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Background: Extracellular vesicles (EVs) represent a sophisticated mechanism of intercellular communication that is implicated in health and disease. Specifically, the role of EVs in metabolic regulation and their implications in metabolic pathologies, such as obesity and its comorbidities, remain unclear., Methods: Extracellular vesicles (EVs) were isolated through serial ultracentrifugation from murine adipocytes treated with palmitate or oleic acid, whole visceral and subcutaneous adipose tissue (obesesomes) of bariatric surgery obese donors, and human hepatocytes under steatosis (steatosomes) for functional in vitro experiments. Functional effects on inflammation and glucose and lipid metabolism of target cells (human and murine macrophages and hepatocytes) were assessed using ELISA, RT-PCR, and immunodetection. Isolated EVs from human steatotic (steatosomes) and control hepatocytes (hepatosomes) were characterized for quantity, size, and tetraspanin profile by NTA and Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS), and their protein cargo analyzed by qualitative (DDA) and quantitative (DIA-SWATH) proteomics using LC-MS/MS. Proteins identified by proteomics were validated by capturing EVs on functionalized chips by SP-IRIS., Results and Conclusions: In this study, we investigated the role of EVs in the local communication between obese adipocytes and immune cells within adipose tissue, and the interaction of steatotic and healthy hepatocytes in the context of fatty liver disease progression. Furthermore, we analyzed obese adipose tissue-to-liver interactions through EV-obesesomes to elucidate their role in obesity-associated hepatic metabolic dysregulation. Our findings reveal that obesesomes promote inflammation and the secretion of pro-inflammatory cytokines upon interaction with macrophages, exerting a significant impact on reducing insulin resistance and altering lipid and glucose metabolism upon interaction with hepatocytes; in both cases, EVs from palmitate-loaded adipocytes and obesesomes from human visceral adipose depots demonstrated the most deleterious effect. Additionally, EVs secreted by steatotic hepatocytes (steatosomes) induced insulin resistance and altered lipid and glucose metabolism in healthy hepatocytes, suggesting their involvement in MASLD development. Proteomic analysis of steatosomes revealed that these vesicles contain liver disease-associated proteins, rendering them significant repositories of real-time biomarkers for the early stages and progression of MASLD., Competing Interests: Declarations. Ethics approval and consent to participate: All human samples and data were obtained after obtaining written informed consent, in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). All procedures were approved by the Clinical Ethical Committee of Galicia (CEIC), Spain, under code number 2020/497. Competing interests: The authors declare that they have no affiliations with or involvement in any organization or entity with any financial interest in the subject matter or materials discussed in this manuscript., (© 2025. The Author(s).)

Objective: Acute intermittent porphyria (AIP) is a rare metabolic disorder caused by haploinsufficiency of hepatic porphobilinogen deaminase (PBGD), the third enzyme of the heme biosynthesis. Individuals with AIP experience neurovisceral attacks closely associated with hepatic overproduction of potentially neurotoxic heme precursors., Design: We replicated AIP in non-human primates (NHPs) through selective knockdown of the hepatic PBGD gene and evaluated the safety and therapeutic efficacy of human PBGD (hPBGD) mRNA rescue., Results: Intrahepatic administration of a recombinant adeno-associated viral vector containing short hairpin RNA against endogenous PBGD mRNA resulted in sustained PBGD activity inhibition in liver tissue for up to 7 months postinjection. The administration of porphyrinogenic drugs to NHPs induced hepatic heme synthesis, elevated urinary porphyrin precursors and reproduced acute attack symptoms in patients with AIP, including pain, motor disturbances and increased brain GABAergic activity. The model also recapitulated functional anomalies associated with AIP, such as reduced brain perfusion and cerebral glucose uptake, disturbances in hepatic TCA cycle, one-carbon metabolism, drug biotransformation, lipidomic profile and abnormal mitochondrial respiratory chain activity. Additionally, repeated systemic administrations of hPBGD mRNA in this AIP NHP model restored hepatic PBGD levels and activity, providing successful protection against acute attacks, metabolic changes in the liver and CNS disturbances. This approach demonstrated better efficacy than the current standards of care for AIP., Conclusion: This novel model significantly expands our understanding of AIP at the molecular, biochemical and clinical levels and confirms the safety and translatability of multiple systemic administration of hPBGD mRNA as a potential aetiological AIP treatment., Competing Interests: Competing interests: LJ and PGVM are employees of Moderna Inc. focusing on the development of therapeutic approaches for rare diseases. MAA is Editor of Gut Journal. The rest of the authors have no conflict of interest to declare., (© Author(s) (or their employer(s)) 2025. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ Group.)

