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Supplementary Figures S1-S4; Supplementary Table S1.

Background and purpose: Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by the degeneration of upper and lower motor neurons, progressive wasting and paralysis of voluntary muscles is currently incurable despite intense research and numerous unsuccessful clinical trials. Although considered as a pure motor neuron disease, increasing evidence indicates that the sole protection of motor neurons by a single target drug is not sufficient to improve the pathological phenotype. We therefore evaluated the therapeutic potential of the multi-target drug, trimetazidine, in SOD1G93A mice. Experimental approach: Trimetazidine is an anti-ischemic drug used for the treatment of coronary artery disease. As a metabolic modulator, Trimetazidine improves glucose metabolism. Furthermore, Trimetazidine enhances mitochondrial metabolism and promotes nerve regeneration, exerting an anti-inflammatory and antioxidant effect. Here, we orally treated SOD1G93A mice with Trimetazidine, solubilized in drinking water at a dose of 20 mg/kg, from disease onset. We assessed the impact of Trimetazidine on disease progression by studying metabolic parameters, grip strength, and histological alterations in skeletal muscle, peripheral nerve and spinal cord. Key results: Trimetazidine administration delays motor function decline, improves muscle performance and metabolism, and significantly extends overall survival of SOD1G93A mice (increased median survival of 16 days and 12.5 days for male and female respectively). Moreover, Trimetazidine prevents the dismantlement of neuromuscular junctions, attenuates motor neuron loss and reduces neuroinflammation in the spinal cord and in peripheral nerves. Conclusion and implications: In SOD1G93A mice, therapeutic effect of Trimetazidine is underpinned by its action on mitochondrial function in skeletal muscle and spinal cord. Keywords: Amyotrophic Lateral Sclerosis; Hypermetabolism; Mitochondria; Neurodegeneration; SOD1G93A mice; Trimetazidine.

Electronic supplementary material is available online at https://doi.org/10.6084/m9.figshare.c.5713034. Copyright © 2021 The Authors. Amplification of the proto-oncogene MYCN is a key molecular aberration in high-risk neuroblastoma and predictive of poor outcome in this childhood malignancy. We investigated the role of MYCN in regulating the protein cargo of extracellular vesicles (EVs) secreted by tumour cells that can be internalized by recipient cells with functional consequences. Using a switchable MYCN system coupled to mass spectrometry analysis, we found that MYCN regulates distinct sets of proteins in the EVs secreted by neuroblastoma cells. EVs produced by MYCN-expressing cells or isolated from neuroblastoma patients induced the Warburg effect, proliferation and c-MYC expression in target cells. Mechanistically, we linked the cancer-promoting activity of EVs to the glycolytic kinase pyruvate kinase M2 (PKM2) that was enriched in EVs secreted by MYC-expressing neuroblastoma cells. Importantly, the glycolytic enzymes PKM2 and hexokinase II were detected in the EVs circulating in the bloodstream of neuroblastoma patients, but not in those of non-cancer children. We conclude that MYC-activated cancers might spread oncogenic signals to remote body locations through EVs. Neuroblastoma UK to AS and FAPESP SPRINT Award (50356-4); FAPESP (2014/06863-3, 2018/18257-1, 2018/15549-1, 16/50356-4); CNPq “bolsa de produtividade”; Ricerca Finalizzata GR11-172. https://doi.org/10.6084/m9.figshare.c.5713034

Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by the degeneration of upper and lower motor neurons, progressive wasting and paralysis of voluntary muscles and is currently incurable. Although considered to be a pure motor neuron disease, increasing evidence indicates that the sole protection of motor neurons by a single targeted drug is not sufficient to improve the pathological phenotype. We therefore evaluated the therapeutic potential of the multi-target drug used to treatment of coronary artery disease, trimetazidine, in SOD1As a metabolic modulator, trimetazidine improves glucose metabolism. Furthermore, trimetazidine enhances mitochondrial metabolism and promotes nerve regeneration, exerting an anti-inflammatory and antioxidant effect. We orally treated SOD1Trimetazidine administration delays motor function decline, improves muscle performance and metabolism, and significantly extends overall survival of SOD1In SOD1

