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The present study, employing a comparative proteomic approach, analyzes the protein profile of pig claustrum (CLA), putamen (PU), and insula (IN). Pig brain is an interesting model whose key translational features are its similarities with cortical and subcortical structures of human brain. A greater difference in protein spot expression was observed in CLA vs PU as compared to CLA vs IN. The deregulated proteins identified in CLA resulted to be deeply implicated in neurodegenerative (i.e., sirtuin 2, protein disulfide-isomerase 3, transketolase) and psychiatric (i.e., copine 3 and myelin basic protein) disorders in humans. Metascape analysis of differentially expressed proteins in CLA vs PU comparison suggested activation of the α-synuclein pathway and L1 recycling pathway corroborating the involvement of these anatomical structures in neurodegenerative diseases. The expression of calcium/calmodulin-dependent protein kinase and dihydropyrimidinase like 2, which are linked to these pathways, was validated using western blot analysis. Moreover, the protein data set of CLA vs PU comparison was analyzed by Ingenuity Pathways Analysis to obtain a prediction of most significant canonical pathways, upstream regulators, human diseases, and biological functions. Interestingly, inhibition of presenilin 1 (PSEN1) upstream regulator and activation of endocannabinoid neuronal synapse pathway were observed. In conclusion, this is the first study presenting an extensive proteomic analysis of pig CLA in comparison with adjacent areas, IN and PUT. These results reinforce the common origin of CLA and IN and suggest an interesting involvement of CLA in endocannabinoid circuitry, neurodegenerative, and psychiatric disorders in humans.

Background/aim The lack of specific parathyroid carcinoma (PC) biomarkers in clinical practice points out the importance of analyzing the proteomic signature of this cancer. We performed a comparative proteomic analysis of PC and parathyroid adenoma (PA) co-existing in the same patient. Patients and methods PC and PA were taken from a 63-year-old patient. Using two-dimensional differential gel electrophoresis (2D-DIGE) coupled to mass spectrometry we examined the differences between PC and PA proteins. For validation, additional PC and PA samples were obtained from 10 patients. Western blot analysis was used to validate the difference of expression observed with 2D-DIGE analysis. Bioinfomatic analysis was performed using QIAGEN's Ingenuity Pathways Analysis (IPA) to determine the predominant canonical pathways and interaction networks involved. Results Thirty-three differentially expressed proteins were identified in PC compared to PA. Among these, ubiquitin C-terminal hydrolase-L1 (UCH-L1) was highly overexpressed in PC. The result was confirmed by Western Blot analysis in additional PC samples. Conclusion Our comparative proteomic analysis of co-existing neoplasms allowed detecting specific and peculiar differences between PC and PA overcoming population biological variability.

The purpose of this study was to define the proteome profile of fine needle aspiration (FNA) samples of malignant major salivary gland tumors (MSGT) compared to benign counterparts, and to evaluate potential clinical correlations and future applications. Patients affected by MSGT (n = 20), pleomorphic adenoma (PA) (n = 37) and Warthin’s tumor (WT) (n = 14) were enrolled. Demographic, clinical and histopathological data were registered for all patients. FNA samples were processed to obtain the protein extracts. Protein separation was obtained by two-dimensional electrophoresis (2-DE) and proteins were identified by mass spectrometry. Western blot analysis was performed to validate the 2-DE results. Statistical differences between groups were calculated by the Mann–Whitney U test for non-normal data. Spearman’s rank correlation coefficient was calculated to evaluate correlations among suggested protein biomarkers and clinical parameters. Twelve and 27 differentially expressed spots were found for MSGT versus PA and MSGT versus WT, respectively. Among these, annexin-5, cofilin-1, peptidyl-prolyl-cis–trans-isomerase-A and F-actin-capping-alpha-1 were able to differentiate MSGT from PA, WT, and healthy samples. Moreover, STRING analysis suggested cofilin-1 as a key node of protein interactions. Some of the overexpressed proteins are related to some clinical factors of our cohort, such as survival and outcome. Our results suggest potential protein biomarkers of MSGT, which could allow for more appropriate treatment plans, as well as shedding light on the molecular pathways involved.

Matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) of intact proteins is mostly performed using time-of-flight (TOF) based mass spectrometers, operated in linear mode. Linear MALDI-TOF systems provide limited mass resolving power and mass accuracy, which complicates assigning identities to the peaks in the MSI datasets. In this work we report ultra-high mass resolution MALDI-MSI based on 15T Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for the analysis of intact proteins directly from non-embedded and OCT-embedded mouse and human (control and type 2 diabetes) pancreas so that small endocrine compartments (islets of Langerhans) may be analyzed in control and pathological tissues. Sample preparation methods, in terms of increased sensitivity while limiting lateral diffusion of analytes, have been investigated. By combining protein localization, high mass accuracy, and the clearly resolved isotope patterns we were able to assign protein identities with additional confidence, including proteins of similar average mass and with interspersed isotopomers. These capabilities allowed us to ascertain the presence of many protein adducts that, with a low resolving power instrument, could be misinterpreted as distinct protein ions. (C) 2017 Published by Elsevier B.V

Neurodegenerative diseases are driven by several mechanisms such as inflammation, abnormal protein aggregation, excitotoxicity, mitochondrial dysfunction and oxidative stress. So far, no therapeutic strategies are available for neurodegenerative diseases and in recent years the research is focusing on bioactive molecules present in food. In particular, extra-virgin olive oil (EVOO) phenols have been associated to neuroprotection. In this study, we investigated the potential antioxidant and neuroprotective activity of two different EVOO extracts obtained from Quercetano cultivar trees grown in two different areas (plain and hill) of the Tuscany region (Italy). The different geographical origin of the orchards influenced phenol composition. Plain extract presented a higher content of phenyl ethyl alcohols, cinnammic acids, oleacein, oleocanthal and flavones, meanwhile, hill extract was richer in lignans. Hill extract was more effective in protecting differentiated SH-SY5Y cells from peroxide stress thanks to a marked upregulation of the antioxidant enzymes heme oxygenase 1, NADPH quinone oxidoreductase 1, thioredoxin Reductase 1 and glutathione reductase. Proteomic analysis revealed that hill extract plays a role in the regulation of proteins involved in neuronal plasticity and activation of neurotrophic factors such as BDNF. In conclusion, these data demonstrate that EVOOs can have important neuroprotective activities, but these effects are strictly related to their specific phenol composition.

Neurodegenerative diseases represent an increasingly public health problem, especially in the aging population, still in search of an effective therapy. The consumption of extra-virgin olive oil represents the main source of phenols from Mediterranean diet. In vitro and in vivo studies demonstrated preventive and protective properties of extra-virgin olive oil against neurodegeneration. In particular, the specific phenolic pattern of different extra virgin olive oils could influence their biological activity such as neuroprotection. To deepen this hypothesis, we investigated the potential differences in neuroprotective effects of four extra virgin olive oil extracts against H2O2-induced oxidative damage in differentiated SH-SY5Y cells by determining their effect in modulating antioxidant enzymes and pro-survival pathways. The different olive oil extracts were characterized for their content in tyrosol, hydroxytyrosol, oleacein and oleocanthal by HPLC. SH-SY5Y were treated with different concentrations (1-500 µg/mL) of the four extracts (corniolo, collina, pianura and spes) in the presence and absence of H2O2. Interestingly, collina is the most effective in counteracting oxidative damage as evaluated by MTT and LDH assays. Collina, pianura and spes reduced the H2O2-induced release of reactive oxygen species level as measured by dichloro-dihydro-fluorescein diacetate assay. Moreover, collina, pianura and spes increased GSH level measured by monochlorobimane assay. The extracts were also able to modulate phase II antioxidant enzymes and BDNF mRNA levels as measured by RT-PCR. Our findings confirm that extra-virgin olive oil possesses beneficial effects in counteracting neurodegeneration and these effects are strongly related to the specific pattern of olive oil phenols presents in the different olive oils. This work was supported by MIUR-PRIN 2015 (No. 20152HKF3Z)

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.

