Plasma Extracellular Vesicles Research Articles

Unlocking the diagnostic power of plasma extracellular vesicle miR-200 family in pancreatic ductal adenocarcinoma

BackgroundDistinguishing benign from malignant pancreaticobiliary disease is challenging because of the absence of reliable biomarkers. Circulating extracellular vesicles (EVs) have emerged as functional mediators between cells. Their cargos, including microRNAs (miRNAs), are increasingly acknowledged as an important source of potential biomarkers. This multicentric, prospective study aimed to establish a diagnostic plasma EV-derived miRNA signature to discriminate pancreatic ductal adenocarcinoma (PDAC) from benign pancreaticobiliary disease.MethodsPlasma EVs were isolated using size exclusion chromatography (SEC) and characterised using nanoparticle tracking analysis, electron microscopy and Western blotting. EV-RNAs underwent small RNA sequencing to discover differentially expressed markers for PDAC (n = 10 benign vs. 10 PDAC). Candidate EV-miRNAs were then validated in a cohort of 61 patients (n = 31 benign vs. 30 PDAC) by RT-qPCR. Logistic regression and optimal thresholds (Youden Index) were used to develop an EV-miR-200 family model to detect cancer. This model was tested in an independent cohort of 95 patients (n = 30 benign, 33 PDAC, and 32 cholangiocarcinoma).ResultsSmall RNA sequencing and RT-qPCR showed that EV-miR-200 family members were significantly overexpressed in PDAC vs. benign disease. Combined expression of the EV-miR-200 family showed an AUC of 0.823. In an independent validation cohort, application of this model showed a sensitivity, specificity and AUC of 100%, 88%, and 0.97, respectively, for diagnosing PDAC.ConclusionsThis is the first study to validate plasma EV-miR-200 members as a clinically-useful diagnostic biomarker for PDAC. Further validation in larger cohorts and clinical trials is essential. These findings also suggest the potential utility in monitoring response and/or recurrence.Graphical

Extracellular Vesicle MicroRNAs as Predictive Biomarkers in Postoperative Delirium After Spine Surgery: Preliminary Study.

Postoperative delirium (POD) can cause poor patient outcomes in older adults who undergo surgery. In this study, we tested plasma extracellular vesicle (EV) miRNAs obtained before the delirium event to find predictive POD biomarkers after spine surgery. We recruited patients who are more than 70 years old and have undergone spine surgery. Finally, POD patients (n = 31) were included, with no-POD patients matched in age, sex, medical history, and type of surgery (n = 31). Peripheral blood was collected from patients in the operating room after the operation was completed. EVs were isolated from plasma, and the 798 miRNA expression level from EVs was measured using a NanoString platform. Sixty-two patients were included in the study; all were Korean, 67.7% were females, and the median age was 75 years. Preoperative medical history was not statistically different between no-POD and POD patients except for hypertension and the American Society of Anesthesiologists physical status. From the miRNA profiling, we identified 142 significantly differentially expressed miRNAs in POD patients compared with no-POD patients, which are associated with psychological/neurological disorders. The top 10 differentially expressed miRNAs including miR-548ar-5p and miR-627-5p were all upregulated in POD patients and the results were validated using qRT-PCR from the independent sets of samples (n = 96). We demonstrated the potential of plasma EV-miRNAs as predictive biomarkers to identify the risk group of POD after spine surgery. It also provides opportunities for future studies investigating the role of EV-miRNAs in delirium pathology.

Increased secretion of adipocyte-derived extracellular vesicles is associated with adipose tissue inflammation and the mobilization of excess lipid in human obesity

