EV Concentration Research Articles

Abstract 284: Transcriptional Profiling Of Human Abdominal Aortic Aneurysm Tissue Reveals Distinct Extracellular Vesicle-Derived MicroRNA Cargo

Aim: Abdominal aortic aneurysm (AAA) contributes to significant post-rupture mortality in aging populations. AAA management with watchful waiting and surgical repair is based on our limited understanding of disease processes, and current research foci including extracellular vesicles (EVs; nano-sized packages of proteins, RNAs, and lipids that facilitate intercellular communication) may aid in developing novel AAA therapies. To characterize this regulatory cargo, we isolated EVs from human AAA tissue or control aortic punch biopsies and profiled EV content with microRNA (miRNA) sequencing to identify dysregulated pathways. Methods: The study was approved by the University Health Network Research Ethics Board. EVs were isolated from human AAA or aortic punch tissue and enriched using size exclusion chromatography (SEC; qEVoriginal columns 70 nm, Izon Science Ltd.) (n=3). EV size and concentration were determined using nanoparticle tracking analysis (NTA; NanoSight NS300, Malvern Panalytical Ltd.). EV-miRNA sequencing was performed with Illumina NextSeq (HTG Molecular Diagnostics Inc.) and analyzed using Partek Genomics Suite (v.10) and MIENTURNET (19-11-25). Results: Patients were selected for infrarenal AAA requiring surgical repair (AAA) or coronary artery disease requiring bypass graft surgery (control). EV size and concentration were confirmed with NTA. Principal components and gene set analyses revealed distinct clustering of tissue types with 901 and 687 miRNAs enriched in AAA and control samples, respectively. Pathway prediction using established AAA miRNAs (e.g., miR-122, miR-146a, and miR-503) identified significant interactions with proaneurysmal signaling pathways (e.g., PI3K-AKT, JAK-STAT, and HIF-1) as well as cell senescence and adhesion processes (FDR < 0.05). Conclusion: EV-derived miRNAs from patients with AAA prominently associate with cell signaling, senescence, and adhesion pathways in aneurysm pathogenesis. To our knowledge, this is the first study to profile the EV-miRNA landscape in human AAA tissue. Further investigation will explore EV-miRNAs as mediators of communication between distinct vascular cell populations that contribute to AAA development.

Quantitative and Multiplex Detection of Extracellular Vesicle-Derived MicroRNA via Rolling Circle Amplification within Encoded Hydrogel Microparticles.

Extracellular vesicle-derived microRNA (EV-miRNA) represent a promising cancer biomarker for disease diagnosis and monitoring. However, existing techniques to detect EV-miRNA rely on complex, bias-prone strategies, and preprocessing steps, making absolute quantification highly challenging. This work demonstrates the development and application of a method for quantitative and multiplex detection of EV-miRNA, via rolling circle amplification within encoded hydrogel particles. By a one-pot extracellular vesicle lysis and microRNA capture step, the bias and losses associated with standard RNA extraction techniques is avoided. The system offers a large dynamic range (3 orders of magnitude), ease of multiplexing, and a limit of detection down to 2.3 zmol (46 × 10-18 m), demonstrating its utility in clinical applications based on liquid biopsy tests. Furthermore, orthogonal measurements of EV concentrations coupled with the direct, absolute quantification of miRNA in biological samples results in quantitative measurements of miRNA copy numbers per volume sample, and per extracellular vesicle.

Proteomic analysis of extracellular vesicles secreted by primary human epithelial endometrial cells reveals key proteins related to embryo implantation