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood characterized by the inability to exit the proliferative myoblast-like stage. The alveolar fusion positive subtype (FP-RMS) is the most aggressive and is mainly caused by the expression of PAX3/7-FOXO1 oncoproteins, which are challenging pharmacological targets. Here, we show that the DEAD box RNA helicase 5 (DDX5) is overexpressed in alveolar RMS cells and that its depletion and pharmacological inhibition decrease FP-RMS viability and slow tumor growth in xenograft models. Mechanistically, we provide evidence that DDX5 functions upstream of the EHMT2/AKT survival signaling pathway, by directly interacting with EHMT2 mRNA, modulating its stability and consequent protein expression. We show that EHMT2 in turns regulates PAX3-FOXO1 activity in a methylation-dependent manner, thus sustaining FP-RMS myoblastic state. Together, our findings identify another survival-promoting loop in FP-RMS and highlight DDX5 as a potential therapeutic target to arrest RMS growth.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by a complex epigenetic mechanism finally leading to the misexpression of DUX4 in skeletal muscle. Detecting DUX4 and quantifying disease progression in FSHD is extremely challenging, thus increasing the need for surrogate biomarkers. We applied a shotgun proteomic approach with two different setups to analyze the protein repertoire of interstitial fluids obtained from 20 muscles in different disease stages classified by magnetic resonance imaging (MRI) and serum samples from 10 FSHD patients. A total of 1156 proteins were identified in the microdialysates by data independent acquisition, 130 of which only found in muscles in active disease stage. Proteomic profiles were able to distinguish FSHD patients from controls. Two innate immunity mediators (S100-A8 and A9) and Dermcidin were upregulated in muscles with active disease and selectively present in the sera of FSHD patients. Structural muscle and plasminogen pathway proteins were downregulated. Together with the upstream inhibition of myogenic factors, this suggests defective muscle regeneration and increased fibrosis in early/active FSHD. Our MRI targeted exploratory approach confirmed that inflammatory response has a prominent role, together with impaired muscle regeneration, before clear muscle wasting occurs. We also identified three proteins as tissue and possibly circulating biomarkers in FSHD.

Leukodystrophies are a heterogeneous group of rare inherited disorders that mostly involve the white matter of the CNS. These conditions are characterized by primary glial cell and myelin sheath pathology of variable aetiology, which causes secondary axonal degeneration, generally emerging with disease progression. Whole exome sequencing performed in five large consanguineous nuclear families allowed us to identify homozygosity for two recurrent missense variants affecting highly conserved residues of RNF220 as the causative event underlying a novel form of leukodystrophy with ataxia and sensorineural deafness. We report these two homozygous missense variants (p.R363Q and p.R365Q) in the ubiquitin E3 ligase RNF220 as the underlying cause of this novel form of leukodystrophy with ataxia and sensorineural deafness that includes fibrotic cardiomyopathy and hepatopathy as associated features in seven consanguineous families. Mass spectrometry analysis identified lamin B1 as the RNF220 binding protein and co-immunoprecipitation experiments demonstrated reduced binding of both RNF220 mutants to lamin B1. We demonstrate that RNF220 silencing in Drosophila melanogaster specifically affects proper localization of lamin Dm0, the fly lamin B1 orthologue, promotes its aggregation and causes a neurodegenerative phenotype, strongly supporting the functional link between RNF220 and lamin B1. Finally, we demonstrate that RNF220 plays a crucial role in the maintenance of nuclear morphology; mutations in primary skin fibroblasts determine nuclear abnormalities such as blebs, herniations and invaginations, which are typically observed in cells of patients affected by laminopathies. Overall, our data identify RNF220 as a gene implicated in leukodystrophy with ataxia and sensorineural deafness and document a critical role of RNF220 in the regulation of nuclear lamina. Our findings provide further evidence on the direct link between nuclear lamina dysfunction and neurodegeneration.

