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Prostate cancer has heterogeneous growth patterns, and its prognosis is the poorest when it progresses to a neuroendocrine phenotype. Using bioinformatic analysis, we evaluated RNA expression of neuroendocrine genes in a panel of five different cancer types: prostate adenocarcinoma, breast cancer, kidney chromophobe, kidney renal clear cell carcinoma and kidney renal papillary cell carcinoma. Our results show that specific neuroendocrine genes are significantly dysregulated in these tumors, suggesting that they play an active role in cancer progression. Among others, synaptophysin (SYP), a conventional neuroendocrine marker, is upregulated in prostate adenocarcinoma (PRAD) and breast cancer (BRCA). Our analysis shows that SYP is enriched in small extracellular vesicles (sEVs) derived from plasma of PRAD patients, but it is absent in sEVs derived from plasma of healthy donors. Similarly, classical sEV markers are enriched in sEVs derived from plasma of prostate cancer patients, but weakly detectable in sEVs derived from plasma of healthy donors. Overall, our results pave the way to explore new strategies to diagnose these diseases based on the neuroendocrine gene expression in patient tumors or plasma sEVs.

Cancer cells communicate within the tumor microenvironment (TME) through extracellular vesicles (EVs), which act as crucial messengers in intercellular communication, transporting biomolecules to facilitate cancer progression. Ubiquitin-like 3 (UBL3) facilitates protein sorting into small EVs as a post-translational modifier. However, the effect of UBL3 overexpression in EV-mediated protein secretion has not been investigated yet. This study aimed to investigate the effect of UBL3 overexpression in enhancing EV-mediated Achilles protein secretion in MDA-MB-231 (MM) cells by a dual-reporter system integrating Akaluc and Achilles tagged with Ubiquitin where self-cleaving P2A linker connects Akaluc and Achilles. MM cells stably expressing Ubiquitin-Akaluc-P2A-Achilles (Ubi-Aka/Achi) were generated. In our study, both the bioluminescence of Ubiquitin-Akaluc (Ubi-Aka) and the fluorescence of Achilles secretion were observed. The intensity of Ubi-Aka was thirty times lower, while the Achilles was four times lower than the intensity of corresponding cells. The ratio of Ubi-Aka and Achilles in conditioned media (CM) was 7.5. They were also detected within EVs using an EV uptake luciferase assay and fluorescence imaging. To investigate the effect of the UBL3 overexpression in CM, Ubi-Aka/Achi was transiently transfected into MM-UBL3-KO, MM, and MM-Flag-UBL3 cells. We found that the relative fluorescence expression of Achilles in CM of MM-UBL3-KO, MM, and MM-Flag-UBL3 cells was 30 %, 28 %, and 45 %, respectively. These findings demonstrated that UBL3 overexpression enhances EV-mediated Achilles protein secretion in CM of MM cells. Targeting UBL3 could lead to novel therapies for cancer metastasis by reducing the secretion of pro-metastatic proteins, thereby inhibiting disease progression., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The accurate diagnosis of non-obstructive azoospermia and obstructive azoospermia is crucial for selecting appropriate clinical treatments. This study aimed to investigate the pivotal role of microRNAs in circulating plasma extracellular vesicles in distinguishing between non-obstructive azoospermia and obstructive azoospermia, as well as uncovering the signaling pathways involved in azoospermia pathogenesis. In this study, differential expression of extracellular vesicle miR-513c-5p and miR-202-5p was observed between non-obstructive azoospermia and obstructive azoospermia patients, while the selenocompound metabolism pathway could be affected in azoospermia through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. The predictive power of these microRNAs was evaluated using receiver characteristic operator-area under the curve analysis, demonstrating promising sensitivity, specificity, and area under the curve values. A binomial regression equation incorporating circulating plasma levels of extracellular vesicles miR-202-5p and miR-513c-5p along with follicle-stimulating hormone was calculated to provide a clinically applicable method for diagnosing non-obstructive azoospermia and obstructive azoospermia. This study presents a potentially non-invasive testing approach for distinguishing between non-obstructive azoospermia and obstructive azoospermia, offering a possibly valuable tool for clinical practice., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aims: To describe tDRs in human milk EVs and their associations with maternal body mass index, age, dietary indices, breastfeeding frequency, season and time of milk collection in a Latina population., Materials & Methods: We sequenced small RNAs from EVs from 109 mature human milk samples collected at 1 month after delivery in the Southern California Mother's Milk Study. We grouped tDRs using hierarchical clustering and clusters were compared across tDR characteristics. We analyzed associations of tDRs with intrinsic maternal variables (body mass index, age), maternal nutrition (caloric intake, Healthy Eating Index, Dietary Inflammatory Index), and variables related to feeding and milk collection (breastfeeding frequency, season and time of milk collection) using negative binomial models., Results: We identified 338 tDRs expressed in 90% or more of milk EV samples, of which 113 were identified in all samples. tDR-1:26-Gly-CCC-1-M4 accounted for most reads (79%). Pathway analysis revealed a wide array of biological processes and disease mechanisms across the four tDR clusters. tDRs were associated with season of collection, time of collection, breastfeeding frequency, and the dietary inflammatory index., Conclusions: tDRs are abundant in milk EVs and may be sensitive to maternal diet, seasonality, time of day, and breastfeeding frequency.

Background: In liquid biopsy, mutation detection is primarily performed using cell-free DNA (cfDNA). However, the numerous advantages of extracellular vesicle (EV) DNA for mutation detection have gradually garnered the attention of researchers in recent years. This study aimed to compare the differences between EV DNA and cfDNA in mutation detection and explore the role of plasma androgen receptor (AR) mutations in the prognosis of prostate cancer (PCa)., Methods: We compared the biological characteristics of plasma extracellular vesicle DNA (p-EV DNA) and cfDNA by capillary electrophoresis and concentration detection. Subsequently, we performed pan-oncogene-targeted sequencing in paired tissue and plasma samples from five patients with PCa to verify the feasibility of mutation detection using p-EV DNA and cfDNA. Further, we conducted AR mutation detection in expanded samples to compare the differences between EV DNA and cfDNA in mutation detection and to analyse their role in PCa., Results: p-EV DNA fragments were larger than plasma cell-free DNA (p-cfDNA) fragments; however, there was no significant difference in their concentrations in the plasma of patients with PCa. Feasibility analysis revealed that major mutations associated with PCa detected in tissue samples could be identified in both p-EV DNA and p-cfDNA. Advantage comparison found that, although cfDNA could detect more mutations, AR mutations in EV DNA were more strongly associated with a poor prognosis of PCa than cfDNA., Conclusion: Mutation detection using either EV DNA or cfDNA is both feasible in PCa liquid biopsies, and EV DNA AR mutations have an advantage in prognostic assessment for PCa. This study lays the foundation for future research on EV DNA-related biomarkers.