BackgroundSuccessful implantation is dependent on coordination between maternal endometrium and embryo, and the role of EVs in the required cross-talk cell-to-cell has been recently established. In this regard, it has been reported that EVs secreted by the maternal endometrium can be internalized by human trophoblastic cells transferring their contents and enhancing their adhesive and invasive capacity. This is the first study to comprehensively evaluate three EV isolation methods on human endometrial epithelial cells in culture and to describe the proteomic content of EVs secreted by pHEECs from fertile women.MethodsIshikawa cells and pHEECs were in vitro cultured and hormonally treated; subsequently, conditioned medium was collected and EVs isolated. Ishikawa cells were used for the comparison of EVs isolation methods ultracentrifugation, ExoQuick-TC and Norgen Cell Culture Media Exosome Purification Kit (n = 3 replicates/isolation method). pHEECs were isolated from endometrial biopsies (n = 8/replicate; 3 replicates) collected from healthy oocyte donors with confirmed fertility, and protein content of EVs isolated by the most efficient methodology was analysed using liquid chromatography–tandem mass spectrometry. EV concentration and size were analyzed by nanoparticle tracking analysis, EV morphology visualized by transmission electron microscopy and protein marker expression was determined by Western blotting.ResultsUltracentrifugation was the most efficient methodology for EV isolation from medium of endometrial epithelial cells. EVs secreted by pHEECs and isolated by ultracentrifugation were heterogeneous in size and expressed EV protein markers HSP70, TSG101, CD9, and CD81. Proteomic analysis identified 218 proteins contained in these EVs enriched in biological processes involved in embryo implantation, including cell adhesion, differentiation, communication, migration, extracellular matrix organization, vasculature development, and reproductive processes. From these proteins, 82 were selected based on their functional relevance in implantation success as possible implantation biomarkers.ConclusionsEV protein cargos are implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development, supporting the concept of a communication system between the embryo and the maternal endometrium via EVs. Identified proteins may define new biomarkers of endometrial receptivity and implantation success.

Serum CD203c+ Extracellular Vesicle Serves as a Novel Diagnostic and Prognostic Biomarker for Succinylated Gelatin Induced Perioperative Hypersensitive Reaction.

BackgroundPerioperative hypersensitivity reaction (HR) is an IgE-FcϵRI-mediated hypersensitivity reaction with degranulation and activation of mast cells and basophils. Several studies have focused on assessing the degranulation and activation of mast cells and basophils to diagnose and predict the prognosis of drug induced HR. However, it is challenging to isolate sufficiently pure mast cells and basophils from human sources to investigate. Effective biomarkers to assess mast cells and basophils activation in vivo could potentially have high diagnostic and prognostic values. In the present study, we investigated EVs pelleted from serum in patients with succinylated gelatin induced HR.MethodsExtracellular vesicles (EVs) were isolated using a total exosome isolation kit and ultracentrifugation, characterized by Western blot, transmission electron microscopy, and nanoparticle tracking analysis. Basophils were isolated from fresh peripheral blood by negative selection using Basophil Isolation Kit II. Human mast cell line was stimulated with IL4. The expression levels of proteins related to the hypersensitive response were evaluated by Western blotting and flow Cytometer. Histamine and tryptase levels were tested using a commercial ELISA kit, and gene expression of inflammatory mediators was evaluated by qRT-PCR. The receiver operating characteristic (ROC) curve was used to evaluate the specificity and sensitivity of biomarker in predicting HR.ResultsThe concentration of EVs and protein expression level of CD63, FcϵRI, CD203c and tryptase were significantly (p< 0.05) increased in HR samples. The expression level of mast cell/basophil specific CD203c were significantly increased in EVs derived from serum and basophils of HR patients, and the CD203c+-EVs production in mast cells is dramatically increased in the presence of IL4, which positively correlated with histamine, tryptase and inflammatory mediators. Moreover, the ROC curve of EVs concentration and CD203c expression indicated that CD203c+-EVs had a strong diagnostic ability for HR.ConclusionSerum CD203c+-EVs serves as a novel diagnostic and prognostic biomarker for HR.

P-262 Staining plasma with lipophilic dye followed by size-exclusion chromatography, immune-capturing and on-bead flow cytometry is a highly sensitive approach to quantifying colorectal cancer derived extracellular nanovesicles

