Journal of extracellular biology最新文献

{"title":"Optimization of ultracentrifugation-based method to enhance the purity and proteomic profiling depth of plasma-derived extracellular vesicles and particles","authors":"Zurong Wan,&nbsp;Jinghua Gu,&nbsp;Uthra Balaji,&nbsp;Linda Bojmar,&nbsp;Henrik Molina,&nbsp;Søren Heissel,&nbsp;Alexandra E. Pagano,&nbsp;Christopher Peralta,&nbsp;Lee Shaashua,&nbsp;Dorina Ismailgeci,&nbsp;Hope K. Narozniak,&nbsp;Yi Song,&nbsp;William R. Jarnagin,&nbsp;David P. Kelsen,&nbsp;Jaqueline Bromberg,&nbsp;Virginia Pascual,&nbsp;Haiying Zhang","doi":"10.1002/jex2.167","DOIUrl":"10.1002/jex2.167","url":null,"abstract":"<p>Circulating extracellular vesicles and particles (EVPs) are being investigated as potential biomarkers for early cancer detection, prognosis, and disease monitoring. However, the suboptimal purity of EVPs isolated from peripheral blood plasma has posed a challenge of in-depth analysis of the EVP proteome. Here, we compared the effectiveness of different methods for isolating EVPs from healthy donor plasma, including ultracentrifugation (UC)-based protocols, phosphatidylserine-Tim4 interaction-based affinity capture (referred to as “PS”), and several commercial kits. Modified UC methods with an additional UC washing or size exclusion chromatography step substantially improved EVP purity and enabled the detection of additional proteins via proteomic mass spectrometry, including many plasma membrane and cytoplasmic proteins involved in vesicular regulation pathways. This improved performance was reproduced in cancer patient plasma specimens, resulting in the identification of a greater number of differentially expressed EVP proteins, thus expanding the range of potential biomarker candidates. However, PS and other commercial kits did not outperform UC-based methods in improving plasma EVP purity. PS yielded abundant contaminating proteins and a biased enrichment for specific EVP subsets, thus unsuitable for proteomic profiling of plasma EVPs. Therefore, we have optimized UC-based protocols for circulating EVP isolation, which enable further in-depth proteomic analysis for biomarker discovery.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 7","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11263976/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141753473","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of resazurin phenoxazine dye as a highly sensitive cell viability potency assay for natural killer cell-derived extracellular vesicle-based cancer biotherapeutics","authors":"Frederic St-Denis-Bissonnette,&nbsp;Shirley Qiu,&nbsp;Sarah E. Cummings,&nbsp;Melanie Kirkby,&nbsp;Yohannes Haile,&nbsp;Sarah Wassmer,&nbsp;Gauri Muradia,&nbsp;Jelica Mehic,&nbsp;Andrew Stalker,&nbsp;Amit Shrestha,&nbsp;Michele Ardolino,&nbsp;Seung-Hwan Lee,&nbsp;Dylan Burger,&nbsp;Lisheng Wang,&nbsp;Jessie R. Lavoie","doi":"10.1002/jex2.166","DOIUrl":"10.1002/jex2.166","url":null,"abstract":"<p>Natural killer cell-derived extracellular vesicles (NK-EVs) are candidate biotherapeutics against various cancers. However, standardised potency assays are necessary for a reliable assessment of NK-EVs' cytotoxicity. This study aims to thoroughly evaluate a highly sensitive resazurin phenoxazine-based cell viability potency assay (measurement of the cellular redox metabolism) for quantifying the cytotoxicity of NK-EVs against leukaemia K562 cells (suspension model) and breast cancer MDA-MB-231 cells (adherent model) in vitro. The assay was evaluated based on common analytical parameters setforth by regulatory guidelines, including specificity, selectivity,accuracy, precision, linearity, range and stability. Our results revealed that this resazurin-based cell viability potency assay reliably and reproducibly measured a dose-response of NK-EVs’ cytotoxic activity against both cancer models. The assay showed precision with 5% and 20% variation for intra-run and inter-run variability. The assay signal showed specificity and selectivity of NK-EVs against cancer target cells, as evidenced by the diminished viability of cancer cells following a 5-hour treatment with NK-EVs, without any detectable interference or background. The linearity analysis of target cancer cells revealed strong linearity for densities of 5000 K562 and 1000 MDA-MB-231 cells per test with a consistent range. Importantly, NK-EVs’ dose-response for cytotoxicity showed a strong correlation (|<i>ρ</i>| ∼ 0.8) with the levels of known cytotoxic