Introduction: Preeclampsia (PE) is a pregnancy complication marked by high blood pressure, posing risk to maternal and fetal health. "Genomic imprinting", an epigenetic phenomenon regulated by DNA methylation at Differently Methylated Regions (DMR's), influences placental development. Research on circulating extracellular vesicles (EVs) in PE suggests them as potential source for early biomarkers, but methylation status of EV-DNA in Preeclampsia is not reported yet., Methods: This study examines the methylation and expression profile of imprinted genes - PEG10, PEG3, MEST, and DLK1 in circulating EVs of 1 st and 3 rd trimester control and early onset preeclampsia (EOPE) pregnant women (n = 15) using pyrosequencing and qRT-PCR respectively., Results: In 1 st trimester, PEG3 was significantly hypermethylated, whereas no significant methylation changes were noted in PEG10 and MEST in EOPE. In 3 rd trimester, significant hypomethylation in PEG10, PEG3 and IGDMR was observed whereas significant hypermethyaltion noted in MEST. mRNA expression of PEG10, PEG3 and DLK1 was not affected in circulating EVs of 1 st trimester EOPE. However, in 3 rd trimester significant increased expression in PEG10, PEG3 and DLK1 noted. MEST expression was reduced in 3 rd trimester EOPE. No correlation was observed between average DNA methylation and gene expression in PEG10 and PEG3 in 1 st trimester. However, in 3 rd trimester, significant negative correlation was noted in PEG10 (r = -0.426, p = 0.04), PEG3 (r = -0.496, p = 0.01), MEST (r = -0.398, p = 0.03) and DLK1 (r = -0.403, p = 0.03)., Discussion: The results of our study strengthen the potential of circulating EVs from maternal serum as non-invasive indicators of placental pathophysiology, including preeclampsia., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Cancer-associated myofibroblasts (mCAFs) represent a significant component of the tumor microenvironment due to their contributions to extracellular matrix (ECM) remodeling. The pro-tumor mechanisms of extracellular vesicles (EVs) by regulating mCAFs and related collagens remain poorly understood in oral squamous cell carcinoma (OSCC). In this study, through analysis of single-cell sequencing data and immunofluorescence staining, we confirmed the increased presence of mCAFs and enrichment of specific collagen types in OSCC tissues. Furthermore, we demonstrated that OSCC-derived EVs promote the transformation of fibroblasts into mCAFs, leading to tumor invasion. Proteomic analysis identified the presence of TGF-β1 in EVs and revealed its role in inducing mCAFs via the TGF-β1/SMAD signaling pathway. Experiments in vivo confirmed that EVs, particularly those carrying TGF-β1, trigger COL18 high COL5 high matrix deposition, thereby forming the pro-tumor ECM in OSCC. In summary, our investigation unveils the significant involvement of OSCC-derived EVs in orchestrating the differentiation of fibroblasts into mCAFs and modulating specific collagen types within the ECM. Therefore, this study provides a theoretical basis for targeting the EV-mediated TGF-β1 signaling pathway as a potential therapeutic strategy for OSCC., Competing Interests: Declaration of competing interest The authors deny any conflicts of interest related to this study., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Maternal hyperglycemia during pregnancy adversely affects maternal and child outcomes. While mechanisms are not fully understood, maternal circulating miRNAs may play a role. We examined whether continuous glucose levels and hyperglycemia subtypes (gestational diabetes, type 2 diabetes, and glucose intolerance) were associated with circulating miRNAs during late pregnancy. Seven miRNAs (hsa-miR-107, hsa-let-7b-5p, hsa-miR-126-3p, hsa-miR-181a-5p, hsa-miR-374a-5p, hsa-miR-382-5p, and hsa-miR-337-5p) were associated ( p  < 0.05) with either hyperglycemia or continuous glucose levels prior to multiple testing correction. These miRNAs target genes involved in pathways relevant to maternal and child health, including insulin signaling, placental development, energy balance, and appetite regulation.

Background: Imatinib (IM) is the primary treatment for patients with chronic-phase CML (CML-CP). However, an increasing number of CML-CP patients have developed resistance to IM. Our study aims to explore the expression of miR-629-5p in extracellular vesicles (EVs) from both IM-sensitive (K562) and resistant (K562-Re) CML cell lines and to investigate the impact of regulating miR-629-5p expression on the biological characteristics of K562 and K562-Re cells., Methods: Assess miR-629-5p expression levels in IM-sensitive and resistant CML cell lines. Separate EVs and verify it. EVs from K562-Re cells were co-cultured with K562 cells to detect the expression level of miR-629-5p. Target genes of miR-629-5p were determined and validated through luciferase experiments. Examined by manipulating miR-629-5p expression in cells using transfection techniques. The expression level of phosphorylated proteins in the PI3K/AKT/mTOR signaling pathway after IM was detected in CML cell lines. In K562-Re cells, the expression level of phosphorylated protein in the PI3K/AKT/mTOR signaling pathway was detected after single transfection of miR-629-5p inhibitor and cotransfection of miR-629-5p inhibitor and siSENP2., Results: Increasing concentrations of EVs from K562-Re cells elevated miR-629-5p expression levels. The expression levels of miR-629-5p in CML cells varied with IM concentration and influenced the biological characteristics of cells. SENP2 was identified as a target gene of miR-629-5p. Furthermore, miR-629-5p was found to modulate the SENP2/PI3K/AKT/mTOR pathway, impacting IM resistance in CML cells., Conclusion: EVs from IM-resistant CML cells alter the expression of miR-629-5p in sensitive cells, activating the SENP2/PI3K/AKT/mTOR pathway and leading to IM resistance.

The migratory and invasive capabilities of melanoma cells contribute to metastasis. Therefore, targeting the genes driving these processes can support melanoma therapy. Rab27A and Rab27B contribute to tumor formation progression in many types of cancer through various mechanisms, including the secretion of small extracellular vesicles (sEVs). We explored the role of these GTPases in melanoma cell functioning in three RAB27A knockout (KO) cell lines (A375, DMBC12, and SkMel28) and a double RAB27A/B KO A375 cell line. The loss of RAB27A impaired the migration and invasion of DMBC12 and SkMel28 cells; however, the behavior of highly aggressive A375 cells was unaffected. The RAB27A/B double knockout moderately decreased the migratory capacity of A375 cells without disturbing their invasiveness. Additionally, the silencing of RAB27A did not affect the number and mean size of the sEVs, despite some alterations in the protein content of the vesicles. Both Rab27 isoforms can, at least partially, act independently. The potential role of Rab27A in the functioning of melanoma cells depends on the individual character of the cell line, but not on its basal expression, and seems to be unrelated to the secretion of sEVs.

Small extracellular vesicles (sEVs) are important intercellular information transmitters in various biological contexts, but their release processes remain poorly understood. Herein, we describe a high-throughput assay platform, CRISPR-assisted individually barcoded sEV-based release regulator (CIBER) screening, for identifying key players in sEV release. CIBER screening employs sEVs barcoded with CRISPR-gRNA through the interaction of gRNA and dead Cas9 fused with an sEV marker. Barcode quantification enables the estimation of the sEV amount released from each cell in a massively parallel manner. Barcoding sEVs with different sEV markers in a CRISPR pooled-screening format allows genome-wide exploration of sEV release regulators in a subpopulation-specific manner, successfully identifying previously unknown sEV release regulators and uncovering the exosomal/ectosomal nature of CD63 + /CD9 + sEVs, respectively, as well as the synchronization of CD9 + sEV release with the cell cycle. CIBER should be a valuable tool for detailed studies on the biogenesis, release, and heterogeneity of sEVs., Competing Interests: Competing interests K.K., Y.U., R.K. have filed a patent for the screening system of sEV release regulators (WO2021095842 (published), application by the University of Tokyo). The other authors report no competing interests., (© 2024. The Author(s).)

