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Goat cheese is an important element of the Mediterranean diet, appreciated for its health-promoting features and unique taste. A pivotal role in the development of these characteristics is attributed to the microbiota and its continuous remodeling over space and time. Nevertheless, no thorough study of the cheese-associated microbiota using two metaomics approaches has previously been conducted. Here, we employed 16S rRNA gene sequencing and metaproteomics to explore the microbiota of a typical raw goat milk cheese at various ripening timepoints and depths of the cheese wheel. The 16S rRNA gene-sequencing and metaproteomics results described a stable microbiota ecology across the selected ripening timepoints, providing evidence for the microbiologically driven fermentation of goat milk products. The important features of the microbiota harbored on the surface and in the core of the cheese mass were highlighted in both compositional and functional terms. We observed the rind microbiota struggling to maintain the biosafety of the cheese through competition mechanisms and/or by preventing the colonization of the cheese by pathobionts of animal or environmental origin. The core microbiota was focused on other biochemical processes, supporting its role in the development of both the health benefits and the pleasant gustatory nuances of goat cheese.

Volatile organic compounds (VOCs) are a differentiated class of molecules, continuously generated in the human body and released as products of metabolic pathways. Their concentrations vary depending on pathophysiological conditions. They are detectable in a wide variety of biological samples, such as exhaled breath, faeces, and urine. In particular, urine represents an easily accessible specimen widely used in clinics. The most used techniques for VOCs detections are expensive and time-consuming, thus not allowing for rapid clinical analysis. In this perspective, the aim of this study is a comprehensive characterisation of the urine volatilome by the development of an alternative rapid analytical method. Briefly, 115 urine samples are collected; sample treatment is not needed. VOCs are detected in the urine headspace using gas chromatography coupled to ion mobility spectrometry (GC–IMS) by an extremely fast analysis (10 min). The method is analytically validated; the analysis is sensitive and robust with results comparable to those reported with other techniques. Twenty-three molecules are identified, including ketones, aldehydes, alcohols, and sulphur compounds, whose concentration is altered in several pathological states such as cancer and metabolic disorders. Therefore, it opens new perspectives for fast diagnosis and screening, showing great potential for clinical applications.

Background Goat cheese is an important element of the Mediterranean diet, appreciated for its health-promoting features and the typical gustatory essences. A pivotal role in the development of these characteristics is attributed to the hosted microbiota and its continuous remodeling over the space and time. Nevertheless, a thorough study of the cheese-associated microbiota by two meta-omics approaches is still missing. Also, the study of these model systems is important in the One Health context as they enable the development of systems for monitoring environmental micro-organisms and their biological variability. In this study we employed 16S rRNA gene sequencing and metaproteomics to explore the microbiota of a typical raw goat milk cheese at diverse ripening timepoints and depths of the cheese wheel. Results findings from 16S rRNA gene sequencing and metaproteomics described a stable microbiota ecology across the selected ripening timepoints, providing evidence on the microbiologically driven fermentation of the goat milk products. On the other hand, important modeling of the microbiota harbored in the surface and core of the cheese mass are highlighted both in compositional and functional terms. Conclusion observed outcomes portrait the rind microbiota struggling for the maintenance of the cheese biosafety through competition mechanisms and/or preventing the cheese colonization by pathobionts of animal or environmental origin. Efforts in microbial competition are also accomplished in the core microbiota, although its further focusing on other biochemical routes supports the role of this microbiota in the development of both the health beneficial effects and the pleasant gustatory nuances of the goat cheeses.

