Abstract Study question Could Extracellular Vesicles be employed as a new tool to characterize the male fertility potential? Summary answer Extracellular Vesicles cargo is involved in mechanisms strictly linked to reproductive processes. What is known already Extracellular vesicle contain a plethora of bioactive molecules, such as lipids, DNA and RNA, mainly proteins, which are delivered to target cells to elicit a specific response. This multivarious EV functionality is accomplished by the transfer of their content to the sperm cell, followed by either downregulating or upregulating a series of proteins expressed throughout different sites of sperm cell structure. Despite various studies exploring the proteomic composition of these vesicles, our comprehension of their specific influence on sperm maturation, motility, and the attainment of fertilizing capacity is currently restricted. Study design, size, duration Semen samples were collected from subjects undergoing semen analysis at the UOSA PMA, Siena University Hospital, Italy. All participants were of Caucasian origin and signed a written informed consent to be enrolled in this study. The median age of the patients was 36.7 years (range: 20-45 years); the BMI ranged between 18 and 25 (normal weight). Patients were divided on three groups based on seminal parameters (WHO, 2021) into: Normozoospermic (NORMO); OligoAsthenoTeratozoospermic (OAT); Azoospermic (AZO). Participants/materials, setting, methods EVs from human semen samples were isolated following an in-house optimized protocol using a chemical precipitation reagent ev-GAG®. Samples were subjected to Gel-free and gel-based proteomic analysis. Comprehensive analysis of the vesicles involved acquiring a detailed proteomic profile through the use of 2-D electrophoresis and mass spectrometry. Subsequent inquiries included functional Omics analysis, utilizing bioinformatic tools like Metacore and STRING. We have combined and compared results from several proteomic approaches, both gel-free and gel-based ones. Main results and the role of chance A total of 1133 proteins were identified in NORMO EV samples from the Gel-free approach by LC-MS/MS. These proteins are involved in numerous functions important for reproduction activity. GO enrichment analysis identified involvement of pathways such as cytoskeleton regulation, cell adhesion, cell cycle, development and neurophysiology, proteostasis mechanisms, immune response, and inflammation, reproduction as progesterone signalling and spermatogenesis, protein translation, and signal transduction. 2DE analysis extrapolated a total of 80 differentially abundant spots among the three conditions. Biological Process analysis report that highly abundant proteins in NORMO EVs were significantly involved in chaperone-mediated protein complex assembly, autophagy and in protein folding, in negative regulation of intrinsic apoptotic signaling pathway, in positive regulation of protein- containing complex assembly, positive regulation of tau-protein kinase activity, in regulation of apoptotic processes, in protein stabilization and in regulation of protein ubiquitination. Biological processes related to the three group of proteins suggested that in OAT and even more in AZO EVs, the altered molecular cargo is involved in mechanism strictly linked to reproductive processes, protein folding, inflammation, immune responses, proteolysis, signal transduction. All these mechanisms, in healthy conditions, cooperate for a successful pregnancy. Limitations, reasons for caution To enhance characterization, it is essential to conduct a confirmatory investigation focused on the distinct group of different EVs such as epididymosomes and prostasomes, aiming to offer more precise insight. Wider implications of the findings EVs released along the male reproductive tract, specifically in seminal fluid, have been shown to play a role in sperm maturation. They have the potential to serve as an attractive diagnostic and therapeutic tool in the realm of human reproduction, particularly for issues related to male fertility and infertility. Trial registration number NA

