Publications

The critical role of neutrophils in pathological inflammation, notably in various autoimmune disorders, is currently the focus of renewed interest. Here, we demonstrate for the first time that activation of neutrophils with various inflammatory stimuli induces the release of extracellular vesicles (EVs) that are internalized by endothelial cells (ECs), thus leading to the transfer of miR-223, miR-142-3p and miR-451 and subsequent endothelial damage. Indeed, while miR-223 has little effect on EC responses, we show that the induced expression of miR-142-3p and miR-451 in ECs results in profound cell damage, especially in inflammatory conditions, characterized by a dramatic increase in cell apoptosis, impaired angiogenic repair responses, and the induction of IL-6, IL-8, CXCL10 and CXCL11 expression. We show that the strong deleterious effect of miR-142-3p may be due in part to its ability to block the activation of ERK1/2 and eNOS-mediated signals in ECs. miR-142-3p also inhibits the expression of RAC1, ROCK2 and CLIC4, three genes that are critical for EC migration and angiogenic responses. Importantly, miR-223, miR-142-3p and miR-451 are markedly increased in kidney biopsies from patients with active ANCA-associated vasculitis, a severe autoimmune disease that is prototypical of a neutrophil-induced microvascular damage. Taken together, our results suggest that miR-142-3p and miR-451 released in EVs by activated neutrophils can target EC to trigger an inflammatory cascade and induce direct vascular damage, and that therapeutic strategies based on the inhibition of these miRNAs in ECs will have implications for neutrophil-mediated inflammatory diseases.

People living with HIV (PLH) have significantly higher rates of cognitive impairment (CI) and major depressive disorder (MDD) versus the general population. The enzyme neutral sphingomyelinase 2 (nSMase2) is involved in the biogenesis of ceramide and extracellular vesicles (EVs), both of which are dysregulated in PLH, CI, and MDD. Here we evaluated EcoHIV-infected mice for behavioral abnormalities relevant to depression and cognition deficits, and assessed the behavioral and biochemical effects of nSMase2 inhibition. Mice were infected with EcoHIV and daily treatment with either vehicle or the nSMase2 inhibitor (R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-carbamate (PDDC) began 3 weeks post-infection. After 2 weeks of treatment, mice were subjected to behavior tests. EcoHIV-infected mice exhibited behavioral abnormalities relevant to MDD and CI that were reversed by PDDC treatment. EcoHIV infection significantly increased cortical brain nSMase2 activity, resulting in trend changes in sphingomyelin and ceramide levels that were normalized by PDDC treatment. EcoHIV-infected mice also exhibited increased levels of brain-derived EVs and altered microRNA cargo, including miR-183-5p, miR-200c-3p, miR-200b-3p, and miR-429-3p, known to be associated with MDD and CI; all were normalized by PDDC. In conclusion, inhibition of nSMase2 represents a possible new therapeutic strategy for the treatment of HIV-associated CI and MDD.

Background Previous work has demonstrated that microRNAs (miRNAs) change as a function of antidepressant treatment (ADT) response. However, it is unclear how representative these peripherally detected miRNA changes are to those occurring in the brain. Our goal was to use peripherally extracted neuron-derived extracellular vesicles (NDEV) to investigate neuronal miRNA changes associated with antidepressant response. Methods Samples were collected at two time points (baseline and after 8 weeks of follow-up) from depressed patients who responded (N=20) and did not respond (N=20) to escitalopram treatment, as well as controls (N=20). Total extracellular vesicles (EVs) were extracted from plasma, and then further enriched for NDEV by immunoprecipitation with L1CAM. EV size was measured using tunable resistive pulse sensing, and exosomal miRNA cargo was extracted and sequenced. Subsequently, studies in cell lines and postmortem tissue were conducted. Results Characterization of NDEVs revealed they were smaller than other EVs isolated from plasma (p<0.0001), had brain-specific neuronal markers, and contained miRNAs enriched for brain functions (p<0.0001) Furthermore, NDEVs from depressed patients were smaller than controls (p<0.05), and NDEV size increased with ADT response (p<0.01). Finally, changes in NDEV cargo, specifically changes in miR-21-5p, miR-30d-5p and miR-486-5p together (p<0.01), were associated with ADT response. Targets of these three miRNAs were altered in brain tissue from depressed individuals (p<0.05). Conclusions Together, this study indicates that changes in peripherally isolated NDEV can act as both a clinically accessible and informative biomarker of ADT response specifically through size and cargo.