During the last 6 months, the world has been unsettled by the diffusion of a virus that has peculiar properties, causing alarm not only for the direct pathologic effect of the virus on affected individuals, but, mostly, for its only partially clarified ways of propagation [[1, 2]]. The medical, social, and economic impact of the new disease is extraordinary and also deeply touches our ethical and moral values. The public confidence in science is perturbed because the medical response is dominated by uncertainty, and the requests to science for a fast remedy are left unmet

Pro-inflammatory cytokines (CYT) lead to several changes in human islet (HI) protein expression, but few data are available on CYT-induced alterations at the whole proteome level, including post-translational modifications (PTM). We used label-free shotgun analysis (SG) and bi-dimensional gel electrophoresis (2DE) to explore how CYT modify the proteome of isolated HI. For this purpose, HI were isolated from the pancreas of 5 nondiabetic organ donors (age: 74±6 years; gender: 3M/2F; BMI: 27.9±3.6 kg/m2) and then cultured for 48h in the presence of 50 U/ml interleukin (IL)-1β + 1000 U/ml interferon (IFN)-γ. Then, 40 or 250 μg protein extracts were used respectively for SG or 2DE (to assess lysine-acetylation) analysis, with the SG mass spectrometry data acquired by nano-liquid chromatography tandem mass spectrometry. SG identified around 3000 proteins, of which 286 were differentially expressed after CYT exposure. Of these, 176 were upregulated and 110 downregulated. Among the former, we found mediators of inflammation, proteins of the ER and the immunoproteasome system and oxidative stress modulators (e.g., CXCL2, CXCL10, PSMB8, PSMB9, PSMB10, SOD2). Among the downregulated proteins, there were cathepsins, antioxidant molecules and enzymes of glycolysis and Krebs’ cycle (e.g., CTSH (a candidate gene for T1D), PRDX2, MDH). Ingenuity Pathway Analysis indicated that several upstream regulators were predicted to be differentially affected. Of them, 118 were presumably activated, including STAT1 and STAT2, NF-κB, JAK1, IRF1, HMGB1 and 36 inhibited, such as ACKR2, TRIM24, MAPK1, SUMO3. Bi-dimensional gel electrophoresis showed 151 spots that were lysine-acetylated, of which 2 were upregulated (PRDX3 and SOD1) and 4 downregulated (GDH, CTSD, ACAD9 and EIF4A1) by CYT. The present study shows novel proteomic changes, including specific PTM, induced in human islets by cytokine treatment that could play a role in beta cell dysfunction and demise and be the target for specific treatments. Disclosure F. Grano: None. F. Ciregia: None. M. Ronci: None. M. Mazzoni: None. L. Overbergh: None. C. De Luca: None. M. Suleiman: None. L. Marselli: None. P. De Simone: None. U. Boggi: None. C. Mathieu: Advisory Panel; Self; Boehringer Ingelheim International GmbH, Eli Lilly and Company, Merck & Co., Inc., Novo Nordisk A/S, Roche Diabetes Care, Sanofi. Speaker's Bureau; Self; AstraZeneca, Novartis AG, Novo Nordisk A/S, Sanofi. D.L. Eizirik: None. A. Lucacchini: None. L. Giusti: None. P. Marchetti: None. M. Bugliani: None. Funding INNODIA

Creatine (Cr) is a small metabolite with a central role in energy metabolism and mitochondrial function. Creatine deficiency syndromes are inborn errors of Cr metabolism causing Cr depletion in all body tissues and particularly in the nervous system. Patient symptoms involve intellectual disability, language and behavioral disturbances, seizures and movement disorders suggesting that brain cells are particularly sensitive to Cr depletion. Cr deficiency was found to affect metabolic activity and structural abnormalities of mitochondrial organelles; however a detailed analysis of molecular mechanisms linking Cr deficit, energy metabolism alterations and brain dysfunction is still missing. Using a proteomic approach we evaluated the proteome changes of the brain mitochondrial fraction induced by the deletion of the Cr transporter (CrT) in developing mutant mice. We found a marked alteration of the mitochondrial proteomic landscape in the brain of CrT deficient mice, with the overexpression of many proteins involved in energy metabolism and response to oxidative stress. Moreover, our data suggest possible abnormalities of dendritic spines, synaptic function and plasticity, network excitability and neuroinflammatory response. Intriguingly, the alterations occurred in coincidence with the developmental onset of neurological symptoms. Thus, cerebral mitochondrial alterations could represent an early response to Cr deficiency that could be targeted for therapeutic intervention.