Development of new and effective methods for screening and early diagnosis of colon cancer is an important scientific and technological issue. There is a tendency to move from invasive methods to liquid biopsy. Extracellular nanovesicles (ENVs) secreted by colon cancer cells may serve as a marker of disease. Standard approaches to analyze cancer-derived ENVs in plasma suppose consequent steps of ENV isolation followed by immune-based labeling and quantification. However, at the early stage of cancer development, the amount of ENVs derived from cancer cells is supposedly not sufficient to be detected by available methods. Low sensitivity of currently available methods and low concentration of colon cancer-derived ENVs (cc-ENVs) compromise overall ENV- based approach for early cancer diagnosis. To solve this issue, we explored ‘inverse’ technology of cc-ENV quantification. This approach supposes three consequent steps: firstly, the labelling of all membranous components of plasma with lipophilic dye (CM-Dil); secondly, the isolation of population of ENVs formed by endosomal route (exosomes) by size-exclusion chromatography; thirdly, the quantification of tumor-specific fraction of these vesicles by flow cytometry with immune-beads. To prove the concept of this approach, we isolated ENVs from colon cancer cells (COLO 320) by ultracentrifugation according to the official rules of MISEV2018. EV concentrations were estimated by the NTA (NanoSight NS300). 20*1010, 10*1010 and 1*1010 EVs were added to the same amount ml of healthy donor blood plasma. The mixtures were colored and purified using the developed method. To isolate and quantify ccENVs, we first defined markers present in surface of ENVs secreted by COLO320 cells (GPA33 and RegIV) and created immune-bead decorated with antibodies specific to these markers. EVs were analyzed by flow cytometry (CytoFLEX) for the above markers with immune beads. GPA33(+) and RegIV (+) ENVs were isolated from mixture of CM-Dil-labeled ENVs and assayed by flow cytometry. The analysis of PE-positive events was carried out by FlowJo 10.7.1 software. The amount of GPA33(+) and RegIV (+) ENVs derived from colon cancer cells and estimated by developed method had obvious dose-dependency. The 20*1010, 10*1010 and 1*1010 cc-ENVs diluted in 2 mL of healthy donor’s plasma (which had a background signal without cc-ENVs) were quantified as 272, 177, 119 (CV, %) and 346, 179, 83 (CV, %), respectively. Since the portion of artificially mixed cc-ENVs could be estimated in a range of 0,1 – 1% of the total amount of plasma ENVs, proposed methods provide exceptional sensitivity for cc-ENV detection and might by applied for cancer screening. The proposed technique allowed for the quantification of minimal numbers of specific colon cancer-derived ENVs in plasma and can be applied in the frame of ENV-based methods of colon cancer diagnosis. The new technology may also be used to improve sensitivity of ENV-based diagnostic methods of other cancer types.

Size Determination and Phenotypic Analysis of Urinary Extracellular Vesicles using Flow Cytometry.

Extracellular vesicles, EVs, are a heterogeneous complex of lipidic membranes, secreted by any cell type, in any fluid such as urine. EVs can be of different sizes ranging from 40-100 nm in diameter such as in exosomes to 100-1000 nm in microvesicles. They can also contain different molecules that can be used as biomarkers for the prognosis and diagnosis of many diseases. Many techniques have been developed to characterize these vesicles. One of these is flow cytometry. However, there are no existing reports to show how to quantify the concentration of EVs and differentiate them by size, along with biomarker detection. This work aims to describe a procedure for the isolation, quantification, and phenotypification of urinary extracellular vesicles, uEVs, using a conventional cytometer for the analysis without any modification to its configuration. The method's limitations include staining a maximum of four different biomarkers per sample. The method is also limited by the amount of EVs available in the sample. Despite these limitations, with this protocol and its subsequent analysis, we can obtain more information on the enrichment of EVs markers and the abundance of these vesicles present in urine samples, in diseases involving kidney and brain damage.

Analytical approach for description of deuterium content in deuterium-beryllium co-deposited layers

Abstract Available literature data for beryllium content in D-Be co-deposited layers was modeled using newly developed model. It was shown that the model adequately describes the experimental results. An empirical scaling for the available trap concentrations as a function of deuteron energy was introduced in order to improve the agreement between the experimental data and model predictions. The limited recombination rate describes the co-deposition better than the unlimited one. Assuming two types of traps in the film with detrapping energies of 0.8–0.9 and 1.2–1.3 eV and trap concentrations of 10 at. % and 1 at. %, respectively, and the deuterium implantation energy of 15.6 eV, the average relative deviation between experimental and predicted D/Be values of 1.9 was observed, which is better than the empirical D/Be scalings.

Importance of between and within Subject Variability in Extracellular Vesicle Abundance and Cargo when Performing Biomarker Analyses.