Military operational stress is known to increase adrenal hormones and inflammatory cytokines, while decreasing hormones associated with the anabolic milieu and neuroendocrine system. Less is known about the role of extracellular vesicles (EVs), a form of cell-to-cell communication, in military operational stress and their relationship to circulating hormones. The purpose of this study was to characterize the neuroendocrine, cytokine, and EV response to an intense. 24-h selection course known as the Naval Special Warfare (NSW) Screener and identify associations between EVs and cytokines. Blood samples were collected the morning of and following the NSW Screener in 29 men (18-26 yr). Samples were analyzed for concentrations of cortisol, insulin-like growth factor I (IGF-I), neuropeptide-Y (NPY), brain-derived neurotrophic factor (BDNF), α-klotho, tumor necrosis factor-α (TNFα), and interleukins (IL) -1β, -6, and -10. EVs stained with markers associated with exosomes (CD63), microvesicles (VAMP3), and apoptotic bodies (THSD1) were characterized using imaging flow cytometry and vesicle flow cytometry. The selection event induced significant changes in circulating BDNF (-43.2%), IGF-I (-24.6%), TNFα (+17.7%), and IL-6 (+13.6%) accompanied by increases in intensities of THSD1+ and VAMP3+ EVs (all P < 0.05). Higher concentrations of IL-1β and IL-10 were positively associated with THSD1+ EVs (P < 0.05). Military operational stress altered the EV profile. Surface markers associated with apoptotic bodies were positively correlated with an inflammatory response. Future studies should consider a multiomics assessment of EV cargo to discern canonical pathways that may be mediated by EVs during military stress.

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.

Extracellular vesicle (EV) secretion is an important mechanism used by cells to release biomolecules. A common necroptosis effector-mixed lineage kinase domain like (MLKL)-was recently found to participate in the biogenesis of small and large EVs independent of its function in necroptosis. The objective of the current study is to gain mechanistic insights into EV biogenesis during necroptosis. Assessing EV number by nanoparticle tracking analysis revealed an increased number of EVs released during necroptosis. To evaluate the nature of such vesicles, we performed a newly adapted, highly sensitive mass spectrometry-based proteomics on EVs released by healthy or necroptotic cells. Compared to EVs released by healthy cells, EVs released during necroptosis contained a markedly higher number of unique proteins. Receptor interacting protein kinase-3 (RIPK3) and MLKL were among the proteins enriched in EVs released during necroptosis. Further, mouse embryonic fibroblasts (MEFs) derived from mice deficient of Rab27a and Rab27b showed diminished basal EV release but responded to necroptosis with enhanced EV biogenesis as the wildtype MEFs. In contrast, necroptosis-associated EVs were sensitive to Ca2+ depletion or lysosomal disruption. Neither treatment affected the RIPK3-mediated MLKL phosphorylation. An unbiased screen using RIPK3 immunoprecipitation-mass spectrometry on necroptotic EVs led to the identification of Rab11b in RIPK3 immune-complexes. Our data suggests that necroptosis switches EV biogenesis from a Rab27a/b dependent mechanism to a lysosomal mediated mechanism.

