Serum Small Extracellular Vesicles Research Articles

Rapid and Highly Efficient Purification of Extracellular Vesicles Enabled by a TiO2 Hybridized Spongy-like Polymer.

We developed a novel purification medium of extracellular vesicles (EVs) by constructing a spongy-like monolithic polymer kneaded with TiO2 microparticles (TiO2-hybridized spongy monolith, TiO2-SPM). TiO2-SPM was applied in a solid-phase extraction format and enabled simple, rapid, and highly efficient purification of EVs. This is due to the high permeability caused by the continuous large flow-through pores of the monolithic skeleton (median pore size; 5.21 μm) and the specific interaction of embedded TiO2 with phospholipids of the lipid bilayers. Our method also excels in efficiency and comprehensiveness, collecting small EVs (SEVs) from the same volume of a cell culture medium 130.7 times more than typical ultracentrifugation and 4.3 times more than affinity purification targeting surface phosphatidylserine by magnetic beads. The purification method was completed within 1 h with simple operations and was directly applied to serum SEVs. Finally, we demonstrated flexibility toward the shape and size of our method by depleting EVs from fetal bovine serum (FBS), which is a necessary process to prevent contamination of culture cell-derived EVs with exogenous FBS-derived EVs. Our method will eliminate the tedious and difficult purification processes of EVs, providing a universal purification platform for EV-based drug discovery and pathological diagnosis.

Identifying Treatment Resistance Related Pathways by Analyzing Serum Extracellular Vesicles of Patients With Resistant Versus Regressed Retinoblastoma.

To identify the genes and pathways responsible for treatment resistance (TR) in retinoblastoma (RB) by analyzing serum small extracellular vesicles (sEVs) of patients with TR active RB (TR-RB) and completely regressed RB (CR-RB). Serum-derived sEVs were characterized by transmission electron microscopy and nanoparticle tracking analysis. sEV transcriptome profiles of two TR-RB and one CR-RB with good response (>20 years tumor free) were compared to their age-matched controls (n = 3). Gene expression data were analyzed by the R Bioconductor package. The CD9 protein and mRNA expression of CD9, CD63, and CD81 were studied in five RB tumors and two control retinae by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. The isolated serum sEVs were round shaped and within the expected size (30-150 nm), and they had zeta potentials ranging from -10.8 to 15.9 mV. The mean ± SD concentrations of sEVs for two adults and four children were 1.1 × 1012 ± 0.1 and 5.8 × 1011 ± 1.7 particles/mL. Based on log2 fold change of ±2 and P < 0.05 criteria, there were 492 dysregulated genes in TR-RB and 184 in CR-RB. KAT2B, VWA1, CX3CL1, MLYCD, NR2F2, USP46-AS1, miR6724-4, and LINC01257 genes were specifically dysregulated in TR-RB. Negative regulation of apoptotic signaling, cell growth, and proton transport genes were greater than fivefold expressed only in TR-RB. CD9, CD63, and CD81 mRNA levels were high in RB tumors versus control retina, with increased and variable CD9 immunoreactivity in the invasive areas of the tumor. Serum sEVs could serve as a potential liquid biopsy source for understanding TR mechanisms in RB.

Decreased in n-3 DHA enriched triacylglycerol in small extracellular vesicles of diabetic patients with cardiac dysfunction.

Diabetic cardiomyopathy is the leading cause of death in diabetic patients, and the mechanism by which factors other than hyperglycemia contribute to the development of diabetic cardiomyopathy is unknown. Serum small extracellular vesicles (sEVs) carry bioactive proteins or nuclei, which enter into remote tissues and modulate cell functions. However, in diabetic conditions, the changes of lipids carried by sEVs has not been identified. Our study aims to explore the changes of lipids in sEVs in diabetic patients with cardiovascular disease, we hope to provide new ideas for understanding the role of lipid metabolism in the pathogenesis of diabetic cardiomyopathy. SEVs samples derived from serum of health controls (Ctrl), diabetic patients without cardiovascular diseases (DM), and diabetic patients with cardiovascular diseases (DM-CAD) were used for lipidomics analysis. Because AC16 cells are also treated with those sEVs to confirm the entrance of cells and effects on insulin sensitivity, a lipidomics analysis on cells was also performed. In this study, we found that docosahexaenoic acid (DHA)-triacylglycerides of sEVs from serums of DM-CAD patients decreased significantly, and those sEVs could enter into AC16 cells and diminish insulin sensitivity. In addition, DHA-triacylglycerides were also decreased in cells treated with sEVs from DM-CAD. Therefore, DHA-triacylglycerides carried by sEVs may mediate intercellular signaling and be associated with the incidence of diabetic cardiovascular complications.