Small extracellular vesicles (sEV) have emerged as a potential rich source of biomarkers in human blood and present the intriguing potential for a ‘liquid biopsy’ to track disease and the effectiveness of interventions. Recently, we have further demonstrated the potential for EV derived biomarkers to account for variability in drug exposure. This study sought to evaluate the variability in abundance and cargo of global and liver-specific circulating sEV, within (diurnal) and between individuals in a cohort of healthy subjects (n = 10). We present normal ranges for EV concentration and size and expression of generic EV protein markers and the liver-specific asialoglycoprotein receptor 1 (ASGR1) in samples collected in the morning and afternoon. EV abundance and cargo was generally not affected by fasting, except CD9 which exhibited a statistically significant increase (p = 0.018). Diurnal variability was observed in the expression of CD81 and ASGR1, which significantly decreased (p = 0.011) and increased (p = 0.009), respectively. These results have potential implications for study sampling protocols and normalisation of biomarker data when considering the expression of sEV derived cargo as a biomarker strategy. Specifically, the novel finding that liver-specific EVs exhibit diurnal variability in healthy subjects should have broad implications in the study of drug metabolism and development of minimally invasive biomarkers for liver disease.

Spherical nanoflower-like bimetallic (Mo,Ni)(S,O)3-x sulfo-oxide catalysts for efficient hydrogen evolution under visible light

Photocatalytic H2O splitting by sulfide-based materials is a great challenge, because of the poor resilience of such materials against hole oxidation. Although sulfide ion of catalyst negatively shifts the valence band-edge relative to its oxide ion, the instability of sulfide ions during H2O oxidation is a critical obstacle to simultaneous evolution of H2 and O2. Here, active, stable, and spherical nanoflower-like bimetal (Mo,Ni)(S,O)3-x sulfo-oxide catalysts with a band gap of ∼2.1 eV and different concentrations of oxygen vacancy defects were synthesized for H2O splitting. (Mo,Ni)(S,O)3-x of 25 mg with a suitable amount of oxygen vacancy defects could evolve 587.5 μmol/h H2 under visible-light irradiation. This work demonstrated an example of converting an oxidation photocatalyst into a reduction one. Microstructure analysis showed that surface oxygen vacancy defects and the multiple-valence charges in Ni and Mo not only promoted effective separation, interface transfer, and reactions of photo-carriers but also reduced the charge build-up to avoid photo-corrosion during photocatalytic water decomposition.

Biologically Active Tissue Factor-Bearing Larger Ectosome-Like Extracellular Vesicles in Malignant Effusions from Ovarian Cancer Patients: Correlation with Incidence of Thrombosis.

The development of malignant effusions such as ascites reflects a massive progression of a malignant disease. In patients with ovarian carcinoma, a high amount of ascites (>500 mL) is an independent negative prognostic marker. The composition and constituents of ascites reflect the inflammatory environment of the underlying tumor. Increased cellular resistance of ascites-derived tumor cells and the development of venous thromboembolic events (VTE) are major risks for these patients, especially in patients with advanced ovarian carcinoma. In this study, we discuss the release of tissue factor-bearing extracellular vesicles (TF+ EVs) from tumor cells into the environment (ascites fluid) and their systemic spreading as a possible causal explanation of the pathologic coagulation status in these patients. We obtained ascites from patients with advanced ovarian carcinoma, collected during surgery or therapeutic paracentesis (n = 20). Larger ectosome-like EVs were isolated using sequential centrifugation, quantified by high-resolution flow cytometry and analyzed using nanoparticle tracking analysis. Furthermore, the pro-coagulant properties (TF activity) of EVs were determined. Compared to published TF activities of EVs from healthy persons, TF activities of EVs derived from ascites of patients with ovarian cancer were very high, with a median of 80 pg/mL. The rate of VTE, as reported in the patient files, was high as well (35%, 7 out of 20). Furthermore, all but one patient with VTE had EV concentrations above the median within their ascetic fluid (p < 0.02). Since VTE continues to be a frequent cause of death in cancer patients, prophylactic antithrombotic treatment might be worth considering in these patients. However, given the risk of bleeding, more clinical data are warranted. Although the study is too small to enable reaching a conclusion on direct clinical implementation, it can well serve as a proof of principle and a rationale to initiate a prospective clinical study with different patient subgroups. We also show ex vivo that these larger ectosome-like EVs induce intracellular ERK phosphorylation and tumor cell migration, which is not directly related to their pro-coagulative potency, but might help to understand why cancer patients with thromboembolic events have a poorer prognosis.

Urinary Exosomes in Prostate Cancer.

The analysis of liquid biopsy as a source of diagnostic, prognostic, and predictive biomarkers is still object of the main research in the prostate cancer field. Many advantages, such as less invasiveness compared to plasma or serum analysis and the rich content, confer to urine a role as an interesting fluid to be analysed especially in urological diseases. Here we report a workflow focused on profile, concentration, and protein surface characterization of EVs from urinary supernatant.