Natural killer (NK) cells recognize and kill target cells undergoing different types of stress. NK cells are also capable of modulating immune responses. In particular, they regulate T cell functions. Small RNA next-generation sequencing of resting and activated human NK cells and their secreted extracellular vesicles (EVs) led to the identification of a specific repertoire of NK-EV-associated microRNAs and their post-transcriptional modifications signature. Several microRNAs of NK-EVs, namely miR-10b-5p, miR-92a-3p, and miR-155-5p, specifically target molecules involved in Th1 responses. NK-EVs promote the downregulation of GATA3 mRNA in CD4+ T cells and subsequent TBX21 de-repression that leads to Th1 polarization and IFN-γ and IL-2 production. NK-EVs also have an effect on monocyte and moDCs (monocyte-derived dendritic cells) function, driving their activation and increased presentation and costimulatory functions. Nanoparticle-delivered NK-EV microRNAs partially recapitulate NK-EV effects in mice. Our results provide new insights on the immunomodulatory roles of NK-EVs that may help to improve their use as immunotherapeutic tools.

Hydroxyapatite (HA), as the main mineral component in hard tissues, has good biocompatibility. In particular, HA films are widely used as bioactive coatings for artificial bones and dental implants in biomedical fields. However, it is currently difficult to prepare a nanostructure-controlled HA film by a wet process for further applications. Herein, we report the synthesis of HA nanoparticles coordinated by citric acid (Cit/HA) based on the interactions between carboxylate and calcium ions to control the sizes and shapes of the hybrid nanoparticles, to improve their dispersibility in water and to eventually form uniform transparent films with nanospaces, and investigated the film formation mechanism. As compared with the well-known rod-like HA nanoparticles (size: 48 × 15 nm2), we successfully synthesized spherical and negatively charged Cit/HA nanoparticles (size: 25 × 23 nm2) to achieve highly transparent Cit/HA films using the spin-coating technique. The Cit/HA films had uniform and crack-free appearance. About the nanostructures, we found that the Cit/HA film surfaces had meso-scaled nanospaces with a diameter of 4.2 nm based on the regular arrangement of spherical nanoparticles, instead of the HA film with a nanospace diameter of 24.5 nm formed by non-uniform accumulation. Therefore, we successfully achieved the control of the nanospace sizes of the films with the nanoparticle arrangement and realized transparent nanoparticle film formation in a very simple way, which will provide more convenient bioceramic films for biomedical applications.

Small extracellular vesicles (sEVs) secreted from cancer cells play pivotal roles in cancer metastasis and malignancy by transferring biomolecules and conditioning future metastatic sites. Studies have elucidated structures and functions of glycans on sEVs; however, whether sEVs remodel glycans in recipient cells remains poorly understood. Here, we examined the enzyme activity of glycosyltransferases for complex N-glycan biosynthesis in cancer-derived sEVs and discovered that cancer-related glycosyltransferase, N-acetylglucosaminyltransferase-V (GnT-V, a.k.a. MGAT5), is selectively enriched in sEVs among various glycosyltransferases. GnT-V in sEVs is a cleaved form, and cleavage by SPPL3 protease is necessary for loading GnT-V in sEVs. Fractionation experiments and single-particle imaging further revealed that GnT-V was enriched in non-exosomal sEVs. Strikingly, we found that enzymatically active GnT-V in sEVs was transferred to recipient cells and the N-glycan structures of recipient cells were remodeled to express GnT-V-produced glycans. Our results suggest GnT-V-enriched sEVs’ role in glycan remodeling in cancer metastasis.