Background In recent years, mechanistic, epidemiologic, and interventional studies have indicated beneficial effects of omega-3 polyunsaturated fatty acids (n-3 PUFA) against brain aging and age-related cognitive decline, with the most consistent effects against Alzheimer’s disease (AD) confined especially in the early or prodromal stages of the pathology. In the present study, we investigated the action of n-3 PUFA supplementation on behavioral performances and hippocampal neurogenesis, volume, and astrogliosis in aged mice subjected to a selective depletion of basal forebrain cholinergic neurons. Such a lesion represents a valuable model to mimic one of the most reliable hallmarks of early AD neuropathology. Methods Aged mice first underwent mu-p75-saporin immunotoxin intraventricular lesions to obtain a massive cholinergic depletion and then were orally supplemented with n-3 PUFA or olive oil (as isocaloric control) for 8 weeks. Four weeks after the beginning of the dietary supplementation, anxiety levels as well as mnesic, social, and depressive-like behaviors were evaluated. Subsequently, hippocampal morphological and biochemical analyses and n-3 PUFA brain quantification were carried out. Results The n-3 PUFA treatment regulated the anxiety alterations and reverted the novelty recognition memory impairment induced by the cholinergic depletion in aged mice. Moreover, n-3 PUFA preserved hippocampal volume, enhanced neurogenesis in the dentate gyrus, and reduced astrogliosis in the hippocampus. Brain levels of n-3 PUFA were positively related to mnesic abilities. Conclusions The demonstration that n-3 PUFA are able to counteract behavioral deficits and hippocampal neurodegeneration in cholinergically depleted aged mice promotes their use as a low-cost, safe nutraceutical tool to improve life quality at old age, even in the presence of first stages of AD.

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).

Amplification of the proto-oncogene MYCN is a key molecular aberration in high-risk neuroblastoma and predictive of poor outcome in this childhood malignancy. We investigated the role of MYCN in regulating the protein cargo of extracellular vesicles (EVs) secreted by tumour cells that can be internalized by recipient cells with functional consequences. Using a switchable MYCN system coupled to mass spectrometry analysis, we found that MYCN regulates distinct sets of proteins in the EVs secreted by neuroblastoma cells. EVs produced by MYCN expressing cells or isolated from neuroblastoma patients induced the Warburg effect, proliferation and c-MYC expression in target cells. Mechanistically, we linked the cancer promoting activity of extracellular vesicles to the glycolytic kinase pyruvate kinase M2 (PKM2) that was enriched in EVs secreted by MYCN expressing neuroblastoma cells. Importantly, the glycolytic enzymes PKM2 and Hexokinase II were detected in the EVs circulating in the blood stream of neuroblastoma patients, but not in those of non-cancer children. We conclude that MYC activated cancers might spread oncogenic signals to remote body locations through extracellular vesicles.

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.

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 order to effectively develop personalized medicine for kidney diseases we urgently need to develop highly accurate biomarkers for use in the clinic, since current biomarkers of kidney damage (changes in serum creatinine and/or urine albumin excretion) apply to a later stage of disease, lack accuracy, and are not connected with molecular pathophysiology. Analysis of urine peptide content (urinary peptidomics) has emerged as one of the most attractive areas in disease biomarker discovery. Urinary peptidome analysis allows the detection of short and long-term physiological or pathological changes occurring within the kidney. Urinary peptidomics has been applied extensively for several years now in renal patients, and may greatly improve kidney disease management by supporting earlier and more accurate detection, prognostic assessment, and prediction of response to treatment. It also promises better understanding of kidney disease pathophysiology, and has been proposed as a “liquid biopsy” to discriminate various types of renal disorders. Furthermore, proteins being the major drug targets, peptidome analysis may allow one to evaluate the effects of therapies at the protein signaling pathway level. We here review the most recent findings on urinary peptidomics in the setting of the most common kidney diseases.