Intercellular communication is a cell-type and stimulus-dependent event driven not only by soluble factors but also by extracellular vesicles (EVs). EVs include vesicles of different size and origin that contain a myriad of molecules. Among them, small EVs (sEV; <200 nm) have been shown to modulate not just regional cell responses but also distant organ behavior. In cancer, distant organ modulation by sEVs has been associated to disease dissemination, which is one of the main concerns in melanoma. Description of broadly conserved alterations in sEV-contained molecules represents a strategy to identify key modifications in cellular communication as well as new disease biomarkers. Here, we characterize proteomes of cutaneous melanocyte and melanoma-derived sEVs to deepen on the landscape of normal and disease-related cell communication. Results reveal the presence of unique protein signatures for melanocytes and melanoma cells that reflect cellular transformation-related profound modifications. Melanocyte-derived sEVs are enriched in oxidative metabolism (e.g., aconitase 2, ACO2) or pigmentation (e.g., tyrosinase, TYR) related proteins while melanoma-derived sEVs reflect a generalized decrease in mature melanocytic markers (e.g., melanoma antigen recognized by T-cells 1, MART-1, also known as MLANA) and an increase in epithelial to mesenchymal transition (EMT)-related adhesion molecules such as tenascin C (TNC)., (© 2023 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd.)

The prostate gland is a complex and heterogeneous organ composed of epithelium and stroma. Whilst many studies into prostate cancer focus on epithelium, the stroma is known to play a key role in disease with the emergence of a cancer-associated fibroblasts (CAF) phenotype associated upon disease progression. In this work, we studied the metabolic rewiring of stromal fibroblasts following differentiation to a cancer-associated, myofibroblast-like, phenotype. We determined that CAFs were metabolically more active compared to normal fibroblasts. This corresponded with a heightened lipogenic metabolism, as both reservoir species and building block compounds. Interestingly, lipid metabolism affects mitochondria functioning yet the mechanisms of lipid-mediated functions are unclear. Data showing oxidised fatty acids and glutathione system are elevated in CAFs, compared to normal fibroblasts, strengthens the hypothesis that increased metabolic activity is related to mitochondrial activity. This manuscript describes mechanisms responsible for the altered metabolic flux and shows that prostate cancer-derived extracellular vesicles can increase basal respiration in normal fibroblasts, mirroring that of the disease-like phenotype. This indicates that extracellular vesicles derived from prostate cancer cells may drive an altered oxygen-dependent metabolism associated to mitochondria in CAFs., 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 B.V. All rights reserved.)

Seminal plasma (SP) is rich in extracellular vesicles (EVs), which are still poorly studied, especially in livestock species. To better understand their functional role in both spermatozoa and endometrial epithelial cells, proper characterization of EVs is an essential step. The objective was to phenotypically characterize porcine seminal EVs (sEVs) using cryogenic electron microscopy (cryo-EM), which allows visualization of EVs in their native state. Porcine ejaculates are released in fractions, each containing SP from different source. This allows characterization sEVs released from various male reproductive tissues. Two experiments were performed, the first with SP from the entire ejaculate (n:6) and the second with SP from three ejaculate fractions (n:15): the first 10 mL of the sperm-rich ejaculate fraction (SRF-P1) with SP mainly from the epididymis, the remainder of the SRF (SRF-P2) with SP mainly from the prostate, and the post-SRF with SP mainly from the seminal vesicles. The sEVs were isolated by size exclusion chromatography and 1840 cryo-EM sEV images were acquired using a Jeol-JEM-2200FS/CR-EM. The size, electron density, complexity, and peripheral corona layer were measured in each sEV using the ImageJ software. The first experiment showed that sEVs were structurally and morphologically heterogeneous, although most (83.1%) were small (less than 200 nm), rounded, and poorly electrodense, and some have a peripheral coronal layer. There were also larger sEVs (16.9%) that were irregularly shaped, more electrodense, and few with a peripheral coronal layer. The second experiment showed that small sEVs were more common in SRF-P1 and SRF-P2, indicating that they originated mainly from the epididymis and prostate. Large sEVs were more abundant in post-SRF, indicating that they originated mainly from seminal vesicles. Porcine sEVs are structurally and morphologically heterogeneous. This would be explained by the diversity of reproductive organs of origin., (© 2024. The Author(s).)