factors associated with the NK-EVs’ corona (FasL, GNLY, GzmB, PFN and IFN-γ), thereby validating the accuracy of the assay. The assay also distinguished cytotoxicity changes in degraded NK-EVs, indicating the ability of the assay to detect the potential loss of sample integrity. Compared to other commonly reported bioassays (i.e., flow cytometry, cell counting, lactate dehydrogenase release assay, DNA-binding reporter assay and confluence assay), our results support this highly sensitive resazurin-based viability potency assay as a high-throughput and quantitative method for assessing NK-EVs’ cytotoxicity against both suspension and adherent cancer models for evaluating NK-EVs’ biotherapeutics.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 7","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11253028/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141636023","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mechanical property estimation of sarcoma-relevant extracellular vesicles using transmission electron microscopy","authors":"Premanshu Kumar Singh,&nbsp;Patricia Sarchet,&nbsp;Catherine Hord,&nbsp;Lucia Casadei,&nbsp;Raphael Pollock,&nbsp;Shaurya Prakash","doi":"10.1002/jex2.158","DOIUrl":"10.1002/jex2.158","url":null,"abstract":"<p>Analysis of single extracellular vesicles (EVs) has the potential to yield valuable label-free information on their morphological structure, biomarkers and therapeutic targets, though such analysis is hindered by the lack of reliable and quantitative measurements of the mechanical properties of these compliant nanoscale particles. The technical challenge in mechanical property measurements arises from the existing tools and methods that offer limited throughput, and the reported elastic moduli range over several orders of magnitude. Here, we report on a flow-based method complemented by transmission electron microscopy (TEM) imaging to provide a high throughput, whole EV deformation analysis for estimating the mechanical properties of liposarcoma-derived EVs as a function of their size. Our study includes extracting morphological data of EVs from a large dataset of 432 TEM images, with images containing single to multiple EVs, and implementing the thin-shell deformation theory. We estimated the elastic modulus, <i>E</i> = 0.16 ± 0.02 MPa (mean±SE) for small EVs (sEVs; 30–150 nm) and <i>E</i> = 0.17 ± 0.03 MPa (mean±SE) for large EVs (<i>l</i>EVs; &gt;150 nm). To our knowledge, this is the first report on the mechanical property estimation of LPS-derived EVs and has the potential to establish a relationship between EV size and EV mechanical properties.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 7","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11222873/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141536086","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterisation of extracellular vesicles in baculovirus infection of Spodoptera frugiperda cells","authors":"Lex J. C. Van Es,&nbsp;Robert D. Possee,&nbsp;Linda A. King","doi":"10.1002/jex2.163","DOIUrl":"10.1002/jex2.163","url":null,"abstract":"<p><i>Autographa californica</i> multiple nucleopolyhedrovirus (AcMNPV) is an enveloped DNA virus of the <i>Baculoviridae</i> family. This baculovirus is widely exploited for the biological control of insect pest species and as an expression platform to produce recombinant proteins in insect cells. Extracellular vesicles (EVs) are secreted by all cells and are involved in key roles in many biological processes through their cargo consisting of proteins, RNA or DNA. In viral infections, EVs have been found to transfer both viral and cellular cargo that can elicit either a pro- or antiviral response in recipient cells. Here, small EVs (sEVs) released by <i>Spodoptera frugiperda</i> (Sf) insect cells were characterised for the first time. Using <i>S. frugiperda</i> (SfC1B5) cells stably expressing the baculovirus <i>gp64</i>, the viral envelope protein GP64 was shown to be incorporated into sEVs. Sf9 cells were also transfected with a bacmid AcMNPV genome lacking <i>p6.9</i> (AcΔP6.9) to prevent budded virus production. The protein content of sEVs from both mock- and AcΔP6.9-transfected cells were analysed by mass spectrometry. In addition to GP64, viral proteins Ac-F, ME-53 and viral ubiquitin were identified, as well as many host proteins including TSG101—which may be useful as a protein marker for sEVs.