Mutations in PARK2 gene are the most frequent cause of familial forms of Parkinson's disease (PD). This gene encodes Parkin, an E3 ubiquitin ligase involved in several cellular mechanisms, including mitophagy. Parkin loss-of-function is responsible for the cellular accumulation of damaged mitochondria, which in turn determines an increment of reactive oxygen species (ROS) levels, lower ATP production, and apoptosis activation. Given the importance of mitochondrial dysfunction and mitophagy impairment in PD pathogenesis, the aim of the present study was to investigate both total and mitochondrial proteome alterations in human skin fibroblasts of PARK2-mutated patients. To this end, both total and mitochondria-enriched protein fractions from fibroblasts of five PARK2-mutated patients and five control subjects were analyzed by quantitative shotgun proteomics to identify proteins specifically altered by Parkin mutations (mass spectrometry proteomics data have been submitted to ProteomeXchange with the identifier PXD015880). Both the network-based and gene set enrichment analyses pointed out pathways in which Rab GTPase proteins are involved. To have a more comprehensive view of the mitochondrial alterations due to PARK2 mutations, we investigated the impact of Parkin loss on mitochondrial function and network morphology. We unveiled that the mitochondrial membrane potential was reduced in PARK2-mutated patients, without inducing PINK1 accumulation, even when triggered with the ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Lastly, the analysis of the mitochondrial network morphology did not reveal any significant alterations in PARK2-mutated patients compared to control subjects. Thus, our results suggested that the network morphology was not influenced by the mitochondrial depolarization and by the lack of Parkin, revealing a possible impairment of fission and, more in general, of mitochondrial dynamics. In conclusion, the present work highlighted new molecular factors and pathways altered by PARK2 mutations, which will unravel possible biochemical pathways altered in the sporadic form of PD.

Background: The uremic syndrome mimes a systemic poisoning with the retention of numerous compounds which are normally removed by the kidney. The study of proteins and peptides, or proteomics, represents an important field of research for the investigation of blood and blood diseases. Methods and Materials: We focused our review on the results of proteomic investigations on blood cells of uremic patients with particular regard to the study of red blood cells, platelets, and monocytes. Results: In literature there are few, preliminary studies on platelets and monocytes while the knowledge on uremic erythrocytes is much wider. Proteomic investigations showed that erythrocyte membrane proteome of uremic patients, differs significantly from the proteome of healthy subjects, being characterized by an extensive remodeling which may influence visco-elastic properties of RBC such as deformability and involve diverse molecular pathways driving red blood cell signaling and removal. Conclusion: Proteomic technologies emerged as a useful tool in defining and characterizing both physiological and disease processes being able, among others, to give important insights into uremic anemia.

The success and the quality of hemodialysis therapy are mainly related to both clearance and biocompatibility properties of the artificial membrane packed in the hemodialyzer. Performance of a membrane is strongly influenced by its interaction with the plasma protein repertoire during the extracorporeal procedure. Recognition that a number of medium-high molecular weight solutes, including proteins and protein-bound molecules, are potentially toxic has prompted the development of more permeable membranes. Such membrane engineering, however, may cause loss of vital proteins, with membrane removal being nonspecific. In addition, plasma proteins can be adsorbed onto the membrane surface upon blood contact during dialysis. Adsorption can contribute to the removal of toxic compounds and governs the biocompatibility of a membrane, since surface-adsorbed proteins may trigger a variety of biologic blood pathways with pathophysiologic consequences. Over the last years, use of proteomic approaches has allowed polypeptide spectrum involved in the process of hemodialysis, a key issue previously hampered by lack of suitable technology, to be assessed in an unbiased manner and in its full complexity. Proteomics has been successfully applied to identify and quantify proteins in complex mixtures such as dialysis outflow fluid and fluid desorbed from dialysis membrane containing adsorbed proteins. The identified proteins can also be characterized by their involvement in metabolic and signaling pathways, molecular networks, and biologic processes through application of bioinformatics tools. Proteomics may thus provide an actual functional definition as to the effect of a membrane material on plasma proteins during hemodialysis. Here, we review the results of proteomic studies on the performance of hemodialysis membranes, as evaluated in terms of solute removal efficiency and blood-membrane interactions. The evidence collected indicates that the information provided by proteomic investigations yields improved molecular and functional knowledge and may lead to the development of more efficient membranes for the potential benefit of the patient.

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.

Traumatic spinal cord injury has dramatic consequences and a huge social impact. We propose a new mouse model of spinal trauma that induces a complete paralysis of hindlimbs, still observable 30 days after injury. The contusion, performed without laminectomy and deriving from the pressure exerted directly on the bone, mimics more closely many features of spinal injury in humans. Spinal cord was injured at thoracic level 10 (T10) in adult anesthetized female CD1 mice, mounted on stereotaxic apparatus and connected to a precision impactor device. Following severe injury, we evaluated motor and sensory functions, and histological/morphological features of spinal tissue at different time points. Moreover, we studied the effects of early and subchronic administration of Docosahexaenoic acid, investigating functional responses, structural changes proximal and distal to the lesion in primary and secondary injury phases, proteome modulation in injured spinal cord. Docosahexaenoic acid was able i) to restore behavioural responses and ii) to induce pro-regenerative effects and neuroprotective action against demyelination, apoptosis and neuroinflammation. Considering the urgent health challenge represented by spinal injury, this new and reliable mouse model together with the positive effects of docosahexaenoic acid provide important translational implications for promising therapeutic approaches for spinal cord injuries.

