Publications

Indocyanine green (ICG) J-aggregates (IJA) are a unique form of aggregation that exhibits superior properties to monomeric ICG. Despite their higher photoacoustic (PA) signals for imaging and heating stability during photothermal therapy (PTT), they exhibit low stability under a biological milieu. Our group previously proposed a simple procedure for in-situ preparation of IJA into liposomes, accelerating their formation and optical properties. To comprehend their potential applications, we systematically investigated the effect of the lipid bilayer composition on ICG J-aggregation and stability. Moreover, their in vitro compatibility and photothermal toxicity in monolayers and cancer spheroids, besides their in vivo biodistribution and clearance were evaluated. Our findings revealed the importance of high cholesterol and PEG-lipid content and low charged lipids (∼ 5 mol %) in liposomes to promote a high IJA/ICG ratio and, thus, high heating stability. More importantly, IJA-liposomes revealed high biocompatibility in monolayer and cancer spheroids with efficient photothermal toxicity. Finally, IJA-liposomes were cleared from the body without toxicity. Interestingly, IJA-liposomes mainly showed lower affinity to the liver than monomeric ICG, resulting in higher renal clearance. Overall, our biodegradable IJA-liposomes could be an excellent alternative to gold-based agents suitable for PA imaging and cancer PTT.

Background: Cytological examination is of suboptimal sensitivity but high specificity for the diagnosis of malignant pleural effusions (MPEs). Pleural fluid extracellular vesicles (PFEVs) are enriched with disease-specific microRNAs (miRNAs) which may improve the diagnostic yield for MPE. Our previous study demonstrated the feasibility of isolating miRNAs from PFEVs and profiling PFEV miRNAs by Nanostring nCounter® Human v3 miRNA expression assay. Here, we interrogated in a small cohort to evaluate the diagnostic potential of PFEV miRNAs to differentiate between benign pleural effusion and MPE. Methods: Extracellular vesicles (EVs) from pleural fluids were isolated by two sequential ultracentrifugation steps. PFEVs were extracted and characterised by western blotting analysis, particle analysis by tunable resistive pulse sensing (TRPS) technology, and transmission electron microscopy (TEM). Total RNAs (including miRNAs) were extracted from PFEVs and profiled by the Nanostring nCounter® 827 probe miRNA expression assay. Differential expression analysis of the miRNA expression assays on PFEV samples was performed using the Bioconductor DESeq2 package. Results: EVs from pleural fluids were evident by staining of positive EV-associated protein markers, particle size distribution within the expected parameters, and the cup-shaped morphology by TEM. Employing Nanostring nCounter® Human v3 miRNA expression assay, this proof-of-principle study demonstrated PFEV miRNAs were differentially expressed between benign effusions and malignant effusions [malignant pleural mesothelioma (MPM) or lung adenocarcinoma metastatic to pleura (metLUAD)]. The expression of six miRNAs (hsa-miR-1246, hsa-miR-136-5p, hsa-miR-141-3p, hsa-miR-145-5p, hsa-miR-200c-3p, and hsa-miR-9-5p) significantly differed between benign and malignant effusions, or between MPM and metLUAD, at adjusted P<0.05 and log2fold change ≥1.0. Conclusions: The miRNAs identified from this study could be interrogated further for their utility as a single biomarker candidate or to be tested simultaneously in a panel to complement pleural effusion diagnostics. PFEV miRNAs represent a novel bioresource with potential to aid in the diagnosis of pleural effusions. Larger prospective studies are needed to confirm their diagnostic utility.

