Vesicle Contents Research Articles

COPD Airway Epithelial Cell-derived Extracellular Vesicles Spread Cellular Senescence via MicroRNA-34a.

Chronic obstructive pulmonary disease (COPD) is associated with the acceleration of lung aging, and the accumulation of senescent cells in lung tissue. MicroRNA (miR)-34a induces senescence by suppressing the anti-aging molecule, sirtuin-1 (SIRT1). Senescent cells spread senescence to neighbouring and distant cells, favouring COPD progression and its comorbidities. Mechanisms for spreading senescence remain undetermined but may be mediated by the transfer of microRNAs in extracellular vesicles. We analysed the miRNA content of extracellular vesicles in COPD and explored their effect on cellular senescence of healthy cells. EVs were isolated from small airway epithelial cells (SAEC) from healthy donors or COPD patients. Recipient healthy SAEC were cultured with EVs and the expression of miR-34a and markers of cellular senescence, p21CIP1 and SIRT1, were measured. We have shown that EVs from COPD cells induce senescence in healthy recipient cells via the selective transfer of miR-34a. COPD SAEC produce increased numbers of EVs enriched with miR-34a. EVs are taken up by healthy cells, resulting in reduced expression of the anti-aging molecule sirtuin-1 and increased expression of markers of senescence, like p21CIP1 and positive staining for senescence-associated β-galactosidase, which were blocked by a specific miR-34a antagomir. Our findings provide evidence of the mechanism by which EVs spread cellular senescence in human primary cells via miR-34a, rather than via soluble mediators. EVs enriched with miR-34a may spread senescence locally, accounting for disease progression, but also provide a mechanism for distant spread to account for comorbidities and multimorbidity of the elderly.

Determining the Rates of α-Tocopherol Movement in DPPC Vesicles Using Small Angle Neutron Scattering.

α-Tocopherol (αtoc, vitamin E) is an essential nutrient sufficiently acquired through a balanced diet. This fat-soluble vitamin is most known for its antioxidative properties, however, its fundamental mechanism of action in cellular membranes remains unknown. To this end, we use time-resolved small angle neutron scattering (TR-SANS) and a contrast matching scheme to determine intervesicular exchange (kex) and intrabilayer flip-flop (kf) rates of αtoc in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles. Moreover, we investigate the role of vesicle concentration and various types of cyclodextrins in affecting these rates. For the 25 mg/mL sample concentration it was determined that kex and kf were 1.35 ± 0.03 x 10-3 min-1 and 0.54 ± 0.10 x 10-3 min-1, which represent half-lives (T1/2) of 513.4 ± 11.7 min and 1285.1 ± 242.7 min, respectively. Differential scanning calorimetry confirmed the observed timescales of αtoc movement.

Characteristics of the monkeypox virus isolate obtained from the first patient in Russia and its sensitivity to 7-[N-(4-trifluoromethylbenzoyl)-hydrazinocarbonyl]-tricyclo-[3.2.2.0^2,4]non-8-en-6-carboxylic acid

Introduction. Since early May 2022, more than 90,000 cases of monkeypox virus infection have been reported in more than 70 countries around the World. This is the largest outbreak of monkeypox ever recorded outside of Africa. The aim of the study is to confirm the first case of monkeypox in Russia, to isolate and sequence a new strain of monkeypox virus (MPXV), and to assess its sensitivity to the 7-[N-(4-trifluoromethylbenzoyl)-hydrazinocarbonyl]-tricyclo-[3.2.2.0^2,4]non-8-en-6-carboxylic acid (NIOCH-14) antipox drug. Materials and methods. The biological materials obtained from the affected area of the skin (contents of vesicles), a nasopharyngeal smear, sputum and venous blood from a patient with suspected monkeypox were used. The disease was confirmed by PCR followed by determination of the nucleotide sequence of viral DNA by sequencing. Isolation of the new MPXV strain from clinical samples was carried out in Vero E6 cells. The antiviral effectiveness of NIOCH-14 against the new MPXV strain was assessed using an adapted spectrophotometric method. Results. A diagnostic study of the biological samples of a patient who returned from a tourist trip to European countries with complaints of skin rashes all over the body revealed MPXV DNA. A new strain of MPXV was isolated from vesicles in Vero E6 cells, and the genomic sequence MPXV-pustule S45 was assembled using high-throughput parallel sequencing (NGS). Discussion. The effectiveness of the finished dosage form of NIOCH-14 against the new strain of MPXV based on the results of determining the 50% virus inhibitory concentration (IC50) was 0.02 μg/mL, and the selectivity index (SI) was 15,000. Conclusion. In this study, the pathogen of monkeypox was detected and identified using real-time PCR, NGS and electron microscopy, and the first imported case of this disease in Russia was confirmed. It has been proven that the drug NIOCH-14 exhibits high antiviral activity in vitro against the new MPXV strain.

