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Aging is a multifactorial phenomenon characterized by degenerative processes closely connected to oxidative damage and chronic inflammation. Recently, many studies have shown that natural bioactive compounds are useful in delaying the aging process. In this work, we studied the effects of an in vivo supplementation of the stilbenoid pterostilbene on lifespan extension in Drosophila melanogaster. We found that the average lifespan of flies of both sexes was increased by pterostilbene supplementation with a higher effect in females. The expression of longevity related genes (Sir2, Foxo, and Notch) was increased in both sexes but with different patterns. Pterostilbene counteracted oxidative stress induced by ethanol and paraquat and up-regulated the antioxidant enzymes Ho e Trxr-1 in male but not in female flies. On the other hand, pterostilbene decreased the inflammatory mediators dome and egr only in female flies. Proteomic analysis revealed that pterostilbene modulates 113 proteins in male flies and only 9 in females. Only one of these proteins was modulated by pterostilbene in both sexes: vacuolar H[+] ATPase 68 kDa subunit 2 (Vha68-2) that was strongly down-regulated. These findings suggest a potential role of pterostilbene in increasing lifespan both in male and female flies by mechanisms that seem to be different in the two sexes, highlighting the need to conduct nutraceutical supplementation studies on males and females separately in order to give more reliable results.

Pompe disease (PD) is caused by deficiency of the enzyme acid α-glucosidase resulting in glycogen accumulation in lysosomes. Clinical symptoms include skeletal myopathy, respiratory failure, and cardiac hypertrophy. We studied plasma proteomic and lipidomic profiles in 12 PD patients compared to age-matched controls. The proteomic profiles were analyzed by nLC-MS/MS SWATH method. Wide-targeted lipidomic analysis was performed by LC-IMS/MS, allowing to quantify >1100 lipid species, spanning 13 classes. Significant differences were found for 16 proteins, with four showing the most relevant changes (GPLD1, PON1, LDHB, PKM). Lipidomic analysis showed elevated levels of three phosphatidylcholines and of the free fatty acid 22:4, and reduced levels of six lysophosphatidylcholines. Up-regulated glycolytic enzymes (LDHB and PKM) are involved in autophagy and glycogen metabolism, while down-regulated PON1 and GPLD1 combined with lipidomic data indicate an abnormal phospholipid metabolism. Reduced GPLD1 and dysregulation of lipids with acyl-chains characteristic of GPI-anchor structure suggest the potential involvement of GPI-anchor system in PD. Results of proteomic analysis displayed the involvement of multiple cellular functions affecting inflammatory, immune and antioxidant responses, autophagy, Ca2+ -homeostasis, and cell adhesion. The combined multi-omic approach revealed new biosignatures in PD, providing novel insights in disease pathophysiology with potential future clinical application.

Alterations of gut microbiota have been identified before clinical manifestation of type 1 diabetes (T1D). To identify the associations amongst gut microbiome profile, metabolism and disease markers, the 16S rRNA-based microbiota profiling and 1H-NMR metabolomic analysis were performed on stool samples of 52 T1D patients at onset, 17 T1D siblings and 57 healthy subjects (CTRL). Univariate, multivariate analyses and classification models were applied to clinical and -omic integrated datasets. In T1D patients and their siblings, Clostridiales and Dorea were increased and Dialister and Akkermansia were decreased compared to CTRL, while in T1D, Lachnospiraceae were higher and Collinsella was lower, compared to siblings and CTRL. Higher levels of isobutyrate, malonate, Clostridium, Enterobacteriaceae, Clostridiales, Bacteroidales, were associated to T1D compared to CTRL. Patients with higher anti-GAD levels showed low abundances of Roseburia, Faecalibacterium and Alistipes and those with normal blood pH and low serum HbA1c levels showed high levels of purine and pyrimidine intermediates. We detected specific gut microbiota profiles linked to both T1D at the onset and to diabetes familiarity. The presence of specific microbial and metabolic profiles in gut linked to anti-GAD levels and to blood acidosis can be considered as predictive biomarker associated progression and severity of T1D.

Type 1 diabetes (T1D) is a chronic autoimmune metabolic disorder with onset in pediatric/adolescent age, characterized by insufficient insulin production, due to a progressive destruction of pancreatic β-cells. Evidence on the correlation between the human gut microbiota (GM) composition and T1D insurgence has been recently reported. In particular, 16S rRNA-based metagenomics has been intensively employed in the last decade in a number of investigations focused on GM representation in relation to a pre-disease state or to a response to clinical treatments. On the other hand, few works have been published using alternative functional omics, which is more suitable to provide a different interpretation of such a relationship. In this work, we pursued a comprehensive metaproteomic investigation on T1D children compared with a group of siblings (SIBL) and a reference control group (CTRL) composed of aged matched healthy subjects, with the aim of finding features in the T1D patients’ GM to be related with the onset of the disease. Modulated metaproteins were found either by comparing T1D with CTRL and SIBL or by stratifying T1D by insulin need (IN), as a proxy of β-cells damage, showing some functional and taxonomic traits of the GM, possibly related to the disease onset at different stages of severity.