To improve the treatment outcome and survival of patients with advanced high-grade serous carcinoma (HGSC), prognostic biomarkers for assessing the feasibility of complete (R0) or optimal (R1) primary cytoreductive surgery are needed. Additionally, biomarkers for predicting the response to neoadjuvant chemotherapy (NACT) in patients with primary inoperable disease could help stratify patients for tailored therapy and improve personalised approach. Such promising biomarkers are extracellular vesicles (EVs), which are present in ascites and plasma and are available for minimally invasive liquid biopsy. EV concentration and EV molecular profile have been at the forefront of research in the field of biomarkers for many years now, but recent studies have highlighted the importance of EV size distribution. Our study aimed to evaluate the potential of the EV concentration and size distribution in pretreatment ascites and plasma samples from patients with advanced HGSC as prognostic biomarkers. In our prospective cohort study, nanoparticle tracking analysis (NTA) was used to determine EV characteristics in paired pretreatment ascites and plasma samples from 37 patients with advanced HGSC. Patients were treated with primary cytoreductive surgery followed by adjuvant chemotherapy (ACT) (N = 15) or NACT followed by interval debulking surgery (IDS) when optimal cytoreduction was not feasible (N = 22). The correlations of the EV concentration and size distribution in ascites and plasma with treatment outcome, progression-free survival (PFS) and overall survival (OS) were analysed. We found a significant correlation between the EV size distribution in ascites and residual disease after primary cytoreductive surgery. Larger EVs in ascites correlated with worse resection success after primary cytoreductive surgery. A significant correlation between the D10 value of EVs in plasma and the chemotherapy response score (CRS) after NACT was observed. A smaller D10 value of plasma EVs was correlated with a better chemotherapy response. Receiver operating characteristic (ROC) curve analysis revealed excellent performance for D10 value in ascites for the prediction of suboptimal (R2) resection at primary debulking surgery and excellent performance for D10 value in plasma for the prediction of complete or near-complete chemotherapy response score (CRS 3) at interval debulking surgery. There was a significant correlation between the mean diameter, D90 value and proportion of medium/large (&gt; 200 nm) EVs in ascites and those in plasma. On the other hand, there was no correlation of the EV concentration or D10 and D50 values between the ascites fluid and plasma samples. Our results indicate that the EV size distribution in ascites has the potential to predict resection success after primary cytoreductive surgery and that the EV size distribution of the smallest EVs in plasma might help predict the chemotherapy response of patients treated with NACT. In the future, molecular analyses of size-dependent EV cargo could provide more insight into their biological functions and potential as predictive biomarkers.

In neurons, the acquisition of a polarized morphology is achieved upon the outgrowth of a single axon from one of several neurites. Small extracellular vesicles (sEVs), such as exosomes, from diverse sources are known to promote neurite outgrowth and thus may have therapeutic potential. However, the effect of fibroblast-derived exosomes on axon elongation in neurons of the central nervous system under growth-permissive conditions remains unclear. Here, we show that fibroblast-derived sEVs promote axon outgrowth and a polarized neuronal morphology in mouse primary embryonic cortical neurons. Mechanistically, we demonstrate that the sEV-induced increase in axon outgrowth requires endogenous Wnts and core PCP components including Prickle, Vangl, Frizzled, and Dishevelled. We demonstrate that sEVs are internalized by neurons, colocalize with Wnt7b, and induce relocalization of Vangl2 to the distal axon during axon outgrowth. In contrast, sEVs derived from neurons or astrocytes do not promote axon outgrowth, while sEVs from activated astrocytes inhibit elongation. Thus, our data reveal that fibroblast-derived sEVs promote axon elongation through the Wnt-PCP pathway in a manner that is dependent on endogenous Wnts.

Extracellular vesicles (EVs) are important mediators of intercellular communication and have various roles in physiological and pathological processes. Discovery of regulators of EV biogenesis and release has led to significant improvements in our understanding of EV biology and has highlighted disease-specific pathways. Large scale discovery studies of EV regulators are limited by conventional methods of EV analysis with limited throughput and sensitivity. To address this, this study presents a high-throughput flow cytometry-based platform for the quantification of EVs released from cells. Here, a system was developed using the MDA–MB-231 cell line stably expressing ZsGreen, which passively loads ZsGreen proteins into EVs, and nanoscale flow cytometry. EV detection and quantitation was optimized and validated for a 96-well format. The high-throughput flow cytometry screening platform quantified the effect of 156 kinase inhibitors on EV number and identified AZ191 – a DYRK1b inhibitor – as a potent EV inhibitor. DYRK1b inhibition and knockdown confirmed a significant reduction in total EV number, with small EVs demonstrating the largest reduction. DYRK1b knockdown altered the intracellular distribution of EV marker CD63, suggesting a role for DYRK1b in EV trafficking. In conclusion, our study establishes a platform for high-throughput analysis of EV dynamics and introduces DYRK1b kinase as a novel EV-regulator.

