[Show abstract][Hide abstract] ABSTRACT: Infectious Leptospira colonize the kidneys of reservoir (e.g. rats) and accidental hosts such as humans. The renal response to persistent leptospiral colonization, as measured by urinary protein biosignatures, has not been systematically studied. Urinary exosomes--bioactive membrane-bound nanovesicles--contain cell-state specific cargo that additively reflect formation all along the nephron. We hypothesized that Leptospira-infection will alter the content of urine exosomes, and further, that these Leptospira-induced alterations will hold clues to unravel novel pathways related to bacterial-host interactions.
Exosome protein content from 24 hour urine samples of Leptospira-infected rats was compared with that of uninfected rats using SDS-PAGE and liquid chromatography/tandem mass spectrometry (LC-MS/MS). Statistical models were used to identify significantly dysregulated proteins in Leptospira-infected and uninfected rat urine exosomes. In all, 842 proteins were identified by LC-MS/MS proteomics of total rat urine and 204 proteins associated specifically with exosomes. Multivariate analysis showed that 25 proteins significantly discriminated between uninfected control and infected rats. Alanyl (membrane) aminopeptidase, also known as CD13 topped this list with the highest score, a finding we validated by Western immunoblotting. Whole urine analysis showed Tamm-Horsfall protein level reduction in the infected rat urine. Total urine and exosome proteins were significantly different in male vs. female infected rats.
We identified exosome-associated renal tubule-specific responses to Leptospira infection in a rat chronic colonization model. Quantitative differences in infected male and female rat urine exosome proteins vs. uninfected controls suggest that urine exosome analysis identifies important differences in kidney function that may be of clinical and pathological significance.
[Show abstract][Hide abstract] ABSTRACT: The rapid development of mass spectrometry-based proteomic technologies has allowed the quantification and validation of protein biomarkers toward detection of signature molecules, called proteotypic peptides. To facilitate their extraction from experimental protein and peptide lists, we present here a friendly computational tool called Experimental Proteotypic Peptides Investigator (EPPI). In this study, it was used for extracting proteotypic peptides from two collections of experimental data obtained by MudPIT analysis of adipose and gut human tissue. In particular, EPPI allows the selection of peptides presenting higher occurrence, evaluates their uniqueness by molecular weight and amino-acid sequence, and takes into consideration combinations of multiple proteotypic peptides (proteotypic peptide sets) for evaluating their capacity to target a single protein. In fact, in combination with high-resolution MS instruments, it could be a starting point for targeting proteins by following only precursor ions in full MS scan mode. The software is available under the permissive Apache 2.0 open-source license, and the code can be accessed from https://github.com/ITB-ProtMet/eppi.git.
[Show abstract][Hide abstract] ABSTRACT: Nasu-Hakola disease (NHD) is a recessively inherited rare disorder characterized by a combination of neuropsychiatric and bone symptoms which, while being unique to this disease, do not provide a rationale for the unambiguous identification of patients. These individuals, in fact, are likely to go unrecognized either because they are considered to be affected by other kinds of dementia or by fibrous dysplasia of bone. Given that dementia in NHD has much in common with Alzheimer's disease and other neurodegenerative disorders, it cannot be expected to achieve the differential diagnosis of this disease without performing a genetic analysis. Under this scenario, the availability of protein biomarkers would indeed provide a novel context to facilitate interpretation of symptoms and to make the precise identification of this disease possible. The work here reported was designed to generate, for the first time, protein profiles of lymphoblastoid cells from NHD patients. Two-dimensional electrophoresis (2-DE) and nano liquid chromatography-tandem mass spectrometry (nLC-MS/MS) have been applied to all components of an Italian family (seven subjects) and to five healthy subjects included as controls. Comparative analyses revealed differences in the expression profile of 21 proteins involved in glucose metabolism and information pathways as well as in stress responses.
