Identification of PRMT5 as a therapeutic target in cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CCA) is a very difficult-to-treat cancer. Chemotherapies are little effective and response to immune checkpoint inhibitors is limited. Therefore, new therapeutic strategies need to be identified. OBJECTIVE: We characterised the enzyme protein arginine-methyltransferase 5 (PRMT5) as a novel therapeutic target in CCA. DESIGN: We evaluated the expression of PRMT5, its functional partner MEP50 and methylthioadenosine phosphorylase (MTAP)-an enzyme that modulates the sensitivity of PRMT5 to pharmacological inhibitors-in human CCA tissues. PRMT5-targeting drugs, currently tested in clinical trials for other malignancies, were assessed in human CCA cell lines and organoids, as well as in two immunocompetent CCA mouse models. Transcriptomic, proteomic and functional analyses were performed to explore the underlying antitumoural mechanisms. RESULTS: PRMT5 and MEP50 proteins were correlatively overexpressed in most CCA tissues. MTAP was absent in 25% of intrahepatic CCA. PRMT5-targeting drugs markedly inhibited CCA cell proliferation, synergising with cisplatin and gemcitabine and hindered the growth of cholangiocarcinoma organoids. PRMT5 inhibition blunted the expression of oncogenic genes involved in chromatin remodelling and DNA repair, consistently inducing the formation of RNA loops and promoting DNA damage. Treatment with PRMT5-targeting drugs significantly restrained the growth of experimental CCA without adverse effects and concomitantly induced the recruitment of CD4 and CD8 T cells to shrinking tumourous lesions. CONCLUSION: PRMT5 and MEP50 are frequently upregulated in human CCA, and PRMT5-targeting drugs have significant antitumoural efficacy in clinically relevant CCA models. Our findings support the evaluation of PRMT5 inhibitors in clinical trials, including their combination with cytotoxic and immune therapies.

Development of bioactive solid-foam scaffolds from decellularized cartilage with chondrogenic and osteogenic properties.

Full osteochondral regeneration remains a major clinical challenge. Among other experimental cartilage regenerative approaches, decellularized cartilage (DCC) is considered a promising material for generating potentially implantable scaffolds useful as cartilage repair strategy. In this work, we focus on screening and comparing different decellularization methods, aiming to generate DCC potentially useful in biomedical context, and therefore, with biological activity and functional properties in terms of induction of differentiation and regeneration. Data indicates that enzymatic and detergents-based decellularization methods differentially affect ECM components, and that it has consequences in further biological behavior. SDS-treated DCC powder is not useful to be further processed in 2D or 3D structures, because these structures tend to rapidly solubilize, or disaggregate, in physiologic media conditions. Conversely, Trypsin-treated DCC powders can be processed to mechanically stable 2D films and 3D solid-foam scaffolds, presumably due to partial digestion of collagens during decellularization, which would ease crosslinking at DCC during solubilization and processing. In vitro cell culture studies indicate that these structures are biocompatible and induce and potentiate chondrogenic differentiation. In vivo implantation of DCC derived 3D porous scaffolds in rabbit osteochondral defects induce subchondral bone regeneration and fibrocartilage tissue formation after implantation. Therefore, this work defines an optimal cartilage tissue decellularization protocol able to generate DCC powders processable to biocompatible and bioactive 2D and 3D structures. These structures are useful for in vitro cartilage research and in vivo subchondral bone regeneration, while hyaline cartilage regeneration with DCC alone as implantable material remains elusive.

Combining sandblasting and pink anodisation of Ti implants as a promising method for improving fibroblast adhesion and immune response.