In vitro maturation (IVM) of oocytes is clinically used in horses to produce blastocysts but current conditions used for horses are suboptimal. We analyzed the composition of equine preovulatory follicular fluid (FF) secretome and tested its effects on meiotic competence and gene expression in oocytes subjected to IVM. Preovulatory FF was obtained, concentrated using ultrafiltration with cut-off of 10 kDa, and stored at -80 °C. The metabolic and proteomic composition was analyzed, and its ultrastructural composition was assessed by cryo-transmission microscopy. Oocytes obtained post-mortem or by ovum pick up (OPU) were subjected to IVM in the absence (control) or presence of 20 or 40 μg/ml (S20 or S40) of secretome. Oocytes were then analyzed for chromatin configuration or snap frozen for gene expression analysis. Proteomic analysis detected 255 proteins in the Equus caballus database, mostly related to the complement cascade and cholesterol metabolism. Metabolomic analysis yielded 14 metabolites and cryo-transmission electron microscopy analysis revealed the presence of extracellular vesicles (EVs). No significant differences were detected in maturation rates among treatments. However, the expression of GDF9 and BMP15 significantly increased in OPU-derived oocytes compared to post-mortem oocytes (fold increase ± SEM: 9.4 ± 0.1 vs. 1 ± 0.5 for BMP15 and 9.9 ± 0.3 vs. 1 ± 0.5 for GDF9, respectively; p < 0.05). Secretome addition increased the expression of TNFAIP6 in S40 regardless of the oocyte source. Further research is necessary to fully understand whether secretome addition influences the developmental competence of equine oocytes., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background and Aims: Alcohol-associated liver disease (ALD) accounts for 70% of liver-related deaths in Europe, with no effective approved therapies. Although mitochondrial dysfunction is one of the earliest manifestations of alcohol-induced injury, restoring mitochondrial activity remains a problematic strategy due to oxidative stress. Here, we identify methylation-controlled J protein (MCJ) as a mediator for ALD progression and hypothesize that targeting MCJ may help in recovering mitochondrial fitness without collateral oxidative damage., Approach and Results: C57BL/6 mice [wild-type (Wt)] Mcj knockout and Mcj liver-specific silencing (MCJ-LSS) underwent the NIAAA dietary protocol (Lieber-DeCarli diet containing 5% (vol/vol) ethanol for 10 days, plus a single binge ethanol feeding at day 11). To evaluate the impact of a restored mitochondrial activity in ALD, the liver, gut, and pancreas were characterized, focusing on lipid metabolism, glucose homeostasis, intestinal permeability, and microbiota composition. MCJ, a protein acting as an endogenous negative regulator of mitochondrial respiration, is downregulated in the early stages of ALD and increases with the severity of the disease. Whole-body deficiency of MCJ is detrimental during ALD because it exacerbates the systemic effects of alcohol abuse through altered intestinal permeability, increased endotoxemia, and dysregulation of pancreatic function, which overall worsens liver injury. On the other hand, liver-specific Mcj silencing prevents main ALD hallmarks, that is, mitochondrial dysfunction, steatosis, inflammation, and oxidative stress, as it restores the NAD + /NADH ratio and SIRT1 function, hence preventing de novo lipogenesis and improving lipid oxidation., Conclusions: Improving mitochondrial respiration by liver-specific Mcj silencing might become a novel therapeutic approach for treating ALD., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) is a feature of many solid tumours and is a key pathogenic driver in the inherited condition Tuberous Sclerosis Complex (TSC). Modulation of the tumour microenvironment by extracellular vesicles (EVs) is known to facilitate the development of various cancers. The role of EVs in modulating the tumour microenvironment and their impact on the development of TSC tumours, however, remains unclear. This study, therefore, focuses on the poorly defined contribution of EVs to tumour growth in TSC. We characterised EVs secreted from TSC2-deficient and TSC2-expressing cells and identified a distinct protein cargo in TSC2-deficient EVs, containing an enrichment of