Feline Idiopathic Cystitis (FIC), is a chronic lower urinary tract condition in cats analogous to PBS/IC in women, which presents significant treatment challenges due to its idiopathic nature. Recent advancements in regenerative medicine highlight the potential of Adipose Tissue-Derived Stem Cells (ADSCs), particularly through their secretome, which includes mediators, bioactive molecules, and extracellular vesicles (EVs). Notably, exosomes, a subset of EVs, facilitate cell-to-cell communication and, when derived from ADSCs, exhibit anti-inflammatory properties and contribute to tissue regeneration. In this work, we aim to characterize the content of exosomes derived from feline ADSCs (fADSCs) to elucidate their mechanisms of action on recipient cells and assess their therapeutic potential for FIC. Exosomes were isolated from fADSCs and their microRNA (miRNA) content sequenced using Illumina technology. Our findings demonstrate that fADSC-derived exosomes harbor miRNAs that can induce regenerative processes, such as cell proliferation, immune modulation, angiogenesis, and anti-inflammatory responses. Key miRNAs identified include fca-miR-221, fca-let-7f-5p, fca-miR-337-5p, fca-miR-542-5p, fca-miR-24-3p, fca-miR-205, and fca-miR-23a, which promote proliferative, angiogenic, differentiation, and regenerative mechanisms. Additionally, miRNAs with anti-inflammatory effects, such as fca-miR-193a-5p and fca-miR-127-3p, and those positively regulating the immune system, including fca-let-7a-5p and fca-miR-chrC1_18846-5p, were identified. Of particular interest, fca-miR-219-5p (has-miR-6766-3p) has been reported to suppress liver fibrosis.These results underline the therapeutic potential of fADSC-derived exosomes in treating FIC and suggest innovative strategies for feline veterinary medicine.

Background: Cancer treatments are still limited by various challenges, such as off-target drug delivery, posttreatment inflammation, and the hypoxic conditions in the tumor microenvironment; thus, the development of effective therapeutics remains highly desirable. Exosomes are extracellular vesicles with a size of 30– 200 nm that have been widely applied as drug carriers over the last decade. In this study, melanoma-derived exosomes were used to develop a perfluorocarbon (PFC) drug nanocarriers loaded with indocyanine green (ICG) and camptothecin (CPT) (ICFESs) for targeted cancer photochemotherapy. Methods: The ICFESs were fabricated by emulsification approach and characterized through instrumental detection. The capabilities of the ICFESs on tumor targeting, intratumoral retention, and cancer photochemotherapy were evaluated using melanoma tumor-bearing mice in association with histological studies and serum marker analyses. Results: ICFESs can be rapidly internalized by homologous melanoma cells, induce hyperthermia and increase the yield of singlet oxygen upon exposure to near-infrared (NIR) irradiation. After 5 min of NIR exposure and 24 h of in vitro culture, ICFESs encapsulating ≥ 10/10 μM [ICG]/[CPT] effectively killed more than 70% of the cancer cells, inducing greater mortality than that caused by a 4-fold higher dose of CPT alone. In a murine melanoma model, we demonstrated that ICFESs indeed targeted homologous tumors with prolonged intratumoral retention compared with free ICG in vivo. Moreover, tumor growth was significantly arrested by ICFESs containing 40/40 μM [ICG]/[CPT] in combination with 30 sec of NIR exposure without systemic toxicity, and the resulting tumors were approximately 15-fold smaller than those treated for 14 days with 40 μM free CPT alone. Conclusion: We suggest that the aforementioned anticancer efficacy was achieved via a dual-stage mechanism, phototherapy followed by chemotherapy. Taken together, the developed ICFESs are anticipated to be highly applicable for clinical cancer treatment.