Antimicrobial resistance (AMR) represents one of the most critical challenges that humanity will face in the following years. In this context, a “One Health” approach with an integrated multidisciplinary effort involving humans, animals and their surrounding environment is needed to tackle the spread of AMR. One of the most common ways for bacteria to live is to adhere to surfaces and form biofilms. Staphylococcus aureus (S. aureus) can form biofilm on most surfaces and in a wide heterogeneity of environmental conditions. The biofilm guarantees the survival of the S. aureus in harsh environmental conditions and represents an issue for the food industry and animal production. The identification and characterization of biofilm-related proteins may provide interesting insights into biofilm formation mechanisms in S. aureus. In this regard, the aims of this study were: (i) to use proteomics to compare proteomes of S. aureus growing in planktonic and biofilm forms in order to investigate the common features of biofilm formation properties of different strains, (ii) to identify specific biofilm mechanisms that may be involved in AMR. The proteomic analysis showed 14 differentially expressed proteins among biofilm and planktonic forms of S. aureus. Moreover, three proteins, such as alcohol dehydrogenase, ATP-dependent 6-phosphofructokinase, and fructose-bisphosphate aldolase, were only differentially expressed in strains classified as high biofilm producers. Differentially regulated catabolites metabolisms and the switch to lower oxygen-related metabolisms were related to the sessile conformation analyzed.

Brucellosis caused by Brucella melitensis is a zoonosis frequently reported in the Mediterranean and Middle-East regions and responsible for important economic losses and reduced animal welfare. To date, current strategies applied to control or eradicate the disease relies on diagnostic tests that suffer from limited specificity in non-vaccinated animals; while prophylactic measures, when applied, use a live attenuated bacterial strain characterized by residual virulence on adult pregnant animals and difficulties in distinguishing vaccinated from infected animals. To overcome these issues, studies are desired to elucidate the bacterial biology and the pathogenetic mechanisms of both the vaccinal strain and the pathogenic strains. Proteomics has a potential in tackling issues of One-Health concern; here, we employed label-free shotgun proteomics to investigate the protein repertoire of the vaccinal strain B. melitensis Rev.1 and compare it with the proteome of the Brucella melitensis 16 M, a reference strain representative of B. melitensis field strains. Comparative proteomics profiling underlines common and diverging traits between the two strains. Common features suggest the potential biochemical routes responsible for the residual virulence of the vaccinal strain, whilst the diverging traits are suggestive biochemical signatures to be further investigated to provide an optimized diagnostic capable of discriminating the vaccinated from infected animals. The data presented in this study are openly available in PRIDE data repository at https://www.ebi.ac.uk/pride/ , reference number PXD022472.

Over the past 30 years a considerable amount of data has accumulated on the multifaceted role of hydrogen sulfide (H2S) in the central nervous system. Depending on its concentrations, H2S has opposite actions, ranging from neuromodulator to neurotoxic. Nowadays, accurate determination of H2S is still an important challenge to understand its biochemistry and functions. In this perspective, this study aims to explore H2S levels in cerebrospinal fluid (CSF), key biofluid for neurological studies, and to assess alleged correlations with neuroinflammatory and neurodegenerative mechanisms. A validated analytical determination combining selective electrochemical detection with ion chromatography was developed to measure free and bound sulfur forms of H2S. A first cohort of CSF samples (n = 134) was analyzed from patients with inflammatory and demyelinating disorders (acute disseminated encephalomyelitis, multiple sclerosis), chronic neurodegenerative diseases (Alzheimer disease, Parkinson disease), and motor neuron disease (Amyotrophic lateral sclerosis). Given its analytical features, the chromatographic method resulted sensitive, reproducible and robust. We also explored low molecular weight-proteome linked to sulphydration by proteomics analysis on matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). This study is a first clinical report on CSF H2S concentrations from neurological diseases and opens up new perspectives on the potential clinical relevance of H2S and its potential therapeutic application.

