During adulthood, the neurotrophin Nerve Growth Factor (NGF) sensitizes nociceptors, thereby increasing the response to noxious stimuli. The relationship between NGF and pain is supported by genetic evidence: mutations in the NGF TrkA receptor in patients affected by an hereditary rare disease (Hereditary Sensory and Autonomic Neuropathy type IV, HSAN IV) determine a congenital form of severe pain insensitivity, with mental retardation, while a mutation in NGFB gene, leading to the aminoacid substitution R100W in mature NGF, determines a similar loss of pain perception, without overt cognitive neurological defects (HSAN V). The R100W mutation provokes a reduced processing of proNGF to mature NGF in cultured cells and a higher percentage of neurotrophin secreted is in the proNGF form. Moreover, using Surface Plasmon Resonance we showed that the R100W mutation does not affect NGF binding to TrkA, while it abolishes NGF binding to p75NTR receptors. However, it remains to be clarified whether the major impact of the mutation is on the biological function of proNGF or of mature NGF and to what extent the effects of the R100W mutation on the HSAN V clinical phenotype are developmental, or whether they reflect an impaired effectiveness of NGF to regulate and mediate nociceptive transmission in adult sensory neurons. Here we show that the R100 mutation selectively alters some of the signaling pathways activated downstream of TrkA NGF receptors. NGFR100 mutants maintain identical neurotrophic and neuroprotective properties in a variety of cell assays, while displaying a significantly reduced pain-inducing activity in vivo (n = 8-10 mice/group). We also show that proNGF has a significantly reduced nociceptive activity, with respect to NGF. Both sets of results jointly contribute to elucidating the mechanisms underlying the clinical HSAN V manifestations, and to clarifying which receptors and intracellular signaling cascades participate in the pain sensitizing action of NGF.

Parkinson's disease (PD) is a complex neurodegenerative disease whose etiology has not been completely characterized. Many cellular processes have been proposed to play a role in the neuronal damage and loss: defects in the proteosomal activity, altered protein processing, increased reactive oxygen species burden. Among them, the involvement of a decreased activity and an altered disposal of mitochondria is becoming more and more evident. The mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP(+)), an inhibitor of complex I, has been widely used to reproduce biochemical alterations linked to PD in vitro and its precursor, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), to induce a Parkinson-like syndrome in vivo. Therefore, we performed a meta-analysis of the literature of all the proteomic investigations of neuronal alterations due to MPP(+) treatment and compared it with our results obtained with a mitochondrial proteomic analysis of SH-SY5Y cells treated with MPP(+). By using open-source bioinformatics tools, we identified the biochemical pathways and the molecular functions mostly affected by MPP(+), i.e., ATP production, the mitochondrial unfolded stress response, apoptosis, autophagy, and, most importantly, the synapse funcionality. Eventually, we generated protein networks, based on physical or functional interactions, to highlight the relationships among the molecular actors involved. In particular, we identified the mitochondrial protein HSP60 as the central hub in the protein-protein interaction network. As a whole, this analysis clarified the cellular responses to MPP(+), the specific mitochondrial proteome alterations induced and how this toxic model can recapitulate some pathogenetic events of PD.

No abstract available

In recent years a growing debate is about whether botulinum neurotoxins are retrogradely transported from the site of injection. Immunodetection of cleaved SNAP-25 (cl-SNAP-25), the protein of the SNARE complex targeted by botulinum neurotoxin serotype A (BoNT/A), could represent an excellent approach to investigate the mechanism of action on the nociceptive pathways at peripheral and/or central level. After peripheral administration of BoNT/A, we analyzed the expression of cl-SNAP-25, from the hindpaw's nerve endings to the spinal cord, together with the behavioral effects on neuropathic pain. We used the chronic constriction injury of the sciatic nerve in CD1 mice as animal model of neuropathic pain. We evaluated immunostaining of cl-SNAP-25 in the peripheral nerve endings, along the sciatic nerve, in dorsal root ganglia and in spinal dorsal horns after intraplantar injection of saline or BoNT/A, alone or colocalized with either glial fibrillar acidic protein, GFAP, or complement receptor 3/cluster of differentiation 11b, CD11b, or neuronal nuclei, NeuN, depending on the area investigated. Immunofluorescence analysis shows the presence of the cl-SNAP-25 in all tissues examined, from the peripheral endings to the spinal cord, suggesting a retrograde transport of BoNT/A. Moreover, we performed in vitro experiments to ascertain if BoNT/A was able to interact with the proliferative state of Schwann cells (SC). We found that BoNT/A modulates the proliferation of SC and inhibits the acetylcholine release from SC, evidencing a new biological effect of the toxin and further supporting the retrograde transport of the toxin along the nerve and its ability to influence regenerative processes. The present results strongly sustain a combinatorial action at peripheral and central neural levels and encourage the use of BoNT/A for the pathological pain conditions difficult to treat in clinical practice and dramatically impairing patients' quality of life.

