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Treatment of anxiety and depression predominantly centres around pharmacological interventions, which have faced criticism for their associated side effects, lack of efficacy and low tolerability. Saffron, which is reportedly well tolerated in humans, has been recognised for its antidepressant and anti-anxiety properties. Indeed, we previously reported upon the efficacy of saffron extract supplementation in healthy adults with subclinical anxiety. However, the molecular aetiology remains unclear. In a rodent model of low-grade chronic inflammation, we explored the impact of a saffron extract (Safr'Inside (TM)) supplemented at a physiological dose, which equated to 22 +/- 1.2 mg per day human equivalent dose for a person of 60 kg. Behavioural tests (Open Field task, Y maze, Novel object recognition), caecal 16S rRNA microbial sequencing, caecal H-1 NMR metabolomic analysis and 2DE brain proteomic analyses were completed to probe gut-brain axis interactions. Time occupying the centre of the Open Field maze (OF) was increased by 62% in saffron supplemented animals. This improvement in anxiety-related behaviour coincided with gut microbial shifts, notably Akkermansia, Muribaculaceae, Christensenellacae and Alloprevotella which significantly increased in response to saffron supplementation. Akkermansia and Muribaculaceae abundance negatively correlated with the neurotoxic metabolite dimethylamine which was reduced in saffron supplemented animals. Brain proteomic analysis highlighted several significantly altered proteins including ketimine reductase mu-crystallin which also correlated with dimethylamine concentration. Both dimethylamine and ketimine reductase mu-crystallin were associated with OF performance. This may be indicative of a novel interaction across the gut-brain axis which contributes to anxiety-related disorders.

Recent studies have highlighted the mechanisms controlling the formation of cerebral cholesterol, which is synthesized in situ primarily by astrocytes, where it is loaded onto apolipoproteins and delivered to neurons and oligodendrocytes through interactions with specific lipoprotein receptors. The “cholesterol shuttle” is influenced by numerous proteins or carbohydrates, which mainly modulate the lipoprotein receptor activity, function and signaling. These molecules, provided with enzymatic/proteolytic activity leading to the formation of peptide fragments of different sizes and specific sequences, could be also responsible for machinery malfunctions, which are associated with neurological, neurodegenerative and neurodevelopmental disorders. In this context, we have pointed out that purines, ancestral molecules acting as signal molecules and neuromodulators at the central nervous system, can influence the homeostatic machinery of the cerebral cholesterol turnover and vice versa. Evidence gathered so far indicates that purine receptors, mainly the subtypes P2Y2, P2X7 and A2A, are involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer’s and Niemann–Pick C diseases, by controlling the brain cholesterol homeostasis; in addition, alterations in cholesterol turnover can hinder the purine receptor function. Although the precise mechanisms of these interactions are currently poorly understood, the results here collected on cholesterol–purine reciprocal control could hopefully promote further research.

The low-density-lipoprotein receptors represent a family of pleiotropic cell surface receptors involved in lipid homeostasis, cell migration, proliferation and differentiation. The family shares common structural features but also has significant differences mainly due to tissue-specific interactors and to peculiar proteolytic processing. Among the receptors in the family, recent studies place low-density lipoprotein receptor-related protein 8 (LRP8) at the center of both neurodegenerative and cancer-related pathways. From one side, its overexpression has been highlighted in many types of cancer including breast, gastric, prostate, lung and melanoma; from the other side, LRP8 has a potential role in neurodegeneration as apolipoprotein E (ApoE) and reelin receptor, which are, respectively, the major risk factor for developing Alzheimer’s disease (AD) and the main driver of neuronal migration, and as a γ-secretase substrate, the main enzyme responsible for amyloid formation in AD. The present review analyzes the contributions of LDL receptors, specifically of LRP8, in both cancer and neurodegeneration, pointing out that depending on various interactions and peculiar processing, the receptor can contribute to both proliferative and neurodegenerative processes.