Fourier Transform Infrared Spectroscopy Analysis as a Tool to Address Aβ Impact on Extracellular Vesicles.

Alzheimer's disease is a challenge in modern healthcare due to its complex etiology and increasing prevalence. Despite advances, further understanding of Alzheimer's disease pathophysiology is needed, particularly the role of Aβ neurotoxic peptide. Fourier transform infrared spectroscopy (FTIR) has shown potential as a screening tool for several pathologies, including Alzheimer's disease. Nonetheless, limited research has explored Aβ direct effects on neurons and extracellular vesicles metabolic profiles. Hence, this study aims to investigate Aβ impact on the spectroscopic profiles of neuronal-like cells (N2a) and N2a-derived extracellular vesicles, employing FTIR spectroscopy and focusing on the 1280-900 cm-1 region. A comprehensive analysis of spectral data was carried out, including multivariate partial least squares (PLS) analysis and peak intensities analysis. PLS analysis revealed moderate to strong correlations within this spectral region for both N2a and N2a-derived extracellular vesicles. The peak intensity analysis revealed additional peaks with significant differences in EVs' spectra relative to N2a, following Aβ treatment. Specifically, Aβ seems to cause alterations in protein phosphorylation and in the nucleic acids content of extracellular vesicles. These findings support that Aβ's role in Alzheimer's disease pathology may be mediated by extracellular vesicles and highlight FTIR's potential for advancing Alzheimer's disease research and clinical applications.

Effect of osmotic pressure on membrane permeation through antimicrobial peptide-induced pores.

Most antimicrobial peptides (AMPs) induce membrane damage such as pore formation in bacterial cells, resulting in rapid cell death. On the other hand, bacterial cells have a large intracellular turgor pressure, i.e., an osmotic pressure (Π) due to higher osmolarity inside bacterial cells, but the effects of Π on the membrane permeation of the internal contents of lipid vesicles and cells through AMP-induced pores are unknown. Here, we investigated the effect of Π on the membrane permeability of a water-soluble fluorescent probe, AlexaFluor 488 hydrazide (AF488), when passing through peptidyl-glycylleucine-carboxyamide (PGLa)- or magainin 2 (Mag)-induced nanopores in giant unilamellar vesicles (GUVs). For the interaction of PGLa with single GUVs under Π, the onset of pore formation was followed by a gradual increase in the membrane permeability coefficient, MP, until MP reached a steady value, Ps. On the other hand, for the interaction of Mag with single GUVs under Π, the onset of pore formation was rapidly followed by a change of MP to Ps. Small Π values enhanced the Ps values of AF488 passing through the PGLa- or Mag-induced nanopores. The mechanisms underlying the increase of Ps at small Π values were discussed. Based on these results and our previous results that the membrane tension (due to Π) enhances rate of AMP-induced pore formation, we consider the role of turgor pressure in AMP-induced damage in bacterial membranes and the efflux of internal contents.

Circulating large extracellular vesicles as diagnostic biomarkers of indeterminate thyroid nodules: multi-platform omics analysis.