Background: Extracellular vesicles (EVs) are a group of nanoscale cell-derived membranous structures secreted by all cell types, containing molecular cargoes involved in intercellular communication. EVs can be used to mimic "nature's delivery system" to transport nucleic acids, peptides, lipids, and metabolites to target recipient cells. EVs offer a range of advantages over traditional synthetic carriers, thus paving the way for innovative drug delivery approaches that can be used in different diseases, including cancer. Here, by using breast cancer (BC) cells treated with the multi-kinase inhibitor sorafenib, we generated EVs enriched in specific non-coding RNAs (miR-23b-3p, miR-126-3p, and the long ncRNA GAS5) and investigated their potential impact on the aggressive properties of the BC in vitro and in vivo using zebrafish., Methods: EVs were collected from 4 different BC cell lines (HCC1937, MDA-MB-231, MCF-7, and MDA-MB-453) and characterized by western blotting, transmission electron microscopy and nanoparticle tracking analysis. Levels of encapsulated miR-23b-3p, miR-126-3p, and GAS5 were quantified by ddPCR. The role of the EVs as carriers of ncRNAs in vivo was established by injecting MDA-MB-231 and MDA-MB-453 cells into zebrafish embryos followed by EV-based treatment of the xenografts with EVs rich in miR-23b-3p, miR-126-3p and GAS5., Results: ddPCR analysis revealed elevated levels of miR-23b-3p, miR-126-3p, and GAS5, encapsulated in the EVs released by the aforementioned cell lines, following sorafenib treatment. The use of EVs as carriers of these specific ncRNAs in the treatment of BC cells resulted in a significant increase in the expression levels of the three ncRNAs along with the inhibition of cellular proliferation in vitro. In vivo experiments demonstrated a remarkable reduction of xenograft tumor area, suppression of angiogenesis, and decreased number of micrometastasis in the tails after administration of EVs enriched with these ncRNAs., Conclusions: Our study demonstrated that sorafenib-induced EVs, enriched with specific tumor-suppressor ncRNAs, can effectively inhibit the aggressive BC characteristics in vitro and in vivo. Our findings indicate an alternative way to enrich EVs with specific tumor-suppressor ncRNAs by treating the cells with an anticancer drug and support the development of new potential experimental molecular approaches to target the aggressive properties of cancer cells., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Polycystic ovary syndrome (PCOS) is highly prevalent in women of reproductive age worldwide, exhibits highly heterogeneous clinical presentation and biochemical parameters, and has no cure. This study aimed to investigate the role of miR-34a-5p in PCOS, its effect on glycolysis in granulosa cells (GCs), and its potential contribution to follicular dysregulation., Methods: Herein, Follicular follicular fluid (FF) samples were collected from six patients with PCOS and six healthy controls undergoing in vitro fertilization-embryo transfer. The isolated extracellular vesicles were characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Additionally, miRNA sequencing was performed to identify differentially expressed microRNAs, and their functions were analyzed through transcriptomics. The In vitro effects of miR-34a-5p on glycolysis, cell proliferation, and apoptosis were assessed in human ovarian granulosa tumour cell line (KGN cells). Targets of miR-34a-5p were identified by dual-luciferase reporter assays, and quantitative real-time polymerase chain reaction and western blotting were performed to determine gene and protein expression., Results: The level of miR-34a-5p in FF-derived extracellular vesicles derived from patients with PCOS was significantly higher than that of the control group. Transcriptomic analysis highlighted miR-34a-5p as a key regulator of glycolysis and apoptosis. Furthermore, in vitro analysis showed that miR-34a-5p targeted lactate dehydrogenase A (LDHA), inhibited glycolysis, reduced energy supply to GCs, and promoted apoptosis of KGN cells. Conversely, miR-34a-5p inhibition restored glycolysis function and cell viability., Conclusion: The findings of this study show that miR-34a-5p mediates GC apoptosis in PCOS by targeting LDHA and inhibiting glycolysis, suggesting its crucial role in PCOS pathophysiology, and offering potential therapeutic targets for improving follicular development and fertility outcomes in patients with PCOS. Further research is needed to explore the clinical implications of miR-34a-5p and its use as a biomarker for early diagnosis and prognosis of PCOS., Competing Interests: Declarations Ethics approval and consent to participate Ethical approval was provided by the Clinical Ethics Review Committee of Affiliated Hospital of Guilin Medical College (No.GZR202152). All methods were performed in accordance with the relevant guidelines and the standardized methods section. All participants signed an informed consent form. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Small extracellular vesicles (sEVs) are crucial mediators of cell-cell communication, transporting diverse cargoes like proteins, lipids, and nucleic acids (microRNA, mRNA, DNA). The microRNA sEV cargo has potential utility as a powerful non-invasive disease biomarker due to sEV's ability to traverse biological barriers (e.g., blood-brain barrier) and become accessible through various body fluids. Despite numerous studies on sEV biomarkers in body fluids, identifying tissue or cell-specific sEV subpopulations remains challenging, particularly from the brain. Our study addresses this challenge by adapting existing methods to isolate sEVs from minimal amounts of frozen human brain sections using size exclusion chromatography (SEC). After ethical approval, approximately 250 µg of fresh-frozen human brain tissue (obtained from Manchester Brain Bank [UK]) was sliced from the 3 donor tissues and incubated in collagenase type 3/Hibernate-E solution, with intermediate agitation, followed by serial centrifugation and filtration steps. Then, sEVs were isolated using the SEC method and characterized by following MISEV guidelines. Before isolating RNA from within these sEVs, the solution was treated with Proteinase-K and RNase-A to remove any non-sEV extracellular RNA. The RNA quantity and quality were checked and processed further for qPCR and small RNA sequencing experiments. The presence of sEVs was confirmed through fluorescence nanoparticle tracking analysis (fNTA) and western blot for surface markers (CD9, CD63, CD81). Size distribution (50-200 nm) was confirmed by NTA and electron microscopy. The total RNA concentration within lysed sEVs ranged from 3-9 ng/µL and was used for successful quantification by qPCR for selected candidate microRNAs. Small RNA sequencing on MiSeq provided high-quality data (Q >32) with 1.4-5 million reads per sample. This method enables efficient isolation and characterization of sEVs from minimal brain tissue volumes, facilitating non-invasive biomarker research and holds promise for equitable disease biomarker studies, offering insights into neurodegenerative diseases and potentially other disorders.

Background: The physiological relevance of cell-to-cell communication mediated by small extracellular vesicle-encapsulated microRNAs (sEV-miRNAs) remains debated because of the limiting representativity of specific miRNAs within the extracellular pool. We hypothesize that sEV-miRNA non-canonical function consisting of the stimulation of Toll-like receptor 7 (TLR7) may rely on a global shift of the sEV cargo rather than on the induction of one or few specific miRNAs. Psoriasis represents an ideal model to test such hypothesis as it is driven by overt activation of TLR7-expressing plasmacytoid dendritic cells (pDCs) following keratinocyte damage., Methods: To mimic the onset of psoriasis, keratinocytes were treated with a cocktail of psoriatic cytokines or UV-irradiated. SmallRNA sequencing was performed on sEVs released by healthy and UV-treated keratinocytes. sEV-miRNAs were analyzed for nucleotide composition as well as for the presence of putative TLR7-binding triplets. Primary human pDCs where stimulated with sEVs +/- inhibitors of TLR7 (Enpatoran), of sEV release (GW4869 + manumycin) and of TLR7-mediated pDC activation (anti-BDCA-2 antibody). Secretion of type I IFNs and activation of CD8 + T cells were used as readouts. qPCR on psoriatic and healthy skin biopsies was conducted to identify induced miRNAs., Results: sEV-miRNAs released by damaged keratinocytes revealed a significantly higher content of TLR7-activating sequences than healthy cells. As expected, differential expression analysis confirmed the presence of miRNAs upregulated in psoriatic skin, including miR203a. More importantly, 76.5% of induced miRNAs possessed TLR7-binding features and among these we could detect several previously demonstrated TLR7 ligands. In accordance with this in silico analysis, sEVs from damaged keratinocytes recapitulated key events of psoriatic pathogenesis by triggering pDCs to release type I interferon and activate cytotoxic CD8 + T cells in a TLR7- and sEV-dependent manner., Discussion: Our results demonstrate that miR203a is just one paradigmatic TLR7-activating miRNA among the hundreds released by UV-irradiated keratinocytes, which altogether trigger pDC activation in psoriatic conditions. This represents the first evidence that cell damage shifts the miRNA content of sEVs towards a TLR7-activating cargo capable to propagate inflammation and immunity, offering strong support to the physiological role of systemic miRNA-based cell-to-cell communication., (© 2024. The Author(s).)

Purpose: Oxidative stress in the retinal pigmented epithelium (RPE) has been implicated in age-related macular degeneration by impacting endocytic trafficking, including the formation, content, and secretion of extracellular vesicles (EVs). Using our model of polarized primary porcine RPE (pRPE) cells under chronic subtoxic oxidative stress, we tested the hypothesis that RPE miRNAs packaged into EVs are secreted in a polarized manner and contribute to maintaining RPE homeostasis., Methods: Small EVs (sEVs) enriched for exosomes were isolated from apical and basal conditioned media from pRPE cells grown for up to four weeks with or without low concentrations of hydrogen peroxide using two sEV isolation methods, leading to eight experimental groups. The sEV miRNA expression was profiled using miRNA-Seq with Illumina MiSeq, followed by quality control and bioinformatics analysis for differential expression using the R computing environment. Expression of selected miRNAs were validated using qRT-PCR., Results: We identified miRNA content differences carried by sEVs isolated using two ultracentrifugation-based methods. Regardless of the sEV isolation method, miR-182 and miR-183 were enriched in the cargo of apically secreted sEVs, and miR-122 in the cargo of basally secreted sEVs from RPE cells during normal homeostatic conditions. After oxidative stress, miR-183 levels were significantly decreased in the cargo of apically released sEVs from stressed RPE cells., Conclusions: We curated RPE sEV miRNA datasets based on cell polarity and oxidative stress. Unbiased miRNA analysis identified differences based on polarity, stress, and sEV isolation methods. These findings suggest that miRNAs in sEVs may contribute to RPE homeostasis and function in a polarized manner.