Current treatments for chronic pain have limited efficacy and significant side effects, warranting research on alternative strategies for pain management. One approach involves using small extracellular vesicles (sEVs), or exosomes, to transport beneficial biomolecular cargo to aid pain resolution. Exosomes are 30–150 nm sEVs that can be beneficial or harmful depending on their source and cargo composition. We report a comprehensive multi-modal analysis of different aspects of sEV characterization, miRNAs, and protein markers across sEV sources. To investigate the short- and long-term effects of mouse serum-derived sEVs in pain modulation, sEVs from naïve control or spared nerve injury (SNI) model male donor mice were injected intrathecally into naïve male recipient mice. These sEVs transiently increased basal mechanical thresholds, an effect mediated by opioid signaling as this outcome was blocked by naltrexone. Mass spectrometry of sEVs detected endogenous opioid peptide leu-enkephalin. sEVs from naïve female mice have higher levels of leu-enkephalin compared to male, matching the analgesic onset of leu-enkephalin in male recipient mice. In investigating the long-term effect of sEVs, we observed that a single prophylactic intrathecal injection of sEVs two weeks prior to induction of the pain model in recipient mice accelerated recovery from inflammatory pain after complete Freund’s adjuvant (CFA) injection. Our exploratory studies examining immune cell populations in spinal cord and dorsal root ganglion using ChipCytometry suggested alterations in immune cell populations 14 days post-CFA. Flow cytometry confirmed increases in CD206+ macrophages in the spinal cord in sEV-treated mice. Collectively, these studies demonstrate multiple mechanisms by which sEVs can attenuate pain.

The early detection of neurodegenerative diseases necessitates the identification of specific brain-derived biomolecules in peripheral blood. In this context, our investigation delineates the role of amyloid precursor-like protein 1 (APLP1)—a protein predominantly localized in oligodendrocytes and neurons—as a previously unidentified biomarker in extracellular vesicles (EVs). Through rigorous analysis, APLP1+ EVs from human sera were unequivocally determined to be of cerebral origin. This assertion was corroborated by distinctive small RNA expression patterns of APLP1+ EVs. The miRNAs’ putative targets within these EVs manifested pronounced expression in the brain, fortifying their neurospecific provenance. We subjected our findings to stringent validation using Thy-1 GFP M line mice, transgenic models wherein GFP expression is confined to hippocampal neurons. An amalgamation of these results with an exhaustive data analysis accentuates the potential of APLP1+ EVs as cerebrally originated biomarkers. Synthesizing our findings, APLP1+ EVs are postulated not merely as diagnostic markers but as seminal entities shaping the future trajectory of neurodegenerative disease diagnostics.

IntroductionDiffuse parenchymal lung diseases (DPLD) cover heterogeneous types of lung disorders. Among many pathological phenotypes, pulmonary fibrosis is the most devastating and represents a characteristic sign of idiopathic pulmonary fibrosis (IPF). Despite a poor prognosis brought by pulmonary fibrosis, there are no specific diagnostic biomarkers for the initial development of this fatal condition. The major hallmark of lung fibrosis is uncontrolled activation of lung fibroblasts to myofibroblasts associated with extracellular matrix deposition and the loss of both lung structure and function.MethodsHere, we used this peculiar feature in order to identify specific biomarkers of pulmonary fibrosis in bronchoalveolar lavage fluids (BALF). The primary MRC-5 human fibroblasts were activated with BALF collected from patients with clinically diagnosed lung fibrosis; the activated fibroblasts were then washed rigorously, and further incubated to allow secretion. Afterwards, the secretomes were analysed by mass spectrometry.ResultsIn this way, the CD44 protein was identified; consequently, BALF of all DPLD patients were positively tested for the presence of CD44 by ELISA. Finally, biochemical and biophysical characterizations revealed an exosomal origin of CD44. Receiver operating characteristics curve analysis confirmed CD44 in BALF as a specific and reliable biomarker of IPF and other types of DPLD accompanied with pulmonary fibrosis.

Candida albicans is a prevalent fungal pathogen responsible for infections in humans. As described recently, nanometer-sized extracellular vesicles (EVs) produced by C. albicans play a crucial role in the pathogenesis of infection by facilitating host inflammatory responses and intercellular communication. This study investigates the functional properties of EVs released by biofilms formed by two C. albicans strains—3147 (ATCC 10231) and SC5314—in eliciting host responses. We demonstrate the capability of C. albicans EVs to trigger reactions in human epithelial and immune cells. The involvement of EVs in pathogenesis was evidenced from the initial stages of infection, specifically in adherence to epithelial cells. We further established the capacity of these EVs to induce cytokine production in the epithelial A549 cell line, THP-1 macrophage-like cells, and blood-derived monocytes differentiated into macrophages. Internalization of EVs by THP-1 macrophage-like cells was confirmed, identifying macropinocytosis and phagocytosis as the most probable mechanisms, as demonstrated using various inhibitors that target potential vesicle uptake pathways in human cells. Additionally, C. albicans EVs and their cargo were identified as chemoattractants for blood-derived neutrophils. After verification of the in vivo effect of biofilm-derived EVs on the host, using Galleria mellonella larvae as an alternative model, it was demonstrated that vesicles from C. albicans SC5314 increased mortality in the injected larvae. In conclusion, for both types of EVs a predominantly pro-inflammatory effect on host was observed, highlighting their significant role in the inflammatory response during C. albicans infection.

