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Petti C.,Candiolo Cancer Institute FPO IRCCS | Picco G.,Candiolo Cancer Institute FPO IRCCS | Picco G.,University of Turin | Martelli M.L.,Candiolo Cancer Institute FPO IRCCS | And 8 more authors.
Oncotarget | Year: 2015

Constitutively active receptor tyrosine kinases (RTKs) are known oncogenic drivers and provide valuable therapeutic targets in many cancer types. However, clinical efficacy of RTK inhibitors is limited by intrinsic and acquired resistance. To identify genes conferring resistance to inhibition of the MET RTK, we conducted a forward genetics screen in the GTL-16 gastric cancer cell line, carrying METamplification and exquisitely sensitive to MET inhibition. Cells were transduced with three different retroviral cDNA expression libraries and selected for growth in the presence of the MET inhibitor PHA-665752. Selected cells displayed robust and reproducible enrichment of library-derived cDNAs encoding truncated forms of RAF1 and BRAF proteins, whose silencing reversed the resistant phenotype. Transduction of naïve GTL-16 cells with truncated, but not full length, RAF1 and BRAF conferred in vitro and in vivo resistance to MET inhibitors, which could be reversed by MEK inhibition. Induction of resistance by truncated RAFs was confirmed in other METaddicted cell lines, and further extended to EGFR-addicted cells. These data show that truncated RAF1 and BRAF proteins, recently described as products of genomic rearrangements in gastric cancer and other malignancies, have the ability to render neoplastic cells resistant to RTK-targeted therapy.


Zanini C.,University of Turin | Ercole E.,University of Turin | Mandili G.,University of Turin | Salaroli R.,University of Bologna | And 5 more authors.
PLoS ONE | Year: 2013

Background:Medulloblastoma (MB) is an aggressive pediatric tumor of the Central Nervous System (CNS) usually treated according to a refined risk stratification. The study of cancer stem cells (CSC) in MB is a promising approach aimed at finding new treatment strategies.Methodology/Principal Findings:The CSC compartment was studied in three characterized MB cell lines (DAOY, UW228 and ONS-76) grown in standard adhesion as well as being grown as spheres, which enables expansion of the CSC population. MB cell lines, grown in adherence and as spheres, were subjected to morphologic analysis at the light and electron microscopic level, as well as cytofluorimetric determinations. Medullospheres (MBS) were shown to express increasingly immature features, along with the stem cells markers: CD133, Nestin and β-catenin. Proteomic analysis highlighted the differences between MB cell lines, demonstrating a unique protein profile for each cell line, and minor differences when grown as spheres. In MBS, MALDI-TOF also identified some proteins, that have been linked to tumor progression and resistance, such as Nucleophosmin (NPM). In addition, immunocytochemistry detected Sox-2 as a stemness marker of MBS, as well as confirming high NPM expression.Conclusions/Significance:Culture conditioning based on low attachment flasks and specialized medium may provide new data on the staminal compartment of CNS tumors, although a proteomic profile of CSC is still elusive for MB. © 2013 Zanini et al.


PubMed | University of Turin, University of Bologna, Center for Experimental and Clinical Studies and BioDigitalValley srl
Type: | Journal: Molecular and cellular therapies | Year: 2015

Medulloblastoma (MB) is the most common malignant childhood brain tumor with the propensity to disseminate at an early stage, and is associated with high morbidity. New treatment strategies are needed to improve cure rates and to reduce life-long cognitive and functional deficits associated with current therapies. Extracellular Vesicles (EVs) are important players in cell-to-cell communication in health and diseases. A clearer understanding of cell-to-cell communication in tumors can be achieved by studying EV secretion in medullospheres. This can reveal subtle modifications induced by the passage from adherent to non-adherent growth, as spheres may account for the adaptation of tumor cells to the mutated environment.Formation of medullospheres from MB cell lines stabilized in adherent conditions was obtained through culture conditioning based on low attachment flasks and specialized medium. EVs collected by ultracentrifugation, in adherent conditions and as spheres, were subjected to electron microscopy, NanoSight measurements and proteomics.Interestingly, iron carrier proteins were only found in EVs shed by CSC-enriched tumor cell population of spheres. We used iron chelators when culturing MB cell lines as spheres. Iron chelators induced a decrease in number/size of spheres and in stem cell populations able to initiate in vitro spheres formation.This work suggests a not yet identified role of iron metabolism in MB progression and invasion and opens the possibility to use chelators as adjuvants in anti-tumoral chemotherapy.