Cells secrete extracellular vesicles (EVs) carrying cell-of-origin markers to communicate with surrounding cells. EVs regulate physiological processes ranging from intercellular signaling to waste management. However, when senescent cells (SnCs) secrete EVs, the EVs, which are newly regarded as senescence-associated secretory phenotype (SASP) factors, can evoke inflammation, senescence induction, and metabolic disorders in neighboring cells. Unlike other soluble SASP factors, the biophysical properties of EVs, including small EVs (sEVs), derived from SnCs have not yet been investigated. In this study, sEVs were extracted from a human IMR90 lung fibroblast in vitro senescence model. Their biomechanical properties were mapped using atomic force microscopy-based quantitative nanomechanical techniques, surface potential microscopy, and Raman spectroscopy. The surfaces of sEVs derived from SnCs are slightly stiffer but their cores are softer than those of sEVs secreted from non-senescent cells (non-SnCs). This inversely proportional relationship between deformation and stiffness, attributed to a decrease in the concentration of genetic and protein materials inside the vesicles and the adsorption of positively charged SASP factors onto the vesicle surfaces, respectively, was found to be a peculiar characteristic of SnC-derived sEVs. Our results demonstrate that the biomechanical properties of SnC-derived sEVs differ from those of non-SnC-derived sEVs and provide insight into the mechanisms underlying their formation and composition.

Tuberculosis (TB), which is caused by the bacterium Mycobacterium tuberculosis (Mtb), is still one of the deadliest infectious diseases. Understanding how the host and pathogen interact in active TB will have a significant impact on global TB control efforts. Exosomes are increasingly recognized as a means of cell-to-cell contact and exchange of soluble mediators. In the case of TB, exosomes are released from the bacillus and infected cells. In the present study, a comprehensive lipidomics and proteomics analysis of size exclusion chromatography-isolated plasma-derived exosomes from patients with TB lymphadenitis (TBL) and treated as well as untreated pulmonary TB (PTB) was performed to elucidate the possibility to utilize exosomes in diagnostics and knowledge building. According to our findings, exosome-derived lipids and proteins originate from both the host and Mtb in the plasma of active TB patients. Exosomes from all patients are mostly composed of sphingomyelins (SM), phosphatidylcholines, phosphatidylinositols, free fatty acids, triacylglycerols (TAG), and cholesterylesters. Relative proportions of, e.g., SMs and TAGs, vary depending on the disease or treatment state and could be linked to Mtb pathogenesis and dormancy. We identified three proteins of Mtb origin: DNA-directed RNA polymerase subunit beta (RpoC), Diacyglycerol O-acyltransferase (Rv2285), and Formate hydrogenase (HycE), the latter of which was discovered to be differently expressed in TBL patients. Furthermore, we discovered that Mtb infection alters the host protein composition of circulating exosomes, significantly affecting a total of 37 proteins. All TB patients had low levels of apolipoproteins, as well as the antibacterial proteins cathelicidin, Scavenger Receptor Cysteine Rich Family Member (SSC5D), and Ficolin 3 (FCN3). When compared to healthy controls, the protein profiles of PTB and TBL were substantially linked, with 14 proteins being co-regulated. However, adhesion proteins (integrins, Intercellular adhesion molecule 2 (ICAM2), CD151, Proteoglycan 4 (PRG4)) were shown to be more prevalent in PTB patients, while immunoglobulins, Complement component 1r (C1R), and Glutamate receptor-interacting protein 1 (GRIP1) were found to be more abundant in TBL patients, respectively. This study could confirm findings from previous reports and uncover novel molecular profiles not previously in focus of TB research. However, we applied a minimally invasive sampling and analysis of circulating exosomes in TB patients. Based on the findings given here, future studies into host-pathogen interactions could pave the way for the development of new vaccines and therapies.

Biological scaffolds derived from decellularized tissues are being investigated as a promising approach to repair volumetric muscle losses (VML). Indeed, extracellular matrix (ECM) from decellularized tissues is highly biocompatible and mimics the original tissue. However, the development of fibrosis and the muscle stiffness still represents a major problem. Intercellular signals mediating tissue repair are conveyed via extracellular vesicles (EVs), biologically active nanoparticles secreted by the cells. This work aimed at using muscle ECM and human EVs derived from Wharton Jelly mesenchymal stromal cells (MSC EVs) to boost tissue regeneration in a VML murine model. Mice transplanted with muscle ECM and treated with PBS or MSC EVs were analyzed after 7 and 30 days. Flow cytometry, tissue analysis, qRT-PCR and physiology test were performed. We demonstrated that angiogenesis and myogenesis were enhanced while fibrosis was reduced after EV treatment. Moreover, the inflammation was directed toward tissue repair. M2-like, pro-regenerative macrophages were significantly increased in the MSC EVs treated group compared to control. Strikingly, the histological improvements were associated with enhanced functional recovery. These results suggest that human MSC EVs can be a naturally-derived boost able to ameliorate the efficacy of tissue-specific ECM in muscle regeneration up to the restored tissue function.