This protocol paper describes how to extract small extracellular vesicles (sEVs) from dried blood spots (DBS). The methodology is described in detail and offers further evidence that the extracted particles are sEVs using western blotting (anti-CD9, CD63 and CD81) and fluorescence nanoparticle tracking analysis (fNTA). In addition, we present evidence that approximately 40% of the sEVs were recovered from DBS compared with EVs analyzed from plasma directly. The protocol proves to be robust, reliable and displays very interesting performances even after several weeks (up to 3 weeks) of storage of the DBS when analyzing the sEVs using protein microarray for the presence of the markers CD9, CD63, CD81, EpCAM, Flotilin-1, CD62E/P, CD142 and CD235a. These findings have important implications for using sEVs as future potential diagnostic tools by supporting the validity of less-invasive methods that can be implemented within vulnerable populations or in the field.

Extracellular vesicles (EVs) have been demonstrated to own the advantages in evading phagocytosis, crossing biological barriers, and possessing excellent biocompatibility and intrinsic stability. Based on these characteristics, EVs have been used as effective therapeutic carriers for drug delivery, but the low drug loading capacity greatly limits further applications. Herein, we developed a drug loading method based on cell-penetrating peptide (CPP) to enhance the encapsulation of therapeutic reagents in EVs, and EVs-based drug delivery system achieved higher killing efficacy to tumor cells. Urinary EVs and chemotherapy reagent doxorubicin (DOX) were used as model. It is easy to conjugate CPP with DOX (CPP-DOX) through the linker N-succinimidyl 3-maleimidopropionate (SMP). CPP-DOX was incubated with EVs under a mild condition, promoting the encapsulation of DOX into EV cavities. CPP-DOX-EVs showed strong anticancer ability since EVs delivery facilitated the uptake by cancer cells. EVs loading of CPP-DOX exhibited higher drug loading efficiency at 37.18%, presenting about 2.5 times increase in efficiency over EVs loading of DOX through passive incubation. Easy operation and controllable condition further reinforce the advantages compared with other loading methods. CPP-based drug loading method provides an effective strategy for EVs-based drug delivery system.

The effects of chronically stressing male mice can be transmitted across generations by stress-specific changes in their sperm miRNA content, which induce stress-specific phenotypes in their offspring. However, how each stress paradigm alters the levels of distinct sets of sperm miRNAs is not known. We showed previously that exposure of male mice to chronic social instability (CSI) stress results in elevated anxiety and reduced sociability specifically in their female offspring across multiple generations because it reduces miR-34c levels in sperm of stressed males and their unstressed male offspring. Here, we describe evidence that astrocyte-derived exosomes (A-Exos) carrying miR-34c mediate how CSI stress has this transgenerational effect on sperm. We found that CSI stress decreases miR-34c carried by A-Exos in the prefrontal cortex and amygdala, as well as in the blood of males. Importantly, miR-34c A-Exos levels are also reduced in these tissues in their F1 male offspring, who despite not being exposed to stress, exhibit reduced sperm miR-34c levels and transmit the same stress-associated traits to their male and female offspring. Furthermore, restoring A-Exos miR-34c content in the blood of CSI-stressed males by intravenous injection of miR-34c-containing A-Exos restores miR-34c levels in their sperm. These findings reveal an unexpected role for A-Exos in maintaining sperm miR-34c levels by a process that when suppressed by CSI stress mediates this example of transgenerational epigenetic inheritance.