While most thyroid nodules are benign, 7-15% are malignant. Patients with indeterminate thyroid nodules (specifically Bethesda IV/Thy3f) often undergo diagnostic hemithyroidectomy to reach a diagnosis on final histology. The aim of this study was to assess the feasibility of circulating large extracellular vesicles as diagnostic biomarkers in patients presenting with Thy3f thyroid nodules. This was a two-gate diagnostic accuracy study; patients with Thy3f thyroid nodules were age, sex and body mass index matched to healthy individuals. Final histology confirmed benign and malignant diagnoses. Plasma large extracellular vesicle counts were quantified using flow cytometry. Large extracellular vesicle microRNA and protein profiles were identified using next generation sequencing and mass spectrometry, respectively. A total of 42 patients with Thy3f nodules (22 with cancer, 20 with non-cancer diagnosis) and 16 healthy controls were included. Total large extracellular vesicle concentrations and the concentrations of extracellular vesicles expressing epithelial cell adhesion molecule and the cancer markers atypical chemokine receptor type 7, extracellular matrix metalloproteinase inducer and syndecan-4 were significantly higher in patients with Thy3f nodules (cancer and non-cancer) compared with healthy individuals. In patients with cancerous versus non-cancer Thy3f nodules, one microRNA was upregulated: mir-195-3p (P < 0.001). Five were downregulated: mir-3176 (P < 0.001), mir-205-5p (P < 0.001), novel-hsa-mir-208-3p (P < 0.001), mir-3529-3p (P = 0.01) and let-7i-3p (P = 0.02). Furthermore, three large extracellular vesicle proteins (kallikrein-related peptidase11 (KLK11) (P = 0.001), alpha-1-acid glycoprotein 2 (A1AG2) (P <0.001) and small integral membrane protein 1 (SMIM1) (P = 0.04)) were significantly upregulated, while 20 large extracellular vesicle proteins were significantly downregulated (most downregulated: chemokine (C-X-C motif) ligand 7 (CXCL7), tubulin beta chain 1 (TBB1), binding immunoglobulin protein (BIP) and actinin alpha 1 (ACTN1) (P < 0.001)) in cancerous compared with non-cancer Thy3f nodules. Circulating large extracellular vesicle miRNA and protein profiles have a high diagnostic value to discriminate between benign and malignant nodules for patients with Thy3f cytology. Further validation for clinical performance will be needed.

Accelerating the Discovery of Abiotic Vesicles with AI-Guided Automated Experimentation.

The first protocells are speculated to have arisen from the self-assembly of simple abiotic carboxylic acids, alcohols, and other amphiphiles into vesicles. To study the complex process of vesicle formation, we combined laboratory automation with AI-guided experimentation to accelerate the discovery of specific compositions and underlying principles governing vesicle formation. Using a low-cost commercial liquid handling robot, we automated experimental procedures, enabling high-throughput testing of various reaction conditions for mixtures of seven (7) amphiphiles. Multitemplate multiscale template matching (MMTM) was used to automate confocal microscopy image analysis, enabling us to quantify vesicle formation without tedious manual counting. The results were used to create a Gaussian process surrogate model, and then active learning was used to iteratively direct the laboratory experiments to reduce model uncertainty. Mixtures containing primarily trimethyl decylammonium and decylsulfate in equal amounts formed vesicles at submillimolar critical vesicle concentrations, and more than 20% glycerol monodecanoate prevented vesicles from forming even at high total amphiphile concentrations.

Identifying the composition of large vesicles in the cytoplasm of oocytes.

Context Oocyte vesicles, or vacuoles, have been described using transmission electron microscopy in most species. In sheep and cow oocytes, vesicles constitute up to 30% of the cytoplasm, their volume decreases during maturation and is lower in poorer quality oocytes, suggesting they are important for oocyte competence. However, the composition and function of these organelles is unknown. Aim This study aimed to ascertain the content of oocyte vesicles and examine the effect of different fixation methods on the size and preservation of these organelles. Methods Sheep oocytes were centrifuged to segregate organelles then stained with organelle-specific fluorescent dyes (Nile Red, LysoTracker, Fluo-4-AM and TMRM) and imaged by live cell confocal microscopy. The oocytes were fixed with either glutaraldehyde or paraformaldehyde and prepared for electron microscopy to confirm the distribution of organelles and compare ultrastructure and organelle size. Key results Nile Red staining has identified that vesicles contain lipid that is different to that in the osmium-stained lipid droplets observed by electron microscopy. Lipid droplets and vesicles were significantly smaller when prepared for electron microscopy compared to live cell imaging. Organelles were less likely to be fully segregated following centrifugation in oocytes prior to maturation (20%) compared to oocytes after maturation (77%; P Conclusions Oocyte vesicles are lipid storing organelles that may be important for oocyte quality. Implications This study highlights the importance of lipid for oocyte quality and the need for further research to identify the optimal fatty acid content for in vitro maturation media and oocyte competence.