proteins thought to be involved in tumour-supporting signalling pathways. We show EVs from TSC2-deficient cells promote cell viability, proliferation and growth factor secretion from recipient fibroblasts within the tumour microenvironment. Rapalogs (mTORC1 inhibitors) are the current therapy for TSC tumours. Here, we demonstrate a previously unknown intercellular therapeutic effect of rapamycin in altering EV cargo and reducing capacity to promote cell proliferation in the tumour microenvironment. Furthermore, EV cargo proteins have the potential for clinical applications as TSC biomarkers, and we reveal three EV-associated proteins that are elevated in plasma from TSC patients compared to healthy donor plasma., (© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

The standardization of clinical studies using extracellular vesicles (EVs) has mainly focused on the procedures employed for their isolation and characterization; however, preanalytical aspects of sample collection, handling and storage also significantly impact the reproducibility of results. We conducted an online survey based on SPREC (Standard PREanalytical Code) among members of GEIVEX (Grupo Español de Investigación en Vesiculas Extracelulares) to explore how different laboratories handled fluid biospecimens destined for EV analyses. We received 70 surveys from forty-three different laboratories: 44% focused on plasma, 9% on serum and 16% on urine. The survey indicated that variability in preanalytical approaches reaches 94%. Moreover, in some cases, researchers had no access to all relevant preanalytical details of samples, with some sample aspects with potential impact on EV isolation/characterisation not coded within the current version of SPREC. Our study highlights the importance of working with common standard operating procedures (SOP) to control preanalytical conditions. The application of SPREC represents a suitable approach to codify and register preanalytical conditions. Integrating SPREC into the SOPs of laboratories/biobanks will provide a valuable source of information and constitute an advance for EV research by improving reproducibility and credibility., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors. Journal of Extracellular Biology published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

The incidence of colorectal cancer (CRC) is increasing, and currently it is the third most common cancer. Early CRC diagnosis is still difficult and relies on an invasive colonoscopy and tissue biopsy. The globally observed tendency demands non-invasive, specific, and accurate diagnostic tools for early diagnosis and prognosis. In this work, the main aim was to evaluate for the first time the feasibility of using extracts from the non-invasive sample collection from faecal occult blood (FOB) kits for its use in metabolomics studies taking advantage in this way of the high sensitivity of this technology. Then, a cohort of 131 samples from control individuals (CTL), adenoma (AD) and CRC patients were analysed using a semitargeted approach by ultra-high-performance liquid chromatography-time-of-flight-mass spectrometry (UHPLC-ToF-MS). Multivariate and univariate statistical analysis revealed that cholesteryl esters (ChoE) with polyunsaturated fatty acids (PUFAs) together with FOB were relevant metabolites that could clearly separate CRC patients from AD and CTL individuals, whereas the metabolic profiles of CTL and AD were very similar. These results are in agreement with previous findings and reveal the advantage of using the same FOBT samples for several analyses, which would facilitate sample collection and improve direct connection between FOB measurements and metabolomics analysis. Although the sample size and the number of metabolites should be enhanced to cover a wider range of metabolites, alterations in lipid metabolism clearly point out for future perspectives., Competing Interests: O.E.A. and J.M.F.-P. are employees of CICbioGUNE-BRTA. J.C. is employee of Complexo Hospitalario Universitario de Orense. L.B. and B.N. are employees of Biodonostia Health Research Institute, and Mar&iacute;a Encarnacion, B.L. and C.A. are employees of OWL Metabolomics. The paper reflects the views of the scientists and not the company. The authors declare no conflict of interest.