Actinobacillus pleuropneumoniae (APP) is a significant pathogen in the swine industry, leading to substantial economic losses and highlighting the need for effective vaccines. This study evaluates the potential of APP-derived extracellular vesicles (APP-EVs) as a vaccine candidate compared to the commercial Coglapix vaccine. APP-EVs, isolated using tangential flow filtration (TFF) and cushioned ultracentrifugation, exhibited an average size of 105 nm and a zeta potential of −17.4 mV. These EVs demonstrated stability under external stressors, such as pH changes and enzymatic exposure and were found to contain 86 major metabolites. Additionally, APP-EVs induced dendritic cell (DC) maturation in a Toll-like receptor 4 (TLR4)-dependent manner without cytotoxicity. APP-EVs predominantly elicited Th1-mediated IgG responses in immunized mice without significant liver and kidney toxicity. Contrarily, unlike Coglapix, which induced stronger Th2-mediated responses and notable toxicity. In addition, APP-EVs triggered APP-specific Th1, Th17, and cytotoxic T lymphocyte (CTL) responses and promoted the activation of multifunctional T-cells. Notably, APP-EV immunization enhanced macrophage phagocytosis and improved survival rates in mice challenged with APP infection compared to those treated with Coglapix. These findings suggest that APP-EVs are promising vaccine candidates, capable of inducing potent APP-specific T-cell responses, particularly Th1, Th17, CTL, and multifunctional T-cells, thereby enhancing the protective immune response against APP infection.

Amniotic epithelial cells (AECs) exhibit significant immunomodulatory and pro-regenerative properties, largely due to their intrinsic paracrine functions that are currently harnessed through the collection of their secretomes. While there is increasing evidence of the role of bioactive components freely secreted or carried by exosomes, the bioactive cargo of AEC microvesicles (MVs) and their crosstalk with the immune cells remains to be fully explored. We showed that under intrinsic conditions or in response to LPS, AEC-derived MV carries components such as lipid-mediated signaling molecules, ER, and mitochondria. They foster the intra/interspecific mitochondrial transfer into immune cells (PBMCs and Jurkat cells) in vitro and in vivo on the zebrafish larvae model of injury. The internalization of MV cargoes through macropinocytosis induces hyperpolarization of PBMC mitochondrial membranes and triggers MV-mediated apoptosis. This powerful immune suppressive mechanism triggered by AEC-MV cargo delivery paves the way for controlled and targeted cell-free therapeutic approaches.

Background: Natural photosensitizers hold potential for photodynamic therapy (PDT) but are often limited by poor visible light absorption. Plant-derived exosome-like nanovesicles offer an innovative platform for enhancing photosensitizer performance. Methods: Hypericum perforatum-derived nanovesicles (HPDENs) were characterized using electron microscopy, dynamic light scattering, and proteomic and miRNA sequencing. High-performance liquid chromatography confirmed hypericin content. PDT efficacy was assessed in vitro and in vivo. Results: HPDENs exhibited robust photosensitizing properties, generating reactive oxygen species (ROS) through both Type I and Type II pathways upon light activation. In vitro, HPDENs showed light dose-dependent cytotoxicity against human melanoma cells, characterized by elevated ROS production and apoptosis induction. In vivo, HPDEN-mediated PDT significantly suppressed tumor growth and induced extensive tumor necrosis, with no observable toxicity to major organs. Conclusion: HPDENs represent a novel plant-derived photosensitizer with dual ROS generation pathways and significant therapeutic efficacy, providing a promising platform for enhancing photodynamic therapy.

SARS-CoV-2 can infect cells through endocytic uptake, a process that is targeted by inhibition of lysosomal proteases. However, clinically this approach to treat viral infections has afforded mixed results, with some studies detailing an oral regimen of hydroxychloroquine accompanied by significant off-target toxicities. We rationalized that an organelle-targeted approach will avoid toxicity while increasing the concentration of the drug at the target. Here, we describe a lysosome-targeted, mefloquine-loaded poly(glycerol monostearate-co-ε-caprolactone) nanoparticle (MFQ-NP) for pulmonary delivery via inhalation. Mefloquine is a more effective inhibitor of viral endocytosis than hydroxychloroquine in cellular models of COVID-19. MFQ-NPs are less toxic than molecular mefloquine, are 100–150 nm in diameter, and possess a negative surface charge, which facilitates uptake via endocytosis allowing inhibition of lysosomal proteases. MFQ-NPs inhibit coronavirus infection in mouse MHV-A59 and human OC43 coronavirus model systems and inhibit SARS-CoV-2 WA1 and its Omicron variant in a human lung epithelium model. Organelle-targeted delivery is an effective means to inhibit viral infection.