Natale M.,BioDigitalValley S.r.l. | Bonino D.,BioDigitalValley S.r.l. | Consoli P.,BioDigitalValley S.r.l. | Alberio T.,University of Insubria | And 4 more authors.
Bioinformatics | Year: 2010

Motivation: The two-dimensional electrophoresis (2-DE) pattern of proteins is thought to be specifically related to the physiological or pathological condition at the moment of sample preparation. On this ground, most proteomic studies move to identify specific hallmarks for a number of different conditions. However, the information arising from these investigations is often incomplete due to inherent limitations of the technique, to extensive protein post-translational modifications and sometimes to the paucity of available samples. The meta-analysis of proteomic data can provide valuable information pertinent to various biological processes that otherwise remains hidden. Results: Here, we show a meta-analysis of the PD protein DJ-1 in heterogeneous 2-DE experiments. The protein was shown to segregate into specific clusters associated with defined conditions. Interestingly, the DJ-1 pool from neural tissues displayed a specific and characteristic molecular weight and isoelectric point pattern. Moreover, changes in this pattern have been related to neurodegenerative processes and aging. These results were experimentally validated on human brain specimens from control subjects and PD patients. Availability: ImageJ is a public domain image processing program developed by the National Institutes of Health and is freely available at http://rsbweb.nih.gov/ij. All the ImageJ macros used in this study are available as supplementary material and upon request at info@biodigitalvalley.com. XLSTAT can be purchased online at http://www.xlstat.com/en/home/ at a current cost of ~300 EUR. Contact: enrico.bucci@biodigitalvalley.com. Supplementary information: Supplementary data are available at Bioinformatics online. © The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org.


Alberio T.,University of Insubria | Bucci E.M.,BioDigitalValley s.r.l. | Natale M.,BioDigitalValley s.r.l. | Natale M.,Polytechnic University of Turin | And 4 more authors.
Journal of Proteomics | Year: 2013

Diagnosis of Parkinson's disease (PD) is currently assessed by the clinical evaluation of extrapyramidal signs. The identification of specific biomarkers would be advisable, however most studies stop at the discovery phase, with no biomarkers reaching clinical exploitation. To this purpose, we developed an automated literature analysis procedure to retrieve all the background knowledge available in public databases. The bioinformatic platform allowed us to analyze more than 51,000 scientific papers dealing with PD, containing information on 4121 proteins. Out of these, we could track back 35 PD-related proteins as present in at least two published 2-DE maps of human plasma. Then, 9 different proteins (haptoglobin, transthyretin, apolipoprotein A-1, serum amyloid P component, apolipoprotein E, complement factor H, fibrinogen γ, thrombin, complement C3) split into 32 spots were identified as a potential diagnostic pattern. Eventually, we compared the collected literature data to experimental gels from 90 subjects (45 PD patients, 45 non-neurodegenerative control subjects) to experimentally verify their potential as plasma biomarkers of PD. This article is part of a Special Issue entitled: From Genome to Proteome: Open Innovations. © 2013 Elsevier B.V.


Bucci E.M.,Biodigitalvalley S.r.l. | Bucci O.M.,University of Naples Federico II | Sorrentino R.,University of Perugia
Proceedings of the IEEE | Year: 2014

Discusses the technologies and applications supported by nanotechnology, considered one of the 21st century's most promising technologies. Nanotechnology deals with the design, development, and manipulation of materials and devices with at least one dimension sized on a nanometer scale. It involves fields of science as diverse as surface science, organic chemistry, molecular biology, semiconductor physics, microfabrication, with a vast range of applications, such as in medicine, electronics, biomaterials, and energy production. Its ultimate goal is to be able to predictably design, construct, and control nanosystems, tailoring them to specified needs. © 2014 IEEE.


Natale M.,BioDigitalValley S.r.l. | Natale M.,Polytechnic University of Turin | Maresca B.,University of Naples Federico II | Abrescia P.,University of Naples Federico II | And 2 more authors.
Proteomics Insights | Year: 2011

A number of commercial software packages are currently available to perform digital two-dimensional electrophoresis (2D-GE) gel analysis. However, both the high cost of the commercial packages and the unavailability of a standard data analysis workflow, have prompted several groups to develop freeware systems to perform certain steps of gel analysis. Unfortunately, to the best of our knowledge none of them offer a package that performs all the steps envisaged in a 2D-GE gel analysis. Here we describe an ImageJ-based procedure, able to manage all the steps of a 2D-GE gel analysis. ImageJ is a free available image processing and analysis application developed by National Institutes of Health (NIH) and widely used in different life sciences fields as medical imaging, microscopy, western blotting and PAGE. Nevertheless no one has yet developed a procedure enabled to compare spots on 2D-GE gels. We collected all used ImageJ tools in a plug-in that allows us to perform the whole 2D-GE analysis. To test it, we performed a set of 2D-GE experiments on plasma samples from 9 patients victims of acute myocardial infarction and 8 controls, and we compared the results obtained by our procedure to those obtained using a widely diffuse commercial package, finding similar performances. © the author(s), publisher and licensee Libertas Academica Ltd.