Glypican-3 (GPC-3) Structural Analysis and Cargo in Serum Small Extracellular Vesicles of Hepatocellular Carcinoma Patients.

Glypican-3 (GPC-3) is a heparin sulfate proteoglycan located extracellularly and anchored to the cell membrane of transformed hepatocytes. GPC-3 is not expressed in normal or cirrhotic liver tissue but is overexpressed in hepatocellular carcinoma (HCC). Because of this, GPC-3 is one of the most important emerging immunotargets for treatment and as an early detection marker of HCC. To determine if GPC-3 domains associated with serum small extracellular vesicles (sEVs) could be used as an HCC diagnostic marker, we predicted in silico GPC-3 structural properties and tested for the presence of its full-length form and/or cleaved domains in serum sEVs isolated from patients with HCC. Structural analysis revealed that the Furin cleavage site of GPC-3 is exposed and readily accessible, suggesting the facilitation of GPC-3 cleavage events. Upon isolation of sEVs from both hepatocytes, culture media and serum of patients with HCC were studied for GPC-3 content. This data suggests that Furin-dependent GPC-3 cleaved domains could be a powerful tool for detection of initial stages of HCC and serve as a predictor for disease prognosis.

Extracellular vesicle microRNA-mediated transcriptional regulation may contribute to dementia with Lewy bodies molecular pathology.

Dementia with Lewy bodies (DLB) is the second most common dementia. Advancing our limited understanding of its molecular pathogenesis is essential for identifying novel biomarkers and therapeutic targets for DLB. DLB is an α-synucleinopathy, and small extracellular vesicles (SEV) from people with DLB can transmit α-synuclein oligomerisation between cells. Post-mortem DLB brains and serum SEV from those with DLB share common miRNA signatures, and their functional implications are uncertain. Hence, we aimed to investigate potential targets of DLB-associated SEV miRNA and to analyse their functional implications. We identified potential targets of six previously reported differentially expressed miRNA genes in serum SEV of people with DLB (MIR26A1, MIR320C2, MIR320D2, MIR548BA, MIR556, and MIR4722) using miRBase and miRDB databases. We analysed functional implications of these targets using EnrichR gene set enrichment analysis and analysed their protein interactions using Reactome pathway analysis. These SEV miRNA may regulate 4278 genes that were significantly enriched among the genes involved in neuronal development, cell-to-cell communication, vesicle-mediated transport, apoptosis, regulation of cell cycle, post-translational protein modifications, and autophagy lysosomal pathway, after Benjamini-Hochberg false discovery rate correction at 5%. The miRNA target genes and their protein interactions were significantly associated with several neuropsychiatric disorders and with multiple signal transduction, transcriptional regulation, and cytokine signalling pathways. Our findings provide in-silico evidence that potential targets of DLB-associated SEV miRNAs may contribute to Lewy pathology by transcriptional regulation. Experimental validation of these dysfunctional pathways is warranted and could lead to novel therapeutic avenues for DLB.

Proteomic profiling of serum small extracellular vesicles reveals immune signatures of children with pneumonia.

BackgroundPneumonia is the leading cause of death in young children globally. However, the underlying pathological mechanism of pediatric pneumonia remains unclear. In infection disease contexts, small extracellular vesicles (sEVs) have been shown to be a useful source of markers for pathogenesis and immune response. We hypothesized that functional molecules such as protein harbored by sEVs would provide mechanistic insights into the immune response in children with pneumonia.MethodsWe isolated sEVs from serum collected from children with and without pneumonia, performed proteomic analysis of the sEVs with label-free mass spectrometry, and then conducted functional enrichment analysis of proteomic data.ResultsWe identified fifteen differentially expressed proteins and ten unique proteins in children with pneumonia as compared to healthy children. In the pneumonia group, immune-related processes and pathways were positively enriched as upregulated proteins were involved in neutrophil activation, complement regulation, defense against bacteria, humoral immune response and regulation of immune effector processes However, pathways associated with tissue development and extracellular matrix remodeling were negatively enriched, as downregulated proteins were linked to extracellular matrix structure and cell adhesions.ConclusionsOur findings provided insights into host responses to pathogen infection, which has contributed to understanding the pathogenesis of children with pneumonia. Furthermore, our studies suggested that serum sEVs proteins could be considered a potential source of biomarkers for diagnosing pediatric pneumonia.