Interferon-stimulated gene 15 (ISG15) is a 15-kDa ubiquitin-like modifier that binds to target proteins in a process termed ISGylation. ISG15, first described as an antiviral molecule against many viruses, participates in numerous cellular processes, from immune modulation to the regulation of genome stability. Interestingly, the role of ISG15 as a regulator of cell metabolism has recently gained strength. We previously described ISG15 as a regulator of mitochondrial functions in bone marrow-derived macrophages (BMDMs) in the context of Vaccinia virus (VACV) infection. Here, we demonstrate that ISG15 regulates lipid metabolism in BMDMs and that ISG15 is necessary to modulate the impact of VACV infection on lipid metabolism. We show that Isg15 -/- BMDMs demonstrate alterations in the levels of several key proteins of lipid metabolism that result in differences in the lipid profile compared with Isg15 +/+ (wild-type [WT]) BMDMs. Specifically, Isg15 -/- BMDMs present reduced levels of neutral lipids, reflected by decreased lipid droplet number. These alterations are linked to increased levels of lipases and are independent of enhanced fatty acid oxidation (FAO). Moreover, we demonstrate that VACV causes a dysregulation in the proteomes of BMDMs and alterations in the lipid content of these cells, which appear exacerbated in Isg15 -/- BMDMs. Such metabolic changes are likely caused by increased expression of the metabolic regulators peroxisome proliferator-activated receptor-γ (PPARγ) and PPARγ coactivator-1α (PGC-1α). In summary, our results highlight that ISG15 controls BMDM lipid metabolism during viral infections, suggesting that ISG15 is an important host factor to restrain VACV impact on cell metabolism. IMPORTANCE The functions of ISG15 are continuously expanding, and growing evidence supports its role as a relevant modulator of cell metabolism. In this work, we highlight how the absence of ISG15 impacts macrophage lipid metabolism in the context of viral infections and how poxviruses modulate metabolism to ensure successful replication. Our results open the door to new advances in the comprehension of macrophage immunometabolism and the interaction between VACV and the host.

This is a meeting report of the 3rd Translational Hepatology Meeting held in Alicante, Spain, in October 2021. The meeting, which was organized by the Spanish Association for the Study of the Liver (AEEH), provided an update on the recent advances in the field of basic and translational hepatology, with a particular focus on the molecular and cellular mechanisms and therapeutic targets involved in metabolic-associated fatty liver disease (MAFLD), metabolic-associated steatohepatitis (MASH), cirrhosis and end-stage hepatocellular carcinoma (HCC)., (Copyright © 2022 The Authors. Publicado por Elsevier España, S.L.U. All rights reserved.)

Study Question: Is it possible to use free and extracellular vesicle-associated microRNAs (miRNAs) from human endometrial fluid (EF) samples as non-invasive biomarkers for implantative endometrium?, Summary Answer: The free and extracellular vesicle-associated miRNAs can be used to detect implantative endometrium in a non-invasive manner., What Is Known Already: miRNAs and extracellular vesicles (EVs) from EF have been described as mediators of the embryo-endometrium crosstalk. Therefore, the analysis of miRNA from this fluid could become a non-invasive technique for recognizing implantative endometrium. This analysis could potentially help improve the implantation rates in ART., Study Design, Size, Duration: In this prospective study, we first optimized different protocols for EVs and miRNA analyses using the EF of a setup cohort (n = 72). Then, we examined differentially expressed miRNAs in the EF of women with successful embryo implantation (discovery cohort n = 15/validation cohort n = 30) in comparison with those for whom the implantation had failed (discovery cohort n = 15/validation cohort n = 30). Successful embryo implantation was considered when pregnancy was confirmed by vaginal ultrasound showing a gestational sac 4 weeks after embryo transfer (ET)., Participants/materials, Setting, Methods: The EF of the setup cohort was obtained before starting fertility treatment during the natural cycle, 16-21 days after the beginning of menstruation. For the discovery and validation cohorts, the EF was collected from women undergoing frozen ET on Day 5, and the samples were collected immediately before ET. In this study, we compared five different methods; two of them based on direct extraction of RNA and the other three with