Protein Cargo Of Extracellular Vesicles Research Articles

Comparative Extracellular Vesicles Proteomics unravels host-pathogen interactions: New insights in bacterial and fungal endophthalmitis in murine models

PurposeEndophthalmitis, an intraocular infection, often results in devastating outcomes due to frequent misdiagnosis and delayed treatment initiation. Extracellular Vesicles (EVs) have long been lauded for its potential as a biomarker in several infections. Previously, we have profiled EVs in bacterial and fungal infected murine model of endophthalmitis. In this study, we reported a comparative analysis of bacterial and fungal endophthalmitis to understand the disease pathobiology and identify a potential signature molecule for diagnosis of infectious endophthalmitis. MethodsEndophthalmitis was induced by intravitreal injection of bacteria (S. aureus and P. aeruginosa) and fungus (A. flavus and C. albicans) in C57BL/6 mice followed by enucleation at 24 hours. EVs were isolated by ultracentrifugation and total proteins were subjected to LC-MS/MS. A comparative analysis of the EV protein cargo during bacterial and fungal endophthalmitis was performed in this study along with the pathway assessment by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology. ResultsProteome analysis of EVs revealed the presence of over 1500 proteins in each of the infected and control groups. Comparative (bacteria versus fungal) study revealed that the protein cargo of the EVs was very distinct among the infected sets with a very few common proteins. Crucial differentially expressed proteins (DEPs) common to both infections were Complement Cascade 8-alpha (C8α), chymase, sorting nexin-29 and glutathione-S-transferase and common pathways were metabolic pathways, Staphylococcus aureus infection, PPAR signalling pathway, cAMP signalling pathway, TGFβ signalling pathway and HIF-1 signalling pathway. ConclusionThis study comprehensively investigates the global proteome of EVs derived from mice model of bacterial and fungal endophthalmitis. Proteomic analysis reveals the disparate EV protein cargo underlining the differences in the pathogenesis of bacterial and fungal endophthalmitis with C8α, chymase, sorting nexin 29 and glutathione-S-transferase common to both infections.

A Comparative Analysis of the Protein Cargo of Extracellular Vesicles from Helminth Parasites.

Helminth parasites cause debilitating-sometimes fatal-diseases in humans and animals. Despite their impact on global health, mechanisms underlying host-parasite interactions are still poorly understood. One such mechanism involves the exchange of extracellular vesicles (EVs), which are membrane-enclosed subcellular nanoparticles. To date, EV secretion has been studied in helminth parasites, including EV protein content. However, information is highly heterogeneous, since it was generated in multiple species, using varied protocols for EV isolation and data analysis. Here, we compared the protein cargo of helminth EVs to identify common markers for each taxon. For this, we integrated published proteomic data and performed a comparative analysis through an orthology approach. Overall, only three proteins were common in the EVs of the seven analyzed species. Additionally, varied repertoires of proteins with moonlighting activity, vaccine antigens, canonical and non-canonical proteins related to EV biogenesis, taxon-specific proteins of unknown function and RNA-binding proteins were observed in platyhelminth and nematode EVs. Despite the lack of consensus on EV isolation protocols and protein annotation, several proteins were shown to be consistently detected in EV preparations from organisms at different taxa levels, providing a starting point for a selective biochemical characterization.

Circulating extracellular vesicles are associated with pathophysiological condition including metabolic syndrome-related dysmetabolism in children and adolescents with obesity.

Obesity of children and adolescents (OCA) is often accompanied by metabolic syndrome (MetS), which often leads to adult obesity and subsequent complications, yet the entire pathophysiological response is not fully understood. The number and composition of circulating extracellular vesicles (EV) reflect overall patient condition; therefore, we investigated the pathophysiological condition of OCA, including MetS-associated dysmetabolism, using circulating EVs. In total, 107 children and adolescents with or without obesity (boys, n = 69; girls, n = 38; median age, 10years) were enrolled. Circulating EV number and EV protein composition were assessed via flow cytometry and liquid chromatography tandem-mass spectrometry, respectively. In a multivariate analysis, relative body weight (standardized partial regression coefficient (SPRC) 0.469, P = 0.012) and serum triglyceride level (SPRC 0.548, P < 0.001) were detected as independent parameters correlating with circulating EV number. Proteomic analysis identified 31 upregulated and 45 downregulated EV proteins in OCA. Gene ontology analysis revealed upregulated proteins to be involved in various biological processes, including intracellular protein transport, protein folding, stress response, leukocyte activation, innate immune response, and platelet degranulation, which can modulate lipid and glucose metabolism, skeletal and cardiac muscle development, inflammation, immune response, carcinogenesis, and cancer progression. Notably, several identified EV proteins are involved in neuro-development, neurotransmitter release, and neuro-protective agents in OCA. Circulating EVs were derived from adipocytes, hepatocytes, B cell lymphocytes, and neurons. Circulating EV number is significantly associated with MetS-related dysmetabolism and the EV protein cargo carries a special "signature" that reflects the alteration of various biological processes under the pathophysiological condition of OCA. KEY MESSAGES: Circulating EV number correlates with physical and laboratory parameters for obesity in children and adolescents. Relative body weight and triglyceride are independent factors for increased circulating EVs. EV composition is significantly changed in obesity of children and adolescents. Identified EV composition changes associated with obesity and involves in metabolism, immune response, and cancer progression. Circulating EVs are partially derived from adipocyte, hepatocytes, B cells, and neurons.