PLoS ONE 12/2014; 12(9). DOI:10.1371/journal.pone.0110073 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Asthma and chronic obstructive pulmonary disease (COPD) are multifactorial respiratory diseases, characterized by reversible and irreversible airway obstruction, respectively. Even If the primary causes of these diseases remain unknown, inflammation is a central feature which leads to progressive and permanent pulmonary tissue damage (airway remodelling) up to the total loss of lung function. Therefore, the elucidation of the inflammation mechanisms and the characterization of the biological pathways, involved in asthma and COPD pathogenesis, are relevant in finding new possible diagnostic/prognostic biomarkers and for the validation of new drug targets. In this context, current advances in proteomic approaches, especially those based on mass spectrometry, provide new tools to facilitate the discovery-driven studies of new biomarkers in respiratory diseases and improve the clinical reliability of the next generation of biomarkers for these diseases consisting of multiple phenotypes. This review will report an overview of the current proteomic methods applied to the discovery of candidate biomarkers for asthma and COPD, giving a special emphasis to emerging mass spectrometry-based techniques.This article is protected by copyright. All rights reserved
[Show abstract][Hide abstract] ABSTRACT: Asthma is a chronic inflammatory disease. Reticular basement membrane (RBM) thickening is considered feature of airway remodelling (AR) particularly in severe asthma (SA). Omalizumab, mAb to IgE is effective in SA and can modulate AR. Herein we describe protein profiles of bronchial biopsies to detect biomarkers of anti-IgE effects on AR and to explain potential mechanisms/pathways. We defined the bronchial biopsy protein profiles, before and after treatment. Unsupervised clustering of baseline proteomes resulted in very good agreement with the morphometric analysis of AR. Protein profiles of omalizumab responders (ORs) were significantly different from those of non-omalizumab responders (NORs). The major differences between ORs and NORs lied to smooth muscle and extra cellular matrix proteins. Notably, an IgE-binding protein (galectin-3) was reliable, stable and predictive biomarker of AR modulation. Omalizumab down-regulated bronchial smooth muscle proteins in SA. These findings suggest that omalizumab may exert disease-modifying effects on remodelling components.
[Show abstract][Hide abstract] ABSTRACT: The enterobacterium Escherichia coli can utilize a variety of molecules as sulfur sources, including cysteine, sulfate, thiosulfate and organosulfonates. An intermediate of the sulfate assimilation pathway, adenosine 5'-phosphosulfate (APS), also acts as a signal molecule regulating the utilization of different sulfur sources. In this work, we show that inactivation of the cysH gene, leading to accumulation of phosphoadenosine 5'-phosphosulfate (PAPS), also an intermediate of the sulfate assimilation pathway, results in increased surface adhesion and cell aggregation by activating the expression of the curli-encoding csgBAC operon. In contrast, curli production was unaffected by the inactivation of any other gene belonging to the sulfate assimilation pathway. Overexpression of the cysH gene down-regulated csgBAC transcription, further suggesting a link between intracellular PAPS levels and curli gene expression. In addition to curli components, the Flu, OmpX and Slp proteins were also found in increased amounts in the outer membrane compartment of the cysH mutant; deletion of the corresponding genes suggested that these proteins also contribute to surface adhesion and cell surface properties in this strain. Our results indicate that, similar to APS, PAPS also acts as a signal molecule, albeit with distinct mechanism and role: while APS regulates organosulfonate utilization, PAPS would couple availability of sulfur sources to re-modulation of the cell surface, as part of a more global effect on cell physiology.
[Show abstract][Hide abstract] ABSTRACT: The cell envelope of Gram-negative bacteria is a complex multi-layered structure comprising an inner cytoplasmic membrane and an additional asymmetric lipid bilayer, the outer membrane, which functions as a selective permeability barrier and is essential for viability. Lipopolysaccharide, an essential glycolipid located in the outer leaflet of the outer membrane, greatly contributes to the peculiar properties exhibited by the outer membrane. This complex molecule is transported to the cell surface by a molecular machine composed of seven essential proteins LptABCDEFG that form a transenvelope complex and function as a single device. While advances in understanding the mechanisms that govern the biogenesis of the cell envelope have been recently made, only few studies are available on how bacterial cells respond to severe envelope biogenesis defects on a global scale. Here we report the use of differential proteomics based on Multidimensional Protein Identification Technology (MudPIT) to investigate how Escherichia coli cells respond to a block of lipopolysaccharide transport to the outer membrane. We analysed the envelope proteome of a lptC conditional mutant grown under permissive and non permissive conditions and identified 123 proteins whose level is modulated upon LptC depletion. Most such proteins belong to pathways implicated in cell envelope biogenesis, peptidoglycan remodelling, cell division and protein folding. Overall these data contribute to our understanding on how E. coli cells respond to LPS transport defects to restore outer membrane functionality.
