A multiplier peroxiporin signal transduction pathway powers piscine spermatozoa.

The primary task of a spermatozoon is to deliver its nuclear payload to the egg to form the next-generation zygote. With polyandry repeatedly evolving in the animal kingdom, however, sperm competition has become widespread, with the highest known intensities occurring in fish. Yet, the molecular controls regulating spermatozoon swimming performance in these organisms are largely unknown. Here, we show that the kinematic properties of postactivated piscine spermatozoa are regulated through a conserved trafficking mechanism whereby a peroxiporin ortholog of mammalian aquaporin-8 (Aqp8bb) is inserted into the inner mitochondrial membrane to facilitate H2O2 efflux in order to maintain ATP production. In teleosts from more ancestral lineages, such as the zebrafish (Danio rerio) and the Atlantic salmon (Salmo salar), in which spermatozoa are activated in freshwater, an intracellular Ca2+-signaling directly regulates this mechanism through monophosphorylation of the Aqp8bb N terminus. In contrast, in more recently evolved marine teleosts, such the gilthead seabream (Sparus aurata), in which spermatozoa activation occurs in seawater, a cross-talk between Ca2+- and oxidative stress-activated pathways generate a multiplier regulation of channel trafficking via dual N-terminal phosphorylation. These findings reveal that teleost spermatozoa evolved increasingly sophisticated detoxification pathways to maintain swimming performance under a high osmotic stress, and provide insight into molecular traits that are advantageous for postcopulatory sexual selection.

Inflammatory capacity of exosomes released in the early stages of acute pancreatitis predicts the severity of the disease.

As acute pancreatitis progresses to the severe form, a life-threatening systemic inflammation is triggered. Although the mechanisms involved in this process are not yet well understood, it has been proposed that circulating exosomes may be involved in the progression of inflammation from the pancreas to distant organs. Here, the inflammatory capacity and protein profile of plasma exosomes obtained during the first 24 h of hospitalization of patients diagnosed with acute pancreatitis were characterized and compared with the final severity of the disease. We found that the final severity of the disease strongly correlates with the inflammatory capacity of exosomes in the early stages of acute pancreatitis. Exosomes isolated from patients with mild pancreatitis had no effect on macrophages, while exosomes isolated from patients with severe pancreatitis triggered NFκB activation, TNFα and IL1ß expression, and free radical generation. To delve deeper into the mechanism involved, we performed a proteomic analysis of the different exosomes that allowed us to identify different groups of proteins whose concentration was also correlated with the clinical classification of pancreatitis. In particular, an increase in the amount of S100A8 and S100A9 carried by exosomes of severe pancreatitis suggests that the mechanism of action of exosomes is mediated by the effect of these proteins on NADPH oxidase. This enzyme is activated by S100A8/S100A9, thus generating free radicals and promoting an inflammatory response. Along these lines, we observed that inhibition of this enzyme abolished all the pro-inflammatory effects of exosomes from severe pancreatitis. All this suggests that the systemic effects, and therefore the final severity of acute pancreatitis, are determined by the content of circulating exosomes generated in the early hours of the process. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Sewage Protein Information Mining: Discovery of Large Biomolecules as Biomarkers of Population and Industrial Activities.

Wastewater-based epidemiology has been revealed as a powerful approach for surveying the health and lifestyle of a population. In this context, proteins have been proposed as potential biomarkers that complement the information provided by currently available methods. However, little is known about the range of molecular species and dynamics of proteins in wastewater and the information hidden in these protein profiles is still to be uncovered. In this study, we investigated the protein composition of wastewater from 10 municipalities in Catalonia with diverse populations and industrial activities at three different times of the year. The soluble fraction of this material was analyzed using liquid chromatography high-resolution tandem mass spectrometry using a shotgun proteomics approach. The complete proteomic profile, distribution among different organisms, and semiquantitative analysis of the main constituents are described. Excreta (urine and feces) from humans, and blood and other residues from livestock were identified as the two main protein sources. Our findings provide new insights into the characterization of wastewater proteomics that allow for the proposal of specific bioindicators for wastewater-based environmental monitoring. This includes human and animal population monitoring, most notably for rodent pest control (immunoglobulins (Igs) and amylases) and livestock processing industry monitoring (albumins).