Multiple Sclerosis (MS) is a neurodegenerative autoimmune demyelinating disease affecting young adults. The aetiology still remains a mystery and diagnosis is impaired by the lack of defined molecular markers. Autoimmune response remains the main topic under investigation and recent studies suggest additional non-proteic mediators of brain inflammation such as lipids. We carried out an LC-MS based lipidomics approach to highlight serum lipids profiling in MS. Method was optimised and applied in a preliminary clinical cross-sectional investigation of MS patients vs Healthy Controls (HC) and patients with Other Neurological Diseases (OND). Ten significant metabolites were highlighted and tentatively identified by accurate mass and MS/MS experiments. Our most relevant data show altered level of lyso-glycerophosphatidylcholine (lysoPC) and glycerophosphatidylcholine (PC) species. Total lysoPC/PC ratio showed significant decrease in pathological groups (MS, OND) and, in addition, MS subjects had a relevant decrease of this ratio also in respect to OND. These findings suggest that there may be an altered phospholipid metabolism in MS that can be evaluated in serum. Some of these features are distinctive and may be considered specific for MS. Our lipidomics data show, for the first time, evidence in serum of a relationship between LysoPC/PC ratio and MS.

Cow serum proteome was evaluated by three different complementary approaches in the control group, subclinical and clinical mastitis in order to possibly find differential protein expression useful for a better understanding of the pathophysiology of mastitis as well as for an early diagnosis of the disease. The systemic inflammatory and oxidative stress response in cows with subclinical and clinical mastitis were observed. The collected evidence shows a differential protein expression of serpin A3-1, vitronectin-like protein and complement factor H in subclinical mastitis in comparison with the control. It was also found a differential protein expression of inter-alpha-trypsin inhibitor heavy chain H4, serpin A3-1, C4b-binding protein alpha chain, haptoglobin and apolipoprotein A-I in clinical mastitis compared to the control. Among the inflammatory proteins up-regulated in clinical mastitis, vitronectin is over-expressed in both subclinical and clinical mastitis indicating a strong bacterial infection. This suggests vitronectin as an important mediator in the pathogenesis of the onset of mastitis as well as a valuable marker for diagnosis of the subclinical form of the disease. Obtained data could be useful for the detection of mastitis during the subclinical phase and for a better comprehension of the pathophysiological mechanisms involved in the onset of the disease.

Mass spectrometry (MS) is becoming the gold standard for biomarker discovery. Several MS-based bioinformatics methods have been proposed for this application, but the divergence of the findings by different research groups on the same MS data suggests that the definition of a reliable method has not been achieved yet. In this work, we propose an integrated software platform, MASCAP, intended for comparative biomarker detection from MALDI-TOF MS data.