Abstract Study question Could Extracellular Vesicles such as Epididymosomes and Prostasomes be employed as a new tool to characterize the male fertility potential? Summary answer Extracellular vesicles (EVs) content represents a new approach to the discovery of novel molecular mechanisms involved in sperm cell maturation and acquisition of fertilizing ability. What is known already Cells throughout the reproductive tract release EVs that are sorted by the endosomal compartment. These vesicles attract great attention because they contain lipids, proteins, nucleic acids that have been shown to be transferred to maturing sperm cells. EVs in seminal plasma, such as epididymosomes and prostasomes, contribute about 3% of the total associated seminal plasma proteins and are recognized as being responsible for sperm maturation, positively influencing actual fertilization. Several studies have documented the proteomic profile of these vesicles, but little is known about their specific contribution to sperm maturation, motility and acquisition of fertilizing capacity. Study design, size, duration 48 Caucasian males underwent semen evaluation at the IVF Unit, Siena University Hospital, from October 2020 to November 2021. According to seminal parameters (WHO,2010), they were divided into Normozoospermic (N; n = 18); OligoAsthenoTeratozoospermic (OAT; n = 15); Azoospermic (A; n = 15). We excluded patients with an intake of spermiotoxic drugs, smoking, alcohol, or drugs abuse. The mean age was 33 years; the BMI ranged between 18-25. All participants signed a written informed consent, approved by the ethics committee. Participants/materials, setting, methods EVs from seminal plasma were isolated by an in-house modified EvGAG®-polymer precipitation-based protocol and characterized for their size and morphology by Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), respectively. Full proteomic profiling of these vesicles was obtained by 2-D electrophoresis and mass spectrometry, and further investigations by functional Omics analysis by bioinformatic tools (as. Metacore, STRING). Gene expression was evaluated by digital PCR (ddPCR). Main results and the role of chance The present work highlights the impact of EVs on male fertility due to their ability to transport selected molecules to maturing sperm cells, thus providing a specific proteomic and transcriptomic profile based on their fertilizing capacity. Proteomic profiling of OAT and A vs N samples provided crucial information regarding differential protein expression, thus obtaining information on the correlation between the encapsulated content of EVs and sperm parameters. The ddPCR analysis of specific genes encoding for proteins expressed differently among patients confirmed that EVs may not only be potential biomarkers for male infertility, but at the same time contribute as an important regulator of specific pathways concerning sperm cell modification through the male reproductive tract. Genes involved in sperm-egg binding and zona pellucida interactions were overexpressed in N compared to OAT and A. Congruent results were obtained for genes involved in sperm cell metabolism. Meanwhile, regarding the acquisition of motility, some genes were found overexpressed in the OAT groups compared to N, indicating that some proteins delivered by EVs have a negative impact on sperm cell motility and function. Limitations, reasons for caution This study represents a preliminary experiment. A confirmatory study on an enlarged number of samples is needed to provide more statistical strength by reducing the effect of inter-sample variability. Wider implications of the findings Seminal extracellular vesicles secreted along the male reproductive tract have been demonstrated to be involved in the process of sperm maturation and could represent a new appealing therapeutic and diagnostic tool in the field of human reproduction for male fertility/infertility. Trial registration number Not applicable

BACKGROUND: Mepolizumab and benralizumab are humanized IgG antibodies directed against IL-5 signaling, acting on eosinophils count and activity, approved for the treatment of severe eosinophilic asthma (SEA). Aside clinical efficacy, little is known about their biomolecular and metabolic effects. AIM: to investigate and compare the serum proteomic profiles variations of SEA patients treated with anti-IL5 drugs METHODS: we prospectively performed differential proteomic analysis on serum samples of healthy controls (HC) and SEA patients at baseline (T0), after 1 month (T1) and 6 months (T6) of treatment with mepolizumab and benralizumab. Statistical analysis by PCA and heatmap analyses were performed and identified proteins were used for enrichment analysis by MetaCore software. RESULTS: At T6, we observed that serum protein profile of both SEA subgroups was similar to that of HC. Identified proteins were mainly related to lipid metabolism and transport, blood coagulation and antioxidant processes. APOAI, APOC-II and APOC-III were up-regulated only in benralizumab patients, while plasminogen and ceruloplasmin were up-regulated at T6 of both treatment subgroups. CONCLUSIONS: mepolizumab and benralizumab were able to remodulate serum protein profile of SEA patients, which appeared similar to HC after 6 months of therapy. Our results suggest that biomolecular effects of anti-IL5 drugs goes beyond eosinophils9 depletion.