Multi-Omic Temporal Landscape of Plasma and Synovial Fluid-Derived Extracellular Vesicles Using an Experimental Model of Equine Osteoarthritis.

Extracellular vesicles (EVs) contribute to osteoarthritis pathogenesis through their release into joint tissues and synovial fluid. Synovial fluid-derived EVs have the potential to be direct biomarkers in the causal pathway of disease but also enable understanding of their role in disease progression. Utilizing a temporal model of osteoarthritis, we defined the changes in matched synovial fluid and plasma-derived EV small non-coding RNA and protein cargo using sequencing and mass spectrometry. Data exploration included time series clustering, factor analysis and gene enrichment interrogation. Chondrocyte signalling was analysed using luciferase-based transcription factor activity assays. EV protein cargo appears to be more important during osteoarthritis progression than small non-coding RNAs. Cluster analysis revealed plasma-EVs represented a time-dependent response to osteoarthritis induction associated with supramolecular complexes. Clusters for synovial fluid-derived EVs were associated with initial osteoarthritis response and represented immune/inflammatory pathways. Factor analysis for plasma-derived EVs correlated with day post-induction and were primarily composed of proteins modulating lipid metabolism. Synovial fluid-derived EVs factors represented intermediate filament and supramolecular complexes reflecting tissue repair. There was a significant interaction between time and osteoarthritis for CRE, NFkB, SRE, SRF with a trend for osteoarthritis synovial fluid-derived EVs at later time points to have a more pronounced effect.

Proteomic and functional characterisation of extracellular vesicles from collagen VI deficient human fibroblasts reveals a role in cell motility

Extracellular vesicles (EVs) are key mediators of cell-to-cell communication. Their content reflects the state of diseased cells representing a window into disease progression. Collagen-VI Related Muscular Dystrophy (COL6-RD) is a multi-systemic disease involving different cell types. The role of EVs in this disease has not been explored. We compared by quantitative proteomics the protein cargo of EVs released from fibroblasts from patients with COL6-RD and controls. Isolated EVs contained a significant proportion of the most frequently reported proteins in EVs according to Exocarta and Vesiclepedia. We identified 67 differentially abundant proteins associated with vesicle transport and exocytosis, actin remodelling and the cytoskeleton, hemostasis and oxidative stress. Treatment of control fibroblasts with EVs from either patient or healthy fibroblasts altered significantly the motility of cells on a cell migration assay highlighting the functional relevance of EVs. In parallel, we analysed the secretome from the same cells and found a distinctly different set of 48 differentially abundant proteins related to extracellular matrix organisation and remodelling, growth factor response, RNA metabolism and the proteasome. The EVs and secretome sets of proteins only shared two identifiers indicating that the sorting of proteins towards EVs or the secretory pathway is tightly regulated for different functions.

The Adaptation of Botrytis cinerea Extracellular Vesicles Proteome to Surrounding Conditions: Revealing New Tools for Its Infection Process.

Extracellular vesicles (EVs) are membranous particles released by different organisms. EVs carry several sets of macromolecules implicated in cell communication. EVs have become a relevant topic in the study of pathogenic fungi due to their relationship with fungal-host interactions. One of the essential research areas in this field is the characterization protein profile of EVs since plant fungal pathogens rely heavily on secreted proteins to invade their hosts. However, EVs of Botrytis cinerea are little known, which is one of the most devastating phytopathogenic fungi. The present study has two main objectives: the characterization of B. cinerea EVs proteome changes under two pathogenic conditions and the description of their potential role during the infective process. All the experimental procedure was conducted in B. cinerea growing in a minimal salt medium supplemented with glucose as a constitutive stage and deproteinized tomato cell walls (TCW) as a virulence inductor. The isolation of EVs was performed by differential centrifugation, filtration, ultrafiltration, and sucrose cushion ultracentrifugation. EVs fractions were visualised by TEM using negative staining. Proteomic analysis of EVs cargo was addressed by LC-MS/MS. The methodology used allowed the correct isolation of B. cinerea EVs and the identification of a high number of EV proteins, including potential EV markers. The isolated EVs displayed differences in morphology under both assayed conditions. GO analysis of EV proteins showed enrichment in cell wall metabolism and proteolysis under TCW. KEGG analysis also showed the difference in EVs function under both conditions, highlighting the presence of potential virulence/pathogenic factors implicated in cell wall metabolism, among others. This work describes the first evidence of EVs protein cargo adaptation in B. cinerea, which seems to play an essential role in its infection process, sharing crucial functions with the conventional secretion pathways.