PLoS ONE 06/2014; 9(6):e100941. DOI:10.1371/journal.pone.0100941 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Burkholderia thailandensis, although normally avirulent for mammals, can infect macrophages in vitro and has occasionally been reported to cause pneumonia in humans. It is therefore used as a model organism for the human pathogen B. pseudomallei, to which it is closely related phylogenetically. We characterized the B. thailandensis clinical isolate CDC2721121 (BtCDC272) at the genome level and studied its response to environmental cues associated with human host colonization, namely, temperature and oxygen limitation. Effects of the different growth conditions on BtCDC272 were studied through whole genome transcription studies and analysis of proteins associated with the bacterial cell surface. We found that growth at 37°C, compared to 28°C, negatively affected cell motility and flagella production through a mechanism involving regulation of the flagellin-encoding fliC gene at the mRNA stability level. Growth in oxygen-limiting conditions, in contrast, stimulated various processes linked to virulence, such as lipopolysaccharide production and expression of genes encoding protein secretion systems. Consistent with these observations, BtCDC272 grown in oxygen limitation was more resistant to phagocytosis and strongly induced the production of inflammatory cytokines from murine macrophages. Our results suggest that, while temperature sensing is important for regulation of B. thailandensis cell motility, oxygen limitation has a deeper impact on its physiology and constitutes a crucial environmental signal for the production of virulence factors.
PLoS ONE 03/2014; 9(3):e93009. DOI:10.1371/journal.pone.0093009 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Myocardial hibernation (MH) is a well-known feature of human ischaemic cardiomyopathy (ICM), whereas its presence in human idiopathic dilated cardiomyopathy (DCM) is still controversial. We investigated the histological and molecular features of MH in left ventricle (LV) regions of failing DCM or ICM hearts. We examined failing hearts from DCM (n = 11; 41.9 ± 5.45 years; left ventricle-ejection fraction (LV-EF), 18 ± 3.16%) and ICM patients (n = 12; 58.08 ± 1.7 years; LVEF, 21.5 ± 6.08%) undergoing cardiac transplantation, and normal donor hearts (N, n = 8). LV inter-ventricular septum (IVS) and antero-lateral free wall (FW) were transmurally (i.e. sub-epicardial, mesocardial and sub-endocardial layers) analysed. LV glycogen content was shown to be increased in both DCM and ICM as compared with N hearts (P < 0.001), with a U-shaped transmural distribution (lower values in mesocardium). Capillary density was homogenously reduced in both DCM and ICM as compared with N (P < 0.05 versus N), with a lower decrease independent of the extent of fibrosis in sub-endocardial and sub-epicardial layers of DCM as compared with ICM. HIF1-α and nestin, recognized ischaemic molecular hallmarks, were similarly expressed in DCM-LV and ICM-LV myocardium. The proteomic profile was overlapping by ~50% in DCM and ICM groups. Morphological and molecular features of MH were detected in end-stage ICM as well as in end-stage DCM LV, despite epicardial coronary artery patency and lower fibrosis in DCM hearts. Unravelling the presence of MH in the absence of coronary stenosis may be helpful to design a novel approach in the clinical management of DCM.
Journal of Cellular and Molecular Medicine 01/2014; 18(3). DOI:10.1111/jcmm.12198 · 3.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In systemic amyloidosis, accumulation of misfolded proteins as extracellular amyloid fibrils in tissues causes severe organ dysfunction, but the molecular events of tissue damage related to amyloid deposition are still largely unknown. Through the use of MudPIT proteomic approach, comprehensive protein profiles of human amyloid-affected adipose tissue from patients and its control (non amyloid affected) counterpart were acquired. Label-free comparison between patients and controls enabled to highlight differences related to the presence of amyloid, by describing up- and down-represented proteins, connected into interacting networks. In particular, extracellular matrix (ECM), protein folding, lipid metabolism and mitochondrial functions were among the most affected structural/functional pathways. The reported results, obtained with no a-priori hypothesis, represent a significant step forward in the clarification of the molecular mechanisms involved in amyloidoses at tissue level and are the premise for understanding protein misfolding diseases.