Neuroinflammation is one of the main contributors to the onset and progression of neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Microglial and astrocyte activation is a brain defense mechanism to counteract harmful pathogens and damaged tissues, while their prolonged activation induces neuroinflammation that can trigger or exacerbate neurodegeneration. Unfortunately, to date there are no pharmacological therapies able to slow down or stop the progression of neurodegeneration. For this reason, research is turning to the identification of natural compounds with protective action against these diseases. Considering the important role of neuroinflammation in the onset and development of neurodegenerative pathologies, natural compounds with anti-inflammatory activity could be good candidates for developing effective therapeutic strategies. Marine organisms represent a huge source of natural compounds, and among them, algae are appreciated sources of important bioactive components such as antioxidants, proteins, vitamins, minerals, soluble dietary fibers, polyunsaturated fatty acids, polysaccharides, sterols, carotenoids, tocopherols, terpenes, phycobilins, phycocolloids, and phycocyanins. Recently, numerous anti-inflammatory compounds have been isolated from marine algae with potential protective efficacy against neuroinflammation. This review highlights the key inflammatory processes involved in neurodegeneration and the potential of specific compounds from marine algae to counteract neuroinflammation in the CNS.

Introduction: This review is an update on recent progress in proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues, which open the opportunity to investigate diseases and research potential biomarkers, particularly when availability of fresh/frozen tissues is low. Areas covered: We described improvement of existing protocols or the new ones regarding deparaffinization and protein extraction of FFPE samples published from 2014 to today. Moreover, the growing interest to use FFPE tissues for mass spectrometry imaging approach is presented together with the search of post-translational modifications. Expert opinion: In the last few years, the number of papers using FFPE tissues in proteomic analysis is growing. The interest to apply proteomic analysis to FFPE tissues lies in the easy accessibility of a great number of samples from archives. Nevertheless, standardization in the approach among the different researchers is not achieved, making essentially incomparable the results obtained. This limit should be overcome.

Fibromyalgia (FM) is a chronic pain disorder characterized by widespread pain and associated with unspecific symptoms. So far, no laboratory tests have been validated. The aim of the present study was to investigate the presence in saliva of potential diagnostic and/or prognostic biomarkers which could be useful for the management of FM patients. Specifically, the salivary profile of FM patients was compared with those of healthy subjects, subjects suffering migraine (model of non-inflammatory chronic pain), and patients affected by rheumatoid arthritis (model of inflammatory chronic pain). For proteomics analysis 2-DE and SELDI-TOF-MS were applied. From 2-DE serotransferrin and alpha-enolase were found differentially expressed in FM. Hence, their expression was validated by ELISA together with phosphoglycerate-mutase-I and transaldolase, which were found in a previous work. Moreover, ROC curve was calculated by comparing FM patients versus control subjects (healthy plus migraine) to investigate the discriminative power of biomarkers. The best performance was obtained by combining alpha-enolase, phosphoglycerate-mutase-I and serotransferrin. On the other hand, none of the candidate proteins showed a statistical correlation with clinical features. Finally, preliminary SELDI analysis highlighted two peaks whose identification need to be validated. Overall, these results could be useful in supporting the clinical diagnosis of FM. SIGNIFICANCE: FM is one of the most common chronic pain condition which is associated with significant disability. The fibromyalgic pain is a peculiar characteristic of this disease and FM patients suffer from reduced quality of life, daily functioning and productivity. Considering the deep complexity of FM, the discovery of more objective markers is crucial for supporting clinical diagnosis. Therefore, the aim of the present study was the selection of biomarkers effectively associated with fibromyalgic pain which will enable clinicians to achieve an unambiguous diagnosis, and to improve approaches to patients' management. We defined a panel of 3 salivary proteins which could be one of the criteria to be taken into account. Consequently, the identification of disease salivary biomarkers could be helpful in detecting FM clusters and targeted treatment. Actually, our future perspective foresees to develop a simple, rapid and not invasive point-of-care testing which will be of use during the diagnostic process. In addition, the present results can offer a clue for shedding light upon the complex entity of such a disease like FM.

Neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease are the largest growing area of neurological research still in search of an effective therapy. Virgin olive oil is a rich source of phenolic compounds that have been demonstrated to interfere with different mechanisms involved in neurodegenerative disorders1. Olive oil polyphenols can be divided into three categories: secoiridoids such as oleocanthal, simple phenols such as hydroxytyrosol and lignanes. Here, we evaluated the neuroprotective effect of four different virgin olive oil extracts against neurodegeneration by determining their effect in modulating oxidative stress and pro-survival pathways. The different olive oil extracts were characterized for their content in tyrosol, hydroxytyrosol, oleacein and oleocanthal by HPLC. SH-SY5Y cells were treated with different concentrations (1-10 µg/mL) of the extracts. Cell viability was evaluated by MTT assay and the results showed that three of the tested extracts significantly increased cell viability. Furthermore, two of these extracts decreased basal reactive oxygen species level measured by dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. Interestingly all the extracts increased reduced GSH level measured by monochlorobimane (MCB) assay. These extracts were also able to modulate phase II antioxidant enzymes and MAPK signalling pathways as measured by RT-PCR and immunoblotting. Our findings support the idea that virgin olive oil has a beneficial health effect in counteracting neurodegeneration and these effects are strongly related to the specific pattern of olive oil phenols.

Graphical abstract, Highlights ⿢ We performed a comparative proteomic analysis of biphasic pleural mesothelioma (BPM) by 2D/MS approach. ⿢ Twenty-two proteins were found differentially expressed in significant manner. ⿢ In B-PM the peculiarity of expression of serum amyloid A1 and gamma enolase was confirmed by western blot analysis. ⿢ Heat map obtained by Ingenuity software analysis suggests a profile of activation typical of B-PM., Malignant pleural mesothelioma (MPM) is a rare cancer originated from pleural mesothelial cells. MPM has been associated with long-term exposure to asbestos. In this work we performed a comparative proteomic analysis of biphasic pleural mesothelioma (B-PM). Tissue biopsies were obtained from 61 patients who were subjected to a diagnostic thoracoscopy. 2D/MS based approach was used for proteomic analysis. The 22 proteins found differentially expressed in B-PM, with respect to benign, were analyzed by Ingenuity Pathways Analysis and compared with those obtained for epitheliod pleural mesothelioma (E-PM). A different activation of transcription factors, proteins and cytokines were observed between two subtypes.

The aim of the present study was to appraise retrospectively the influence of valproate (VPA) and antidepressants (ADs) on the steady-state plasma concentrations of clozapine (CLZ), the prototype of various second-generation antipsychotics, norclozapine (NCLZ, its main metabolite), and their ratio (NCLZ:CLZ).Sixty-seven psychotic patients with a prevalent diagnosis of bipolar disorder were studied. We then analyzed data altogether and subdivided them into 4 groups, according to pharmacological treatments: #1 CLZ (n = 21), #2 CLZ plus ADs (n = 13), #3 CLZ plus VPA (n = 16), and #4 CLZ plus ADs plus VPA (n = 17).First, significant positive between CLZ and NCLZ plasma levels (in nanograms/milliliter) and the drug daily dosages (in milligrams/kilogram of body weight) (n = 67) were observed (Spearman: rCLZ = 0.49; rNCLZ = 0.61; P0.001). We then normalized by given doses CLZ and NCLZ plasma levels, natural log transformed them, and performed analysis of variance factor analyses followed by pairwise comparisons, performed on the 4 groups and the 3 CLZ parameters. We identified significant drug effects on (1) CLZ plasma levels, significantly higher in group #2 versus group #1, and (2) NCLZ:CLZ ratio, lower in group #2 versus groups #1 and #3. Significant drug × gender interactions were observed in group #3, showing higher NCLZ levels and NCLZ:CLZ ratios in men compared with women.Despite its inherent limitations, this observational study confirms the significant increase in plasma CLZ concentrations and reduction in NCLZ:CLZ ratio when this drug was coadministered with ADs (group #2), an effect apparently counteracted by VPA (group #4). The drug × gender interactions in patients taking both CLZ and VPA (group #3) warrant further prospective study.