Mepolizumab and Benralizumab are biological drugs for severe asthma patients able to reduce moderate-tosevere exacerbation rate (peripheral eosinophilial % mepolizumab 1.6 +/- 1.2; benralizumab 0; p < 0.0001), improving the quality of life and lung function parameters (FEV1%: mepolizumab 87.1 +/- 21.5; benralizumab 89.7 +/- 15, p < 0.04). Here we report a preliminary redox proteomic study highlighting the level of oxidative burst present in serum from patients before and after one month of both treatments. Our results highlighted apolipoprotein A1 oxidation after Mepolizumab treatment, that could be related to HDL functionality and could represent a potential biomarker for the treatment. On the other hand, after one month of Benralizumab we detected higher oxidation levels of ceruloplasmin and transthyretin, considered an important oxidative stress biomarker which action help to maintain redox homeostasis.

Abstract Study question Do exosomes from seminal plasma have a role in male fertility? Summary answer Exosomes isolated from seminal plasma have a pivotal role during spermatogenesis and sperm maturation and may represent eligible biomarkers for male fertility/infertility. What is known already During their journey along the male reproductive tract, exosomes contained in seminal fluid are involved in the transfer of several molecules to the maturing sperm. Exosomes are extracellular vesicles (EVs) released by all the cells; they carry a cargo of nucleic acids, proteins and lipids. In the male genital tract, they are released at various levels and their composition differs between men of proven fertility and infertile male patients. Recent studies reported the proteomic profile of exosomes, revealing the presence of several proteins with a well know role in sperm maturation and fertilizing ability acquiring. Study design, size, duration This prospective study consisted of 36 Caucasian men; according to seminal parameters (WHO 2010) they were divided in normozoospermic (N; n = 12), oligoasthenoteratozoospermic (OAT: n = 12) and azoospermic (A; n = 12). Semen samples were collected between October 2020 and January 2021 at the Assisted Reproductive Unit, Siena University Hospital (Italy) after institutional ethical approval and signed written consent from all the participants. Participants/materials, setting, methods Ejaculated sperm were analyzed according to WHO–2010 criteria and divided into the three groups: N, OAT and A. Exosomes were isolated by an in-house modified ExoGAG®-polymer precipitation-based protocol and characterized for size and ultrastructure by Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The exosomal proteins were extracted and analyzed by 2D-electrophoresis and the identified profiles were examined by applying bioinformatic tools. The expression of selected genes was evaluated by digital droplets PCR (ddPCR). Main results and the role of chance The present work is readily providing an improvement of the standard ExoGAG® protocol and underlines its advantages over more conventional EVs isolation protocols used to date for recovery from seminal fluid: the number of recovered EVs and their size were finely included in the range of exosomes. This isolation protocol provides samples suitable for proteomic analyses, representing the first 2D-electrophoresis reference map of exosome-pay loaded proteins in N respect to OAT/A groups and providing an innovative and comprehensive functional overview of its proteins. Moreover, the STRING protein-protein interaction analysis revealed the deregulation of specific pathways (e.g. signaling proteins, chromatin packaging and/or remodeling, protein folding and apoptosis) in A and OAT in comparison with N group. Gene expression by ddPCR analysis highlighted that most of the analyzed genes are modulated in according to seminal parameters, in particular: GAPDHS (Glyceraldehyde–3-Phosphate Dehydrogenase, Spermatogenic); SPAM1 (Sperm Adhesion Molecule–1) encoding a members of hyaluronidase family; ADAM2 (ADAM Metallopeptidase Domain–2) that plays an important role in sperm-egg interactions; CRISP1,2,3 (Cysteine Rich Secretory Protein 1,2,3) expressed in the epididymis and secreted into the epididymal lumen; CLGN (Calmegin) encoding a testis-specific chaperone protein and PGK2 (Phosphoglycerate Kinase–2) expressed in the later stages of spermatogenesis. Limitations, reasons for caution This study represents a preliminary experiment. We suggest further comparative studies in larger study cohorts. Wider implications of the findings: This pilot study, demonstrating the unique proteomic and transcriptomic pattern of exosomes in N/OAT/A groups, supports the importance of exosomes in sperm production and maturation. This methodological set-up is expected to open new ways for advancement in the use of exosomes as fertility biomarkers, making possible personalized approaches in ART. Trial registration number Not applicable

