Protein Co-Enrichment Analysis of Extracellular Vesicles

Extracellular Vesicles (EVs) carry cell-derived proteins that confer functionality and selective cell uptake. However, whether proteins are packaged stochastically or co-enriched within individual EVs, and whether co-enrichment fluctuates under homeostasis and disease, has not been measured. EV abundance and protein global relative expression have been qualified by bulk analysis. Meanwhile, co-enrichment is not directly accessible via bulk measurement and has not been reported for single EV analysis. Here, we introduce the normalized index of co-enrichment (NICE) to measure protein co-enrichment. NICE was derived by (i) capturing EVs based on the expression of a membrane-bound protein, (ii) probing for the co-expression of a second protein at the population level - EV integrity underwrites the detection of single EV co-expression without the need to resolve single EVs - and (iii) normalizing measured values using two universal normalization probes. Axiomatically, NICE = 1 for stochastic inclusion or no overall co-enrichment, while for positive and negative co-enrichment NICE > 1 or < 1, respectively. We quantified the NICE of tetraspanins, growth factor receptors and integrins in EVs of eight breast cancer cell lines of varying metastatic potential and organotropism, combinatorially mapping up to 104 protein pairs. Our analysis revealed protein enrichment and co-expression patterns consistent with previous findings. For the organotropic cell lines, most protein pairs were co-enriched on EVs, with the majority of NICE values between 0.2 to 11.5, and extending from 0.037 to 80.4. Median NICE were either negative, neutral or positive depending on the cells. NICE analysis is easily multiplexed and is compatible with microarrays, bead-based and single EV assays. Additional studies are needed to deepen our understanding of the potential and significance of NICE for research and clinical uses

Impactos da menopausa na saúde da mulher / Impacts of menopause on women’s health

A menopausa corresponde ao último ciclo menstrual, sendo somente reconhecida depois de 12 meses, e ocorre, em média, aos 51 anos. Dessa forma, ela pode ser caracterizada como de início precoce (antes dos 40 anos) ou tardio (depois dos 55 anos). Neste cenário, as flutuações hormonais levam a mudanças, não só na função reprodutiva, mas em outras áreas corporais importantes. Vale saliente que a amenorreia pode ser, também, de causa induzida, decorrente da remoção de partes do sistema reprodutivo feminino, por exemplo. Assim, a parada da atividade ovariana induz um caráter inflamatório crônico, aumento a suscetibilidade da mulher a diversas doenças. Nessa perspectiva, o presente estudo tem como objetivo analisar as principais características da menopausa, bem como os aspectos psicológicos, o seu impacto na vida da mulher e a qualidade da assistência proporcionada pela Atenção Primária à saúde feminina. Realizou-se uma revisão integrativa da literatura nas bases de dados Medline (via PubMed) e Lilacs (via BVS), a partir da utilização de descritores (MeSH e DeCS). Aplicou-se os filtros de artigos publicados durante o período de 2015 a 2020, texto completo gratuito, pesquisas realizadas em humanos e sexo feminino. Dessa forma, foram encontrados um total de 3.548 artigos, dos quais, após a leitura dos títulos, resumos e texto na íntegra, 27 foram selecionados para compor a revisão. A menopausa pode ser assintomática ou sintomática e seus impactos levam a mudanças na função reprodutiva e em outras áreas do corpo e da mente, atingindo os aspectos hormonais, imunológicos, emocionais, estressantes e sociais

Extracellular Vesicle Antibody Microarray for Multiplexed Inner and Outer Protein Analysis

Proteins are found both outside and inside of extracellular vesicles (EVs) and govern the properties and functions of EVs, while also constituting a signature of the cell of origin and of biological function and disease. Outer proteins on EVs can be directly bound by antibodies to either enrich EVs, or probe the expression of a protein on EVs, including in a combinatorial manner. However, co-profiling of inner proteins remains challenging. Here, we present the high-throughput, multiplexed analysis of EV inner and outer proteins (EVPio). We describe the optimization of fixation and heat-induced protein epitope retrieval for EVs, along with oligo-barcoded antibodies and branched DNA signal amplification for sensitive, multiplexed, and high-throughput assays. We captured four subpopulations of EVs from colorectal cancer (CRC) cell lines HT29 and SW403 based on EpCAM, CD9, CD63, and CD81 expression, and quantified the co-expression of eight outer [integrins (ITGs) and tetraspanins] and four inner (heat shock, endosomal, and inner leaflet) proteins. The differences in co-expression patterns were consistent with the literature and known biological function. In conclusion, EVPio analysis can simultaneously detect multiple inner and outer proteins in EVs immobilized on a surface, opening the way to extensive combinatorial protein profiles for both discovery and clinical translation