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Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression.
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Retrovirus Endógenos , Vesículas Extracelulares , Neoplasias , Animales , Ratones , Vesículas Extracelulares/metabolismo , Línea Celular Tumoral , Diferenciación Celular , Células Dendríticas , Neoplasias/metabolismoRESUMEN
SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less likely to occur in vitro for reasons unknown. Extracellular vesicles (EVs) are secreted by the embryo into the culture medium. Yet the role that embryonic EVs and their cargo microRNAs (miRNAs) play in blastocyst hatching has not been elucidated, partially due to the difficulties of isolating them from low amounts of culture medium. Here, we optimized EV-miRNA isolation from medium conditioned by individually cultured bovine embryos and subsequently showed that miR-378a-3p, which was up-regulated in EVs secreted by blastocysts, plays a crucial role in promoting blastocyst hatching. This demonstrates the regulatory effect of miR-378-3p on hatching, which is an established embryo quality parameter linked with implantation.
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Vesículas Extracelulares , MicroARNs , Animales , Blastocisto , Bovinos , Medios de Cultivo , Técnicas de Cultivo de Embriones , Embrión de Mamíferos , Vesículas Extracelulares/genética , MicroARNs/genéticaRESUMEN
Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.
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Biomarcadores de Tumor/genética , Proliferación Celular , Bases del Conocimiento , Neoplasias/patología , Programas Informáticos , Esferoides Celulares/patología , Microambiente Tumoral , Técnicas de Cultivo de Célula/métodos , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/clasificación , Neoplasias/metabolismo , RNA-Seq , Reproducibilidad de los Resultados , Esferoides Celulares/inmunología , Esferoides Celulares/metabolismo , Células Tumorales CultivadasRESUMEN
BACKGROUND: Substantial research has been devoted to elucidating the role of extracellular vesicles (EVs) in the different hallmarks of cancer. Consequently, EVs are increasingly explored as a source of cancer biomarkers in body fluids. However, the heterogeneity in EVs, the complexity of body fluids, and the diversity in methods available for EV analysis, challenge the development and translation of EV-based biomarker assays. CONTENT: Essential steps in EV-associated biomarker development are emphasized covering biobanking, biomarker discovery, verification and validation, and clinical implementation. A meticulous study design is essential and ideally results from close interactions between clinicians and EV researchers. A plethora of different EV preparation protocols exists which warrants quality control and transparency to ensure reproducibility and thus enable verification of EV-associated biomarker candidates identified in the discovery phase in subsequent independent cohorts. The development of an EV-associated biomarker assay requires thorough analytical and clinical validation. Finally, regulatory affairs must be considered for clinical implementation of EV-based biomarker assays. SUMMARY: In this review, the current challenges that prevent us from exploiting the full potential of EV-based biomarker assays are identified. Guidelines and tools to overcome these hurdles are highlighted and are crucial to advance EV-based biomarker assays into clinical use.
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Vesículas Extracelulares , Neoplasias , Humanos , Bancos de Muestras Biológicas , Reproducibilidad de los Resultados , Biomarcadores de Tumor , Neoplasias/diagnósticoRESUMEN
PURPOSE: The aim of the study was to compare the difference in survival between invasive ductal (IDC) and lobular carcinoma (ILC). METHODS: Data of patients (n = 1843) with a hormone receptor-positive, HER2-negative, pT1-3 IDC or ILC cancer without distant metastasis, treated at the Ghent University Hospital over the time period 2001-2015, were analyzed. RESULTS: ILC represented 13.9% of the tumors, had a higher percentage of pT3 and pN3 stages than IDC, lymphovascular space invasion (LVSI) was less present and Ki-67 was mostly low. 73.9% of ILCs were grade 2, whereas IDC had more grade 1 and grade 3 tumors. Kaplan-Meier curves and log-rank testing showed a significant worse DFS for ILC with pN ≥ 1 than for their IDC counterpart. In a multivariable Cox regression analysis the histologic tumor type, ductal or lobular, was a determinant of DFS over 120 months (IDC as reference; hazard ratio for ILC 1.77, 95% CI 1.08-2.90) just as the ER Allred score (hazard ratio 0.84, 95% CI 0.78-0.91), LVSI (hazard ratio 1.75, 95% CI 1.12-2.74) and pN3 (hazard ratio 2.29, 95% CI 1.03-5.09). Determinants of OS over ten years were age (hazard ratio 1.05, 95% CI 1.02-1.07), LVSI (hazard ratio 3.62, 95% CI 1.92-6.82) and the ER Allred score (hazard ratio 0.80, 95% CI 0.73-0.89). CONCLUSION: The histologic tumor type, ductal or lobular, determines DFS in hormone receptor-positive, HER2-negative, pT1-3 breast cancer besides the ER Allred score, LVSI and pN3.
