Biological Procedures Online最新文献

{"title":"Development of an In Vitro 3D Model for Investigating Ligamentum Flavum Hypertrophy.","authors":"Cheng-Li Lin,&nbsp;Yi-Ting Kuo,&nbsp;Che-Hao Tsao,&nbsp;Yan-Jye Shyong,&nbsp;Shu-Hsien Shih,&nbsp;Ting-Yuan Tu","doi":"10.1186/s12575-020-00132-6","DOIUrl":"https://doi.org/10.1186/s12575-020-00132-6","url":null,"abstract":"<p><strong>Background: </strong>Ligamentum flavum hypertrophy (LFH) is among the most crucial factors in degenerative lumbar spinal stenosis, which can cause back pain, lower extremity pain, cauda equina syndrome and neurogenic claudication. The exact pathogenesis of LFH remains elusive despite extensive research. Most in vitro studies investigating LFH have been carried out using conventional two-dimensional (2D) cell cultures, which do not resemble in vivo conditions, as they lack crucial pathophysiological factors found in three-dimensional (3D) LFH tissue, such as enhanced cell proliferation and cell cluster formation. In this study, we generated ligamentum flavum (LF) clusters using spheroid cultures derived from primary LFH tissue.</p><p><strong>Results: </strong>The cultured LF spheroids exhibited good viability and growth on an ultra-low attachment 96-well plate (ULA 96-plate) platform according to live/dead staining. Our results showed that the 100-cell culture continued to grow in size, while the 1000-cell culture maintained its size, and the 5000-cell culture exhibited a decreasing trend in size as the culture time increased; long-term culture was validated for at least 28 days. The LF spheroids also maintained the extracellular matrix (ECM) phenotype, i.e., fibronectin, elastin, and collagen I and III. The 2D culture and 3D culture were further compared by cell cycle and Western blot analyses. Finally, we utilized hematoxylin and eosin (H&E) staining to demonstrate that the 3D spheroids resembled part of the cell arrangement in LF hypertrophic tissue.</p><p><strong>Conclusions: </strong>The developed LF spheroid model has great potential, as it provides a stable culture platform in a 3D model that can further improve our understanding of the pathogenesis of LFH and has applications in future studies.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"20"},"PeriodicalIF":6.4,"publicationDate":"2020-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12575-020-00132-6","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38438894","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Potential Diagnostic Systems for Coronavirus Detection: a Critical Review.","authors":"Elena Ekrami,&nbsp;Mahdi Pouresmaieli,&nbsp;Fatemeh Barati,&nbsp;Sahar Asghari,&nbsp;Farzad Ramezani Ziarani,&nbsp;Parvin Shariati,&nbsp;Matin Mamoudifard","doi":"10.1186/s12575-020-00134-4","DOIUrl":"https://doi.org/10.1186/s12575-020-00134-4","url":null,"abstract":"<p><strong>Abstract: </strong>Currently there are no effective anti-viral drugs for SARS-CoV-2, so the primary line of defense is to detect infected cases as soon as possible. The high rate of contagion for this virus and the highly nonspecific symptoms of the disease (Coronovirus disease 2019, (Covid-19)) that it causes, such as respiratory symptoms, cough, dyspnea, fever, and viral pneumonia, require the urgent establishment of precise and fast diagnostic tests to verify suspected cases, screen patients, and conduct virus surveillance. Nowadays, several virus detection methods are available for viral diseases, which act on specific properties of each virus or virus family, therefore, further investigations and trials are needed to find a highly efficient and accurate detection method to detect and prevent the outcomes of the disease. Hence, there is an urgent need for more and precise studies in this field. In this review, we discussed the properties of a new generation of coronaviruses (SARS-CoV-2) following routine virus detection methods and proposed new strategies and the use of potential samples for SARS-CoV-2 detection.</p><p><strong>Graphical abstract: </strong></p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"21"},"PeriodicalIF":6.4,"publicationDate":"2020-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12575-020-00134-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38342670","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A comprehensive review of COVID-19 characteristics.","authors":"Hanie Esakandari, Mohsen Nabi-Afjadi, Javad Fakkari-Afjadi, Navid Farahmandian, Seyed-Mohsen Miresmaeili, Elham Bahreini","doi":"10.1186/s12575-020-00128-2","DOIUrl":"10.1186/s12575-020-00128-2","url":null,"abstract":"<p><p>In December 2019, a novel coronavirus, named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) or (2019-nCoV) with unknown