The Extracellular Vesicle Citrullinome and Signature in a Piglet Model of Neonatal Seizures.

Neonatal seizures are commonly associated with acute perinatal brain injury, while understanding regarding the downstream molecular pathways related to seizures remains unclear. Furthermore, effective treatment and reliable biomarkers are still lacking. Post-translational modifications can contribute to changes in protein function, and post-translational citrullination, which is caused by modification of arginine to citrulline via the calcium-mediated activation of the peptidylarginine deiminase (PAD) enzyme family, is being increasingly linked to neurological injury. Extracellular vesicles (EVs) are lipid-bilayer structures released from cells; they can be isolated from most body fluids and act as potential liquid biomarkers for disease conditions and response to treatment. As EVs carry a range of genetic and protein cargo that can be characteristic of pathological processes, the current study assessed modified citrullinated protein cargo in EVs isolated from plasma and CSF in a piglet neonatal seizure model, also following phenobarbitone treatment. Our findings provide novel insights into roles for PAD-mediated changes on EV signatures in neonatal seizures and highlight the potential of plasma- and CSF-EVs to monitor responses to treatment.

Human blastocysts uptake extracellular vesicles secreted by endometrial cells containing miRNAs related to implantation.

Are the extracellular vesicles (EVs) secreted by the maternal endometrium uptaken by human embryos and is their miRNA cargo involved in implantation and embryo development? Data suggest that EVs secreted by human endometrial epithelial cells are internalized by human blastocysts, and transport miRNAs to modulate biological processes related to implantation events and early embryo development. Successful implantation is dependent on coordination between maternal endometrium and embryo, and EVs role in the required cell-to-cell crosstalk has recently been established. In this regard, our group previously showed that protein cargo of EVs secreted by primary human endometrial epithelial cells (pHEECs) is implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development. However, little is known about the regulation of these biological processes through EVs secreted by the endometrium at a transcriptomic level. A prospective descriptive study was performed. Endometrial biopsies were collected from healthy oocyte donors with confirmed fertility on the day of oocyte retrieval, 36 h after the LH surge. pHEECs were isolated from endometrial biopsies (n = 8 in each pool) and cultured in vitro. Subsequently, conditioned medium was collected and EVs were isolated and characterized. Uptake of EVs by human blastocysts and miRNA cargo of these EVs (n = 3 pools) was analyzed. EVs were isolated from the conditioned culture media using ultracentrifugation, and characterization was performed using western blotting, nanoparticle tracking analysis, and transmission electron microscopy. EVs were fluorescently labeled with Bodipy-TR ceramide, and their uptake by human blastocysts was analyzed using confocal microscopy. Analysis of the miRNA cargo of EVs was performed using miRNA sequencing, target genes of the most expressed miRNA were annotated, and functional enrichment analysis was performed. EVs measured 100-300 nm in diameter, a concentration of 1.78 × 1011 ± 4.12 × 1010 (SD) particles/ml and expressed intraluminal protein markers Heat shock protein 70 (HSP70) and Tumor Susceptibility Gene 101 (TSG101), in addition to CD9 and CD81 transmembrane proteins. Human blastocysts efficiently internalized fluorescent EVs within 1-2 h, and more pronounced internalization was observed in the hatched pole of the embryos. miRNA-seq analysis featured 149 annotated miRNAs, of which 37 were deemed most relevant. The latter had 6592 reported gene targets, that in turn, have functional implications in several processes related to embryo development, oxygen metabolism, cell cycle, cell differentiation, apoptosis, metabolism, cellular organization, and gene expression. Among the relevant miRNAs contained in these EVs, we highlight hsa-miR-92a-3p, hsa-let-7b-5p, hsa-miR-30a-5p, hsa-miR-24-3p, hsa-miR-21-5p, and hsa-let-7a-5p as master regulators of the biological processes. This is an in vitro study in which conditions of endometrial cell culture could not mimic the intrauterine environment. This study defines potential biomarkers of endometrial receptivity and embryo competence that could be useful diagnostic and therapeutic targets for implantation success, as well as open insight further investigations to elucidate the molecular mechanisms implicated in a successful implantation. This study was supported by the Spanish Ministry of Education through FPU awarded to M.S.-B. (FPU18/03735), the Health Institute Carlos III awarded to E.J.-B. (FI19/00110) and awarded to H.F. by the Miguel Servet Program 'Fondo Social Europeo «El FSE invierte en tu futuro»' (CP20/00120), and Generalitat Valenciana through VALi+d Programme awarded to M.C.C.-G. (ACIF/2019/139). The authors have no conflicts of interest to disclose. N/A.