Exposure to asbestos is the main cause of malignant pleural mesothelioma (MPM), a highly aggressive cancer of the pleura. Since the only tools for early detection are based on radiological tests, some authors focused on serum markers (i.e., mesothelin). The aim of this study was the evaluation of new serum biomarkers to be used individually or in combination, in order to improve the outcome of patients whose disease would be diagnosed at an earlier stage. Serum and plasma were available from 43 subjects previously exposed to asbestos and 27 MPM patients, all being epithelioid type. All the new markers found differentially expressed in MPM and healthy subjects, by proteomic and genomic approaches, have been validated in the serum by the use of specific ELISA. The combined approach, using tools of genomics and proteomics, is found to be highly innovative for this type of disease and led to the identification of new serum markers in the diagnosis of MPM. These results, if confirmed in a larger series, may have a strong impact in this area, because early detection of this cancer in people at high risk could significantly improve the course of the disease and the clinical approach to an individualized therapy.

Type 2 diabetes is characterized by progressive β cell dysfunction, with lipotoxicity playing a possible pathogenetic role. Palmitate is often used to examine the direct effects of lipotoxicity and it may cause mitochondrial alterations by activating protein acetylation. However, it is unknown whether palmitate influences protein acetylation in β cells. We investigated lysine acetylation in mitochondrial proteins from INS-1E β cells (INS-1E) and in proteins from human pancreatic islets (HPI) after 24 h palmitate exposure. First, we confirmed that palmitate damages β cells and demonstrated that chemical inhibition of deacetylation also impairs INS-1E function and survival. Then, by 2-D gel electrophoresis, Western Blot and Liquid Chromatography-Mass Spectrometry we evaluated the effects of palmitate on protein acetylation. In mitochondrial preparations from palmitate-treated INS-1E, 32 acetylated spots were detected, with 13 proteins resulting over-acetylated. In HPI, 136 acetylated proteins were found, of which 11 were over-acetylated upon culture with palmitate. Interestingly, three proteins, glutamate dehydrogenase, mitochondrial superoxide dismutase, and SREBP-1, were over-acetylated in both INS-1E and HPI. Therefore, prolonged exposure to palmitate induces changes in β cell protein lysine acetylation and this modification could play a role in causing β cell damage. Dysregulated acetylation may be a target to counteract palmitate-induced β cell lipotoxicity., SCOPUS: ar.j, info:eu-repo/semantics/published

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

Formalin-fixed paraffin-embedded (FFPE) tissue specimens represent a valuable informational resource of histologically characterized specimens for proteomic studies. In this article, the authors review the advancement performed in the field of FFPE proteomics focusing on formaldehyde treatment and on strategies addressed to obtain the best recovery in the protein/peptide extraction. A variety of approaches have been used to characterize protein tissue extracts, and many efforts have been performed demonstrating the comparability between fresh/frozen and FFPE proteomes. Finally, the authors report and discuss the large numbers of works aimed at developing new strategies and sophisticated platforms in the analysis of FFPE samples to validate known potential biomarkers and to discover new ones.

Given the paucity of data on the distribution of serotonin (5-HT) receptors of type 6 (5-HT(6)) in the human brain, the aim of this study was to investigate their distribution in postmortem human prefrontal cortex, striatum and hippocampus by either immunohistochemical or immunofluorescence techniques. The brain samples were obtained from 6 subjects who had died for causes not involving primarily or secondarily the CNS. The 5-HT(6) receptor distribution was explored by the [(125)I]SB-258585 binding to brain membranes followed by immunohistochemical and immunofluorescence evaluations. A specific [(125)I]SB-258585 binding was detected in all the regions under investigation, whilst the content in the hippocampus and cortex being about 10-30 times lower than in the striatum. Immunohistochemistry and double-label immunofluorescence microscopy experiments, carried out in the prefrontal cortex and hippocampus only, since data in the striatum were already published, showed the presence of 5-HT(6) receptors in both pyramidal and glial cells of prefrontal cortex, while positive cells were mainly pyramidal neurons in the hippocampus. The heterogeneous distribution of 5-HT(6) receptors provides a preliminary explanation of how they might regulate different functions in different brain areas, such as, perhaps, brain trophism in the cortex and neuronal firing in the hippocampus. This study, taking into account all the limitations due to the postmortem model used, represents the starting point to explore the 5-HT(6) receptor functionality and its sub-cellular distribution.