origin spread in Hubei province of China. The epidemic disease caused by SARS-CoV-2 called coronavirus disease-19 (COVID-19). The presence of COVID-19 was manifested by several symptoms, ranging from asymptomatic/mild symptoms to severe illness and death. The viral infection expanded internationally and WHO announced a Public Health Emergency of International Concern. To quickly diagnose and control such a highly infectious disease, suspicious individuals were isolated and diagnostic/treatment procedures were developed through patients' epidemiological and clinical data. Early in the COVID-19 outbreak, WHO invited hundreds of researchers from around the world to develop a rapid quality diagnosis, treatment and vaccines, but so far no specific antiviral treatment or vaccine has been approved by the FDA. At present, COVID-19 is managed by available antiviral drugs to improve the symptoms, and in severe cases, supportive care including oxygen and mechanical ventilation is used for infected patients. However, due to the worldwide spread of the virus, COVID-19 has become a serious concern in the medical community. According to the current data of WHO, the number of infected and dead cases has increased to 8,708,008 and 461,715, respectively (Dec 2019 -June 2020). Given the high mortality rate and economic damage to various communities to date, great efforts must be made to produce successful drugs and vaccines against 2019-nCoV infection. For this reason, first of all, the characteristics of the virus, its pathogenicity, and its infectious pathways must be well known. Thus, the main purpose of this review is to provide an overview of this epidemic disease based on the current evidence.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"19"},"PeriodicalIF":3.7,"publicationDate":"2020-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402395/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38255428","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isolation and Quantification of Blood Apoptotic Bodies, a Non-invasive Tool to Evaluate Apoptosis in Patients with Ischemic Stroke and Neurodegenerative Diseases.","authors":"Gemma Serrano-Heras,&nbsp;Inmaculada Díaz-Maroto,&nbsp;Beatriz Castro-Robles,&nbsp;Blanca Carrión,&nbsp;Ana B Perona-Moratalla,&nbsp;Julia Gracia,&nbsp;Sandra Arteaga,&nbsp;Francisco Hernández-Fernández,&nbsp;Jorge García-García,&nbsp;Oscar Ayo-Martín,&nbsp;Tomás Segura","doi":"10.1186/s12575-020-00130-8","DOIUrl":"https://doi.org/10.1186/s12575-020-00130-8","url":null,"abstract":"<p><strong>Background: </strong>Improper regulation of apoptosis has been postulated as one of the main factors that contributes to the etiology and/or progression of several prevalent diseases, including ischemic stroke and neurodegenerative pathologies. Consequently, in the last few years, there has been an ever-growing interest in the in vivo study of apoptosis. The clinical application of the tissue sampling and imaging approaches to analyze apoptosis in neurological diseases is, however, limited. Since apoptotic bodies are membrane vesicles that are released from fragmented apoptotic cells, it follows that the presence of these vesicles in the bloodstream is likely due to the apoptotic death of cells in tissues. We therefore propose to use circulating apoptotic bodies as biomarkers for measuring apoptotic death in patients with ischemic stroke and neurodegenerative diseases.</p><p><strong>Results: </strong>Since there is no scientific literature establishing the most appropriate method for collecting and enumerating apoptotic bodies from human blood samples. Authors, here, describe a reproducible centrifugation-based method combined with flow cytometry analysis to isolate and quantify plasma apoptotic bodies of patients with ischemic stroke, multiple sclerosis, Parkinson's disease and also in healthy controls. Electron microscopy, dynamic light scattering and proteomic characterization in combination with flow cytometry studies revealed that our isolation method achieves notable recovery rates of highly-purified intact apoptotic bodies.</p><p><strong>Conclusions: </strong>This easy, minimally time consuming and effective procedure for isolating and quantifying plasma apoptotic bodies could help physicians to implement the use of such vesicles as a non-invasive tool to monitor apoptosis in patients with cerebrovascular and neurodegenerative diseases for prognostic purposes and for monitoring disease activity.