BackgroundObesity is a worldwide epidemic characterized by adipose tissue (AT) inflammation. AT is also a source of extracellular vesicles (EVs) that have recently been implicated in disorders related to metabolic syndrome. However, our understanding of mechanistic aspect of obesity’s impact on EV secretion from human AT remains limited.MethodsWe investigated EVs from human Simpson Golabi Behmel Syndrome (SGBS) adipocytes, and from AT as well as plasma of subjects undergoing bariatric surgery. SGBS cells were treated with TNFα, palmitic acid, and eicosapentaenoic acid. Various analyses, including nanoparticle tracking analysis, electron microscopy, high-resolution confocal microscopy, and gas chromatography–mass spectrometry, were utilized to study EVs. Plasma EVs were analyzed with imaging flow cytometry.ResultsEVs from mature SGBS cells differed significantly in size and quantity compared to preadipocytes, disagreeing with previous findings in mouse adipocytes and indicating that adipogenesis promotes EV secretion in human adipocytes. Inflammatory stimuli also induced EV secretion, and altered EV fatty acid (FA) profiles more than those of cells, suggesting the role of EVs as rapid responders to metabolic shifts. Visceral AT (VAT) exhibited higher EV secretion compared to subcutaneous AT (SAT), with VAT EV counts positively correlating with plasma triacylglycerol (TAG) levels. Notably, the plasma EVs of subjects with obesity contained a higher number of adiponectin-positive EVs than those of lean subjects, further demonstrating higher AT EV secretion in obesity. Moreover, plasma EV counts of people with obesity positively correlated with body mass index and TNF expression in SAT, connecting increased EV secretion with AT expansion and inflammation. Finally, EVs from SGBS adipocytes and AT contained TAGs, and EV secretion increased despite signs of less active lipolytic pathways, indicating that AT EVs could be involved in the mobilization of excess lipids into circulation.ConclusionsWe are the first to provide detailed FA profiles of human AT EVs. We report that AT EV secretion increases in human obesity, implicating their role in TAG transport and association with adverse metabolic parameters, thereby emphasizing their role in metabolic disorders. These findings promote our understanding of the roles that EVs play in human AT biology and metabolic disorders.

P-704 Distinctive proteomic profiling and pathway analysis of plasma extracellular vesicles provided clues for their regulatory roles in the pathogenesis of polycystic ovarian syndrome

Abstract Study question To elucidate the distinctive proteomic profiling of plasma extracellular vesicles (EVs) and the pathogenic contributions in polycystic ovarian syndrome (PCOS). Summary answer In PCOS, differentially expressed proteins in the circulatory exosomes are associated with the several characteristic signaling pathways related to cardiovascular and metabolism risks. What is known already EVs from various tissues or body fluids have been shown to be involved in the pathogenesis of PCOS and might serve as potential biomarkers for the diagnosis and prognostic prediction. Most of previous studies focus on the miRNAs expression of EVs isolated from blood, endometrium and follicular fluid. However, a comprehensive quantitative protein analysis of circulatory EVs has seldom been conducted in PCOS. Whether and how the proteomic profiling of circulatory EVs is related to the characteristic phenotypic expression is also unclear. Study design, size, duration This is a case-control study conducted at the reproductive endocrinologic department of the tertiary medical center from 2019 to 2022. Blood samples were collected from sixty PCOS and thirty non-PCOS control subjects. Participants/materials, setting, methods Plasma EVs were isolated by sucrose gradient-based ultracentrifugation. Quantitative proteomic analysis was performed by tandem mass tag labeling and liquid chromatography tandem mass spectrometry (TMT) to identify differentially expressed proteins. The functions and pathways of identified proteins were analyzed with Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The correlation between the identified proteins and the clinical, hormonal, and biochemical parameters were also analyzed. Main results and the role of chance Nanoparticle tracking analysis suggested the majority of EVs were 70-190 nm in diameter, which were corresponsive to the size of exosome. Differential expressed protein (DEP) analysis revealed distinctive upregulated proteins in the circulatory exosomes of PCOS patients, includes IGF-1, HIST1H1E, PF4, HBA1, HBB, SNC73, which were enriched in nitric oxide transport and negative regulation of extrinsic apoptotic signaling pathway. Proteins which were downregulated in PCOS patients, including RAA1, RARRES2, CFHR1, LBP, IGFBP3, TPI1, SERPINA5, SNC73, were enriched in lipid transport and complement and coagulation cascades. There is significant correlation between the differentially expressed exosome proteins and several clinical phenotypes, including hypertension, hyperandrogenism and iron metabolism. The enriched pathways and characterized phenotypes have been shown to be closely related to cardiovascular and metabolic aberrations of PCOS, which could provide pathogenic clues for how the circulatory EVs proteins contribute to the disease development and progression. Limitations, reasons for caution PCOS is highly heterogeneous and the case number of our study might not be large enough to differentiate distinctive patterns among different subtypes of PCOS. The enrolled population is limited to reproductive-age Asian patients and might not be generalized to other age or ethnic groups. Wider implications of the findings Our results could be apply to further studies for biomarker identification and mechanistic researches, and to elucidate whether and how the proteomic biology of circulatory EVs is related to the pathogenesis of PCOS. Trial registration number not applicable