Cystic fibrosis (CF) is the most common rare disease caused by a mutation of the

Extremely sensitive food-allergic patients may react to very small amounts of allergenic foods. Precautionary allergen labelling (PAL) warns from possible allergenic contaminations. We explored the ability of proteomic methods to identify minute amounts of milk/egg allergens in a brand of PAL-labelled milk- and egg-free biscuits. We evaluated the reactivity of children with severe milk and egg allergy, by oral food challenge. Traces of milk and/or egg allergens in biscuits were measured by two different liquid-chromatography-mass spectrometry methods. The binding of patient's serum with egg/milk proteins was assessed at immunoblotting. None of the patients reacted to biscuits. Egg and milk proteins were found under the limit of detection of 0.6 µg/g for milk and egg (method A), and of 0.1 and 0.3 µg /g for milk and egg, respectively (method B). The immunoblots did not show milk/egg proteins in the studied biscuits. Our biscuits did not contain allergens of clinical significance. Their milk/egg content is far lower than 4 µg of milk or egg protein per gram of product, the minimal doses considered theoretically capable of causing reactions. With high sensitivity, proteomic assessments predict the harmlessness of very small amount of allergenic foods and can be used to avoid unnecessary PAL.

Background Hepatocellular carcinoma (HCC) is one of the most common and lethal malignant tumours worldwide. Sorafenib (SOR) is one of the most effective single-drug systemic therapy against advanced HCC, but the identification of novel combination regimens for a continued improvement in overall survival is a big challenge. Recent studies highlighted the crucial role of focal adhesion kinase (FAK) in HCC growth. The aim of this study was to investigate the antitumor effects of three different FAK inhibitors (FAKi), alone or in combination with SOR, using in vitro and in vivo models of HCC. Methods The effect of PND1186, PF431396, TAE226 on cell viability was compared to SOR. Among them TAE226, emerging as the most effective FAKi, was tested alone or in combination with SOR using 2D/3D human HCC cell line cultures and HCC xenograft murine models. The mechanisms of action were assessed by gene/protein expression and imaging approaches, combined with high-throughput methods. Results TAE226 was the more effective FAKi to be combined with SOR against HCC. Combined TAE226 and SOR treatment reduced HCC growth both in vitro and in vivo by affecting tumour-promoting gene expression and inducing epigenetic changes via dysregulation of FAK nuclear interactome. We characterized a novel nuclear functional interaction between FAK and the NuRD complex. TAE226-mediated FAK depletion and SOR-promoted MAPK down-modulation caused a decrease in the nuclear amount of HDAC1/2 and a consequent increase of the histone H3 lysine 27 acetylation, thus counteracting histone H3 lysine 27 trimethylation. Conclusions Altogether, our findings provide the first evidence that TAE226 combined with SOR efficiently reduces HCC growth in vitro and in vivo. Also, our data highlight that deep analysis of FAK nuclear interactome may lead to the identification of new promising targets for HCC therapy.

Aging is a multifactorial phenomenon characterized by degenerative processes closely connected to oxidative damage and chronic inflammation. Recently, many studies have shown that natural bioactive compounds are useful in delaying the aging process. In this work, we studied the effects of an

Recurrent cystitis (RC) is a common disease, especially in females. Anatomical, behavioral and genetic predisposing factors are associated with the ascending retrograde route, which often causes bladder infections. RC seems to be mainly caused by agents derived from the intestinal microbiota, and most frequently by Escherichia coli. Intestinal contiguity contributes to the etiopathogenesis of RC and an alteration in intestinal permeability could have a major role in RC. The aim of this pilot study is to assess gut microbiome dysbiosis and intestinal permeability in female patients with RC. Patients with RC (n = 16) were enrolled and compared with healthy female subjects (n = 15) and patients with chronic gastrointestinal (GI) disorders (n = 238). We calculated the Acute Cystitis Symptom Score/Urinary Tract Infection Symptom Assessment (ACSS/UTISA) and Gastrointestinal Symptom Rating Scale (GSRS) scores and evaluated intestinal permeability and the fecal microbiome in the first two cohorts. Patients with RC showed an increased prevalence of gastrointestinal symptoms compared with healthy controls. Of the patients with RC, 88% showed an increased intestinal permeability with reduced biodiversity of gut microbiota compared to healthy controls, and 68% of the RC patients had a final diagnosis of gastrointestinal disease. Similarly, GI patients reported a higher incidence of urinary symptoms with a diagnosis of RC in 20%. Gut barrier impairment seems to play a major role in the pathogenesis of RC. Further studies are necessary to elucidate the role of microbiota and intestinal permeability in urinary tract infections.