This study examined the effect of combining the sandblasting and anodising of titanium alloys used in implants on the cell response and protein adsorption patterns. The titanium samples were divided into four groups depending on the surface treatment: machining (MC), pink anodisation (PA), sandblasting (MC04) and a combination of the last two (MC04 + PA). Their physicochemical properties were analysed by SEM/EDX, Raman, contact angle measurements and profilometry. In vitro responses were examined using human gingival fibroblastic (HGF) cells and THP-1 macrophages. Cytokine secretion, macrophage adhesion and gene expression were measured by ELISA, confocal microscopy and RT-PCR. Cell adhesion and collagen secretion were evaluated in HGF cultures. The adsorption of immune and regenerative proteins onto the surfaces was assessed employing nLC-MS/MS. MC04 + PA surfaces exhibited a change in the roughness, chemical composition and hydrophilicity of the material, showing more elongated HGF cells and a considerable increase in the area of cells exposed to the MC04 + PA surfaces. Moreover, cells cultured on MC04 + PA generally showed a reduction in the expression of proinflammatory genes (TNF-α, MCP-1, C5, NF-kB and ICAM-1) and an increase in the secretion of anti-inflammatory cytokines, such as IL-4. These results correlated with the proteomic data; we found preferential adsorption of proteins favouring cell adhesion, such as DSC1 and PCOC1. A considerable reduction in the adsorption of immunoglobulins and proteins associated with acute inflammatory response (including SAA4) was also observed. The study highlights the potential advantages of MC04 + PA surface treatment to modify dental implant abutments; it enhances their compatibility with soft tissues and reduces the inflammatory response.

Neddylation inhibition prevents acetaminophen-induced liver damage by enhancing the anabolic cardiolipin pathway.

Drug-induced liver injury (DILI) is a significant cause of acute liver failure (ALF) and liver transplantation in the Western world. Acetaminophen (APAP) overdose is a main contributor of DILI, leading to hepatocyte cell death through necrosis. Here, we identified that neddylation, an essential post-translational modification involved in the mitochondria function, was upregulated in liver biopsies from patients with APAP-induced liver injury (AILI) and in mice treated with an APAP overdose. MLN4924, an inhibitor of the neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8)-activating enzyme (NAE-1), ameliorated necrosis and boosted liver regeneration in AILI. To understand how neddylation interferes in AILI, whole-body biotinylated NEDD8 (bioNEDD8) and ubiquitin (bioUB) transgenic mice were investigated under APAP overdose with and without MLN4924. The cytidine diphosphate diacylglycerol (CDP-DAG) synthase TAM41, responsible for producing cardiolipin essential for mitochondrial activity, was found modulated under AILI and restored its levels by inhibiting neddylation. Understanding this ubiquitin-like crosstalk in AILI is essential for developing promising targeted inhibitors for DILI treatment.

Effect of Health Status and Heat-Induced Inactivation on the Proteomic Profile of Plasma Rich in Growth Factors Obtained from Donors with Chronic Inflammatory Skin Conditions.

Atopic dermatitis, psoriasis and lichen sclerosus are among the most challenging conditions treated by dermatologists worldwide, with potentially significant physical, social and psychological impacts. Emerging evidence suggests that autologous-platelet-rich plasma could be used to manage skin inflammation. However, the presence of soluble autoimmune components could hinder their therapeutic potential. The aim of this study was to analyze the proteomic profile of plasma rich in growth factors (PRGFs) obtained from donors with inflammatory skin conditions to evaluate the impact of skin health status on the composition and bioactivity of PRGF-based treatments. Venous blood from healthy volunteers and patients with psoriasis, lichen sclerosus and atopic dermatitis was processed to produce PRGF supernatant. Half of the samples were subjected to an additional thermal treatment (56 °C) to inactivate inflammatory and immune molecules. Proteomic analysis was performed to assess the protein profile of PRGFs from healthy and non-healthy patients and the effect of Immunosafe treatment. Differential abundance patterns of several proteins related to key biological processes have been identified, including complement activation, blood coagulation, and glycolysis- and gluconeogenesis-related genes. These results also demonstrate that the thermal treatment (Immunosafe) contributes to the inactivation of the complement system and, as a consequence, reduction in the immunogenic potential of PRGF products.

The PP2A regulator IER5L supports prostate cancer progression.