Recent advances in the field of meta-omics sciences and related bioinformatics tools have allowed a comprehensive investigation of human-associated microbiota and its contribution to achieving and maintaining the homeostatic balance. Bioactive compounds from the microbial community harboring the human gut are involved in a finely tuned network of interconnections with the host, orchestrating a wide variety of physiological processes. These includes the bi-directional crosstalk between the central nervous system, the enteric nervous system, and the gastrointestinal tract (i.e., gut–brain axis). The increasing accumulation of evidence suggest a pivotal role of the composition and activity of the gut microbiota in neurodegeneration. In the present review we aim to provide an overview of the state-of-the-art of meta-omics sciences including metagenomics for the study of microbial genomes and taxa strains, metatranscriptomics for gene expression, metaproteomics and metabolomics to identify and/or quantify microbial proteins and metabolites, respectively. The potential and limitations of each discipline were highlighted, as well as the advantages of an integrated approach (multi-omics) to predict microbial functions and molecular mechanisms related to human diseases. Particular emphasis is given to the latest results obtained with these approaches in an attempt to elucidate the link between the gut microbiota and the most common neurodegenerative diseases, such as multiple sclerosis (MS), Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS).

More than 300 different protein post-translational modifications are currently known, but only a few have been extensively investigated because modified proteoforms are commonly present in sub-stoichiometry amount. For this reason, improvement of specific enrichment techniques is particularly useful for the proteomic characterization of post-translationally modified proteins. Enrichment proteomic strategies could help the researcher in the challenging issue to decipher the complex molecular cross-talk existing between the different factors influencing the cellular pathways. In this review the state of art of the platforms applied for the enrichment of specific and most common post-translational modifications, such as glycosylation and glycation, phosphorylation, sulfation, redox modifications (i.e. sulfydration and nitrosylation), methylation, acetylation, and ubiquitinylation, are described. Enrichments strategies applied to characterize less studied post-translational modifications are also briefly discussed.

The environment, including animals and animal products, is colonized by bacterial species that are typical and specific of every different ecological niche. Natural and human-related ecological pressure promotes the selection and expression of genes related to antimicrobial resistance (AMR). These genes might be present in a bacterial consortium but might not necessarily be expressed. Their expression could be induced by the presence of antimicrobial compounds that could originate from a given ecological niche or from human activity. In this work, we applied (meta)proteomics analysis of bacterial compartment of raw milk in order to obtain a method that provides a measurement of circulating AMR involved proteins and gathers information about the whole bacterial composition. Results from milk analysis revealed the presence of 29 proteins/proteoforms linked to AMR. The detection of mainly &beta, lactamases suggests the possibility of using the milk microbiome as a bioindicator for the investigation of AMR. Moreover, it was possible to achieve a culture-free qualitative and functional analysis of raw milk bacterial consortia.

In the framework of the Human Proteome Project initiative, we aim to improve mapping and characterization of mitochondrial proteome. In this work we implemented an experimental workflow, combining classical biochemical enrichments and mass spectrometry, to pursue a much deeper definition of mitochondrial proteome and possibly mine mitochondrial uncharacterized dark proteins. We fractionated in two compartments mitochondria enriched from HeLa cells in order to annotate 4230 proteins in both fraction by means of a multiple-enzyme digestion (trypsin, chymotrypsin and Glu-C) followed by mass spectrometry analysis using a combination of Data Dependent Acquisition (DDA) and Data Independent Acquisition (DIA). We detected 22 mitochondrial dark proteins not annotated for their function and we provide their relative abundance inside the mitochondrial organelle. Considering this work as a pilot study we expect that the same approach, in different biological system, could represent an advancement in the characterization of the human mitochondrial proteome providing uncharted ground to explore the mitonuclear phenotypic relationships. All spectra have been deposited to ProteomeXchange with PXD014201 and PXD014200 identifier.

Background Malignant pleural mesothelioma (MPM) a rare neoplasm linked to asbestos exposure is characterized by a poor prognosis. Soluble mesothelin is currently considered the most specific diagnostic biomarker. The aim of the study was to identify novel biomarkers by proteomic analysis of two MPM cell lines secretome. Materials and methods The protein patterns of MPM cells secretome were examined and compared to a non-malignant mesothelial cell line using two-dimensional gel electrophoresis coupled to mass spectrometry. Serum levels of candidate biomarkers were determined in MPM patients and control subjects. Results Two up-regulated proteins involved in cancer biology, prosaposin and quiescin Q6 sulfhydryl oxidase 1, were considered candidate biomarkers. Serum levels of both proteins were significantly higher in MPM patients than control subjects. Combining the data of each receiver-operating characteristic analysis predicted a good diagnostic accuracy. Conclusion A panel of the putative biomarkers represents a promising tool for MPM diagnosis.