Prediction, prevention, diagnosis and management of food allergy can take advantage of a series of omics approaches. They can provide (1) information on the disease itself and the affected organism, and (2) information on the environmental factors determining food allergy, including all the exposomic determinants. As to the first objective, data from genetics, epigenetics, transcriptomics, proteomics and metabolomics evaluations can be combined. Their integration can outline the path of sensitization to food allergens with absolute precision, can help in identifying predictive factors and early diagnostic factors, can assist in the phenotyping of food allergy, and, in particular, in the identification of patients who respond positively to oral immunotherapy. The applications for the exposome are even wider. Bacterial genomics can help establish associations between species, genera, and phyla of bacteria in gut microbiota and food allergy. Metabolomic techniques can identify bacterial metabolites in the intestine and their functions in the development of disease and its resolution. The level of environmental exposure to allergens, toxins, bacterial glycoproteins, and pollutants can be important in interpreting the genesis of food allergy. Finally, proteomic methods are becoming protagonists in the context of precautionary food allergen labeling. It is no exaggeration to say that these techniques will revolutionize the field of food allergy in the next 10 years.

Anisakiasis is nowadays a well-known infection, mainly caused by the accidental ingestion of Anisakis larvae, following the consumption of raw or undercooked fishes and cephalopods. Due to the similarity of symptoms with those of common gastrointestinal disorders, this infection is often underestimated, and the need for new specific diagnostic tools is becoming crucial. Given the remarkable impact that MALDI–TOF MS biotyping had in the last decade in clinical routine practice for the recognition of bacterial and fungi strains, a similar scenario could be foreseen for the identification of parasites, such as nematodes. In this work, a MALDI–TOF MS profiling of Anisakis proteome was pursued with a view to constructing a first spectral library for the diagnosis of Anisakis infections. At the same time, a shotgun proteomics approach by LC–ESI–MS/MS was performed on the two main fractions obtained from protein extraction, to evaluate the protein species enriched by the protocol. A set of MALDI–TOF MS signals associated with proteins originating in the ribosomal fraction of the nematode extract was selected as a potential diagnostic tool for the identification of Anisakis spp.

Fragile X-related disorders are due to a dynamic mutation of the CGG repeat at the 5? UTR of the FMR1 gene, coding for the RNA-binding protein FMRP. As the CGG sequence expands from premutation (PM, 56-200 CGGs) to full mutation (> 200 CGGs), FMRP synthesis decreases until it is practically abolished in fragile X syndrome (FXS) patients, mainly due to FMR1 methylation. Cells from rare individuals with no intellectual disability and carriers of an unmethylated full mutation (UFM) produce slightly elevated levels of FMR1-mRNA and relatively low levels of FMRP, like in PM carriers. With the aim of clarifying how UFM cells differ from CTRL and FXS cells, a comparative proteomic approach was undertaken, from which emerged an overexpression of SOD2 in UFM cells, also confirmed in PM but not in FXS. The SOD2-mRNA bound to FMRP in UFM more than in the other cell types. The high SOD2 levels in UFM and PM cells correlated with lower levels of superoxide and reactive oxygen species (ROS), and with morphological anomalies and depolarization of the mitochondrial membrane detected through confocal microscopy. The same effect was observed in CTRL and FXS after treatment with MC2791, causing SOD2 overexpression. These mitochondrial phenotypes reverted after knock-down with siRNA against SOD2-mRNA and FMR1-mRNA in UFM and PM. Overall, these data suggest that in PM and UFM carriers, which have high levels of FMR1 transcription and may develop FXTAS, SOD2 overexpression helps to maintain low levels of both superoxide and ROS with signs of mitochondrial degradation.

Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by behavioral alterations and currently affect about 1% of children. Significant genetic factors and mechanisms underline the causation of ASD. Indeed, many affected individuals are diagnosed with chromosomal abnormalities, submicroscopic deletions or duplications, single-gene disorders or variants. However, a range of metabolic abnormalities has been highlighted in many patients, by identifying biofluid metabolome and proteome profiles potentially usable as ASD biomarkers. Indeed, next-generation sequencing and other omics platforms, including proteomics and metabolomics, have uncovered early age disease biomarkers which may lead to novel diagnostic tools and treatment targets that may vary from patient to patient depending on the specific genomic and other omics findings. The progressive identification of new proteins and metabolites acting as biomarker candidates, combined with patient genetic and clinical data and environmental factors, including microbiota, would bring us towards advanced clinical decision support systems (CDSSs) assisted by machine learning models for advanced ASD-personalized medicine. Herein, we will discuss novel computational solutions to evaluate new proteome and metabolome ASD biomarker candidates, in terms of their recurrence in the reviewed literature and laboratory medicine feasibility. Moreover, the way to exploit CDSS, performed by artificial intelligence, is presented as an effective tool to integrate omics data to electronic health/medical records (EHR/EMR), hopefully acting as added value in the near future for the clinical management of ASD.

Purpose of review We review the laws on labeling in the international community, the difficulties they pose to the food manufacturers to prepare the food labels and the methodologies to determine the concentration of potential allergens in foods. Recent findings European Food Safety Authority and International Life Sciences Institute Europe are evaluating strategies to identify the threshold level of allergen that can trigger a reaction in individuals. The most used techniques to detect the presence of protein in food are Enzyme-linked immunosorbent assay, polymerase chain reaction and real time polymerase chain reaction. Researchers are now trying to apply proteomics to estimate the amount of protein within the food.In order to protect the health of consumers, the Codex Alimentarius Commission updates constantly the list of allergens. In response to these regulations, some industries have also added some precautionary allergen labeling (PAL). It was generally agreed that PAL statements needed to be visible, simple, and safe. It was suggested that PAL be standardized, an action that would occur if the 'Voluntary Incidental Trace Allergen Labelling' process was made mandatory. Summary So far, no laboratory technique is able to reassure the consumers about the composition of foods found on the packaging. International authorities produced increasingly stringent laws, but more is still to do.

