Mediterranean viticulture is increasingly affected by salinity since decades and it was necessary to develop new salt-tolerant varieties and rootstocks to ensure its productivity and sustainability. Among these alternatives, wild grapevine species, revealed to possess untapped genetic diversity and valuable traits that can be effectively utilized for grapevine varieties improvement. Until now, no work focused on the proteomic modifications in wild grapevine plants in response to salt stress. Here, we report a comparative physiological and proteomic analysis carried out on a salt-tolerant Tunisian wild grapevine (Vitis vinifera L. ssp. sylvestris), named "Tebaba", exposed to salt stress for more than two weeks. Physiological analyses demonstrated the ability of “Tebaba” accession to maintain its growth and photosynthetic activity along the salt stress period. The proteomic analysis revealed changes in many proteins associated with various molecular processes, including carbohydrate and energy metabolism, photosynthesis, stress response and defense, protein synthesis/processing/degradation, amino acids and nitrogen metabolism, signal transduction and secondary metabolites. The Tunisian wild grapevine accession "Tebaba" was able to preserve its physiological levels of growth and photosynthesis. It was able to stimulate its antioxidant activities and its Calvin cycle. Its salt tolerance was mainly expressed by a remobilization of energy, an up-regulation of the elongation factor, ubiquitin, and chaperone proteins, as well as an increase in amino acids. To our knowledge, this is the first proteomic study on the mechanisms of salt tolerance in wild grapevine (Vitis sylvestris L. ssp. sylvestris).

Prevention and treatment of obesity using nutritional supplements became a realistic and effective approach. The present work aims at the assessment of the potential protection afforded by grape seed extract (GSE) on the rat cardiac proteome using a proteomic approach. Using a rodent model of obesity, we performed a comparative quantitative proteomic approach of heart samples from standard, high-fat diet (HFD) and HFD+GSE groups. LTQ OrbiTrap technology, followed by bioinformatic tools, allowed the identification of differentially abundant proteins (DAPs). GSE alleviated the HFD-induced energy metabolism alteration by recovering and/or enhancing some DAPs, such as the aconitase and the propionyl-CoA carboxylase. Also, GSE renews the capacity of cardiomyocytes to produce energy by an overabundance of complex I and complex III. Additionally, GSE improved the contractile function and alleviated cardiomyopathy in obesity following an overabundance of key contractile proteins like myosins and actinin. This study demonstrates that GSE confers cardioprotective effects in the context of obesity-induced metabolic disturbances. Through a proteomic approach, we identified key DAPs involved in energy metabolism and contractile function that were positively modulated by GSE. These findings support the potential of GSE as a promising therapeutic anti-obesity agent.

When a biomedical device is implanted into the body, its surface initially encounters biological fluids, resulting in the natural adsorption of various host proteins. This protein-adsorbed layer alters the inherent properties of the biomaterial surface and plays a crucial role in interactions between the implant and bacteria. Here, we investigated the influence of an adsorbed layer of albumin, the most abundant blood protein, on the adhesion and biofilm formation of three different bacterial strains: Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa. We found that the effect of a serum albumin layer on bacterial adhesion was straindependent. Albumin pre-adsorption reduced the initial attachment of S. aureus and S. epidermidis to PDMS surfaces but had no impact on P. aeruginosa. However, with prolonged incubation, albumin-coated surfaces significantly promoted P. aeruginosa attachment and biofilm formation. Additionally, this biofilm alteration was associated with reduced macrophage-mediated bacterial clearance. Proteomic analysis further revealed significant physiological changes in P. aeruginosa upon exposure to albumin-coated surfaces compared to uncoated controls. These alterations were particularly associated with molecular pathways involved in surface colonization, including quorum sensing, motility, adhesion, and biofilm formation. These findings suggest that serum albumin adsorption not only affects the initial adhesion of bacteria but also modifies their adaptive responses upon contact with the biomaterial surface. This study provides a deeper understanding of the complex bacteriasurface interactions, contributing to the development of future strategies for preventing implant-associated infections.