Acute Cerebral Ischemia Increases a Set of Brain-Specific miRNAs in Serum Small Extracellular Vesicles.

Small extracellular vesicles (sEVs) miRNAs are promising diagnosis and prognosis biomarkers for ischemic stroke (IS). This study aimed to determine the impact of IS on the serum sEVs miRNA profile of IS patients and a transient middle cerebral artery occlusion (tMCAO) mouse model. Small RNAseq was used to define the serum sEVs miRNA profile in IS patients and healthy controls (HC), and tMCAO mice and sham controls. Among the 1,444 and 1,373 miRNAs identified in human and mouse serum sEVs, the expression of 424 and 37 miRNAs was significantly altered in the IS patients and tMCAO mice, respectively (| Log2FC| ≥ 1, p < 0.01). Notably, five of the top 25 upregulated miRNAs in IS patients were brain-specific or enriched, including hsa-miR-9-3p, hsa-miR-124-3p, hsa-miR-143-3p, hsa-miR-98-5p, and hsa-miR-93-5p. Upregulation of these four miRNAs was further validated by qPCR. Nine of the 20 upregulated miRNAs in tMCAO mice were also brain-specific or enriched miRNAs. Temporal analysis indicated that the dynamics of mmu-miR-9-5p, mmu-miR-124-3p, mmu-miR-129-5p, and mmu-miR-433-3p were closely correlated with the evolution of ischemic brain injury, as their expression increased at 0.5 days after the onset of ischemia, peaked at day 1 or 3, and returned to normal levels at day 7 and 14. Notably, with the exceptions of mmu-miR-128-3p, the expression of the other eight miRNAs in the mouse serum sEVs was unaffected in the lipopolysaccharide (LPS)-induced neuroinflammation model. Together, in this study, we provided a comprehensive view of the influences of IS on the serum sEVs miRNA profile of IS patients and tMCAO mice and demonstrated the increment of a set of brain-specific miRNAs in serum sEVs after acute cerebral ischemia, which could be promising candidates directly reflecting the ischemic brain injury.

Machine Learning-Assisted Dual-Marker Detection in Serum Small Extracellular Vesicles for the Diagnosis and Prognosis Prediction of Non-Small Cell Lung Cancer.

Small extracellular vesicles (sEVs) carry molecular information from their source cells and are desired biomarkers for cancer diagnosis. We establish a machine learning-assisted dual-marker detection method to analyze the expression of epidermal growth factor receptor (EGFR) and C-X-C chemokine receptor 4 (CXCR4) in serum sEVs for the diagnosis and prognosis prediction of non-small cell lung cancer (NSCLC). We find that the serum sEV EGFR and CXCR4 are significantly higher in advanced stage NSCLC (A/NSCLC) patients compared to early stage NSCLC (E/NSCLC) patients and the healthy donors (HDs). A receiver operating characteristic curve (ROC) analysis demonstrates that the combination of EGFR and CXCR4 in serum sEVs as an efficient diagnostic index and malignant degree indicator for NSCLC. Machine learning further shows a diagnostic accuracy of 97.4% for the training cohort and 91.7% for the validation cohort based on the combinational marker. Moreover, this machine leaning-assisted serum sEV analysis successfully predicts the possibility of tumor relapse in three NSCLC patients by comparing their serum sEVs before and three days after surgery. This study provides an intelligent serum sEV-based assay for the diagnosis and prognosis prediction of NSCLC, and will benefit the precision management of NSCLC.

Isolation and Proteomic Analysis of Mouse Serum Small Extracellular Vesicles for Individual Subject Analysis.