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Neoplasias de la Mama , Carcinoma Ductal de Mama , Carcinoma Lobular , Humanos , Femenino , Carcinoma Lobular/patología , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Resultado del Tratamiento , Modelos de Riesgos Proporcionales , Pronóstico , Estudios RetrospectivosRESUMEN
A phase II study (PRIMMO) of patients with pretreated persistent/recurrent/metastatic cervical or endometrial cancer is presented. Patients received an immunomodulatory five-drug cocktail (IDC) consisting of low-dose cyclophosphamide, aspirin, lansoprazole, vitamin D, and curcumin starting 2 weeks before radioimmunotherapy. Pembrolizumab was administered three-weekly from day 15 onwards; one of the tumor lesions was irradiated (8Gyx3) on days 15, 17, and 19. The primary endpoint was the objective response rate per immune-related response criteria (irORR) at week 26 (a lower bound of the 90% confidence interval [CI] of > 10% was considered efficacious). The prespecified 43 patients (cervical, n = 18; endometrial, n = 25) were enrolled. The irORR was 11.1% (90% CI 2.0-31.0) in cervical cancer and 12.0% (90% CI 3.4-28.2) in endometrial cancer. Median duration of response was not reached in both cohorts. Median interval-censored progression-free survival was 4.1 weeks (95% CI 4.1-25.7) in cervical cancer and 3.6 weeks (95% CI 3.6-15.4) in endometrial cancer; median overall survival was 39.6 weeks (95% CI 15.0-67.0) and 37.4 weeks (95% CI 19.0-50.3), respectively. Grade ≥ 3 treatment-related adverse events were reported in 10 (55.6%) cervical cancer patients and 9 (36.0%) endometrial cancer patients. Health-related quality of life was generally stable over time. Responders had a significantly higher proportion of peripheral T cells when compared to nonresponders (p = 0.013). In conclusion, PRIMMO did not meet its primary objective in both cohorts; pembrolizumab, radiotherapy, and an IDC had modest but durable antitumor activity with acceptable but not negligible toxicity.Trial registration ClinicalTrials.gov (identifier NCT03192059) and EudraCT Registry (number 2016-001569-97).
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Neoplasias Endometriales , Neoplasias del Cuello Uterino , Femenino , Humanos , Neoplasias del Cuello Uterino/tratamiento farmacológico , Calidad de Vida , Anticuerpos Monoclonales Humanizados/uso terapéutico , Neoplasias Endometriales/patologíaRESUMEN
MicroRNAs (miRNAs), which can be carried inside extracellular vesicles (EVs), play a crucial role in regulating embryo development up to the blastocyst stage. Yet, the molecular mechanisms underlying blastocyst development and quality are largely unknown. Recently, our group identified 69 differentially expressed miRNAs in extracellular vesicles (EVs) isolated from culture medium conditioned by bovine embryos that either developed to the blastocyst stage or did not (non-blastocysts). We found miR-146b to be more abundant in the EVs derived from media conditioned by non-blastocyst embryos. Using RT-qPCR, we here confirmed the upregulation of miR-146b in non-blastocyst (arrested at 2-4 cell and morula stage) embryos compared to blastocysts (p<0.005), which coincides with the upregulation of miR-146b in EVs derived from the medium of these non-blastocysts. To evaluate a functional effect, bovine embryo culture media were supplemented with miR-146b mimics, resulting in significantly decreased embryo quality, with lower blastocyst rates at day 7 and lower total cell numbers, while the opposite was found after supplementation with miR-146b inhibitors, which resulted in reduced apoptosis rates (P < 0.01). Transcriptomic analysis of embryos treated with miR-146b mimics or inhibitors showed differential expression (P < 0.01) of genes associated with apoptosis, cell differentiation, and the RNA Pol II transcription complex, including WDR36, MBNL2, ERCC6l2, PYGO1, and SNIP1. Overall, miR-146b is overexpressed in non-blastocyst embryos and in EVs secreted by these embryos, and it regulates genes involved in embryo development and apoptosis, resulting in decreased embryo quality.