Food allergy is the disease where the immune system is elicited by antigens in food. Although innocuous for immune-tolerant individuals, an ever-growing number of food allergenic people are being registered worldwide. To date, no treatment to cure food allergy is available and the disease management relies on the careful exclusion of the allergenic food from the diet of the allergic individuals. Great efforts are ongoing to clarify the allergenic mechanisms of the diverse allergenic proteins of food origin, aimed to both designing suitable therapies and for a timely and precise diagnosis of the allergic condition. Among the other omics sciences, mass spectrometry (MS)-based proteomics is gaining a steadily increasing interest by the whole scientific community acknowledged its high versatility. In the present work, the latest proteomics based-studies on allergenic proteins are reviewed to provide guidance on the different MS-based methodologies adopted in the research on food allergens. Our review points to highlight the strengths of the MS-based proteomics and how these have been exploited to address specific research questions. Also, the most common drawbacks encountered in a proteomic study are discussed, providing an overview that helps novel researchers in choosing the more suitable experimental workflow. SIGNIFICANCE: Wide wealth of knowledge arising from the various MS-based proteomic investigations is improving our understanding of food allergy through molecular characterization of food allergens. The present work reviews the key aspects to be evaluated while investigating food allergens by means of MS-based proteomics and provide guidance to the novel research groups approaching to the fascinating world of MS-based food allergens detection.

Carbapenem-resistant Acinetobacter baumannii strains cause life-threatening infections due to the lack of therapeutic options. Although the main mechanisms underlying antibiotic-resistance have been extensively studied, the general response to maintain bacterial viability under antibiotic exposure deserves to be fully investigated. Since the periplasmic space contains several proteins with crucial cellular functions, besides carbapenemases, we decided to study the periplasmic proteome of the multidrug-resistant (MDR) A. baumannii AB5075 strain, grown in the absence and presence of imipenem (IMP). Through the proteomic approach, 65 unique periplasmic proteins common in both growth conditions were identified: eight proteins involved in protein fate, response to oxidative stress, energy metabolism, antibiotic-resistance, were differentially expressed. Among them, ABUW_1746 and ABUW_2363 gene products presented the tetratricopeptide repeat motif, mediating protein-protein interactions. The expression switch of these proteins might determine specific protein interactions to better adapt to changing environmental conditions. ABUW_2868, encoding a heat shock protein likely involved in protection against oxidative stress, was upregulated in IMP-exposed bacteria. Accordingly, the addition of periplasmic proteins from A. baumannii cultured with IMP increased bacterial viability in an antioxidant activity assay. Overall, this study provides the first insights about the composition of the periplasmic proteins of a MDR A. baumannii strain, its biological response to IMP and suggests possible new targets to develop alternative antibiotic drugs.

The present work aimed to explore the innovative hypothesis that different transcript/protein variants of a pro-neurotrophin may generate different biological outcomes in a cellular system. Nerve growth factor (NGF) is important in the development and progression of neurodegenerative and cancer conditions. Mature NGF (mNGF) originates from a precursor, proNGF, produced in mouse in two major variants, proNGF-A and proNGF-B. Different receptors bind mNGF and proNGF, generating neurotrophic or neurotoxic outcomes. It is known that dysregulation in the proNGF/mNGF ratio and in NGF-receptors expression affects brain homeostasis. To date, however, the specific roles of the two major proNGF variants remain unexplored. Here we attempted a first characterization of the possible differential effects of proNGF-A and proNGF-B on viability, differentiation and endogenous ngf gene expression in the PC12 cell line. We also investigated the differential involvement of NGF receptors in the actions of proNGF. We found that native mouse mNGF, proNGF-A and proNGF-B elicited different effects on PC12 cell survival and differentiation. Only mNGF and proNGF-A promoted neurotrophic responses when all NGF receptors are exposed at the cell surface. Tropomyosine receptor kinase A (TrkA) blockade inhibited cell differentiation, regardless of which NGF was added to culture media. Only proNGF-A exerted a pro-survival effect when TrkA was inhibited. Conversely, proNGF-B exerted differentiative effects when the p75 neurotrophin receptor (p75NTR) was antagonized. Stimulation with NGF variants differentially regulated the autocrine production of distinct proNgf mRNA. Overall, our findings suggest that mNGF and proNGF-A may elicit similar neurotrophic effects, not necessarily linked to activation of the same NGF-receptor, while the action of proNGF-B may be determined by the NGF-receptors balance. Thus, the proposed involvement of proNGF/NGF on the development and progression of neurodegenerative and tumor conditions may depend on the NGF-receptors balance, on specific NGF trancript expression and on the proNGF protein variant ratio.