Proteomics characterization of blood and circulating material has been extensively explored for the study of pathological states. In particular, circulating small extracellular vesicles (sEV, diameter: 30-150nm) are known to play an important role in intercellular communication processes, and proteomics profiling has been explored to develop minimally invasive assays for disease monitoring and diagnosis. Due to the genetic and physiological similarities between the two species, and also on account of their shorter life span and rapid disease progression, rodent models are the most commonly used animal model for many human diseases. Such models have provided invaluable insight into the molecular mechanisms of disease progression, candidate drug efficacy, therapy monitoring, and biomarkers research.Longitudinal investigations, in which individuals are monitored over periods of time, are more able to resolve molecular changes during disease progression because they circumvent the inter-individual variation. Longitudinal investigations of rodent models are challenging because of the limited amount of blood that can be withdrawn at each time; the American Association of Veterinary Science stipulates that fortnightly sampling should be limited to a maximum of 10% of the total blood volume. For adult mice this corresponds to approximately 75μL of serum. We developed an approach for the isolation and characterization of serum sEV proteins from just 50μL of serum, for longitudinal studies of disease mouse models. This chapter describes in detail the steps and considerations involved in the sEV isolation, morphological characterization, and proteome profiling by mass spectrometry.

Liver injury changes the biological characters of serum small extracellular vesicles and reprograms hepatic macrophages in mice.

BACKGROUNDSerum small extracellular vesicles (sEVs) and their small RNA (sRNA) cargoes could be promising biomarkers for the diagnosis of liver injury. However, the dynamic changes in serum sEVs and their sRNA components during liver injury have not been well characterized. Given that hepatic macrophages can quickly clear intravenously injected sEVs, the effect of liver injury-related serum sEVs on hepatic macrophages deserves to be explored.AIMTo identify the characteristics of serum sEVs and the sRNAs during liver injury and explore their effects on hepatic macrophages.METHODSTo identify serum sEV biomarkers for liver injury, we established a CCL4-induced mouse liver injury model in C57BL/6 mice to simulate acute liver injury (ALI), chronic liver injury (CLI) and recovery. Serum sEVs were obtained and characterized by transmission electron microscopy and nanoparticle tracking analysis. Serum sEV sRNAs were profiled by sRNA sequencing. Differentially expressed microRNAs (miRNAs) were compared to mouse liver-enriched miRNAs and previously reported circulating miRNAs related to human liver diseases. The biological significance was evaluated by Ingenuity Pathway Analysis of altered sEV miRNAs and conditioned cultures of ALI serum sEVs with primary hepatic macrophages.RESULTSWe found that both ALI and CLI changed the concentration and morphology of serum sEVs. The proportion of serum sEV miRNAs increased upon liver injury, with the liver as the primary contributor. The altered serum sEV miRNAs based on mouse studies were consistent with human liver disease-related circulating miRNAs. We established serum sEV miRNA signatures for ALI and CLI and a panel of miRNAs (miR-122-5p, miR-192-5p, and miR-22-3p) as a common marker for liver injury. The differential serum sEV miRNAs in ALI contributed mainly to liver steatosis and inflammation, while those in CLI contributed primarily to hepatocellular carcinoma and hyperplasia. ALI serum sEVs decreased both CD86 and CD206 expression in monocyte-derived macrophages but increased CD206 expression in resident macrophages in vitro.CONCLUSIONSerum sEVs acquired different concentrations, sizes, morphologies and sRNA contents upon liver injury and could change the phenotype of liver macrophages. Serum sEVs therefore have good diagnostic and therapeutic potential for liver injury.

Serum small extracellular vesicles promote M1 activation of microglia after cerebral ischemia/reperfusion injury

Microglial M1 activation is detrimental to stroke outcomes. Recent studies have shown that circulating small extracellular vesicles (sEVs) can deliver miRNAs to target cells and regulate recipient cell functions. Herein, we tested the hypothesis that miRNA delivery by serum sEVs after cerebral ischemia/reperfusion (I/R) injury promote microglial M1 activation, demonstrating that serum sEVs from middle cerebral artery occlusion (MCAO) mice promoted proliferation and M1 activation of BV2 microglia. To explore the underlying mechanism of serum sEVs-mediated microglial activation in the early phase of cerebral I/R injury, we examined the effects of ischemic brain injury on the serum sEVs miRNAs profile in a mouse MCAO model using small RNAseq. Of the 1257 detected miRNA replications, the levels of 72 were significantly modulated. Bioinformatics analysis revealed that a panel of miRNAs was closely associated with inflammation, and in vitro experiments demonstrated that serum sEVs from MCAO mice could effectively transfer inflammatory miRNAs to BV2 microglia. Collectively, our data suggested that miRNAs delivered by serum sEVs after cerebral I/R injury promoted microglial M1 activation. The identification of microglial activation regulators in future studies will give rise to more effective treatments for stroke.

