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Ever since its presence was reported in the brain, the nature and role of hydrogen sulfide (H2S) in the Central Nervous System (CNS) have changed. Consequently, H2S has been elected as the third gas transmitter, along with carbon monoxide and nitric oxide, and a number of studies have focused on its neuromodulatory and protectant functions in physiological conditions. The research on H2S has highlighted its many facets in the periphery and in the CNS, and its role as a double-faced compound, switching from protective to toxic depending on its concentration. In this review, we will focus on the bell-shaped nature of H2S as an angiogenic factor and as a molecule released by glial cells (mainly astrocytes) and non-neuronal cells acting on the surrounding environment (paracrine) or on the releasing cells themselves (autocrine). Finally, we will discuss its role in Amyotrophic Lateral Sclerosis, a paradigm of a neurodegenerative disease.

Prostate cancer (PCa) continues to be the second most common malignant tumour and the main cause of oncological death in men. Investigating endogenous volatile organic metabolites (VOMs) produced by various metabolic pathways is emerging as a novel, effective, and non-invasive source of information to establish the volatilomic biosignature of PCa. In this study, headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME/GC-MS) was used to establish the urine volatilomic profile of PCa and identify VOMs that can discriminate between the two investigated groups. This non-invasive approach was applied to oncological patients (PCa group, n = 26) and cancer-free individuals (control group, n = 30), retrieving a total of 147 VOMs from various chemical families. This included terpenes, norisoprenoid, sesquiterpenes, phenolic, sulphur and furanic compounds, ketones, alcohols, esters, aldehydes, carboxylic acid, benzene and naphthalene derivatives, hydrocarbons, and heterocyclic hydrocarbons. The data matrix was subjected to multivariate analysis, namely partial least-squares discriminant analysis (PLS-DA). Accordingly, this analysis showed that the group under study presented different volatomic profiles and suggested potential PCa biomarkers. Nevertheless, a larger cohort of samples is required to boost the predictability and accuracy of the statistical models developed.

African Swine Fever (ASF) is an acute hemorrhagic fever affecting suids with high mortality and morbidity rate. The causal agent of ASF, the African Swine Fever Virus (ASFV), is an icosahedral virus of 200 nm diameter, composed of an outer envelope layer of host derivation and a linear 170-190 kb long dsDNA molecule. As of today, no efficient therapeutic intervention nor prophylactic measures exist to fight ASFV diffusion, underlining the importance of the early diagnosis and the need for efficient in-field screening of ASF. Recommended guidelines for the diagnosis of ASF are unpracticable in the desirable context of the rapid in-farm screening. In this view, the design of innovative diagnostics based on a panel of multiple ASFV epitopes would amend versatility and the analytical performances of the deliverable, ensuring high quality and accuracy standards worth of implementation in rapid in-field monitoring programs. Pursuing this view, we performed epitope prediction from the major AFSV structural proteins holding the potential to be targeted in innovative rapid diagnostic tests. Selected ASFV structural protein sequences were retrieved from data repositories and their tridimensional structure was computed. Linear and 3D protein structures were subjected to the prediction of the epitope sequences, that are likely to elicit antibody production, by independent bioinformatic tools, providing a list of candidate biomarkers whose batch employment held the potential suitability for the unbiased rapid in-field diagnosis and, in turn, might be implemented in screening programs, crowing the current monitoring and control campaigns that are currently running worldwide.

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: 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.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by pro-gressive 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.

To have information on the proteolytic activity of convertases and exo-peptidases on human salivary proteins, this study investigated the relative amounts of the truncated proteoforms in the saliva of preterm newborns and compared them with the relative amounts measured in saliva of at-term newborns, of babies (0–10 years old) and of adults. Results indicated that convertase(s), acting on acidic proline-rich proteins and histatin 3, and carboxypeptidase(s) acting on acidic proline-rich proteins, P–C peptide, histatin 6 and statherin were many folds more active in preterm newborns than in the other groups. Conversely, the aminopeptidase responsible for the removal of the N-terminal Asp residue of statherin was not active in preterm newborns, becoming active only several months after the normal term of delivery. The high activity of convertases determined in preterm newborns suggests that it is required for the molecular events connected to the fetus development, and encourages further studies devoted to the characterization of their specific substrates.