P-040 The characteristic of human seminal plasma extracellular vesicle (SPEVs) profiles with different sperm features in a large cohort of 309 Taiwanese men

Abstract Study question To investigate human seminal plasma extracellular vesicle profiles in different sperm characteristics and their potential clinical application. Summary answer Normozoospermic semen has significantly higher SPEV concentration than asthenozoospermic or oligozoospermic samples. Moreover, SPEV concentration was highly correlated with total motile sperm count. What is known already Seminal plasma extracellular vesicles (SPEVs) are cell-derived membrane structures that carry proteins, nucleic acid, and lipids, which can modify the constitution and biological function of sperm. However, the characteristics of SPEVs in different sperm features remained unclear. Study design, size, duration From January 2021 to September 2023, 309 men who visited the NCKUH andrology clinic agreed to collect semen samples for this study, which the Institutional Review Board has approved. Participants/materials, setting, methods Semen parameters were measured by the X1 CASA system (Bonraybio, Taiwan). SPEVs were isolated by size exclusion chromatography column and ExoQuick™. Next, the SPEVs’ concentration and size distribution were performed with nanoparticle tracking analysis. The Kruskal-Wallis test calculated comparison in multiple groups, and Dunn’s multiple comparison tests accessed group-group comparison. The Pearson correlation coefficient r of exosome concentration with individual sperm parameters was calculated. A p-value less than 0.05 was set as statistical significance. Main results and the role of chance Among them, 59 semen samples were presented with normozoospermia, 36 were oligozoospermia, 73 were asthenozoospermia, and 141 samples had no sperm in semen. Among these 141 azoospermic men, 89 men are characterized by non-obstructive azoospermia (NOA), while the other 52 men had a vasectomy. Our data showed that normozoospermic semen has significantly higher SPEV concentration than asthenozoospermic or oligozoospermic semen samples (26 x109 [IQR 17.25 x109-56.25 x109] versus 12 x109 [IQR 5.6 x109-19.5 x109] versus 15.5 x109 [IQR 8.575 x109-26.75 x109], p = 0.0001). Nevertheless, azoospermic semen has the lowest SPEV concentrations. The concentration of SPEVs from infertile men diagnosed with NOA is slightly lower than vasectomized men (3.9 x109 [IQR 1.875 x109-7.675 x109] versus 5.4 x109 [IQR 2.9 x109-8 x109], p = 0.0001). The Pearson r coefficient of SPEV concentration with sperm concentration, progressive motility, and total motile sperm count was 0.238, 0.303, and 0.451, respectively. (P < 0.0001) Limitations, reasons for caution Because our data were collected from Taiwanese men, it is not yet known whether SPEV profiles differ among races. Wider implications of the findings Human SPEV concentration and its bio-content may be a potential non-invasive biomarker to predict the male reproductive outcome. Trial registration number not applicable

Metformin modifies plasma microbial-derived extracellular vesicles in polycystic ovary syndrome with insulin resistance

IntroductionThis study investigated changes in plasma microbial-derived extracellular vesicles (EVs) in patients with polycystic ovary syndrome and insulin resistance (PCOS-IR) before and after metformin treatment, and aimed to identify bacterial taxa within EVs that were biologically and statistically significant for diagnosis and treatment.MethodsThe case–control study was conducted at Xiamen Chang Gung Hospital, Hua Qiao University. Plasma samples were collected from five PCOS-IR patients of childbearing age before and after 3 months of metformin treatment, and the samples were sequenced. The diversity and taxonomic composition of different microbial communities were analyzed through full-length 16 S glycosomal RNA gene sequencing.ResultsAfter metformin treatment, fasting plasma glucose levels and IR degree of PCOS-IR patients were significantly improved. The 16 S analysis of plasma EVs from metformin-treated patients showed higher microbial diversity. There were significant differences in EVs derived from some environmental bacteria before and after metformin treatment. Notably, Streptococcus salivarius was more abundant in the metformin-treated group, suggesting it may be a potential probiotic.DiscussionThe study demonstrated changes in the microbial composition of plasma EVs before and after metformin treatment. The findings may offer new insights into the pathogenesis of PCOS-IR and provide new avenues for research.