Prostate cancer exhibits high prevalence and accounts for a high number of cancer-related deaths. The discovery and characterization of molecular determinants of aggressive prostate cancer represents an active area of research. The Immediate Early Response (IER) family of genes, which regulate Protein Phosphatase 2A (PP2A) activity, has emerged among the factors that influence cancer biology. Here, we show that the less studied member of this family, Immediate Early Response 5 like (IER5L), is upregulated in aggressive prostate cancer. Interestingly, the upregulation of IER5L expression exhibits a robust association with metastatic disease in prostate and is recapitulated in other cancer types. In line with this observation, IER5L silencing reduces foci formation, migration and invasion ability in a variety of human and murine prostate cancer cell lines. In vivo, using zebrafish and immunocompromised mouse models, we demonstrate that IER5L-silencing reduces prostate cancer tumor growth, dissemination, and metastasis. Mechanistically, we characterize the transcriptomic and proteomic landscapes of IER5L-silenced cells. This approach allowed us to identify DNA replication and monomeric G protein regulators as downstream programs of IER5L through a pathway that is consistent with the regulation of PP2A. In sum, we report the alteration of IER5L in prostate cancer and beyond and provide biological and molecular evidence of its contribution to tumor aggressiveness.

Glucocorticoid receptor controls atopic dermatitis inflammation via functional interactions with P63 and autocrine signaling in epidermal keratinocytes.

Atopic dermatitis (AD), a prevalent chronic inflammatory disease with multifactorial etiology, features epidermal barrier defects and immune overactivation. Synthetic glucocorticoids (GCs) are widely prescribed for treating AD due to their anti-inflammatory actions; however, mechanisms are incompletely understood. Defective local GC signaling due to decreased production of endogenous ligand and/or GC receptor (GR) levels was reported in prevalent inflammatory skin disorders; whether this is a consequence or contributing factor to AD pathology is unclear. To identify the chromatin-bound cell-type-specific GR protein interactome in keratinocytes, we used rapid immunoprecipitation of endogenous proteins and mass spectrometry identifying 145 interactors that increased upon dexamethasone treatment. GR-interacting proteins were enriched in p53/p63 signaling, including epidermal transcription factors with critical roles in AD pathology. Previous analyses indicating mirrored AD-like phenotypes between P63 overexpression and GR loss in epidermis, and our data show an intricate relationship between these transcription factors in human keratinocytes, identifying TP63 as a direct GR target. Dexamethasone treatment counteracted transcriptional up-regulation of inflammatory markers by IL4/IL13, known to mimic AD, causing opposite shifts in GR and P63 genomic binding. Indeed, IL4/IL13 decreased GR and increased P63 levels in cultured keratinocytes and human epidermal equivalents (HEE), consistent with GR down-regulation and increased P63 expression in AD lesions vs normal skin. Moreover, GR knockdown (GRKD) resulted in constitutive increases in P63, phospho-P38 and S100A9, IL6, and IL33. Also, GRKD culture supernatants showed increased autocrine production of TH2-/TH1-/TH17-TH22-associated factors including IL4, CXCL10, CXCL11, and CXCL8. GRKD HEEs showed AD-like features including hyperplasia and abnormal differentiation, resembling phenotypes observed with GR antagonist or IL4/IL13 treatment. The simultaneous GR/P63 knockdown partially reversed constitutive up-regulation of inflammatory genes in GRKD. In summary, our data support a causative role for GR loss in AD pathogenesis via functional interactions with P63 and autocrine signaling in epidermal keratinocytes.

Roughness affects the response of human fibroblasts and macrophages to sandblasted abutments.

BACKGROUND: A strong seal of soft-tissue around dental implants is essential to block pathogens from entering the peri-implant interface and prevent infections. Therefore, the integration of soft-tissue poses a challenge in implant-prosthetic procedures, prompting a focus on the interface between peri-implant soft-tissues and the transmucosal component. The aim of this study was to analyse the effects of sandblasted roughness levels on in vitro soft-tissue healing around dental implant abutments. In parallel, proteomic techniques were applied to study the interaction of these surfaces with human serum proteins to evaluate their potential to promote soft-tissue regeneration. RESULTS: Grade-5 machined titanium discs (MC) underwent sandblasting with alumina particles of two sizes (4 and 8 µm), resulting in two different surface types: MC04 and MC08. Surface morphology and roughness were characterised employing scanning electron microscopy and optical profilometry. Cell adhesion and collagen synthesis, as well as immune responses, were assessed using human gingival fibroblasts (hGF) and macrophages (THP-1), respectively. The profiles of protein adsorption to the surfaces were characterised using proteomics; samples were incubated with human serum, and the adsorbed proteins analysed employing nLC-MS/MS. hGFs exposed to MC04 showed decreased cell area compared to MC, while no differences were found for MC08. hGF collagen synthesis increased after 7 days for MC08. THP-1 macrophages cultured on MC04 and MC08 showed a reduced TNF-α and increased IL-4 secretion. Thus, the sandblasted topography led a reduction in the immune/inflammatory response. One hundred seventy-six distinct proteins adsorbed on the surfaces were identified. Differentially adsorbed proteins were associated with immune response, blood coagulation, angiogenesis, fibrinolysis and tissue regeneration. CONCLUSIONS: Increased roughness through MC08 treatment resulted in increased collagen synthesis in hGF and resulted in a reduction in the surface immune response in human macrophages. These results correlate with the changes in protein adsorption on the surfaces observed through proteomics.