Background: The spread of the SARS-CoV2 virus, which causes COVID-19 disease, profoundly impacted the surgical community. Recommendations have been published to manage patients needing surgery during the COVID-19 pandemic. This survey, under the aegis of the Italian Society of Endoscopic Surgery, aims to analyze how Italian surgeons have changed their practice during the pandemic. Methods: The authors designed an online survey that was circulated for completion to the Italian departments of general surgery registered in the Italian Ministry of Health database in December 2020. Questions were divided into three sections: hospital organization, screening policies, and safety profile of the surgical operation. The investigation periods were divided into the Italian pandemic phases I (March–May 2020), II (June–September 2020), and III (October–December 2020). Results: Of 447 invited departments, 226 answered the survey. Most hospitals were treating both COVID-19-positive and -negative patients. The reduction in effective beds dedicated to surgical activity was significant, affecting 59% of the responding units. 12.4% of the respondents in phase I, 2.6% in phase II, and 7.7% in phase III reported that their surgical unit had been closed. 51.4%, 23.5%, and 47.8% of the respondents had at least one colleague reassigned to non-surgical COVID-19 activities during the three phases. There has been a reduction in elective (> 200 procedures: 2.1%, 20.6% and 9.9% in the three phases, respectively) and emergency (< 20 procedures: 43.3%, 27.1%, 36.5% in the three phases, respectively) surgical activity. The use of laparoscopy also had a setback in phase I (25.8% performed less than 20% of elective procedures through laparoscopy). 60.6% of the respondents used a smoke evacuation device during laparoscopy in phase I, 61.6% in phase II, and 64.2% in phase III. Almost all responders (82.8% vs. 93.2% vs. 92.7%) in each analyzed period did not modify or reduce the use of high-energ

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.

Paratuberculosis is an infectious disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). MAP is an intracellular pathogen with a possible zoonotic potential since it has been successfully isolated from the intestine and blood of Crohn’s disease patients.Since no cure is available, after the detection of the disease, animal culling is the sole applicable containment strategy. However, the difficult detection of the disease in its subclinical form, facilitates its spread raising the need for the development of effective diagnosis and vaccination strategies. The prompt identification and isolation of the infected animals in the subclinical stage would prevent the spread of the infection.In the present study, an immunoinformatic approach has been used to investigate the immunogenic properties of 10 MAP proteins. These proteins were chosen according to a previously published immunoproteomics approach. For each previously-described immunoreactive protein, we predicted the epitopes capable of eliciting an immune response by binding both B-cells and/or class I MHC antigens. The retrieved peptide sequences were analyzed for their specificity and cross-reactivity. The final aim is to employ the discovered peptides sequences as a filtered library useful for early-stage diagnosis and/or to be used in novel multi-subunit or recombinant vaccine formulations.

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 bidirectional 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).

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.

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.

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.

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 on the behavior of quiescent S. epidermidis in mature biofilms is scarce. 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.

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.

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.

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.

Blood proteome analysis for biomarker discovery represents one of the most challenging tasks to be achieved through clinical proteomics due to the sample complexity, such as the extreme heterogeneity of proteins in very dynamic concentrations, and to the observation of proper sampling and storage conditions. Quantitative and qualitative proteomics profiling of plasma and serum could be useful both for the early detection of diseases and for the evaluation of pathological status. Two main sources of variability can affect the precision and accuracy of the quantitative experiments designed for biomarker discovery and validation. These sources are divided into two categories, pre-analytical and analytical, and are often ignored; however, they can contribute to consistent errors and misunderstanding in biomarker research. In this chapter, we review critical pre-analytical and analytical variables that can influence quantitative proteomics. According to guidelines accepted by proteomics community, we propose some recommendations and strategies for a proper proteomics analysis addressed to biomarker studies.

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 < 0.05 (ANOVA) were considered statistically significant and were excised from the gels for analysis by MALDI-TOF-MS for protein identification. RESULTS:Eight proteins resulted differentially expressed among the groups and significantly related to the progression of the disease. In mild affected group versus healthy dogs complement factor H isoform 2, inhibitor of carbonic anhydrase, hemopexin, dystrobrevin beta isoform X7 and CD5 molecule-like resulted to be down-regulated, whereas fibronectin type-III domain-containing protein 3A isoform X4 was up-regulated. In severe affected dogs versus healthy group complement factor H isoform 2, calpain-3 isoform X2, dystrobrevin beta isoform X7, CD5 molecule-like and l-2-hydroxyglutarate dehydrogenase resulted to be down-regulated. Complement factor H isoform 2, calpain-3 isoform X2, dystrobrevin beta isoform X7, CD5 molecule-like and hydroxyglutarate dehydrogenase were found to be down-regulated in mild affected group versus healthy dogs. All of these proteins except complement factor H followed a decreas

Escherichia coli (E. coli) urinary tract infections (UTIs) are becoming a serious problem both for pets and humans (zoonosis) due to the close contact and to the increasing resistance to antibiotics. This study has been performed in order to unravel the mechanism of induced enrofloxacin resistance in canine E. coli isolates that represent a good tool to study this pathology. The isolated E. coli has been induced with enrofloxacin and studied through 2D DICE and shotgun MS. Discovered differentially expressed proteins are principally involved in antibiotic resistance and linked to oxidative stress response, to DNA protection and to membrane permeability. Moreover, since enrofloxacin is an inhibitor of DNA gyrase, the overexpression of DNA starvation/stationary phase protection protein (Dsp) could be a central point to discover the mechanism of this clone to counteract the effects of enrofloxacin. In parallel, the dramatic decrease of the synthesis of the outer membrane protein W, which represents one of the main gates for enrofloxacin entrance, could explain additional mechanism of E. coli defense against this antibiotic. All 20 DICE and MS data have been deposited into the ProteomeXchange Consortium with identifier PXD002000 and DOI http://dx.doi.org/10.601/PXD002000.