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 7","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11212295/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141473245","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Towards IVDR-compliance by implementing quality control steps in a quantitative extracellular vesicle-miRNA liquid biopsy assay for response monitoring in patients with classic Hodgkin lymphoma","authors":"Esther E. E. Drees,&nbsp;Nils J. Groenewegen,&nbsp;Sandra A. W. M. Verkuijlen,&nbsp;Monique A. J. van Eijndhoven,&nbsp;Jip Ramaker,&nbsp;Pepijn Veenstra,&nbsp;Mirjam Hussain,&nbsp;Catharina G. M. Groothuis-Oudshoorn,&nbsp;Daphne de Jong,&nbsp;Josée M. Zijlstra,&nbsp;Johan de Rooij,&nbsp;D. Michiel Pegtel","doi":"10.1002/jex2.164","DOIUrl":"10.1002/jex2.164","url":null,"abstract":"<p>Previously, we showed that quantification of lymphoma-associated miRNAs miR-155-5p, -127-3p and let-7a-5p levels in plasma extracellular vesicles (EVs) report treatment response in patients with classic Hodgkin lymphoma (cHL). Prior to clinical implementation, quality control (QC) steps and validation are required to meet international regulatory standards. Most published EV-based diagnostic assays have yet to meet these requirements. In order to advance the assay towards regulatory compliance (e.g., IVDR 2017/746), we incorporated three QC steps in our experimental EV-miRNA quantitative real-time reverse-transcription PCR (q-RT-PCR) assay in an ISO-13485 certified quality-management system (QMS). Liposomes encapsulated with a synthetic (nematode-derived) miRNA spike-in controlled for EV isolation by automated size-exclusion chromatography (SEC). Additional miRNA spike-ins controlled for RNA isolation and cDNA conversion efficiency. After deciding on quality criteria, in total 107 out of 120 samples from 46 patients passed QC. Generalized linear mixed-effect modelling with bootstrapping determined the diagnostic performance of the quality-controlled data at an area under the curve (AUC) of 0.84 (confidence interval [CI]: 0.76–0.92) compared to an AUC of 0.87 (CI: 0.80–0.94) of the experimental assay. After the inclusion of QC steps, the accuracy of the assay was determined to be 78.5% in predicting active disease status in cHL patients during treatment. We demonstrate that a quality-controlled plasma EV-miRNA assay is technically robust, taking EV-miRNA as liquid biopsy assay an important step closer to clinical evaluation.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 7","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11213689/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141473247","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Acute neuroinflammation promotes a metabolic shift that alters extracellular vesicle cargo in the mouse brain cortex","authors":"Natasha Vassileff,&nbsp;Jereme G. Spiers,&nbsp;Juliani Juliani,&nbsp;Rohan G. T. Lowe,&nbsp;Keshava K. Datta,&nbsp;Andrew F. Hill","doi":"10.1002/jex2.165","DOIUrl":"10.1002/jex2.165","url":null,"abstract":"<p>Neuroinflammation is initiated through microglial activation and cytokine release which can be induced through lipopolysaccharide treatment (LPS) leading to a transcriptional cascade culminating in the differential expression of target proteins. These differentially expressed proteins can then be packaged into extracellular vesicles (EVs), a form of cellular communication, further propagating the neuroinflammatory response over long distances. Despite this, the EV proteome in the brain, following LPS treatment, has not been investigated. Brain tissue and brain derived EVs (BDEVs) isolated from the cortex of LPS-treated mice underwent thorough characterisation to meet the minimal information for studies of extracellular vesicles guidelines before undergoing mass spectrometry analysis to identify the differentially expressed proteins. Fourteen differentially expressed proteins were identified in the LPS brain tissue samples compared to the controls and 57 were identified in the BDEVs isolated from the LPS treated mice compared to the controls. This included proteins associated with the initiation of the inflammatory response, epigenetic regulation, and metabolism. These results allude to a potential link between small EV cargo and early inflammatory signalling.