Neddylation orchestrates the complex transcriptional and posttranscriptional program that drives Schwann cell myelination.

Myelination is essential for neuronal function and health. In peripheral nerves, >100 causative mutations have been identified that cause Charcot-Marie-Tooth disease, a disorder that can affect myelin sheaths. Among these, a number of mutations are related to essential targets of the posttranslational modification neddylation, although how these lead to myelin defects is unclear. Here, we demonstrate that inhibiting neddylation leads to a notable absence of peripheral myelin and axonal loss both in developing and regenerating mouse nerves. Our data indicate that neddylation exerts a global influence on the complex transcriptional and posttranscriptional program by simultaneously regulating the expression and function of multiple essential myelination signals, including the master transcription factor EGR2 and the negative regulators c-Jun and Sox2, and inducing global secondary changes in downstream pathways, including the mTOR and YAP/TAZ signaling pathways. This places neddylation as a critical regulator of myelination and delineates the potential pathogenic mechanisms involved in CMT mutations related to neddylation.

SUMOylation controls Hu antigen R posttranscriptional activity in liver cancer.

The posttranslational modification of proteins critically influences many biological processes and is a key mechanism that regulates the function of the RNA-binding protein Hu antigen R (HuR), a hub in liver cancer. Here, we show that HuR is SUMOylated in the tumor sections of patients with hepatocellular carcinoma in contrast to the surrounding tissue, as well as in human cell line and mouse models of the disease. SUMOylation of HuR promotes major cancer hallmarks, namely proliferation and invasion, whereas the absence of HuR SUMOylation results in a senescent phenotype with dysfunctional mitochondria and endoplasmic reticulum. Mechanistically, SUMOylation induces a structural rearrangement of the RNA recognition motifs that modulates HuR binding affinity to its target RNAs, further modifying the transcriptomic profile toward hepatic tumor progression. Overall, SUMOylation constitutes a mechanism of HuR regulation that could be potentially exploited as a therapeutic strategy for liver cancer.

Characterization of preovulatory follicular fluid secretome and its effects on equine oocytes during in vitro maturation.

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.

Biallelic variants in SNUPN cause a limb girdle muscular dystrophy with myofibrillar-like features.

Alterations in RNA-splicing are a molecular hallmark of several neurological diseases, including muscular dystrophies, where mutations in genes involved in RNA metabolism or characterized by alterations in RNA splicing have been described. Here, we present five patients from two unrelated families with a limb-girdle muscular dystrophy (LGMD) phenotype carrying a biallelic variant in SNUPN gene. Snurportin-1, the protein encoded by SNUPN, plays an important role in the nuclear transport of small nuclear ribonucleoproteins (snRNPs), essential components of the spliceosome. We combine deep phenotyping, including clinical features, histopathology and muscle MRI, with functional studies in patient-derived cells and muscle biopsies to demonstrate that variants in SNUPN are the cause of a new type of LGMD according to current definition. Moreover, an in vivo model in Drosophila melanogaster further supports the relevance of Snurportin-1 in muscle. SNUPN patients show a similar phenotype characterized by proximal weakness starting in childhood, restrictive respiratory dysfunction and prominent contractures, although inter-individual variability in terms of severity even in individuals from the same family was found. Muscle biopsy showed myofibrillar-like features consisting of myotilin deposits and Z-disc disorganization. MRI showed predominant impairment of paravertebral, vasti, sartorius, gracilis, peroneal and medial gastrocnemius muscles. Conservation and structural analyses of Snurportin-1 p.Ile309Ser variant suggest an effect in nuclear-cytosol snRNP trafficking. In patient-derived fibroblasts and muscle, cytoplasmic accumulation of snRNP components is observed, while total expression of Snurportin-1 and snRNPs remains unchanged, which demonstrates a functional impact of SNUPN variant in snRNP metabolism. Furthermore, RNA-splicing analysis in patients' muscle showed widespread splicing deregulation, in particular in genes relevant for muscle development and splicing factors that participate in the early steps of spliceosome assembly. In conclusion, we report that SNUPN variants are a new cause of limb girdle muscular dystrophy with specific clinical, histopathological and imaging features, supporting SNUPN as a new gene to be included in genetic testing of myopathies. These results further support the relevance of splicing-related proteins in muscle disorders.