Upscaling human mesenchymal stromal cell production in a novel vertical-wheel bioreactor enhances extracellular vesicle secretion and cargo profile.

Human mesenchymal stromal cells (hMSCs) are mechanically sensitive undergoing phenotypic alterations when subjected to shear stress, cell aggregation, and substrate changes encountered in 3D dynamic bioreactor cultures. However, little is known about how bioreactor microenvironment affects the secretion and cargo profiles of hMSC-derived extracellular vesicles (EVs) including the subset, "exosomes", which contain therapeutic proteins, nucleic acids, and lipids from the parent cells. In this study, bone marrow-derived hMSCs were expanded on 3D Synthemax II microcarriers in the PBS mini 0.1L Vertical-Wheel bioreactor system under variable shear stress levels at 25, 40, and 64 RPM (0.1-0.3dyn/cm2). The bioreactor system promotes EV secretion from hMSCs by 2.5-fold and upregulates the expression of EV biogenesis markers and glycolysis genes compared to the static 2D culture. The microRNA cargo was also altered in the EVs from bioreactor culture including the upregulation of miR-10, 19a, 19b, 21, 132, and 377. EV protein cargo was characterized by proteomics analysis, showing upregulation of metabolic, autophagy and ROS-related proteins comparing with 2D cultured EVs. In addition, the scalability of the Vertical-Wheel bioreactor system was demonstrated in a 0.5L bioreactor, showing similar or better hMSC-EV secretion and cargo content compared to the 0.1L bioreactor. This study advances our understanding of bio-manufacturing of stem cell-derived EVs for applications in cell-free therapy towards treating neurological disorders such as ischemic stroke, Alzheimer's disease, and multiple sclerosis.

Proteome profiling of salivary small extracellular vesicles in glioblastoma patients.

Extracellular vesicles (EVs) play a critical role in intercellular communication under physiological and pathological conditions, including cancer. EVs cargo reflects their cell of origin, suggesting their utility as biomarkers. EVs are detected in several biofluids, and their ability to cross the blood-brain barrier has highlighted their potential as prognostic and diagnostic biomarkers in gliomas, including glioblastoma (GBM). Studies have demonstrated the potential clinical utility of plasma-derived EVs in glioma. However, little is known about the clinical utility of saliva-derived EVs in GBM. Small EVs were isolated from whole mouth saliva of GBM patients pre- and postoperatively. Isolation was performed using differential centrifugation and/or ultracentrifugation. EVs were characterized by concentration, size, morphology, and EVs cell-surface protein markers. Protein cargo in EVs was profiled using mass spectrometry. There were no statistically significant differences in size and concentration of EVs derived from pre- and post GBM patients' saliva samples. A higher number of proteins were detected in preoperative samples compared to postoperative samples. The authors found four highly abundant proteins (aldolase A, 14-3-3 protein ε, enoyl CoA hydratase 1, and transmembrane protease serine 11B) in preoperative saliva samples from GBM patients with poor outcomes. Functional enrichment analysis of pre- and postoperative saliva samples showed significant enrichment of several pathways, including those related to the immune system, cell cycle and programmed cell death. This study, for the first time, demonstrates the feasibility of isolating and characterizing small EVs from pre- and postoperative saliva samples from GBM patients. Preliminary findings encourage further large cohort validation studies on salivary small EVs to evaluate prognosis in GBM.