Cellular, Extracellular and Extracellular Vesicular miRNA Profiles of Pre-Ovulatory Follicles Indicate Signaling Disturbances in Polycystic Ovaries.

Cell-free RNAs have the potential to act as a means of gene expression regulation between cells and are therefore used as diagnostic markers describing the state of tissue environment. The origin and functions of such RNAs in human ovarian follicle, the environment of oocyte maturation, are unclear. The current study investigates the difference in the microRNA profiles of fertile women and polycystic ovary syndrome (PCOS) patients in three compartments from the same preovulatory follicle: mural granulosa cells (MGC), cell-free follicular fluid (FF), and extracellular vesicles (EV) of the FF by small RNA sequencing. In silico analysis was used for the prediction and over-representation of targeted pathways for the detected microRNAs. PCOS follicles were distinguished from normal tissue by the differential expression of 30 microRNAs in MGC and 10 microRNAs in FF (FDR < 0.1) that commonly regulate cytokine signaling pathways. The concentration of EV-s was higher in the FF of PCOS patients (p = 0.04) containing eight differentially expressed microRNAs (p < 0.05). In addition, we present the microRNA profiles of MGC, FF, and EV in the fertile follicle and demonstrate that microRNAs loaded into EVs target mRNAs of distinct signaling pathways in comparison to microRNAs in FF. To conclude, the three follicular compartments play distinct roles in the signaling disturbances associated with PCOS.

CSF extracellular vesicle proteomics demonstrates altered protein homeostasis in amyotrophic lateral sclerosis

BackgroundExtracellular vesicles (EVs) released by neurons and glia reach the cerebrospinal fluid (CSF). Studying the proteome of CSF-derived EVs offers a novel perspective on the key intracellular processes associated with the pathogenesis of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and a potential source from which to develop biomarkers.MethodsCSF EVs were extracted using ultrafiltration liquid chromatography from ALS patients and controls. EV size distribution and concentration was measured using nanoparticle tracking analysis and liquid chromatography-tandem mass spectrometry proteomic analysis performed.ResultsCSF EV concentration and size distribution did not differ between ALS and control groups, nor between a sub-group of ALS patients with or without an associated hexanucleotide repeat expansion (HRE) in C9orf72. Univariate proteomic analysis identified downregulation of the pentameric proteasome-like protein Bleomycin hydrolase in ALS patients, whilst Gene Ontology enrichment analysis demonstrated downregulation of proteasome core complex proteins (8/8 proteins, normalized enrichment ratio -1.77, FDR-adjusted p = 0.057) in the ALS group. The sub-group of ALS patients associated with the C9orf72 HRE showed upregulation in Ubiquitin-like modifying-activating protein 1 (UBA1) compared to non-C9orf72 cases.ConclusionsProteomic analysis of CSF EVs in ALS detects intracellular alterations in protein homeostatic mechanisms, previously only identified in pathological tissues. This supports the wider use of CSF EVs as a source of novel biomarkers reflecting key and potentially druggable pathological intracellular pathway alterations in ALS.

Effect of thickness, bandgap, and carrier concentration on the basic parameters of Cu2O nanostructures photovoltaics: numerical simulation study

ABSTRACT There is no clear data about the optimum thicknesses, band gaps, and charge densities of Cu2O thin films to fabricate solar cells. Therefore, here, Solar Cell Capacitance Simulator (SCAPS) program was employed to simulate the Cu2O nanostructures solar cells. Effect of window layer thickness, absorber layer thickness, bandgap, and carrier concentration on basic parameters of Cu2O solar cells were studied. Results revealed that window layer thickness in range from 0.3 to 0.4 µm is optimum to produce a higher performance of about 6.5%. Bandgap should be greater than 2.1 eV and donor carrier concentration under 1×1016 cm-3 are required to improve solar cell efficiency of about 8%. Built-in potential, width of the depletion layer, collection length of charge carrier, lifetime of minority carrier, and recombination rate are the main factors directing performance of devices. Consequently, employing our results to fabricate Cu2O solar cells is a step forward to improve efficiencies.