an EV enrichment step before the RNA extraction. Small RNA sequencing was performed to determine the most efficient method and find a predictive model differentiating between implantative and non-implantative endometrium. The models were confirmed using quantitative PCR in two sets of samples (discovery and validation cohorts) with different implantation outcomes., Main Results and the Role of Chance: The protocols using EV enrichment detected more miRNAs than the methods based on direct RNA extraction. The two most efficient protocols (using polymer-based precipitation (PBP): PBP-M and PBP-N) were used to obtain two predictive models (based on three miRNAs) allowing us to distinguish between an implantative and non-implantative endometrium. The first Model 1 (PBP-M) (discovery: AUC = 0.93; P-value = 0.003; validation: AUC = 0.69; P-value = 0.019) used hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-148b-3p. Model 2 (PBP-N) (discovery: AUC = 0.92; P-value = 0.0002; validation: AUC = 0.78; P-value = 0.0002) used hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-99b-5p. Functional analysis of these miRNAs showed strong association with key implantation processes such as in utero embryonic development or transforming growth factor-beta signaling., Large Scale Data: The FASTQ data are available in the GEO database (access number GSE178917)., Limitations, Reasons for Caution: One important factor to consider is the inherent variability among the women involved in the trial and among the transferred embryos. The embryos were pre-selected based on morphology, but neither genetic nor molecular studies were conducted, which would have improved the accuracy of our tests. In addition, a limitation in miRNA library construction is the low amount of input RNA., Wider Implications of the Findings: We describe new non-invasive protocols to analyze miRNAs from small volumes of EF. These protocols could be implemented in clinical practice to assess the status of the endometrium before attempting ET. Such evaluation could help to avoid the loss of embryos transferred to a non-implantative endometrium., Study Funding/competing Interest(s): J.I.-P. was supported by a predoctoral grant from the Basque Government (PRE&#95;2017&#95;0204). This study was partially funded by the Grant for Fertility Innovation (GFI, 2011) from Merck (Darmstadt, Germany). It was also supported by the Spanish Ministry of Economy and Competitiveness MINECO within the National Plan RTI2018-094969-B-I00, the European Union's Horizon 2020 research and innovation program (860303), the Severo Ochoa Centre of Excellence Innovative Research Grant (SEV-2016-0644) and the Instituto de Salud Carlos III (PI20/01131). The funding entities did not play any role in the study design, collection, analysis and interpretation of data, writing of the report or the decision to submit the article for publication. The authors declare no competing interests., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Background: Sorafenib constitutes a suitable treatment alternative for patients with advanced hepatocellular carcinoma (HCC) in whom atezolizumab + bevacizumab therapy is contraindicated. The aim of the study was the identification of a miRNA signature in liquid biopsy related to sorafenib response., Methods: miRNAs were profiled in hepatoblastoma HepG2 cells and tested in animal models, extracellular vesicles (EVs), and plasma from HCC patients., Results: Sorafenib altered the expression of 11 miRNAs in HepG2 cells. miR-200c-3p and miR-27a-3p exerted an anti-tumoral activity by decreasing cell migration and invasion, whereas miR-122-5p, miR-148b-3p, miR-194-5p, miR-222-5p, and miR-512-3p exerted pro-tumoral properties by increasing cell proliferation, migration, or invasion, or decreasing apoptosis. Sorafenib induced a change in EVs population with an increased number of larger EVs, and promoted an accumulation of miR-27a-3p, miR-122-5p, miR-148b-3p, miR-193b-3p, miR-194-5p, miR-200c-3p, and miR-375 into exosomes. In HCC patients, circulating miR-200c-3p baseline levels were associated with increased survival, whereas high levels of miR-222-5p and miR-512-3p after 1 month of sorafenib treatment were related to poor prognosis. The RNA sequencing revealed that miR-200c-3p was related to the regulation of cell growth and death, whereas miR-222-5p and miR-512-3p were related to metabolic control., Conclusions: The study showed that Sorafenib regulates a specific miRNA signature in which miR-200c-3p, miR-222-5p, and miR-512-3p bear prognostic value and contribute to treatment response.

