Bacillus species are frequently used as bacterial workhorses in industrial microbial cultivation for production of probiotic compounds and antibiotics. Bacterial secretory proteins are known to perform "remote-control" functions, such as the provision of nutrients, detoxification of the environment, and killing of potential competitors. A previous proteomic study carried out in our laboratory examined cell proteins extracted from four closely related isogenic B. Clausii strain, used to produce a commercial probiotic preparation and led to the recognition of a number of proteins differentially expressed in the four strains. Here, we report a proteomic study of the secretome, i.e. the complement of extracellular secreted proteins, of the four B. clausii strains. The main objective of this study was to characterize the proteins actively secreted including secreted enzyme that can be useful in industry and domestic life and metabolic bottlenecks that can contribute to improve process optimization of their production. The electropherogram of the four B. clausii secretomes show more than 400 protein spots separated by two-dimensional gel electrophoresis (2-DE). More pronounced differences were revealed at the expression level of specific proteins, some of which represent the most abundant secreted proteins. The different expression pattern of proteins in the four secretomes demonstrates that the B. clausii strains, which are characterized by a notable low level of intraspecific genome diversity, present distinct pattern of protein secretion.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder caused primarily by selective degeneration of the dopaminergic neurons in substantia nigra. In this work the proteomes extracted from primary fibroblasts of two unrelated, hereditary cases of PD patients, with different parkin mutations, were compared with the proteomes extracted from commercial adult normal human dermal fibroblasts (NHDF) and primary fibroblasts from the healthy mother of one of the two patients. The results show that the fibroblasts from the two differen tcases of parkin-mutant patients display analogous alterations in the expression level of proteins involved in different cellular functions, like cytoskeleton structure-dynamics, calcium homeo- stasis, oxidative stress response and protein and RNA processing.

Dendritic cells (DC) are sentinels of the immune system deriving from circulating monocyte precursors recruited to sites of inflammation. In a previous report (Del Prete et al., 2008) we showed that, after differentiation, DC exhibited increased number of condensed mitochondria and dynamic changes in their energy metabolism. A study is presented here showing that the DC differentiation process is characterized by increased expression level and activity of mitochondrial respiratory complexes, as well as by an increased mitochondrial DNA (mtDNA) copy number. Moreover, DC are equipped with more efficient antioxidant protection systems, over expressed most likely to detoxify increased ROS production, as a consequence of the much higher mitochondrial activity. Kinetic analysis of the three main mitochondrial biogenesis-associated genes revealed that the peak in PPAR? coactivator-1alpha (PGC-1?) gene expression was suddenly reached few hours after the onset of the differentiation. While PGC-1? expression rapidly declines, the mitochondrial transcription factor A (TFAM) and nuclear respiratory factor-1 (NRF-1) expression gradually increased. These findings demonstrate that an active mitochondrial biogenesis occurs during DC differentiation and further suggest that an early input by the master regulator of mitochondrial biogenesis PGC-1? is needed to trigger the subsequent activation of the downstream transcription factors, NRF-1 and TFAM in this process.

Protein biomarkers are important diagnostic tools for cancer and several other diseases. To be validated in a clinical context, a biomarker should satisfy some requirements including the ability to provide reliable information on a pathological state by measuring its expression levels. In parallel, the development of an approach capable of detecting biomarkers with high sensitivity and specificity would be ideally suited for clinical applications. Here, we performed an immune-based label free assay using Surface Plasmon Resonance (SPR)-based detection of the soluble form of E-cadherin, a cell-cell contact protein that is involved in the maintaining of tissue integrity. With this approach, we obtained a specific and quantitative detection of E-cadherin from a few hundred microliters of serum of breast cancer patients by obtaining a 10-fold enhancement in the detection limit over a traditional colorimetric ELISA.

Recently has been acknowledged the healthy use of Bacillus and related bacteria as probiotics. A mixture reported to contain four probiotic strains of Bacillus clausii is marketed as an OTC (Over The Counter) medicinal supplement for human use. Their poliantibiotic resistant property, useful for restoring the gut microbiota during antibiotic treatment, raises the question about the risk of resistance transfer. In order to better assess the risk-benefit ratio it is important to always monitoring the pattern and stability of resistance spectra in these bacteria. In this work, we have extensively redefined the antibiotic susceptibility profile of these four probiotic strains. Resistance phenotype has been determined by screening a large number of antibiotics, including natural products (such as penicillin, vancomycin and erythromycin), and completely synthetic molecules (such as fluoroquinolones). Extensive comparison with a wild type strain belonging to the normal intestinal microbiome was carried out. The molecular basis of some resistances was determined. Observed antibiotic resistances were correlated with previous and new data in safety evaluations of these strains for human use.