Journal of Proteome Research 10/2013; 12(12). DOI:10.1021/pr400583h · 5.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neuroblastoma (NB) is the most common extracranial solid tumor in childhood, with grim prognosis in a half of patients. Exosomes are nanometer-sized membrane vesicles derived from the multivesicular bodies (MVBs) of the endocytic pathway and released by normal and neoplastic cells. Tumor-derived exosomes have been shown in different model systems to carry molecules that promote cancer growth and dissemination. In this respect, we have here performed the first characterization and proteomic analysis of exosomes isolated from human NB cell lines by filtration and ultracentrifugation. Electron microscopy demonstrated that NB-derived exosomes exhibited the characteristic cup-shaped morphology. Dynamic light scattering studies showed a bell-shaped curve and a polydispersity factor consistent with those of exosomes. Zeta potential values suggested a good nanoparticle stability. We performed proteomic analysis of NB-derived exosomes by two dimension liquid chromatography separation and mass spectrometry analyses using the multidimensional protein identification technology strategy. We found that the large majority of the proteins identified in NB derived exosomes are present in Exocarta database including tetraspanins, fibronectin, heat shock proteins, MVB proteins, cytoskeleton-related proteins, prominin-1 (CD133), basigin (CD147) and B7-H3 (CD276). Expression of the CD9, CD63 and CD81 tetraspanins, fibronectin, CD133, CD147 and CD276 was validated by flow cytometry. Noteworthy, flow cytometric analysis showed that NB-derived exosomes expressed the GD2 disialoganglioside, the most specific marker of NB. In conclusion, this study shows that NB-derived exosomes express a discrete set of molecules involved in defense response, cell differentiation, cell proliferation and regulation of other important biological process. Thus, NB-derived exosomes may play an important role in the modulation of tumor microenvironment and represent potential tumor biomarkers.
PLoS ONE 09/2013; 8(9):e75054. DOI:10.1371/journal.pone.0075054 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION: Syndecan-1 is a cell membrane protein that, after its shedding by heparanase enzymes, is accumulated in the extracellular matrix of some tumours, e.g. myeloma and lung carcinoma, where it modulates several key processes of tumourigenesis such as cancer cell proliferation and apoptosis, angiogenesis and metastasis. Few studies have focused on syndecan-1 in malignant melanoma, a tumour for which new therapeutic targets are desperately needed. We aimed to investigate the role of syndecan-1 in melanoma and to evaluate the potential therapeutic efficacy of a novel fully human anti-syndecan-1 recombinant antibody in this deadly disease. METHODS: The OC-46F2 recombinant antibody was generated by selecting a human antibody phage display library on human melanoma cells and by its expression in mammalian cells. The specific antigen recognised by the antibody was identified by mass spectrometry. Murine models of human melanoma and ovarian carcinoma were used in the pre-clinical in vivo experiments. RESULTS: The fully human antibody OC-46F2, specific for the extracellular domain of syndecan-1, inhibited vascular maturation and tumour growth in an experimental human melanoma model. The therapeutic efficacy of this antibody was also demonstrated in an experimental ovarian carcinoma model. A co-distribution of syndecan-1 with vascular endothelial growth factor receptor 2 (VEGFR2) observed in the intratumour melanoma microenvironment was absent in the tumours from mice treated with OC-46F2 scFv. CONCLUSION: These findings highlight the role of syndecan-1 as a potential therapeutic target in melanoma and ovarian carcinoma and provide a new tool able to block vessel maturation, one of the mechanisms that underpin the angiogenic process essential for solid tumour growth.
European journal of cancer (Oxford, England: 1990) 01/2013; 49(8). DOI:10.1016/j.ejca.2012.12.019 · 4.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Mass spectrometry is an important analytical tool for clinical proteomics. Primarily employed for biomarker discovery, it is increasingly used for developing methods which may help to provide unambiguous diagnosis of biological samples. In this context, we investigated the classification of phenotypes by applying support vector machine (SVM) on experimental data obtained by MudPIT approach. In particular, we compared the performance capabilities of SVM by using two independent collection of complex samples and different data-types, such as mass spectra (m/z), peptides and proteins.