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EV) are widely considered as a cell-free therapeutic alternative to MSC cell administration, due to their immunomodulatory and regenerative properties. However, the interaction mechanisms between EV and target cells are not fully understood. The surface glycans could be key players in EV-cell communication, being specific molecular recognition patterns that are still little explored. In this study, we focused on the role of N-glycosylation of MSC-EV as mediators of MSC-EV and endothelial cells' interaction for subsequent EV uptake and the induction of cell migration and angiogenesis. For that, EV from immortalized Wharton's Jelly MSC (iWJ-MSC-EV) were isolated by size exclusion chromatography (SEC) and treated with the glycosidase PNGase-F in order to remove wild-type N-glycans. Then, CFSE-labelled iWJ-MSC-EV were tested in the context of in vitro capture, agarose-spot migration and matrigel-based tube formation assays, using HUVEC. As a result, we found that the N-glycosylation in iWJ-MSC-EV is critical for interaction with HUVEC cells. iWJ-MSC-EV were captured by HUVEC, stimulating their tube-like formation ability and promoting their recruitment. Conversely, the removal of N-glycans through PNGase-F treatment reduced all of these functional activities induced by native iWJ-MSC-EV. Finally, comparative lectin arrays of iWJ-MSC-EV and PNGase-F-treated iWJ-MSC-EV found marked differences in the surface glycosylation pattern, particularly in N-acetylglucosamine, mannose, and fucose-binding lectins. Taken together, our results highlight the importance of N-glycans in MSC-EV to permit EV-cell interactions and associated functions.

Steroid hormones play a vital role in the regulation of cellular processes, and dysregulation of these metabolites can provoke or aggravate pathological issues, such as autoimmune diseases and cancer. Regulation of steroid hormones involves different organs and biological compartments. Therefore, it is important to accurately determine their levels in tissues and biofluids to monitor changes after challenge or during disease. In this work, we have developed and optimized the extraction and quantification of 11 key members of the different steroid classes, including androgens, estrogens, progestogens and corticoids. The assay consists of a liquid/liquid extraction step and subsequent quantification by high-resolution liquid chromatography coupled time-of-flight mass spectrometry. The recoveries range between 74.2 to 126.9% and 54.9 to 110.7%, using a cell culture or urine as matrix, respectively. In general, the signal intensity loss due to matrix effect is no more than 30%. The method has been tested in relevant steroidogenic tissues in rat models and it has also been tested in human urine samples. Overall, this assay measures 11 analytes simultaneously in 6 min runtime and it has been applied in adrenal gland, testis, prostate, brain and serum from rats, and urine and extracellular vesicles from humans., Competing Interests: The authors declare no conflict of interest.

Accurate diagnosis of colorectal cancer (CRC) still relies on invasive colonoscopy. Noninvasive methods are less sensitive in detecting the disease, particularly in the early stage. In the current work, a metabolomics analysis of fecal samples was carried out by ultra-high-performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS). A total of 1380 metabolites were analyzed in a cohort of 120 fecal samples from patients with normal colonoscopy, advanced adenoma (AA) and CRC. Multivariate analysis revealed that metabolic profiles of CRC and AA patients were similar and could be clearly separated from control individuals. Among the 25 significant metabolites, sphingomyelins (SM), lactosylceramides (LacCer), secondary bile acids, polypeptides, formiminoglutamate, heme and cytidine-containing pyrimidines were found to be dysregulated in CRC patients. Supervised random forest (RF) and logistic regression algorithms were employed to build a CRC accurate predicted model consisting of the combination of hemoglobin (Hgb) and bilirubin E,E, lactosyl-N-palmitoyl-sphingosine, glycocholenate sulfate and STLVT with an accuracy, sensitivity and specificity of 91.67% (95% Confidence Interval (CI) 0.7753-0.9825), 0.7 and 1, respectively.

