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Exosomes are tiny membrane-enveloped vesicles of endosomal origin, typically 40-120nm in diameter, produced by most cells in both normal and pathological situations. These exosomes can be isolated from all biofluids, including urine. In this context, many researchers have focused on the analysis of urinary exosomes because urine can be collected in large quantities, regularly, and with minimal effort. Exosomes contain phospholipids, cholesterol, proteins, glycoconjugates, nucleic acids, and metabolites. Because all organs and tissues produce exosomes, their molecular cargo can provide first-hand information about the physiological and biological state of the site of origin. Many potential disease biomarker candidates have already been identified in urinary exosomes. In this chapter, we performed a bibliometric analysis of the keywords "exosome(s)" and "urine" to identify related terms, diseases and molecular/biological processes, and other related terms. This yielded interesting results suggesting that exosomes in urine may play a role in the pathogenesis of various diseases. Moreover, this chapter discusses exosomes isolation and characterization methodologies and highlight the importance of urinary exosomes and their role in the diagnosis, prognosis and therapy of various diseases. We offer a bibliometric approach and an in-depth analysis on several exosomes' isolation techniques, diagnostic potential for urogenital and specific non-urogenital diseases, as well as an overview of miRNAs significance on urinary exosomes, conferring a more complete status to this review, something that was still lacking in the current literature.

Exosomes are involved in a wide variety of biochemical processes in human body homeostasis. Exosomes also provide important information regarding communications among several organ systems. Additionally, they can serve as molecular vehicles to deliver drugs. Therefore, exosomes have received wide attention in current biomedical research for unraveling pathogenic mechanisms of diseases, searching for novel biomarkers, and discovering new drugs. This paper reviews and discusses the significance of urinary exosomes for a better understanding of human disease pathophysiology and their potential use as therapeutic targets. Isolation methods of exosomes and the latest technological advances are also discussed. Furthermore, novel urinary exosomal biomarkers are highlighted with special emphasis on their clinical applicability (particularly sensitivity, specificity, reliability, and other aspects). Finally, future trends for this field are analyzed and our perspectives are provided.

Background: Exosome research is a current trend in functional proteomics as it provides im-portant data on the pathophysiology and pathogenesis of diseases. The scientific outputs re-garding these topics often only approach disease-protein/peptide/exosome or mecha-nism-protein/peptide/exosome association. Approaching all three aspects could be the key to a better understanding of the pathophysiology and uncovering novel biomarkers for urogenital diseases. The focus of this work is to study exosome datasets to understand the possible role of underlying proteins in disease manifestation. We also attempt to link 4 different diseases that affect renal functions and are genetically inherited. Methods: For this purpose, the existing literature is consulted to understand the importance of exosomes in disease prediction, diagnosis and therapy. Available biotechnological methods of exosome analysis and the tools of proteomic analysis, data mining and visualization are dis-cussed. The database PRIDE is selected to query the information of several datasets related to urinary exosome analysis. Results: We have obtained a list of 19 proteins/genes involved in the mentioned diseases. On this list, we found a proteomic fingerprint consisting of Rab-7a, PDCD6, and CDC42, among others, and we are exploring their biological significance and underlying processes. Conclusion: APOA1, CD59, CD9, IGHG1, RAB7A, RAP1A, SEMG1 and SEMG2 are common in four urogenital diseases, and are involved in interactions with podosomes and endosomes, re-modeling of chylomicrons, regulation of interleukin production, regulation of endopeptidase activity, and establishment of apical/basal polarity of epithelial cells.

This review focuses on discussing key mechanisms in disease pathogenesis mediated by the protein post-translational modification citrullination. These processes are discussed in depth in the context of complex diseases such as rheumatoid arthritis, cancer, central nervous system disorders, and cardiovascular disease. Additionally, a critical evaluation of challenges in laboratory detection of citrullination sites is also outlined. In this context, the role of mass spectrometry is discussed with a focus on contemporary techniques that offer promising options to detect the exact site of protein citrullination. Novel methods described in the paper have the potential to detect and quantify the occurrence of post-translational modification sites for diagnosis and therapeutic purposes with a high degree of specificity and sensitivity. Furthermore, they offer a much faster performance than traditional techniques making them ideal for large-scale experimentation.

Several anticancer agents have been associated with cardiac toxic effects. The currently proposed mechanisms to explain cardiotoxicity differ among anticancer agents, but in fact, the specific modulation is not completely elucidated. Thus, this systematic review aims to provide an integrative perspective of the molecular mechanisms underlying the toxicity of anticancer agents on heart muscle while using a high-throughput technology, mass spectrometry (MS)-based proteomics. A literature search using PubMed database led to the selection of 27 studies, of which 13 reported results exclusively on animal models, 13 on cardiomyocyte-derived cell lines and only one included both animal and a cardiomyocyte line. The reported anticancer agents were the proteasome inhibitor carfilzomib, the anthracyclines daunorubicin, doxorubicin, epirubicin and idarubicin, the antimicrotubule agent docetaxel, the alkylating agent melphalan, the anthracenedione mitoxantrone, the tyrosine kinase inhibitors (TKIs) erlotinib, lapatinib, sorafenib and sunitinib, and the monoclonal antibody trastuzumab. Regarding the MS-based proteomic approaches, electrophoretic separation using two-dimensional (2D) gels coupled with tandem MS (MS/MS) and liquid chromatography-MS/MS (LC-MS/MS) were the most common. Overall, the studies highlighted 1826 differentially expressed proteins across 116 biological processes. Most of them were grouped in larger processes and critically analyzed in the present review. The selection of studies using proteomics on heart muscle allowed to obtain information about the anticancer therapy-induced modulation of numerous proteins in this tissue and to establish connections that have been disregarded in other studies. This systematic review provides interesting points for a comprehensive understanding of the cellular cardiotoxicity mechanisms of different anticancer drugs.

SIGNIFICANCE Infertility is a major global health problem, with nearly half of the cases being associated with male factors. Although reactive oxygen species (ROS) are crucial for sperm cells capacitation, an imbalance between ROS production and antioxidants can lead to oxidative stress that can impair sperm function. Indeed, high semen ROS levels are reported in 30-80% of infertile men. Recent Advances: Male oxidative stress infertility is an uprising classification for idiopathic infertility. Proteomics approaches, including quantitative mass spectrometry (MS)-based proteomics are being utilized to explore the molecular mechanisms associated with oxidative stress in male infertility. CRITICAL ISSUES In this review, proteome data were collected from articles available on PubMed centered on MS-based proteomics studies, performed in seminal plasma and sperm cells samples, and enrolling men with impaired semen parameters. The bioinformatics analysis of proteome data with Cytoscape (ClueGO+CluePedia) and STRING tools allowed the identification of the biological processes more prevalent in asthenozoospermia, with focus on the ones related to oxidative stress. FUTURE DIRECTIONS The identification of the antioxidant proteins in seminal plasma and sperm cells that can protect sperm cells from oxidative stress is crucial not only for a better understanding of the molecular mechanisms associated with male infertility but especially to guide new therapeutic possibilities.