Mature neurons maintain their distinctive morphology for extended periods in adult life. Compared to developmental neurite outgrowth, axon guidance, and target selection, relatively little is known of mechanisms that maintain the morphology of mature neurons. Loss of function in C. elegans dip-2, a member of the conserved lipid metabolic regulator Dip2 family, results in progressive overgrowth of neurites in adults. We find that dip-2 mutants display specific genetic interactions with sax-2, the C. elegans ortholog of Drosophila Furry and mammalian FRY. Combined loss of dip-2 and sax-2 results in failure to maintain neuronal morphology and elevated release of neuronal extracellular vesicles (EVs). By screening for suppressors of dip-2(0) sax-2(0) double mutant defects, we identified gain-of-function (gf) mutations in the conserved Dopey family protein PAD-1 and its associated phospholipid flippase TAT-5/ATP9A that restore normal neuronal morphology and normal levels of EV release to dip-2(0) sax-2(0) double mutants. Neuron-specific knockdown suggests that PAD-1(gf) can act cell autonomously in neurons. PAD-1(gf) displays increased association with the plasma membrane in oocytes and inhibits EV release in multiple cell types. Our findings uncover a novel functional network of DIP-2, SAX-2, PAD-1, and TAT-5 that maintains neuronal morphology and modulates EV release., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Circular RNAs (circRNAs) are closed RNA loops present in humans and other organisms. Various circRNAs have an essential role in diseases, including cancer. Cells can release circRNAs into the extracellular space of adjacent biofluids and can be present in extracellular vesicles. Due to their circular nature, extracellular circRNAs (excircRNAs) are more stable than their linear counterparts and are abundant in many biofluids, such as blood plasma and urine. circRNAs' link with disease suggests their extracellular counterparts have high biomarker potential. However, circRNAs and the extracellular space are challenging research domains, as they consist of complex biological systems plagued with nomenclature issues and a wide variety of protocols with different advantages and disadvantages. Here, we summarize what is known about excircRNAs, the current challenges in the field, and what is needed to improve extracellular circRNA research., (© 2024 Wiley Periodicals LLC.)

In the free-living nematode Caenorhabditis elegans, the transmembrane protein SID-2 imports double-stranded RNA into intestinal cells to trigger systemic RNA interference (RNAi), allowing organisms to sense and respond to environmental cues such as the presence of pathogens. This process, known as environmental RNAi, has not been observed in the most closely related parasites that are also within clade V. Previous sequence-based searches failed to identify sid-2 orthologues in available clade V parasite genomes. In this study, we identified sid-2 orthologues in these parasites using genome synteny and protein structure-based comparison, following identification of a SID-2 orthologue in extracellular vesicles from the murine intestinal parasitic nematode Heligmosomoides bakeri . Expression of GFP-tagged H. bakeri SID-2 in C. elegans showed similar localization to the intestinal apical membrane as seen for GFP-tagged C. elegans SID-2, and further showed mobility in intestinal cells in vesicle-like structures. We tested the capacity of H. bakeri SID-2 to functionally complement environmental RNAi in a C. elegans SID-2 null mutant and show that H. bakeri SID-2 does not rescue the phenotype in this context. Our work identifies SID-2 as a highly abundant EV protein whose ancestral function may be unrelated to environmental RNAi, and rather highlights an association with extracellular vesicles in nematodes.

Acipenser gueldenstaedtii is one of the most cultured sturgeon species worldwide and of considerable economic value for caviar production. There are though considerable challenges around chronic stress responses due to increased summer temperatures, impacting sturgeons' immune responses and their susceptibility to opportunistic infections. The identification of molecular and cellular pathways involved in stress responses may contribute to identifying novel biomarkers reflective of fish health status, crucial for successful sturgeon aquaculture. Protein citrullination is a calcium-catalysed post-translational modification caused by peptidylarginine deiminases (PADs), altering target protein function and affecting protein interactions in physiological and pathobiological processes. PADs can also modulate extracellular vesicle (EVs) profiles, which play critical roles in cellular communication, via transport of their cargoes (proteins, including post-translationally modified proteins, genetic material and micro-RNAs). This study identified differences in EV signatures, and citrullinated proteins in sera from winter and summer farmed sturegeons. EVs were significantly elevated in sera of the summer chronically stressed group. The citrullinated proteins and associated gene ontology (GO) pathways in sera and serum-EVs of chronically heat stressed A. gueldenstaedtii, showed some changes, with specific citrullinated serum protein targets including alpa-2-macroglobulin, alpha globin, calcium-dependent secretion activator, ceruloplasmin, chemokine XC receptor, complement C3 isoforms, complement C9, plectin, selenoprotein and vitellogenin. In serum-EVs, citrullinated protein cargoes identified only in the chronically stressed summer group included alpha-1-antiproteinase, apolipoprotein B-100, microtubule actin crosslinking factor and histone H3. Biological gene ontology (GO) pathways related to citrullinated serum proteins in the chronically stressed group were associated with innate and adaptive immune responses, stress responses and metabolic processes. In serum-EVs of the heat-stressed group the citrullinome associated with various metabolic GO pathways. In addition to modified citrullinated protein content, Serum-EVs from the stressed summer group showed significantly increased levels of the inflammatory associated miR-155 and the hypoxia-associated miR-210, but significantly reduced levels of the growth-associated miR-206. Our findings highlight roles for protein citrullination and EV signatures in response to chronic heat stress in A. gueldenstaedtii, indicating a trade-off in immunity versus growth and may be of value for sturgeon aquaculture., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Breast cancer remains the leading cause of cancer-related morbidity and mortality among women worldwide, underscoring the urgent need for novel diagnostic and therapeutic strategies. This review explores the emerging roles of circular RNAs (circRNAs) within extracellular vesicles (exosomes) in breast cancer. circRNAs, known for their stability and tissue-specific expression, are aberrantly expressed in breast cancer and regulate critical cellular processes such as proliferation, migration, and apoptosis, positioning them as promising biomarkers. Exosomes facilitate intercellular communication by delivering circRNAs, reflecting the physiological and pathological state of their source cells. This review highlights the multifaceted roles of exosomal circRNAs in promoting tumor growth, metastasis, and drug resistance through their modulation of tumor metabolism, the tumor microenvironment, and immune responses. In particular, we emphasize their contributions to chemotherapy resistance and their potential as both diagnostic markers and therapeutic targets. By synthesizing current research, this review provides novel insights into the clinical applications of exosomal circRNAs, offering a foundation for future studies aimed at improving breast cancer management through non-invasive diagnostics and targeted therapies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Argonaute (AGO), a component of RNA-induced silencing complexes (RISCs), is a representative RNA-binding protein (RBP) known to bind with mature microRNAs (miRNAs) and is directly involved in post-transcriptional gene silencing. However, despite the biological significance of miRNAs, the roles of other miRNA-binding proteins (miRBPs) remain unclear in the regulation of miRNA loading, dissociation from RISCs and extracellular release. In this study, we performed protein arrays to profile miRBPs and identify 118 RBPs that directly bind to miRNAs. Among those proteins, the RBP quaking (QKI) inhibits extracellular release of the mature microRNA let-7b by controlling the loading of let-7b into extracellular vesicles via additional miRBPs such as AUF1 (also known as hnRNPD) and hnRNPK. The enhanced extracellular release of let-7b after QKI depletion activates Toll-like receptor 7 (TLR7) and promotes the production of proinflammatory cytokines in recipient cells, leading to brain inflammation in the mouse cortex. Thus, this study reveals the contribution of QKI to the inhibition of brain inflammation via regulation of extracellular let-7b release., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Skin wound healing is a multifaceted biological process that encompasses a variety of cell types and intricate signaling pathways. Recent research has uncovered that exosomes derived from adipose stem cells, commonly referred to as ADSC exosomes, play a crucial role in facilitating the healing process. Moreover, it has been demonstrated that an anoxic, or low-oxygen, environment significantly enhances the effectiveness of these exosomes in promoting skin repair. The primary objective of this study was to investigate the underlying mechanisms through which ADSC exosomes contribute to Skin wound healing, particularly by regulating the long non-coding RNA known as NORAD under hypoxic conditions. A significant focus of our research was to examine the interplay between the microRNA miR-524-5p and the Pumilio protein, as we aimed to understand how these molecular interactions might influence the overall healing process. In this study, ADSC exosomes were extracted by simulating hypoxia in vitro and their effects on the proliferation and migration of skin fibroblasts (FB) were evaluated. The expression levels of NORAD, miR-524-5p and Pumilio were analyzed by fluorescence quantitative PCR. Pumilio protein was silenced by siRNA technique to evaluate its role in ADSC exosome-mediated wound healing. The experimental results showed that under hypoxia conditions, NORAD levels in ADSC exosomes increased significantly and could effectively regulate the expression of miR-524-5p. After Pumilio protein silencing, the proliferation and migration ability of fibroblasts were significantly reduced, indicating that Pumilio protein played a role in the process of wound healing. By inhibiting miR-524-5p, the expression of Pumilio protein was restored, further confirming its regulatory mechanism., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: Despite the key role of the endothelium in atherosclerosis, there are no direct techniques for its analysis. The study of extracellular vesicles of endothelial origin (EEVs), might lead to the identification of molecular signatures and early biomarkers of atherosclerosis. The aim of this work was to set up the methods for EEVs separation and transcriptomic analysis., Methods: We adapted an antibody-magnetic-bead based immunocapture protocol for plasma EEVs separation from control (G1), subclinical atherosclerosis (G2) and peripheral artery disease subjects (PAD) (G3), and modified an ultra-low input RNASeq method (n=5/group). By bioinformatics analysis we compared the transcriptome of plasma EEVs with that of human aortic endothelial cells (TeloHAECs), and then, searched for differentially expressed genes (DEG) among EEVs of G1, G2 and G3. From those DEG, UCP2 was selected for further validation in plasma EVs (qPCR), and in vitro, in stimulated TeloHAECs (IL-1β, TNFα, oxLDL and hypoxia)., Results: The RNASeq analysis of plasma EEVs rendered 1667 genes enriched in transcripts expressed by TeloHAECs (NES: 1.93, p adjust=1.4 e-73 ). One hundred seventy DEGs were identified between G2 vs G1, and 180 between G3 vs G1, of which 17 were similarly expressed in G2 and G3 vs control, including UCP2. IL-1β and TNFα (10ng/mL, p<0.05), hypoxia (1% O 2 , p=0.05) and oxLDL (100μg/mL, p=0.055) reduced UCP2 expression in TeloHAECs., Conclusions: We set up a protocol for EEVs separation and sequencing that might be useful for the identification of early markers of endothelial dysfunction in atherosclerosis., (Copyright © 2024 The Authors. Publicado por Elsevier España, S.L.U. All rights reserved.)