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"17"},"PeriodicalIF":6.4,"publicationDate":"2020-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12575-020-00130-8","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38248151","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Nasopharyngeal Microbiota Profiling of SARS-CoV-2 Infected Patients.","authors":"Flavio De Maio,&nbsp;Brunella Posteraro,&nbsp;Francesca Romana Ponziani,&nbsp;Paola Cattani,&nbsp;Antonio Gasbarrini,&nbsp;Maurizio Sanguinetti","doi":"10.1186/s12575-020-00131-7","DOIUrl":"https://doi.org/10.1186/s12575-020-00131-7","url":null,"abstract":"<p><p>We analyzed the bacterial communities of the nasopharynx in 40 SARS-CoV-2 infected and uninfected patients. All infected patients had a mild COVID-19 disease. We did not find statistically significant differences in either bacterial richness and diversity or composition. These findings suggest a nasopharyngeal microbiota at least early resilient to SARS-CoV-2 infection.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"18"},"PeriodicalIF":6.4,"publicationDate":"2020-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12575-020-00131-7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38207753","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Expansion of Single Cell Transcriptomics Data of SARS-CoV Infection in Human Bronchial Epithelial Cells to COVID-19.","authors":"Reza Zolfaghari Emameh,&nbsp;Hassan Nosrati,&nbsp;Mahyar Eftekhari,&nbsp;Reza Falak,&nbsp;Majid Khoshmirsafa","doi":"10.1186/s12575-020-00127-3","DOIUrl":"https://doi.org/10.1186/s12575-020-00127-3","url":null,"abstract":"<p><strong>Background: </strong>Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 19 (COVID-19) that was emerged as a new member of coronaviruses since December 2019 in Wuhan, China and then after was spread in all continentals. Since SARS-CoV-2 has shown about 77.5% similarity to SARS-CoV, the transcriptome and immunological regulations of SARS-CoV-2 was expected to have high percentage of overlap with SARS-CoV.</p><p><strong>Results: </strong>In this study, we applied the single cell transcriptomics data of human bronchial epithelial cells (2B4 cell line) infected with SARS-CoV, which was annotated in the Expression Atlas database to expand this data to COVID-19. In addition, we employed system biology methods including gene ontology (GO) and Reactome pathway analyses to define functional genes and pathways in the infected cells with SARS-CoV. The transcriptomics analysis on the Expression Atlas database revealed that most genes from infected 2B4 cell line with SARS-CoV were downregulated leading to immune system hyperactivation, induction of signaling pathways, and consequently a cytokine storm. In addition, GO:0016192 (vesicle-mediated transport), GO:0006886 (intracellular protein transport), and GO:0006888 (ER to Golgi vesicle-mediated transport) were shown as top three GOs in the ontology network of infected cells with SARS-CoV. Meanwhile, R-HAS-6807070 (phosphatase and tensin homolog or PTEN regulation) showed the highest association with other Reactome pathways in the network of infected cells with SARS-CoV. PTEN plays a critical role in the activation of dendritic cells, B- and T-cells, and secretion of proinflammatory cytokines, which cooperates with downregulated genes in the promotion of cytokine storm in the COVID-19 patients.</p><p><strong>Conclusions: </strong>Based on the high similarity percentage of the transcriptome of SARS-CoV with SARS-CoV-2, the data of immunological regulations, signaling pathways, and proinflammatory cytokines in SARS-CoV infection can be expanded to COVID-19 to have a valid platform for future pharmaceutical and vaccine studies.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"16"},"PeriodicalIF":6.4,"publicationDate":"2020-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12575-020-00127-3","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38228419","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Potential Drugs and Remedies for the Treatment of COVID-19: a Critical Review.","authors":"Fatemeh Barati,&nbsp;Mahdi Pouresmaieli,&nbsp;Elena Ekrami,&nbsp;Sahar Asghari,&nbsp;Farzad Ramezani Ziarani,&nbsp;Matin Mamoudifard","doi":"10.1186/s12575-020-00129-1","DOIUrl":"10.1186/s12575-020-00129-1","url":null,"abstract":"<p><strong>Abstract: </strong>COVID-19 disease with a high rate of contagious and highly nonspecific symptoms, is an infectious disease caused by a newly discovered coronavirus. Most people who fall sick with COVID-19 will experience mild to moderate symptoms such as respiratory symptoms, cough, dyspnea, fever, and viral pneumonia and recover without any special cure. However, some others need special and emergency treatment to get rid of this widespread disease. Till now, there are numbers of proposed novel compounds as well as standards therapeutics agent existed for other conditions seems to have efficacy against the 