Enhancing the correlation between in vitro and in vivo experiments in dental implant osseointegration: investigating the role of Ca ions.

This study delves into the osteogenic potential of a calcium-ion modified titanium implant surface, unicCa, employing state-of-the-art proteomics techniques both in vitro (utilizing osteoblasts and macrophage cell cultures) and in vivo (in a rabbit condyle model). When human osteoblasts (Hobs) were cultured on unicCa surfaces, they displayed a marked improvement in cell adhesion and differentiation compared to their unmodified counterparts. The proteomic analysis also revealed enrichment in functions associated with cell migration, adhesion, extracellular matrix organization, and proliferation. The analysis also underscored the involvement of key signalling pathways such as PI3K-Akt and mTOR. In the presence of macrophages, unicCa initially exhibited improvement in immune-related functions and calcium channel activities at the outset (1 day), gradually tapering off over time (3 days). Following a 5-day implantation in rabbits, unicCa demonstrated distinctive protein expression profiles compared to unmodified surfaces. The proteomic analysis highlighted shifts in adhesion, immune response, and bone healing-related proteins. unicCa appeared to influence the coagulation cascade and immune regulatory proteins within the implant site. In summary, this study provides a comprehensive proteomic analysis of the unicCa surface, drawing correlations between in vitro and in vivo results. It emphasizes the considerable potential of unicCa surfaces in enhancing osteogenic behavior and immunomodulation. These findings significantly contribute to our understanding of the intricate molecular mechanisms governing the interplay between biomaterials and bone cells, thereby facilitating the development of improved implant surfaces for applications in bone tissue engineering.

Comparative proteomic analysis of the changes in mare milk associated with different lactation stages and management systems.

Mare milk has traditionally been attributed a number of health promoting properties. However, knowledge on its composition and functionality remains scarce, with particularly limited studies on mare milk proteomics. This study deeply characterized mare milk proteome accounting for both caseins and proteins in the whey fraction, also addressing the impact of lactation stage and different management systems. Milk samples from Basque Mountain Horse breed mares belonging to three different farms and three lactation stages were analysed after in-gel and in-solution digestion using nLC-MS/MS. Among the 469 proteins identified, the content of alpha-1 antitrypsin was significantly higher in pasture-based compared to other systems. Moreover, lactation stage significantly affected the content of beta-lactoglobulin II, immunoglobulin-like domain-containing protein, interferon alpha-inducible protein 27, lactotransferrin, polypeptide N-acetylgalactosaminyltransferase, and transforming acidic coiled-coil containing protein 2. This study contributes to the deep characterization of mare milk proteome and provides new insights into the effect of different production factors.

Maintenance of cell wall remodeling and vesicle production are connected in Mycobacterium tuberculosis.