Isolation of HLA-DR-naturally presented peptides identifies T-cell epitopes for rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) immunopathogenesis revolves around the presentation of poorly characterised self-peptides by human leucocyte antigen (HLA)-class II molecules on the surface of antigen-presenting cells to autoreactive CD4 +T cells. Here, we analysed the HLA-DR-associated peptidome of synovial tissue (ST) and of dendritic cells (DCs) pulsed with synovial fluid (SF) or ST, to identify potential T-cell epitopes for RA. METHODS: HLA-DR/peptide complexes were isolated from RA ST samples (n=3) and monocyte-derived DCs, generated from healthy donors carrying RA-associated shared epitope positive HLA-DR molecules and pulsed with RA SF (n=7) or ST (n=2). Peptide sequencing was performed by high-resolution mass spectrometry. The immunostimulatory capacity of selected peptides was evaluated on peripheral blood mononuclear cells from patients with RA (n=29) and healthy subjects (n=12) by flow cytometry. RESULTS: We identified between 103 and 888 HLA-DR-naturally presented peptides per sample. We selected 37 native and six citrullinated (cit)-peptides for stimulation assays. Six of these peptides increased the expression of CD40L on CD4 +T cells patients with RA, and specifically triggered IFN-γ expression on RA CD4 +T cells compared with healthy subjects. Finally, the frequency of IFN-γ-producing CD4 +T cells specific for a myeloperoxidase-derived peptide showed a positive correlation with disease activity. CONCLUSIONS: We significantly expanded the peptide repertoire presented by HLA-DR molecules in a physiologically relevant context, identifying six new epitopes recognised by CD4 +T cells from patients with RA. This information is important for a better understanding of the disease immunopathology, as well as for designing tolerising antigen-specific immunotherapies.

Phosphoproteomic Analysis of Platelets in Severe Obesity Uncovers Platelet Reactivity and Signaling Pathways Alterations.

OBJECTIVE: Obesity is associated with a proinflammatory and prothrombotic state that supports atherosclerosis progression. The goal of this study was to gain insights into the phosphorylation events related to platelet reactivity in obesity and identify platelet biomarkers and altered activation pathways in this clinical condition. Approach and Results: We performed a comparative phosphoproteomic analysis of resting platelets from obese patients and their age- and gender-matched lean controls. The phosphoproteomic data were validated by mechanistic, functional, and biochemical assays. We identified 220 differentially regulated phosphopeptides, from at least 175 proteins; interestingly, all were up-regulated in obesity. Most of the altered phosphoproteins are involved in SFKs (Src-family kinases)-related signaling pathways, cytoskeleton reorganization, and vesicle transport, some of them validated by targeted mass spectrometry. To confirm platelet dysfunction, flow cytometry assays were performed in whole blood indicating higher surface levels of GP (glycoprotein) VI and CLEC (C-type lectin-like receptor) 2 in platelets from obese patients correlating positively with body mass index. Receiver operator characteristics curves analysis suggested a much higher sensitivity for GPVI to discriminate between obese and lean individuals. Indeed, we also found that obese platelets displayed more adhesion to collagen-coated plates. In line with the above data, soluble GPVI levels-indicative of higher GPVI signaling activation-were almost double in plasma from obese patients. CONCLUSIONS: Our results provide novel information on platelet phosphorylation changes related to obesity, revealing the impact of this chronic pathology on platelet reactivity and pointing towards the main signaling pathways dysregulated.

TCellXTalk facilitates the detection of co-modified peptides for the study of protein post-translational modification cross-talk in T cells.