Optimized AF4 combined with density cushion ultracentrifugation enables profiling of high-purity human blood extracellular vesicles.

Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, the identification of EVs derived from blood samples is hindered by the presence of abundant plasma proteins, which impairs the downstream biochemical analysis of EV-associated proteins and nucleic acids. Here, we employed optimized asymmetric flow field-flow fractionation (AF4) combined with density cushion ultracentrifugation (UC) to obtain high-purity and intact EVs with very low lipoprotein contamination from human plasma and serum. Further proteomic analysis revealed more than 1000 EV-associated proteins, a large proportion of which has not been previously reported. Specifically, we found that cell-line-derived EV markers are incompatible with the identification of plasma-EVs and proposed that the proteins MYCT1, TSPAN14, MPIG6B and MYADM, as well as the traditional EV markers CD63 and CD147, are plasma-EV markers. Benefiting from the high-purity of EVs, we conducted comprehensive miRNA profiling of plasma EVs and nanosized particles (NPs), as well as compared plasma- and serum-derived EVs, which provides a valuable resource for the EV research community. Overall, our findings provide a comprehensive assessment of human blood EVs as a basis for clinical biopsy applications.

Assessing Alzheimer's disease via plasma extracellular vesicle-derived mRNA.

Alzheimer's disease (AD), the most prevalent neurodegenerative disorder globally, has emerged as a significant health concern. Recently it has been revealed that extracellular vesicles (EVs) play a critical role in AD pathogenesis and progression. Their stability and presence in various biofluids, such as blood, offer a minimally invasive window for monitoring AD-related changes. We analyzed plasma EV-derived messenger RNA (mRNA) from 82 human subjects, including individuals with AD, mild cognitive impairment (MCI), and healthy controls. With next-generation sequencing, we profiled differentially expressed genes (DEGs), identifying those associated with AD. Based on DEGs identified in both the MCI and AD groups, a diagnostic model was established based on machine learning, demonstrating an average diagnostic accuracy of over 98% and showed a strong correlation with different AD stages. mRNA derived from plasma EVs shows significant promise as a non-invasive biomarker for the early detection and continuous monitoring of AD. The study conducted next-generation sequencing (NGS) of mRNA derived from human plasma extracellular vesicles (EVs) to assess Alzheimer's disease (AD).Profiling of plasma EV-derived mRNA shows a significantly enriched AD pathway, indicating its potential for AD-related studies.The AD-prediction model achieved a receiver-operating characteristic area under the curve (ROC-AUC) of more than 0.98, with strong correlation to the established Clinical Dementia Rating (CDR).

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.

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.

Decreased Extracellular Vesicle Vasorin in Severe Preeclampsia Plasma Mediates Endothelial Dysfunction.