Longitudinal Bottom-Up Proteomics of Serum, Serum Extracellular Vesicles, and Cerebrospinal Fluid Reveals Candidate Biomarkers for Early Detection of Glioblastoma in a Murine Model.

Glioblastoma Multiforme (GBM) is a brain tumor with a poor prognosis and low survival rates. GBM is diagnosed at an advanced stage, so little information is available on the early stage of the disease and few improvements have been made for earlier diagnosis. Longitudinal murine models are a promising platform for biomarker discovery as they allow access to the early stages of the disease. Nevertheless, their use in proteomics has been limited owing to the low sample amount that can be collected at each longitudinal time point. Here we used optimized microproteomics workflows to investigate longitudinal changes in the protein profile of serum, serum small extracellular vesicles (sEVs), and cerebrospinal fluid (CSF) in a GBM murine model. Baseline, pre-symptomatic, and symptomatic tumor stages were determined using non-invasive motor tests. Forty-four proteins displayed significant differences in signal intensities during GBM progression. Dysregulated proteins are involved in cell motility, cell growth, and angiogenesis. Most of the dysregulated proteins already exhibited a difference from baseline at the pre-symptomatic stage of the disease, suggesting that early effects of GBM might be detectable before symptom onset.

Exploration of Potential Diagnostic Value of Protein Content in Serum Small Extracellular Vesicles for Early-Stage Epithelial Ovarian Carcinoma.

Epithelial ovarian carcinoma (EOC) is one of the most common gynecologic malignancies with a high mortality rate. Serum biomarkers and imaging approaches are insufficient in identifying EOC patients at an early stage. This study is to set up a combination of proteins from serum small extracellular vesicles (sEVs) for the diagnosis of early-stage EOC and to determine its performance. A biomarker for early-stage ovarian cancer (BESOC) cohort was used as a Chinese multi-center population-based biomarker study and registered as a Chinese Clinical Trial ChiCTR2000040136. The sEV protein levels of CA125, HE4, and C5a were measured in 299 subjects. Logistic regression was exploited to calculate the odds ratio and to create the sEV protein model for the predicted probability and subsequently receiver-operating characteristic (ROC) analysis. The combined sEV marker panel of CA125, HE4, and C5a as a sEV model obtained an area under curve (AUC) of 0.912, which was greater than the serum model (0.809), by ROC analysis to identify EOC patients from the whole cohort. With the cutoff of 0.370, the sensitivity and specificity of the sEV model were 0.80 and 0.89, which were much better performance than the serum markers (sensitivity: 0.55~0.66; specificity: 0.59~0.68) and the risk of ovarian malignancy algorithm (ROMA) index approved by the U.S. Food and Drug Administration (sensitivity: 0.65; specificity: 0.61), to identify EOC patients from patients with benign ovarian diseases or other controls. The sEV levels of CA125 significantly differed among early-stage and late-stage EOC (p < 0.001). Moreover, the AUC of ROC to identify early-stage EOC patients was 0.888. Further investigation revealed that the sEV levels of these 3 proteins significantly decreased after cytoreductive surgery (CA125, p = 0.008; HE4, p = 0.025; C5a, p = 0.044). In summary, our study showed that CA125, HE4, and C5a levels in serum sEVs can identify EOC patients at the early stage, elucidating the possibility of using a sEV model for the diagnosis of early-stage EOC.

MiRNA-93 in Serum Extracellular Vesicles Before and After Low Dose Rate Prostate Brachytherapy.