catenin plays an important role as regulatory hub in several cellular processes including cell adhesion, metabolism, and epithelial mesenchymal transition. This is mainly achieved by its dual role as structural component of cadherin-based adherens junctions, and as a key nuclear effector of the Wnt pathway. For this dual role, different classes of proteins are differentially regulated via ?-catenin dependent mechanisms. Here, we applied a liquid chromatography-mass spectrometry (LC-MS/MS) approach to identify proteins modulated after ?-catenin knockdown in the breast cancer cell line MCF-7. We used a label free analysis to compare trypsin-digested proteins from CTR (shCTR) and ?-catenin knockout cells (sh?cat). This led to the identification of 98 differentially expressed proteins, 53 of them were up-regulated and 45 down-regulated. Loss of ?-catenin induced morphological changes and a significant modulation of the expression levels of proteins associated with primary metabolic processes. In detail, proteins involved in carbohydrate metabolism and tricarboxylic acid cycle were found to be down-regulated, whereas proteins associated to lipid metabolism were found up-regulated in sh?cat compared to shCTR. A loss of mitochondrial mass and membrane potential was also assessed by fluorescent probes in sh?cat cells with respect to the controls. These data are consistent with the reduced expression of transcriptional factors regulating mitochondrial biogenesis detected in sh?cat cells. ?-catenin driven metabolic reprogramming resulted also in a significant modulation of lipogenic enzyme expression and activity. Compared to controls, ?-catenin knockout cells showed increased incorporation of [1-14C]acetate and decreased utilization of [U-14C]glucose for fatty acid synthesis. Our data highlight a role of ?-catenin in the regulation of metabolism and energy homeostasis in breast cancer cells.

A comparative proteomic approach, using two dimensional gel electrophoresis and mass spectrometry, has been developed to compare and elucidate the differences among the cellular proteomes of four closely related isogenic O/C, SIN, N/R and T, B. clausii strains during both exponential and stationary phases of growth.Image analysis of the electropherograms reveals a high degree of concordance among the four proteomes, some proteins result, however, differently expressed. The proteins spots exhibiting high different expression level were identified, by mass-spectrometry analysis, as alcohol dehydrogenase (ADHA, EC1.2.1.3; ABC0046 isoform) aldehyde dehydrogenase (DHAS, EC 1.2.1.3; ABC0047 isoform) and flagellin-protein of B. clausii KSM-k16. The different expression levels of the two dehydrogenases were confirmed by quantitative RT- PCR and dehydrogenases enzymatic activity. The different patterns of protein expression can be considered as cell proteome signatures of the different strains.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Genes which have been implicated in autosomal-recessive PD include PARK2 which codes for parkin, an E3 ubiquitin ligase that par- ticipates in a variety of cellular activities. In this study, we compared parkin-mutant primary fibroblasts, from a patient with parkin compound heterozygous mutations, to healthy control cells. Western blot analysis of proteins obtained from patient's fibroblasts showed quantitative differences of many proteins involved in the cytoskeleton organization with respect to control cells. These molecular alterations are accompanied by changes in the organization of actin stress fibers and biomechanical properties, as revealed by confocal laser scanning microscopy and atomic forcemicroscopy. In particular, parkin deficiency is associated with a significant increase of Young's modulus of -cells in comparison to normal fibroblasts. The current study proposes that parkin influences the spatial organization of actin filaments, the shape of human fibroblasts, and their elastic response to an external applied force.

Mitochondrial dysfunction and oxidative stress occur in Parkinson's disease (PD), but the molecular mechanisms controlling these events are not completely understood. Peroxisome proliferator-activated receptor-gamma coactivator-1? (PGC-1?) is a transcriptional coactivator known as master regulator of mitochondrial functions and oxidative metabolism. Recent studies, including one from our group, have highlighted altered PGC-1? activity and transcriptional deregulation of its target genes in PD pathogenesis suggesting it as a new potential therapeutic target. Resveratrol, a natural polyphenolic compound proved to improve mitochondrial activity through the activation of several metabolic sensors resulting in PGC-1? activation. Here we have tested in vitro the effect of resveratrol treatment on primary fibroblast cultures from two patients with early-onset PD linked to different Park2 mutations. We show that resveratrol regulates energy homeostasis through activation of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) and raise of mRNA expression of a number of PGC-1?'s target genes resulting in enhanced mitochondrial oxidative function, likely related to a decrease of oxidative stress and to an increase of mitochondrial biogenesis. The functional impact of resveratrol treatment encompassed an increase of complex I and citrate synthase activities, basal oxygen consumption, and mitochondrial ATP production and a decrease in lactate content, thus supporting a switch from glycolytic to oxidative metabolism. Moreover, resveratrol treatment caused an enhanced macro-autophagic flux through activation of an LC3-independent pathway. Our results, obtained in early-onset PD fibroblasts, suggest that resveratrol may have potential clinical application in selected cases of PD-affected patients. © 2014 Elsevier B.V.