Preeclampsia (PE) is a serious pregnancy complication affecting 5-8% of pregnancies globally. It is a leading cause of maternal and neonatal morbidity and mortality. Despite its prevalence, the underlying mechanisms of PE remain unclear. This study aimed to determine the potential role of vasorin (VASN) in PE pathogenesis by investigating its levels in extracellular vesicles (EV) and its effects on vascular function. We conducted unbiased proteomics on urine-derived EV from severe PE (sPE) and normotensive pregnant women (NTP), identifying differential protein abundances. Out of one hundred and twenty proteins with ≥ ±1.5-fold regulation at P<0.05 between sPE and NTP, we focused on Vasorin (VASN), which is downregulated in sPE in urinary EV, in plasma EV and in the placenta and is a known regulator of vascular function. We generated EV with high VASN content from both human and murine placenta explants (Plex EV), which recapitulated disease-state-dependent effects on vascular function observed when treating murine aorta rings (MAR) or human aortic endothelial cells (HAEC) with murine or human plasma-derived EV. In normal murine pregnancy, VASN increases with gestational age (GA), and VASN is decreased in plasma EV, in placenta tissue and in Plex EV after intravenous administration of adenovirus encoding short FMS-like tyrosine kinase 1 (sFLT-1), a murine model of PE (murine-PE). VASN is decreased in plasma EV, in placenta tissue and in EV isolated from conditioned media collected from placenta explants (Plex EV) in patients with sPE as compared to NTP. Human sPE and murine-PE plasma EV and Plex EV impair migration, tube formation, and induces apoptosis in human aortic endothelial cells (HAEC) and inhibit acetylcholine-induced vasorelaxation in murine vascular rings (MAR). VASN over-expression counteracts the effects of sPE EV treatment in HAEC and MAR. RNA sequencing revealed that over-expression or knock down of VASN in HAEC results in contrasting effects on transcript levels of hundreds of genes associated with vasculogenesis, endothelial cell proliferation, migration and apoptosis. The data suggest that VASN, delivered to the endothelium via EV, regulates vascular function and that the loss of EV VASN may be one of the mechanistic drivers of PE. What is NewVASN in circulating plasma EV in sPE is reduced compared with VASN content in plasma EV of gestational age-matched pregnant women.VASN is encapsulated and transported in EV and plays a pro-angiogenic role during pregnancy.VASN should be explored both for its pro-angiogenic mechanistic role and as a novel biomarker and potential predictive diagnostic marker for the onset and severity of PE.What Are the Clinical Implications?VASN plays a role in maintaining vascular health and the normal adaptive cardiovascular response in pregnancy. A decrease of VASN is observed in sPE patients contributing to cardiovascular maladaptation.Strategies to boost diminished VASN levels and/or to pharmacologically manipulate mechanisms downstream of VASN may be explored for potential therapeutic benefit in PE.The decrease in EV-associated VASN could potentially be used as a (predictive) biomarker for PE.

A fast and sensitive size-exclusion chromatography method for plasma extracellular vesicle proteomic analysis.

Extracellular vesicles (EVs) carry diverse biomolecules derived from their parental cells, making their components excellent biomarker candidates. However, purifying EVs is a major hurdle in biomarker discovery since current methods require large amounts of samples, are time-consuming and typically have poor reproducibility. Here we describe a simple, fast, and sensitive EV fractionation method using size exclusion chromatography (SEC) on a fast protein liquid chromatography (FPLC) system. Our method uses a Superose 6 Increase 5/150, which has a bed volume of 2.9mL. The FPLC system and small column size enable reproducible separation of only 50µL of human plasma in 15min.To demonstrate the utility of our method, we used longitudinal samples from a group of individuals who underwent intense exercise. A total of 838 proteins were identified, of which, 261 were previously characterized as EV proteins, including classical markers, such as cluster of differentiation (CD)9 and CD81. Quantitative analysis showed low technical variability with correlation coefficients greater than 0.9 between replicates. The analysis captured differences in relevant EV proteins involved in response to physical activity.Our method enables fast and sensitive fractionation of plasma EVs with low variability, which will facilitate biomarker studies in large clinical cohorts.

Development of highly multiplex targeted proteomics assays in biofluids using the Stellar mass spectrometer.

The development of targeted assays that monitor biomedically relevant proteins is an important step in bridging discovery experiments to large scale clinical studies. Targeted assays are currently unable to scale to hundreds or thousands of targets. We demonstrate the generation of large-scale assays using a novel hybrid nominal mass instrument. The scale of these assays is achievable with the Stellar™ mass spectrometer through the accommodation of shifting retention times by real-time alignment, while being sensitive and fast enough to handle many concurrent targets. Assays were constructed using precursor information from gas-phase fractionated (GPF) data-independent acquisition (DIA). We demonstrate the ability to schedule methods from an orbitrap and linear ion trap acquired GPF DIA library and compare the quantification of a matrix-matched calibration curve from orbitrap DIA and linear ion trap parallel reaction monitoring (PRM). Two applications of these proposed workflows are shown with a cerebrospinal fluid (CSF) neurodegenerative disease protein PRM assay and with a Mag-Net enriched plasma extracellular vesicle (EV) protein survey PRM assay.