Breastfeeding is nowadays known to be one of the most critical factors contributing to the development of an efficient immune system. In the last decade, a consistent number of pieces of evidence demonstrated the relationship between a healthy organism and its gut microbiota. However, this link is still not fully understood and requires further investigation. We recently adopted a murine model to describe the impact of either maternal milk or parental genetic background, on the composition of the gut microbial population in the first weeks of life. A metaproteomic approach to such complex environments is a big challenge that requires a strong effort in both data production and analysis, including the set-up of dedicated multitasking bioinformatics pipelines. Herein we present an LC-MS/MS based investigation to monitor mouse gut microbiota in the early life, aiming at characterizing its functions and metabolic activities together with a taxonomic description in terms of operational taxonomic units. We provided a quantitative evaluation of bacterial metaproteins, taking into account differential expression results in relation to the functional and taxonomic classification, particularly with proteins from orthologues groups. This allowed the reduction of the bias arising from the presence of a high number of shared peptides, and proteins, among different bacterial species. We also focused on host mucosal proteome and its modulation, according to different microbiota composition. SIGNIFICANCE: This paper would represent a reference work for investigations on gut microbiota in early life, from both a microbiological and a functional proteomic point of view. We focused on the shaping of the mouse gut microbiota in dependence on the feeding modality, defining a reliable taxonomic description, highlighting some functional characteristics of the microbial community, and performing a first quantitative evaluation by data independent analysis in metaproteomics.

Human saliva contains hundreds of small proline-rich peptides originated by the proteolytic cleavage of the salivary basic Proline-Rich Proteins. Nevertheless only for few of them a specific biological activity has been assigned to date. Among them, the 1932 Da peptide (p1932) has been patented as an anti-HIV agent. In order to shed light on the possible mechanism of action of this peptide, we assessed in this study, by means of molecular dynamics calculations, circular dichroism and FTIR spectroscopic techniques, that p1932 has an intrinsic propensity to adopt a polyproline-II helix arrangement. This structural feature combined with the presence of PxxP motifs in its primary structure, represents an essential property for the exploitation of several biological activities. Next to these findings, we recently demonstrated the ability of this peptide to be internalized within cells of the oral mucosa, thus we focused onto a possible intracellular target, represented by the SH3 domains family. Its ability to interact with selected SH3 domains was finally assayed by Surface Plasmon Resonance spectroscopy. As a result, only Fyn, Hck, and c-Src SH3 domains gave positive results in terms of interaction, showing dissociation constants ranging from nanomolar to micromolar values having the best performer a KD of 148 nM. It is noteworthy that all the interacting domains belong to the Src kinases family, suggesting a role for p1932 as a modulator of the signal transduction pathways mediated by these kinases. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 714-725, 2016.

The aim of the present work has been the mass spectrometry characterization of the Nimotuzumab (NIM) antibody chemically modified with the bifunctional chelating agent para-S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraaza cyclododecanetetraacetic acid (p-SCN-Bn-DOTA). The conjugate, upon labeling with the pure β--emitter 90Y3+, could represent a promising candidate as radiotracer for an innovative radio-guided surgery (RGS) technique, developed and patented by researchers of our group, which uses a probe system for intraoperative detection of tumor residues exploiting the selective uptake of β--emitting tracers. The results reported in this study show that multiple DOTA molecules bind to lysine residues of both light and heavy chains of the antibody and, probably, some of them are linked to the variable region of antibody. Moreover, the new mass spectrometric analysis highlights the presence of unreacted NIM in the final product. The information obtained by this work is of fundamental importance in the perspective to utilize this conjugate as a radiocompound after its labeling with 90Y3+ radioisotope. Indeed, the conjugation efficiency and the presence of unreacted NIM affect the specific activity of the final radiotracer which binds specific receptor.

The aim of the present work has been the mass spectrometry characterization of the Nimotuzumab (NIM) antibody chemically modified with the bifunctional chelating agent para-S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraaza cyclododecanetetraacetic acid (p-SCN-Bn-DOTA). The conjugate, upon labeling with the pure β

The human gut has been continuously exposed to a broad spectrum of intestinal organisms, including viruses, bacteria, fungi, and parasites (protozoa and worms), over millions of years of coevolution, and plays a central role in human health. The modern lifestyles of Western countries, such as the adoption of highly hygienic habits, the extensive use of antimicrobial drugs, and increasing globalisation, have dramatically altered the composition of the gut milieu, especially in terms of its eukaryotic “citizens.” In the past few decades, numerous studies have highlighted the composition and role of human intestinal bacteria in physiological and pathological conditions, while few investigations exist on gut parasites and particularly on their coexistence and interaction with the intestinal microbiota. Studies of the gut “parasitome” through “omic” technologies, such as (meta)genomics, transcriptomics, proteomics, and metabolomics, are herein reviewed to better understand their role in the relationships between intestinal parasites, host, and resident prokaryotes, whether pathogens or commensals. Systems biology–based profiles of the gut “parasitome” under physiological and severe disease conditions can indeed contribute to the control of infectious diseases and offer a new perspective of omics-assisted tropical medicine.