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BACKGROUND: Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers. RESULTS: Automated versus manual density-based separation of trackable recombinant extracellular vesicles (rEV) spiked in PBS significantly reduces variability in rEV recovery as quantified by fluorescent nanoparticle tracking analysis and ELISA. To validate automated density-based EV separation from complex body fluids, including blood plasma and urine, we assess reproducibility, recovery, and specificity by mass spectrometry-based proteomics and transmission electron microscopy. Method reproducibility is the highest in the automated procedure independent of the matrix used. While retaining (in urine) or enhancing (in plasma) EV recovery compared to manual liquid handling, automation significantly reduces the presence of body fluid specific abundant proteins in EV preparations, including apolipoproteins in plasma and Tamm-Horsfall protein in urine. CONCLUSIONS: In conclusion, automated liquid handling ensures cost-effective EV separation from human body fluids with high reproducibility, specificity, and reduced hands-on time with the potential to enable larger-scale biomarker studies.
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Vesículas Extracelulares , Humanos , Reproducibilidad de los Resultados , Flujo de Trabajo , Vesículas Extracelulares/metabolismo , Proteínas , Biomarcadores/metabolismoRESUMEN
Extracellular vesicles (EVs) have attracted great attention as potential biomarkers for cancer diagnostics. Although several technologies have been developed for EV detection, many of them are still not applicable to clinical settings as they rely on complex EV isolation processes, while lacking sensitivity, specificity or standardization. To solve this problem, we have developed a sensitive breast cancer-specific EV detection bioassay directly in blood plasma using a fiber-optic surface plasmon resonance (FO-SPR) biosensor, previously calibrated with recombinant EVs. First, we established a sandwich bioassay to detect SK-BR-3 EVs by functionalizing the FO-SPR probes with anti-HER2 antibodies. A calibration curve was built using an anti-HER2/Banti-CD9 combination, resulting in an LOD of 2.1 × 107 particles/mL in buffer and 7 × 108 particles/mL in blood plasma. Next, we investigated the potential of the bioassay to detect MCF7 EVs in blood plasma using an anti-EpCAM/Banti-mix combination, obtaining an LOD of 1.1 × 10 8 particles/mL. Finally, the specificity of the bioassay was proven by the absence of signal when testing plasma samples from 10 healthy people unknown to be diagnosed with breast cancer. The remarkable sensitivity and specificity of the developed sandwich bioassay together with the advantages of the standardized FO-SPR biosensor highlight outstanding potential for the future of EV analysis.
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Técnicas Biosensibles , Neoplasias de la Mama , Vesículas Extracelulares , Femenino , Humanos , Biomarcadores , Técnicas Biosensibles/métodos , Neoplasias de la Mama/diagnóstico , Resonancia por Plasmón de Superficie/métodosRESUMEN
Extracellular vesicles (EVs) are membranous vesicles that are released by both prokaryotic and eukaryotic cells into the extracellular microenvironment. EVs can be categorised as exosomes, ectosomes or shedding microvesicles and apoptotic bodies based on the mode of biogenesis. EVs contain biologically active cargo of nucleic acids, proteins, lipids and metabolites that can be altered based on the precise state of the cell. Vesiclepedia (http://www.microvesicles.org) is a web-based compendium of RNA, proteins, lipids and metabolites that are identified in EVs from both published and unpublished studies. Currently, Vesiclepedia contains data obtained from 1254 EV studies, 38 146 RNA entries, 349 988 protein entries and 639 lipid/metabolite entries. Vesiclepedia is publicly available and allows users to query and download EV cargo based on different search criteria. The mode of EV isolation and characterization, the biophysical and molecular properties and EV-METRIC are listed in the database aiding biomedical scientists in assessing the quality of the EV preparation and the corresponding data obtained. In addition, FunRich-based Vesiclepedia plugin is incorporated aiding users in data analysis.