The resistance of Chronic Lymphocytic Leukemia (CLL) B-cells to cell death is mainly attributed to interactions within their microenvironment, where they interact with various types of cells. Within this microenvironment, CLL-B-cells produce and bind cytokines, growth factors, and extracellular vesicles (EVs). In the present study, EVs purified from nurse-like cells and M2-polarized THP1 cell (M2-THP1) cultures were added to CLL-B-cells cultures. EVs were rapidly internalized by B-cells, leading to a decrease in apoptosis (P = 0.0162 and 0.0469, respectively) and an increased proliferation (P = 0.0335 and 0.0109). Additionally, they induced an increase in the resistance of CLL-B-cells to Ibrutinib, the Bruton kinase inhibitor in vitro (P = 0.0344). A transcriptomic analysis showed an increase in the expression of anti-apoptotic gene BCL-2 (P = 0.0286) but not MCL-1 and an increase in the expression of proliferation-inducing gene APRIL (P = 0.0286) following treatment with EVs. Meanwhile, an analysis of apoptotic protein markers revealed increased amounts of IGFBP-2 (P = 0.0338), CD40 (P = 0.0338), p53 (P = 0.0219) and BCL-2 (P = 0.0338). Finally, exploration of EVs protein content by mass spectrometry revealed they carry various proteins involved in known oncogenic pathways and the RNAseq analysis of CLL-B-cells treated or not with NLCs EVs show various differentially expressed genes.

Abstract Idiopathic inflammatory myopathies (IIMs) are severe autoimmune diseases with poorly understood pathogenesis and unmet medical needs. Here, we examine the role of interferon γ (IFNγ) using NOD female mice deficient in the inducible T cell co-stimulator (Icos), which have previously been shown to develop spontaneous IFNγ-driven myositis mimicking human disease. Using muscle proteomic and spatial transcriptomic analyses we reveal profound myofiber metabolic dysregulation in these mice. In addition, we report muscle mitochondrial abnormalities and oxidative stress in diseased mice. Supporting a pathogenic role for oxidative stress, treatment with a reactive oxygen species (ROS) buffer compound alleviated myositis, preserved muscle mitochondrial ultrastructure and respiration, and reduced inflammation. Mitochondrial anomalies and oxidative stress were diminished following anti-IFNγ treatment. Further transcriptomic analysis in IIMs patients and human myoblast in vitro studies supported the link between IFNγ and mitochondrial dysfunction observed in mice. These results suggest that mitochondrial dysfunction, ROS and inflammation are interconnected in a self-maintenance loop, opening perspectives for mitochondria therapy and/or ROS targeting drugs in myositis.

Raine syndrome (RNS) is a rare autosomal recessive osteosclerotic dysplasia. RNS is caused by loss-of-function disease-causative variants of the FAM20C gene that encodes a kinase that phosphorylates most of the secreted proteins found in the body fluids and extracellular matrix. The most common RNS clinical features are generalized osteosclerosis, facial dysmorphism, intracerebral calcifications and respiratory defects. In non-lethal RNS forms, oral traits include a well-studied hypoplastic amelogenesis imperfecta (AI) and a much less characterized gingival phenotype. We used immunomorphological, biochemical, and siRNA approaches to analyze gingival tissues and primary cultures of gingival fibroblasts of two unrelated, previously reported RNS patients. We showed that fibrosis, pathological gingival calcifications and increased expression of various profibrotic and pro-osteogenic proteins such as POSTN, SPARC and VIM were common findings. Proteomic analysis of differentially expressed proteins demonstrated that proteins involved in extracellular matrix (ECM) regulation and related to the TGFβ/SMAD signaling pathway were increased. Functional analyses confirmed the upregulation of TGFβ/SMAD signaling and subsequently uncovered the involvement of two closely related transcription cofactors important in fibrogenesis, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Knocking down of FAM20C confirmed the TGFβ-YAP/TAZ interplay indicating that a profibrotic loop enabled gingival fibrosis in RNS patients. In summary, our in vivo and in vitro data provide a detailed description of the RNS gingival phenotype. They show that gingival fibrosis and calcifications are associated with, and most likely caused by excessed ECM production and disorganization. They furthermore uncover the contribution of increased TGFβ–YAP/TAZ signaling in the pathogenesis of the gingival fibrosis.