In the last decade, it has become clear that extracellular vesicles (EVs) are a ubiquitous component of living systems. These small membrane-enclosed particles can confer diverse functions to the cells that release, capture, or coexist with them in an environment. We use examples across living systems to produce a conceptual framework that classifies three modes by which EVs exert functions: ( a ) EV release that serves a function for producing cells, ( b ) EV modification of the extracellular environment, and ( c ) EV interactions with, and alteration of, receiving cells. We provide an overview of the inherent properties of EVs (i.e., their nature) as well as factors in the environment and receiving cell (i.e., nurture) that determine whether transmission of EV cargo leads to functional cellular responses. This review broadens the context for ruminating on EV functions and highlights the emergent properties of EVs that define their role in biology and will shape their applications in medicine.

Mitochondria are dynamic, intracellular organelles with a separate genome originating from prokaryotes. They perform numerous functions essential for cellular metabolism and energy production. Mitochondrial-derived vesicles (MDVs) are single or double membrane-enclosed vesicles, formed and released from the mitochondrial sub-compartments into the cytosol, in response to various triggers. MDVs interact with other organelles such as lysosomes and peroxisomes or may be incorporated and excreted via extracellular vesicles (EVs). MDVs selectively incorporate diverse protein and lipid cargoes and are involved in various functions such as mitochondrial quality control, immunomodulation, energy complementation, and compartmentalization and transport. This review aims to provide a summary of the current knowledge of MDVs biogenesis, release, cargoes, and roles., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Circular RNAs (circRNAs) have emerged as a prominent class of covalently closed single-stranded RNA molecules that exhibit tissue-specific expression and potential as biomarkers in extracellular vesicles (EVs) derived from liquid biopsies. Still, their characteristics and applications in EVs remain to be unveiled., Methods: We performed a comprehensive analysis of EV-derived circRNAs (EV-circRNAs) using transcriptomics data obtained from 1082 human body fluids, including plasma, urine, cerebrospinal fluid (CSF), and bile. Our validation strategy utilized RT-qPCR and RNA immunoprecipitation assays, complemented by computational techniques for analyzing EV-circRNA features and RNA-binding protein interactions., Results: We identified 136,327 EV-circRNAs from various human body fluids. Significantly, a considerable amount of circRNAs with a high back-splicing ratio are highly enriched in EVs compared to linear RNAs. Additionally, we discovered brain-specific circRNAs enriched in plasma EVs and cancer-associated EV-circRNAs linked to clinical outcomes. Moreover, we demonstrated that EV-circRNAs have the potential to serve as biomarkers for evaluating immunotherapy efficacy in non-small cell lung cancer (NSCLC). Importantly, we identified the involvement of RBPs, particularly YBX1, in the sorting mechanism of circRNAs into EVs., Conclusions: This study unveils the extensive repertoire of EV-circRNAs across human biofluids, offering insights into their potential as disease biomarkers and their mechanistic roles within EVs. The identification of specific circRNAs and the elucidation of RBP-mediated sorting mechanisms open new avenues for the clinical application of EV-circRNAs in disease diagnostics and therapeutics., (© 2024. The Author(s).)

UVB radiation induces oxidative stress, DNA damage, and inflammation, leading to skin wrinkling, compromised barrier function, and an increased risk of carcinogenesis. Addressing or preventing photoaging may offer a promising therapeutic avenue for these conditions. Recent research indicated that mesenchymal stem cells (MSCs) exhibit significant therapeutic potential for various skin diseases. Given that extracellular vesicles (EV) can deliver diverse cargo to recipient cells and elicit similar therapeutic effects, we investigated the roles and underlying mechanisms of both adipose-derived MSC-derived EV (AMSC-EV) and umbilical cord-derived MSC-derived EV (HUMSC-EV) in photoaging. Our findings indicated that in vivo, treatment with AMSC-EV and HUMSC-EV resulted in improvements in wrinkles and skin hydration while also mitigating skin inflammation and thickness alterations in both the epidermis and dermis. Additionally, in vitro studies using human keratinocytes (HaCaTs), human dermal fibroblast cells (HDFs), and T-Skin models revealed that AMSC-EV and HUMSC-EV attenuated senescence, reduced levels of reactive oxygen species (ROS) and DNA damage, and alleviated inflammation induced by UVB. Furthermore, EV treatment enhanced cell viability and migration capacity in the epidermis and promoted extracellular matrix (ECM) remodeling in the dermis in photoaged cell models. Mechanistically, proteomics results showed that TIMP1 was highly expressed in both AMSC-EV and HUMSC-EV and could exert similar effects as MSC-EV. In addition, we found that EV and TIMP1 could inhibit Notch1 and downstream targets Hes1, P16, P21, and P53. Collectively, our data suggests that both AMSC-EV and HUMSC-EV attenuate skin photoaging through TIMP1/Notch1., (© 2024. The Author(s).)

Last decade, extracellular vesicles (EVs) attracted a lot of attention as potent versatile drug delivery vehicles. We reported earlier the development of EV-based delivery systems for therapeutic proteins and small molecule chemotherapeutics. In this work, we first time engineered EVs with multivalent cationic lipids for the delivery of nucleic acids. Stable, small size cationized EVs were loaded with plasmid DNA (pDNA), or mRNA, or siRNA. Nucleic acid loaded EVs were efficiently taken up by target cells as demonstrated by confocal microscopy and delivered their cargo to the nuclei in triple negative breast cancer (TNBC) cells and macrophages. Efficient transfection was achieved by engineered cationized EVs formulations of pDNA- and mRNA in vitro. Furthermore, siRNA loaded into cationized EVs showed significant knockdown of the reporter gene in Luc-expressing cells. Overall, multivalent cationized EVs represent a promising strategy for gene delivery., (© 2024. The Author(s).)