To identify novel biomarkers for prostate cancer (PC), we evaluated changes of miRNAs contained in serum small extracellular vesicles (EVs) in patients who received low dose rate prostate brachytherapy (BT). EVs were isolated from the pooled serum of 10 PC patients prior to and 1 month after BT. miRNA profiling and quantitation in EVs was performed by microarray analysis and RT-digital PCR, respectively. Expression of miRNA-93 in prostate tissue was evaluated using the TCGA database and its level in EVs was determined in 25 patients before and 1, 3, 6 and 12 months after BT. Profiling and quantitation identified miRNA-93 as significantly down-regulated in EVs after BT. TCGA database analysis showed that miRNA-93 was increased in PC tissue. miRNA-93 in EVs significantly decreased in 3, 6 and 12 months after BT. miRNA-93 contained in serum EVs may be a novel diagnostic and monitoring biomarker for PC.

Liquid biopsy based on small extracellular vesicles predicts chemotherapy response of canine multicentric lymphomas

Lymphoma is the most common type of canine hematological malignancy where the multicentric (cMCL) form accounts for 75% of all cases. The standard treatment is the CHOP chemotherapy protocols that include cyclophosphamide, doxorubicin, vincristine and prednisone, where the majority of dogs achieve complete/partial response; however, it is very important to predict non-responsive cases to improve treatment and to develop new targeted therapies. Here we evaluate a liquid biopsy approach based on serum Small Extracellular Vesicles enriched for exosomes (SEVs) to predict cMCL chemotherapy response. Nineteen dogs at the end of the 19-week chemotherapy protocol (8 Complete Response and 11 Progressive Disease) were evaluated for serum SEVs size, concentration and screened for 95 oncomirs. PD patients had higher SEVs concentration at the diagnosis than CR patients (P = 0.034). The ROC curve was significant for SEVs concentration to predict the response to CHOP (AUC = 0.8011, P = 0.0287). A potential molecular signature based on oncomirs from SEVs (caf-miR-205, caf-miR-222, caf-mir-20a and caf-miR-93) is proposed. To the best of our knowledge, this is the first study demonstrating the potential of a liquid biopsy based on SEVs and their miRNAs content to predict the outcome of chemotherapy for canine multicentric lymphomas.

Next-Generation RNA-Sequencing of Serum Small Extracellular Vesicles Discovers Potential Diagnostic Biomarkers for Dementia With Lewy Bodies

ObjectiveThere is an urgent clinical need for identifying blood-based diagnostic biomarkers for Dementia with Lewy Bodies (DLB). Transcriptomic studies have reported unique RNA changes in postmortem DLB brains. Small extracellular vesicles (SEV) that transport RNA between brain and peripheral circulation enable identifying molecular changes in living human brain. Hence, we aimed to identify differentially expressed RNA in serum SEVs from people with DLB. MethodsWe investigated serum SEV total RNA profiles in people with DLB (n = 10) and age and gender matched comparisons (n = 10) using next-generation RNA-sequencing. SEVs were separated by ultracentrifugation with density gradient and were characterized by nanoparticle analysis and western blotting. We verified the differential expression levels of identified differentially expressed genes (DEG) using high-throughput qPCR. Functional implications of identified DEG were evaluated using Ingenuity pathway analyses. ResultsWe identified 846 nominally significant DEG including 30 miRNAs in DLB serum SEVs. We identified significant downregulation of proinflammatory genes, IL1B, CXCL8, and IKBKB. Previously reported postmortem DLB brain DEGs were significantly enriched (χ2=4.99; df=1; p = 0.03) among the identified DEGs, and the differential expression of 40 postmortem DLB brain DEGs could be detected in serum SEVs of people living with DLB. Functional pathway and network analyses highlighted the importance of immunosenescence, ubiquitin proteasome system (UPS) dysfunction, DNA repair, and RNA post-transcriptional modification deficits in DLB pathology. ConclusionIdentified DEGs, especially reduced expression levels of inflammation, and UPS-associated RNA, may aid diagnosing DLB, and their biomarker potential warrants further investigation in larger clinical cohorts. Our findings corroborate the absence of chronic neuroinflammation in DLB.