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease (ILD) sharing various genetic, molecular and cell processes with lung cancer (LC). Nintedanib, a tyrosine-kinase inhibitor, was first developed as an anticancer drug because it suppresses angiogenesis. It was then recognized as an anti-fibrotic agent and approved for the treatment of IPF. On the basis of in vitro studies of the drug, we performed a bioinformatic analysis of all targeted tyrosine kinases with the aim of highlighting common molecular pathways modulated by the drug in LC and IPF. The results show that MAPK, PI3K/AKT, JAK/STAT, TGF-β, VEGF and WNT/β-catenin signalling are the main molecular pathways modulated by the drug. Interestingly, these pathways include that controlled by intercellular adherence junctions (compromised in LC and IPF), and by central carbon metabolism (usually studied more in relation to the pathogenesis of cancer than IPF). On the basis of the tyrosine kinases considered, our bioinformatic analysis highlighted five microRNAs influencing VEGF-A signalling and epithelial to mesenchymal transition mechanisms. Comparison of our results with those of previous studies highlighted correlations between microRNAs and the development of LC and IPF.

Background The pathogenetic and regulatory roles of natural killer (NK) and natural killer T-like cells in interstitial lung diseases (ILDs), fibrotic and granulomatous of unknown etiology are unclear. Objectives Here we investigated NK and NKT-like cells in peripheral blood (PB) and Bronchoalveolar lavage (BAL) from patients with ILDs. Method 190 patients (94 male mean age 61 ± 14.3 years) and 8 controls undergoing bronchoscopy for ILD diagnostic work-up were enrolled consecutively; 115 patients sarcoidosis, 24 chronic fibrotic hypersensitivity pneumonitis and 43 patients other ILDs [32 idiopathic pulmonary fibrosis (IPF) and 11 non-specific interstitial pneumonia (NSIP)]. PB and BAL were processed by flow cytometry using monoclonal antibodies to differentiate NK and NKT-like cells. Results NK% in BAL was significantly different among ILDs (p = 0.02). Lower NK% was observed in BAL from sarcoidosis than other ILDs (p < 0.05). Similar findings were observed for NKT-like, whereas no differences were found for PB NK%. Difference of NK% was observed between BAL and PB in all groups (p < 0.001). Sarcoidosis patients reported the best area under the curve for NKT-like (AUC = 0.678, p = 0.0015) and NK cells (AUC = 0.61, p = 0.001). In the IPF-NSIP subgroup, NK% cell was inversely correlated with FVC% (r = − 0.34, p = 0.03) and DLCO% (r = − 0.47, p = 0.0044). Conclusions NK and NKT-like were expressed differently in BAL from patients with different ILD and were significantly depleted in sarcoidosis respect to other ILDs. This suggests that these cells may play a protective role in the pathogenesis of sarcoidosis.

One of the crucial and unsolved problems of the airborne carbon nanoparticles is the role played by the adsorbed environmental pollutants on their toxicological effect. Indeed, in the urban areas, the carbon nanoparticles usually adsorb some atmospheric contaminants, whose one of the leading representatives is the benzo(α)pyrene. Herein, we used the proteomics to investigate the alteration of toxicological pathways due to the carbon nanopowder-benzo(α)pyrene complex in comparison with the two contaminants administered alone on human skin-derived fibroblasts (hSDFs) exposed for 8 days in semi-static conditions. The preliminary confocal microscopy observations highlighted that carbon-nanopowder was able to pass through the cell membranes and accumulate into the cytoplasm both when administered alone and with the adsorbed benzo(α)pyrene. Proteomics revealed that the effect of carbon nanopowder-benzo(α)pyrene complex seems to be related to a new toxicological behavior instead of simple additive or synergistic effects. In detail, the cellular pathways modulated by the complex were mainly related to energy shift (glycolysis and pentose phosphate pathway), apoptosis, stress response and cellular trafficking.