Down syndrome (DS) is the most common chromosomal disorder and the leading genetic cause of intellectual disability in humans, which results from the triplication of chromosome 21. To search for biomarkers for the early detection and exploration of the disease mechanisms, here, we investigated the protein expression signature of peripheral blood mononuclear cells (PBMCs) in DS children compared with healthy donors (HD) by using an in-depth label-free shotgun proteomics approach. Identified proteins are found associated with metabolic pathways, cellular trafficking, DNA structure, stress response, cytoskeleton network, and signaling pathways. The results showed that a well-defined number of dysregulated pathways retain a prominent role in mediating DS pathological features. Further, proteomics results are consistent with published study in DS and provide evidences that increased oxidative stress and the increased induction of stress related response, is a participant in DS pathology. In addition, the expression levels of some key proteins have been validated by Western blot analysis while protein carbonylation, as marker of protein oxidation, was investigated. The results of this study propose that PBMCs from DS children might be in an activated state where endoplasmic reticulum stress and increased production of radical species are one of the primary events contributing to multiple DS pathological features.

There is increasing evidence to suggest that, in addition to their regenerative effect, mesenchymal stromal cells (MSCs), and their secretome have an anti-inflammatory and antimicrobial role in the innate immune response in conditions such as sepsis. However, there is no published information on the effect of MSCs in bovine mastitis. Mastitis often results in extensive tissue damage due to multi-microorganism co-infection. This study investigated the ability of amniotic-derived conditioned medium (CM), in vitro and in vivo, to counteract microbial action and restore healthy tissue capable of milk production. Following determination of a dose-response curve, 10,000 colony-forming units (CFU) of Staphylococcus aureus (S. aureus) were inoculated into bovine mammary epithelial cell culture with and without 10% CM (supplemented either at the time of bacteria inoculation or after 4 h). Acridine orange staining was used to assess cell viability/apoptosis. Additionally, an in vivo study was performed using 48 dairy cows with acute and chronic mastitis, treated with CM (treated group) or antibiotics (control group). In vitro results showed that CM can attenuate bacterial growth, as evaluated by the number of CFU. After 24 h of culture with S. aureus, 89.67% of mammary epithelial cells treated with CM were still alive, whereas all cells cultured without CM were dead. Rates of epithelial cell survival (60.67%) were similar when CM was added 4 h after bacteria inoculation. There was no difference in somatic cell count between cases of acute mastitis in the CM-treated or control group in the in vivo study. However, relapses in chronic mastitis were less common in the group receiving CM. Our results show that CM is able to mitigate bacterial growth in vitro and may be particularly useful in the treatment of chronic mastitis, aiding restoration of milk production in cows that would otherwise be removed from the production cycle.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of the corticospinal motor neurons, which ultimately leads to death. The repeat expansion in chromosome 9 open reading frame 72 (C9ORF72) represents the most common genetic cause of ALS and it is also involved in the pathogenesis of other neurodegenerative disorders. To offer insights into C9ORF72-mediated pathogenesis, we quantitatively analyzed the proteome of patient-derived primary skin fibroblasts from ALS patients carrying the C9ORF72 mutation compared with ALS patients who tested negative for it. Differentially expressed proteins were identified, used to generate a protein-protein interaction network and subjected to a functional enrichment analysis to unveil altered molecular pathways. ALS patients were also compared with patients affected by frontotemporal dementia carrying the C9ORF72 repeat expansion. As a result, we demonstrated that the molecular pathways mainly altered in fibroblasts (e.g., protein homeostasis) mirror the alterations observed in C9ORF72-mutated neurons. Moreover, we highlighted novel molecular pathways (nuclear and mitochondrial transports, vesicle trafficking, mitochondrial bioenergetics, glucose metabolism, ER-phagosome crosstalk and Slit/Robo signaling pathway) which might be further investigated as C9ORF72-specific pathogenetic mechanisms. Data are available via ProteomeXchange with the identifier PXD023866.