INTRODUCTION With available genomic data and related information, it is becoming possible to better highlight mutations or genomic alterations associated with a particular disease or disorder. The advent of high-throughput sequencing technologies has greatly advanced diagnostics, prognostics, and drug development. AREAS COVERED Peptidomics and proteogenomics are the two post-genomic technologies that enable the simultaneous study of peptides and proteins/transcripts/genes. Both technologies add a remarkably large amount of data to the pool of information on various peptides associated with gene mutations or genome remodeling. Literature search was performed in the PubMed database and is up to date. EXPERT OPINION This article lists various techniques used for peptidomic and proteogenomic analyses. It also explains various bioinformatics workflows developed to understand differentially expressed peptides/proteins and their role in disease pathogenesis. Their role in deciphering disease pathways, cancer research, and biomarker discovery using biofluids is highlighted. Finally, the challenges and future requirements to overcome the current limitations for their effective clinical use are also discussed.

Recently, proteomics has emerged as an important tool for understanding biological systems, protein–protein interactions, and networks that ultimately lead to a deeper understanding of the underlying mechanisms of certain diseases. More recently, the study of secretomes, a type of proteomics, has also been highlighted as a potential next step in the field of diagnosis/prognosis. The secretome is the set of proteins expressed by an organism and secreted into the extracellular space, comprising 13–20% of all proteins. Since almost all, if not all, organs produce secretomes, this means that it is possible to study secretomes and trace these proteins back to their origin, supporting the idea that this could indeed be very important in diagnosing certain diseases. This is often combined with techniques such as mass spectrometry to measure the secretome of, for example, a particular tissue, and bioinformatics tools and databases to give us an idea of what to expect (prediction). In this paper, we will give a general overview of this world, but with a focus on the new bioinformatics tools and databases, their advantages and disadvantages, as well as a deeper look at isolation systems for proteomes, specifically salivary secretomes. Indeed, the salivary secretome represents a valuable new tool capable of providing insights into immunopathology and potentially aiding in diagnostics. Furthermore, we will explore applications of these methods and give an idea of what the future holds for such promising techniques: Salivary secretome in conjunction with bioinformatics tools/databases in the diagnosis of diseases (such as diabetes, Sjogren’s syndrome, and cardiovascular disease).

Introduction Peptidomics is an emerging field of omics sciences using advanced isolation, analysis, and computational techniques that enable qualitative and quantitative analyses of various peptides in biological samples. Peptides can act as useful biomarkers and as therapeutic molecules for diseases. Areas covered The use of therapeutic peptides can be predicted quickly and efficiently using data-driven computational methods, particularly artificial intelligence (AI) approach. Various AI approaches are useful for peptide-based drug discovery, such as support vector machine, random forest, extremely randomized trees, and other more recently developed deep learning methods. AI methods are relatively new to the development of peptide-based therapies, but these techniques already become essential tools in protein science by dissecting novel therapeutic peptides and their functions (Figure 1).[Figure: see text]. Expert opinion Researchers have shown that AI models can facilitate the development of peptidomics and selective peptide therapies in the field of peptide science. Biopeptide prediction is important for the discovery and development of successful peptide-based drugs. Due to their ability to predict therapeutic roles based on sequence details, many AI-dependent prediction tools have been developed (Figure 1).

Simple Summary Acquired resistance to antiestrogenic therapy remains the major obstacle to curing luminal subtype breast cancer. While current treatment in acquired endocrine-resistant settings includes combined therapy with receptor tyrosine kinase or cyclin-dependent kinase inhibitors, progression to incurable disease remains possible. In recent years, the antioxidant system and the protein quality control network have been associated with the enhanced resistance of breast cancer cells to hormonal therapy. In this work, we raise the hypothesis that antiestrogen treatment induces the accumulation of protein aggregates in sensitive cells, which in turn could hinder the activation of survival pathways. We present evidence concerning a novel way to identify antiestrogen response and disclose a novel protein, RTBC, that controls acquired antiestrogen resistance. This work opens a new avenue for research towards finding breast cancer prognostic markers and therapeutic targets, where the identification of proteins prone to aggregate could help to identify antiestrogen response and understand mechanisms of disease. Abstract The protein quality control network, including autophagy, the proteasome and the unfolded protein response (UPR), is triggered by stress and is overactive in acquired antiestrogen therapy resistance. We show for the first time that the aggresome load correlates with apoptosis and is increased in antiestrogen-sensitive cells compared to endocrine-resistant variants. LC-MS/MS analysis of the aggregated proteins obtained after 4OH-tamoxifen and Fulvestrant treatment identified proteins with essential function in protein quality control in antiestrogen-sensitive cells, but not in resistant variants. These include the UPR modulators RTCB and PDIA6, as well as many proteasome proteins such as PSMC2 and PSMD11. RTCB is a tRNA and XBP1 ligase and its aggregation induced by antiestrogens correlated with impaired XBP1s expression in sensitive cells. Knock down of RTCB was sufficient to restore sensitivity to tamoxifen in endocrine-resistant cells and increased the formation of aggresomes, leading to apoptotic cell death. Analysis of primary human breast cancer samples and their metastases appearing after endocrine treatment showed that RTCB is only localized to aggresomes in the primary tumors, while total aggresomes, including aggregated RTCB, were significantly reduced in the metastases. Therefore, different protein aggregation patterns may indicate loss of function of essential proteins resulting in enhanced protein aggregation that can be used to identify antiestrogen-resistant breast cancer cells and improve the response to antiestrogenic therapy.