WOS: 000482303000065, PubMed: 31315236, Ethyl acetate (EA), methanol (MeOH), and aqueous extracts of aerial parts of Anthemis tinctoria var. pallida (ATP) and A. cretica subsp. tenuiloba (ACT) were investigated for their phenol and flavonoid content, antioxidant, and key enzyme inhibitory potentials. All extracts displayed antiradical effects, with MeOH and aqueous extracts being a superior source of antioxidants. On the other hand, EA and MeOH extracts were potent against AChE and BChE. Enzyme inhibitory effects against tyrosinase and alpha-glucosidase were observed, as well. We also studied Anthemis extracts in an ex vivo experimental neurotoxicity paradigm. We assayed extract influence on oxidative stress and neurotransmission biomarkers, including lactate dehydrogenase (LDH) and serotonin (5-HT), in isolated rat cortex challenged with K+ 60 mM Krebs-Ringer buffer (excitotoxicity stimulus). An untargeted proteomic analysis was finally performed in order to explore the putative mechanism in the brain. The pharmacological study highlighted the capability of ACT water extract to blunt K+ 60 mM increase in LDH level and 5-HT turnover, and restore physiological activity of specific proteins involved in neuron morphology and neurotransmission, including NEFMs, VAMP-2, and PKC gamma, thus further supporting the neuroprotective role of ACT water extract., Italian Ministry of UniversityMinistero dell' Istruzione, dell' Universita e della Ricerca (MIUR), This research was supported by Italian Ministry of University Grant (FAR 2017 granted to Claudio Ferrante; FAR 2017 granted to Giustino Orlando).

Pseudoexfoliation (PEX) syndrome is a systemic disease involving the extracellular matrix. It increases the risk of glaucoma, an irreversible cause of blindness, and susceptibility to heart disease, stroke and hearing loss. Single nucleotide polymorphisms (SNPs) in the LOXL1 (Lysyl oxidase-like 1) gene are the major known genetic risk factor for PEX syndrome. Two coding SNPs, rs1048861 (G > T; Arg141Leu) and rs3825942 (G > A; Gly153Asp), in the LOXL1 gene are strongly associated with the disease risk in multiple populations worldwide. In the present study, we investigated functional effects of these SNPs on the LOXL1 protein. We show through molecular modelling that positions 141 and 153 are likely surface residues and hence possible recognition sites for protein-protein interactions; the Arg141Leu and Gly153Asp substitutions cause charge changes that would lead to local differences in protein electrostatic potential and in turn the potential to modify protein-protein interactions. In RFL-6 rat fetal lung fibroblast cells ectopically expressing the LOXL1 protein variants related to PEX (Arg141_Gly153, Arg141_Asp153 or Leu141_Gly153), immunoprecipitation of the secreted variants showed differences in their processing by endogenous proteins, possibly Bone morphogenetic protein-1 (BMP-1) that cleaves and leads to enzymatic activation of LOXL1. Immunofluorescence labelling of the ectopically expressed protein variants in RFL-6 cells showed no significant difference in their extracellular accumulation tendency. In conclusion, this is the first report of a biological effect of the coding SNPs in the LOXL1 gene associated with PEX syndrome, on the LOXL1 protein. The findings indicate that the disease associated coding variants themselves may be involved in the manifestation of PEX syndrome. Refereed/Peer-reviewed