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Bases de Datos Factuales , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Lípidos/análisis , Proteínas/análisis , ARN/análisisRESUMEN
Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AOF), using different isolation methods. However, it is not clear whether different purification methods can affect the functionality of resulting EVs. Here, we compared two methods (OptiPrep™ density gradient ultracentrifugation (ODG UC) and single-step size exclusion chromatography (SEC) (qEV IZON™ single column)) for the isolation of EVs from bovine FF and AOF. Additionally, we evaluated whether the addition of EVs derived either by ODG UC or SEC from FF or AOF during oocyte maturation would yield extra benefits for embryo developmental competence. The characterization of EVs isolated using ODG UC or SEC from FF and AOF did not show any differences in terms of EV sizes (40-400 nm) and concentrations (2.4 ± 0.2 × 1012-1.8 ± 0.2 × 1013 particles/mL). Blastocyst yield and quality was higher in groups supplemented with EVs isolated from FF and AOF by ODG UC, with higher total cell numbers and a lower apoptotic cell ratio compared with the other groups (p < 0.05). Supplementing in vitro maturation media with EVs derived by ODG UC from AOF was beneficial for bovine embryo development and quality.
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Técnicas de Cultivo de Embriones , Desarrollo Embrionario/genética , Vesículas Extracelulares/metabolismo , Oogénesis/efectos de los fármacos , Animales , Blastocisto/efectos de los fármacos , Bovinos , Centrifugación por Gradiente de Densidad , Medios de Cultivo Condicionados , Femenino , Líquido Folicular/química , Líquido Folicular/metabolismo , Células Ciliadas Ampollares/química , Células Ciliadas Ampollares/metabolismo , Humanos , Oviductos/efectos de los fármacosRESUMEN
We investigated the potential of tumor-infiltrating immune cells (ICs) as predictive or prognostic biomarkers for cervical cancer patients. In total, 38 patients treated with (chemo)radiotherapy and subsequent surgery were included in the current study. This unique treatment schedule makes it possible to analyze IC markers in pretreatment and posttreatment tissue specimens and their changes during treatment. IC markers for T cells (CD3, CD4, CD8 and FoxP3), macrophages (CD68 and CD163) and B cells (CD20), as well as IL33 and PD-L1, were retrospectively analyzed via immunohistochemistry. Patients were grouped in the low score or high score group based on the amount of positive cells on immunohistochemistry. Correlations to pathological complete response (pCR), cause-specific survival (CSS) and metastasis development during follow-up were evaluated. In analysis of pretreatment biopsies, significantly more pCR was seen for patients with CD8 = CD3, CD8 ≥ CD4, positive IL33 tumor cell (TC) scores, IL33 IC < TC and PD-L1 TC ≥5%. Besides patients with high CD8 scores, also patients with CD8 ≥ CD4, CD163 ≥ CD68 or PD-L1 IC ≥5% had better CSS. In the analysis of posttreatment specimens, less pCR was observed for patients with high CD8 or CD163 scores. Patients with decreasing CD8 or CD163 scores between pretreatment and posttreatment samples showed more pCR, whereas those with increasing CD8 or decreasing IL33 IC scores showed a worse CSS. Meanwhile, patients with an increasing CD3 score or stable/increasing PD-L1 IC score showed more metastasis during follow-up. In this way, the intratumoral IC landscape is a promising tool for prediction of outcome and response to (chemo)radiotherapy.
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Antígeno B7-H1/metabolismo , Complejo CD3/metabolismo , Quimioradioterapia/métodos , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias del Cuello Uterino/terapia , Adulto , Anciano , Anciano de 80 o más Años , Linfocitos B/inmunología , Femenino , Humanos , Macrófagos/inmunología , Persona de Mediana Edad , Medicina de Precisión , Pronóstico , Estudios Retrospectivos , Linfocitos T/inmunología , Resultado del Tratamiento , Microambiente Tumoral , Procedimientos Quirúrgicos Urogenitales , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/patologíaRESUMEN
Extracellular vesicles (EVs) have been identified as one of the communication mechanisms amongst embryos. They are secreted into the embryo culture medium and, as such, represent a source of novel biomarkers for identifying the quality of cells and embryos. However, only small amounts of embryo-conditioned medium are available, which represents a challenge for EV enrichment. Our aim is to assess the suitability of different EV separation methods to retrieve EVs with high specificity and sufficient efficiency. Bovine embryo-conditioned medium was subjected to differential ultracentrifugation (DU), OptiPrepTM density gradient (ODG) centrifugation, and size exclusion chromatography. Separated EVs were characterized by complementary characterization methods, including Western blot, electron microscopy, and nanoparticle tracking analysis, to assess the efficiency and specificity. OptiPrepTM density gradient centrifugation outperformed DU and SEC in terms of specificity by substantial removal of contaminating proteins such as ribonucleoprotein complexes (Argonaute-2 (AGO-2)) and lipoproteins (ApoA-I) from bovine embryo-derived EVs (density: 1.02-1.04, 1.20-1.23 g/mL, respectively). In conclusion, ODG centrifugation is the preferred method for identifying EV-enriched components and for improving our understanding of EV function in embryo quality and development.