Carbon-based nanoparticles (CBNs) are largely distributed worldwide due to fossil fuel combustion and their presence in many consumer products. In addition to their proven toxicological effects in several biological models, attention in recent years has focussed on the role played by CBNs as Trojan-horse carriers for adsorbed environmental pollutants. This role has not been conclusively determined to date because CBNs can decrease the bioavailability of contaminants or represent an additional source of intake. Herein, we evaluated the intake, transport and distribution of one of the carbon-based powders, the so-called carbon nanopowder (CNPW), and benzo(α)pyrene, when administered alone and in co-exposure to Danio rerio embryos. Data obtained by means of advanced microscopic techniques illustrated that the "particle-specific" effect induced a modification in the accumulation of benzo(α)pyrene, which is forced to follow the distribution of the physical pollutant instead of its natural bioaccumulation. The combined results from functional proteomics and gene transcription analysis highlighted the different biochemical pathways involved in the action of the two different contaminants administered alone and when bound together. In particular, we observed a clear change in several proteins involved in the homeostatic response to hypoxia only after exposure to the CNPW or co-exposure to the mixture, whereas exposure to benzo(α)pyrene alone mainly modified structural proteins. The entire dataset suggested a Trojan-horse mechanism involved in the biological impacts on Danio rerio embryos especially due to different bioaccumulation pathways and cellular targets.

Over the last years the zebrafish imposed itself as a powerful model to study skeletal diseases, but a limit to its use is the poor characterization of collagen type I, the most abundant protein in bone and skin. In tetrapods collagen type I is a trimer mainly composed of two α1 chains and one α2 chain, encoded by COL1A1 and COL1A2 genes, respectively. In contrast, in zebrafish three type I collagen genes exist , col1a1a , col1a1b and col1a2 coding for α1(I), α3(I) and α2(I) chains. During embryonic and larval development the three collagen type I genes showed a similar spatio-temporal expression pattern, indicating their co-regulation and interdependence at these stages. In both embryonic and adult tissues, the presence of the three α(I) chains was demonstrated, although in embryos α1(I) was present in two distinct glycosylated states, suggesting a developmental-specific collagen composition. Even though in adult bone, skin and scales equal amounts of α1(I), α3(I) and α2(I) chains are present, the presented data suggest a tissue-specific stoichiometry and/or post-translational modification status for collagen type I. In conclusion, this data will be useful to properly interpret results and insights gained from zebrafish models of skeletal diseases.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by progressive deterioration of the alveolar integrity. Among IPF identified phenotypes, that of familial (f-)IPF is usually associated with several gene mutations which are seldom observed in sporadic (s-)IPF. This study aimed at investigating the molecular patterns and variability in f-IPF and s-IPF patients through a differential proteomic analysis. Protein patterns of bronchoalveolar lavage fluid (BALF) samples from 10 familial and 17 sporadic IPF patients were compared using 2D electrophoresis and mass spectrometry. Principal component analysis (PCA) was applied to proteomic data and an enrichment analysis was also performed to characterize specific pathogenic mechanisms and to identify potential biomarkers. BALF samples from f-IPF showed 87 protein spots differentially expressed than those from s-IPF samples; once identified, these spots revealed 22 unique proteins. The functional analysis showed that the endothelial reticulum stress probably plays a central pathogenetic role in f-IPF with an up-regulation of proteins involved in wounding and immune responses, coagulation system, and ion homeostasis. Up-regulated proteins in the s-IPF group were those involved in the oxidative stress response. PCA analysis of differentially expressed proteins clearly distinguished f-IPF from s-IPF patients, and in agreement with radiological and histological patterns, pointed out a higher heterogeneity in f-IPF than s-IPF samples. The 'Slit/Robo signaling', 'clathrin-coated vesicle' and 'cytoskeleton remodelling', were extrapolated by 'pathways analysis' and the results of 'diseases (by biomarkers)' highlighted a 'connective tissue and autoimmune disease', two aspects of increasing interest in IPF.

Pulmonary sarcoidosis (Sar) is an idiopathic disease histologically typified by non-caseating epitheliod cell sarcoid granulomas. A cohort of 37 Sar patients with chronic persistent pulmonary disease was described in this study. BAL protein profiles from 9 of these Sar patients were compared with those from 8 smoker (SC) and 10 no-smoker controls (NSC) by proteomic approach. Principal Component Analysis was performed to clusterize the samples in the corresponding conditions highlighting a differential pattern profiles primarily in Sar than SC. Spot identification reveals thirty-four unique proteins involved in lipid, mineral, and vitamin Dmetabolism, and immuneregulation of macrophage function. Enrichment analysis has been elaborated by MetaCore, revealing 14-3-3ε, α1-antitrypsin, GSTP1, and ApoA1 as "central hubs". Process Network as well as Pathway Maps underline proteins involved in immune response and inflammation induced by complement system, innate inflammatory response and IL-6signalling. Disease Biomarker Network highlights Tuberculosis and COPD as pathologies that share biomarkers with sarcoidosis. In conclusion, Sar protein expression profile seems more similar to that of NSC than SC, conversely to other ILDs. Moreover, Disease Biomarker Network revealed several common features between Sar and TB, exhorting to orientate the future proteomics investigations also in comparative BALF analysis of Sar and TB.