A facile “one-material” strategy for tandem enrichment of small extracellular vesicles phosphoproteome

Small extracellular vesicles (SEVs), are cell-derived, membrane-enclosed nanometer-sized vesicles that play vital roles in many biological processes. Recent years, more and more evidences proved that small EVs have close relationship with many diseases such as cancers and Alzheimer's disease. The use of phosphoproteins in SEVs as potential biomarkers is a promising new choice for early diagnosis and prognosis of cancer. However, current techniques for SEVs isolation still facing many challenges, such as highly instrument dependent, time consuming and insufficient purity. Furthermore, complex enrichment procedures and low microgram amounts of proteins available from clinical sources largely limit the throughput and the coveage depth of SEVs phosphoproteome mapping. Here, we synthesized Ti4+-modified magnetic graphene-oxide composites (GFST) and developed a “one-material” strategy for facile and efficient phosphoproteome enrichment and identification in SEVs from human serum. By taking advantage of chelation and electrostatic interactions between metal ions and phosphate groups, GFST shows excellent performance in both SEVs isolation and phosphopeptide enrichment. Close to 85% recovery is achieved within a few minutes by simple incubation with GFST and magnetic separation. Proteome profiling of the isolated serum SEVs without phosphopeptide enrichment results in 515 proteins, which is approximately one-fold more than those otained by ultracentrifugation or coprecipitation kits. Further application of GFST in one-material-based enrichment led to identification of 859 phosphosites in 530 phosphoproteins. Kinase-substrate correlation analysis reveals enriched substrates of CAMK in serum SEVs phosphoproteome. Therefore, we expect that the low instrument dependency and the limited sample requirement of this new strategy may facilitate clinical investigations in SEV-based transportation of abnormal kinases and substrates for drug target discovery and cancer monitoring.

Characterization of serum small extracellular vesicles and their small RNA contents across humans, rats, and mice

Serum small extracellular vesicles (sEVs) have recently drawn considerable interest because of the diagnostic and therapeutic potential of their miRNAs content. However, the characteristics of human, mouse and rat serum sEVs and their differences in small RNA contents are still unknown. In this study, through nanoparticle tracking analysis and small RNA sequencing, we found that human, rat, and mouse serum sEVs exhibited distinct sizes and particle numbers as well as small RNA contents. Serum sEVs contained not only abundant miRNAs but also a large number of tRNA fragments. Most serum miRNAs existed both inside and outside of sEVs but were enriched in sEVs. Common serum sEV miRNAs (188 miRNAs) and species-specific serum sEV miRNAs (265, 58, and 159 miRNAs, respectively) were identified in humans, rats, or mice. The serum sEVs contained miRNAs from tissues and organs throughout the body, with blood cells as the main contributors. In conclusion, our findings confirmed the rationality of exploring serum sEV miRNAs as noninvasive diagnostic markers and revealed great differences in serum sEV small RNAs between humans, rats, and mice. Inadequate attention to these differences and the contribution of blood cells to serum sEV miRNAs could hinder the clinical translation of basic studies.

Front Cover: Serum Small Extracellular Vesicles Proteome of Tuberculosis Patients Demonstrated Deregulated Immune Response

Front Cover: Serum Small Extracellular Vesicles Proteome of Tuberculosis Patients Demonstrated Deregulated Immune Response

Serum Small Extracellular Vesicles Proteome of Tuberculosis Patients Demonstrated Deregulated Immune Response.

Detailed understanding of host pathogen interaction in tuberculosis is an important avenue for identifying novel therapeutic targets. Small extracellular vesicles (EVs) like exosomes that are rich in proteins, nucleic acids and lipids, act as messengers and may show altered composition in disease conditions. In this case control study, small EVs are isolated from serum of 58 subjects (all male, 33 (15-70) in years) including drug naïve active tuberculosis (ATB: n = 22), non-tuberculosis (NTB: n = 18), and healthy subjects (n = 18). Serum small EVs proteome analysis is carried out using isobaric tag for relative and absolute quantification (iTRAQ) experiments and an independent sample (n = 36) is used for validation. A set of 132 and 68 proteins are identified in iTRAQ-I (ATB/Healthy) and iTRAQ-II (ATB/NTB) experiments, respectively. Four proteins (KYAT3, SERPINA1, HP, and APOC3) show deregulation (log2 -fold change >±0.48, p<0.05) in ATB with respect to healthy controls and Western blot data corroborated mass spectrometry findings. These important proteins, involved in neutrophil degranulation, plasma heme scavenging, kynurenine, and lipid metabolism, show deregulation in ATB patients. Identification of such a protein panel in circulating small EVs besides providing novel insights into their role in tuberculosis may prove to be useful targets to develop host-directed therapeutic intervention.