BACKGROUND: The analysis of liquid biopsies brings new opportunities in the precision oncology field. Under this context, extracellular vesicle circular RNAs (EV-circRNAs) have gained interest as biomarkers for lung cancer (LC) detection. However, standardized and robust protocols need to be developed to boost their potential in the clinical setting. Although nCounter has been used for the analysis of other liquid biopsy substrates and biomarkers, it has never been employed for EV-circRNA analysis of LC patients. METHODS: EVs were isolated from early-stage LC patients (n = 36) and controls (n = 30). Different volumes of plasma, together with different number of pre-amplification cycles, were tested to reach the best nCounter outcome. Differential expression analysis of circRNAs was performed, along with the testing of different machine learning (ML) methods for the development of a prognostic signature for LC. RESULTS: A combination of 500 μL of plasma input with 10 cycles of pre-amplification was selected for the rest of the study. Eight circRNAs were found upregulated in LC. Further ML analysis selected a 10-circRNA signature able to discriminate LC from controls with AUC ROC of 0.86. CONCLUSIONS: This study validates the use of the nCounter platform for multiplexed EV-circRNA expression studies in LC patient samples, allowing the development of prognostic signatures.

Acoustofluidicly manipulated microbubbles (MBs) and echogenic liposomes (ELIPs) have been suggested as drug delivery systems for the 'on demand' release of drug in target tissue. This requires a clear understanding of their behaviour during ultrasonication and after ultrasonication stops. The main focus of this study is to investigate the behaviour of MBs and ELIPs clusters after ultrasonication stops and the underlaying cause of cluster diffusion considering electrostatic repulsion, steric repulsion and Brownian motion. It also examines the capability of existing models used to predict MBs' attraction velocity due to secondary radiation force, on predicting ELIPs' attraction velocity. Tunable resistive pulse sensing (TRPS) and phase analysis light scattering (PALS) techniques were used to measure zeta potentials of the agents and the size distributions were measured using TRPS. The zeta potentials were found to be -2.43 mV and -0.62 mV for Definity™ MBs, and -3.62 mV and -2.35 mV for ELIPs using TRPS and PALS, respectively. Both agents were shown to have significant cluster formation at pressures as low as 6 kPa. Clusters of both agents were shown to diffuse as sonication stops at a rate that approximately equals the sum of the diffusion coefficients of the agents forming them. The de-clustering behaviours are due to Brownian motion as no sign of electrostatic repulsion was observed and particles movements were observed to be faster for smaller diameters. These findings are important to design and optimise effective drug delivery systems using acoustofluidically manipulated MBs and ELIPs.

Liquid biopsies based on extracellular vesicles (EVs) represent a promising tool for treatment monitoring of tumors, including non-small-cell lung cancers (NSCLC). In this study, we report on a multiplexed electrokinetic sensor for surface protein profiling of EVs from clinical samples. The method detects the difference in the streaming current generated by EV binding to the surface of a functionalized microcapillary, thereby estimating the expression level of a marker. Using multiple microchannels functionalized with different antibodies in a parallel fluidic connection, we first demonstrate the capacity for simultaneous detection of multiple surface markers in small EVs (sEVs) from NSCLC cells. To investigate the prospects of liquid biopsies based on EVs, we then apply the method to profile sEVs isolated from the pleural effusion (PE) fluids of five NSCLC patients with different genomic alterations (ALK, KRAS or EGFR) and applied treatments (chemotherapy, EGFR- or ALK-tyrosine kinase inhibitors). The vesicles were targeted against CD9, as well as EGFR and PD-L1, two treatment targets in NSCLC. The electrokinetic signals show detection of these markers on sEVs, highlighting distinct interpatient differences, e.g., increased EGFR levels in sEVs from a patient with EGFR mutation as compared to an ALK-fusion one. The sensors also detect differences in PD-L1 expressions. The analysis of sEVs from a patient prior and post ALK-TKI crizotinib treatment reveals significant increases in the expressions of some markers (EGFR and PD-L1). These results hold promise for the application of the method for tumor treatment monitoring based on sEVs from patient liquid biopsies.