Human follicular fluid (HFF) has been proven to contain biologically active molecules and proteins that may affect follicle growth and oocyte fertilization. Based on this concept, HFF proteomic characterization is having a significant impact in the delineation of a biomarkers' profile for oocyte quality estimation and, maybe, for in vitro fertilization (IVF) success improvement.Follicular fluid is characterized by a vast protein complexity and a broad dynamic range of protein abundances that hinder its analysis. In this study we determined a proper solubilization and resolution method of HFF in 2-DE, minimizing sample manipulation, protein loss, and experimental artifacts. According to our methodology some low-abundance proteins were detected and identified by MS. Identified proteins were then functionally cross-linked by a pathway analysis. The generated path highlighted the occurrence in HFF of a tight functional-network in which effectors and inhibitors control and balance a space- and time-dependent induction/inhibition of inflammation, coagulation, and ECM degradation/remodeling. Such fine modulation of enzymatic activities exerts a fundamental role in follicle development and in oocyte competence acquiring. Alpha-1-antitypsin resulted in the core protein of the delineated net and we interestingly detected its differential incidence in FF and serum from two small cohorts of patients who underwent IVF.Biological significanceHuman ovarian follicular fluid (HFF) is the in vivo microenvironment for oocyte during folliculogenesis. It contains biologically active molecules that may affect oocyte quality, fertilization, and embryo development. HIT is also one of the most abundant "waste product" in assisted reproduction. This makes HFF a readily accessible source of biomolecules for competence evaluation of collected oocytes. The methodological improvement we obtained in proteomics characterization of HFF lead to a wide overview on the functional correlation existing between several fluid components and on how their aberrant occurrence/activity may affect oocyte quality and ovulation. This article is part of a Special Issue entitled: From Genome to Proteome: Open Innovations. (C) 2013 Elsevier B.V. All rights reserved.

Bronchoalveolar lavage fluid of patients with four interstitial lung diseases (sarcoidosis, idiopathic pulmonary fibrosis, pulmonary Langerhans cell histiocytosis, fibrosis associated to systemic sclerosis) and smoker and non smoker control subjects were compared in a proteomic study. Principal component analysis was used to statistically verify the association between differentially expressed proteins and the conditions analyzed. Pathway and functional analysis by MetaCore and DAVID software revealed possible regulatory factors involved in specific "process networks" like regulation of stress and inflammatory responses. Immune response by alternative complement pathways, protein folding, Slit-Robo signaling and blood coagulation were "pathway maps" possibly associated with interstitial lung diseases pathogenesis. Four interesting proteins plastin 2, annexin A3, 14-3-3 epsilon and 510A6 (calcyclin) were validated by Western blot analysis. In conclusion, we identified proteins that could be directly or indirectly linked to the pathophysiology of the different interstitial lung diseases. Multivariate analysis allowed us to classify samples in groups corresponding to the different conditions analyzed and based on their differential protein expression profiles. Finally, functional and pathway analysis defined the potential function and relations among identified proteins, including low abundance molecules present in the MetaCore database.Biological significanceThis is the first study where different interstitial lung diseases such as sarcoidosis, idiopathic pulmonary fibrosis, pulmonary Langerhans cell histiocytosis, fibrosis associated to systemic sclerosis and smoker and non smoker control subjects were compared in a proteomic study to highlight their common pathways. We decided to report not only principal component analysis, used to statistically verify the association between differentially expressed proteins and the conditions analyzed, but also functional analysis general results, considering all differential proteins potentially involved in these conditions, to speculate about possible common pathogenetic pathways involved in fibrotic lung damage. (C) 2013 Elsevier B.V. All rights reserved.