2019-nCoV. Some which are being tested for MERS-CoV and SARS-CoV are validated that could be also efficient against this new coronavirus. However, there are currently no effective specific antivirals or drug combinations introduced for 2019-nCoV specifically that be supported by high-level evidence. The main purpose of this paper is to review typical and ongoing treatments for coronavirus disease including home remedies, herbal medicine, chemical drugs, plasma therapy, and also vaccinies. In this regards, famous herbal medicines and common chemical drugs which are routinely to be prescribed for patients are introduced. Moreover, a section is assigned to the drug interactions and some outdated drugs which have been proved to be inefficient. We hope that this work could pave the way for researchers to develop faster and more reliable methods for earlier treatment of patients and rescue more people.</p><p><strong>Graphical abstract: </strong></p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"15"},"PeriodicalIF":6.4,"publicationDate":"2020-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12575-020-00129-1","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38228418","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Efficient In Vivo Introduction of Point Mutations Using ssODN and a Co-CRISPR Approach.","authors":"Tgst Levi,&nbsp;Anna Sloutskin,&nbsp;Rachel Kalifa,&nbsp;Tamar Juven-Gershon,&nbsp;Offer Gerlitz","doi":"10.1186/s12575-020-00123-7","DOIUrl":"https://doi.org/10.1186/s12575-020-00123-7","url":null,"abstract":"<p><strong>Background: </strong>The generation of point mutations is a major tool for evaluating the roles of specific nucleotides or amino acids within the regulatory or functional landscape. However, examination of these mutations in vivo requires the generation of animals carrying only the relevant point mutations at the endogenous genomic loci, which is technically challenging. The CRISPR-Cas9 based genome editing greatly facilitates the generation of such genetically modified animals; however, most of the described methods use double-strand DNA (dsDNA) as the donor template. The dsDNA plasmids frequently undergo undesired integration events into the targeted genomic locus. The use of a single-strand oligodeoxynucleotide (ssODN) as the donor template prevents this complication and is therefore the preferred choice for introducing point mutations, as well as short sequences such as protein tags.</p><p><strong>Results: </strong>We successfully applied the CRISPR-based <i>white</i> co-conversion strategy with a ssODN template, instead of the originally described dsDNA plasmid, to create genetically modified <i>Drosophila melanogaster</i> strains. We used the technique to easily introduce point mutations in two distinct chromosomes. Using the generated flies, we were able to demonstrate the in vivo importance of the respective mutations. For the <i>Nucleoporin107</i> (<i>Nup107</i>) gene, the 1090G > A mutation was confirmed to affect ovarian development, while for the <i>tinman</i> (<i>tin</i>) gene, the regulatory role of the downstream core promoter element (DPE) was demonstrated within the developing <i>Drosophila melanogaster</i> embryo.</p><p><strong>Conclusions: </strong>The described approach has facilitated the successful generation of point mutations in two different chromosomes, by two different labs. Distinct phenotypes associated with the newly-generated genotype were identified, thus exemplifying the importance of investigating the in vivo role of specific nucleotides. In addition, detailed guidelines, recommendations and crossing schemes are provided in order to support the generation of additional genetically modified animals by the scientific community.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"14"},"PeriodicalIF":6.4,"publicationDate":"2020-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12575-020-00123-7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38176548","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bimodal Whole-Mount Imaging of Tendon Using Confocal Microscopy and X-ray Micro-Computed Tomography.","authors":"Neil Marr,&nbsp;Mark Hopkinson,&nbsp;Andrew P Hibbert,&nbsp;Andrew A Pitsillides,&nbsp;Chavaunne T Thorpe","doi":"10.1186/s12575-020-00126-4","DOIUrl":"https://doi.org/10.1186/s12575-020-00126-4","url":null,"abstract":"<p><strong>Background: </strong>Three-dimensional imaging modalities for optically dense connective tissues such as tendons are limited and typically have a single imaging methodological endpoint. Here, we have developed a bimodal procedure utilising fluorescence-based confocal microscopy and x-ray micro-computed tomography for the imaging of adult tendons to visualise and analyse extracellular sub-structure and cellular composition in small and large animal species.