Approaches and Challenges in Characterizing the Molecular Content of Extracellular Vesicles for Biomarker Discovery.

Extracellular vesicles (EVs) are lipid bilayer nanoparticles released from all known cells and are involved in cell-to-cell communication via their molecular content. EVs have been found in all tissues and body fluids, carrying a variety of biomolecules, including DNA, RNA, proteins, metabolites, and lipids, offering insights into cellular and pathophysiological conditions. Despite the emergence of EVs and their molecular contents as important biological indicators, it remains difficult to explore EV-mediated biological processes due to their small size and heterogeneity and the technical challenges in characterizing their molecular content. EV-associated small RNAs, especially microRNAs, have been extensively studied. However, other less characterized RNAs, including protein-coding mRNAs, long noncoding RNAs, circular RNAs, and tRNAs, have also been found in EVs. Furthermore, the EV-associated proteins can be used to distinguish different types of EVs. The spectrum of EV-associated RNAs, as well as proteins, may be associated with different pathophysiological conditions. Therefore, the ability to comprehensively characterize EVs' molecular content is critical for understanding their biological function and potential applications in disease diagnosis. Here, we set out to provide an overview of EV-associated RNAs and proteins as well as approaches currently being used to characterize them.

103 Evaluating the IFN-t content in extracellular vesicles released by bovine embryos produced in vitro in the blastocyst stage

103 Evaluating the IFN-t content in extracellular vesicles released by bovine embryos produced in vitro in the blastocyst stage

Endothelial Cell-Derived Extracellular Vesicles Allow to Differentiate Between Various Endotypes of INOCA: A Multicentre, Prospective, Cohort Study.

Ischemia and non-obstructive coronary artery disease (INOCA) might be due to coronary microvascular dysfunction (CMD), vasospastic angina (VSA) or both. We compared plasma concentration of various extracellular vesicles (EVs) in patients with different INOCA endotypes. Patients were divided into those with INOCA (CMD, VSA, mixed CMD + VSA) and non-anginal chest pain. Plasma concentrations of EVs were measured using flow cytometry. Out of 96 patients included, 34 had CMD (35%), 15 VSA (16%), 24 mixed endotype (25%) and 23 non-anginal chest pain (24%). Patients with INOCA had lower ratio of endothelial EVs (CD144 +) to total EVs, compared to patients with non-anginal pain (p = 0.027). Patients with mixed endotype had lower ratio of endothelial EVs (CD144 +) to total EVs, compared to CMD (p = 0.008), VSA (p = 0.014) and non-anginal pain (p < 0.001). Decreased ratio of endothelial EVs (CD144 +) to total EVs might serve as a "circulating footprint" of the mixed INOCA endotype.

Monitoring concentration and lipid signature of plasma extracellular vesicles from HR+ metastatic breast cancer patients under CDK4/6 inhibitors treatment.

Extracellular vesicles (EVs) are cell-derived small membrane structures that transport various molecules. They have emerged as potential circulating biomarkers for monitoring responses to cancer therapies. This study aimed to comprehensively characterize plasma-carried EVs in hormone receptor-positive (HR+) metastatic breast cancer (MBC) patients treated with first-line CDK4/6 inhibitors (iCDK4/6) combined with endocrine therapy. MBC patients were classified into three groups based on their response to therapy: resistant, intermediate or sensitive. In a prospective cohort, we monitored the concentration of circulating EVs, analyzed their lipid signature and correlated these factors with treatment response. To facilitate the translation of EV research to clinical practice, we established a three-step procedure: (1) EVs were isolated from plasma using semi-automatized size exclusion chromatography (SEC); (2) EV concentration, termed vesiclemia, was determined by drop counting via interferometric light microscopy (ILM); and (3) EV lipid composition was analyzed by mass spectrometry. ILM-based vesiclemia values were highly fluctuating upon iCDK4/6 treatment, while early increase associated with accelerated progression. Of note, vesiclemia remained a steady parameter over a 1-year period in age-matched healthy women. Additionally, analysis of the EV cargo unveiled a distinct sphingolipid profile, characterized by increased levels of ceramides and sphingomyelins in resistant patients within the first 2 months of treatment. Based on 16 sphingolipid species, sensitive and resistant patients were correctly classified with an overall accuracy of 82%. This specific sphingolipid pattern was exclusively discernible within EVs, and not in plasma, highlighting the significance of EVs in the early prediction of individual responses to iCDK4/6 and disease progression. Overall, this study provides insights of the longitudinal characterization of plasma-borne EVs in both a healthy group and HR+ MBC patients under iCDK4/6 therapies. Combined vesiclemia and EV sphingolipid profile emphasize the promising potential of EVs as non-invasive biomarkers for monitoring early treatment response.