A series of imidazo[1,5-a]quinoline derivatives was designed and synthesized as central benzodiazepine receptor (CBR) ligands. Most of the compounds showed high CBR affinity with Ki values within the submicromolar and subnanomolar ranges with interesting modulations in their structure-affinity relationships. In particular, fluoroderivative 7w (Ki = 0.44 nM) resulted in the most potent ligand among the imidazo[1,5-a]quinoline derivatives described so far. Overall, these observations confirmed the assumption concerning the presence of a large though apparently saturable lipophilic pocket in the CBR binding site region interacting with positions 4 and 5 of the imidazo[1,5-a]quinoline nucleus. The in vivo biological characterization revealed that compounds 7a,c,d,l,m,q,r,w show anxiolytic and antiamnestic activities without the unpleasant myorelaxant side effects of the classical 1,4-BDZ. Furthermore, the effect of 7l,q,r, and 8i in lowering lactate dehydrogenase (LDH) release induced by ischemia-like conditions in rat brain slices suggested neuroprotective properties for these imidazo[1,5-a]quinoline derivatives.

Background Systemic sclerosis (SSc) is an autoimmune disease characterized by progressive fibrosis of the skin and the internal organs. In a previous work we suggested a correlation between levels of salivary psoriasin (S100A7) and pulmonary involvement in SSc patients. The goals of this study are to determine the distribution characteristics of psoriasin in whole saliva (WS) of SSc and healthy donor populations and define its predictive value on diffusion capacity of carbon monoxide (DLCO), along with others clinical parameters. Methods Salivary level of psoriasin was determined by ELISA kit in 134 SSc patients, 63 Raynaud syndrome patients, 40 patients affected by other connective diseases (non-case) and 74 healthy control subjects. Results A significant increase of salivary psoriasin was observed in SSc patients when compared with other healthy and pathological controls. Moreover, we confirmed the efficacy of salivary psoriasin to correlate with DLCO in a large cohort of SSc patients. Conclusions Overall our results suggest a rapid, non invasive and low costing method which can help clinicians in the evaluation of SSc pulmonary involvement.

Thyroid nodules are common in the general population and vary widely in their propensity to harbor thyroid malignancies. The category of follicular lesion of undetermined significance, for instance, carries only a 15% risk of malignancy. The overarching aim of this work was the proteomic study of thyroid cancer because more effort needs to be placed on differentiating malignant thyroid nodules to avoid unnecessary thyroidectomy. We used 2-dimensional electrophoresis coupled to nano-liquid chromatography electrospray ionization tandem mass spectrometry, to examine fine-needle aspiration (FNA), which was easily attainable from the wash of the syringe used for classical FNA biopsy. Overall, we found 25 different proteins able to discriminate benign from malignant samples. The different expression of moesin; annexin A1 (ANXA1); cornulin (CRNN); lactate dehydrogenase; enolase; protein DJ-1; and superoxide dismutase was confirmed in FNA by enzyme-linked immunosorbent assay or Western blot. Receiver operating characteristic curves were calculated to investigate the discriminative power of our marker. The best performance in diagnosis was obtained by combining ANXA1, enolase, protein DJ-1, superoxide dismutase, and CRNN. In addition, the most highly ranked proteins, from the perspective of follicular lesion of undetermined significance, were ANXA1 and CRNN. The research of these candidate biomarkers has then been widened to other biological fluids, such as serum and whole saliva. In conclusion, we believe that when a decision by a thyroid nodule biopsy cannot be distinctly made, the combination of our biomarkers may be one of the criteria to be taken into account for the final decision, together with the identification of ANXA1 in serum and saliva.