The tissue inhibitor of metalloproteinase TIMP3 is a stromal protein that restrains the activity of both protease and receptor in the extracellular matrix and has been found to be down-regulated in diabetic nephropathy (DN), the leading cause of end-stage renal disease in developed countries. In order to gain deeper insights on the association of loss of TIMP3 and DN, we performed differential proteomic analysis of kidney and blood metabolic profiling of wild-type and Timp3-knockout mice before and after streptozotocin (STZ) treatment, widely used to induce insulin deficiency and hyperglycemia. Kidney proteomic data and blood metabolic profiles suggest significant alterations of peroxisomal and mitochondrial fatty acids β-oxidation in Timp3-knockout mice compared to wild-type mice under basal condition. These alterations were exacerbated in response to STZ treatment. Proteomic and metabolomic approaches showed that loss of TIMP3 alone or in combination with STZ treatment results in significant alterations of kidney lipid metabolism and peripheral acylcarnitine levels, supporting the idea that loss of TIMP3 may generate a phenotype more prone to DN.

In this research, salicylic acid is proposed as an alternative biocide-free agent suitable for a preventive or integrative anti-biofilm approach. Salicylic acid has been proved to: (1) reduce bacterial adhesion up to 68.1 ± 5.6%; (2) affect biofilm structural development, reducing viable biomass by 97.0 ± 0.7% and extracellular proteins and polysaccharides by 83.9 ± 2.5% and 49.5 ± 5.5% respectively; and (3) promote biofilm detachment 3.4 ± 0.6-fold. Moreover, salicylic acid treated biofilm showed an increased amount of intracellular (2.3 ± 0.2-fold) and extracellular (2.1 ± 0.3-fold) reactive oxygen species, and resulted in increased production of the quorum sensing signal indole (7.6 ± 1.4-fold). For the first time, experiments revealed that salicylic acid interacts with proteins that play a role in quorum sensing, reactive oxygen species accumulation, motility, extracellular polymeric matrix components, transport and metabolism.

Purpose: Among the different types of kidney stones, matrix stones are uncommon urinary calculi composed of a soft, pliable, amorphous substance with little crystalline content. To gain insight into the pathogenesis we investigated the protein component by analyzing the proteomic profiles of surgically removed matrix stones. Materials and Methods: A total of 5 stones were harvested from 4 patients who underwent surgery for medical reasons at 3 clinical centers during a 7-year period. Matrix stone proteome characterization was performed by mass spectrometry based techniques using an integrated top-down/bottom-up proteomic platform. Results: We identified 142 nonredundant proteins and peptides across all samples. Neutrophil defensin 1, and proteins S100-A8 and S100-A9 were the main components of these renal calculi. Conclusions: The abundance of identified inflammatory molecules points to an inflammatory process as the event that initializes soft calculi formation rather than as a consequence of such formation. The post-translational oxidative changes in S100-A8 and A9, and the presence of thymosin ?-4, granulins and ubiquitin also suggest the intervention of host defenses through a superimposed, vigorous counter inflammatory process. The post-translational changes seen in the proteins and peptides, and the known self-assembling capability of S100-A8 and S100-A9 probably explain the gelatinous consistency of these stones.

Human saliva contains hundreds of small proline-rich peptides originated by the proteolytic cleavage of the salivary basic Proline-Rich Proteins. Nevertheless only for few of them a specific biological activity has been assigned to date. Among them, the 1932 Da peptide (p1932) has been patented as an anti-HIV agent. In order to shed light on the possible mechanism of action of this peptide, we assessed in this study, by means of molecular dynamics calculations, circular dichroism and FTIR spectroscopic techniques, that p1932 has an intrinsic propensity to adopt a polyproline-II helix arrangement. This structural feature combined with the presence of PxxP motifs in its primary structure, represents an essential property for the exploitation of several biological activities. Next to these findings, we recently demonstrated the ability of this peptide to be internalized within cells of the oral mucosa, thus we focused onto a possible intracellular target, represented by the SH3 domains family. Its ability to interact with selected SH3 domains was finally assayed by Surface Plasmon Resonance spectroscopy. As a result, only Fyn, Hck, and c-Src SH3 domains gave positive results in terms of interaction, showing dissociation constants ranging from nanomolar to micromolar values having the best performer a KD of 148 nM. It is noteworthy that all the interacting domains belong to the Src kinases family, suggesting a role for p1932 as a modulator of the signal transduction pathways mediated by these kinases. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 714-725, 2016.

A salivary proline-rich peptide of 1932 Da showed a dose-dependent antagonistic effect on the cytosolic Ca2+ mobilization induced by progesterone in a tongue squamous carcinoma cell line. Structure-activity studies showed that the activity of the peptide resides in the C-terminal region characterized by a proline stretch flanked by basic residues. Furthermore, lack of activity of the retro-inverso peptide analogue suggested the involvement of stereospecific recognition. Mass spectrometry-based shotgun analysis, combined with Western blotting tests and biochemical data obtained with the Progesterone Receptor Membrane Component 1 (PGRMC1) inhibitor AG205, showed strong evidence that p1932 performs its modulatory action through an interaction with the progesterone receptor PGRMC1, which is predominantly expressed in this cell line and, clearly, plays a role in progesterone induced Ca2+ response. Thus, our results point to p1932 as a modulator of the transduction signal pathway mediated by this protein and, given a well-established involvement of PGRMC1 in tumorigenesis, highlight a possible therapeutic potential of p1932 for the treatment of oral cancer.