Exosomes and extracellular vesicles (EV) are increasingly being explored as circulating biomarkers, but their heterogenous composition will likely mandate the development of single EV technologies. Highly multiplexed analyses of single EVs have been challenging to implement beyond a few colors during spectral sensing. We use a multiplexed analysis of the single EV technique (MASEV) to interrogate thousands of individual EVs during 5 cycles of multi-channel fluorescence staining for 15 EV biomarkers. Contrary to the common belief, we show that i) several “ubiquitous” markers are much less common than believed; ii) that multiple biomarkers concur in single vesicles but only in small fractions, iii) that affinity purification can lead to loss of rare EV subtypes, and iv) that deep profiling allows detailed analysis of EV potentially improving the diagnostic content. These findings establish the potential of MASEV for uncovering fundamental EV biology and heterogeneity and increasing diagnostic specificity. Graphical abstract Multiplexed analysis of single EV (MASEV) allows robust protein profiling at the single EV level, a prerequisite for early cancer detection or organ of origin determination. Multiplexed analysis of single EV (MASEV) allows robust protein profiling at the single EV level, a prerequisite for early cancer detection or organ of origin determination.

Detecting protein markers in extracellular vesicles (EVs) is becoming a useful tool for basic research and clinical diagnoses. Most EV protein assays, however, require lengthy processes─conjugating affinity ligands onto sensing substrates and affixing EVs with additional labels to maximize signal generation. Here, we present an iPEX (impedance profiling of extracellular vesicles) system, an all-electrical strategy toward fast, multiplexed EV profiling. iPEX adopts one-step electropolymerization to rapidly functionalize sensor electrodes with antibodies; it then detects EV proteins in a label-free manner through impedance spectroscopy. The approach streamlines the entire EV assay, from sensor preparation to signal measurements. We achieved (i) fast immobilization of antibodies (<3 min) per electrode; (ii) high sensitivity (500 EVs/mL) without secondary labeling; and (iii) parallel detection (quadruple) in a single chip. A potential clinical utility was demonstrated by directly analyzing plasma samples from glioblastoma multiforme patients.

Background: MSC-derived extracellular vehicles (EVs) exhibit a protective functional role in renal ischemia/reperfusion injury (RIRI). Recent studies have revealed that mitophagy could be a potential target process in the treatment of RIRI. However, whether MSC-derived EVs are involved in the regulation of mitophagy in RIRI remains largely unknown to date. Methods: RIRI model was established in vivo in mice by subjecting them to renal ischemia/reperfusion. TCMK-1 cells were subjected to hypoxia/reoxygenation (H/R) stimulation to mimic RIRI in vitro. BMSCs and BMSC-derived EVs were isolated and identified. Renal injury was assessed using H&E staining. The qPCR and western blot analyses were conducted to detect the mRNA and protein levels. Apoptosis was evaluated using the TUNEL assay and flow cytometry analysis. The EVs, autophagosomes, and mitochondria were observed using TEM. The colocalization of autophagosomes with mitochondria was confirmed through the confocal assay. The direct binding of miR-223-3p to NLRP3 was validated through the dual-luciferase assay. Results: BMSCs and BMSC-derived EVs were successfully isolated from mice and identified. The protective effect of BMSC-derived EVs against RIRI was validated both in vitro and in vivo, which was indicated by a decrease in apoptosis and inflammasome activation and an increase in mitophagy. However, this protective effect was impaired in the miR-223-3p-depleted EVs, suggesting that miR-223-3p mediated this protective effect. Further mechanistic investigation revealed that miR-223-3p suppressed inflammasome activation to enhance mitophagy by directly targeting NLRP3. Conclusion: In conclusion, the protective role of BMSC-derived EVs and exosome-delivered miR-223-3p in RIRI was validated. Exogenous miR-223-3p directly targeted NLRP3 to attenuate inflammasome activation, thereby promoting mitophagy.