MOTIVATION: Protein function is regulated by post-translational modifications (PTMs) that may act individually or interact with others in a phenomenon termed PTM cross-talk. Multiple databases have been dedicated to PTMs, including recent initiatives oriented towards the in silico prediction of PTM interactions. The study of PTM cross-talk ultimately requires experimental evidence about whether certain PTMs coexist in a single protein molecule. However, available resources do not assist researchers in the experimental detection of co-modified peptides. RESULTS: Herein, we present TCellXTalk, a comprehensive database of phosphorylation, ubiquitination and acetylation sites in human T cells that supports the experimental detection of co-modified peptides using targeted or directed mass spectrometry. We demonstrate the efficacy of TCellXTalk and the strategy presented here in a proof of concept experiment that enabled the identification and quantification of 15 co-modified (phosphorylated and ubiquitinated) peptides from CD3 proteins of the T-cell receptor complex. To our knowledge, these are the first co-modified peptide sequences described in this widely studied cell type. Furthermore, quantitative data showed distinct dynamics for co-modified peptides upon T cell activation, demonstrating differential regulation of co-occurring PTMs in this biological context. Overall, TCellXTalk facilitates the experimental detection of co-modified peptides in human T cells and puts forward a novel and generic strategy for the study of PTM cross-talk. AVAILABILITY AND IMPLEMENTATION: TCellXTalk is available at https://www.tcellxtalk.org. Source Code is available at https://bitbucket.org/lp-csic-uab/tcellxtalk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A Combination of Proteomic Approaches Identifies A Panel of Circulating Extracellular Vesicle Proteins Related to the Risk of Suffering Cardiovascular Disease in Obese Patients.

Plasma-derived extracellular vesicles (EVs) have been extensively described as putative biomarkers in different diseases. Interestingly, increased levels of EVs subpopulations are well known to associate with obesity. The goal of this study is to identify EVs-derived biomarkers in plasma from obese patients in order to predict the development of pathological events associated with obesity. Samples are obtained from 22 obese patients and their lean-matched controls are divided into two cohorts: one for a 2D fluorescence difference gel electrophoresis (2D-DIGE)-based study, and the other one for a label free LC-MS/MS-based approach. EVs are isolated following a serial ultracentrifugation protocol. Twenty-two and 23 differentially regulated features are detected from 2D-DIGE and label free LC-MS/MS, respectively; most of them involve in the coagulation and complement cascades. Remarkably, there is an upregulation of complement C4, complement C3, and fibrinogen in obese patients following both approaches, the latter two also validated by 2D-western-blotting in an independent cohort. These results correlate with a proinflammatory and prothrombotic state of those individuals. On the other hand, a downregulation of adiponectin leading to an increased risk of suffering cardiovascular diseases has been shown. The results suggest the relevance of plasma-derived-EVs proteins as a source of potential biomarkers for the development of atherothrombotic events in obesity.

Large-Scale Filter-Aided Sample Preparation Method for the Analysis of the Ubiquitinome.

Protein ubiquitination regulates key cellular functions, including protein homeostasis and signal transduction. The digestion of ubiquitinated proteins with trypsin yields a glycine-glycine remnant bound to the modified lysine residue (K-ε-GG) that can be recognized by specific antibodies for immunoaffinity purification (IAP) and subsequent identification of ubiquitination sites by mass spectrometry. Previous ubiquitinome studies based on this strategy have consistently digested milligram amounts of protein as starting material using in-solution digestion protocols prior to K-ε-GG enrichment. Filter-aided sample preparation (FASP) surpasses in-solution protein digestion in cleavage efficiency, but its performance has thus far been shown for digestion of sample amounts on the order of micrograms. Because cleavage efficiency is pivotal in the generation of the K-ε-GG epitope recognized during IAP, here we developed a large-scale FASP method (LFASP) for digestion of milligram amounts of protein and evaluated its applicability to the study of the ubiquitinome. Our results demonstrate that LFASP-based tryptic digestion is efficient, robust, reproducible, and applicable to the study of the ubiquitinome. We benchmark our results with state-of-the-art ubiquitinome studies and show a ∼3-fold reduction in the proportion of miscleaved peptides with the method presented here. Beyond ubiquitinome analysis, LFASP overcomes the general limitation in sample capacity of standard FASP-based protocols and can therefore be used for a variety of applications that demand a large(r) amount of starting material.