Fluoro-magnetic nanoparticles play an important role in biomedical applications since their size and concentration in tumors allow a very high resolution and an accurate mapping of lesions. Fluorescein isothiocyanate (FITC) has been entrapped inside crystals of magnetic nanoparticles (MNPs) during crystallization. This causes changes of nanoparticle crystal architecture towards elongated rods. TEM and SEM-EDX show elongated crystals with high iron concentration. The intensity of fluoro-MNP fluorescence was detected by fluorescence spectrophotometry and confocal microscopy. The benzene ring structure of FITC and its carboxylic group were clearly detected in the fluoro-MNP spectrum by using FTIR, compared to MNPs prepared in the absence of FITC. Rods were functionalized by hydrogel cross-linking structure (PEG-CMC) onto the fluoro-MNPs surface by using alternate layer-by-layer (LbL) adsorption. These hydrogel properties are used as a preserver for protein delivery. ALK1fc as specific TGF beta 1 inhibitor, was encapsulated inside (PEG-CMC) layers during LbL assembly. Zeta potential measurement, X-ray diffraction and SDS PAGE-silver staining results confirmed the encapsulation of ALK1fc. The efficiency of encapsulated ALK1fc was quantified by immunofluorescence assay against localization of TGF beta 1. Stained TGF beta 1 appeared a purple color and is distributed in the cytoplasm of untreated HLF (a liver cancer invasive cell line), whereas it disappeared in a HLF sample treated with encapsulated ALK1fc.

Mutations in the parkin gene are expected to play an essential role in autosomal recessive Parkinson's disease. Recent studies have established an impact of parkin mutations on mitochondrial function and autophagy. In primary skin broblasts from two patients affected by an early onset Parkinson's disease, we identi ed a hitherto unreported compound heterozygous mutation del exon2-3/del exon3 in the parkin gene, leading to the complete loss of the full-length protein. In both patients, but not in their heterozygous parental control, we observed severe ultrastructural abnormalities, mainly in mitochondria. This was associated with impaired energy metabolism, deregulated reactive oxygen species (ROS) production, resulting in lipid oxidation, and peroxisomal alteration. In view of the involvement of parkin in the mitochondrial quality control system, we have investigated upstream events in the organelles' biogenesis. The expression of the peroxisome proliferator-activated receptor gamma-coactivator 1-alpha (PGC-1 ), a strong stimulator of mitochondrial biogenesis, was remarkably upregulated in both patients. However, the function of PGC-1 was blocked, as revealed by the lack of its downstream target gene induction. In conclusion, our data con rm the role of parkin in mitochondrial homeostasis and suggest a potential involvement of the PGC-1 pathway in the pathogenesis of Parkinson's disease. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

Chitosan nanoparticles (CS NPs) have been widely exploited for the delivery of various types of drugs due to their biocompatibility, availability, ease of functionalization and other advantages. Nevertheless, despite their wide use, their mechanism of action is not very clear and many aspects still need to be investigated in detail, with only a few studies having studied the behavior of this polymer. We prepared CS NPs encapsulating dopamine (DA) and studied the generation of reactive oxygen species (ROS) and the antioxidant effect of the neurotransmitter in detail. Encapsulation of the drug and its subsequent sustained release significantly reduced the oxidation rate in vitro, thus potentially exerting neuroprotective effects. ROS production in SH-SY5Y cells was investigated through a H2O2 assay, while a deeper study of the enzymatic activity allowed us to determine the significant contribution of both GPx and SOD enzymes in preventing oxidative stress.

LY2157299 (LY), which is very small molecule bringing high cancer diffusion, is a pathway antagonist against TGFbeta. LY dosage can be diluted by blood plasma, can be captured by immune system or it might be dissolved during digestion in gastrointestinal tract. The aim of our study is to optimize a "nano-elastic" carrier to avoid acidic pH of gastrointestinal tract, colon alkaline pH, and anti-immune recognition. Polygalacturonic acid (PgA) is not degradable in the gastrointestinal tract due to its insolubility at acidic pH. To avoid PgA solubility in the colon, we have designed its conjugation with Polyacrylic acid (PAA). PgA-PAA conjugation has enhanced their potential use for oral and injected dosage. Following these pre-requisites, novel polymeric nano-micelles derived from PgA-PAA conjugation and loading LY2157299 are developed and characterized. Efficacy, uptake and targeting against a hepatocellular carcinoma cell line (HLF) have also been demonstrated.