Background: Extracellular vesicles (EVs), containing microRNAs (miRNAs) and other molecules, play a central role in intercellular communication, especially in viral infections caused by SARS-CoV-2. This study explores the miRNA profiles in plasma-derived EVs from patients with severe COVID-19 vs controls, identifying potential mortality predictors., Methods: This prospective study included 36 patients with severe COVID-19 and 33 controls without COVID-19. EV-derived miRNAs were sequenced, and bioinformatics and differential expression analysis between groups were performed. The plasma miRNA profile of an additional cohort of patients with severe COVID-19 (n = 32) and controls (n = 12) was used to compare with our data. Survival analysis identified potential mortality predictors among the significantly differentially expressed (SDE) miRNAs in EVs., Results: Patients with severe COVID-19 showed 50 SDE miRNAs in plasma-derived EVs. These miRNAs were associated with pathways related to inflammation and cell adhesion. Fifteen of these plasma-derived EV miRNAs were SDE in the plasma of severe cases vs controls. Two miRNAs, hsa-miR-1469 and hsa-miR-6124, were identified as strong mortality predictors with an area under the receiver operating characteristic curve of 0.938., Conclusions: This research provides insights into the role of miRNAs within EVs in severe COVID-19 and their potential as clinical biomarkers for mortality., Competing Interests: Potential conflicts of interest . All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Ischemia-reperfusion (IR) injury is associated with neurological disorders such as stroke. The therapeutic potential of human umbilical cord mesenchymal stem cells (hUC-MSCs) and their secreted extracellular vesicles (EVs) in alleviating IR injury across various cell types including neuronal cells has been documented. However, the underlying mechanisms through which hUC-MSCs and hUC-MSC-EVs protect neuronal cells from IR-triggered damage are not well understood. In this study, we co-cultured SH-SY5Y neuroblastoma cells with hUC-MSCs or hUC-MSC-EVs and subjected them to oxygen-glucose deprivation/reperfusion (OGD/R) treatment. Our findings indicate that both hUC-MSCs and hUC-MSC-EVs significantly improved viability, reduced apoptosis, promoted autophagy of OGD/R-induced SH-SY5Y cells, and decreased mitochondrial reactive oxygen species levels within them. Furthermore, the neuroprotective effect of hUC-MSCs and hUC-MSC-EVs in OGD/R-induced SH-SY5Y cells was enhanced by overexpressing USP35, a deubiquitinase. Mechanistically, USP35 interacted with and stabilized FUNDC1, a positive regulator of mitochondrial metabolism. Knockdown of FUNDC1 in USP35-overexpressing hUC-MSCs and their secreted EVs eliminated the augmented neuroprotective function induced by excess USP35. In conclusion, these findings underscore the crucial role of USP35 in enhancing the neuroprotective function of hUC-MSCs and their secreted EVs, achieved through the stabilization of FUNDC1 in OGD/R-induced SH-SY5Y cells., (© 2024. The Author(s).)

Extracellular vesicle-derived DNA (evDNA) encapsulates the complete genome and mutational status of cells; however, whether cancer cell-derived evDNA mirrors the epigenetic features of parental genomic DNA remains uncertain. This study aimed to assess and compare the DNA methylation patterns of evDNA from cancer cell lines and primary cancer tissues with those of the nuclear genomic DNA. We isolated evDNA secreted by two cancer cell lines (HCT116 and MDA-MB-231) from various subcellular compartments, including the nucleus and cytoplasm. Additionally, we obtained evDNA and nuclear DNA (nDNA) from the primary cancer tissues of colon cancer patients. We conducted a comprehensive genome-wide DNA methylation analysis using the Infinium Methylation EPIC BeadChip, examining > 850,000 CpG sites. Remarkable similarities were observed between evDNA and nDNA methylation patterns in cancer cell lines and patients. This concordance extended to clinical cancer tissue samples, showcasing the potential utility of evDNA methylation patterns in deducing cellular origin within heterogeneous populations through methylation-based deconvolution. The observed concordance underscores the potential of evDNA as a noninvasive surrogate marker for discerning tissue origin, particularly in cancer tissues, offering a promising future for cancer diagnostics. This finding enhances our understanding of cellular origins and would help develop innovative diagnostic and therapeutic strategies for cancer., (© 2024. The Author(s).)

The cascade of metastasis in tumor cells, exhibiting organ-specific tendencies, may occur at numerous phases of the disease and progress under intense evolutionary pressures. Organ-specific metastasis relies on the formation of pre-metastatic niche (PMN), with diverse cell types and complex cell interactions contributing to this concept, adding a new dimension to the traditional metastasis cascade. Prior to metastatic dissemination, as orchestrators of PMN formation, primary tumor-derived extracellular vesicles prepare a fertile microenvironment for the settlement and colonization of circulating tumor cells at distant secondary sites, significantly impacting cancer progression and outcomes. Obviously, solely intervening in cancer metastatic sites passively after macrometastasis is often insufficient. Early prediction of metastasis and holistic, macro-level control represent the future directions in cancer therapy. This review emphasizes the dynamic and intricate systematic alterations that occur as cancer progresses, illustrates the immunological landscape of organ-specific PMN creation, and deepens understanding of treatment modalities pertinent to metastasis, thereby identifying some prognostic and predictive biomarkers favorable to early predict the occurrence of metastasis and design appropriate treatment combinations., (© 2024. The Author(s).)

Tissue-specific gene knockout by CRISPR/Cas9 is a powerful approach for characterizing gene functions during development. However, this approach has not been successfully applied to most Drosophila tissues, including the Drosophila neuromuscular junction (NMJ). To expand tissue-specific CRISPR to this powerful model system, here we present a CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) toolkit for knocking out genes in motoneurons, muscles, and glial cells. We validated the efficacy of CRISPR-TRiM by knocking out multiple genes in each tissue, demonstrated its orthogonal use with the Gal4/UAS binary expression system, and showed simultaneous knockout of multiple redundant genes. We used CRISPR-TRiM to discover an essential role for SNARE components in NMJ maintenance. Furthermore, we demonstrate that the canonical ESCRT pathway suppresses NMJ bouton growth by downregulating retrograde Gbb signaling. Lastly, we found that axon termini of motoneurons rely on ESCRT-mediated intra-axonal membrane trafficking to release extracellular vesicles at the NMJ. Thus, we have successfully developed an NMJ CRISPR mutagenesis approach which we used to reveal genes important for NMJ structural plasticity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma, with embryonal (ERMS) and alveolar (ARMS) representing the two most common histological subtypes. ARMS shows poor prognosis, being often metastatic at diagnosis. Thus, the discovery of novel biomarkers predictive of tumor aggressiveness represents one of the most important challenges to overcome and may help the development of tailored therapies. In the last years, miRNAs carried in extracellular vesicles (EVs), small vesicles of endocytic origin, have emerged as ideal candidate biomarkers due to their stability in plasma and their tissue specificity., Methods: EVs miRNAs were isolated from plasma of 21 patients affected by RMS and 13 healthy childrens (HC). We performed a miRNA profile using the Serum/Plasma Focus microRNA PCR panels (Qiagen), and RT-qPCR for validation analysis. Statistically significant (p < 0.05) miRNAs were obtained by ANOVA test., Results: We identified nine EVs miRNAs (miR-483-5p, miR-132-3p, miR-766-3p, miR-454-3p miR-197-3p, miR-335-3p, miR-17-5p, miR-486-5p and miR-484) highly upregulated in RMS patients compared to HCs. Interestingly, 4 miRNAs (miR-335-5p, miR-17-5p, miR-486-5p and miR-484) were significantly upregulated in ARMS samples compared to ERMS. In the validation analysis performed in a larger group of patients only three miRNAs (miR-483-5p, miR-335-5p and miR-484) were differentially significantly expressed in RMS patients compared to HC. Among these, mir-335-5p was significant also when compared ARMS to ERMS patients. MiR-335-5p was upregulated in RMS tumor tissues respect to normal tissues (p = 0.00202) and upregulated significantly between ARMS and ERMS (p = 0.04). Furthermore, the miRNA expression correlated with the Intergroup Rhabdomyosarcoma Study (IRS) grouping system, (p = 0.0234), and survival (OS, p = 0.044; PFS, p = 0.025). By performing in situ hybridization, we observed that miR-335-5p signal was exclusively in the cytoplasm of cancer cells., Conclusion: We identified miR-335-5p as significantly upregulated in plasma derived EVs and tumor tissue of patients affected by ARMS. Its expression correlates to stage and survival in patients. Future studies are needed to validate miR-335-5p as prognostic biomarker and to deeply elucidate its biological role., (© 2024. The Author(s).)

Milk-derived extracellular vesicles (mEVs) are beneficial to the health of infants. However, the effect of mEVs on early intestinal inflammation is not well established. Herein, weaned colitic mice were used to explore the potential effects and underlying mechanisms of porcine mEVs (pmEVs) on intestinal inflammation during early life. We found that pmEVs administration attenuated early life intestinal inflammation and promoted colonic barrier integrity in mice. The anti-inflammatory effect of pmEVs was achieved by shifting a proinflammatory macrophage (M1) toward an anti-inflammatory macrophage (M2). Moreover, pmEVs can be absorbed by macrophages and reduce proinflammatory polarization (stimulated by LPS) in vitro. Noteworthily, ssc-let- 7 c was found to be highly expressed in pmEVs that can regulate the polarization of macrophages by targeting the tensin homologue deleted on chromosome ten (PTEN), thereby activating the PI3K/Akt pathway. Collectively, our findings revealed a crucial role of mEVs in early intestinal immunity and its underlying mechanism.