Purpose: Pseudoexfoliation (PEX) syndrome is an age-related progressive disease of the extracellular matrix with ocular manifestations. PEX is clinically diagnosed by the presence of extracellular exfoliative deposits on the anterior surface of the ocular lens. PEX syndrome is a major risk factor for developing glaucoma the leading cause of irreversible blindness in the world and is often associated with the development of cataract. PEX reportedly coexists with Alzheimer disease and increases the risk of heart disease and stroke. PEX material deposited on the anterior surface of the ocular lens is highly proteinaceous complex and insoluble making deciphering the protein composition of the material challenging. Thus to date only a small proportion of the protein composition of PEX material is known. The aim of this study was to decipher the protein composition of pathological PEX material deposited on the ocular lens in patients and advance the understanding of pathophysiology of PEX syndrome. Methods: Liquid-chromatography and tandem mass spectrometry (LC-MS/MS) was employed to discover novel proteins in extracts of neat PEX material surgically isolated from patients (n = 4) with PEX syndrome undergoing cataract surgery. A sub-set of the identified proteins was validated with immunohistochemistry using lens capsule specimens from independent patients (n=3) lens capsules from patients with cataract but without PEX syndrome were used as controls (n=4). Expression of transcripts of the validated proteins in the human lens epithelium was analyzed with reverse transcription PCR (RT-PCR). Functional relationships among the proteins identified in this study and genes and proteins previously implicated in the disease were bioinformatically determined using InnateDB. Results: Peptides corresponding to 66 proteins including ten proteins previously known to be present in PEX material were identified. Thirteen newly identified proteins were chosen for validation. Of those proteins 12 were found to be genuine components of the material. The novel protein constituents include apolipoproteins (APOA1 and APOA4) stress response proteins (CRYAA and PRDX2) and blood-related proteins (fibrinogen and hemoglobin subunits) including iron-free hemoglobin. The gene expression data suggest that the identified stress-response proteins and hemoglobin are contributed by the lens epithelium and apolipoproteins and fibrinogen by the aqueous humor to the PEX material. Pathway analysis of the identified novel protein constituents and genes or proteins previously implicated in the disease reiterated the involvement of extracellular matrix organization and degradation elastic fiber formation and complement cascade in PEX syndrome. Network analysis suggested a central role of fibronectin in the pathophysiology of the disease. The identified novel protein constituents of PEX material also shed light on the molecular basis of the association of PEX syndrome with heart disease stroke and Alzheimer disease. Conclusions: This study expands the understanding of the protein composition of pathological PEX material deposited on the ocular lens in patients with PEX syndrome and provides useful insights into the pathophysiology of this disease. This study together with the previous study by our group (Sharma et al. Experimental Eye Research 2009;89(4):479-85) demonstrate that using neat PEX material devoid of the underlying lens capsule for proteomics analysis is an effective approach for deciphering the protein composition of complex and highly insoluble extracellular pathological ocular deposits present in patients with PEX syndrome Refereed/Peer-reviewed

The exposure of blood to an artificial surface such as the haemodialysis membrane results in the nearly instantaneous deposition of a layer of plasma proteins. The composition of the protein layer profoundly influences all subsequent events, and to a large extent determines the biocompatibility of the biomaterial. In the present study, we examine the protein adsorption capacity and coagulation profiles of the polysulfone-based helixone material in comparison to cellulose triacetate. A differential profiling investigation using shotgun proteomics data-independent analysis was applied to eluates obtained with each membrane after a dialysis session, in order to assess the function of desorbed proteins. Functional classification and network analysis performed using bioinformatics tools shed light on the involvement of adsorbed proteins into important molecular processes, such as lipid transport and metabolism, cell growth differentiation and communication, and the coagulation cascade. The collected evidence was further validated by targeted mass spectrometry using selected reaction monitoring on proteotypic transitions of key protein effectors, confirming the different panels of adsorbed protein on each membrane. The coagulation profile during haemodialysis of patients under polysulfone-based helixone filter cartridges was also assessed showing a slightly higher platelet activation profile after the dialysis session. The overall collected evidence highlights a modulation of the coagulation biological pathway during haemodialysis, which is largely influenced by the biomaterial used. Refereed/Peer-reviewed

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.

Desorption/ionization on porous silicon-mass spectrometry (DIOS-MS) is an attractive alternative to conventional matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of low molecular weight compounds. Porous silicon (pSi) chips are also suitable as support for mass spectrometry imaging (MSI). Here, we report an implementation of DIOS-MSI using the biosynthetic organs of a marine mollusc for proof of principle. The tissue section is stamped onto a fluorocarbon-functionalized pSi chip, which extracts and traps small hydrophobic molecules from the tissue under retention of their relative spatial distribution. The section is subsequently removed and the chip is imaged without any remaining tissue. We apply this novel tissue contact printing approach to investigate the distribution of biologically active brominated precursors to Tyrian purple in the hypobranchial gland of the marine mollusc, Dicathais orbita, using DIOS-MSI. The tissue contact printing is also compatible with other types of desorption/ionization surfaces, such as nanoassisted laser desorption/ionization (NALDI) targets. Refereed/Peer-reviewed

Porous silicon (pSi) surfaces have been chemically patterned via a UV initiated hydrosilylation reaction of an alkene through a photomask, introducing chemical functionality in the exposed surface areas. A secondary, UV initiated hydrosilylation reaction with a second alkene of different functionality is performed to backfill the silicon hydride terminated regions on the surface, thereby affording patterned porous films with dual, surface chemistry. UV initiated hydrosilylations were performed using the alkene undecylenic acid N-hydroxysuccinimide (NHS) ester, and the pSi surfaces were stabilized by a second hydrosilylation reaction with a polyethylene glycol (PEG) appended alkene. NHS ester and PEG functionalized surfaces were used for the selective immobilization of the cell adhesion mediator protein fibronectin (FN), in the NHS-functional regions. Matrix-assisted laser desorption/ionization mass spectrometry imaging on the protein functionalized pSi surface confirmed the patterned conjugation of the FN to the NHS functionalized regions. Mammalian cells cultured on these surfaces showed attachment that was confined to the patterned areas of FN on the pSi surface.