Extracellular vesicles secreted by Echinococcus multilocularis: important players in angiogenesis promotion

The involvement of Echinococcus multilocularis, and other parasitic helminths, in regulating host physiology is well recognized, but molecular mechanisms remain unclear. Extracellular vesicles (EVs) released by helminths play important roles in regulating parasite-host interactions by transferring materials to the host. Analysis of protein cargo of EVs from E. multilocularis protoscoleces in the present study revealed a unique composition exclusively associated with vesicle biogenesis. Common proteins in various Echinococcus species were identified, including the classical EVs markers tetraspanins, TSG101 and Alix. Further, unique tegumental antigens were identified which could be exploited as Echinococcus EV markers. Parasite- and host-derived proteins within these EVs are predicted to support important roles in parasite–parasite and parasite-host communication. In addition, the enriched host-derived protein payloads identified in parasite EVs in the present study suggested that they can be involved in focal adhesion and potentially promote angiogenesis. Further, increased angiogenesis was observed in livers of mice infected with E. multilocularis and the expression of several angiogenesis-regulated molecules, including VEGF, MMP9, MCP-1, SDF-1 and serpin E1 were increased. Significantly, EVs released by the E. multilocularis protoscolex promoted proliferation and tube formation by human umbilical vein endothelial cells (HUVECs) in vitro. Taken together, we present the first evidence that tapeworm-secreted EVs may promote angiogenesis in Echinococcus-infections, identifying central mechanisms of Echinococcus-host interactions.

Human milk extracellular vesicles protect protein cargo from human digestion and are taken up by neonatal human enteroids

Background: Human milk (HM) is the ideal infant nutrition and reduces infant death and disease. For example, HM is the best-known preventative for the deadly neonatal intestinal inflammatory disease, necrotizing enterocolitis (NEC), for which there is no cure. How HM reduces NEC risk is relatively unknown. HM contains thousands of molecular components, including extracellular vesicles (EVs). EVs are lipid bilayer-encased particles released from cells that carry biological cargo. EVs are putative regulators of intestinal function and HM EVs offer a mechanism for the transfer of proteins from the mom to the infant’s gut. It is unknown if milk EVs or protein cargo survive digestion. This limits the ability to leverage HM EV proteins as treatments for NEC, additives to infant nutrition, or therapeutics for other diseases. The objective of this proposal is to examine if HM EV protein cargo survives in vivo human digestion and if surviving cargo confers anti-inflammatory benefits in neonatal human enteroids. Hypothesis: HM EVs transport key protein cargo to intercellular targets and protect against intestinal inflammation. Methods: All studies were conducted under approved IRB protocols. Neonatal intestinal contents (digesta) were collected after gastric feeding from naso- or orojejunal sampling tubes. EVs were isolated from HM and digesta by density-gradient ultracentrifugation. EVs were validated by electron microscopy, nanoparticle tracking analysis, and western blot. EV protein cargo from n=3 paired HM and digesta samples were profiled by a C18-UPLC with Orbitrap mass spectrometry. Apical-out neonatal human enteroids were validated by qPCR for proliferation and differentiation markers (mean ± SEM). Enteroid EV uptake was assessed with CMPTX dye-labeled EVs. Effects of digesta EVs on inflammation were assessed by qPCR in LPS-treated enteroids. Results: EVs are enriched for markers CD63, CD81, CD9, and TSG101 and de-enriched for casein, milk fat globules. Only 4.68% ± 0.02 (p=0.0012 vs HM) of EV cargo proteins from HM reach the human intestine, but nearly half 64.08% ± 0.02 (p=0.007 vs HM) of the protein diversity is preserved. Human mammary-derived protein BTN1A1 is present in digesta EVs. Apical-out enteroids gene expression is consistent with a more differentiated epithelium, e.g. elevated ChgA (7.60 ± 4.04 vs 1.37 ± 0.39, p=0.0026) and down-regulated Ki67 (0.13 ± 0.04 vs 1.00 ± 0.04, p&lt;0.0001) Enteroids take up milk EVs. Filtered (0.22μm) neonatal digesta (contains EVs) attenuates LPS-induced TNFα gene expression. Conclusion: These novel findings demonstrate that a mostly pure population of EVs can be isolated from a 1mL starting volume. A subset of HM EVs reaches the neonatal human intestine where they can be taken up by the epithelium and may act to reduce inflammation. These data demonstrate the importance of examining EV cargo that survives to the human intestine when investigating potential HM-mediated mechanisms of disease prevention. NIH K01DK129401, USDA NIFA, Collins Medical Trust, Medical Research Foundation, OHSU Exploratory Research Seed Grant This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Extracellular vesicles secreted from bone metastatic renal cell carcinoma promote angiogenesis and endothelial gap formation in bone marrow in a time-dependent manner in a preclinical mouse model.