Background: With the rising global incidence and mortality rates of breast cancer, early diagnosis is becoming increasingly crucial. The World Health Organization (WHO) recommends mammography as a primary screening tool. However, despite its clinical benefits, mammography has potential risks including radiation exposure, unnecessary follow-up, and overdiagnosis due to false positives, particularly in cases of early cancer or dense breast tissue. In this study, we aimed to address these concerns by introducing an innovative diagnostic method that employs circulating biomarkers to enhance the existing screening techniques Methods: Breast cancer-derived extracellular vesicles (BEVs) were isolated from the bloodstream using advanced immunoaffinity capture techniques. Subsequently, we analyzed the microRNA (miRNA) profiles of BEVs in plasma samples from 120 patients with breast cancer, 46 with benign tumors, and 45 healthy controls. Results: This retrospective study identified a distinct signature of five EV miRNAs (miR-21, miR-106b, miR-181a, miR-484, and miR-1260b) that effectively differentiated patients with breast cancer from healthy controls. This signature provides essential insights into tumor progression, metastasis, and the risk of recurrence. Notably, overexpression of this signature correlated with poorer survival outcomes. Conclusions: Our novel gene signature-based approach not only complements existing diagnostic methods with high accuracy but also provides a deeper understanding of the molecular aspects of breast cancer, heralding a significant advancement in precision medicine and personalized cancer care., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Human epithelial growth factor receptor 2 (HER2)-targeted therapies are effective in patients with HER2-positive breast cancer. Recent advances have shown that HER2-targeted therapies can also be of benefit when treating tumors expressing low levels of HER2, highlighting the importance of identifying the HER2-low subgroup. This clinical trend has opened new therapeutic avenues for patients who were previously ineligible for HER2-targeted therapies. Thus, the development of new diagnostic methods for real-time HER2 profiling is crucial for accurately tailoring the treatment for these patients. We hypothesized that tumor-derived extracellular vesicles (TEVs) could reflect the HER2 profiles of primary tumors and potentially serve as diagnostic tools for HER2 status. This approach was validated using six breast cancer cell lines, which confirmed that the TEVs accurately reflected the HER2 profiles of the tumor cells. TEVs were isolated using an immunoaffinity method, and copy number variation (CNV) in the ERBB2/EIF2C ratio was assessed using droplet digital PCR of DNA from these vesicles. Clinical validation using plasma samples from 33 breast cancer patients further reinforced the diagnostic potential of our method. Pearson's correlation coefficient analysis of the flow cytometry results demonstrated that TEVs reflected HER2 expression in primary cells. To distinguish between HER2-negative and HER2-low patients, the area under the curve (AUC) of the ROC curve in our method was 0.796, with a sensitivity of 53.8% and a specificity of 100%. These findings suggest the clinical utility of extracellular vesicles derived from plasma and emphasize the need for further research to distinguish HER2-negative from HER2-low patients., (© 2024 The Author(s). Genes, Chromosomes and Cancer published by Wiley Periodicals LLC.)

Despite the advances in the understanding of Huntington's disease (HD), there is a need for molecular biomarkers to categorize mutation carriers during the preclinical stage of the disease preceding functional decline. Small RNAs (sRNAs) are a promising source of biomarkers since their expression levels are highly sensitive to pathobiological processes. Here, using an optimized method for plasma extracellular vesicles (EVs) purification and an exhaustive analysis pipeline of sRNA sequencing data, we show that EV-sRNAs are downregulated early in mutation carriers and that this deregulation is associated with premanifest cognitive performance. Seven candidate sRNAs (tRF-Glu-CTC, tRF-Gly-GCC, miR-451a, miR-21-5p, miR-26a-5p, miR-27a-3p and let7a-5p) were validated in additional subjects, showing a significant diagnostic accuracy at premanifest stages. Of these, miR-21-5p was significantly decreased over time in a longitudinal study; and miR-21-5p and miR-26a-5p levels correlated with cognitive changes in the premanifest cohort. In summary, the present results suggest that deregulated plasma EV-sRNAs define an early biosignature in mutation carriers with specific species highlighting the progression and cognitive changes occurring at the premanifest stage., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Intercellular communication is an important mechanism for the development and maintenance of normal biological processes in all organs, including the female reproductive system. Extracellular vesicles, as important carriers of intercellular communication, contain a variety of biologically active molecules, such as mRNAs, miRNAs, lncRNAs, and circRNAs, which are involved in cell-to-cell exchanges as well as in many physiological and pathological processes in the body. Compared with biomarkers found in tissues or body fluids, extracellular vesicles show better stability due to the presence of their envelope membrane which prevents the degradation of the RNA message in their vesicles. Therefore, the genomic and proteomic information contained in extracellular vesicles can serve as important markers and potential therapeutic targets for female reproductive system-related diseases or placental function. Moreover, changes in the expression of non-coding RNAs (mainly miRNAs, lncRNAs, and circRNAs) in maternal extracellular vesicles can accurately and promptly reflect the progress of female reproductive system diseases. The aim of this review is to collect information on different types of non-coding RNAs with key molecular carriers in female pathologic pregnancies (preeclampsia and recurrent spontaneous abortion), so as to explore the relevant molecular mechanisms in female pathologic pregnancies and provide a theoretical basis for clinical research on the pathogenesis and therapeutic approaches of reproductive system diseases. The current state of the art of exosome isolation and extraction is also summarized., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Neurodegenerative diseases are characterized by progressive deterioration of the nervous system. Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD) are prominently life-threatening examples of neurodegenerative diseases. The complexity of the pathophysiology in neurodegenerative diseases causes difficulties in diagnosing. Although the drugs temporarily help to correct specific symptoms including memory loss and degeneration, a complete treatment has not been found yet. New therapeutic approaches have been developed to understand and treat the underlying pathogenesis of neurodegenerative diseases. With this purpose, clustered-regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) technology has recently suggested a new treatment option. Editing of the genome is carried out by insertion and deletion processes on DNA. Safe delivery of the CRISPR/Cas system to the targeted cells without affecting surrounding cells is frequently investigated. Extracellular vesicles (EVs), that is exosomes, have recently been used in CRISPR/Cas studies. In this review, CRISPR/Cas and EV approaches used for diagnosis and/or treatment in AD, PD, ALS, and HD are reviewed. CRISPR/Cas and EV technologies, which stand out as new therapeutic approaches, may offer a definitive treatment option in neurodegenerative diseases., (© 2024 The Author(s). European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Tumor-derived extracellular vesicles (EVs) are potential biomarkers for tumors, but their reliable molecular targets have not been identified. The previous study confirms that ubiquitin-specific protease 22 (USP22) promotes lung adenocarcinoma (LUAD) metastasis in vivo and in vitro. Moreover, USP22 regulates endocytosis of tumor cells and localizes to late endosomes. However, the role of USP22 in the secretion of tumor cell-derived EVs remains unknown. In this study, it demonstrates that USP22 increases the secretion of tumor cell-derived EVs and accelerates their migration and invasion, invadopodia formation, and angiogenesis via EV transfer. USP22 enhances EV secretion by upregulating myosin IB (MYO1B). This study further discovers that USP22 activated the SRC signaling pathway by upregulating the molecule KDEL endoplasmic reticulum protein retention receptor 1 (KDELR1), thereby contributing to LUAD cell progression. The study provides novel insights into the role of USP22 in EV secretion and cell motility regulation in LUAD., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Small extracellular vesicles (sEV) transfer cargos between cells and participate in various physiological and pathological processes through their autocrine and paracrine effects. However, the pathological mechanisms employed by sEV-encapsulated microRNA (miRNA) in acute myeloid leukemia (AML) are still obscure. In this study, we aimed to investigate the effects of AML cell-derived sEV (AML-sEV) on AML cells and delineate the underlying mechanisms. We initially used high-throughput sequencing to identify miR-221-3p as the miRNA prominently enriched in AML-sEV. Our findings revealed that miR-221-3p promoted AML cell proliferation and leukemogenesis by accelerating cell cycle entry and inhibiting apoptosis. Furthermore, Gbp2 was confirmed as a target gene of miR-221-3p by dual luciferase reporter assays and rescue experiments. Additionally, AML-sEV impaired the clonogenicity, particularly the erythroid differentiation ability, of hematopoietic stem and progenitor cells. Taken together, our findings reveal how sEV-delivered miRNA contribute to AML pathogenesis, which can be exploited as a potential therapeutic target to attenuate AML progression.