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

Despite significant advances in chemical ecology, the biodistribution, temporal changes and ecological function of most marine secondary metabolites remain unknown. One such example is the association between choline esters and Tyrian purple precursors in muricid molluscs. Mass spectrometry imaging (MSI) on nano-structured surfaces has emerged as a sophisticated platform for spatial analysis of low molecular mass metabolites in heterogeneous tissues, ideal for low abundant secondary metabolites. Here we applied desorption-ionisation on porous silicon (DIOS) to examine in situ changes in biodistribution over the reproductive cycle. DIOS-MSI showed muscle-relaxing choline ester murexine to co-localise with tyrindoxyl sulfate in the biosynthetic hypobranchial glands. But during egg-laying, murexine was transferred to the capsule gland and then to the egg capsules, where chemical ripening resulted in Tyrian purple formation. Murexine was found to tranquilise the larvae and may relax the reproductive tract. This study shows that DIOS-MSI is a powerful tool that can provide new insights into marine chemo-ecology.

Prolonged exposure to palmitate impairs insulin secretion and leads to beta-cell death. Some evidence suggests that palmitate could induce these effects through defects in mitochondrial function. However, the mechanisms of lipotoxicity are not well understood. In particular, little is known about mitochondrial response to induced-palmitate stress and the mechanisms through which glucagon-like peptide-1 (GLP-1) exerts its potential protective effect in beta-cell mitochondrial dysfunction. The aim of this study was to analyze the protein expression profiles of enriched mitochondrial preparations of INS-1E beta-cells treated with palmitate in the presence and in the absence of GLP-1 using gel-based and gel-free proteomic approaches. INS1E beta-cells were incubated in the presence of 0.5 mM palmitate for 24 h, in the presence and in the absence of 10 nM GLP-1, and mitochondria were isolated. Co-incubation of palmitate-treated beta-cell lines with GLP-1 identified several GLP-1 responsive mitochondrial proteins from different functional classes indicating major changes in ATP production, oxidative stress, apoptosis, lipid and amino acid metabolism. Moreover, an interaction network analysis of proteins and metabolites found to be differentially expressed has been performed to understand the pathways involved in the palmitate and GLP-1 activity at the mitochondrial level. In summary, our results provided a snapshot of mitochondrial proteins and potential pathways affected by palmitate treatment and gave us information on the potential protective role of GLP-1. Refereed/Peer-reviewed

Human medulloblastoma (MB) is a malignant brain tumor that comprises four distinct molecular subgroups including the Sonic Hedgehog (SHH)-MB group. A leading cause of the SHH subgroup is an aberrant activation of the SHH pathway, a developmental signaling that regulates postnatal development of the cerebellum by promoting the mitotic expansion of granule neural precursors (GNPs) in the external granule layer (EGL). The abnormal SHH signaling pathway drives not only SHH-MB but also its cancer stem-like cells (SLCs), which represent a fraction of the tumor cell population that maintain cancer growth and have been associated with high grade tumors. Here, we report the first proteomic analysis of human SHH-MB SLCs before and after Retinoic Acid (RA)-induced differentiation. A total of 994 nLC-MS buckets were statistically analysed returning 68 modulated proteins between SLCs and their differentiated counterparts. Heat Shock Protein 70 (Hsp70) was one of the proteins that characterized the protein profile of SLCs. By means of Ingenuity Pathway Analysis (IPA), Genomatix analysis and extending the network obtained using the differentially expressed proteins we found a correlation between Hsp70 and the NF-κB complex. A key driver of the SHH-MB group is cMET whose downstream proliferation/survival signalling is indeed via PI3K/Akt/NF-κB. We confirmed the results of the proteomic analysis by western blot, underlining that a P-p65/NF-κB activatory complex is highly expressed in SLCs. Taking together these results we define a new protein feature of SHH-MB SLCs. Refereed/Peer-reviewed