Natale M.,Polytechnic University of Turin | Natale M.,BioDigitalValley S.r.l. | Caiazzo A.,WIAS Berlin | Bucci E.M.,BioDigitalValley S.r.l. | And 2 more authors.
Genomics, Proteomics and Bioinformatics | Year: 2012

Analysis of images obtained from two-dimensional gel electrophoresis (2D-GE) is a topic of utmost importance in bioinformatics research, since commercial and academic software available currently has proven to be neither completely effective nor fully automatic, often requiring manual revision and refinement of computer generated matches. In this work, we present an effective technique for the detection and the reconstruction of over-saturated protein spots. Firstly, the algorithm reveals overexposed areas, where spots may be truncated, and plateau regions caused by smeared and overlapping spots. Next, it reconstructs the correct distribution of pixel values in these overexposed areas and plateau regions, using a two-dimensional least-squares fitting based on a generalized Gaussian distribution. Pixel correction in saturated and smeared spots allows more accurate quantification, providing more reliable image analysis results. The method is validated for processing highly exposed 2D-GE images, comparing reconstructed spots with the corresponding non-saturated image, demonstrating that the algorithm enables correct spot quantification. © 2012 Beijing Institute of Genomics, Chinese Academy of Sciences and Genetics Society of China.


Acquadro E.,BioIndustry Park Silvano Fumero S.p.A. | Caron I.,Mario Negri Institute for Pharmacological Research | Tortarolo M.,Mario Negri Institute for Pharmacological Research | Bucci E.M.,BioIndustry Park Silvano Fumero S.p.A. | And 3 more authors.
Journal of Proteome Research | Year: 2014

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease caused by the degeneration of motor neurons. The transgenic mouse model carrying the human SOD1G93A mutant gene (hSOD1G93A mouse) represents one of the most reliable and widely used model of this pathology. In the present work, the innovative technique of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was applied in the study of pathological alterations at the level of small brain regions such as facial and trigeminal nuclei, which in rodents are extremely small and would be difficult to analyze with classical proteomics approaches. Comparing slices from three mice groups (transgenic hSOD1G93A, transgenic hSOD1WT, and nontransgenic, Ntg), this technique allowed us to evidence the accumulation of hSOD1G93A in the facial and trigeminal nuclei, where it generates aggregates. This phenomenon is likely to be correlated to the degeneration observed in these regions. Moreover, a statistical analysis allowed us to highlight other proteins as differentially expressed among the three mice groups analyzed. Some of them were identified by reverse-phase HPLC fractionation of extracted proteins and mass spectrometric analysis before and after trypsin digestion. In particular, the 40S ribosomal protein S19 (RPS19) was upregulated in the parenkyma and reactive glial cells in facial nuclei of hSOD1G93A mice when compared to transgenic hSOD1WT and nontransgenic ones. © 2014 American Chemical Society.


Giordano M.,BioDigitalValley S.r.l | Natale M.,BioDigitalValley S.r.l | Natale M.,Polytechnic University of Turin | Cornaz M.,BioDigitalValley S.r.l | And 4 more authors.
Electrophoresis | Year: 2013

iMole is a platform that automatically extracts images and captions from biomedical literature. Images are tagged with terms contained in figure captions by means of a sophisticate text-mining tool. Moreover, iMole allows the user to upload directly their own images within the database and manually tag images by curated dictionary. Using iMole the researchers can develop a proper biomedical image database, storing the images extracted from paper of interest, image found on the web repositories, and their own experimental images. In order to show the functioning of the platform, we used iMole to build a 2DE database. Briefly, tagged 2DE gel images were collected and stored in a searchable 2DE gel database, available to users through an interactive web interface. Images were obtained by automatically parsing 16608 proteomic publications, which yielded more than 16500 images. The database can be further expanded by users with images of interest trough a manual uploading process. iMole is available with a preloaded set of 2DE gel data at http://imole.biodigitalvalley.com. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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