Thermoelectric properties, efficiency and thermal expansion of ZrNiSn half-Heusler by first-principles calculations

In this work, we try to understand the experimental thermoelectric (TE) properties of a ZrNiSn sample with DFT and semiclassical transport calculations using SCAN functional. SCAN and mBJ provide the same band gap Eg of ∼0.54 eV. This Eg is found to be inadequate to explain the experimental data. The better explanation of experimental Seebeck coefficient S is done by considering Eg of 0.18 eV which suggests the non-stoichiometry and/or disorder in the sample. In the calculation of S and other TE properties temperature dependence on chemical potential is included. In order to look for the possible enhanced TE properties obtainable in ZrNiSn with Eg of ∼0.54 eV, power factor and optimal carrier concentrations are calculated. The optimal electron and hole concentrations required to attain highest power factors are ∼7.6 × 1019 cm−3 and ∼1.5 × 1021 cm−3, respectively. The maximum figure of merit ZT calculated at 1200 K for n-type and p-type ZrNiSn are ∼0.5 and ∼0.6, respectively. The % efficiency obtained for n-type ZrNiSn is ∼4.2% while for p-type ZrNiSn is ∼5.1%. The ZT are expected to be further enhanced to ∼1.1 (n-type) and ∼1.2 (p-type) at 1200 K by doping with heavy elements for thermal conductivity reduction. The phonon properties are also studied by calculating dispersion, total and partial density of states. The calculated Debye temperature of 382 K is in good agreement with experimental value of 398 K. The thermal expansion behaviour in ZrNiSn is studied under quasi-harmonic approximation. The average linear thermal expansion coefficient αave(T) of ∼7.8 × 10−6 K−1 calculated in our work is quite close to the experimental values. The calculated linear thermal expansion coefficient will be useful in designing the thermoelectric generators for high temperature applications.

P1265INTRA-DIALYTIC RELEASE OF EXTRACELLULAR VESICLES (EVS) IS INDEPENDENT OF THE MODE OF DIALYSIS A COMPARISON BETWEEN STANDARD HEMODIALYSIS (SHD), COOL HD (CHD), LOW-VOLUME HEMODIAFILTRATION (LVHDF) AND HIGH-VOLUME HDF (HVHDF): THE HOLLANT STUDY

Abstract Background and Aims Treatment with high-volume HDF is associated with a better survival than standard HD. The underlying mechanism is unknown, but may depend on a lower level of intra-dialytic tissue damage. We compared tissue injury, as measured by the release of EVs during four dialysis treatment modalities. Method Chronic hemodialysis (CHD) patients were included in an open, cross-over, randomized fashion (n=14). EV release was measured by a calibrated flow cytometer (Apogee A-60) after antibody labeling. EVs are defined as cell-derived particles with a diameter &amp;lt;1.000nm and a refraction index (RI) &amp;lt;1.42. Two HD modalities were studied: sHD, t dialysate 36,5°C (sHD) and cHD, t dialysate 35,5°C and two HDF modalities (t dialysate 36,5°C): lvHDF with a convection volume (CV) &amp;lt;15 L/session and hvHDF with a CV &amp;gt;23 L/session.To establish the cellular origin of EVs, EVs were labelled with (anti-) CD45 (leukocytes), CD62e (activated endothelial cells), CD62p (activated platelets), CD144 (vascular endothelial cadherin), CD61 (platelets), CD235a (erythrocytes) and connexin-43 (myocardial and endothelial cells), and with the general EV marker lactadherin. Differences between the 4 dialysis modalities were investigated by comparing pre- and post-dialysis and the delta (post- minus pre-dialysis) EV concentrations by Friedman’s test. Results As shown in the figures below, CD61, lactadherin and CD144 increased during the 4 modalities (1=sHD,2=cHD,3=lvHDF,4=hvHDF). The overall increase (sum of all modalities) in connexin-43. In Table 1, the concentrations of EVs before- and after dialysis are shown with a p-value for the difference (post- minus pre-dialysis). No significant differences were found in pre- and post-dialysis and delta EV concentrations between the 4 dialysis modalities (data not shown). Conclusion From this analysis the following conclusions can be drawn: 1) except for CD62e, which declined, all measured EVs increased significantly during dialysis (Table 1). 2) Although not all EVs increased similarly during the 4 modalities (Fig 1), in terms of tissue damage no modality appears more advantageous than the other.