As major components of neuronal membranes, omega-3 polyunsaturated acids (n-3 PUFA) exhibit a wide range of regulatory functions, modulating from synaptic plasticity to neuroinflammation, from oxidative stress to neuroprotection. Recent human and animal studies indicated the n-3 PUFA neuroprotective properties in aging, with a clear negative correlation between n-3 PUFA levels and hippocampal deficits. The present multidimensional study was aimed at associating cognition, hippocampal neurogenesis, volume, neurodegeneration and metabolic correlates to verify n-3 PUFA neuroprotective effects in aging. To this aim 19 month-old mice were given n-3 PUFA mixture, or olive oil or no dietary supplement for 8 weeks during which hippocampal-dependent mnesic functions were tested. At the end of behavioral testing morphological and metabolic correlates were analyzed. n-3 PUFA supplemented aged mice exhibited better object recognition memory, spatial and localizatory memory, and aversive response retention, without modifications in anxiety levels in comparison to controls. These improved hippocampal cognitive functions occurred in the context of an enhanced cellular plasticity and a reduced neurodegeneration. In fact, n-3 PUFA supplementation increased hippocampal neurogenesis and dendritic arborization of newborn neurons, volume, neuronal density and microglial cell number, while it decreased apoptosis, astrocytosis and lipofuscin accumulation in the hippocampus. The increased levels of some metabolic correlates (blood Acetyl-L-Carnitine and brain n-3 PUFA concentrations) found in n-3 PUFA supplemented mice also pointed towards an effective neuroprotection. On the basis of the present results n-3 PUFA supplementation appears to be a useful tool in health promotion and cognitive decline prevention during aging. © 2014 Cutuli, De_bartolo, Caporali, Laricchiuta, Foti, Ronci, Rossi, Neri, Spalletta, Caltagirone, Farioli_vecchioli and Petrosini.

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is an excellent analytical technique for the rapid and sensitive analysis of macromolecules (>700 Da), such as peptides, proteins, nucleic acids, and synthetic polymers. However, the detection of smaller organic molecules with masses below 700 Da using MALDI-MS is challenging due to the appearance of matrix adducts and matrix fragment peaks in the same spectral range. Recently, nanostructured substrates have been developed that facilitate matrix-free laser desorption ionization (LDI), contributing to an emergi ng analytical paradigm referred to as surface-assisted laser desorption ionization (SALDI) MS. Since SALDI enables the detection of small organic molecules, it is rapidly growing in popularity, including in the field of forensics. At the same time, SALDI also holds significant potential as a high throughput analytical tool in roadside, work place and athlete drug testing. In this review, we discuss recent advances in SALDI techniques such as desorption ionization on porous silico n (DIOS), nano-initiator mass spectrometry (NIMS) and nano assisted laser desorption io nization (NALDI TM) and compare their strengths and weaknesses with particular focus on forensic applicat ions. These include the detection of illicit drug molecules and their metabolites in biological matrices and small molecule detection from forensic samples including banknotes and fingerprints. Finally, the review highlights recent advances in mass spectrometry imaging (MSI) using SALDI techniques. Refereed/Peer-reviewed

The antiproliferative and differentiation potential of anthocyanin-rich strawberry fruit crude extracts (SE) were investigated on B16-F10 murine melanoma cells. Treatment of melanoma cells with SE produced a remarkable reduction of cell proliferation, paralleled with both the lowering of the intracellular levels of polyamine, and the enhancement of tissue transglutaminase (TG2, EC 2.3.2.13) activity (used as a differentiation marker). To gain further insight into profiling altered protein expression as a potential biomarker of the SE action on melanoma cells, analysis of the proteomic profile was performed on the treated B16-F10 cells, compared to the control. Following SE treatment, 30 proteins resulted up-regulated, and 87 proteins were down-regulated. In particular proteins overexpressed in cancer cells, involved in tumor progression and metabolism, were down-regulated. The possibility that SE may affect the Warburg effect in B16-F10 melanoma cells is discussed. Refereed/Peer-reviewed