Native biofluid peptides offer important information about diseases, holding promise as biomarkers. Particularly, the non-invasive nature of urine sampling, and its high peptide concentration, make urine peptidomics a useful strategy to study the pathogenesis of renal conditions. Moreover, the high number of detectable peptides as well as their specificity set the ground for the expansion of urine peptidomics to the identification of surrogate biomarkers for extra-renal diseases. Peptidomics further allows the prediction of proteases (degradomics), frequently dysregulated in disease, providing a complimentary source of information on disease pathogenesis and biomarkers. Then, what does urine peptidomics tell us so far? In this paper, we appraise the value of urine peptidomics in biomarker research through a comprehensive analysis of all datasets available to date. We have mined > 50 papers, addressing > 30 different conditions, comprising > 4700 unique peptides. Bioinformatic tools were used to reanalyze peptide profiles aiming at identifying disease fingerprints, to uncover hidden disease-specific peptides physicochemical properties and to predict the most active proteases associated with their generation. The molecular patterns found in this study may be further validated in the future as disease biomarker not only for kidney diseases but also for extra-renal conditions, as a step forward towards the implementation of a paradigm of predictive, preventive and personalized (3P) medicine.

Periodontitis is a complex immune-inflammatory condition characterized by the disruption of the periodontal ligament and subsequent formation of periodontal pockets, and by alveolar bone loss, often resulting in tooth loss. A myriad of factors, namely, genetic, metabolic, immunological, and inflammatory, is associated with progression of periodontitis. Periodontitis is also associated with systemic conditions such as neoplastic disorders, obesity, and diabetes. The current diagnosis of this disease relies on clinical measurements such as clinical attachment loss and probing depth, which have poor precision due to patient, operator and probe-related factors. Thus, there is a need to develop reliable, objective, and reproducible biomarkers for early diagnosis of periodontitis. In this regard, saliva, with contributions from the gingival crevicular fluid, holds great potential. However, most of the information on biomarkers of periodontium-related salivary proteins has come from studies on the molecular pathogenesis of periodontitis. In periodontitis, a more holistic approach, such as the use of -omics technologies, for biomarker discovery, is needed. Herein, we review the biomarkers proposed to date for the assessment of periodontitis, with emphasis on the role of salivary peptides in periodontitis and their assessment by high-throughput saliva proteomics. We also discuss the challenges pertaining to the identification of new periodontitis biomarkers in saliva.

Heart failure (HF) is growing in prevalence, due to an increase in aging and comorbidities. Heart failure with reduced ejection fraction (HFrEF) is more common in men, whereas heart failure with preserved ejection fraction (HFpEF) has a higher prevalence in women. However, the reasons for these epidemiological trends are not clear yet. Since HFpEF affects mostly postmenopausal women, sex hormones should play a pivotal role in HFpEF development. Furthermore, for HFpEF, contrary to HFrEF, effective therapeutic approaches are missing. Interestingly, studies evidenced that some therapies can have better results in women than in HFpEF men, emphasizing the necessity of understanding these observations at a molecular level. Thus, herein, we review the molecular mechanisms of estrogen and androgen actions in the heart in physiological conditions and explain how its dysregulation can lead to disease development. This clarification is essential in the road for an effective personalized management of HF, particularly HFpEF, towards the development of sex-specific therapeutic approaches.

The human body has many different hormones that interact with each other and with other factors such as proteins, cell receptors and metabolites. There is still a limited understanding of some of the underlying biological mechanisms of some hormones. In the past decades, science and technology have made major advancements in regard to innovation and knowledge in fields such as medicine. However, some conditions are complex and have many variables that their full picture is still unclear, even though some of these conditions have an alarming rate of incidence and serious health consequences. Conditions such as type 2 diabetes, obesity, nonalcoholic liver disease (NAFLD), cancer in its different forms and even mental conditions, such as Alzheimer's disease, are some of the most common diseases in the 21st century. These conditions are relevant not only because of their high incidence on the general population, but also because of their severity. In this chapter, we present an overview of cardiovascular (CV) diseases. According to the World Health Organization (WHO), cardiovascular diseases, such as coronary artery disease (CAD), heart attack, cardiomyopathy and heart failure (among others), are the number one cause of death worldwide. In 2016, it was estimated that 17.9 million people died from CV diseases, representing more than 30% of all global deaths. Approximately 95% of people who died from CV diseases were so-called "premature deaths" because were referenced to individuals under the age of 70 years old. In this chapter we described some of the hormones that may have an impact on CV diseases, including ghrelin, a peptide that is mostly produced in the stomach, known to induce hunger. Ghrelin is linked to an increase in body fat, i.e., adipose tissue in animals. For this reason, we also included the adipokines leptin, adiponectin and resistin. The main objectives of this chapter are to present the state of the art knowledge concerning the mechanisms of each hormone relevant to CV diseases; to compile data and results that further elucidate the relevance of these peptides for several physiological events, conditions and diseases; and to discuss the metabolic impact of each hormone. We established connections between multiple peptides and the underlying condition/disease with tools such as STRING, referring to research using databases, such as UniProt, DisGeNET and Proteomics DB. Fig. 1 shows a network that summarizes the information presented in this chapter, which serves as a visual representation.

Introduction: With the worldwide decline on male fertility potential, the importance of the insight of the spermatogenic process has been increasing. In recent years, proteomic methodologies have been applied to seminal fluid of infertile men to search for infertility potential biomarkers. However, to understand the spermatogenic event and to search for treatment to spermatogenic impairment, comparative analysis of testicular proteomics is considered a powerful methodology.Areas covered: Herein, we present a critical overview of the studies addressing proteomic alterations in the development of spermatogenesis during puberty, as well as during the different phases of the spermatogenic event. The comparative studies of the proteomic testicular profile of men with and without spermatogenic impairment are also discussed and key proteins and pathways involved highlighted.Expert opinion: The usage of whole human testicular tissue with its heterogeneous cellular composition makes proteome data interpretation particularly challenging. This may be minimized by controlled experiments involving the collection of testicular tissue and sperm from the same individuals, integrated in a clinically characterized cohort of healthy and infertile men. The analysis of specific subcellular proteomes can add more information to the proteomic puzzle, opening new treatment possibilities for infertile/subfertile men.