Bone is a major metastatic site of renal cell carcinoma (RCC). Recently, it is well recognized that bone metastatic tumor cells remodel bone marrow vasculature. However, the precise mechanism underlying cell-cell communication between bone metastatic RCC and the cells in bone marrow remains unknown. Extracellular vesicles (EVs) reportedly play crucial roles in intercellular communication between metastatic tumor cells and bone marrow. Therefore, we conducted the current study to clarify the histological alteration in vascular endothelium in bone marrow induced by EVs secreted from bone metastatic RCC cells as well as association between angiogenesis in bone marrow and bone metastasis formation. We established a bone metastatic RCC cell line (786-O BM) by in vivo selection and observed phenotypic changes in tissues when EVs were intravenously injected into immunodeficient mice. Proteomic analysis was performed to identify the protein cargo of EVs that could contribute to histological changes in bone. Tissue exudative EVs (Te-EVs) from cancer tissues of patients with bone metastatic RCC (BM-EV) and those with locally advanced disease (LA-EV) were compared for in vitro function and protein cargo. Treatment of mice with EVs from 786-O BM promoted angiogenesis in the bone marrow in a time-dependent manner and increased the gaps of capillary endothelium. 786-O BM EVs also promoted tube formation in vitro. Proteomic analysis of EVs identified aminopeptidase N (APN) as a candidate protein that enhances angiogenesis. APN knockdown in 786-O BM resulted in reduced angiogenesis in vitro and in vivo. When parental 786-O cells were intracardially injected 12 weeks after treatment with786-O BM EVs, more bone metastasis developed compared to those treated with EVs from parental 786-O cells. In patient samples, BM-EVs contained higher APN compared to LA-EV. In addition, BM-EVs promoted tube formation in vitro compared to LA-EVs. EVs from bone metastatic RCC promote angiogenesis and gap formation in capillary endothelium in bone marrow in a time-dependent manner.

Quantitative proteomic analysis of extracellular vesicles in response to baculovirus infection of a Trichoplusia ni cell line.

Due to its outstanding suitability to produce complex biopharmaceutical products including virus-like particles and subunit vaccines, the baculovirus/insect cell expression system has developed into a highly popular production platform in the biotechnological industry. For high productivity, virus-cell communication and an efficient spreading of the viral infection are crucial, and, in this context, extracellular vesicles (EVs) might play a significant role. EVs are small particles, utilized by cells to transfer biologically active compounds such as proteins, lipids as well as nucleic acids to recipient cells for intracellular communication. Studies in mammalian cells showed that the release of EVs is altered in response to infection with many viruses, ultimately either limiting or fostering infection spreading. In this study we isolated and characterized EVs, from both uninfected and baculovirus infected Tnms42 insect cells. Via quantitative proteomic analysis we identified more than 3000 T. ni proteins in Tnms42 cell derived EVs, of which more than 400 were significantly differentially abundant upon baculovirus infection. Subsequent gene set enrichment analysis revealed a depletion of proteins related to the extracellular matrix in EVs from infected cultures. Our findings show a significant change of EV protein cargo upon baculovirus infection, suggesting a major role of EVs as stress markers. Our study might serve in designing new tools for process monitoring and control to further improve biopharmaceutical production within the baculovirus/insect cell expression system.

Proteomic Profiling of Extracellular Vesicles Released by Leptin-Treated Breast Cancer Cells: A Potential Role in Cancer Metabolism.

Tumor extracellular vesicles (EVs), as endocytic vesicles able to transport nucleic acids, proteins, and metabolites in recipient cells, have been recognized fundamental mediators of cell-to-cell communication in breast cancer. The biogenesis and release of EVs are highly regulated processes and both the quantity of EVs and their molecular cargo might reflect the metabolic state of the producing cells. We recently demonstrated that the adipokine leptin, whose circulating levels correlate with adipose tissue expansion, is an inducer of EV release from breast cancer cells. Here, we show a specific proteomic signature of EVs released by MCF-7 breast cancer cells grown in the presence of leptin (Lep-EVs), in attempt to find additional molecular effectors linking obesity to breast cancer biology. An analysis of the proteomic profile of Lep-EVs by LC-MS/MS revealed a significant enrichment in biological processes, molecular functions, and cellular components mainly related to mitochondrial machineries and activity, compared to protein content of EVs from untreated breast cancer cells. Metabolic investigations, carried out to assess the autocrine effects of these vesicles on breast cancer cells, revealed that Lep-EVs were able to increase ATP levels in breast cancer cells. This result is associated with increased mitochondrial respiration evaluated by Seahorse analyzer, supporting the concept that Lep-EVs can modulate MCF-7 breast cancer cell oxidative metabolism. Moreover, taking into account the relevance of tumor immune cell crosstalk in the tumor microenvironment (TME), we analyzed the impact of these vesicles on macrophage polarization, the most abundant immune component in the breast TME. We found that tumor-derived Lep-EVs sustain the polarization of M0 macrophages, derived from the human THP-1 monocytic cells, into M2-like tumor-associated macrophages, in terms of metabolic features, phagocytic activity, and increased expression of CD206-positive population. Overall, our results indicate that leptin by inducing the release of EV-enriched in mitochondrial proteins may control the metabolism of MCF-7 breast cancer cells as well as that of macrophages. Characterization of tumor-derived EV protein cargo in an obesity-associated milieu, such as in the presence of elevated leptin levels, might allow identifying unique features and specific metabolic mechanisms useful to develop novel therapeutic approaches for treatment of breast cancer, especially in obese patients.