Involvement of exosomes in lung inflammation associated with experimental acute pancreatitis.

A frequent complication of acute pancreatitis is the lung damage associated with the systemic inflammatory response. Although various pro-inflammatory mediators generated at both local and systemic levels have been identified, the pathogenic mechanisms of the disease are still poorly understood. In recent years, exosomes have emerged as a new intercellular communication system able to transfer encapsulated proteins and small RNAs and protect them from degradation. Using an experimental model of taurocholate-induced acute pancreatitis in rats, we aimed to evaluate the role of exosomes in the extent of the systemic inflammatory response. Induction of pancreatitis increased the concentration of circulating exosomes, which showed a different proteomic profile to those obtained from control animals. A series of tracking experiments using PKH26-stained exosomes revealed that circulating exosomes effectively reached the alveolar compartment and were internalized by macrophages. In vitro experiments revealed that exosomes obtained under inflammatory conditions activate and polarize these alveolar macrophages towards a pro-inflammatory phenotype. Interestingly, the proteomic analysis of circulating exosomes during acute pancreatitis suggested a multi-organ origin with a relevant role for the liver as a source of these vesicles. Tracking experiments also revealed that the liver retains the majority of exosomes from the peritoneal cavity. We conclude that exosomes are involved in the lung damage associated with experimental acute pancreatitis and could be relevant mediators in the systemic effects of pancreatitis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Central T cell tolerance: Identification of tissue-restricted autoantigens in the thymus HLA-DR peptidome.

Promiscuous gene expression (pGE) of tissue-restricted self-antigens (TRA) in medullary thymic epithelial cells (mTECs) is in part driven by the Autoimmune Regulator gene (AIRE) and essential for self-tolerance. The link between AIRE functional mutations and multi-organ autoimmunity in human and mouse supports the role of pGE. Deep sequencing of the transcriptome revealed that mouse mTECs potentially transcribe an unprecedented range of >90% of all genes. Yet, it remains unclear to which extent these low-level transcripts are actually translated into proteins, processed and presented by thymic APCs to induce tolerance. To address this, we analyzed the HLA-DR-associated thymus peptidome. Within a large panel of peptides from abundant proteins, two TRA peptides were identified: prostate-specific semenogelin-1 (an autoantigen in autoimmune chronic prostatitis/chronic pelvic pain syndrome) and central nervous system-specific contactin-2 (an autoantigen in multiple sclerosis). Thymus expression of both genes was restricted to mTECs. SEMG1 expression was confined to mature HLA-DR(hi) mTECs of male and female donors and was AIRE-dependent, whereas CNTN2 was apparently AIRE-independent and was expressed by both populations of mTECs. Our findings establish a link between pGE, MHC-II peptide presentation and autoimmunity for bona fide human TRAs.

A collection of open source applications for mass spectrometry data mining.

We present several bioinformatics applications for the identification and quantification of phosphoproteome components by MS. These applications include a front-end graphical user interface that combines several Thermo RAW formats to MASCOT™ Generic Format extractors (EasierMgf), two graphical user interfaces for search engines OMSSA and SEQUEST (OmssaGui and SequestGui), and three applications, one for the management of databases in FASTA format (FastaTools), another for the integration of search results from up to three search engines (Integrator), and another one for the visualization of mass spectra and their corresponding database search results (JsonVisor). These applications were developed to solve some of the common problems found in proteomic and phosphoproteomic data analysis and were integrated in the workflow for data processing and feeding on our LymPHOS database. Applications were designed modularly and can be used standalone. These tools are written in Perl and Python programming languages and are supported on Windows platforms. They are all released under an Open Source Software license and can be freely downloaded from our software repository hosted at GoogleCode.

