a unified vocabulary for the field. Such definitions would facilitate laying the foundations of this field, streamlining communication and resolving semantic controversies. This opinion, written by a panel of experts in the field, aims to provide this burgeoning area with guidelines for the experimental definition and analysis of contact sites. It also includes suggestions on how to operationally and tractably measure and analyze them with the hope of ultimately facilitating knowledge production and dissemination within and outside the field of contact-site research.


Assuntos
Membrana Celular/metabolismo , Células Eucarióticas/metabolismo , Membranas Intracelulares/metabolismo , Organelas/metabolismo , Terminologia como Assunto , Animais , Fracionamento Celular/métodos , Membrana Celular/ultraestrutura , Células Eucarióticas/ultraestrutura , Humanos , Membranas Intracelulares/ultraestrutura , Microscopia/instrumentação , Microscopia/métodos , Organelas/ultraestrutura , Proteínas/genética , Proteínas/metabolismo , Coloração e Rotulagem/métodos
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