Mesenchymal stem cell exosome characterisation and high-throughput quantification by fluorescence polarisation spectroscopy

Background & Aim Mesenchymal Stem Cells (MSC) are a heterogenous subpopulation of adherent cells that are isolated from adult connective tissue, multipotent and capable of self-renewal. They are an attractive tool in cell therapy for multiple diseases and recent studies have demonstrated a therapeutic effect using MSC-derived exosomes instead of the cells themselves. Exosomes play an important role in cell-to-cell communication and have diverse biological roles; however, the production of EVs as a medicinal product presents serious challenges in terms of isolation, quantification and release. Current methods for quantification and testing rely on either the expression of specific membrane-associated proteins, such as CD81 and CD63, or on the measurement of physical properties such as size or density. The aim of this work is to develop a novel and high-throughput EV quantification platform for MSCs-derived exosomes using fluorescent polarisation (FP). FP is an accessible spectroscopic method that relies on the interaction between a small labeled probe and a target protein, taking advantage of differences in rotational energy of the free vs. bound probe. The change in polarisation between the unbound and bound probe can be used the devise a quantitative test of the concentration of EVs. Methods, Results & Conclusion EV-binding probes should exhibit strong and specific binding to the target and be highly stable in solution. Based on these characteristics, different probes have been identified in the current study. As prominent exosomes surface markers, the CD81 and CD63 tetraspanins have been targeted and candidate probes have been chosen based on their interaction with these markers. An enzyme-linked immunosorbent assay (ELISA) system has been established to assess the ability of the candidate probes to detect surface markers and capture exosomes. Following probe validation, candidate probes will be labeled with a suitable fluorophore and their utility in an FP assay format assessed in comparison with gold standard techniques for exosome quantification. As part of this study, MSCs-derived exosomes have been harvested from various culture conditions using ultracentrifugation and characterised through standard methods including western blot, Dynamic Light Scattering and Transmission Electron Microscopy. This assay will provide a rapid and high-throughput solution for exosome quantification directly in cell culture conditioned media, in a cost effective and timely manner.

Giardia extracellular vesicles disrupt intestinal epithelial junctions and inhibit the growth of commensal bacteria while increasing their swimming motility

IntroductionExtracellular vesicles (EVs) have emerged as important mediators of host‐parasite interactions. The protozoan parasite Giardia duodenalis, the most common cause of parasitic diarrheal diseases worldwide, produces EVs in response to changes in the environment. The exact role of EVs in the pathogenesis of giardiasis remains unclear. In this study, we investigate the effects of EVs on the intestinal barrier such as the intestinal epithelium and the gut commensal bacteria. Disruption of the intestinal barrier has been implicated in the development of post‐giardiasis gut disorders and EVs may represent a novel mechanism of this disruption.AimsThe aim of this research is to characterize Giardia EVs and investigate their effect on the host epithelium and commensal gut bacteria.MethodsG. duodenalis (isolate NF) trophozoites were either incubated with 10X bile for 1 hour or left untreated and EVs were isolated using Qiagen Exo‐Easy Maxi Kit. The concentration and size of EVs were assessed using Nanosight track analysis (NTA). Proteomic analysis of EVs was conducted using liquid chromatography with tandem mass spectrometry. The effects of Giardia EVs on the host epithelium were assessed by exposing epithelial primary cell line (SCBN) to EVs for 24 hours and examining the tight junction proteins ZO‐1 and claudin‐4 by fluorescence microscopy. Antibacterial effect of Giardia EVs was assessed on gut commensals such as Escherichia coli strain HB101 and strain K12, Enterobacter cloacae (human isolate) and Enterococcus faecalis (human isolate) by measuring bacterial kinetic growth for 12 hours. Swimming motility of non‐invasive commensals was assessed on 0.3% agar after incubating with EVs for 24 hours and the halo sizes were measured as a marker of swimming behavior.ResultsOur findings indicate that bile treated Giardia trophozoites produce more EVs as compared to untreated trophozoites. NTA revealed that Giardia EVs range around 200 nm. Proteomic analysis of EVs revealed the presence of well characterized Giardia virulence factors such as cysteine proteases, tenascins, oxidative defense enzymes, variant surface proteins as well as metabolic enzymes such as arginine deaminase and ornithine carbamoyl transferase. Additionally, exposure of SCBN cells to EVs resulted in the disruption and/or rearrangement of tight junction proteins Zo‐1 and Claudin‐4. Moreover, our results also indicate that Giardia EVs exert bacteriostatic effects on E. coli strains HB101, K12, E. cloacae and E. faecalis. Finally, Giardia EVs also significantly increased the swimming motility of non‐invasive commensals E. coli strain HB101 and E. cloacae.ConclusionOur research highlights a novel mechanism of Giardia’s interaction with the intestinal barrier. Giardia EVs contain virulence factors that disrupt the host’s intestinal epithelium and modify the growth and motility of commensal bacteria. These effects may have significant implications in disease pathophysiology.Support or Funding InformationNatural Sciences and Engineering Research Council (NSERC)