Secretome analysis of human mesenchymal stem cells undergoing chondrogenic differentiation.

Human mesenchymal stem cells (hMSCs) can be triggered to differentiate toward chondrocytes and thus harbor great therapeutic potential for the repair of cartilage defects in osteoarthritis (OA) and other articular diseases. However, the molecular mechanisms underlying the chondrogenesis process are still in part unknown. In this work, we followed a double-stable isotope labeling by amino acids in cell culture (SILAC) strategy to evaluate the quantitative modulation of the secretome of stem cells isolated from bone marrow (hBMSCs) during the first steps of their chondrogenic differentiation. Analysis by LC-ESI-MS/MS led to the identification of 221 proteins with a reported extracellular localization. Most of them were characteristic of cartilage extracellular matrix, and 34 showed statistically significant quantitative alterations during chondrogenesis. These include, among others, cartilage markers such as Proteoglycan 4 or COMP, anticatabolic markers (TIMP1), reported markers of cartilage development (Versican), and a suggested marker of chondrogenesis, CRAC1. Altogether, our work demonstrates the usefulness of secretome analysis for understanding the mechanisms responsible for cartilage matrix formation, and it reports a panel of extracellular markers potentially useful for the evaluation of tissue development in cell therapy- or tissue engineering-based approaches for cartilage repair.

General statistical framework for quantitative proteomics by stable isotope labeling.

The combination of stable isotope labeling (SIL) with mass spectrometry (MS) allows comparison of the abundance of thousands of proteins in complex mixtures. However, interpretation of the large data sets generated by these techniques remains a challenge because appropriate statistical standards are lacking. Here, we present a generally applicable model that accurately explains the behavior of data obtained using current SIL approaches, including (18)O, iTRAQ, and SILAC labeling, and different MS instruments. The model decomposes the total technical variance into the spectral, peptide, and protein variance components, and its general validity was demonstrated by confronting 48 experimental distributions against 18 different null hypotheses. In addition to its general applicability, the performance of the algorithm was at least similar than that of other existing methods. The model also provides a general framework to integrate quantitative and error information fully, allowing a comparative analysis of the results obtained from different SIL experiments. The model was applied to the global analysis of protein alterations induced by low H2O2 concentrations in yeast, demonstrating the increased statistical power that may be achieved by rigorous data integration. Our results highlight the importance of establishing an adequate and validated statistical framework for the analysis of high-throughput data.

Composition of the HLA-DR-associated human thymus peptidome.

Major histocompatibility complex class II (MHC-II) molecules bind to and display antigenic peptides on the surface of antigen-presenting cells (APCs). In the absence of infection, MHC-II molecules on APCs present self-peptides and interact with CD4(+) T cells to maintain tolerance and homeostasis. In the thymus, self-peptides bind to MHC-II molecules expressed by defined populations of APCs specialised for the different steps of T-cell selection. Cortical epithelial cells present peptides for positive selection, whereas medullary epithelial cells and dendritic cells are responsible for peptide presentation for negative selection. However, few data are available on the peptides presented by MHC molecules in the thymus. Here, we apply mass spectrometry to analyse and identify MHC-II-associated peptides from five fresh human thymus samples. The data show a diverse self-peptide repertoire, mostly consisting of predicted MHC-II high binders. Despite technical limitations preventing single cell population analyses of peptides, these data constitute the first direct assessment of the HLA-II-bound peptidome and provide insight into how this peptidome is generated and how it drives T-cell repertoire formation.

Lipoprotein lipase isoelectric point isoforms in humans.