Because of the distinctive features of the oral cavity, the determination of the proteins involved in the formation of the "oral protein pellicle" is demanding. The present study investigated the susceptibility of several human basic proline-rich peptides, named P-H, P-D, P-F, P-J, and II-2, as substrates of transglutaminase-2. The reactivity of the P-C peptide and statherin was also investigated. Peptides purified from human whole saliva were incubated with the enzyme in the presence or in the absence of monodansyl-cadaverine. Mass spectrometry analyses of the reaction products highlighted that P-H and P-D (P32 and A32 variants) were active substrates, II-2 was less reactive, and P-F and P-J showed very low reactivity. P-C and statherin were highly reactive. All of the peptides formed cyclo derivatives, and only specific glutamine residues were involved in the cycle formation and reacted with monodansyl-cadaverine: Q29 of P-H, Q37 of P-D, Q21 of II-2, Q41 of P-C, and Q37 of statherin were the principal reactive residues. One or two secondary glutamine residues of only P-H, P-D P32, P-C, and statherin were hierarchically susceptible to the reaction with monodansyl-cadaverine. MS and MS/MS data were deposited to the ProteomeXchange Consortium ( http://www.ebi.ac.uk/pride ) via the PRIDE partner repository with the data set identifier PXD014658.

Mutations in PARK2 gene are the most frequent cause of familial forms of Parkinson’s disease (PD). This gene encodes Parkin, an E3 ubiquitin ligase involved in several cellular mechanisms, including mitophagy. Parkin loss-of-function is responsible for the cellular accumulation of damaged mitochondria, which in turn determines an increment of reactive oxygen species (ROS) levels, lower ATP production, and apoptosis activation. Given the importance of mitochondrial dysfunction and mitophagy impairment in PD pathogenesis, the aim of the present study was to investigate both total and mitochondrial proteome alterations in human skin fibroblasts of PARK2-mutated patients. To this end, both total and mitochondria-enriched protein fractions from fibroblasts of five PARK2-mutated patients and five control subjects were analyzed by quantitative shotgun proteomics to identify proteins specifically altered by Parkin mutations (mass spectrometry proteomics data have been submitted to ProteomeXchange with the identifier PXD015880). Both the network-based and gene set enrichment analyses pointed out pathways in which Rab GTPase proteins are involved. To have a more comprehensive view of the mitochondrial alterations due to PARK2 mutations, we investigated the impact of Parkin loss on mitochondrial function and network morphology. We unveiled that the mitochondrial membrane potential was reduced in PARK2-mutated patients, without inducing PINK1 accumulation, even when triggered with the ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Lastly, the analysis of the mitochondrial network morphology did not reveal any significant alterations in PARK2-mutated patients compared to control subjects. Thus, our results suggested that the network morphology was not influenced by the mitochondrial depolarization and by the lack of Parkin, revealing a possible impairment of fission and, more in general, of mitochondrial dynamics. In conclusion, the present work highlighted new molecular factors and pathways altered by PARK2 mutations, which will unravel possible biochemical pathways altered in the sporadic form of PD.

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.

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.

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.

Traumatic spinal cord injury has dramatic consequences and a huge social impact. We propose a new mouse model of spinal trauma that induces a complete paralysis of hindlimbs, still observable 30 days after injury. The contusion, performed without laminectomy and deriving from the pressure exerted directly on the bone, mimics more closely many features of spinal injury in humans. Spinal cord was injured at thoracic level 10 (T10) in adult anesthetized female CD1 mice, mounted on stereotaxic apparatus and connected to a precision impactor device. Following severe injury, we evaluated motor and sensory functions, and histological/morphological features of spinal tissue at different time points. Moreover, we studied the effects of early and subchronic administration of Docosahexaenoic acid, investigating functional responses, structural changes proximal and distal to the lesion in primary and secondary injury phases, proteome modulation in injured spinal cord. Docosahexaenoic acid was able i) to restore behavioural responses and ii) to induce pro-regenerative effects and neuroprotective action against demyelination, apoptosis and neuroinflammation. Considering the urgent health challenge represented by spinal injury, this new and reliable mouse model together with the positive effects of docosahexaenoic acid provide important translational implications for promising therapeutic approaches for spinal cord injuries.