ADP-ribosylation is a posttranslational modification that modulates the functions of many target proteins. We previously showed that the fungal toxin brefeldin A (BFA) induces the ADP-ribosylation of C-terminal-binding protein-1 short-form/BFA-ADP-ribosylation substrate (CtBP1-S/BARS), a bifunctional protein with roles in the nucleus as a transcription factor and in the cytosol as a regulator of membrane fission during intracellular trafficking and mitotic partitioning of the Golgi complex. Here, we report that ADP-ribosylation of CtBP1-S/BARS by BFA occurs via a nonconventional mechanism that comprises two steps: (i) synthesis of a BFA-ADP-ribose conjugate by the ADP-ribosyl cyclase CD38 and (ii) covalent binding of the BFA-ADP-ribose conjugate into the CtBP1-S/BARS NAD+-binding pocket. This results in the locking of CtBP1-S/BARS in a dimeric conformation, which prevents its binding to interactors known to be involved in membrane fission and, hence, in the inhibition of the fission machinery involved in mitotic Golgi partitioning. As this inhibition may lead to arrest of the cell cycle in G2, these findings provide a strategy for the design of pharmacological blockers of cell cycle in tumor cells that express high levels of CD38., We thank all colleagues who kindly provided antibodies and reagents; Dr. J. Donaldson (National Institutes of Health) for BFA analogs; Dr. C. P. Berrie for editorial assistance; and Drs. C. Limina, A. Tamburro, M. G. Silletta, R. Weigert, and S. Spanò (Negri Sud Institute) for performing initial experiments. We also acknowledge financial support from Italian Association for Cancer Research (AIRC) through the Grants IG4664 and IG10341 (to D.C.), IG4700 (to A.L.), and IG6074 (to A.C.); and from the Liguria Region and the Ministry of Education, University, and Research (Fund for Investments in Basic Research Project; A.D.F.). G.G. and C.V. received fellowships from AIRC (Italian Foundation for Cancer Research). Financial support from Technological Innovation Fund DM 24/09/2009, Legge 46/82-MEF, and Project FaReBio di Qualità also is acknowledged

This article appeared in a journal published by Elsevier Ltd. Under Elsevier's copyright, mandated authors are not permitted to make work available in an institutional repository., Pseudoexfoliation (PEX) syndrome is an age-related systemic disease of the extracellular matrix, characterized by the presence of amyloid-like fibrillar deposits on the anterior lens capsule. The pathological deposits (PEX material) can obstruct aqueous outflow leading to increased intraocular pressure that in turn can result in glaucoma. PEX syndrome is the most common risk factor for glaucoma. In our previous work, we reported a protocol for the analysis of human lens capsules by MALDI MS imaging. Here, we extend our previous work applying the developed protocol to the analysis of human lens capsules affected by PEX syndrome. We focus our investigation on known components of the PEX material, namely lysyl oxidase-like 1 (LOXL1) and apolipoprotein E (APOE). Our results show that LOXL1 is more abundant in the deposits in the iris region and, alternatively APOE is concentrated in the PEX material accumulated in the pupillary area of the anterior lens capsule. Furthermore, we identify potentially relevant post-translational modifications which may have an important role in promoting the cross-linking processes in PEX syndrome and stabilize aggregate structures within the proteinaceous PEX material., Australian National Health & Medical Research Council

The ability to detect illicit drugs directly in oral fluids is of major interest for roadside, workplace and athlete drug testing. For example, roadside testing for popular drugs of abuse is being rolled out by law enforcement agencies following the introduction of legislation in several countries all over the world. This paper reports on the direct analysis of methamphetamine, cocaine and 3,4-methylenedioxymethamphetamine in oral fluids using a hydrophobic porous silicon array as a combined drug extraction and concentration medium. Analysis by laser desorption/ionization time-of-flight mass spectrometry (MS) identified these drugs with a sensitivity in line with the suggested confirmatory cut-off concentrations, and 300 times faster. In addition, MS imaging demonstrated good spot-to-spot reproducibility of the signal. Our analytical approach is compatible with multiplexing and is therefore suitable for high-throughput analysis of samples obtained from drug testing in the field. Furthermore, the application of this analytical technology is not limited to illicit drugs or oral fluids. Indeed, we believe that this platform technology could be applied to the high-throughput analysis of diverse metabolites in body fluids. Refereed/Peer-reviewed