International audience; Simple Summary This work described the analysis of differential protein expression of subcutaneous adipose tissue of cows that went under negative energy balance during peripartum. In particular, to the best of our knowledge, it represented an original proteomics study that was able to discriminate cows in negative energy balance up to one month before calving. We believed that our findings would open new perspectives to improve animal welfare during peripartum. To know in advance, the metabolic status of cows would permit to correct the status with appropriate measures, like diet or management. Abstract Fat mobilization in high-yielding dairy cows during early lactation occurs to overcome negative energy balance (NEB), caused by insufficient feed intake and the concomitant increased nutritional requirements. For this reason, adipose tissue represents an essential organ for healthy and performant lactation. However, only a few data are known about adipose tissue proteome and its metabolic status during peripartum. The aim of this study was to analyze the differential proteomics profiles of subcutaneous adipose tissue belonging to cows with different NEB scores (low NEB and severe NEB). Both groups were analyzed at three different time points (one month before calving, one and sixteen weeks after calving) that were related to different levels and rates of adipose tissue mobilization. The dataset highlighted the differential expression of the same four key proteins (annexin A2, actin-related protein 10, glyceraldehyde-3-phosphate dehydrogenase, and fatty acid-binding protein) involved in lipid metabolism during all time points and of other 22 proteins typical of the other comparisons among remaining time points. The obtained dataset suggested that the individual variability in adipose tissue metabolism/mobilization/energy availability could be linked to the different outcomes in levels of energy balance and related physical complications among dairy cows during peripartum.

Prosthetic joint replacement failure has a huge impact on quality of life and hospitalization costs. A leading cause of prosthetic joint infection is bacteria-forming biofilm on the surface of orthopedic devices. Staphylococcus epidermidis is an emergent, low-virulence pathogen implicated in chronic infections, barely indistinguishable from aseptic loosening when embedded in a mature matrix. The literature is scarce on the behavior of quiescent S. epidermidis in mature biofilms. To fill this gap, we performed comparative analysis of the whole proteomic profiles of two methicillin-resistant S. epidermidis strains growing in planktonic and in sessile form to investigate the molecular mechanisms underlying biofilm stability. After 72-h culture of biofilm-forming S. epidermidis, overexpression of proteins involved in the synthesis of nucleoside triphosphate and polysaccharides was observed, whereas planktonic bacteria expressed proteins linked to stress and anaerobic growth. Cytological analysis was performed to determine why planktonic bacteria unexpectedly expressed proteins typical of sessile culture. Images evidenced that prolonged culture under vigorous agitation can create a stressful growing environment that triggers microorganism aggregation in a biofilm-like matrix as a mechanism to survive harsh conditions. The choice of a unique late time point provided an important clue for future investigations into the biofilm-like behavior of planktonic cells. Our preliminary results may inform comparative proteomic strategies in the study of mature bacterial biofilm. Finally, there is an increasing number of studies on the aggregation of free-floating bacteria embedded in an extracellular matrix, prompting the need to gain further insight into this mode of bacterial growth.

Background Probiotics are defined as live, nonpathogenic bacteria that confer health benefits beyond their nutritional value. In particular, VSL#3 exhibits demonstrated efficacy in the management of diseases characterized by an increased intestinal permeability. Our study aimed to understand how VSL#3 promotes gut health by secreting bioactive factors and identify which human pathways are modulated by secretome derived from the VSL#3 formula. Methods Two different lots of VSL#3 were used, and Caco-2 cell line was treated with conditioned media (CM) prepared using 1 g of the probiotic formula. We evaluated the effects of the probiotics on cellular proliferation and apoptosis by cytometry and the expression of tight junction proteins by western blotting. A proteomics analysis of both culture media and the whole proteome of Caco-2 cells treated with VSL#3-CM was performed by nano-ultra performance liquid chromatography - tandem mass (nUPLC MS/MS) spectrometry. Results The probiotic formula increased cell proliferation, decreased cellular apoptosis cells, and increased re-epithelialization in the scratch assay. Several peptides specifically synthetized by all the species within the probiotic preparation were recognized in the proteomics analysis. Human proteins synthesized by CaCo-2 cells were also identified. Conclusions To our knowledge, this manuscript describes the first evaluation of the probiotic secretome, and the results showed that the improvement in intestinal barrier functions induced by probiotics seems to be accompanied by the modulation of some human cellular pathways.