Extracellular vesicles (EVs) participate in cell-stroma crosstalk within the tumor microenvironment and fibroblasts (Fb) contribute to tumor promotion in thyroid cancer. However, the role of tumor-stroma derived EVs still needs to be deciphered. We hypothesized that the interaction of thyroid tumor cells with Fb would liberate EVs with a specific proteomic profile, which would have an impact on EV-functionality in thyroid tumor progression-related events. Tumor (TPC-1, 8505c) and non-tumor (NThyOri) thyroid cells were co-cultured with human Fb. EVs, obtained by ultracentrifugation of conditioned media, were characterized by nanoparticle tracking analysis and western blotting. EV-proteomic analysis was performed by mass-spectrometry, and metalloproteinases (MMPs) were studied by zymography. EV-exchange was evaluated using immunofluorescence, confocal microscopy and FACS. EVs expressed classical exosome markers, with EVs from thyroid tumor cell-Fb co-cultures showing a proteomic profile related to extracellular matrix (ECM) remodeling. Bidirectional crosstalk between Fb and TPC-1 cells produced significantly more EVs than their isolated cells, and potentiated EV-functionality. In line with this, Fb-TPC-1 derived EVs induced MMP2 activation in NThyOri supernatants, and MMP2 activity could be evidenced in Fb and TPC-1 contact-independent co-cultures. Besides, MMP2 interactors allowed us to discriminate between EVs from thyroid tumoral and non-tumoral milieus. Interestingly, Fb internalized more EVs from TPC-1 than from NThyOri producing cells. Fb and thyroid tumor cell crosstalk produces specialized EVs with an ECM remodeling proteomic profile, enabling activation of MMP2 and possibly facilitating ECM-degradation, which is potentially linked with thyroid tumor progression., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Cells release membrane-delimited particles into the environment. These particles are called "extracellular vesicles" (EVs), and EVs are present in fluids contacting cells, including body fluids and conditioned culture media. Because EVs change and contribute to health and disease, EVs have become a hot topic. From the thousands of papers now published on EVs annually, one easily gets the impression that EVs provide biomarkers for all diseases, and that EVs are carriers of all relevant biomolecules and are omnipotent therapeutics. At the same time, EVs are heterogeneous, elusive and difficult to study due to their physical properties and the complex composition of their environment. This overview addresses the current challenges encountered when working with EVs, and how we envision that most of these challenges will be overcome in the near future. Right now, an infrastructure is being developed to improve the reproducibility of EV measurement results. This infrastructure comprises expert task forces of the International Society of Extracellular Vesicles (ISEV) developing guidelines and recommendations, instrument calibration, standardized and transparent reporting, and education. Altogether, these developments will support the credibility of EV research by introducing robust reproducibility, which is a prerequisite for understanding their biological significance and biomarker potential., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: Cholangiocarcinoma (CCA) is still a deadly tumour. Histological and molecular aspects of thioacetamide (TAA)-induced intrahepatic CCA (iCCA) in rats mimic those of human iCCA. Carcinogenic changes and therapeutic vulnerabilities in CCA may be captured by molecular investigations in bile, where we performed bile proteomic and metabolomic analyses that help discovery yet unknown pathways relevant to human iCCA., Methods: Cholangiocarcinogenesis was induced in rats (TAA) and mice (Jnk Δhepa  + CCl 4  + DEN model). We performed proteomic and metabolomic analyses in bile from control and CCA-bearing rats. Differential expression was validated in rat and human CCAs. Mechanisms were addressed in human CCA cells, including Huh28-KRAS G12D cells. Cell signaling, growth, gene regulation and [U- 13 C]-D-glucose-serine fluxomics analyses were performed. In vivo studies were performed in the clinically-relevant iCCA mouse model., Results: Pathways related to inflammation, oxidative stress and glucose metabolism were identified by proteomic analysis. Oxidative stress and high amounts of the oncogenesis-supporting amino acids serine and glycine were discovered by metabolomic studies. Most relevant hits were confirmed in rat and human CCAs (TCGA). Activation of interleukin-6 (IL6) and epidermal growth factor receptor (EGFR) pathways, and key genes in cancer-related glucose metabolic reprogramming, were validated in TAA-CCAs. In TAA-CCAs, G9a, an epigenetic pro-tumorigenic writer, was also increased. We show that EGFR signaling and mutant KRAS G12D can both activate IL6 production in CCA cells. Furthermore, phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in serine-glycine pathway, was upregulated in human iCCA correlating with G9a expression. In a G9a activity-dependent manner, KRAS G12D promoted PHGDH expression, glucose flow towards serine synthesis, and increased CCA cell viability. KRAS G12D CAA cells were more sensitive to PHGDH and G9a inhibition than controls. In mouse iCCA, G9a pharmacological targeting reduced PHGDH expression., Conclusions: In CCA, we identified new pro-tumorigenic mechanisms: Activation of EGFR signaling or KRAS mutation drives IL6 expression in tumour cells; Glucose metabolism reprogramming in iCCA includes activation of the serine-glycine pathway; Mutant KRAS drives PHGDH expression in a G9a-dependent manner; PHGDH and G9a emerge as therapeutic targets in iCCA., (© 2022. The Author(s).)