The human body has many different hormones that interact with each other and with other factors such as proteins, cell receptors and metabolites. There is still a limited understanding of some of the underlying biological mechanisms of some hormones. In the past decades, science and technology have made major advancements in regard to innovation and knowledge in fields such as medicine. However, some conditions are complex and have many variables that their full picture is still unclear, even though some of these conditions have an alarming rate of incidence and serious health consequences. Conditions such as type 2 diabetes, obesity, nonalcoholic liver disease (NAFLD), cancer in its different forms and even mental conditions, such as Alzheimer's disease, are some of the most common diseases in the 21st century. These conditions are relevant not only because of their high incidence on the general population, but also because of their severity. In this chapter, we present an overview of cardiovascular (CV) diseases. According to the World Health Organization (WHO), cardiovascular diseases, such as coronary artery disease (CAD), heart attack, cardiomyopathy and heart failure (among others), are the number one cause of death worldwide. In 2016, it was estimated that 17.9 million people died from CV diseases, representing more than 30% of all global deaths. Approximately 95% of people who died from CV diseases were so-called "premature deaths" because were referenced to individuals under the age of 70 years old. In this chapter we described some of the hormones that may have an impact on CV diseases, including ghrelin, a peptide that is mostly produced in the stomach, known to induce hunger. Ghrelin is linked to an increase in body fat, i.e., adipose tissue in animals. For this reason, we also included the adipokines leptin, adiponectin and resistin. The main objectives of this chapter are to present the state of the art knowledge concerning the mechanisms of each hormone relevant to CV diseases; to compile data and results that further elucidate the relevance of these peptides for several physiological events, conditions and diseases; and to discuss the metabolic impact of each hormone. We established connections between multiple peptides and the underlying condition/disease with tools such as STRING, referring to research using databases, such as UniProt, DisGeNET and Proteomics DB. Fig. 1 shows a network that summarizes the information presented in this chapter, which serves as a visual representation.

This chapter will mainly focus on the general assets and applications of sulfur- or H2S-bearing thermal mineral waters on a world scale. It deals with basic information on hydrogen sulfide properties, on biological effects, and on the therapies traditionally used in medical hydrology as treatment for skin, respiratory, and musculoskeletal disorders. The authors provide information on additional therapeutic benefits of sulfurous waters in diseases such as myalgia (muscle pain), rheumatic diseases (osteoarthritis, rheumatoid arthritis, fibromyalgia), respiratory diseases (upper and lower respiratory tracts), dermatological/skin diseases, cardiovascular diseases, and other diseases (cancer, wound healing in diabetic patients). Possible side effects or situations of diseases for which sulfurous thermal waters are contraindicated are reported too.

Micropeptides are small polypeptides coded by small open-reading frames. Progress in computational biology and the analyses of large-scale transcriptomes and proteomes have revealed that mammalian genomes produce a large number of transcripts encoding micropeptides. Many of these have been previously annotated as long noncoding RNAs. The role of micropeptides in cellular homeostasis maintenance has been demonstrated. This review discusses different types of micropeptides as well as methods to identify them, such as computational approaches, ribosome profiling, and mass spectrometry.

Exosomes are involved in a wide variety of biochemical processes in human body homeostasis. Exosomes also provide important information regarding communications among several organ systems. Additionally, they can serve as molecular vehicles to deliver drugs. Therefore, exosomes have received wide attention in current biomedical research for unraveling pathogenic mechanisms of diseases, searching for novel biomarkers, and discovering new drugs. This paper reviews and discusses the significance of urinary exosomes for a better understanding of human disease pathophysiology and their potential use as therapeutic targets. Isolation methods of exosomes and the latest technological advances are also discussed. Furthermore, novel urinary exosomal biomarkers are highlighted with special emphasis on their clinical applicability (particularly sensitivity, specificity, reliability, and other aspects). Finally, future trends for this field are analyzed and our perspectives are provided.

Signal transduction is essential for skeletal muscle functionality, a highly plastic tissue able to respond to mechanical and chemical stimuli by altering its metabolism, size and myogenic status. Several signaling pathways involving membrane and nuclear receptors, and secondary messengers as Ca2+, cAMP, inositol triphosphate (IP3) and diacylglycerol (DAG), contribute to such plasticity. The contribution of these transduction systems to muscle remodeling is well characterized; however, their input depends on several issues such as type of muscle (slow-twitch or fast-twitch, postural or locomotor), gender, age, muscle performance, duration and intensity of stimulus exposure, among others. All these issues together with the crosstalk between cell signaling pathways make difficult to correlate a unique signaling pathway or molecular player to a specific effect on muscle remodeling. This chapter overviews the mechanical and chemical transduction pathways, and the molecular players involved in the regulation of skeletal muscle mass, contractile activity and metabolism.

Introduction Proteins are crucial for every cellular activity and unraveling their sequence and structure is a crucial step to fully understand their biology. Early methods of protein sequencing were mainly based on the use of enzymatic or chemical degradation of peptide chains. With the completion of the human genome project and with the expansion of the information available for each protein, various databases containing this sequence information were formed. Areas covered De novo protein sequencing, shotgun proteomics and other mass-spectrometric techniques, along with the various software are currently available for proteogenomic analysis. Emphasis is placed on the methods for de novo sequencing, together with potential and shortcomings using databases for interpretation of protein sequence data. Expert opinion As mass-spectrometry sequencing performance is improving with better software and hardware optimizations, combined with user-friendly interfaces, de-novo protein sequencing becomes imperative in shotgun proteomic studies. Issues regarding unknown or mutated peptide sequences, as well as, unexpected post-translational modifications (PTMs) and their identification through false discovery rate searches using the target/decoy strategy need to be addressed. Ideally, it should become integrated in standard proteomic workflows as an add-on to conventional database search engines, which then would be able to provide improved identification. published

Cerebrospinal fluid (CSF) is a source of valuable information concerning brain disorders. The technical advances of high-throughput omics platforms to analyze body fluids can generate a huge amount of data, whose translation to biological meaning is a challenge. Several bioinformatic tools have emerged to help handling this data into systems biology comprehensively. Herein, we describe a step-by-step tutorial for CSF proteome data analysis in the set of neurodegenerative diseases using (1) ClueGO+CluePedia tool to perform cluster-based analysis envisioning the characterization of the biological processes dysregulated in neurodegenerative diseases including Alzheimer's and Parkinson's diseases; (2) Cytoscape to map disease-specific proteins; (3) SecretomeP to inquire the secretion pathway of CSF proteins; and (4) STRING to identify biological processes modulated by secreted CSF proteins based on protein-protein interaction analysis. This step-by-step guide might help researchers to better characterize disease pathogenesis and to identify putative disease biomarkers.