Lipoprotein lipase (LPL) hydrolyzes circulating triacylglycerols (TAG) into free fatty acids and glycerol. It is present in almost all tissues and its tissue-specific regulation directs the flow of circulating TAG in the body. We demonstrated in a previous study that, in rat heart and post-heparin plasma (PHP), LPL consists of a pattern of more than 8 forms of the same apparent molecular weight, but different isoelectric point (pI). In the present study we describe, for the first time, the existence of at least nine LPL pI isoforms in human PHP, with apparent pI between 6.8 and 8.6. Separation and characterization of these forms was carried out by 2DE combined with Western blotting and mass spectrometry (MALDI-TOF/MS and LC-MS/MS). Further studies are needed to discover their molecular origin, the pattern of pI isoforms in human tissues, their possible physiological functions and possible modifications of their pattern in different pathologies.

Increased CD38 expression in T cells and circulating anti-CD38 IgG autoantibodies differentially correlate with distinct cytokine profiles and disease activity in systemic lupus erythematosus patients.

CD38 is a multifunctional protein possessing ADP-ribosyl cyclase activity responsible for both the synthesis and the degradation of several Ca(2+)-mobilizing second messengers. In mammals, CD38 also functions as a receptor. In this study CD38 expression in CD4(+), CD8(+), or CD25(+) T cells was significantly higher in systemic lupus erythematosus (SLE) patients than in Normal controls. Increased CD38 expression in SLE T cells correlated with plasma levels of Th2 (IL-4, IL-10, IL-13) and Th1 (IL-1ß, IL-12, IFN-γ, TNF-α) cytokines, and was more prevalent in clinically active SLE patients than in Normal controls. In contrast, elevated anti-CD38 IgG autoantibodies were more frequent in clinically quiescent SLE patients (SLEDAI=0) than in Normal controls, and correlated with moderate increased plasma levels of IL-10 and IFN-γ. However, clinically active SLE patients were mainly discriminated from quiescent SLE patients by increased levels of IL-10 and anti-dsDNA antibodies, with odds ratios (ORs) of 3.7 and 4.8, respectively. Increased frequency of anti-CD38 autoantibodies showed an inverse relationship with clinical activity (OR=0.43), and in particular with the frequency of anti-dsDNA autoantibodies (OR=0.21). Increased cell death occurred in CD38(+) Jurkat T cells treated with anti-CD38(+) SLE plasmas, and not in these cells treated with anti-CD38(-) SLE plasmas, or Normal plasmas. This effect did not occur in CD38-negative Jurkat T cells, suggesting that it could be attributed to anti-CD38 autoantibodies. These results support the hypothesis that anti-CD38 IgG autoantibodies or their associated plasma factors may dampen immune activation by affecting the viability of CD38(+) effector T cells and may provide protection from certain clinical SLE features.

Shotgun proteomics to characterize wastewater proteins.

Classically, the characterization of wastewater components has been restricted to the measurement of indirect parameters (chemical and biological oxygen demand, total nitrogen) and small molecules of interest in epidemiology or for environmental control. Despite the fact that metaproteomics has provided important knowledge about the microbial communities in these waters, practically nothing is known about other non-microbial proteins transported in the wastewater. The method described here has allowed us to perform a large-scale characterization of the wastewater proteome. Wastewater protein profiles have shown to be very different in different collection sites probably reflecting their human population and industrial activities. We believe that wastewater proteomics is opening the doors to the discovery of new environmental and health biomarkers and the development of new, more effective monitoring devices for issues like monitorization of population health, pest control, or control of industry discharges. The method developed is relatively simple and combines procedures for the separation of the soluble and particulate fractions of wastewater and their concentration, and conventional shotgun proteomics using high-resolution mass spectrometry for protein identification. •Unprecedented method for wastewater proteome characterization.•Proteins as new potential biomarkers for sewage chemical-information mining, wastewater epidemiology and environmental monitoring.•Wastewater protein profiles reflect human and industrial activities.

The response to fasting and refeeding reveals functional regulation of lipoprotein lipase proteoforms.