Previously, we pointed out in P. aeruginosa PAO1 biofilm cells the accumulation of a hypothetical protein named PA3731 and showed that the deletion of the corresponding gene impacted its biofilm formation capacity. PA3731 belongs to a cluster of 4 genes (pa3732 to pa3729) that we named bac for “Biofilm Associated Cluster.” The present study focuses on the PA14_16140 protein, i.e., the PA3732 (BacA) homolog in the PA14 strain. The role of BacA in rhamnolipid secretion, biofilm formation and virulence, was confirmed by phenotypic experiments with a bacA mutant. Additional investigations allow to advance that the bac system involves in fact 6 genes organized in operon, i.e., bacA to bacF. At a molecular level, quantitative proteomic studies revealed an accumulation of the BAC cognate partners by the bacA sessile mutant, suggesting a negative control of BacA toward the bac operon. Finally, a first crystallographic structure of BacA was obtained revealing a structure homologous to chaperones or/and regulatory proteins.

The resistance of leukemic B cells to cell death is mainly associated with interactions within the tumor microenvironment, where they interact with various types of cells. Within this microenvironment, CLL B cells produce and bind cytokines, growth factors, and microvesicles. In the present study, extracellular vesicles (EVs) purified from nurse-like cells and M2-polarized THP1 cell (M2-THP1) cultures were added to CLL B cell cultures. The vesicles were rapidly internalized by B cells, leading to a decrease in spontaneous apoptosis and an increase in proliferation. Additionally, the vesicles induced an increase in the resistance of CLL B cells to Bruton kinase inhibitor Ibrutinib. A transcriptomic analysis showed an increase in the expression of anti-apoptotic gene Bcl-2 but not Mcl-1 and an increase in the expression of proliferation-inducing gene APRIL following treatment of CLL B cells with EVs derived from both NLCs and M2-THP1 cells. Meanwhile, an analysis of apoptotic protein markers revealed increased amounts of IGFBP-2, CD40, p53 and Bcl-2 following EVs treatment. Finally, exploration of EVs protein content by mass spectrometry revealed they carry various proteins involved in known oncogenic pathways and the RNAseq analysis of B CLL cells treated or not with NLCs EVs show various differentially expressed genes.

AIMS:Idiopathic inflammatory myopathies (IIM) are autoimmune inflammatory disorders leading to skeletal muscle weakness and disability. The pathophysiology of IIM is poorly understood due to the scarcity of animal disease models. Genetic deletion of Icos or Icosl (inducible T cell co-stimulator/ligand) in non-obese diabetic (NOD) mice leads to muscle disease. Our aim was to characterise Icos-/- NOD myopathy and to search for novel autoantibodies (aAbs) in this model.METHODS:Diabetes, weight, myopathy incidence/clinical score and grip strength were assessed over time. Locomotor activity was analysed with the Catwalk XT gait analysis system. Muscle histology was evaluated in haematoxylin/eosin and Sirius red-stained sections, and immune infiltrates were characterised by immunofluorescence and flow cytometry. 2D gel electrophoresis of muscle protein extracts and mass spectrometry were used to identify novel aAbs. NOD mice were immunised with troponin T3 (TNNT3) in incomplete Freund's adjuvant (IFA) and R848. An addressable laser bead immunoassay (ALBIA) was developed to measure aAb IgG serum levels.RESULTS:Icos-/- NOD mice did not exhibit diabetes but developed spontaneous progressive myositis with decreased muscle strength and altered locomotor activity. Muscle from these mice exhibited myofibre necrosis, myophagocytosis, central nuclei, fibrosis and perimysial and endomysial cell infiltrates with macrophages and T cells. We identified anti-TNNT3 aAbs in diseased mice. Immunisation of NOD mice with murine TNNT3 protein led to myositis development, supporting its pathophysiological role.CONCLUSIONS:These data show that Icos-/- NOD mice represent a spontaneous model of myositis and the discovery of anti-TNNT3 aAb suggests a new autoantigen in this model.