The preanalytical phase of saliva proteomics/peptidomics, which includes sample collection, handling, and storage, represents a major challenge for any researcher that envisions sensitive and high-throughput analyses, coupled to well-controlled study design. The methodology used to collect saliva determines the contribution of each salivary gland to saliva composition with impact on data retrieved from proteomics/peptidomics. The awareness of the importance of this step in the analysis of saliva has prompted the proposal of several collection strategies. Moreover, numerous commercial devices are available in an attempt to routine the procedures. However, whatever the chosen method, procedures should be kept simple, standardized to get better reproducibility and repeatability on saliva proteomics analysis. Sample preservation is also a key step in saliva proteomics/peptidomics, and the implemented lab procedures should avoid posttranslational modifications such as proteolysis, as well as protein precipitation.In this chapter, we provide recommendations for saliva sampling and preservation, envisaging to standardize procedures that facilitate the use of saliva in clinical applications and the translation of proteomics data to diagnosis and/or definition of therapeutic targets.

Progression to hormone-independent growth leading to endocrine therapy resistance occurs in a high proportion of patients with estrogen receptor alpha (ERα) and progesterone receptors (PR) positive breast cancer. We and others have previously shown that estrogen- and progestin-induced tumor growth requires ERα and PR interaction at their target genes. Here, we show that fibroblast growth factor 2 (FGF2)-induces cell proliferation and tumor growth through hormone-independent ERα and PR activation and their interaction at the MYC enhancer and proximal promoter. MYC inhibitors, antiestrogens or antiprogestins reverted FGF2-induced effects. LC?MS/MS identified 700 canonical proteins recruited to MYC regulatory sequences after FGF2 stimulation, 397 of which required active ERα (ERα-dependent). We identified ERα-dependent proteins regulating transcription that, after FGF2 treatment, were recruited to the enhancer as well as proteins involved in transcription initiation that were recruited to the proximal promoter. Also, among the ERα-dependent and independent proteins detected at both sites, PR isoforms A and B as well as the novel protein product PRBΔ4 were found. PRBΔ4 lacks the hormone-binding domain and was able to induce reporter gene expression from estrogen-regulated elements and to increase cell proliferation when cells were stimulated with FGF2 but not by progestins. Analysis of the Cancer Genome Atlas data set revealed that PRBΔ4 expression is associated with worse overall survival in luminal breast cancer patients. This discovery provides a new mechanism by which growth factor signaling can engage nonclassical hormone receptor isoforms such as PRBΔ4, which interacts with growth-factor activated ERα and PR to stimulate MYC gene expression and hence progression to endocrine resistance. Fil: Giulianelli, Sebastian Jesus. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biología de Organismos Marinos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Riggio, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Guillardoy, Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Pérez Piñero, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Gorostiaga, María A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Sequeira, Gonzalo Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Pataccini, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Abascal, María F.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Toledo, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Jacobsen, Britta M.. University of Colorado; Estados Unidos Fil: Guerreiro, Ana C.. Universidade de Aveiro; Portugal Fil: Barros, António. Universidade de Aveiro; Portugal Fil: Novaro, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Monteiro, Fátima L.. Universidade de Aveiro; Portugal Fil: Amado, Francisco. Universidade de Aveiro; Portugal Fil: Gass, Hugo. Hospital Zonal General de Agudos Magdalena V de Martínez; Argentina Fil: Abba, Martin. Universidad Nacional de La Plata; Argentina Fil: Helguero, Luisa A.. Universidade de Aveiro; Portugal Fil: Lanari, Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina

Cerebrospinal fluid (CSF) is a source of valuable information concerning brain disorders. The technical advances of high-throughput omics platforms to analyze body fluids can generate a huge amount of data, whose translation to biological meaning is a challenge. Several bioinformatic tools have emerged to help handling this data into systems biology comprehensively. Herein, we describe a step-by-step tutorial for CSF proteome data analysis in the set of neurodegenerative diseases using (1) ClueGO+CluePedia tool to perform cluster-based analysis envisioning the characterization of the biological processes dysregulated in neurodegenerative diseases including Alzheimer's and Parkinson's diseases; (2) Cytoscape to map disease-specific proteins; (3) SecretomeP to inquire the secretion pathway of CSF proteins; and (4) STRING to identify biological processes modulated by secreted CSF proteins based on protein-protein interaction analysis. This step-by-step guide might help researchers to better characterize disease pathogenesis and to identify putative disease biomarkers.

The preanalytical phase of saliva proteomics/peptidomics, which includes sample collection, handling, and storage, represents a major challenge for any researcher that envisions sensitive and high-throughput analyses, coupled to well-controlled study design. The methodology used to collect saliva determines the contribution of each salivary gland to saliva composition with impact on data retrieved from proteomics/peptidomics. The awareness of the importance of this step in the analysis of saliva has prompted the proposal of several collection strategies. Moreover, numerous commercial devices are available in an attempt to routine the procedures. However, whatever the chosen method, procedures should be kept simple, standardized to get better reproducibility and repeatability on saliva proteomics analysis. Sample preservation is also a key step in saliva proteomics/peptidomics, and the implemented lab procedures should avoid posttranslational modifications such as proteolysis, as well as protein precipitation.

Estrogen-like metabolites have been identified in S. haematobium, the helminth parasite that causes urogenital schistosomiasis (UGS) and in patients´ blood and urine during UGS. Estrogen receptor (ER) activation is enriched in the luminal molecular subtype bladder cancer (BlaCa). To date, the significance of ER to these diseases remains elusive. We evaluated ERα and ERβ expression in UGS-related BlaCa (n = 27), UGS-related non-malignant lesions (n = 35), and noninfected BlaCa (n = 80). We investigated the potential of ERα to recognize S. haematobium-derived metabolites by docking and molecular dynamics simulations and studied ERα modulation in vitro using 3 BlaCa cell lines, T24, 5637 and HT1376. ERα was expressed in tumor and stromal cells in approximately 20% noninfected cases and in 30% of UGS-related BlaCa, predominantly in the epithelial cells. Overall, ERα expression was associated with features of tumor aggressiveness such as high proliferation and p53 positive expression. ERα expression correlated with presence of schistosome eggs. ERβ was widely expressed in both cohorts but weaker in UGS-related cases. molecular dynamics simulations of the 4 most abundant S. haematobium-derived metabolites revealed that smaller metabolites have comparable affinity for the ERα active state than 17β-estradiol, while the larger metabolites present higher affinity. Our in vitro findings suggested that ERα activation promotes proliferation in ERα expressing BlaCa cells and that this can be reverted with anti-estrogenic therapy. In summary, we report differential ER expression between UGS-related BlaCa and noninfected BlaCa and provide evidence supporting a role of active ERα during UGS and UGS-induced carcinogenesis. Highlights: Estrogen receptor (ERs) are expressed in both urogenital schistosomiasis and bladder cancer; ERα was associated with aggressive tumor features and presence of schistosome eggs; In silico analysis shows an agonistic putative action of S. haematobium-derived metabolites for ERα; ERα induced proliferation in bladder cancer cells can be reverted by anti-estrogens; Evidence suggests that activation of ERα could play a role in urogenital schistosomiasis and urogenital schistosomiasis-induced carcinogenesis. PJB acknowledges support from award R01CA164719 from the National Cancer Institute (NCI), National Institutes of Health (NIH), USA. info:eu-repo/semantics/publishedVersion