Small Extracellular Vesicle-Associated MiRNAs in Polarized Retinal Pigmented Epithelium.

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. 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. 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. 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.

Identification of the P24 Gene Family Related to Vesicular Transport in Cyclocarya paliurus and Their Expression Analysis Under Salt Stress

Vesicle transport is a fundamental mechanism for intracellular substance transfer and signal transduction, involving the formation, transport, fusion with target membranes, and release of vesicle contents within cells. Issues such as disruption of water balance, difficulty in regulating osmotic pressure, and oxidative stress caused by salt stress can lead to disturbances in the secretion system of plants, thus affecting plant growth and development. Cyclocarya paliurus (C. paliurus), widely used in traditional Chinese medicine, has not been previously reported in terms of how the vesicle transport P24 gene regulates its adaptation to salt stress. In this research project, a total of eight CpP24 genes were successfully identified. Upon examination of gene architecture and conserved sequence elements, the CpP24 genes exhibited a variation in exon count, ranging from 4 to 6. Moreover, the CpP24 gene family’s reaction to salt stress and specific stressors including methyl jasmonate (MeJA), sodium hydrosulfide (NaHS), and sodium nitroprusside (SNP) was further explored in our study. Comprehensive analysis of the expression patterns of CpP24 genes under various conditions showed that salt stress induced the expression of these genes, and the combined treatment of salt stress with specific stresses caused changes in their expression. This study lays a theoretical foundation for further probing into the physiological functions of C. paliurus and the underlying mechanisms regarding its response to environmental stress.

Alleviation of ulcerative colitis via curcumin-fortified orange juice: Utilization of orange-derived extracellular vesicles as active nanocarrier

There is evidence that consumption of fruit and vegetable juices may alleviate ulcerative colitis. However, their application is limited because of their relatively weak and uncertain health benefits. In this study, curcumin-fortified orange juice (Cur-OJ) was produced using orange extracellular vesicles as natural active nanocarriers. The concentration, composition, morphology and size of extracellular vesicles were first determined. It was shown that curcumin was incorporated into the lipid bilayer of extracellular vesicles in an amorphous form. The influences of oral administration of Cur-OJ on DSS-triggered colitis mice was evaluated. Cur-OJ reduced the pathological symptoms in mice, affected the expression of inflammatory cytokine, and improved the integrity of the colon tissue through enhancing the levels of three tight junction proteins. The gut microflora and colonic metabolites of colitis mice in Cur-OJ group were close to those in control group. Noticeably, the treatment of colitis was better in the Cur-OJ group than those in curcumin or orange juice groups, suggesting that curcumin and orange juice can work together to relieve colitis symptoms. In summary, this study showed that Cur-OJ can reduce colitis, which may be useful for the progress of novel functional fruit or vegetable juice and foods designed to improve body health and human wellbeing.

Comparative quantification of varicella-zoster virus in air, pharyngeal swabs, and vesicle content in patients with varicella, disseminated zoster, and localized herpes zoster.

We evaluated the viral load of varicella-zoster virus (VZV) in ambient air, vesicle, and pharyngeal swabs in VZV-infected patients. Of 46 cases, 6 had VZV detected in indoor air samples from patient rooms. Results suggest an association between viral load in the pharyngeal swab and indoor air.