Pathogenic and nonpathogenic mycobacteria secrete extracellular vesicles (EVs) under various conditions. EVs produced by Mycobacterium tuberculosis ( Mtb ) have raised significant interest for their potential in cell communication, nutrient acquisition, and immune evasion. However, the relevance of vesicle secretion during tuberculosis infection remains unknown due to the limited understanding of mycobacterial vesicle biogenesis. We have previously shown that a transposon mutant in the LCP-related gene virR ( virR mut ) manifested a strong attenuated phenotype during experimental macrophage and murine infections, concomitant to enhanced vesicle release. In this study, we aimed to understand the role of VirR in the vesicle production process in Mtb . We employ genetic, transcriptional, proteomics, ultrastructural and biochemical methods to investigate the underlying processes explaining the enhanced vesiculogenesis phenomenon observed in the virR mut . Our results establish that VirR is critical to sustain proper cell permeability via regulation of cell envelope remodeling possibly through the interaction with similar cell envelope proteins, which control the link between peptidoglycan and arabinogalactan. These findings advance our understanding of mycobacterial extracellular vesicle biogenesis and suggest that these set of proteins could be attractive targets for therapeutic intervention.

The impact of glycosylation on the structure, function, and interactions of CD14.

CD14 is an innate immune receptor that senses pathogen-associated molecular patterns, such as lipopolysaccharide, to activate the innate immune response. Although CD14 is known to be glycosylated, detailed understanding about the structural and functional significance of this modification is still missing. Herein, an NMR and MS-based study, assisted by MD simulations, has provided a 3D-structural model of glycosylated CD14. Our results reveal the existence of a key N-glycosylation site at Asn282 that exclusively contains unprocessed oligomannnose N-glycans that perfectly fit the concave cavity of the bent-solenoid shaped protein. This site is not accessible to glycosidases and is fundamental for protein folding and secretion. A second N-site at Asn151 displays mostly complex N-glycans, with the typical terminal epitopes of the host cell-line expression system (i.e. ßGal, α2,3 and α2,6 sialylated ßGal, here), but also particularities, such as the lack of core fucosylation. The glycan at this site points outside the protein surface, resulting in N-glycoforms fully exposed and available for interactions with lectins. In fact, NMR experiments show that galectin-4, proposed as a binder of CD14 on monocytes to induce their differentiation into macrophages-like cells, interacts in vitro with CD14 through the recognition of the terminal glycoepitopes on Asn151. This work provides key information about CD14 glycosylation, which helps to better understand its functional roles and significance. Although protein glycosylation is known to be dynamic and influenced by many factors, some of the features found herein (presence of unprocessed N-glycans and lack of core Fuc) are likely to be protein specific.

Next-Generation Proteomics of Brain Extracellular Vesicles in Schizophrenia Provide New Clues on the Altered Molecular Connectome.

Extracellular vesicles (EVs) are tiny membranous structures that mediate intercellular communication. The role(s) of these vesicles have been widely investigated in the context of neurological diseases; however, their potential implications in the neuropathology subjacent to human psychiatric disorders remain mostly unknown. Here, by using next-generation discovery-driven proteomics, we investigate the potential role(s) of brain EVs (bEVs) in schizophrenia (SZ) by analyzing these vesicles from the three post-mortem anatomical brain regions: the prefrontal cortex (PFC), hippocampus (HC), and caudate (CAU). The results obtained indicate that bEVs from SZ-affected brains contain region-specific proteins that are associated with abnormal GABAergic and glutamatergic transmission. Similarly, these vesicles from the analyzed regions were implicated in synaptic decay, abnormal brain immunity, neuron structural imbalances, and impaired cell homeostasis. Our findings also provide evidence, for the first time, that networks of molecular exchange (involving the PFC, HC, and CAU) are potentially active and mediated by EVs in non-diseased brains. Additionally, these bEV-mediated networks seem to have become partially reversed and largely disrupted in the brains of subjects affected by SZ. Taken as a whole, these results open the door to the uncovering of new biological markers and therapeutic targets, based on the compositions of bEVs, for the benefit of patients affected by SZ and related psychotic disorders.

Müller glial cells located in the peripheral retina are more susceptible to high pressure: implications for glaucoma.

BACKGROUND: Glaucoma, a progressive neurodegenerative disease, is a leading cause of irreversible vision loss worldwide. This study aims to elucidate the critical role of Müller glia (MG) in the context of retinal ganglion cell (RGC) death, particularly focusing on the influence of peripheral MG sensitivity to high pressure (HP). METHODS: Co-cultures of porcine RGCs with MG were isolated from both the central and peripheral regions of pig retinas and subjected to both normal and HP conditions. Mass spectrometry analysis of the MG-conditioned medium was conducted to identify the proteins released by MG under all conditions. RESULTS: Peripheral MG were found to secrete a higher quantity of neuroprotective factors, effectively promoting RGC survival under normal physiological conditions. However, under HP conditions, co-cultures with peripheral MG exhibited impaired RGC survival. Moreover, under HP conditions, peripheral MG significantly upregulated the secretion of proteins associated with apoptosis, oxidative stress, and inflammation. CONCLUSIONS: This study provides robust evidence suggesting the involvement of MG in RGC death in glaucoma, thus paving the way for future therapeutic investigations.