Lipoprotein lipase (LPL) is responsible for the intravascular catabolism of triglyceride-rich lipoproteins and plays a central role in whole-body energy balance and lipid homeostasis. As such, LPL is subject to tissue-specific regulation in different physiological conditions, but the mechanisms of this regulation remain incompletely characterized. Previous work revealed that LPL comprises a set of proteoforms with different isoelectric points, but their regulation and functional significance have not been studied thus far. Here we studied the distribution of LPL proteoforms in different rat tissues and their regulation under physiological conditions. First, analysis by two-dimensional electrophoresis and Western blot showed different patterns of LPL proteoforms (i.e., different pI or relative abundance of LPL proteoforms) in different rat tissues under basal conditions, which could be related to the tissue-specific regulation of the enzyme. Next, the comparison of LPL proteoforms from heart and brown adipose tissue between adults and 15-day-old rat pups, two conditions with minimal regulation of LPL in these tissues, yielded virtually the same tissue-specific patterns of LPL proteoforms. In contrast, the pronounced downregulation of LPL activity observed in white adipose tissue during fasting is accompanied by a prominent reconfiguration of the LPL proteoform pattern. Furthermore, refeeding reverts this downregulation of LPL activity and restores the pattern of LPL proteoforms in this tissue. Importantly, this reversible proteoform-specific regulation during fasting and refeeding indicates that LPL proteoforms are functionally diverse. Further investigation of potential differences in the functional properties of LPL proteoforms showed that all proteoforms exhibit lipolytic activity and have similar heparin-binding affinity, although other functional aspects remain to be investigated. Overall, this study demonstrates the ubiquity, differential distribution and specific regulation of LPL proteoforms in rat tissues and underscores the need to consider the existence of LPL proteoforms for a complete understanding of LPL regulation under physiological conditions.

Diabetes mellitus and aortic stenosis head to head: toward personalized medicine in patients with both pathologies.

Diabetes mellitus (DM) and calcific aortic stenosis (CAS) are common morbidities in the elderly, which are both chronic, progressive and often concomitant diseases. Several studies revealed that DM increases the risk of developing severe CAS, yet clear information about the relationship between both these diseases and the influence of DM on the progression of CAS is currently lacking. To evaluate the effect of DM on aortic valves and on the process of calcification, and to achieve better patient management in daily clinical practice, we analysed calcified and noncalcified valve tissue from patients with severe CAS, with or without DM. A proteomic strategy using isobaric tags was adopted and the plasma concentrations of nine proteins were studied using 3 orthogonal methods and in a separate cell model. The differentially expressed proteins identified are implicated in biological processes like endopeptidase activity, lipid metabolism, coagulation, and fibrinolysis. The results obtained provide evidence that DM provokes changes in the proteome of aortic valves, affecting valve calcification. This finding may help enhance our understanding of the pathogenesis of CAS and how DM affects the evolution of this condition, an important step in identifying targets to personalize the treatment of these patients.

Noncanonical splicing junctions between exons and transposable elements represent a source of immunogenic recurrent neo-antigens in patients with lung cancer.

Although most characterized tumor antigens are encoded by canonical transcripts (such as differentiation or tumor-testis antigens) or mutations (both driver and passenger mutations), recent results have shown that noncanonical transcripts including long noncoding RNAs and transposable elements (TEs) can also encode tumor-specific neo-antigens. Here, we investigate the presentation and immunogenicity of tumor antigens derived from noncanonical mRNA splicing events between coding exons and TEs. Comparing human non-small cell lung cancer (NSCLC) and diverse healthy tissues, we identified a subset of splicing junctions that is both tumor specific and shared across patients. We used HLA-I peptidomics to identify peptides encoded by tumor-specific junctions in primary NSCLC samples and lung tumor cell lines. Recurrent junction-encoded peptides were immunogenic in vitro, and CD8+ T cells specific for junction-encoded epitopes were present in tumors and tumor-draining lymph nodes from patients with NSCLC. We conclude that noncanonical splicing junctions between exons and TEs represent a source of recurrent, immunogenic tumor-specific antigens in patients with NSCLC.