In the context of improving the efficacy of autologous fat grafts (AFGs) in reconstructive surgery, this study delineates the novel use of adipose-derived mesenchymal stem cells (ADSCs) and their extracellular vesicles (EVs) as vehicles for delivering delta-like ligand 4 (DLL4) siRNA. The aim was to inhibit DLL4, a gene identified through transcriptome analysis as a critical player in the vascular endothelial cells of AFG tissues, thereby negatively affecting endothelial cell functions and graft survival through the Notch signaling pathway. By engineering ADSC EVs to carry DLL4 siRNA (ADSC EVs-siDLL4), the research demonstrated a marked improvement in endothelial cell proliferation, migration, and lumen formation, and enhanced angiogenesis in vivo, leading to a significant increase in the survival rate of AFGs. This approach presents a significant advancement in the field of tissue engineering and regenerative medicine, offering a potential method to overcome the limitations of current fat grafting techniques. NEW & NOTEWORTHY This study introduces a groundbreaking method for enhancing autologous fat graft survival using adipose-derived stem cell extracellular vesicles (ADSC EVs) to deliver DLL4 siRNA. By targeting the delta-like ligand 4 (DLL4) gene, crucial in endothelial cell dynamics, this innovative approach significantly improves endothelial cell functions and angiogenesis, marking a substantial advancement in tissue engineering and regenerative medicine.

Background: Extracellular vesicles (EVs) facilitate cell-cell interactions in the tumour microenvironment. However, standard and efficient methods to isolate tumour tissue-derived EVs are lacking, and their biological functions remain elusive., Methods: To determine the optimal method for isolating tissue-derived EVs, we compared the characterization and concentration of EVs obtained by three previously reported methods using transmission electron microscopy, nanoparticle tracking analysis, and nanoflow analysis (Nanoflow). Additionally, the differential content of small RNAs, especially tsRNAs, between hepatocellular carcinoma (HCC) and adjacent normal liver tissues (ANLTs)-derived EVs was identified using Arraystar small RNA microarray. The targets of miRNAs and tsRNAs were predicted, and downstream functional analysis was conducted using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, non-negative matrix factorization and survival prediction analysis., Results: A differential centrifugation-based protocol without cell cultivation (NC protocol) yielded higher EV particles and higher levels of CD9 + and CD63 + EVs compared with other isolation protocols. Interestingly, the NC protocol was also effective for isolating frozen tissue-derived EVs that were indistinguishable from fresh tissue. HCC tissues showed significantly higher EV numbers compared with ANLTs. Furthermore, we identified different types of small RNAs in HCC tissue-derived EVs, forming a unique multidimensional intercellular communication landscape that can differentiate between HCC and ANLTs. ROC analysis further showed that the combination of the top 10 upregulated small RNAs achieved better diagnostic performance (AUC = .950 [.895-1.000]). Importantly, most tsRNAs in HCC tissue-derived EVs were downregulated and mitochondria-derived, mainly involving in lipid-related metabolic reprogramming., Conclusion: The NC protocol was optimal for isolating EVs from HCC, especially from frozen tissues. Our study emphasized the different roles of small-RNA in regulating the HCC ecosystem, providing insights into HCC progression and potential therapeutic targets., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Circular RNAs (circRNAs) are endogenous biological macromolecules that regulate various biological processes including embryo development. However, little is known about which circRNAs are present in bovine preimplantation embryos and their respective roles. Here, we characterized the expression profile of circRNAs in bovine blastocysts for the first time. We detected 25,700 circRNAs in total, with 12,630 circRNAs uniquely expressed in blastocysts compared to degenerated embryos. CircRNA alternative splicing (AS) events were also found more frequently in blastocysts than in degenerated embryos (299 vs 258). Additionally, 410 circRNAs, among which 11 circRNAs with a high potential to encode polypeptides, were found differentially expressed between blastocysts and degenerated embryos. We further predicted and constructed a circRNA-miRNA-mRNA network, wherein differentially expressed circRNAs were shown to bind to bovine preimplantation embryo development-related miRNAs. Employing bioinformatic algorithms we found that differentially expressed circRNAs are associated with differentially expressed miRNAs and transfer RNA-derived small RNAs (tsRNAs) enclosed in embryonic extracellular vesicles (EVs). Furthermore, functional analysis revealed that knockdown of the evolutionarily conserved circAGO2 can inhibit blastocyst hatching. Overall, our study provides the first landscape of circRNAs in bovine preimplantation embryos and highlights the novel role of circRNAs as tsRNA binding partners influencing small RNA sorting and loading into EVs, with circAGO2 playing a regulatory role in bovine blastocyst hatching., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Progesterone (P4) is predicted to act as a negative regulatory hormone for oocyte maturation events; however, its local effects during follicular development remain poorly understood in bovine. The complex process of oocyte meiosis progression is dependent on cellular communication among follicular cells. Besides, the breakdown of this communication, mainly between cumulus cells (CC) and oocyte, through the retraction of cumulus projections connecting these cells can impact oocyte maturation. In our study, we observed that follicles from the ovary ipsilateral to the corpus luteum (CL) containing high intrafollicular P4 concentrations enhance the abundance of proteins detected in follicular-derived small extracellular vesicles (sEVs) predicted to be involved in the retraction of membrane projections based on actin filaments, such as transzonal projections (TZPs). Conversely, we found that follicles from the ovary contralateral to the CL, which contained low intrafollicular P4 concentrations, had a high detection of proteins predicted to regulate the maintenance of TZPs. We also performed RNAseq analysis which demonstrated that 177 genes were differentially expressed in CC under the different P4 environments. Bioinformatic analysis points to changes associated to cell metabolism in cells from follicles ipsilateral to the CL in comparison to genes involved in cell communication in CC from follicles contralateral to the CL. Our functional analysis experiment confirmed that supplementation of cumulus-oocyte complexes during in vitro maturation with P4 at concentration similar to ipsilateral follicles reduces the number of TZPs. In summary, our study underscores a direct association between P4 concentration and cumulus-oocyte interaction, with potential consequences for the acquisition of oocyte competence., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Juliano Coelho da Silveira reports financial support was provided by São Paulo Research Foundation - FAPESP. Juliano Coelho da Silveira reports financial support was provided by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES). Juliano Coelho da Silveira reports financial support was provided by National Council for Scientific and Technological Development. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) are bio-active elements cargoed by seminal plasma extracellular vesicles extracellular vesicles (SPEVs) which are crucial for sperm function and fertility modulation. This study aimed to isolate, characterize, and identify the miRNA expression profiles in the SPEVs from high (HSM) and low sperm motility (LSM) groups that could serve as fertility biomarkers and explain the underlying mechanisms. The isolated SPEVs were round spherical structures of approximately 50-200 nm in diameter expressing molecular markers. A total of 1006 and 1084 miRNAs were detected in HSM and LSM, respectively, with 34 being differentially expressed. Their targeted genes involved in SNARE interactions in vesicular transport, Metabolic pathways, and Apelin signaling pathway, etc. The joint analysis with mRNAs of sperm and sperm storage tubules cells highlighted the cellular communication mediated by SPEVs miRNAs, where they may rule fertility by affecting sperm maturation and amino acid metabolism. SPEVs as additives could improve fertility of fresh and frozen sperm, while the knockdown of one of the differentially expressed miRNAs, miR-24-3p, diminished this effect, indicating its crucial roles. This study expands our understanding of SPEVs miRNAs mediated sperm maturation and fertility modulation, and may help to develop new therapeutic strategies for infertility and sperm storage., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Prostate cancer (PCa) is the second most prevalent cancer in men worldwide, presenting a significant global public health challenge that necessitates early detection and personalized treatment. Recently, non-invasive liquid biopsy methods have emerged as promising tools to provide insights into the genetic landscape of PCa and monitor disease progression, aiding decision-making at all stages. Research efforts have concentrated on identifying liquid biopsy biomarkers to improve PCa diagnosis, prognosis, and treatment prediction. This article reviews recent research advances over the last five years utilizing extracellular vesicles (EVs) as a natural biomarker library for PCa, and discusses the clinical translation of EV biomarkers, including ongoing trials and key implementation challenges. The findings underscore the transformative role of liquid biopsy, particularly EV-based biomarkers, in revolutionizing PCa diagnosis, prediction, and treatment., Competing Interests: Declaration of competing interest All authors declare that there is no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)