Botulinum neurotoxin type A (BoNT/A) and minocycline are potent drugs used in clinical therapies. The primary molecular mechanism of BoNT/A is the cleavage of SNARE proteins, which prevents cells from releasing neurotransmitters from vesicles, while the effects of minocycline are related to the inhibition of p38 activation. Both BoNT/A and minocycline exhibit analgesic effects, however, their direct impact on glial cells is not fully known. Therefore, the aim of the present study was to determine the effects of those drugs on microglial and astroglial activity after lipopolysaccharide (LPS) stimulation and their potential synergistic action. Our results show that BoNT/A and minocycline influenced primary microglial cells by inhibiting intracellular signaling pathways, such as p38, ERK1/2, NF-κB, and the release of pro-inflammatory factors, including IL-1β, IL-18, IL-6, and NOS2. We have revealed that, in contrast to minocycline, BoNT/A treatment did not decrease LPS-induced release of pro-inflammatory factors in the astroglia. In addition, BoNT/A decreased SNAP-23 in both types of glial cells and also SNAP-25 expressed only in astrocytes. Moreover, BoNT/A increased TLR2 and its adaptor protein MyD88, but not TLR4 exclusively in microglial cells. Furthermore, we have shown the impact of BoNT/A on microglial and astroglial cells, with a particular emphasis on its molecular target, TLR2. In contrast, minocycline did not affect any of those factors. We have revealed that despite of different molecular targets, minocycline, and BoNT/A reduced the release of microglia-derived pro-inflammatory factors. In conclusion, we have shown that BoNT/A and minocycline are effective drugs for the management of neuroinflammation by dampening the activation of microglial cells, with minocycline also affecting astroglial activity.

Although histologically benign, adamantinomatous craniopharyngioma (AC) pediatric brain tumor is a locally aggressive disease that frequently determines symptoms and hormonal dysfunctions related to the mass effect on the surrounding structures. Another typical feature of this benign neoplasm is the presence of voluminous liquid cysts frequently associated with the solid component. Even if studies have been devoted to the proteomic characterization of the tumor intracystic fluid, poor explorations have been performed on its solid part, principally investigated by transcriptomics technologies. In the present study, seven specimens of AC whole tumor tissue have been analyzed by LC-MS for a preliminary assessment of the proteomic profile by a top-down/bottom-up integrated approach. Thymosin beta 4, ubiquitin, calmodulin, S100 proteins, prothymosin α isoform 2, alpha-defensins 1-4, and fragments largely belonging to vimentin, hemoglobin, and glial fibrillary acidic protein characterized the intact proteome. The identification of alpha-defensins, formerly characterized in AC intracystic fluid, reinforces the hypothesis of a role for inflammation in tumor pathogenesis. A total number of 1798 unique elements were identified by a bottom-up approach with a special focus on the 433 proteins commonly characterized in the 85.7% of the samples analyzed. Their gene ontology classification evidenced the involvement of the adherence system, intermediate filaments, and actin cytoskeleton in tumor pathogenesis and of elements part of the Wnt, FGF, and EGFR signaling pathways. In addition, proteins involved in calcium modulation, innate immunity, inflammation, CCKR and integrin signaling, and gonadotropin-releasing hormone receptor pathways were also outlined. Further than confirming proteomic data previously obtained on AC intracystic fluid, these results offer a preliminary overview of the AC whole tissue protein phenotype, adding new hints towards the comprehension of this still obscure pediatric brain tumor.

During the last decade, the evidences on the relationship between neurodevelopmental disorders and the microbial communities of the intestinal tract have considerably grown. Particularly, the role of gut microbiota (GM) ecology and predicted functions in Autism Spectrum Disorders (ASD) has been especially investigated by 16S rRNA targeted and shotgun metagenomics, trying to assess disease signature and their correlation with cognitive impairment or gastrointestinal (GI) manifestations of the disease. Herein we present a metaproteomic approach to point out the microbial gene expression profiles, their functional annotations, and the taxonomic distribution of gut microbial communities in ASD children. We pursued a LC-MS/MS based investigation, to compare the GM profiles of patients with those of their respective relatives and aged-matched controls, providing a quantitative evaluation of bacterial metaproteins by SWATH analysis. All data were managed by a multiple step bioinformatic pipeline, including network analysis. In particular, comparing ASD subjects with CTRLs, up-regulation was found for some metaproteins associated with Clostridia and with carbohydrate metabolism (glyceraldehyde-3-phosphate and glutamate dehydrogenases), while down-regulation was observed for others associated with Bacteroidia (SusC and SusD family together with the TonB dependent receptor). Moreover, network analysis highlighted specific microbial correlations among ASD subgroups characterized by different functioning levels and GI symptoms. SIGNIFICANCE: To the best of our knowledge, this study represents the first metaproteomic investigation on the gut microbiota of ASD children compared with relatives and age-matched CTRLs. Remarkably, the applied SWATH methodology allowed the attribution of differentially regulated functions to specific microbial taxa, offering a novel and complementary point of view with respect to previous studies.

