MicroRNAs (miRNAs), small non-coding molecules targeting messenger RNAs and inhibiting protein translation, modulate key biological processes, including cell growth and development, energy utilization, and homeostasis. In particular, miRNAs control the differentiation, survival, and activation of CD4+ T conventional (Tconv) cells, key players of the adaptive immunity, and regulate the physiological response to infections and the pathological loss of immune homeostasis in autoimmunity. Upon T-cell receptor (TCR) stimulation, the described global miRNA quantitative decrease occurring in T cells is believed to promote the acquisition of effector functions by relaxing the post-transcriptional repression of genes associated with proliferation and cell activity. MiRNAs were initially thought to get downregulated uniquely by intracellular degradation; on the other hand, miRNA secretion via extracellular vesicles (EVs) represents an additional mechanism of rapid downregulation. By focusing on molecular interactions by means of graph theory, we have found that miRNAs released by TCR-stimulated Tconv cells are significantly enriched for targeting transcripts upregulated upon stimulation, including those encoding for crucial proteins associated with Tconv cell activation and function. Based on this computational approach, we present our perspective based on the following hypothesis: a stimulated Tconv cell will release miRNAs targeting genes associated with the effector function in the extracellular space in association with EVs, which will thus possess a suppressive potential toward other Tconv cells in the paracrine environment. We also propose possible future directions of investigation aimed at taking advantage of these phenomena to control Tconv cell effector function in health and autoimmunity.

In the last decades, it has been demonstrated that the regenerative therapeutic efficacy of mesenchymal stromal cells is primarily due to the secretion of soluble factors and extracellular vesicles, collectively known as secretome. In this context, our work described the preparation and characterization of a freeze-dried secretome (Lyosecretome) from adipose tissue-derived mesenchymal stromal cells for the therapy of equine musculoskeletal disorder. An intraarticular injectable pharmaceutical powder has been formulated, and the technological process has been validated in an authorized facility for veterinary clinical-use medicinal production. Critical parameters for quality control and batch release have been identified regarding (i) physicochemical properties; (ii) extracellular vesicle morphology, size distribution, and surface biomarker; (iii) protein and lipid content; (iv) requirements for injectable pharmaceutical dosage forms such as sterility, bacterial endotoxin, and Mycoplasma; and (v) in vitro potency tests, as anti-elastase activity and proliferative activity on musculoskeletal cell lines (tenocytes and chondrocytes) and mesenchymal stromal cells. Finally, proteins putatively responsible for the biological effects have been identified by Lyosecretome proteomic investigation: IL10RA, MXRA5, RARRES2, and ANXA1 modulate the inflammatory process RARRES2, NOD1, SERPINE1, and SERPINB9 with antibacterial activity. The work provides a proof-of-concept for the manufacturing of clinical-grade equine freeze-dried secretome, and prototypes are now available for safety and efficacy clinical trials in the treatment of equine musculoskeletal diseases

Plant mitoviruses belong to Mitoviridae family and consist of positive single-stranded RNA genomes replicating exclusively in host mitochondria. We previously reported the biological characterization of a replicating plant mitovirus, designated Chenopodium quinoa mitovirus 1 (CqMV1), in some Chenopodium quinoa accessions. In this study, we analyzed the mitochondrial proteome from leaves of quinoa, infected and not infected by CqMV1. Furthermore, by protein–protein interaction and co-expression network models, we provided a system perspective of how CqMV1 affects mitochondrial functionality. We found that CqMV1 is associated with changes in mitochondrial protein expression in a mild but well-defined way. In quinoa-infected plants, we observed up-regulation of functional modules involved in amino acid catabolism, mitochondrial respiratory chain, proteolysis, folding/stress response and redox homeostasis. In this context, some proteins, including BCE2 (lipoamide acyltransferase component of branched-chain alpha-keto acid dehydrogenase complex), DELTA-OAT (ornithine aminotransferase) and GR-RBP2 (glycine-rich RNA-binding protein 2) were interesting because all up-regulated and network hubs in infected plants; together with other hubs, including CAT (catalase) and APX3 (L-ascorbate peroxidase 3), they play a role in stress response and redox homeostasis. These proteins could be related to the higher tolerance degree to drought we observed in CqMV1-infected plants. Although a specific causative link could not be established by our experimental approach at this stage, the results suggest a new mechanistic hypothesis that demands further in-depth functional studies.

Extracellular vesicles (EVs) are receiving increasing attention for their role in spreading both beneficial and harmful information during cell–cell communication. The complexity and heterogeneity of the origin of EVs make integrated molecular, structural, and functional studies extremely challenging but necessary at the same time. In fact, a comprehensive interdisciplinary approach is needed to correlate the features of EVs, target cells/organs, and the pathophysiological outcomes exerted by the EVs’ actions. Based on these premises, after introducing a brief state-of-the-art outline on the current analytical approaches exploited to characterize EVs, this review aims to highlight the effectiveness of those studies where authors put in correlation the diverse EV data collected from different points of view. Although these examples are still just a few, they still represent an excellent starting point to be taken as a reference in the perspective for improving the correlation among EV-related clinical aspects. Of course, to fully reach this goal, several points need to be further improved and developed. Undoubtedly, new avenues in diagnostic, prognostic, and therapeutic applications by EVs will be initiated by integrative strategies, combining biophysical approaches, high-throughput omics technologies, and computational models.