Bladder Cancer (BC) presents one of the highest recurrence rates amongst solid tumours and constitutes the second deadliest disease of the genitourinary track. Non-invasive identification of patients facing disease recurrence and/or progression remains one of the most critical and challenging aspects in disease management. To contribute to this goal, we demonstrate the potential of glycan-affinity glycoproteomics nanoplatforms for urinary biomarkers discovery in bladder cancer. Briefly, magnetic nanoprobes (MNP) coated with three broad-spectrum lectins, namely Concanavalin A (ConA; MNP@ConA), Wheat Germ Agglutinin (WGA; MNP@WGA), and Sambucus nigra (SNA; MNP@SNA), were used to selectively capture glycoproteins from the urine of low-grade and high-grade non-muscle invasive as well as muscle-invasive BC patients. Proteins were identified by nano-LC MALDI-TOF/TOF and data was curated using bioinformatics tools (UniProt, NetOGlyc, NetNGlyc, ClueGO app for Cytoscape and Oncomine) to highlight clinically relevant species. Accordingly, 63 glycoproteins were exclusively identified in cancer samples compared with healthy controls matching in age and gender. Specific glycoprotein sets exclusively found in low-grade non-muscle invasive bladder tumours may aid early diagnosis, while those only found in high-grade non-invasive and muscle-invasive tumours hold potential for accessing progression. Amongst these proteins is bladder cancer stem-cell marker CD44, which has been associated with poor prognosis. Orthogonal validation studies by slot-blotting demonstrated an elevation in urine CD44 levels of high-grade patients, which became more pronounced upon muscle-invasion, in mimicry of the primary tumour. These observations demonstrate the potential of MNP@lectins for identification of clinically relevant glycoproteomics signatures in bladder cancer. Future clinical validation in a larger and well characterized patient subset is required envisaging clinical translation of the results. published

The comprehension of how protease networks sculpt proteomes might help to disclose the functional annotation of the peptidome in health and disease. Envisioning to add new insights on the protease networks involved in the regulation of body fluid peptidomes, the authors apply Proteasix software to predict the proteases involved in the generation of the naturally occurring peptides present in six of the most studied human body fluids. Peptidome data is collected from the databases and from experimental studies. The analysis highlights 132 putative proteases from four families with the predominance of serine proteases and metalloproteases. From these, 49 proteases seem to be common to all fluids and are mostly associated to extracellular matrix organization as well as protein/peptide hormone processing. Data analysis also emphasizes: i) the similarity between plasma and CSF protease profiles; ii) that saliva and tears share proteases involved in the generation of peptides with antimicrobial activity; iii) that urine is the body fluid with the highest number of unique putative proteases, precluding an easy tracing of proteolytic events in this case. Taken together, the analysis emphasizes the intricate modus operandi of proteases, challenged by the interconnected pathways and amplification cascades in which they are involved.

Oral sensorial perception is a key aspect in food choices and knowing the mechanisms modulating such perception is of major importance in the context of child obesity, which is reaching high rates in Mediterranean countries. Salivary proteome has been linked to taste sensitivity in adults. The aim of this study was to search for differences in salivary proteomes of children with different bitter or sweet taste sensitivities and to assess if these potential differences are associated with their body mass index percentile (BMI percentile). 387 children aged 8–9 years old were assessed for BMI percentile and classified according to their sensitivity to bitter and sweet tastes, according to their caffeine and sucrose detection thresholds, respectively. Saliva protein composition was compared among taste sensitivity groups, taking into account BMI percentile and gender, using gel-based proteomics approaches, coupled to mass spectrometry for protein identification. Among the salivary proteins related to bitter taste sensitivity, higher levels of cystatins were observed in bitter-sensitive children, in the case of those of normal weight, and in bitter low-sensitive, in the case of overweight children. For sweetness, the relationship between saliva and taste perception was also dependent on BMI percentile, with several proteins (including salivary cystatins) differing between taste sensitivity groups, with disparities arising between normal-weight and overweight children. Cystatin isoforms A, B and SA were observed to be considerably increased in saliva from obese children. Salivary proteome is related with sensitivities to bitter and sweet tastes in children, but the association is dependent on BMI percentile and gender.

Objective To study the effect of the satiety hormone, leptin, in saliva proteome and salivary gland histology and ultrastructure. Design Increases in blood leptin levels were induced through mini-pump infusion in male Wistar rats, during a period of 7 days. Saliva was collected before and at the end of the experimental period, for proteomic analysis, and major salivary glands were collected, at the end, for biochemical, histological and ultrastructural analysis. Results Immunohistochemistry revealed the presence of leptin receptors in major salivary glands. Salivary amylase levels and enzymatic activity were decreased in saliva, whereas the enzymatic activity of this protein was increased in the cytosol of parotid gland cells. Transmission electron microscopy allowed the observation of high number of electron-dense granules in cytosol of parotid acinar cells, from leptin treated animals. Conclusions Increased levels of plasmatic leptin result in changes in saliva composition and salivary glands function. To our knowledge, this is the first study providing evidences for a potential role of leptin in salivary gland secretion and saliva composition. An understanding of how appetite/satiety factors influence saliva composition and how this composition influences food processing in mouth may be relevant in understanding ingestivebehaviour.