Amniotic epithelial cells (AECs) exhibit significant immunomodulatory and pro-regenerative properties, largely due to their intrinsic paracrine functions that are currently harnessed through the collection of their secretomes. While there is increasing evidence of the role of bioactive components freely secreted or carried by exosomes, the bioactive cargo of AEC microvesicles (MVs) and their crosstalk with the immune cells remains to be fully explored. We showed that under intrinsic conditions or in response to LPS, AEC-derived MV carries components such as lipid-mediated signaling molecules, ER, and mitochondria. They foster the intra/interspecific mitochondrial transfer into immune cells (PBMCs and Jurkat cells) in vitro and in vivo on the zebrafish larvae model of injury. The internalization of MV cargoes through macropinocytosis induces hyperpolarization of PBMC mitochondrial membranes and triggers MV-mediated apoptosis. This powerful immune suppressive mechanism triggered by AEC-MV cargo delivery paves the way for controlled and targeted cell-free therapeutic approaches.

Exercise improves cognitive function in Alzheimer’s disease (AD) via mechanism that are not fully clear. Here, we first examined the effect of voluntary exercise training (VET) on energy metabolism and cognitive function in the APP/PS1 transgenic mouse (Tg) model of familial AD. Next, we profiled extracellular vesicles (EVs) and examined whether they may play a role in the protective effects of VET via intranasal administration of EVs, purified from the blood of sedentary (sEV) and/or acutely exercised (eEV) donor wild-type mice into APP/PS1Tg mice. We show that VET reduced resting energy expenditure (REE) and improved cognition in APP/PS1 Tg mice. Administration of eEV, but not sEV, also reduced REE, but had no effect on cognition. Taken together, these data show that exercise is effective intervention to improve symptoms of AD in APP/PS1Tg mice. In addition, eEVs mediate some of these effects, implicating EVs in the treatment of age-related neurodegenerative diseases.

Transposons are parasitic genome elements that can also serve as raw material for the evolution of new cellular functions. However, how retrotransposons are selected and domesticated by host organisms to modulate synaptic plasticity remains largely unknown. Here, we show that the Ty1 retrotransposon Copia forms virus-like capsids in vivo and transfers between cells. Copia is enriched at the Drosophila neuromuscular junction (NMJ) and transported across synapses, and disrupting its expression promotes both synapse development and structural synaptic plasticity. We show that proper synaptic plasticity is maintained in Drosophila by the balance of Copia and the Arc1 (activity-regulated cytoskeleton-associated protein) homolog. High-resolution cryogenic-electron microscopy imaging shows that the structure of the Copia capsid has a large capacity and pores like retroviruses but is distinct from domesticated capsids such as dArc1. Our results suggest a fully functional transposon mediates synaptic plasticity, possibly representing an early stage of domestication of a retrotransposon.

Microbiota-released extracellular vesicles (MEVs) have emerged as a key player in intercellular signaling. However, their involvement in the gut-brain axis has been poorly investigated. We hypothesize that MEVs cross host cellular barriers and deliver their cargoes of bioactive compounds to the brain. In this study, we aimed to investigate the cargo capacity of MEVs for bioactive metabolites and their interactions with the host cellular barriers. First, we conducted a multi-omics profiling of MEVs’ contents from ex vivo and stool samples. Metabolomics analysis identified various neuro-related compounds encapsulated within MEVs, such as arachidonyl-dopamine, gabapentin, glutamate, and N-acylethanolamines. Metaproteomics unveiled an enrichment of enzymes involved in neuronal metabolism, primarily in the glutamine/glutamate/gamma-aminobutyric acid (GABA) pathway. These neuro-related proteins and metabolites were correlated with Bacteroides spp. We isolated 18 Bacteroides strains and assessed their GABA production capacity in extracellular vesicles (EVs) and culture supernatant. A GABA-producing Bacteroides finegoldii , released EVs with a high GABA content (4 µM) compared to Phocaeicola massiliensis . Upon testing the capacity of MEVs to cross host barriers, MEVs exhibited a dose-dependent paracellular transport and were endocytosed by Caco-2 and hCMEC/D3 cells. Exposure of Caco-2 cells to MEVs did not alter expression of genes related to intestinal barrier integrity, while affected immune pathways and cell apoptosis process as revealed by RNA-seq analyses. In vivo , MEVs biodistributed across mice organs, including the brain, liver, stomach, and spleen. Our results highlight the ability of MEVs to cross the intestinal and blood-brain barriers to deliver their cargoes to distant organs, with potential implication for the gut-brain axis. IMPORTANCE Microbiota-released extracellular vesicles (MEVs) have emerged as a key player in intercellular signaling. In this study, a multi-level analysis revealed presence of a diverse array of biologically active molecules encapsulated within MEVs, including neuroactive metabolites, such as arachidonyl-dopamine, gabapentin, glutamate, and N-acylethanolamines, and gamma-aminobutyric acid (GABA). Metaproteomics also unveiled an enrichment of neural-related proteins, mainly the glutamine/glutamate/GABA pathway. MEVs were able to cross epithelial and blood-brain barriers in vitro . RNA-seq analyses showed that MEVs stimulate several immune pathways while suppressing cell apoptosis process. Furthermore, MEVs were able to traverse the intestinal barriers and reach distal organs, including the brain, thereby potentially influencing brain functionality and contributing to mental and behavior.