In this study, we investigated by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) analysis the effects of resveratrol treatment on skin primary fibroblasts from a healthy subject and from a parkin-mutant early onset Parkinson's disease patient. Parkin, an E3 ubiquitin ligase, is the most frequently mutated gene in hereditary Parkinson's disease. Functional alteration of parkin leads to impairment of the ubiquitin-proteasome system, resulting in the accumulation of misfolded or aggregated proteins accountable for the neurodegenerative process. The identification of proteins differentially expressed revealed that resveratrol treatment can act on deregulated specific biological process and molecular function such as cellular redox balance and protein homeostasis. In particular, resveratrol was highly effective at restoring the heat-shock protein network and the protein degradation systems. Moreover, resveratrol treatment led to a significant increase in GSH level, reduction of GSSG/GSH ratio, and decrease of reduced free thiol content in patient cells compared to normal fibroblasts. Thus, our findings provide an experimental evidence of the beneficial effects by which resveratrol could contribute to preserve the cellular homeostasis in parkin-mutant fibroblasts

Strain SPC-1T was isolated from the phyllosphere of Cynara cardunculus L. var. sylvestris (Lamk) Fiori (wild cardoon), a Mediterranean native plant considered the wild ancestor of the globe artichoke and cultivated cardoon. This Gram-negative, catalase-positive, oxidase-negative, non-spore-forming, rod-shaped and non-motile strain secreted copious amounts of an exopolysaccharide and formed slimy, viscous, orange-pigmented colonies and grew optimally at around pH 6.0-6.5 and 26-30°C in the presence of 0-0.5% NaCl. Phylogenetic analysis based on comparisons of 16S rRNA gene sequences demonstrated that SPC-1T clustered together with species of the genus Sphingomonas sensu strictu. The G+C content of the DNA (66.1 mol%), the presence of Q-10 as the predominant ubiquinone, sym-homospermidine as the predominant polyamine, and 2-hydroxymiristic acid (14:0 2-OH) as the major hydroxylated fatty acid, the absence of 3-hydroxy fatty acids and the presence of sphingoglycolipid supported this taxonomical position. 16S rRNA gene sequence analysis showed that SPC-1 was most closely related to Sphingomonas hankookensis ODN7T, Sphingomonas insulae DS-28T and Sphingomonas panni C52T (98.19%, 97.91% and 97.11% similarity, respectively). However, DNA-DNA hybridization analysis did not reveal any relatedness at the species level. Further differences were apparent in biochemical traits, and fatty acid, quinone and polyamine profiles leading us to conclude that strain SPC-1T (JCM 17498; ITEM 13494) represents a new species of Sphingomonas, for which the name Sphingomonas cynarae sp. nov. is proposed. A component analysis of the exopolysaccharide (named SPC-1T EPS) suggested that it represents a novel type of sphingan containing glucose, rhamnose, mannose and galactose, while glucuronic acid, which is commonly found in sphingans, was not detected.

n contrast to the widely accepted consensus of the existence of a single RNA polymerase in bacteria, several actinomycetes have been recently shown to possess two forms of RNA polymerases due theto co-existence of two rpoB paralogs in their genome. However, the biological signi cance of the rpoB duplication is obscure. In this study we have determined the genome sequence of the lipoglycopeptide antibiotic A40926 producer Nonomuraea gerenzanensis ATCC 39727, an actinomycete with a large genome and two rpoB genes, i.e. rpoB(S) (the wild-type gene) and rpoB(R) (the mutant-type gene). We next analyzed the transcriptional and metabolite pro les in the wild-type gene and in two derivative strains over-expressing either rpoB(R) or a mutated form of this gene to explore the physiological role and biotechnological potential of the "mutant-type" RNA polymerase. We show that rpoB(R) controls antibiotic production and a wide range of metabolic adaptive behaviors in response to environmental pH. This may give interesting perspectives also with regard to biotechnological applications.

A comparative analysis of terminal respiratory enzymes has been performed on four strains of Bacillus clausii used for preparation of a European probiotic. These four strains originated most probably from a common ancestor through early selection of stable clones for industrial propagation. They exhibit a low level of intra-specific diversity and a high degree of genomic conservation, making them an attractive model to study the different bioenergetics behaviors of alkaliphilic bacilli. The analysis of the different bioenergetics responses has been carried out revealing striking differences among the strains. Two out of the four strains have shown a functional redundancy of the terminal part of the respiratory chain. The biochemical data correlate with the expression level of the mRNA of cytochrome c oxidase and quinol oxidase genes (heme-copper type). The consequences of these different bioenergetics behaviors are also discussed.

The growing understanding of the molecular mechanisms underlying epithelial-to-mesenchymal transition (EMT) may represent a potential source of clinical markers. Despite EMT drivers have not yet emerged as candidate markers in the clinical setting, their association with established clinical markers may improve their specificity and sensitivity. Mass spectrometry-based platforms allow analyzing multiple samples for the expression of EMT candidate markers, and may help to diagnose diseases or monitor treatment efficiently. This review highlights proteomic approaches applied to elucidate the differences between epithelial and mesenchymal tumors and describes how these can be used for target discovery and validation.