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.

The retention of a number of solutes that may cause adverse biochemical/biological effects, called uremic toxins, characterizes uremic syndrome. Uremia therapy is based on renal replacement therapy, hemodialysis being the most commonly used modality. The membrane contained in the hemodialyzer represents the ultimate determinant of the success and quality of hemodialysis therapy. Membrane's performance can be evaluated in terms of removal efficiency for unwanted solutes and excess fluid, and minimization of negative interactions between the membrane material and blood components that define the membrane's bio(in)compatibility. Given the high concentration of plasma proteins and the complexity of structural functional relationships of this class of molecules, the performance of a membrane is highly influenced by its interaction with the plasma protein repertoire. Proteomic investigations have been increasingly applied to describe the protein uremic milieu, to compare the blood purification efficiency of different dialyzer membranes or different extracorporeal techniques, and to evaluate the adsorption of plasma proteins onto hemodialysis membranes. In this article, we aim to highlight investigations in the hemodialysis setting making use of recent developments in proteomic technologies. Examples are presented of why proteomics may be helpful to nephrology and may possibly affect future directions in renal research.

The development of precision medicine requires advanced technologies to address the multifactorial disease stratification and to support personalized treatments. Among omics techniques, proteomics based on Mass Spectrometry (MS) is becoming increasingly relevant in clinical practice allowing a phenotypic characterization of the dynamic functional status of the organism. From this perspective, Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) MS is a suitable platform for providing a high-throughput support to clinics. Areas covered: This review aims to provide an updated overview of MALDI-TOF MS applications in clinical proteomics. The most relevant features of this analysis have been discussed, highlighting both pre-analytical and analytical factors that are crucial in proteomics studies. Particular emphasis is placed on biofluids proteomics for biomarkers discovery and on recent progresses in clinical microbiology, drug monitoring, and minimal residual disease (MRD). Expert commentary: Despite some analytical limitations, the latest technological advances together with the easiness of use, the low time and low cost consuming and the high throughput are making MALDI-TOF MS instruments very attractive for the clinical practice. These features offer a significant potential for the routine of the clinical laboratory and ultimately for personalized medicine.

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

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 Many tumor-related factors have shown the ability to affect metabolic pathways by paving the way for cancer-specific metabolic features. Here, we investigate the regulation of mTORC1 by MDM4, a p53-inhibitor with oncogenic or anti-survival activities depending on cell growth conditions. Method MDM4-mTOR relationship was analysed through experiments of overexpression or silencing of endogenous proteins in cell culture and using purified proteins in vitro. Data were further confirmed in vivo using a transgenic mouse model overexpressing MDM4. Additionally, the Cancer Genome Atlas (TCGA) database (N = 356) was adopted to analyze the correlation between MDM4 and mTOR levels and 3D cultures were used to analyse the p53-independent activity of MDM4. Results Following nutrient deprivation, MDM4 impairs mTORC1 activity by binding and inhibiting the kinase mTOR, and contributing to maintain the cytosolic inactive pool of mTORC1. This function is independent of p53. Inhibition of mTORC1 by MDM4 results in reduced phosphorylation of the mTOR downstream target p70S6K1 both in vitro and in vivo in a MDM4-transgenic mouse. Consistently, MDM4 reduces cell size and proliferation, two features controlled by p70S6K1, and, importantly, inhibits mTORC1-mediated mammosphere formation. Noteworthy, MDM4 transcript levels are significantly reduced in breast tumors characterized by high mTOR levels. Conclusion Overall, these data identify MDM4 as a nutrient-sensor able to inhibit mTORC1 and highlight its metabolism-related tumor-suppressing function. Electronic supplementary material The online version of this article (doi:10.1186/s12943-017-0626-7) contains supplementary material, which is available to authorized users.

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.