The present study aimed to test whether a chronic intermittent workload could induce an adaptive cardiac phenotype Chronic intermittent workload induced features of adaptive hypertrophy This was paralleled by protection against acute pressure overload insult The heart may adapt favourably to balanced demands, regardless of the nature of the stimuli. The present study aimed to test whether submitting the healthy heart to intermittent and tolerable amounts of workload, independently of its nature, could result in an adaptive cardiac phenotype. Male Wistar rats were subjected to treadmill running (Ex) (n = 20), intermittent cardiac overload with dobutamine (ITO) (2 mg kg(-1) , s.c.; n = 20) or placebo administration (Cont) (n = 20) for 5 days week(-1) for 8 weeks. Animals were then killed for histological and biochemical analysis or subjected to left ventricular haemodynamic evaluation under baseline conditions, in response to isovolumetric contractions and to sustained LV acute pressure overload (35% increase in peak systolic pressure maintained for 2 h). Baseline cardiac function was enhanced only in Ex, whereas the response to isovolumetric heartbeats was improved in both ITO and Ex. By contrast to the Cont group, in which rats developed diastolic dysfunction with sustained acute pressure overload, ITO and Ex showed increased tolerance to this stress test. Both ITO and Ex developed cardiomyocyte hypertrophy without fibrosis, no overexpression of osteopontin-1 or β-myosin heavy chain, and increased expression of sarcoplasmic reticulum Ca(2+) protein. Regarding hypertrophic pathways, ITO and Ex showed activation of the protein kinase B/mammalian target of rapamycin pathway but not calcineurin. Mitochondrial complex IV and V activities were also increased in ITO and Ex. Chronic submission to controlled intermittent cardiac overload, independently of its nature, results in an adaptive cardiac phenotype. Features of the cardiac overload, such as the duration and magnitude of the stimuli, may play a role in the development of an adaptive or maladaptive phenotype.

Saliva is essential to interact with microorganisms in the oral cavity. Therefore, the interest in saliva antimicrobial properties is on the rise. Here, we used an immunoproteomic approach, based on protein separation of Staphylococcus epidermidis biofilms by 2DE, followed by Western-blotting, to compare human serum and saliva reactivity profile. A total of 17 proteins were identified by MALDI-TOF/TOF. Serum and saliva presented a distinct pattern of immunoreactive proteins. Our results suggest that saliva seems to have higher propensity to react against S. epidermidis proteins with oxidoreductase activity and proteins involved with L-serine metabolic processes. We show that saliva was a powerful tool for the identification of potential S. epidermidis biofilms proteins.

Strategies to prevent tumour burden-induced cardiac remodelling that might progress to heart failure are necessary to improve patients' health outcomes and tolerability to cancer therapies. Exercise has been suggested as a measure to prevent cardiac damage; however, its effectiveness on regulating cardiac remodelling secondary to cancer was never addressed. Using an animal model of mammary tumorigenesis, we studied the impact of 35weeks of endurance training on heart, focusing on the signalling pathways modulated by pro-inflammatory and wasting cytokines. The cardiac fibrosis and myofiber disorganization induced by tumour burden was paralleled by the increase of myostatin and TWEAK with the activation of signalling pathways involving Smad-3, NF-κB, TRAF-6 and atrogin-1. The activation of Akt/mTOR was observed in heart from rats with tumours, for which contributed the extracellular matrix. Endurance training prevented the increase of serum and cardiac TWEAK promoted by cancer, as well as the activation of NF-κB, TRAF6, atrogin-1 and p70S6K in heart. Data highlight the impact of exercise in the modulation of signalling pathways activated by wasting cytokines and the resulting outcomes on heart adaptation. Future studies focused on the cellular pathways underlying cardiac remodelling will assist in the development of exercise programs targeting cancer-related cardiac alterations.

The salivary peptidome, which can represent up to 20% of total secreted proteins in human saliva, is highly influenced by proteolytic events. However, the development of strategies to understand the dynamics underlying the generation of salivary peptides has been a challenging task. In order to disclose in more detail the proteolytic events taking place in saliva, we aimed to characterize salivary peptidome and predict salivary proteases by applying, for the first time, a filter-aided sample preparation (FASP) approach to saliva. Thus, as a proof-of-concept of this application, harvested saliva samples from healthy individuals were incubated in 30 kDa cut-off spin filters for 18 or 115 h, at 37 °C, to promote saliva autolysis and the attained peptidome was characterized and compared with the naturally occurring one. In ex vivo conditions, proline-rich proteins, P-B peptide, histatin 1 and statherin were found to be the most susceptible salivary proteins to proteolysis. Peptide fragments were mainly attributed to the activity of cathepsin L1 and K at 18 h, whereas at 115 h, the attained peptide fragments were attributed to the activity of cathepsins K and L1, and MEP1A. Overall, the described endoProteoFASP approach makes the most of saliva׳s own protease pool and avoids the use of synthetic peptides and exogenous proteases to understand the proteolytic events occurring in the oral fluid. Hence, it could be very helpful in future studies targeting the characterization of salivary proteases and peptidome from different pathophysiological conditions.

The satiety inducing hormone leptin acts not only at central nervous system but also at peripheral level. Leptin receptors are found in several sense related organs, including the mouth. A role of leptin in sweet taste response has been suggested but, until now, studies have been based on in vitro experiments, or in assessing the levels of the hormone in circulation. The present study investigated whether the levels of leptin in saliva are related to taste perception in children and whether Body Mass Index (BMI) affects such relationship. Sweet and bitter taste sensitivity was assessed for 121 children aged 9-10 years and unstimulated whole saliva was collected for leptin quantification, using ELISA technique. Children females with lower sweet taste sensitivity presented higher salivary leptin levels, but this is only in the normal weight ones. For bitter taste, association between salivary leptin and caffeine threshold detection was observed only in preobese boys, with higher levels of salivary hormone in low sensitive individuals. This study is the first presenting evidences of a relationship between salivary leptin levels and taste perception, which is sex and BMI dependent. The mode of action of salivary leptin at taste receptor level should be elucidated in future studies.

Findings on experimental animals show that ovarian failure is accompanied by a decrease in motor activity. As mechanical loading has a vital role in the maintenance of skeletal health, our aim was to determine to what extent this decrease in motor activity contributes to ovariectomy-induced bone loss. Thirty-two female Wistar rats were ovariectomized or sham-operated and housed in standard cages or with access to running wheels for 36 weeks with their running distance monitored. Markers of bone turnover were assayed in the serum, and bone geometry, trabecular and cortical bone microarchitecture, mineralization degree, and biomechanical properties were assessed in the femur. Differences between groups were determined by one-way ANOVA. Although reduced motor activity and sex steroid deficiency both resulted in decreases in trabecular bone volume, trabecular number decreases were mostly associated with sex steroid deficiency, whereas trabecular thickness decreases were mostly associated with sedentary behavior. Cortical bone appeared to be more sensitive to variations in motor activity, whereas bone turnover rate and bone tissue mineralization degree seemed to be primarily affected by sex steroid deficiency, even though they were further aggravated by sedentary behavior. Increases in femur length were mostly a consequence of sex steroid deficiency, whereas femoral neck length was also influenced by sedentary behavior. Differences in mechanical properties resulted mostly from differences in physical activity. Both the direct effect of sex steroid deficiency and the indirect effect of motor activity changes are implicated in bone loss following ovariectomy.