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 7","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11212288/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141473244","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"P2RX7 plays a critical role in extracellular vesicle-mediated secretion of pathogenic molecules from microglia and astrocytes","authors":"Mohammad Abdullah,&nbsp;Zhi Ruan,&nbsp;Seiko Ikezu,&nbsp;Tsuneya Ikezu","doi":"10.1002/jex2.155","DOIUrl":"10.1002/jex2.155","url":null,"abstract":"<p>Extracellular vesicle (EV) secretion is mediated by purinergic receptor P2X₇ (P2RX7), an ATP-gated cation channel highly expressed in microglia. We have previously shown that administration of GSK1482160, a P2RX7 selective inhibitor, suppresses EV secretion from murine microglia and prevents tauopathy development, leading to the recovery of the hippocampal function in PS19 mice, expressing P301S tau mutant. It is yet unknown, however, whether the effect of GSK1482160 on EV secretion from glial cells is specifically regulated through P2RX7. Here we tested GSK1482160 on primary microglia and astrocytes isolated from C57BL/6 (WT) and <i>P2rx7^–/–</i> mice and evaluated their EV secretion and phagocytotic activity of aggregated human tau (hTau) under ATP stimulation. GSK1482160 treatment and deletion of <i>P2rx7</i> significantly reduced secretion of small and large EVs in microglia and astrocytes in both ATP stimulated or unstimulated condition as determined by nanoparticle tracking analysis, CD9 ELISA and immunoblotting of Tsg101 and Flotilin 1 using isolated EVs. GSK1482160 treatment had no effect on EV secretion from <i>P2rx7</i>^–/– microglia while we observed significant reduction in the secretion of small EVs from <i>P2rx7</i>^–/– astrocytes, suggesting its specific targeting of P2RX7 in EV secretion except small EV secretion from astrocytes. Finally, deletion of <i>P2rx7</i> suppressed IL-1β secretion and phagocytosed misfolded tau from both microglia and astrocytes. Together, these findings show that GSK1482160 suppresses EV secretion from microglia and astrocytes in P2RX7-dependment manner, and P2RX7 critically regulates secretion of IL-1β and misfolded hTau, demonstrating as the viable target of suppressing EV-mediated neuroinflammation and tau propagation.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 7","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11212328/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141473246","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bone marrow-fibroblast progenitor cell-derived small extracellular vesicles promote cardiac fibrosis via miR-21-5p and integrin subunit αV signalling","authors":"Prabhat Ranjan,&nbsp;Roshan Kumar Dutta,&nbsp;Karen Colin,&nbsp;Jing Li,&nbsp;Qinkun Zhang,&nbsp;Hind Lal,&nbsp;Gangjian Qin,&nbsp;Suresh Kumar Verma","doi":"10.1002/jex2.152","DOIUrl":"10.1002/jex2.152","url":null,"abstract":"<p>Cardiac fibrosis is the hallmark of cardiovascular disease (CVD), which is leading cause of death worldwide. Previously, we have shown that interleukin-10 (IL10) reduces pressure overload (PO)-induced cardiac fibrosis by inhibiting the recruitment of bone marrow fibroblast progenitor cells (FPCs) to the heart. However, the precise mechanism of FPC involvement in cardiac fibrosis remains unclear. Recently, exosomes and small extracellular vesicles (sEVs) have been linked to CVD progression. Thus, we hypothesized that pro-fibrotic miRNAs enriched in sEV-derived from IL10 KO FPCs promote cardiac fibrosis in pressure-overloaded myocardium. Small EVs were isolated from FPCs cultured media and characterized as per MISEV-2018 guidelines. Small EV's miRNA profiling was performed using Qiagen fibrosis-associated miRNA profiler kit. For functional analysis, sEVs were injected in the heart following TAC surgery. Interestingly, TGFβ-treated IL10-KO-FPCs sEV increased profibrotic genes expression in cardiac fibroblasts. The exosomal miRNA profiling identified miR-21a-5p as the key player, and its inhibition with antagomir prevented profibrotic signalling and fibrosis. At mechanistic level, miR-21a-5p binds and stabilizes <i>ITGAV (</i>integrin av) mRNA. Finally, miR-21a-5p-silenced in sEV reduced PO-induced cardiac fibrosis and improved cardiac function. Our study elucidates the mechanism by which inflammatory FPC-derived sEV exacerbate cardiac fibrosis through the miR-21a-5p/ITGAV/Col1α signalling pathway, suggesting miR-21a-5p as a potential therapeutic target for treating hypertrophic cardiac remodelling and heart failure.