The root extracellular trap (RET) has emerged as a specialized compartment consisting of root AC-DC and mucilage. However, the RET's contribution to plant defense is still poorly understood. While the roles of polysaccharides and glycoproteins secreted by root AC-DC have started to be elucidated, how the low-molecular-weight exudates of the RET contribute to root defense is poorly known. In order to better understand the RET and its defense response, the transcriptomes, proteomes and metabolomes of roots, root AC-DC and mucilage of soybean (Glycine max (L.) Merr, var. Castetis) upon elicitation with the peptide PEP-13 were investigated. This peptide is derived from the pathogenic oomycete Phytophthora sojae. In this study, the root and the RET responses to elicitation were dissected and sequenced using transcriptional, proteomic and metabolomic approaches. The major finding is increased synthesis and secretion of specialized metabolites upon induced defense activation following PEP-13 peptide elicitation. This study provides novel findings related to the pivotal role of the root extracellular trap in root defense.

Drought is one of the major environmental constraints threatening viticulture worldwide. Therefore, it is critical to reveal the molecular mechanisms underlying grapevine (Vitis vinifera L.) drought stress tolerance useful to select new species with higher tolerance/resilience potentials. Drought-tolerant Tunisian local grapevine cultivar Razegui was exposed to water deficit for 16days. Subsequent proteomic analysis revealed 49 differentially accumulated proteins in leaves harvested on the drought-stressed vines. These proteins were mainly involved in photosynthesis, stress defence, energy and carbohydrate metabolism, protein synthesis/turnover and amino acid metabolism. Physiological analysis revealed that reduction of photosynthesis under drought stress was attributed to the downregulation of the light-dependent reactions, Calvin cycle and key enzymes of the photorespiration pathway. The accumulation of proteins involved in energy and carbohydrate metabolism indicate enhanced need of energy during active stress acclimation. Accumulation of protein amino acids seems to play a protective role under drought stress due to their osmoprotectant and ROS scavenging potential. Reduced protein synthesis and turnover help plants preserving energy to fight drought stress. Proteins related to stress defence might scavenge ROS and transmit the ROS signal as an oxidative signal transducer in drought-stress signalling. All of these original results represent valuable information towards improving drought tolerance of grapevine and promoting sustainable viticulture under climate change conditions.

Background To validate the ability of PROS (vitamin K-dependent protein S) and CO7 (complement component C7) to predict response to the methotrexate (MTX)/etanercept (ETA) combination in rheumatoid arthritis (RA) patients who received this therapeutic combination in a well-documented cohort. Method From the ESPOIR cohort, RA patients having received the MTX/ETA or MTX/adalimumab (ADA) combination as a first-line biologic treatment were included. Serum concentrations of PROS and CO7 were measured by ELISA prior to the initiation of ETA or ADA, at a time where the disease was active (DAS28 ESR > 3.2). The clinical efficacy (response/non-response) of both combinations has been evaluated after at least 6 months of treatment, according to the EULAR response criteria with some modifications. Results Thirty-two were treated by MTX/ETA; the numbers of responders and non-responders were 24 and 8, respectively. Thirty-three patients received the MTX/ADA combination; 27 and 5 patients were respectively responders and non-responders. While there were no differences for demographic, clinical, biological, and X-rays data, as well as for CO7, serum levels of PROS tended to be significantly higher in responders to the MTX/ETA combination ( p = 0.08) while no difference was observed in the group receiving MTX/ADA. For PROS, the best concentration threshold to differentiate both groups was calculated at 40 μg/ml using ROC curve. The theranostic performances of PROS appeared better for the ETA/MTX combination. When considering the response to this combination, analysis of pooled data from ESPOIR and SATRAPE (initially used to validate PROS and CO7 as potential theranostic biomarkers) cohorts led to a higher theranostic value of PROS that became significant ( p = 0.009). Conclusion PROS might be one candidate of a combination of biomarkers capable of predicting the response to MTX/ETA combination in RA patients refractory to MTX. Trial registration ClinicalTrials.gov identifiers: NCT03666091 and NCT00234234 .