Objective To preliminary investigate the link between the darkness hormone melatonin (MLT) and the pro-hypnotic effectiveness of the atypical antidepressant Trazodone (TRZ) in a group of mood disorder patients suffering of insomnia. Design and methods The study's design comprised: i) the enrolment of insomniac outpatients, ii) baseline (t 0 ) psychiatric and biochemical examinations; iii) the subsequent patients' introduction into a treatment with TRZ for 3–4 weeks, followed by post-therapy re-evaluations (t 1 ). The MLT function was investigated by t 0 /t 1 ELISA determinations of 6-hydroxy-MLT sulfate (6-OH-MLTs) levels in early-morning urines and HPLC analysis of morning MLT serum amount. Concomitantly, TRZ and its metabolite m -chloro-phenylpiperazine ( m- CPP) were measured by HPLC in serum to monitor patients' compliance/metabolism. Results Seventeen insomniac outpatients, displaying mild symptoms of depression/anxiety resistant to antidepressants, completed TRZ therapy (dose:10–20 mg/day, bedtime). Serum TRZ levels (127 ± 57 ng ml − 1 , mean ± SD) confirmed patients' compliance, while the anxiogenic metabolite m- CPP resulting almost undetectable. Moreover, the 6-OH-MLTs output was found increased at t 1 vs. baseline values (t 1 : 58.4 ± 45.02 ng ml − 1 ; t 0 : 28.6 ± 15.8 ng ml − 1 ; mean ± SD, P P 1 : 48.53 ± 50.70 ng ml − 1 ; t 0 : 49.80 ± 66.53 ng ml − 1 ). Morning MLT in serum slightly diminished at t 1 without reaching the statistical significance, not allowing therefore to define the patients' outcome. Conclusions This initial investigation encourages to explore MLT networks as possible correlates of TRZ pro-hypnotic responses.

Introduction: Psychiatric disorders are severe, debilitating and heterogeneous diseases with a high impact on public health. In this review we address state of the art clinical approaches to diagnose psychiatric disorders and underline the necessity to found new tools to help clinicians.Areas covered: We provide an update on proteomic studies and suggest potential biomarkers focusing on schizophrenia (SCZ), bipolar disorder (BD), and major depression (MD). In particular, we direct our attention to proteomic results obtained from studies on biological fluids. We also show an interaction analysis of differentially expressed proteins found in SCZ, BD and MD.Expert commentary: To date, there is a need to find molecular biomarkers for psychiatric disorders. The use of a proteomic approach allows protein fingerprints to be defined in normal and pathological states. We believe that saliva is an intriguing biological fluid, whose proteomic study in psychiatric disorders is still in its early stages.

Chronic fatigue syndrome (CFS) is a debilitating and complex disorder characterized by unexplained fatigue not improved by rest. An area of investigation is the likely connection of CFS with defective mitochondrial function. In a previous work, we investigated the proteomic salivary profile in a couple of monozygotic twins discordant for CFS. Following this work, we analyzed mitochondrial proteins in the same couple of twins. Nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-MS) was used to study the mitochondria extracted from platelets of the twins. Subsequently, we selected three proteins that were validated using western blot analysis in a big cohort of subjects (n=45 CFS; n=45 healthy), using whole saliva (WS). The selected proteins were as follows: aconitate hydratase (ACON), ATP synthase subunit beta (ATPB) and malate dehydrogenase (MDHM). Results for ATPB and ACON confirmed their upregulation in CFS. However, the MDHM alteration was not confirmed. Thereafter, seeing the great variability of clinical features of CFS patients, we decided to analyze the expression of our proteins after splitting patients according to clinical parameters. For each marker, the values were actually higher in the group of patients who had clinical features similar to the ill twin. In conclusion, these results suggest that our potential markers could be one of the criteria to be taken into account for helping in diagnosis. Furthermore, the identification of biomarkers present in particular subgroups of CFS patients may help in shedding light upon the complex entity of CFS. Moreover, it could help in developing tailored treatments.