Maturity-onset diabetes of the young (MODY) is a rare form of non-autoimmune diabetes with an autosomal dominant inheritance. To date, 14 genes have been reported as genetic basis of MODY. GCK gene, encoding the glucokinase enzyme, was the first MODY gene to be identified. GCK heterozygous inactivating variants cause the GCK-MODY or MODY2 subtype. However, partial or whole gene deletions have been rarely identified, showing it to be a rare cause of GCK-MODY. We reported the molecular evaluation of a Ukrainian patient with clinical diagnosis of MODY2. We performed the Next generation sequencing of the clinical exome using the Clinical Exome Solution® kit (SOPHiA Genetics), followed by the design of a 14 genes virtual panel related to the suggestive diagnosis of MODY. Bioinformatics analysis was performed using the SOPHiA DDM platform (SOPHiA Genetics). The SALSA MLPA kit for MODY (MRC-Holland) was used for relative quantification of GCK exons. From the molecular evaluation, no pathogenic sequence variants were detected in the investigated genes. Copy Number Variation analysis was able to identify a large deletion involving the last three exons of the GCK gene. This result was confirmed by MLPA. To the best of our knowledge, the identified rearrangement has never been reported in the literature.

In the present study, we investigated the water extract of Harpagophytum procumbens DC. ex Meisn. in an experimental model of inflammatory bowel diseases (IBDs). Additionally, a microbiological investigation was carried out to discriminate the efficacy against bacterial and fungal strains involved in IBDs. Finally, an untargeted proteomic analysis was conducted on more than one hundred colon proteins involved in tissue morphology and metabolism. The extract was effective in blunting the production of oxidative stress and inflammation, including serotonin, prostaglandins, cytokines, and transcription factors. Additionally, the extract inhibited the growth of Candida albicans and C. tropicalis. The extract was also able to exert a pro-homeostatic effect on the levels of a wide plethora of colon proteins, thus corroborating a protective effect. Conversely, the supraphysiological downregulation of cytoskeletal-related proteins involved in tissue morphology and antimicrobial barrier function suggests a warning in the use of food supplements containing H. procumbens extracts.

The present investigation aimed to characterize the protein profile of cavitating ultrasound aspirator fluid of newly diagnosed and recurrent glioblastoma comparing diverse zones of collection, i.e., tumor core and tumor periphery, with the aid of 5-aminolevulinic acid fluorescence. The samples were pooled and analyzed in triplicate by LC-MS following the shotgun proteomic approach. The identified proteins were then grouped to disclose elements exclusive and common to the tumor state or tumor zones and submitted to gene ontology classification and pathway overrepresentation analysis. The proteins common to the distinct zones were further investigated by relative quantitation, following a label free approach, to disclose possible differences of expression. Nine proteins, i.e., tubulin 2B chain, CD59, far upstream element-binding, CD44, histone H1.4, caldesmon, osteopontin, tropomyosin chain and metallothionein-2, marked the core of newly diagnosed glioblastoma with respect to tumor periphery. Considering the tumor zone, including the core and the fluorescence positive periphery, the serine glycine biosynthesis, pentose phosphate, 5-hydroxytryptamine degredation, de novo purine biosynthesis and huntington disease pathways resulted statistically significantly overrepresented with respect to the human genome of reference. The fluorescence negative zone shared several protein elements with the tumor zone, possibly indicating the presence of pathological aspects of glioblastoma rather than of normal brain parenchyma. On the other hand, its exclusive protein elements were considered to represent the healthy zone and, accordingly, exhibiting no pathways overrepresentation. On the contrary to newly diagnosed glioblastoma, pathway overrepresentation was recognized only in the healthy zone of recurrent glioblastoma. The TGFβ signaling pathway, exclusively classified in the fluorescence negative periphery in newly diagnosed glioblastoma, was instead the exclusive pathway classified in the tumor core of recurrent glioblastoma. These results, preliminary obtained on sample pools, demonstrated the potential of cavitron ultrasonic sur.