Background Microvesicles are vesicles shed by plasma membranes following cell activation and apoptosis. The role of lymphocyte‐derived microvesicles in endothelial function remains poorly understood. Methods CD4+ T cells isolated from peripheral blood of healthy human donors were stimulated using anti‐CD3/anti‐CD28‐coated beads. Proteomic profiling of microvesicles was performed using linear discriminant analysis (LDA) from activated T cells (MV.Act) and nonactivated T cells (MV.NAct). In addition, data processing analysis was performed using MaxQUANT workflow. Differentially expressed proteins found in MV.Act or MV.NAct samples with identification frequency = 100%, which were selected by both LDA (p < .01) and MaxQUANT (p < .01) workflows, were defined as “high‐confidence” differentially expressed proteins. Functional effects of MV.Act on human primary microvascular endothelial cells were analysed. Results T cells released large amounts of microvesicles upon stimulation. Proteomic profiling of microvesicles using LDA identified 2279 proteins (n = 2110 and n = 851 proteins in MV.Act and MV.NAct, respectively). Protein–protein interaction network models reconstructed from both differentially expressed proteins (n = 594; LDA p ≤ .01) and “high‐confidence” differentially expressed proteins (n = 98; p ≤ .01) revealed that MV.Act were enriched with proteins related to immune responses, protein translation, cytoskeleton organisation and TNFα‐induced apoptosis. For instance, MV.Act were highly enriched with IFN‐γ, a key proinflammatory pathway related to effector CD4+ T cells. Endothelial cell incubation with MV.Act induced superoxide generation, apoptosis, endothelial wound healing impairment and endothelial monolayer barrier disruption. Conclusions T cell receptor‐mediated activation of CD4+ T cells stimulates the release of microvesicles enriched with proteins involved in immune responses, inflammation and apoptosis. T cell‐derived microvesicles alter microvascular endothelial function and barrier permeability, potentially promoting tissue inflammation.

Background: The cause of chronic liver diseases (CLD) remains undiagnosed in up to 30% of adult patients. Whole-Exome Sequencing (WES) can improve the diagnostic rate of genetic conditions, but it is not yet widely available, due to the costs and the difficulties in results interpretation. Targeted panel sequencing (TS) represents an alternative more focused diagnostic approach.Aims: To validate a customized TS for hereditary CLD diagnosis.Methods: We designed a customized panel including 82 CLD-associated genes (iron overload, lipid metabolism, cholestatic diseases, storage diseases, specific hereditary CLD and susceptibility to liver diseases). DNA samples from 19 unrelated adult patients with undiagnosed CLD were analyzed by both TS (HaloPlex) and WES (SureSelect Human All Exon kit v5) and the diagnostic performances were compared.Results: The mean depth of coverage of TS-targeted regions was higher with TS than WES (300x vs. 102x; p &lt; 0.0001). Moreover, TS yielded a higher average coverage per gene and lower fraction of exons with low coverage (p &lt; 0.0001). Overall, 374 unique variants were identified across all samples, 98 of which were classified as “Pathogenic” or “Likely Pathogenic” with a high functional impact (HFI). The majority of HFI variants (91%) were detected by both methods; 6 were uniquely identified by TS and 3 by WES. Discrepancies in variant calling were mainly due to variability in read depth and insufficient coverage in the corresponding target regions. All variants were confirmed by Sanger sequencing except two uniquely detected by TS. Detection rate and specificity for variants in TS-targeted regions of TS were 96.9% and 97.9% respectively, whereas those of WES were 95.8% and 100%, respectively.Conclusion: TS was confirmed to be a valid first-tier genetic test, with an average mean depth per gene higher than WES and a comparable detection rate and specificity.

Distant metastasis occurs when cancer cells adapt to a tissue microenvironment that is different from the primary organ. This process requires genetic and epigenetic changes in cancer cells and the concomitant modification of the tumor stroma to facilitate invasion by metastatic cells. In this study, we analyzed differences in the epigenome of brain metastasis from the colon (n = 4) and lung (n = 14) cancer and we compared these signatures with those found in primary tumors. Results show that CRC tumors showed a high degree of genome-wide methylation compared to lung cancers. Further, brain metastasis from lung cancer deeply activates neural signatures able to modify the brain microenvironment favoring tumor cells adaptation. At the protein level, brain metastases from lung cancer show expression of the neural/glial marker Nestin. On the other hand, colon brain metastases show activation of metabolic signaling. These signatures are specific for metastatic tumors since primary cancers did not show such epigenetic derangements. In conclusion, our data shed light on the epi/molecular mechanisms that colon and lung cancers adopt to thrive in the brain environment.

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