Characterization of spinal cord tissue-derived extracellular vesicles in neuroinflammation

Extracellular vesicles (EVs) are released by all cells, can cross the blood–brain barrier, and have been shown to play an important role in cellular communication, substance shuttling, and immune modulation. In recent years EVs have shifted into focus in multiple sclerosis (MS) research as potential plasma biomarkers and therapeutic vehicles. Yet little is known about the disease-associated changes in EVs in the central nervous system (CNS). To address this gap, we characterized the physical and proteomic changes of mouse spinal cord-derived EVs before and at 16 and 25 days after the induction of experimental autoimmune encephalomyelitis (EAE), a neuroinflammatory model of MS. Using various bioinformatic tools, we found changes in inflammatory, glial, and synaptic proteins and pathways, as well as a shift in the predicted contribution of immune and glial cell types over time. These results show that EVs provide snapshots of crucial disease processes such as CNS-compartmentalized inflammation, re/de-myelination, and synaptic pathology, and might also mediate these processes. Additionally, inflammatory plasma EV biomarkers previously identified in people with MS were also altered in EAE spinal cord EVs, suggesting commonalities of EV-related pathological processes during EAE and MS and overlap of EV proteomic changes between CNS and circulating EVs.

Non-targeted analysis of the plasma proteome between early and late rugby playing career

Plasma proteomics can offer objective biological insight into the physiological responses of athletes. Rugby players may be repeatedly exposed to high-impact collisions that could predispose them to a higher risk of neurodegenerative conditions. The potential mechanisms underlying the heightened risk are unclear and discovery proteomics could provide unbiased insight into the pathways involved. Plasma extracellular vesicles (EV) are a novel system of intercellular communication which have been implicated in the propagation of misfolded proteins in the brain and could carry putative diagnostic biomarkers. This project investigates whether differences in plasma EV proteome exist between early and late career rugby players. Twenty-four male participants were recruited, including 8 academy players (18 ± 1 years), 8 rugby players (33 ± 5 years) with 10-year professional rugby career and 8 CrossFit athletes (32 ± 5 years) with no history of collision-related sports injuries. Participants gave their informed consent to the ethically approved procedures. Venous blood samples were collected in EDTA blood sample tubes, according to standard operating procedures. Hyper-porous strong-anion exchange magnetic microparticles (MagReSyn® SAX) were used to capture membrane-bound particles (i.e. EV) from plasma. EV-enriched samples were digested with trypsin and analysed using nano-flow liquid chromatography and high-resolution tandem mass spectrometry (Thermo, U3000 and Q-Exactive mass spectrometer). Label free quantitation was performed using Progenesis QI-P (Waters Corp) and proteins were identified by Mascot (Matrix Sci) searches of the UniProt Knowledge base. Statistical differences were investigated by one-way analysis of variance (with correction for multiple testing) in R (v4.3.2). Functional enrichment analysis was performed using ShinyGO (0.80). In total, 449 proteins were confidently identified (false discovery rate; FDR &lt;1%) and gene ontology profiling confirmed 414 of these proteins were of EV origin (Jensen COMPARTMENTS). One-way ANOVA highlighted 192 significantly (P &lt; 0.05, q &lt; 0.02) different proteins across the 3 participant groups, of which 24 proteins were specific to professional rugby players. Functional enrichment analysis revealed key pathways related to Vitamin digestion and absorption, complement and coagulation cascade, inflammatory response, neuron death and negative regulation of cell death (FDR=5%).In conclusion, proteomic profiling is able to detect robust differences between the plasma EV proteome of long-serving professional rugby players compared to younger rugby players or age-matched athletes that did not have a history of collision-related sports injuries. Our findings shed new light on processes affected by a professional rugby playing career, further exploitation of this type of analysis could be used to discover sport-specific biomarkers.