Anti-miR-873-5p improves alcohol-related liver disease by enhancing hepatic deacetylation via SIRT1.

Background & Aims: Current therapies for the treatment of alcohol-related liver disease (ALD) have proven largely ineffective. Patients relapse and the disease progresses even after liver transplantation. Altered epigenetic mechanisms are characteristic of alcohol metabolism given excessive acetate and NAD depletion and play an important role in liver injury. In this regard, novel therapeutic approaches based on epigenetic modulators are increasingly proposed. MicroRNAs, epigenetic modulators acting at the post-transcriptional level, appear to be promising new targets for the treatment of ALD. Methods: MiR-873-5p levels were measured in 23 liver tissue from Patients with ALD, and GNMT levels during ALD were confirmed using expression databases (transcriptome n = 62, proteome n = 68). High-resolution proteomics and metabolomics in mice following the Gao-binge model were used to investigate miR-873-5p expression in ALD. Hepatocytes exposed to 50 mM alcohol for 12 h were used to study toxicity. The effect of anti-miR-873-5p in the treatment outcomes of ALD was investigated. Results: The analysis of human and preclinical ALD samples revealed increased expression of miR-873-5p in the liver. Interestingly, there was an inverse correlation with NNMT, suggesting a novel mechanism for NAD depletion and aberrant acetylation during ALD progression. High-resolution proteomics and metabolomics identified miR-873-5p as a key regulator of NAD metabolism and SIRT1 deacetylase activity. Anti-miR-873-5p reduced NNMT activity, fuelled the NAD salvage pathway, restored the acetylome, and modulated the levels of NF-κB and FXR, two known SIRT1 substrates, thereby protecting the liver from apoptotic and inflammatory processes, and improving bile acid homeostasis. Conclusions: These data indicate that targeting miR-873-5p, a repressor of GNMT previously associated with NAFLD and acetaminophen-induced liver failure. is a novel and attractive approach to treating alcohol-induced hepatoxicity. Impact and implications: The role of miR-873-5p has not been explicitly examined in the progression of ALD, a pathology with no therapeutic options. In this study, inhibiting miR-873-5p exerted hepatoprotective effects against ALD through rescued SIRT1 activity and consequently restored bile acid homeostasis and attenuated the inflammatory response. Targeting hepatic miR-873-5p may represent a novel therapeutic approach for the treatment of ALD.

Using osteogenic medium in the in vitro evaluation of bone biomaterials: Artefacts due to a synergistic effect.

In vitro tests using bone cells to evaluate the osteogenic potential of biomaterials usually employ the osteogenic medium (OM). The lack of correlation frequently reported between in vitro and in vivo studies in bone biomaterials, makes necessary the evaluation of the impact of osteogenic supplements on these results. This study analysed the proteomic profiles of human osteoblasts (HOb) cultured in the media with and without osteogenic agents (ascorbic acid and ß-glycerol phosphate). The cells were incubated for 1 and 7 days, on their own or in contact with Ti. The comparative Perseus analysis identified 2544 proteins whose expression was affected by osteogenic agents. We observed that the OM strongly alters protein expression profiles with a complex impact on multiple pathways associated with adhesion, immunity, oxidative stress, coagulation, angiogenesis and osteogenesis. OM-triggered changes in the HOb intracellular energy production mechanisms, with key roles in osteoblast maturation. HOb cultured with and without Ti showed enrichment in the skeletal system development function due to the OM. However, differentially expressed proteins with key regenerative functions were associated with a synergistic effect of OM and Ti. This synergy, caused by the Ti-OM interaction, could complicate the interpretation of in vitro results, highlighting the need to analyse this phenomenon in biomaterial testing.