Zinc is required for a wide variety of cellular functions and plays a key role in bacterial metabolism and virulence. However, Zn can also be toxic and, therefore, its influx is tightly regulated. The high affinity zinc uptake transporter ZnuABC is the main Zn influx system in Salmonella enterica under conditions of Zn starvation. It has been shown that deletion of the gene encoding for its periplasmic subunit ZnuA significantly affects S. Typhimurium growth rate and virulence, highlighting the importance of this system in the host-pathogen interaction. To gain further insight into the mechanisms involved in Zn influx regulation, we characterized the main alterations in the ionome and proteome of S. Typhimurium wild type and znuA mutant strains grown either under Zn starvation or under Zn-replete conditions. We found significant differences in the element profile and protein expression that were reversed by Zn supplementation.In particular, several of the differentially regulated proteins are predicted to be metal-binding proteins. Interestingly, their over-expression in the znuA mutant strain strictly depends on Zn starvation and correlates with the differences found at the ionome level. In conclusion, our data demonstrate that inhibition of Zn influx has relevant effects either on the bacterial ionome orproteome and shed new light on the role of the ZnuABC system and Zn influx in S. Typhimurium pathogenicity. Refereed/Peer-reviewed

The ocular lens capsule is a smooth, transparent basement membrane that encapsulates the lens and is composed of a rigid network of interacting structural proteins and glycosaminoglycans. During cataract surgery, the anterior lens capsule is routinely removed in the form of a circular disk. We considered that the excised capsule could be easily prepared for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-MSI) analysis. MALDI-MSI is a powerful tool to elucidate the spatial distribution of small molecules, peptides, and proteins within tissues. Here, we apply this molecular imaging technique to analyze the freshly excised human lens capsule en face. We demonstrate that novel information about the distribution of proteins by MALDI-MSI can be obtained from this highly compact connective tissue, having no evident histo-morphological characteristics. Trypsin digestion carried out on-tissue is shown to improve MALDI-MSI analysis of human lens capsules and affords high repeatability. Most importantly, MALDI-MSI analysis reveals a concentric distribution pattern of proteins such as apolipoprotein E (ApoE) and collagen IV alpha-1 on the anterior surface of surgically removed lens capsule, which may indicate direct or indirect effects of environmental and mechanical stresses on the human ocular lens. Refereed/Peer-reviewed

Tumor necrosis factor α–converting enzyme (TACE, also known as ADAM17) was recently involved in the pathogenesis of insulin resistance. We observed that TACE activity was significantly higher in livers of mice fed a high-fat diet (HFD) for 1 month, and this activity was increased in liver > white adipose tissue > muscle after 5 months compared with chow control. In mouse hepatocytes, C2C12 myocytes, and 3T3F442A adipocytes, TACE activity was triggered by palmitic acid, lipolysaccharide, high glucose, and high insulin. TACE overexpression significantly impaired insulin-dependent phosphorylation of AKT, GSK3, and FoxO1 in mouse hepatocytes. To test the role of TACE activation in vivo, we used tissue inhibitor of metalloproteinase 3 (Timp3) null mice, because Timp3 is the specific inhibitor of TACE and Timp3−/− mice have higher TACE activity compared with wild-type (WT) mice. Timp3−/− mice fed a HFD for 5 months are glucose-intolerant and insulin-resistant; they showed macrovesicular steatosis and ballooning degeneration compared with WT mice, which presented only microvesicular steatosis. Shotgun proteomics analysis revealed that Timp3−/− liver showed a significant differential expression of 38 proteins, including lower levels of adenosine kinase, methionine adenosysltransferase I/III, and glycine N-methyltransferase and higher levels of liver fatty acid-binding protein 1. These changes in protein levels were also observed in hepatocytes infected with adenovirus encoding TACE. All these proteins play a role in fatty acid uptake, triglyceride synthesis, and methionine metabolism, providing a molecular explanation for the increased hepatosteatosis observed in Timp3−/− compared with WT mice. Conclusion: We have identified novel mechanisms, governed by the TACE–Timp3 interaction, involved in the determination of insulin resistance and liver steatosis during overfeeding in mice. (HEPATOLOGY 2009.)