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.

Objective 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 (H2S), as a glial-released inflammatory factor, contributes to ALS-mediated motor neuron death. Methods H2S 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 H2S on motor neurons cultures was analyzed immunohistochemically and by patch clamp recordings and microfluorometry. Results We found a significantly high level of H2S in the spinal fluid of the ALS patients. Consistently, we found increased levels of H2S in the tissues and in the media from mice spinal cord cultures bearing the fALS mutation SOD1G93A. In addition, NaHS, an H2S donor, added to spinal culture, obtained from control C57BL/6J mice, is toxic for motor neurons, and induces an intracellular Ca2+ increase, attenuated by the intracytoplasmatic application of adenosine triphosphate. We further show that H2S is mainly released by astrocytes and microglia. Interpretation This study unravels H2S as an astroglial mediator of motor neuron damage possibly involved in the cellular death characterizing ALS. Ann Neurol 2015;77:697-709

Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of a chronic enteritis of ruminants (bovine paratuberculosis (PTB)-Johne's disease) that is associated with enormous worldwide economic losses for the animal production. Diagnosis is based on observation of clinical signs, the detection of antibodies in milk or serum, or evaluation of bacterial culture from feces. The limit of these methods is that they are not able to detect the disease in the subclinical stage and are applicable only when the disease is already advanced. For this reason, the main purpose of this study is to use the MAP proteome to detect novel immunoreactive proteins that may be helpful for PTB diagnoses. 2DE and 2D immunoblotting of MAP proteins were performed using sera of control cattle and PTB-infected cattle in order to highlight the specific immunoreactive proteins. Among the assigned identifiers to immunoreactive spots it was found that most of them correspond to surface-located proteins while three of them have never been described before as antigens. The identification of these proteins improves scientific knowledge that could be useful for PTB diagnoses. The sequence of the identified protein can be used for the synthesis of immunoreactive peptides that could be screened for their immunoreaction against bovine sera infected with MAP. All MS data have been deposited in the ProteomeXchange consortium with identifier PXD001159 and DOI 10.6019/PXD001159.

Measurement of biochemical markers represents an important aid to clinicians in the early diagnosis and prognosis of neurological diseases. Many factors can contribute to increase the chances that a biomarker study becomes successful. In a cerebrospinal fluid analysis (CSF), more than 84% of laboratory errors can be attributed to several preanalytical variables that include CSF collection, storage, and freeze thawing cycles. In this concept paper, we focus on some critical issues arising from basic proteomics investigation in order to highlight some key elements of CSF quality control. Furthermore, we propose a direct assessment of sample quality (DASQ) by applying a fast MALDI-TOF-MS methodology to evaluate molecular features of sample degradation and oxidation. We propose DASQ as a fast and simple initial step to be included in large-scale projects for neurological biomarker studies. In fact, such a procedure will improved the development of standardized protocols in order to have well-characterized CSF biobanks.

Transthyretin (TTR) is a homotetrameric protein of the CNS that plays a role of as the major thyroxine (T4) carrier from blood to cerebrospinal fluid (CSF). T4 physiologically helps oligodendrocyte precursor cells to turn into myelinating oligodendrocytes, enhancing remyelination after myelin sheet damage. We investigated post-translational oxidative modifications of serum and CSF TTR in multiple sclerosis subjects, highlighting high levels of S-sulfhydration and S-sulfonation of cysteine in position ten only in the cerebral TTR, which correlate with an anomalous TTR protein folding as well as with disease duration. Moreover, we found low levels of free T4 in CSF of multiple sclerosis patients, suggestive of a potential role of these modifications in T4 transport into the brain.

Introduction: Spinobulbar muscular atrophy (SBMA) is an inherited adult-onset motor neuron disease caused by the expansion of a polyglutamine tract within the androgen receptor. Autonomic nervous system involvement (ANS) is not considered part of SBMA. The aim of this study was to assess autonomic cardiovascular function in 5 SBMA patients. Methods: Five quantitative autonomic function tests (AFTs) were performed in 5 SBMA patients. Plasma noradrenaline (NA) concentration in patients and in 5 healthy subjects was also measured. Results: AFTs were abnormal in 4 of the 5 patients, and plasma NA concentration was significantly reduced in patients with respect to controls. Conclusion: The impairment of cardiovascular responses to AFTs in addition to reduced plasma NA concentration observed in our patients suggests subclinical involvement of the ANS in Kennedy disease. © 2011 Wiley Periodicals, Inc.