Urinary extracellular vesicle-derived miR-126-3p predicts lymph node invasion in patients with high-risk prostate cancer

To investigate extracellular vesicles (EVs), biomarkers for predicting lymph node invasion (LNI) in patients with high-risk prostate cancer (HRPCa), plasma, and/or urine samples were prospectively collected from 45 patients with prostate cancer (PCa) and five with benign prostatic hyperplasia (BPH). Small RNA sequencing was performed to identify miRNAs in the EVs. All patients with PCa underwent radical prostatectomy and extended pelvic lymph node dissection. Differentially expressed miRNAs were identified in patients with and without pathologically-verified LNI. The candidate miRNAs were validated in low-risk prostate cancer (LRPCa) and BPH. Four miRNA species (e.g., miR-126-3p) and three miRNA species (e.g., miR-27a-3p) were more abundant in urinary and plasma EVs, respectively, of patients with PCa. None of these miRNA species were shared between urinary and plasma EVs. miR-126-3p was significantly more abundant in patients with HR PCa with LNI than in those without (P = 0.018). miR-126-3p was significantly more abundant in the urinary EVs of patients with HRPCa than in those with LRPCa (P = 0.017) and BPH (P = 0.011). In conclusion, urinary EVs-derived miR-126-3p may serve as a good biomarker for predicting LNI in patients with HRPCa.

A Mesoporous Gold Sensor Unveils Phospho PD-L1 in Extracellular Vesicles as a Proxy for PD-L1 Expression in Lung Cancer Tissue.

Immune checkpoint inhibitors (ICIs) targeting programmed cell death ligand 1 (PD-L1), or its receptor, PD-1 have improved survival in patients with non-small-cell lung cancer (NSCLC). Assessment of PD-L1 expression requires tissue biopsy or fine needle aspiration that are currently used to identify patients most likely to respond to single agent anti-PD-1/PD-L1 therapy. However, obtaining sufficient tissue to generate a PD-L1 tissue proportion score (TPS) ≥ 50% using immunohistochemistry remains a challenge that potentially may be overcome by liquid biopsies. This study utilized a mesoporous gold sensor (MGS) assay to examine the phosphorylation status of PD-L1 in plasma extracellular vesicles (EV pPD-L1) and PD-L1 levels in plasma from NSCLC patient samples and their association with tumor PD-L1 TPS. The 3-dimensional mesoporous network of the electrodes provides a large surface area, high signal-to-noise ratio, and a superior electro-conductive framework, thereby significantly improving the detection sensitivity of PD-L1 nanosensing. Test (n = 20) (Pearson's r = 0.99) and validation (n = 45) (Pearson's r = 0.99) cohorts show that EV pPD-L1 status correlates linearly with the tumor PD-L1 TPS assessed by immunohistochemistry irrespective of the tumor stage, with 64% of patients overall showing detectable EV pPD-L1 levels in plasma. In contrast to the EV pPD-L1 results, plasma PD-L1 levels did not correlate with the tumor PD-L1 TPS score or EV pPD-L1 levels. These data demonstrate that EV pPD-L1 levels may be used to select patients for appropriate PD-1 and PD-L1 ICI therapy regimens in early, locally advanced, and advanced NSCLC and should be tested further in randomized controlled trials. Most importantly, the assay used has a less than 24h turnaround time, facilitating adoption of the test into the routine diagnostic evaluation of patients prior to therapy.

Identification of potential candidate miRNAs related to semen quality in seminal plasma extracellular vesicles and sperms of male duck (Anas Platyrhynchos)