Ataxia Telangiectasia Mutated (ATM) protein kinase is a key effector in the modulation of the functionality of some important stress responses, including DNA damage and oxidative stress response, and its deficiency is the hallmark of Ataxia Telangiectasia (A-T), a rare genetic disorder. ATM modulates the activity of hundreds of target proteins, essential for the correct balance between proliferation and cell death. The aim of this study is to evaluate the phenotypic adaptation at the protein level both in basal condition and in presence of proteasome blockage in order to identify the molecules whose level and stability are modulated through ATM expression. We pursued a comparative analysis of ATM deficient and proficient lymphoblastoid cells by label-free shotgun proteomic experiments comparing the panel of proteins differentially expressed. Through a non-supervised comparative bioinformatic analysis these data provided an insight on the functional role of ATM deficiency in cellular carbohydrate metabolism's regulation. This hypothesis has been demonstrated by targeted metabolic fingerprint analysis SRM (Selected Reaction Monitoring) on specific thermodynamic checkpoints of glycolysis. This article is part of a Special Issue entitled: Translational Proteomics., Graphical abstract Highlights ► Identification of the repertoire of protein adaptions in A-T cells ► Evident impairment of carbohydrate metabolism in absence of ATM expression ► ATM deficiency affects the control of protein stability

Mitochondria play an important role on the entire cellular copper homeostatic mechanisms.Alteration of cellular copper levels may thus influence mitochondrial proteome and its investigation represents an important contribution to the general understanding of copper related cellular effects. In these study we have performed an organelle targeted proteomic investigation focusing our attention on the effect of non-lethal 1 mM copper concentration on Saccharomyces cerevisiae mitochondrial proteome. Functional copper effects on yeast mitochondrial proteome were evaluated by using both 2D electrophoresis (2-DE) and liquid chromatography coupled with tandem mass spectrometry. Proteomic data have been then analyzed by different unsupervised meta-analysis approaches that highlight the impairment of mitochondrial functions and the activation of oxidative stress response.Interestingly, our data have shown that stress response generated by 1 mM copper treatment determines the activation of S. cerevisiae survival pathway. To investigate these findings we have treated yeast cells responsiveness to copper with hydrogen peroxide and observed a protective role of this metal. These results are suggestive of a copper role in the protection from oxidative stress possibly due to the activation of mechanisms involved in cellular survival and growth. Refereed/Peer-reviewed

Aliment Pharmacol Ther 2011; 34: 982–993 Summary Background Erosive reflux disease (ERD) and non-erosive reflux disease (NERD) are often regarded as part of the spectrum of the same disease. Aim To elucidate molecular features that characterise NERD and ERD at the protein level. Methods A total of 56 consecutive subjects were enrolled: 10 healthy subjects, 24 with NERD and 22 with ERD. Eight specimens were taken from macroscopically normal mucosa at 5 cm of gastro-oesophageal junction. Four were processed for the proteins extraction and four for evaluation using haematoxylin-eosin and immunohistochemistry. We used shotgun proteomics to identify tentative disease molecular features for ERD or NERD. Candidate distinctive proteins were verified using immunohistochemistry. Results Shotgun proteomics analysis revealed 33 differentially expressed proteins in NERD vs. ERD samples, involved in cellular proliferation, keratinisation, stress responses and sugar metabolism. Based on a gene ontology meta-analysis, seven of them were further analysed using Western blotting (WB) and four also using immunohistochemistry. We identified novel candidate disease molecular features for GERD and few distinctive proteins to discriminate NERD and ERD. In particular, Transitional Endoplasmic Reticulum ATPase (TER ATPase), GAPDH, Alpha 1 Acid Glycoprotein 1, Annexin A1, Calmodulin and 14-3-3 proteins were confirmed at WB analysis. Conclusions Non-erosive reflux disease and ERD are distinct disease entities at the protein level. This study proposes an array of candidate biomarkers possibly useful to discriminate between NERD and ERD.

Anandamide (AEA) is an endogenous agonist of type 1 cannabinoid receptors (CB1R) that, along with metabolic enzymes of AEA and congeners, compose the "endocannabinoid system." Here we report the biochemical, morphological, and functional characterization of the endocannabinoid system in human neuroblastoma SH-SY5Y cells that are an experimental model for neuronal cell damage and death, as well as for major human neurodegenerative disorders. We also show that AEA dose-dependently induced apoptosis of SH-SY5Y cells. Through proteomic analysis, we further demonstrate that AEA-induced apoptosis was paralleled by an approximately 3 to approximately 5-fold up-regulation or down-regulation of five genes; IgG heavy chain-binding protein, stress-induced phosphoprotein-1, and triose-phosphate isomerase-1, which were up-regulated, are known to act as anti-apoptotic agents; actin-related protein 2/3 complex subunit 5 and peptidylprolyl isomerase-like protein 3 isoform PPIL3b were down-regulated, and the first is required for actin network formation whereas the second is still function-orphan. Interestingly, only the effect of AEA on BiP was reversed by the CB1R antagonist SR141716, in SH-SY5Y cells as well as in human neuroblastoma LAN-5 cells (that express a functional CB1R) but not in SK-NBE cells (which do not express CB1R). Silencing or overexpression of BiP increased or reduced, respectively, AEA-induced apoptosis of SH-SY5Y cells. In addition, the expression of BiP and of the BiP-related apoptotic markers p53 and PUMA was increased by AEA through a CB1R-dependent pathway that engages p38 and p42/44 mitogen-activated protein kinases. Consistently, this effect of AEA was minimized by SR141716. In conclusion, we identified BiP as a key protein in neuronal apoptosis induced by AEA.