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.152","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141441300","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Amniotic fluid-derived small extracellular vesicles for predicting postnatal severe outcome of congenital diaphragmatic hernia","authors":"Seiko Matsuo,&nbsp;Akira Yokoi,&nbsp;Kosuke Yoshida,&nbsp;Masami Kitagawa,&nbsp;Eri Asano-Inami,&nbsp;Mayo Miura,&nbsp;Takao Yasui,&nbsp;Sho Tano,&nbsp;Takafumi Ushida,&nbsp;Kenji Imai,&nbsp;Hiroaki Kajiyama,&nbsp;Tomomi Kotani","doi":"10.1002/jex2.160","DOIUrl":"10.1002/jex2.160","url":null,"abstract":"<p>Congenital diaphragmatic hernia (CDH) is a life-threatening condition with high morbidity and mortality rates. The survival rate of neonates with severe CDH is reportedly only 10%–15%. However, prenatal prediction of severe cases is difficult, and the discovery of new predictive markers is an urgent issue. In this study, we focused on microRNAs (miRNAs) in amniotic fluid-derived small EVs (AF-sEVs). We identified four miRNAs (hsa-miR-127-3p, hsa-miR-363-3p, hsa-miR-493-5p, and hsa-miR-615-3p) with AUC &gt; 0.8 to classify good prognosis group and poor prognosis group in human study. The AUC for hsa-miR-127-3p and hsa-miR-615-3p, for predicting the poor prognosis, were 0.93 and 0.91, respectively. In addition, in the in vivo study, the miRNA profiles of the lung tissues of CDH rats were different from those of control rats. Additionally, two elevated miRNAs (rno-miR-215-5p and rno-miR-148a-3p) in the lung tissues of CDH rats were increased in the AF-sEVs of CDH rats. Our results suggest that severe CDH neonates can be predicted prenatally with high accuracy using miRNAs contained in AF-sEVs. Furthermore, miRNA profile changes in AF-sEVs reflected the lung status in CDH. Our findings may contribute to the development of advanced perinatal care for patients with CDH.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.160","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141439550","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterisation of sRNAs enriched in outer membrane vesicles of pathogenic Flavobacterium psychrophilum causing Bacterial Cold Water Disease in rainbow trout","authors":"Pratima Chapagain,&nbsp;Ali Ali,&nbsp;Destaalem T. Kidane,&nbsp;Mary Farone,&nbsp;Mohamed Salem","doi":"10.1002/jex2.161","DOIUrl":"10.1002/jex2.161","url":null,"abstract":"<p><i>Flavobacterium psychrophilum</i> (<i>Fp</i>) causes Bacterial Cold Water Disease in salmonids. During host-pathogen interactions, gram-negative bacteria, such as <i>Fp</i>, release external membrane vesicles (OMVs) harbouring cargos, such as DNA, RNA and virulence factors. This study aimed to characterise the potential role of the OMVs’ small RNAs (sRNAs) in the <i>Fp</i>-rainbow trout host-pathogen interactions. sRNAs carried within OMVs were isolated from <i>Fp</i>. RNA-Seq datasets from whole-cell <i>Fp</i> and their isolated OMVs indicated substantial enrichment of specific sRNAs in the OMVs compared to the parent cell. Many of the OMV-packaged sRNAs were located in the pathogenicity islands of <i>Fp</i>. Conservation of sRNAs in 65 strains with variable degrees of virulence was reported. Dual RNA-Seq of host and pathogen transcriptomes on day 5 post-infection of <i>Fp</i> -resistant and -susceptible rainbow trout genetic lines revealed correlated expression of OMV-packaged sRNAs and their predicted host's immune gene targets. In vitro, treatment of the rainbow trout epithelial cell line RTgill-W1 with OMVs showed signs of cytotoxicity accompanied by dynamic changes in the expression of host genes when profiled 24 h following treatment. The OMV-treated cells, similar to the <i>Fp</i> -resistant fish, showed downregulated expression of the suppressor of cytokine signalling 1 (SOCS1) gene, suggesting induction of phagosomal maturation. Other signs of modulating the host gene expression following OMV-treatment include favouring elements from the phagocytic, endocytic and antigen presentation pathways in addition to HSP70, HSP90 and cochaperone proteins, which provide evidence for a potential role of OMVs in boosting the host immune response. In conclusion, the study identified novel microbial targets and inherent characteristics of OMVs that could open up new avenues of treatment and prevention of fish infections.</p>","PeriodicalId":73747,"journal":{"name":"Journal of extracellular biology","volume":"3 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jex2.161","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141435646","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}