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Medios de Cultivo Condicionados/metabolismo , Embrión de Mamíferos/metabolismo , Vesículas Extracelulares/metabolismo , Animales , Bovinos , Centrifugación por Gradiente de Densidad , Fraccionamiento Químico/métodos , Cromatografía en Gel , Técnicas de Cultivo de Embriones , Vesículas Extracelulares/ultraestructura , Fracciones Subcelulares , UltracentrifugaciónRESUMEN
Owing to the relationship between extracellular vesicles (EVs) and physiological and pathological conditions, the interest in EVs is exponentially growing. EVs hold high hopes for novel diagnostic and translational discoveries. This review provides an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs. Common misconceptions and methodological pitfalls are highlighted. Although EVs are found in all body fluids, in this review, we will focus on EVs from human blood, not only our most complex but also the most interesting body fluid for cardiovascular research.
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Recolección de Muestras de Sangre/métodos , Recolección de Muestras de Sangre/normas , Vesículas Extracelulares/metabolismo , Biomarcadores/sangre , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/diagnóstico , Exosomas/metabolismo , Citometría de Flujo/métodos , HumanosRESUMEN
Extracellular vesicles (EVs) play a possible role in cellâ»cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrepTM density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25â»230 nm with an average concentration of 236.5 ± 1.27 × 108 particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group.
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Fraccionamiento Celular , Medios de Cultivo Condicionados/metabolismo , Vesículas Extracelulares/metabolismo , Animales , Bovinos , Fraccionamiento Celular/métodos , Centrifugación por Gradiente de Densidad , Técnicas de Cultivo de Embriones , Embrión de Mamíferos , Vesículas Extracelulares/ultraestructuraRESUMEN
Exosome-like vesicles (ELVs) are a novel class of biomarkers that are receiving a lot of attention for the detection of cancer at an early stage. In this study the feasibility of using a surface enhanced Raman spectroscopy (SERS) based method to distinguish between ELVs derived from different cellular origins is evaluated. A gold nanoparticle based shell is deposited on the surface of ELVs derived from cancerous and healthy cells, which enhances the Raman signal while maintaining a colloidal suspension of individual vesicles. This nanocoating allows the recording of SERS spectra from single vesicles. By using partial least squares discriminant analysis on the obtained spectra, vesicles from different origin can be distinguished, even when present in the same mixture. This proof-of-concept study paves the way for noninvasive (cancer) diagnostic tools based on exosomal SERS fingerprinting in combination with multivariate statistical analysis.
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Exosomas/química , Espectrometría Raman/métodos , Algoritmos , Oro/química , Nanopartículas del Metal/químicaRESUMEN
MOTIVATION: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. RESULTS: We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. AVAILABILITY AND IMPLEMENTATION: The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info.
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Biología Computacional , Sistemas de Administración de Bases de Datos , Bases de Datos Factuales , Exosomas/metabolismo , Espacio Extracelular/metabolismo , Programas Informáticos , Investigación Biomédica , Humanos , Interfaz Usuario-ComputadorRESUMEN
Extracellular RNA (exRNA) is functionally transferrable from donor to recipient cells and is protected from RNAses by electrostatic interactions with proteins or by membrane encapsulation. In addition to bioactive RNA, extracellular vesicles (EVs) contain intraluminal and membrane-associated proteins. The cellular context and fitness affect the composition of EVs and thus the outcome of the communication between the EV-producer and recipient cells. Adaptive communication through EVs is particularly important between cancer cells and their local and distant environment and drives life-threatening metastatic progression. Small noncoding RNAs (miRNAs) have been reported in EV isolations and play a role in local invasion, angiogenesis, immune modulation, metastatic niche preparation, colonization and dormancy. The metastasis-related functions attributed to EV-associated miRNAs are currently increasing exponentially in the scientific literature. We must be aware that the correct and efficient separation of non-vesicular entities (soluble proteins, RNA-protein complexes and RNA-lipoprotein complexes) from EVs is necessary to determine the true contribution of EVs in any experiment that describes the molecular content or the functional consequences of the isolated material.