A number of factors can trigger amyotrophic lateral sclerosis (ALS), although its precise pathogenesis is still uncertain. In a previous study done by us, poisonous liquoral levels of hydrogen sulphide (H2S) in sporadic ALS patients were reported. In the same study very high concentrations of H2S in the cerebral tissues of the familial ALS (fALS) model of the SOD1G93A mouse, were measured. The objective of this study was to test whether decreasing the levels of H2S in the fALS mouse could be beneficial. Amino-oxyacetic acid (AOA)&mdash, a systemic dual inhibitor of cystathionine-&beta, synthase and cystathionine-&gamma, lyase (two key enzymes in the production of H2S)&mdash, was administered to fALS mice. AOA treatment decreased the content of H2S in the cerebral tissues, and the lifespan of female mice increased by approximately ten days, while disease progression in male mice was not affected. The histological evaluation of the spinal cord of the females revealed a significant increase in GFAP positivity and a significant decrease in IBA1 positivity. In conclusion, the results of the study indicate that, in the animal model, the inhibition of H2S production is more effective in females. The findings reinforce the need to adequately consider sex as a relevant factor in ALS.

Proteins oxidation by reactive species is implicated in the aetiology or progression of a panoply of disorders and diseases such as neurodegenerative disorders. It is becoming increasingly evident that redox imbalance in the brain mediates neurodegeneration. Free radicals, as reactive species of oxygen (ROS) but also reactive nitrogen species (RNS) and reactive sulfur species (RSS), are generated in vivo from several sources. Within the cell the mitochondria represent the main source of ROS and mitochondrial dysfunction is both the major contributor to oxidative stress (OS) as well its major consequence.To date there are no doubts that a condition of OS added to other factors as mitochondrial damage in mtDNA or mitochondrial respiratory chain, may contribute to trigger or amplify mechanisms leading to neurodegenerative disorders.In this chapter, we aim at illustrate the molecular interplay occurring between mitochondria and OS focusing on Amyotrophic Lateral Sclerosis, describing a phenotypic reprogramming mechanism of mitochondria in complex neurological disorder.

Purpose The artificial membrane inside the haemodialyzer is the main determinant of the quality and success of haemodialysis therapy. The performances of haemodialysis membranes are highly influenced by the interactions with plasma proteins, which in turn are related to the physical and chemical characteristics of the membrane material. The present cross-over study is aimed to analyse the haemodialysis performance of a newly developed asymmetric cellulose triacetate membrane (ATA) in comparison to the conventional parent symmetric polymer (CTA). Experimental design In four chronic non diabetic haemodialysis patients, the protein constituents of the adsorbed material from the filters after the haemodialysis session, and the proteins recovered in the ultrafiltrate during the session, are identified using a bottom-up shotgun proteomics approach. Results The ATA membrane shows a lower protein adsorption rate and a lower mass distribution pattern of the proteinaceous material. Conclusions and clinical relevance By highlighting the differences between the two haemodialysis filters in terms of adsorbed proteins and flow through, it is demonstrated the higher biocompatibility of the novel ATA membrane, that fulfils the indications for the development of more performant membranes and may represent a step forward for the treatment of patients on chronic haemodialysis.