Discovering the role of microRNAs and exosomal microRNAs in chest and pulmonary diseases: a spotlight on chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a progressive respiratory condition and ranks as the fourth leading cause of mortality worldwide. Despite extensive research efforts, a reliable diagnostic or prognostic tool for COPD remains elusive. The identification of novel biomarkers may facilitate improved therapeutic strategies for patients suffering from this debilitating disease. MicroRNAs (miRNAs), which are small non-coding RNA molecules, have emerged as promising candidates for the prediction and diagnosis of COPD. Studies have demonstrated that dysregulation of miRNAs influences critical cellular and molecular pathways, including Notch, Wnt, hypoxia-inducible factor-1α, transforming growth factor, Kras, and Smad, which may contribute to the pathogenesis of COPD. Extracellular vesicles, particularly exosomes, merit further investigation due to their capacity to transport various biomolecules such as mRNAs, miRNAs, and proteins between cells. This intercellular communication can significantly impact the progression and severity of COPD by modulating signaling pathways in recipient cells. A deeper exploration of circulating miRNAs and the content of extracellular vesicles may lead to the discovery of novel diagnostic and prognostic biomarkers, ultimately enhancing the management of COPD. The current review focus on the pathogenic role of miRNAs and their exosomal counterparts in chest and respiratory diseases, centering COPD.

Development of High-Production Bacterial Biomimetic Vesicles for Inducing Mucosal Immunity Against Avian Pathogenic Escherichia coli.

To evaluate the immunoprotective effect of bacterial biomimetic vesicles (BBVs) against avian pathogenic Escherichia coli (APEC), a ΔtolA J11 mutant strain was generated by deleting the tolA gene in the low pathogenic O78 serotype J11 strain. The total protein content of outer membrane vesicles (OMVs) derived from the ΔtolA J11 strain exhibited a sevenfold increase compared to the wild-type strain. Additionally, high-pressure homogenization technology was employed to produce BBVs, resulting in a sixfold increase in total protein content compared to spontaneously secreted OMVs from ΔtolA J11. The immunogenicity of both OMVs and BBVs was assessed through intranasal or intramuscular immunization in specific pathogen-free (SPF) chickens. Results demonstrated that intranasal immunization with OMVs or BBVs in chickens elicited specific IgY antibodies against APEC outer membrane proteins and specific sIgA antibodies in the nasal cavity and trachea, as well as a significant increase in the proliferation response of chicken peripheral blood lymphocytes. The bacterial load in the blood and various organs of the challenged chickens were significantly reduced, resulting in a 66.67% and 58.30% survival rate against a high pathogenic serotype O78 strain challenge, while the control group exhibited only a 16.67% survival rate. The intramuscular immunization with OMVs or BBVs in chickens only induced specific IgY antibodies, with a survival rate of only 33.33% for challenged chickens during the same period. Therefore, intranasal vaccination of the highly productive BBVs is capable of eliciting an immune response similar to that of OMVs and providing protection against APEC infection, thus offering innovative insights for the advancement of APEC vaccines.

Cell damage shifts the microRNA content of small extracellular vesicles into a Toll-like receptor 7-activating cargo capable to propagate inflammation and immunity

BackgroundThe 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.MethodsTo 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.ResultssEV-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.DiscussionOur 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.

Diversity of the Circulating Tumor Markers: Perspectives of a Multimodal Liquid Biopsy.

Over the past decade, liquid biopsy(LB) has become a routine diagnostic test essential for the treatment of malignant tumors of various localizations. Its capabilities include early diagnosis, molecular genotyping, prognosis, prediction, and monitoring of tumor response. Typically, liquid biopsy involves the extraction of a single type of tumor-derived molecules or cellular elements from blood and subsequent molecular analysis. These elements may include circulating tumor DNA(ctDNA), circulating tumor cells(CTCs), circulating tumor RNA(ctRNA), or contents of extracellular vesicles (exosomes). Despite the technical sophistication of molecular analysis methods for circulating biomarkers, this diagnostic approach has limited relevance. In a significant proportion of cancer patients (ranging from 10 to 50%, depending on the tumor type), none of these analytes can be detected and analyzed, even in the presence of large, progressing neoplastic foci in the body. It seems reasonable to suggest that heterogeneous fractions of the circulating tumor-specific biomarkers complement each other, thus simultaneous analysis of several fractions will not only increase sensitivity of the method but also more accurately characterize and predict the clinical situation. This review examines the possibilities and advantages of applying a combined multiparametric approach to liquid biopsy, which involves testing multiple circulating analytes in a single blood sample.