</p><p><strong>Results: </strong>Using fluorescent immunolabelling and optical clearing, we visualised the expression of the novel cross-species marker of tendon basement membrane, laminin-α4 in 3D throughout whole rat Achilles tendons and equine superficial digital flexor tendon 5 mm segments. This revealed a complex network of laminin-α4 within the tendon core that predominantly localises to the interfascicular matrix compartment. Furthermore, we implemented a chemical drying process capable of creating contrast densities enabling visualisation and quantification of both fascicular and interfascicular matrix volume and thickness by x-ray micro-computed tomography. We also demonstrated that both modalities can be combined using reverse clarification of fluorescently labelled tissues prior to chemical drying to enable bimodal imaging of a single sample.</p><p><strong>Conclusions: </strong>Whole-mount imaging of tendon allowed us to identify the presence of an extensive network of laminin-α4 within tendon, the complexity of which cannot be appreciated using traditional 2D imaging techniques. Creating contrast for x-ray micro-computed tomography imaging of tendon using chemical drying is not only simple and rapid, but also markedly improves on previously published methods. Combining these methods provides the ability to gain spatio-temporal information and quantify tendon substructures to elucidate the relationship between morphology and function.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"13"},"PeriodicalIF":6.4,"publicationDate":"2020-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12575-020-00126-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38118374","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Combination of Size-Exclusion Chromatography and Ultracentrifugation Improves the Proteomic Profiling of Plasma-Derived Small Extracellular Vesicles.","authors":"Rui Wei,&nbsp;Libo Zhao,&nbsp;Guanyi Kong,&nbsp;Xiang Liu,&nbsp;Shengtao Zhu,&nbsp;Shutian Zhang,&nbsp;Li Min","doi":"10.1186/s12575-020-00125-5","DOIUrl":"https://doi.org/10.1186/s12575-020-00125-5","url":null,"abstract":"<p><strong>Background: </strong>Circulating small extracellular vesicles (sEVs) and its associated proteins are of great interest in the early detection of many diseases. However, there is no gold standard for plasma sEVs isolation, especially for proteomic profiling which could be largely affected by contamination such as lipoproteins and plasma proteins. Previous studies suggested combinations of different sEVs isolation methods could improve the yield and purity of the isolated fractions. Nevertheless, there is no systematic evaluation of size-exclusion chromatography (SEC), ultracentrifugation (UC), and their combination in a proteomic perspective.</p><p><strong>Results: </strong>Plasma samples were collected from healthy individuals, and sEVs were separated by one-step SEC, one-step UC, and combining SEC with UC, respectively. Here we exhibited that the purity of sEVs was improved by SEC in contrast to traditional UC. Furthermore, by conducting a SEC procedure followed by UC, we separated sEVs with the highest purity. In the proteomic analysis, 992 protein species were identified in the plasma sEVs isolated by our novel separation method, of which several proteins are sEVs-associated proteins but hitherto never been identified in the previous studies and database, much more than plasma sEVs isolated by UC (453) or SEC (682) alone. As compared to Vesiclepedia and Exocarta databases, plasma sEVs isolated by the new procedure kept 584 previously identified sEVs-associated proteins and 360 other proteins that have not been detected before. Detailed analysis suggested that more kinds of sEVs biomarkers, such as CD9, ALIX, and FLOT1, could be identified in plasma sEVs isolated by the novel isolation method as compared to one-step UC/SEC. Furthermore, the lower abundance ranks of common contaminants, such as lipoproteins and IgG chains, in the sEVs fractions obtained by our new method as compared to one-step UC/SEC also demonstrated the purity of sEVs had been improved.</p><p><strong>Conclusions: </strong>Combining SEC with UC could significantly improve the performance of mass spectrometry-based proteomic profiling in analyzing plasma-derived sEVs.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"22 ","pages":"12"},"PeriodicalIF":6.4,"publicationDate":"2020-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12575-020-00125-5","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38092057","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}