Mutations in CHRNE encoding the epsilon subunit of acetylcholine receptor result in impaired neuromuscular transmission and congenital myasthenic syndrome (CMS) with variying severity of symptoms. Although the pathophysiology is well-known, blood biomarker signatures enabling a patient-stratification are lacking. This retrospective two-center-study includes 19 recessive CHRNE-patients (AChR deficiency; mean age 14.8 years) from 13 families which were clinically characterized according to disease severity. 15 patients were classified as mildly and 4 patients as moderate to severely affected. Seven known pathogenic and one unreported variant (c.1032 + 2_1032 + 3delinsGT) were identified. Biomarker discovery was carried out on blood samples: proteomics was performed on white blood cells (WBC; n = 12) and on extracellular vesicles (EV) purified from serum samples (n = 7) in addition to amino acid profiling (n = 9) and miRNA screening (n = 18). For miRNA studies, 7 patients with other CMS-subtypes were moreover included. WBC-proteomics unveiled a significant increase of 7 and a decrease of 36 proteins. In silico studies of these proteins indicated affection of secretory granules and the extracellular space. Comparison across patients unveiled increase of two vesicular transport proteins (SCAMP2 and SNX2) in severely affected patients and indeed EV-proteomics revealed increase of 7 and decrease of 13 proteins. Three of these proteins (TARSH, ATRN &amp; PLEC) are known to be important for synaptogenesis and synaptic function. Metabolomics showed decrease of seven amino acids/ amino acid metabolites (aspartic and glutamic acids, phosphoserine, amino adipate, citrulline, ornithine, and 1-methyhistidine). miRNA-profiling showed increase miR − 483 − 3p, miR-365a-3p, miR − 365b − 3p and miR-99a, and decrease of miR-4433b-3p, miR-6873-3p, miR-182-5p and let-7b-5p in CHRNE-patients whereas a comparison with other CMS subtypes showed increase of miR − 205 − 5p, miR − 10b − 5p, miR-125a-5p, miR-499-5p, miR-3120-5p and miR − 483 − 5p and decrease of miR − 1290. Our combined data introduce a molecular fingerprint on protein, metabolic and miRNA level with some of those playing different roles along the neuromuscular axis.

Staphylococcus aureus (S. aureus) is one of the most common causative agents of mammary gland infection and mastitis, but the specific role of S. aureus-derived extracellular vesicles (SaEVs) in mastitis has been poorly studied to date. Here, we aimed to investigate the response of bovine monocyte-derived macrophages (boMdM) to SaEVs of the genotype B (GTB) mastitis-related strain M5512B. Specifically, we evaluated the effects on the actin cytoskeleton, gene expression, and the SaEV proteomic cargo. Furthermore, we assessed to what extent the cellular and molecular response of boMdM to SaEVs differed from peripheral mononuclear blood cells (PBMCs) used for in vitro derivation of the former. We observed that SaEVs induced morphological changes in boMdM, leading to a pro-inflammatory and pyroptosis-related increased gene expression. Additionally, our study revealed that boMdM and PBMCs exhibited stimulus-specific differing responses. The proteomic analysis of SaEVs identified clusters of proteins related to virulence and antibiotic resistance, supporting the theory that S. aureus might use EVs to evade host defences and colonize the mammary gland. Our results bring new insights into how SaEVs might impact the host during an S. aureus infection, which can be useful for future S. aureus vaccine development.