Watermelon-Derived Extracellular Vesicles Influence Human Ex Vivo Placental Cell Behavior by Altering Intestinal Secretions.

During pregnancy, mother-to-fetus transfer of nutrients is mediated by the placenta; sub-optimal placental development and/or function results in fetal growth restriction (FGR), and the attendant risk of stillbirth, neurodevelopmental delay, and non-communicable diseases in adulthood. A maternal diet high in fruit and vegetables lowers the risk of FGR but the association cannot be explained fully by known macro- and micronutrients. This study investigates if dietary-derived extracellular vesicles (EVs) can regulate placental function. The study characterizes the microRNA and protein cargo of EVs isolated from watermelon, show they are actively internalized by human intestinal epithelial cells in vitro, use mass spectrometry to demonstrate that they alter the intestinal secretome and bioinformatic analyses to predict the likely affected pathways in cells/tissues distal to gut. Application of the watermelon EV-modified intestinal secretome to human placental trophoblast cells and ex vivo tissue explants affects the trophoblast proteome and key aspects of trophoblast behavior, including migration and syncytialization. Dietary-derived plant EVs can modify intestinal communication with distal tissues, including the placenta. Harnessing the beneficial properties of dietary-derived plant EVs and/or exploiting their potential as natural delivery agents may provide new ways to improve placental function and reduce rates of FGR.

Proteome profiling of Extracellular Vesicles in Pseudomonas aeruginosa endophthalmitis: Prognostic and therapeutic significance in a mouse model

Endophthalmitis is a sight-threatening infection and a serious consequence of complications during intraocular surgery or penetrating injury of which Pseudomonas aeruginosa is an important etiology. Extracellular vesicles (EVs) have evolved as a promising entity for developing diagnostic and therapeutic biomarkers due to their involvement in intracellular communication and pathogenesis of diseases. We aimed to characterise the protein cargo of extracellular vesicles, isolated from a murine (C57BL/6) model of P. aeruginosa endophthalmitis by LC-MS/MS at 24 h post infection (p.i). EVs were extracted by ultracentrifugation, characterized by Dynamic Light Scattering (DLS) and western blotting with tetraspannin markers, CD9 and CD81 and quantified by the ExoCet quantification kit. Multiplex ELISA was performed to estimate the levels of TNF-α, IL-6, IFN-γ and IL-1β. Proteomic analysis identified 2010 proteins (FDR ≤0.01) in EVs from infected mice eyes, of which 137 were differentially expressed (P-value ≤ 0.05). A total of 101 proteins were upregulated and 36 were downregulated. Additionally, 43 proteins were exclusive to infection set. KEGG and Gene Ontology revealed, Focal adhesion, Phagosome pathway, Complement cascade and IL-17 signalling pathway are crucial upregulated pathways involving proteins such as Tenascin, caveolin 1, caveolin 2, glutamine synthetase, microtubule-associated protein, C1, C8 and IL-17. Tenascin and caveolins are known to suppress anti-inflammatory cytokines further exacerbating the disease. The result of this study provides insight into the global extracellular vesicle proteome of P. aeruginosa endophthalmitis with their functional correlation and distinctive pattern of expression and tenascin, caveolin 1 and caveolin 2 are attractive biomarkers for P. aeruginosa endophthalmitis.

Proteomic Analysis of Extracellular Vesicles From Fasciola hepatica Hatching Eggs and Juveniles in Culture.