Exercise‐enriched extracellular vesicles benefit brain health by decreasing protein aggregation

Aerobic exercise results in improved brain health during both healthy aging and dementia. However, the precise biological mechanisms that drive exercise‐induced benefits for brain health remain unknown. One possible mechanism by which exercise may benefit the brain is by facilitating the delivery of molecular mediators within extracellular vesicles (EVs; exosomes and microvesicles). EVs are bilayer‐phospholipid enclosed vesicles that carry protein and microRNA cargo and are released into the blood from many organs and cells. EVs have been shown to regulate nerve regeneration, synaptic function and behavior. Importantly, when directly injected into rodent brains, EVs can effectively eliminate protein aggregates like amyloid‐β‐peptide (Aβ), which are closely linked to Alzheimer’s Disease (AD) pathology. We hypothesize that EVs isolated from human serum post‐exercise could effectively clear Aβ aggregates in neuronal cells. To test this hypothesis, we isolated EVs from cognitively healthy subjects and early onset AD subjects (8 non‐dementia subjects, ND and 4 early AD subjects) 15 minutes after maximal aerobic exercise (EX). EV size did not change with exercise in either ND or AD subjects, while EV concentration increased by 20% post‐EX in both groups. EV function was tested by incubating serum EVs with human neuronal SY5Y cells over expressing β‐amyloid precursor protein (APPswe). We found that post‐EX‐derived EVs were more effective at alleviating Aβ protein aggregation (40% decrease in aggregation compared to pre‐EX‐derived EVs). Heat Shock Proteins (HSPs), molecular chaperones that play a critical role in protein folding/misfolding, were increased in post‐EX EVs. These findings demonstrate a novel way in which exercise could benefit the brain, through the cell‐to‐cell signaling network of extracellular vesicle communication.Support or Funding InformationResearch support by the University of Kansas Alzheimer’s Disease Center.

Disuse Atrophy Elevates the Release of Skeletal Muscle‐derived Extracellular Vesicles but Lowers Serum EV Concentration in Rats

Extracellular vesicles (EVs) have gained appreciation as important mediators of intercellular communication and physiological functions across almost any cell type in which they have been studied. However, how muscle disuse and atrophy alter production of EVs is largely unknown, and may have a role in dictating the poor systemic outcomes that occur with muscle atrophy and physical inactivity. The purpose of this study was to determine how muscle disuse and atrophy alter the concentration of serum EVs and the production of muscle‐derived EVs. Adult 10 month old male Fisher 344/BN rats underwent hindlimb suspension (HS) for either 4 hours (4h HS; n=4), 24 hours (24h HS; n=4), or 7 days (7d HS; n=5). Serum was collected and EVs were isolated using ExoQuickTM Precipitation solution. For collection of muscle‐derived EVs, soleus muscles were excised and incubated in continuously gassed (95%O2/5% CO2) Kreb’s Henseleit Buffer (118.5mM NaCl, 1.2mM MgSO4, 4.7mM KCl, 1.2mM KH2PO4, 25mM NaHCO3, 2.5mM CaCl2; pH 7.4) supplemented with 5mM glucose at 37°C for 1 hour. Compared with weight bearing rats (WB; n=7), all HS groups exhibited a drastically lower (70–75%; p&lt;0.05) serum EV concentration. The much lower EV concentration is not likely due to reduced production of muscle EVs however, as muscle‐derived EV release (vesicles/mg muscle) was 28% higher from 4h HS and 66% and 60% higher (p&lt;0.05) from 24h and 7d HS muscle, respectively, compared with WB. Altogether, these data suggest that there are rapid systemic changes in EV biology occurring very early with muscle disuse and atrophy as evidenced by the robust and rapid changes in EV serum concentration. There is also a rapid increase in the release of EVs from muscle during disuse that is maintained as the muscle atrophies, which may be important for communication of muscle with other organs. Finally, the lack of association in serum EV concentration and the changes in the rate of EV release from muscle emphasizes the complications and pitfalls of assuming the contribution and behavior of muscle EVs based on serum measurements alone.Support or Funding InformationFunded by NIH grant AT009268.