Semen quality is an important indicator that can directly affect fertility. In mammals, miRNAs in seminal plasma extracellular vesicles (SPEVs) and sperms can regulate semen quality. However, relevant regulatory mechanism in duck sperms remains largely unclear. In this study, duck SPEVs were isolated and characterized by transmission electron microscopy (TEM), western blot (WB), and nanoparticle tracking analysis (NTA). To identify the important molecules affecting semen quality, we analysed the miRNA expression in sperms and SPEVs of male ducks in high semen quality group ((DHS, DHSE) and low semen quality group (DLS, DLSE). We identified 94 differentially expressed (DE) miRNAs in the comparison of DHS vs. DLS, and 21 DE miRNAs in DHSE vs. DLSE. Target genes of SPEVs DE miRNAs were enriched in ErbB signaling pathway, glycometabolism, and ECM-receptor interaction pathways (P < 0.05), while the target genes of sperm DE miRNAs were enriched in ribosome (P < 0.05). The miRNA-target-pathway interaction network analyses indicated that 5 DE miRNAs (miR-34c-5p, miR-34b-3p, miR-449a, miR-31-5p, and miR-128-1-5p) targeted the largest number of target genes enriched in MAPK, Wnt and calcium signaling pathways, of which FZD9 and ANAPC11 were involved in multiple biological processes related to sperm functions, indicating their regulatory effects on sperm quality. The comparison of DE miRNAs of SPEVs and sperms found that mir-31-5p and novel-273 could potentially serve as biomarkers for semen quality detection. Our findings enhance the insight into the crucial role of SPEV and sperm miRNAs in regulating semen quality and provide a new perspective for subsequent studies.

Plasma and synovial fluid extracellular vesicles display altered microRNA profiles in horses with naturally occurring post-traumatic osteoarthritis: an exploratory study.

The objective of this study was to characterize extracellular vesicles (EVs) in plasma and synovial fluid obtained from horses with and without naturally occurring post-traumatic osteoarthritis (PTOA). EVs were isolated from plasma and synovial fluid from horses with (n = 6) and without (n = 6) PTOA. Plasma and synovial fluid EVs were characterized with respect to quantity, size, and surface markers. Small RNA sequencing was performed, and differentially expressed microRNAs (miRNAs) underwent bioinformatic analysis to identify putative targets and to explore potential associations with specific biological processes. Plasma and synovial fluid samples from horses with PTOA had a significantly higher proportion of exosomes and a lower proportion of microvesicles compared to horses without PTOA. Small RNA sequencing revealed several differentially expressed miRNAs, including miR-144, miR-219-3p, and miR-199a-3l in plasma and miR-199a-3p, miR-214, and miR-9094 in synovial fluid EVs. Bioinformatics analysis of the differentially expressed miRNAs highlighted their potential role in fibrosis, differentiation of chondrocytes, apoptosis, and inflammation pathways in PTOA. We have identified dynamic molecular changes in the small noncoding signatures of plasma and synovial fluid EVs in horses with naturally occurring PTOA. These findings could serve to identify promising biomarkers in the pathogenesis of PTOA, to facilitate the development of targeted therapies, and to aid in establishing appropriate translational models of PTOA.

Plasma extracellular vesicle tau and TDP-43 as diagnostic biomarkers in FTD and ALS

Minimally invasive biomarkers are urgently needed to detect molecular pathology in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Here, we show that plasma extracellular vesicles (EVs) contain quantifiable amounts of TDP-43 and full-length tau, which allow the quantification of 3-repeat (3R) and 4-repeat (4R) tau isoforms. Plasma EV TDP-43 levels and EV 3R/4R tau ratios were determined in a cohort of 704 patients, including 37 genetically and 31 neuropathologically proven cases. Diagnostic groups comprised patients with TDP-43 proteinopathy ALS, 4R tauopathy progressive supranuclear palsy, behavior variant FTD (bvFTD) as a group with either tau or TDP-43 pathology, and healthy controls. EV tau ratios were low in progressive supranuclear palsy and high in bvFTD with tau pathology. EV TDP-43 levels were high in ALS and in bvFTD with TDP-43 pathology. Both markers discriminated between the diagnostic groups with area under the curve values >0.9, and between TDP-43 and tau pathology in bvFTD. Both markers strongly correlated with neurodegeneration, and clinical and neuropsychological markers of disease severity. Findings were replicated in an independent validation cohort of 292 patients including 34 genetically confirmed cases. Taken together, the combination of EV TDP-43 levels and EV 3R/4R tau ratios may aid the molecular diagnosis of FTD, FTD spectrum disorders and ALS, providing a potential biomarker to monitor disease progression and target engagement in clinical trials.

KRAS mutations allelic frequency dynamics using plasma extracellular vesicles: Association with survival outcomes in localized pancreatic adenocarcinoma.

KRAS mutations allelic frequency dynamics using plasma extracellular vesicles: Association with survival outcomes in localized pancreatic adenocarcinoma.