Alterations in muscle mitochondrial bioenergetics during cancer cachexia were previously suggested; however, the underlying mechanisms are not known. So, the goal of this study was to evaluate mitochondrial phospholipid remodeling in cancer-related muscle wasting and its repercussions to respiratory chain activity and fiber susceptibility to apoptosis. An animal model of urothelial carcinoma induced by exposition to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) and characterized by significant body weight loss due to skeletal muscle mass decrease was used. Morphological evidences of muscle atrophy were associated to decreased respiratory chain activity and increased expression of mitochondrial UCP3, which altogether highlight the lower ability of wasted muscle to produce ATP. Lipidomic analysis of isolated mitochondria revealed a significant decrease of phosphatidic acid, phosphatidylglycerol and cardiolipin in BBN mitochondria, counteracted by increased phosphatidylcholine levels. Besides the impact on membrane fluidity, this phospholipid remodeling seems to justify, at least in part, the lower oxidative phosphorylation activity observed in mitochondria from wasted muscle and their increased susceptibility to apoptosis. Curiously, no evidences of lipid peroxidation were observed but proteins from BBN mitochondria, particularly the metabolic ones, seem more prone to carbonylation with the consequent implications in mitochondria functionality. Overall, data suggest that bladder cancer negatively impacts skeletal muscle activity specifically by affecting mitochondrial phospholipid dynamics and its interaction with proteins, ultimately leading to the dysfunction of this organelle. The regulation of phospholipid biosynthetic pathways might be seen as potential therapeutic targets for the management of cancer-related muscle wasting.

Moderate physical activity has traditionally been associated with the improvement of cardiac function and, consequently, with the extension of life span. Mitochondria play a key role in the adaptation of heart muscle to exercise-related metabolic demands. In order to disclose the molecular mechanisms underlying the beneficial effect of lifelong physical activity in cardiac function, we performed label-free quantitative mass spectrometry-based proteomics of Sprague-Dawley rat heart mitochondrial proteome and phosphoproteome. Our data revealed that 54 weeks of moderate treadmill exercise modulates the abundance of proteins involved in the generation of precursor metabolites and cellular respiration, suggesting an increase in carbohydrate oxidation-based metabolism. Moreover, from the 1335 phosphopeptides identified in this study, 6 phosphosites were exclusively assigned to heart mitochondria from sedentary rats and 17 to exercised animals, corresponding to 6 and 16 proteins, respectively. Most proteins exhibiting significant alterations in specific phosphorylation sites were involved in metabolism. Analysis of the acquired data led to the identification of several kinases potentially modulated by exercise training, which were selected for further validation. Indeed, higher protein abundance levels of RAF and p38 in mitochondria were confirmed to be modulated by sustained exercise. Our work describes the plasticity of heart mitochondria in response to long exercise programs manifested by the reprogramming of phosphoproteome and provides evidence for the kinases involved in the regulation of metabolic pathways and mitochondrial maintenance.

Diabetes mellitus is a chronic metabolic disease with multiple complications, and its successful management requires early diagnosis, to allow timely interventions. Here, we have comprehensively analyzed the proteome changes in urine of type 1 diabetic subjects with and without complications such as retinopathy and nephropathy. gel electrophoresis combined to liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) analysis of midstream urine highlighted the mechanisms involved in disease pathogenesis as, for instance wound healing and blood coagulation in all diabetics or altered ganglioside metabolism in retinopathy, and also some urinary proteins with potential diagnosis value. From these, gelsolin and antithrombin-III appear as promising diagnosis markers for type 1 diabetes mellitus (T1DM), whereas ephrin type-B receptor 4 and vitamin K-dependent protein Z seem to be promising markers for advanced T1DM disease state presenting retinopathy and nephropathy (T1DM-R + N). Data also suggest urinary ganglioside GM2 activator and beta-hexosaminidase subunit beta as potential urinary markers of retinopathy in diabetics. Taken together, the present exploratory urinary proteomic analysis might be seen as an important starting point for studies targeting specific urinary proteins aimed at the implementation of new biomarkers for the early detection of T1DM-related microvascular complications.

Cardiac cachexia (CC) affects a large proportion of patients with chronic heart failure, a major public health issue in western countries. The pathophysiology of CC is complex and multifactorial, resulting from several factors interacting in a complex system with metabolic, immune and neurohormonal consequences, triggered to protect the heart and the circulation from damage. Despite the adverse clinical effects, CC diagnosis is not straightforward and has not specifically been targeted, with therapeutic strategies only comprising interventions with appetite stimulants, and anti-inflammatory substances. Here we review the molecular pathways underlying CC-related muscle wasting aiming to provide clues for the definition of CC-specific biomarkers and for the development of drugs that prevent and/or counteract muscle impairment, which will certainly impact the management of cardiovascular disorders.

Proliferation and differentiation are controlled through chromatin remodelling. Therefore, there is an enormous biological significance and clinical value in understanding how specific signalling pathways are affected by histone replacement in the nucleosome. In this work, mass spectrometry was used to screen HC11 mammary epithelial cells for changes in histone levels throughout cell differentiation. The canonical histone isoform Histone H2A type 2-C (Hist2h2ac) was found only in undifferentiated/proliferating cells. Hist2h2ac mRNA was induced by EGF, specifically in the CD24+/CD29hi/DC44hi cell subpopulation. Hist2h2ac mRNA was increased by MEK(1/2) or PI3-K activation in HC11 and EpH4 mammary epithelial cells, and in MC4-L2 and T47-D breast cancer cells. Hist2h2ac silencing inhibited EGF-induced Zeb-1 expression and E-cadherin down-regulation, and this effect was reverted by Hist2h2ac re-expression. Notably, silencing of Hist2h2ac increased EGFR, ERBB2, and ERK(1/2) activation but did not allow EGF-induced proliferation. HIST2H2AC was expressed in all breast cancer molecular subtypes and found altered in 17% breast cancers, being 16.8% of the cases related to HIST2H2AC gene amplification and/or mRNA upregulation. In summary, this is the first study that identifies a canonical histone isoform -Hist2h2ac-downstream of the EGFR pathway, regulating oncogenic signalling and thereby contributing to deregulation of target genes. QC 20170322