The identification of extracellular vesicles (EVs) in Fasciola hepatica has provided a new way to understand parasite-host communication. Most of the studies on EVs have focused on the adult stage of F. hepatica, but recently, the presence of EVs from different developmental stages has been reported. To better understand the potential role of EVs in the biology of the parasite and in the infection process, the protein cargo of EVs from embryonated eggs and newly-excysted juvenile (NEJs) flukes cultured up to 28 days, has been analyzed. EVs were isolated by size exclusion chromatography and evaluated by nanoparticle tracking analysis and transmission electron microscopy. LC-MS/MS proteomic analysis of EVs revealed the presence of 23 different proteins from embryonated egg-derived EVs and 29 different proteins from NEJ-derived EVs. Most of the identified proteins had been previously described in EVs from F. hepatica adults, including cytoskeletal proteins, glycolytic enzymes, stress-related proteins and tetraspanins. Nevertheless, EVs from hatching eggs and NEJs exhibited qualitative differences in composition, when compared to EVs form adults, including the absence of cathepsin cysteine peptidases. The differential content of the EVs released by the different developmental stages of the parasite reflect the intense activity of NEJs at this early stage, with several proteins involved in membrane traffic and cell physiology. This new set of identified proteins could help to understand key metabolic, biochemical and molecular mechanisms mediated by EVs that take place upon egg hatching and after parasite excystment.

Characterization and proteome profiling of extracellular vesicles in a murine model of Staphylococcus aureus endophthalmitis

Endophthalmitis is a vision-threatening complication of intraocular surgery or penetrating injury of which Staphylococcus aureus is an important etiological agent. Extracellular vesicles (EVs) hold a tremendous possibility for developing diagnostic and therapeutic biomarkers due to their role in the pathogenesis of various infections. The aim of this study was to characterise the protein cargo of EVs, isolated from a murine (C57BL/6) model of S. aureus endophthalmitis by LC–MS/MS. Contralateral eye injected with sterile media served as control and both eyes were enucleated after 24 h, followed by extraction of EVs by ultracentrifugation. EVs were characterized by DLS and western blotting with tetraspanin markers, CD9 and CD81 and quantified by ExoCet quantification kit. Proteomic analysis identified 1964 proteins (FDR ≤ 0.01) in EVs from infected mice eyes, of which 40 proteins varied significantly in their amounts in comparison to EVs obtained from control eyeballs (P-value ≤ 0.05). The results of this study provide insight into the global EV proteome of S. aureus endophthalmitis with their functional correlation and differential protein amounts between infected and control set. Annexin A5, cathepsin D and C5a play a pivotal role in disease pathogenesis and could possibly play a role as a prognostic marker in endophthalmitis.

Obese Adipose Tissue‐Derived Extracellular Vesicles increase Colorectal Cancer Cell proliferation and glycolysis

Colorectal cancer (CRC) is the second deadliest cancer worldwide and obesity is an established risk factor for both men and women. However, we still have limited knowledge regarding the molecular mechanisms that support obesity‐induced CRC. Obesity substantially modifies the adipose tissue (AT) and increases secretion of various factors including nanometer‐sized extracellular vesicles (EVs). EVs are lipid bilayer vesicles released from most types of cells and contain lipids, proteins, microRNAs and other cytoplasmic components of the parent cell. Considering their ability to travel to distant sites and deliver cargo to recipient cells, EVs could act as intercellular mediators that alter the tumorigenic potential of recipient cancer cells. Therefore, we hypothesized that obese AT secreted EVs carry cargo that enhances tumorigenicity of CRC cells. Here, we aimed to determine the functional and metabolic changes induced by obese human AT‐secreted EVs on human CRC cell lines. Visceral AT tissue from patients with obesity (BMI&gt;35) undergoing bariatric surgery and non‐obese patients (BMI&lt;30) undergoing anti‐reflux surgery was cultured ex vivo in EV‐free DMEM‐H/Ham’s F12 for 48 hrs. Conditioned media was collected and EVs were isolated by ultracentrifugation. EV size and concentration were measured through Nanoparticle Tracking Analysis (NTA). EV protein cargo was characterized by unbiased proteomics analysis using LC‐MS. Human CRC cell lines HCT‐116 and HT‐29 were treated with 10^7 EV/ml for 24 or 48 hrs and proliferation and mitochondrial/glycolytic ATP production were measured by CCK‐8 assay or Seahorse XF ATP Rate Assay, respectively. NTA reported our EV preps were on average 120 nm in size, suggesting we isolated exosomes and/or small microvesicles. Using KEGG Pathway Database and Panther Protein Classification Software, 25% of EV proteins secreted from human AT were associated with metabolism, with 13% of total metabolic proteins in EVs associating with glycolysis. Obese EVs exhibited increased expression of glycolytic enzymes pyruvate kinase, phosphoglycerate kinase 1, and G3P dehydrogenase. Obese EVs increased proliferation and glycolytic ATP production in CRC cell lines, both serving as indicators of cancer aggressiveness. Taken together, our results suggest that obese adipose EVs mediate the tumorigenic potential of CRC cells. Our study is significant since it aims to identify druggable pathways which support tumorigenesis in obese CRC patients.