Context The liver is the organ by which the majority of substances are metabolized, including psychotropic drugs. Lithium (Li) used as drug for many neurological disorders such as bipolar disorders. Objective This study aims to assess lithium toxicity and to evaluate the hepatic-protective properties of a grape skin seed and extract (GSSE). Materials and methods Twenty-four male Wistar rats were exposed for 30 days to either various lithium concentrations, GSSE alone, or lithium supplemented with GSSE. The proteomic analysis revealed alterations of liver protein profiles after lithium treatments that were successfully identified by mass spectrometry. Results Lithium treatment induced an oxidative damage by the alteration of antioxidant enzymes activities such as superoxide dismutase, CAT, and Gpx. The regulated proteins are mainly involved in the respiratory electron transport chain, detoxification processes, ribosomal stress pathway, glycolysis, and cytoskeleton. Proteins were differentially expressed in a dose-dependent manner. Interestingly, GSSE reversed the situation and restored the level of liver proteins whose abundance was modified after lithium treatment, arguing for its protective activity. Conclusion Our data demonstrated the ability of proteomic analysis to underline the toxicity mechanisms of lithium in animal models. Based on these results, GSSE may be envisaged as a nutritional supplement to weaken the liver toxicity of lithium.

Sexual selection is the basis of some of the most striking phenotypic variation in nature.1,2 In animals, sexual selection in males can act on traits that improve access to mates prior to copulation,3-8 but also on sperm traits filtered by sperm competition,9-14 or female choice expressed simply by the morphology and physiology of genital tracts.14-16 Although long overlooked as a mode of selection on plant traits, sexual selection should act on land plants too because they are anisogamous: males produce more, and smaller, gametes than females.17-19 Numerical asymmetry in gamete production is thought to play a central role in selection on traits that affect pollen transfer to mates,20,21 but very little is known about how pollen competition or cryptic female choice might affect the evolution of traits expressed after pollination.22,23 Here, we report the divergence of pollen and pistil traits of the dioecious wind-pollinated annual herb Mercurialis annua during evolution over three generations between populations at low versus high plant density, corresponding to low versus higher levels of polyandry;24 we expected selection under higher polyandry to strengthen competition among pollen donors for fertilizing ovules. We found that populations at high density evolved faster-growing pollen tubes (an equivalent of greater sperm velocity), greater expression of pollen proteins involved in pollen growth, and larger stigmas (a trait likely enhancing the number of pollen donors and thus competition for ovules). Our results identify the post-pollination phase of plant mating as an important arena for the action of sexual selection.

The enamel renal syndrome (ERS) is a rare disorder featured by amelogenesis imperfecta, gingival fibromatosis and nephrocalcinosis. ERS is caused by bi-allelic mutations in the secretory pathway pseudokinase FAM20A. How mutations in FAM20A may modify the gingival connective tissue homeostasis and cause fibromatosis is currently unknown. We here analyzed conditioned media of gingival fibroblasts (GFs) obtained from four unrelated ERS patients carrying distinct mutations and control subjects. Secretomic analysis identified 109 dysregulated proteins whose abundance had increased (69 proteins) or decreased (40 proteins) at least 1.5-fold compared to control GFs. Proteins over-represented were mainly involved in extracellular matrix organization, collagen fibril assembly, and biomineralization whereas those under-represented were extracellular matrix-associated proteins. More specifically, transforming growth factor-beta 2, a member of the TGFβ family involved in both mineralization and fibrosis was strongly increased in samples from GFs of ERS patients and so were various known targets of the TGFβ signaling pathway including Collagens, Matrix metallopeptidase 2 and Fibronectin. For the over-expressed proteins quantitative RT-PCR analysis showed increased transcript levels, suggesting increased synthesis and this was further confirmed at the tissue level. Additional immunohistochemical and western blot analyses showed activation and nuclear localization of the classical TGFβ effector phospho-Smad3 in both ERS gingival tissue and ERS GFs. Exposure of the mutant cells to TGFB1 further upregulated the expression of TGFβ targets suggesting that this pathway could be a central player in the pathogenesis of the ERS gingival fibromatosis.In conclusion our data strongly suggest that TGFβ -induced modifications of the extracellular matrix contribute to the pathogenesis of ERS. To our knowledge this is the first proteomic-based analysis of FAM20A-associated modifications.