Background We recently demonstrated that epicardial progenitor cells participate in the regenerative response to myocardial infarction (MI) and factors released in the pericardial fluid (PF) may play a key role in this process. Exosomes are secreted nanovesicles of endocytic origin, identified in most body fluids, which may contain molecules able to modulate a variety of cell functions. Here, we investigated whether exosomes are present in the PF and their potential role in cardiac repair. Methods and results Early gene expression studies in 3day-infarcted mouse hearts showed that PF induces epithelial-to-mesenchymal transition (EMT) in epicardial cells. Exosomes were identified in PFs from non-infarcted patients (PFC) and patients with acute MI (PFMI). A shotgun proteomics analysis identified clusterin in exosomes isolated from PFMI but not from PFC. Notably, clusterin has a protective effect on cardiomyocytes after acute MI in vivo and is an important mediator of TGFβ-induced. Clusterin addition to the pericardial sac determined an increase in epicardial cells expressing the EMT marker α-SMA and, interestingly, an increase in the number of epicardial cells ckit + /α-SMA + , 7days following MI. Importantly, clusterin treatment enhanced arteriolar length density and lowered apoptotic rates in the peri-infarct area. Hemodynamic studies demonstrated an improvement in cardiac function in clusterin-treated compared to untreated infarcted hearts. Conclusions Exosomes are present and detectable in the PFs. Clusterin was identified in PFMI-exosomes and might account for an improvement in myocardial performance following MI through a framework including EMT-mediated epicardial activation, arteriogenesis and reduced cardiomyocyte apoptosis.

Aims Due to their antimicrobial activity, silver nanoparticles (Ag-NPs) are being increasingly used in a number of industrial products. The accumulation of Ag-NPs in the soil might affect plant growth-promoting rhizobacteria and, in turn, the plants. We describe the effects of Ag-NPs on the soil bacteria Azotobacter vinelandii and Bacillus subtilis. Methods and Results In growth-inhibition studies, A. vinelandii showed extreme sensitivity to Ag-NPs, compared to B. subtilis. We investigated the effects of Ag-NPs at subinhibitory concentrations, both on planktonic and sessile B. subtilis cells. As determined by 2,7-dichlorofluorescein-diacetate assays, Ag-NPs increase the formation of reactive oxygen species in planktonic cells, but not in sessile cells, suggesting the activation of scavenging systems in biofilms. Consistently, proteomic analysis in B. subtilis Ag-NPs-treated biofilms showed increased production of proteins related to quorum sensing and involved in stress responses and redox sensing. Extracellular polysaccharides production and inorganic phosphate solubilization were also increased, possibly as part of a coordinated response to stress. Conclusions At low concentrations, Ag-NPs killed A. vinelandii and affected cellular processes in planktonic and sessile B. subtilis cells. Significance and Impact of the Study Re-direction of gene expression, linked to selective toxicity, suggests a strong impact of Ag-NPs on soil bacterial communities.

The tissue inhibitor of metalloproteinase (TIMP)3, a stromal protein that restrains the activity of proteases and receptors, is reduced in inflammatory metabolic disorders such as type 2 diabetes mellitus (T2DM) and atherosclerosis. We overexpressed Timp3 in mouse macrophages (MacT3) to analyze its potential antidiabetic and antiatherosclerotic effects. Transgenic mice with myeloid cells targeting overexpression of TIMP3 were generated and fed a high-fat diet for 20 weeks. Physical and metabolic phenotypes were determined. Inflammatory markers, lipid accumulation, and insulin sensitivity were measured in white adipose tissue (WAT), liver, and skeletal muscle. In a model of insulin resistance, MacT3 mice were more glucose tolerant and insulin sensitive than wild-type mice in both in vitro and in vivo tests. Molecular and biochemical analyses revealed that increased expression of TIMP3 restrained metabolic inflammation and stress-related pathways, including Jun NH2-terminal kinase and p38 kinase activation, in WAT and liver. TIMP3 overexpression in macrophages resulted in reduced activation of oxidative stress signals related to lipid peroxidation, protein carbonylation, and nitration in WAT and liver. Our data show that macrophage-specific overexpression of TIMP3 protects from metabolic inflammation and related metabolic disorders such as insulin resistance, glucose intolerance, and nonalcoholic steatohepatitis.