Basic and preclinical research founded the progress of personalized medicine by providing a prodigious amount of integrated profiling data and by enabling the development of biomedical applications to be implemented in patient-centered care and cures. If the rapid development of genomics research boosted the birth of personalized medicine, further development in omics technologies has more recently improved our understanding of the functional genome and its relevance in profiling patients' phenotypes and disorders. Concurrently, the rapid biotechnological advancement in diverse research areas enabled uncovering disease mechanisms and prompted the design of innovative biological treatments tailored to individual patient genotypes and phenotypes. Research in stem cells enabled clarifying their role in tissue degeneration and disease pathogenesis while providing novel tools toward the development of personalized regenerative medicine strategies. Meanwhile, the evolving field of integrated omics technologies ensured translating structural genomics information into actionable knowledge to trace detailed patients' molecular signatures. Finally, neuroscience research provided invaluable models to identify preclinical stages of brain diseases. This review aims at discussing relevant milestones in the scientific progress of basic and preclinical research areas that have considerably contributed to the personalized medicine revolution by bridging the bench-to-bed gap, focusing on stem cells, omics technologies, and neuroscience fields as paradigms.

Epithelial ovarian cancer (EOC) is a highly heterogeneous disease with a high death rate mainly due to the metastatic spread. The expression of MDM4, a well-known p53-inhibitor, is positively associated with chemotherapy response and overall survival (OS) in EOC. However, the basis of this association remains elusive. We show that in vivo MDM4 reduces intraperitoneal dissemination of EOC cells, independently of p53 and an immune-competent background. By 2D and 3D assays, MDM4 impairs the early steps of the metastatic process. A 3D-bioprinting system, ad hoc developed by co-culturing EOC spheroids and endothelial cells, showed reduced dissemination and intravasation into vessel-like structures of MDM4-expressing cells. Consistent with these data, high MDM4 levels protect mice from ovarian cancer-related death and, importantly, correlate with increased 15 y OS probability in large data set analysis of 1656 patients. Proteomic analysis of EOC 3D-spheroids revealed decreased protein synthesis and mTOR signaling, upon MDM4 expression. Accordingly, MDM4 does not further inhibit cell migration when its activity towards mTOR is blocked by genetic or pharmacological approaches. Importantly, high levels of MDM4 reduced the efficacy of mTOR inhibitors in constraining cell migration. Overall, these data demonstrate that MDM4 impairs EOC metastatic process by inhibiting mTOR activity and suggest the usefulness of MDM4 assessment for the tailored application of mTOR-targeted therapy.

The current management of lung cancer patients has reached a high level of complexity. Indeed, besides the traditional clinical variables (e.g., age, sex, TNM stage), new omics data have recently been introduced in clinical practice, thereby making more complex the decision-making process. With the advent of Artificial intelligence (AI) techniques, various omics datasets may be used to create more accurate predictive models paving the way for a better care in lung cancer patients.

We synthesized new pyrrole and indole derivatives as human carbonic anhydrase (hCA) inhibitors with the potential to inhibit the Wnt/β-catenin signaling pathway. The presence of both N1-(4-sulfonamidophenyl) and 3-(3,4,5-trimethoxyphenyl) substituents was essential for strong hCA inhibitors. The most potent hCA XII inhibitor 15 (Ki = 6.8 nM) suppressed the Wnt/β-catenin signaling pathway and its target genes MYC, Fgf20, and Sall4 and exhibited the typical markers of apoptosis, cleaved poly(ADP-ribose)polymerase, and cleaved caspase-3. Compound 15 showed strong inhibition of viability in a panel of cancer cells, including colorectal cancer and triple-negative breast cancer cells, was effective against the NCI/ADR-RES DOX-resistant cell line, and restored the sensitivity to doxorubicin (DOX) in HT29/DX and MDCK/P-gp cells. Compound 15 is a novel dual-targeting compound with activity against hCA and Wnt/β-catenin. It thus has a broad targeting spectrum and is an anticancer agent with specific potential in P-glycoprotein overexpressing cell lines.

The results of Aspirin prevention of colorectal adenomas in patients with familial adenomatous polyposis (FAP) are controversial.

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 1mM 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 1mM 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.

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)-a systemic dual inhibitor of cystathionine-β-synthase and cystathionine-γ lyase (two key enzymes in the production of H2S)-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.