Mitochondria are undeniably the cell powerhouse, directly affecting cell survival and fate. Growing evidence suggest that mitochondrial protein repertoire affects metabolic activity and plays an important role in determining cell proliferation/differentiation or quiescence shift. Consequently, the bioenergetic status of a cell is associated with the quality and abundance of the mitochondrial populations and proteomes. Mitochondrial morphology changes in the development of different cellular functions associated with metabolic switches. It is therefore reasonable to speculate that different cell lines do contain different mitochondrial-associated proteins, and the investigation of these pools may well represent a source for mining missing proteins (MPs). A very effective approach to increase the number of IDs through mass spectrometry consists of reducing the complexity of the biological samples by fractionation. The present study aims at investigating the mitochondrial proteome of five phenotypically different cell lines, possibly expressing some of the MPs, through an enrichment-fractionation approach at the organelle and protein level. We demonstrate a substantial increase in the proteome coverage, which, in turn, increases the likelihood of detecting low abundant proteins, often falling in the category of MPs, and resulting, for the present study, in the identification of METTL12, FAM163A, and RGS13. All MS data have been deposited to the MassIVE data repository ( https://massive.ucsd.edu ) with the data set identifier MSV000082409 and PXD010446.

The exposure of blood to an artificial surface such as the haemodialysis membrane results in the nearly instantaneous deposition of a layer of plasma proteins. The composition of the protein layer profoundly influences all subsequent events, and to a large extent determines the biocompatibility of the biomaterial. In the present study, we examine the protein adsorption capacity and coagulation profiles of the polysulfone-based helixone material in comparison to cellulose triacetate. A differential profiling investigation using shotgun proteomics data-independent analysis was applied to eluates obtained with each membrane after a dialysis session, in order to assess the function of desorbed proteins. Functional classification and network analysis performed using bioinformatics tools shed light on the involvement of adsorbed proteins into important molecular processes, such as lipid transport and metabolism, cell growth differentiation and communication, and the coagulation cascade. The collected evidence was further validated by targeted mass spectrometry using selected reaction monitoring on proteotypic transitions of key protein effectors, confirming the different panels of adsorbed protein on each membrane. The coagulation profile during haemodialysis of patients under polysulfone-based helixone filter cartridges was also assessed showing a slightly higher platelet activation profile after the dialysis session. The overall collected evidence highlights a modulation of the coagulation biological pathway during haemodialysis, which is largely influenced by the biomaterial used. Refereed/Peer-reviewed

E. coli is one of the most frequently involved bacteria in uterine diseases. Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria involved in pathogenic processes leading to post-partum metritis and endometritis in cattle. It also causes inflammation of the endometrium. The increase of cell proliferation by LPS is part of the inflammatory process. The aim of this study was to investigate possible changes in protein expression in relation to the proliferative response of bEECs after challenge with E. coli-LPS. In vitro culture of bEECs was performed from cow genital tracts collected at a slaughterhouse. In passage 5, bEECs from each of 9 cows (3 series of 3 cows) were exposed to 0, 8, and 16 μg ml(-1) LPS for 72 h. At time 0 and 72 h later, attached cells/living cells were counted and for each time and LPS dosage, cells were frozen for proteomic analyses. All samples from the 3 series were analyzed by 2-D gel electrophoresis coupled to MALDI-TOF/TOF mass spectrometry. The samples from the first series were subjected to shotgun nLC-MS/MS analysis. From the whole differential proteomics analysis, 38 proteins were differentially expressed (p < 0.05 to p < 0.001) following exposure to LPS. Among them, twenty-eight were found to be up-regulated in the LPS groups in comparison to control groups and ten were down-regulated. Differentially expressed proteins were associated with cell proliferation and apoptosis, transcription, destabilization of cell structure, oxidative stress, regulation of histones, allergy and general cell metabolism pathways. The de-regulations induced by LPS were consistent with the proliferative phenotype and indicated strong alterations of several cell functions. In addition, some of the differentially expressed proteins relates to pathways activated at the time of implantation. The specific changes induced through those signals may have negative consequences for the establishment of pregnancy.

The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.