Extracellular vesicles (EVs) of various types are released or shed from all cells. EVs carry proteins and contain additional protein and nucleic acid cargo that relates to their biogenesis and cell of origin. EV cargo in liquid biopsies is of widespread interest owing to its ability to provide a retrospective snapshot of cell state at the time of EV release. For the purposes of EV cargo analysis and repertoire profiling, multiplex assays are an essential tool in multiparametric analyte studies but are still being developed for high-parameter EV protein detection. Although bead-based EV multiplex analyses offer EV profiling capabilities with conventional flow cytometers, the utilization of EV multiplex assays has been limited by the lack of software analysis tools for such assays. To facilitate robust EV repertoire studies, we developed multiplex analysis post-acquisition analysis (MPAPASS) open-source software for stitched multiplex analysis, EV database-compatible reporting, and visualization of EV repertoires.

Understanding the motion of colloidal particles flowing in small spaces is a general issue in various fields such as thermal engineering and micro/nanofluidics. In the present study, we investigated the motion of fluorescent submicrometer particles in a 3- μm microchannel by defocusing nanoparticle image velocimetry. An optical measurement system with controlled spherical aberration and an algorithm for processing defocused particle images with multiple diffraction rings were developed. By detecting the centroid position and the diameter of the outermost diffraction ring, which is proportional to the distance between the focal plane and the particle, the position of particles was determined with the spatial resolutions of 154–204 nm in the streamwise direction and 76–311 nm in the depthwise direction, which are comparable to or smaller than the optical diffraction limit. A reusable microfluidic device containing a size-regulated microchannel made of glass was developed, which is suitable for optical measurements and precise flow control. By controlling the strength of low-temperature glass bonding, detachment of the bonded glass substrates, washing, and reuse were achieved. Based on this method and technology, the velocity of particles with diameters of 199, 457, and 1114 nm was successfully measured in pressure-driven laminar flow. Results suggested that for larger particles comparable to the channel size, the particle velocity is slowed from the flow velocity by particle–wall hydrodynamic interactions. Therefore, the motion of colloidal particles in 10 0 - μm spaces is considered to be affected by particle–wall hydrodynamic interactions, as well as 10 2 - μm spaces reported previously.

Extracellular vesicles (EVs) are important for intercellular communication and act as vehicles for biological material, such as various classes of coding and non-coding RNAs, a few of which were shown to selectively target into vesicles. However, protein factors, mechanisms, and sequence elements contributing to this specificity remain largely elusive. Here, we use a reporter system that results in different types of modified transcripts to decipher the specificity determinants of RNAs released into EVs. First, we found that small RNAs are more efficiently packaged into EVs than large ones, and second, we determined absolute quantities for several endogenous RNA transcripts in EVs (U6 snRNA, U1 snRNA, Y1 RNA, and GAPDH mRNA). We show that RNA polymerase III (pol III) transcripts are more efficiently secreted into EVs compared to pol II-derived transcripts. Surprisingly, our quantitative analysis revealed no RNA accumulation in the vesicles relative to the total cellular levels, based on both overexpressed reporter transcripts and endogenous RNAs. RNA appears to be EV-associated only at low copy numbers, ranging between 0.02 and 1 molecule per EV. This RNA association may reflect internal EV encapsulation or a less tightly bound state at the vesicle surface.

Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co-isolated entities, a combination of methods is needed. However, this is time-consuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available. Therefore, we compared EVs rendered by five commonly used methods based on different principles from conditioned cell medium and 250 μl or 3 ml plasma, that is, precipitation (ExoQuick ULTRA), membrane affinity (exoEasy Maxi Kit), size-exclusion chromatography (qEVoriginal), iodixanol gradient (OptiPrep), and phosphatidylserine affinity (MagCapture). EVs were characterised by electron microscopy, Nanoparticle Tracking Analysis, Bioanalyzer, flow cytometry, and LC-MS/MS. The different methods yielded samples of different morphology, particle size, and proteomic profile. For the conditioned medium, Izon 35 isolated the highest number of EV proteins followed by exoEasy, which also isolated fewer non-EV proteins. For the plasma samples, exoEasy isolated a high number of EV proteins and few non-EV proteins, while Izon 70 isolated the most EV proteins. We conclude that no method is perfect for all studies, rather, different methods are suited depending on sample type and interest in EV subtype, in addition to sample volume and budget.

Proximal tubular epithelial cells (PTEC) are central players in inflammatory kidney diseases. However, the complex signalling mechanism/s via which polarized PTEC mediate disease progression are poorly understood. Small extracellular vesicles (sEV), including exosomes, are recognized as fundamental components of cellular communication and signalling courtesy of their molecular cargo (lipids, microRNA, proteins). In this study, we examined the molecular content and function of sEV secreted from the apical versus basolateral surfaces of polarized human primary PTEC under inflammatory diseased conditions. PTEC were cultured under normal and inflammatory conditions on Transwell inserts to enable separate collection and isolation of apical/basolateral sEV. Significantly increased numbers of apical and basolateral sEV were secreted under inflammatory conditions compared with equivalent normal conditions. Multi-omics analysis revealed distinct molecular profiles (lipids, microRNA, proteins) between inflammatory and normal conditions for both apical and basolateral sEV. Biological pathway analyses of significantly differentially expressed molecules associated apical inflammatory sEV with processes of cell survival and immunological disease, while basolateral inflammatory sEV were linked to pathways of immune cell trafficking and cell-to-cell signalling. In line with this mechanistic concept, functional assays demonstrated significantly increased production of chemokines (monocyte chemoattractant protein-1, interleukin-8) and immuno-regulatory cytokine interleukin-10 by peripheral blood mononuclear cells activated with basolateral sEV derived from inflammatory PTEC. We propose that the distinct molecular composition of sEV released from the apical versus basolateral membranes of human inflammatory PTEC may reflect specialized functional roles, with basolateral-derived sEV pivotal in modulating tubulointerstitial inflammatory responses observed in many immune-mediated kidney diseases. These findings provide a rationale to further evaluate these sEV-mediated inflammatory pathways as targets for biomarker and therapeutic development.

Parasite derived extracellular vesicles (EVs) have been proposed to play key roles in the establishment and maintenance of infection. Calicophoron daubneyi is a newly emerging parasite of livestock with many aspects of its underpinning biology yet to be resolved. This research is the first in-depth investigation of EVs released by adult C. daubneyi. EVs were successfully isolated using both differential centrifugation and size exclusion chromatography (SEC), and morphologically characterized though transmission electron microscopy (TEM). EV protein components were characterized using a GeLC approach allowing the elucidation of comprehensive proteomic profiles for both their soluble protein cargo and surface membrane bound proteins yielding a total of 378 soluble proteins identified. Notably, EVs contained Sigma-class GST and cathepsin L and B proteases, which have previously been described in immune modulation and successful establishment of parasitic flatworm infections. SEC purified C. daubneyi EVs were observed to modulate rumen bacterial populations by likely increasing microbial species diversity via antimicrobial activity. This data indicates EVs released from adult C. daubneyi have a role in establishment within the rumen through the regulation of microbial populations offering new routes to control rumen fluke infection and to develop molecular strategies to improve rumen efficiency.