Changes in metabolism and protein expression were analysed during cryopreservation of the ancient apple variety Annurca. Our experiments concerned transglutaminase activity, polyamine levels and protein expression associated with shoot tip dehydration. Cryopreserved shoot tips displayed 72% regrowth after treatment in liquid medium with 0.75 M sucrose for 1 day followed by dehydration to 19% moisture content (fresh weight basis). After dehydration, the concentration of polyamines putrescine and spermidine decreased compared with untreated controls, while spermine concentration remained unaffected. Transglutaminase activity was slightly reduced in treated samples, while post-thaw regrowth enzyme activity approached control values. We also detected significant changes in protein expression profiles and identified six proteins related with stress response or involved in the slowing down of the cell cycle. The relationship between biochemical parameters, protein synthesis and cryotolerance is discussed.Changes in metabolism and protein expression were analysed during cryopreservation of the ancient apple variety Annurca. Our experiments concerned transglutaminase activity, polyamine levels and protein expression associated with shoot tip dehydration. Cryopreserved shoot tips displayed 72% regrowth after treatment in liquid medium with 0.75 M sucrose for 1 day followed by dehydration to 19% moisture content (fresh weight basis). After dehydration, the concentration of polyamines putrescine and spermidine decreased compared with untreated controls, while spermine concentration remained unaffected. Transglutaminase activity was slightly reduced in treated samples, while post-thaw regrowth enzyme activity approached control values. We also detected significant changes in protein expression profiles and identified six proteins related with stress response or involved in the slowing down of the cell cycle. The relationship between biochemical parameters, protein synthesis and cryotolerance is discussed.Changes in metabolism and protein expression were analysed during cryopreservation of the ancient apple variety Annurca. Our experiments concerned transglutaminase activity, polyamine levels and protein expression associated with shoot tip dehydration. Cryopreserved shoot tips displayed 72% regrowth after treatment in liquid medium with 0.75 M sucrose for 1 day followed by dehydration to 19% moisture content (fresh weight basis). After dehydration, the concentration of polyamines putrescine and spermidine decreased compared with untreated controls, while spermine concentration remained unaffected. Transglutaminase activity was slightly reduced in treated samples, while post-thaw regrowth enzyme activity approached control values. We also detected significant changes in protein expression profiles and identified six proteins related with stress response or involved in the slowing down of the cell cycle. The relationship between biochemical parameters, protein synthesis and cryotolerance is discussed. Refereed/Peer-reviewed

Performance enhancing agents (PEAs) are illegally used in cattle and other meat producing species to increase food conversion and lean meat production. Due to the very short breeding cycle, veal calves represent the meat producing bovine category mostly subjected to illicit treatments. These chemical agents are difficult to detect by conventional analytical approaches due to the employment of synergistic formulations at very low dosage and given the use of uncharacterized novel compounds. Such a scenario has fostered a strong interest in the discovery of functional molecular biomarkers for the detection of growth promoting agents in meat producing species. A multivariate MALDI-TOF-MS proteomics platform has been developed using bovine serum samples. Analytical performances have been thoroughly evaluated in order to enable reproducible profiles from 10 microL sera samples. We propose univariate and multivariate discrimination models capable to identify calves undergoing illicit treatments. In particular, we found a strong discrimination power associated with a polypeptide fragment from beta2-glycoprotein-I. We provide a fundamental proof of concept in the potential application of MALDI-TOF-MS proteomics profiling in the food safety control.

Aberrations in the Ubiquitin-Proteasome System (UPS) have been recently connected to the pathogenesis of several human protein degradation disorders (e.g., cancer and neurodegenerative diseases), so that proteasome is now considered an important target for drug discovery. Small molecules able to inhibit and modulate UPS have been, in fact, described as novel tools for a new approach in anti-cancer therapy. In particular Proteasome Inhibitors (PIs), blocking activation of nuclear factor-kappa B (NF-kB), trigger a decreased cellular proliferation and angiogenic cytokine production, induce cell death and inhibit tumor cell adhesion to stroma. Furthermore, several studies have demonstrated that PIs potentiate the activity of other anti-cancer treatment, in part by down-regulating chemoresistance pathways. Therefore pharmacologic, preclinical, and clinical data suggested the use of PIs in anticancer strategies, for their potential therapeutic relevance in the treatment of cancer and inflammatory-related diseases. This review focuses on recent advances in the development of PIs anticancer agents highlighting both novel patented compounds and novel therapeutic protocol of intervention.