Cerebrospinal fluid (CSF) biomarkers may support the diagnosis of Alzheimer's disease (AD). We studied if the diagnostic power of AD CSF biomarker concentrations, i.e., Aβ42, total tau (t-tau), and phosphorylated tau (p-tau), is affected by differences in lateral ventricular volume (VV), using CSF biomarker data and magnetic resonance imaging (MRI) scans of 730 subjects, from 13 European Memory Clinics. We developed a Matlab-algorithm for standardized automated segmentation analysis of T1 weighted MRI scans in SPM8 for determining VV, and computed its ratio with total intracranial volume (TIV) as proxy for total CSF volume. The diagnostic power of CSF biomarkers (and their combination), either corrected for VV/TIV ratio or not, was determined by ROC analysis. CSF Aβ42 levels inversely correlated to VV/TIV in the whole study population (Aβ42: r=-0.28; p42 alone (AUC: 0.75 versus 0.81) or in combination with t-tau (AUC: 0.81 versus 0.91). In conclusion, differences in VV may be an important confounder in interpreting CSF Aβ42 levels.

Hydrogen sulfide (H2S) is an endogenous gasotransmitter recognized as an essential body product with a dual, biphasic action. It can function as an antioxidant and a cytoprotective, but also as a poison with a high probability of causing brain damage when present at noxious levels. In a previous study, we measured toxic liquoral levels of H2S in sporadic amyotrophic lateral sclerosis (ALS) patients and in the familial ALS (fALS) mouse model, SOD1G93A. In addition, we experimentally demonstrated that H2S is extremely and selectively toxic to motor neurons, and that it is released by glial cells and increases Ca2+ concentration in motor neurons due to a lack of ATP. The presented study further examines the effect of toxic concentrations of H2S on embryonic mouse spinal-cord cultures. We performed a proteomic analysis that revealed a significant H2S-mediated activation of pathways related to oxidative stress and cell death, particularly the Nrf-2-mediated oxidative stress response and peroxiredoxins. Furthermore, we report that Na2S (a stable precursor of H2S) toxicity is, at least in part, reverted by the Bax inhibitor V5 and by necrostatin, a potent necroptosis inhibitor.

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease whose pathophysiological deficits, causing impairment in motor function, are largely unknown. Here we propose that hydrogen sulfide (H2 S), as a glial-released inflammatory factor, contributes to ALS-mediated motor neuron death.H2 S concentrations were analyzed in the cerebrospinal fluid of 37 sporadic ALS patients and 14 age- and gender-matched controls, in tissues of a familial ALS (fALS) mouse model, and in spinal cord culture media by means of a specific and innovative high-performance liquid chromatography method. The effects of H2 S on motor neurons cultures was analyzed immunohistochemically and by patch clamp recordings and microfluorometry.We found a significantly high level of H2 S in the spinal fluid of the ALS patients. Consistently, we found increased levels of H2 S in the tissues and in the media from mice spinal cord cultures bearing the fALS mutation SOD1G93A. In addition, NaHS, an H2 S donor, added to spinal culture, obtained from control C57BL/6J mice, is toxic for motor neurons, and induces an intracellular Ca(2+) increase, attenuated by the intracytoplasmatic application of adenosine triphosphate. We further show that H2 S is mainly released by astrocytes and microglia.This study unravels H2 S as an astroglial mediator of motor neuron damage possibly involved in the cellular death characterizing ALS.

Background Myxomatous mitral valve disease (MVD) is the most common acquired heart disease in dogs, and the Cavalier King Charles Spaniel (CKCS) is the most studied breed because of the high prevalence, early onset and hereditary component evidenced in the breed. MVD has different severity levels, and there are many practical limitations in identifying its asymptomatic stages. Proteomic techniques are valuable for studying the proteins and peptides involved in cardiovascular diseases, including the period prior to the clinical onset of the disease. The aim of this study was to identify the serum proteins that were differentially expressed in healthy CKCS and those affected by MVD in mild to severe stages. Proteomics analysis was performed using two-dimensional gel electrophoresis separation and a bioinformatics analysis for the detection of differentially expressed spots. In a comparative analysis, protein spots with a p