Globally, protein and peptide data allowed a better discriminant informative content than experimental mass spectra (overall accuracy higher than 87% in both collection 1 and 2). These results indicate that sequencing of peptides and proteins reduces the experimental noise affecting the raw mass spectra, and allows the extraction of more informative features available for the effective classification of samples. In addition, proteins and peptides features selected by SVM matched for 80% with the differentially expressed proteins identified by the MAProMa software.
These findings confirm the availability of the most label-free quantitative methods based on processing of spectral count and SEQUEST-based SCORE values. On the other hand, it stresses the usefulness of MudPIT data for a correct grouping of sample phenotypes, by applying both supervised and unsupervised learning algorithms. This capacity permit the evaluation of actual samples and it is a good starting point to translate proteomic methodology to clinical application.
Journal of Clinical Bioinformatics 01/2013; 3(1):1. DOI:10.1186/2043-9113-3-1
[Show abstract][Hide abstract] ABSTRACT: Amyloidoses are characterized by deposition of misfolded proteins as beta-pleated sheet fibrils in organs. Despite the similar morphologic appearance of fibrils, at least 28 different proteins have been identified as causative agents of amyloidosis in humans, 14 of which responsible for systemic forms. Correct identification of the amyloidogenic proteins in each patient is crucial for clinical management, in order to avoid misdiagnosis, inappropriate treatment and to assess the prognosis. Amyloidosis, being essentially a protein deposition disorder, is an attractive venue for the application of proteomics methodologies; among the different possible analytic goals, the most important is the unequivocal diagnosis and typing of the amyloid deposits. Amyloidosis typing has been traditionally based on a multidisciplinary approach, requiring detailed clinical evaluation and immunohistochemical studies together with biochemical and genetic tests. However, drawbacks of immunohistochemistry-based techniques have driven the search for alternative methods for direct amyloid typing. In particular, mass spectrometry-based proteomics, recently introduced in the clinical practice with or without the previous 2DE separation of proteins, has revolutionized amyloid typing. This review provides a description of current proteomic methods for the identification of the amyloidogenic proteins, with special attention to the most innovative mass spectrometry-based techniques.
[Show abstract][Hide abstract] ABSTRACT: We report on specific magneto-capturing followed by Multidimensional Protein Identification Technology (MudPIT) for the analysis of surface-exposed proteins of intact cells of the bacterial opportunistic pathogen Pseudomonas aeruginosa. The magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction allowed the MudPIT identification of the captured and neighboring proteins. Remarkably, we identified 63 proteins captured directly by nanoparticles and 67 proteins embedded in the cell envelope fragments. For a high number of proteins, our analysis strongly indicates either surface exposure or localization in an envelope district. The localization of most identified proteins was only predicted or totally unknown. This novel approach greatly improves the sensitivity and specificity of the previous methods, such as surface shaving with proteases that was also tested on P. aeruginosa. The magneto-capture procedure is simple, safe, and rapid, and appears to be well-suited for envelope studies in highly pathogenic bacteria.
PLoS ONE 11/2012; 7(11):e51062. DOI:10.1371/journal.pone.0051062 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The antral compartment in the ovary consists of two populations of oocytes that differ by their ability to resume meiosis and to develop to the blastocyst stage. Still for reasons not entirely clear, antral oocytes termed surrounded nucleolus (SN; 70% of the population of antral oocytes) develop to the blastocyst stage, while those called not-surrounded nucleolus (NSN) arrest at two cells. We profiled transcriptomic, proteomic and morphological characteristics of antral oocytes and observed that NSN oocyte arrest is associated with lack of cytoplasmic lattices coincident with reduced expression of MATER and ribosomal proteins. Cytoplasmic lattices have been shown to store maternally-derived mRNA and ribosomes in mammalian oocytes and embryos while MATER has been shown to be required for cytoplasmic lattices formation. Thus, we isolated antral oocytes from a Mater(tm/tm) mouse and we observed that 84% of oocytes are of the NSN type. Our results provide the first molecular evidence to account for inability of NSN-derived embryos to progress beyond the two-cell stage; these results may be relevant to naturally-occurring pre-implantation embryo demise in mammals.
Biology of Reproduction 11/2012; 88(1). DOI:10.1095/biolreprod.112.103887 · 3.45 Impact Factor