We previously showed that the physiological concentration of 17β-estradiol in the vaginal environment is sufficient to affect the membrane dynamics and adhesion phenotype of the Lactobacillus crispatus strain CIP104459. However, L. crispatu s is a heterogeneous species. Here, we investigated the effect of 17β-estradiol on the recently isolated L. crispatus vaginal strain V4, related to a cluster distant from CIP104459 and at the limit of being a different subspecies. Grown in the same medium, the two strains expressed a highly similar pool of proteins. However, in contrast to CIP104459, L. crispatus V4 showed high aggregation potential and 17β-estradiol promoted this phenotype. This effect was associated with large changes in cell-surface polarity and Lewis acid/base properties. In addition, we observed no effect on the membrane dynamics, contrary to CIP104459. These results can be explained by differences in the properties and organization of the S layer between the two strains. However, as for CIP104459, 17β-estradiol increased biosurfactant production of L. crispatus V4 and their adhesion to vaginal cells. This suggests that 17β-estradiol agonists would be valuable tools to favor a stable re-implantation of L. crispatus in the vaginal mucosa.

To identify the targets recognized by anti-carbamylated protein antibodies (anti-CarP) in patients with early Rheumatoid Arthritis (RA), to study the cross-reactivity between anti-CarP and anti-citrullinated protein antibodies (ACPA) and to evaluate their prognostic value. 331 patients (184 RA and 147 other rheumatisms) from the Very Early Arthritis (VErA) French cohort were analyzed. We performed mass spectrometry analysis of RA sera displaying anti-CarP activity and epitope mapping of the carbamylated fibrinogen γ chain to identify immunodominant peptides. The specificity of these targets was studied using competition assays with the major antigens recognized by ACPA. The prognostic value of anti-carbamylated fibrinogen IgG antibodies (ACa-Fib IgG) was compared to that of anti-cyclic citrullinated peptide antibodies (anti-CCP) and anti-CarP using an in-house ELISA. Besides the α chain, the γ chain of fibrinogen, particularly one immunodominant epitope that has a specific reactivity, was identified as a circulating carbamylated target in sera. The prevalence of ACa-Fib was 37% at baseline and 10.9% for anti-CCP-negative RA. In anti-CCP-negative patients, ACa-Fib positivity was associated with a more inflammatory and erosive disease at baseline but not with rapid radiological progression, which remains strongly related to anti-CCP antibodies. Fibrinogen seems to be one of the antigens recognized in vivo by the anti-CarP response, particularly 2 epitopes of the γ chain, one of which is not cross reactive with ACPA. This specificity might be associated with a distinct clinical phenotype since ACa-Fib IgG were shown to be linked to systemic inflammation in very early RA but not to rapid radiological progression.

Acinetobacter baumannii is a problematic nosocomial pathogen owing to its increasing resistance to antibiotics and its great ability to survive in the hospital environment, which is linked to its capacity to form biofilms. Structural and functional investigations of post-translational modifications, such as phosphorylations, may lead to identification of candidates for therapeutic targets against this pathogen. Here, we present the first S/T/Y phosphosecretome of two A. baumannii strains, the reference strain ATCC 17978 and the virulent multi-drug resistant strain AB0057, cultured in two modes of growth (planktonic and biofilm) using TiO2 chromatography followed by high resolution mass spectrometry. In ATCC 17978, we detected a total of 137 (97 phosphoproteins) and 52 (33 phosphoproteins) phosphosites in biofilm and planktonic modes of growth, respectively. Similarly, in AB0057, 155 (119 phosphoproteins) and 102 (74 phosphoproteins) phosphosites in biofilm and planktonic modes of growth were identified, respectively. Both strains in the biofilm mode of growth showed a higher number of phosphosites and phosphoproteins compared to planktonic growth. Several phosphorylated sites are localized in key regions of proteins involved in either drug resistance (β-lactamases), adhesion to host tissues (pilins), or protein secretion (Hcp). Site-directed mutagenesis of the Hcp protein, essential for type VI secretion system-mediated interbacterial competition, showed that four of the modified residues are essential for type VI secretion system activity.

Described herein is a quinoxalinone-based photoaffinity probe with caged fluorescence properties. Upon visible blue LED irradiation (λmax 450 nm), this photo-crosslinker is able to covalently capture proteins with concomitant fluorescence labelling. This process enables monitoring applications under "no wash" conditions.