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ItPA XII Annual Congress

Conference hall of the Rectorate Via Tancredi, 7 - Lecce

Lecce, June 12th-15th, 2017

Italian Proteomics Association

2 XII ItPA National Congress Lecce, June 12th-15th, 2017

Congress president Michele Maffia Università Del Salento SCIENTIFIC COMMITTEE LuigiBonizzi Margherita Ruoppolo Laura Giusti Claudia Desiderio TizianaCabras VincenzoCunsolo MaurizioRonci ORGANIZING COMMITTEE Michele Maffia Luigi Bonizzi Tiziana Cabras Vincenzo Cunsolo Claudia Desiderio Laura Giusti Paola Roncada Maurizio Ronci Margherita Ruoppolo Andrea Urbani Daniele Vergara Loredana Capobianco Anna Giudetti

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ItPA XII Annual Congress Lecce, June 12th-15th, 2017

ABSTRACT VOLUME

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Program at Glance MONDAY 12 JUNE Conference hall of the Rectorate Food & Nutrition (FaN) Proteomics 14:00 – 14:15 Opening 14:15 – 15:00 The central role of microbiota: from environment to food production

Paola Roncada (Italy) 15:00 – 15:30 Ex vivo screening of individual human gut microbiomes

Daniel Figeys (Canada) 15:30 – 16:00 Role of the microbiome in food allergy and asthma

Liam O’ Mahony (Switzerland) 16:00 – 16:30 Microbiota from childhood to adulthood in health and disease

Lorenza Putignani (Italy) 16:30 – 17:15 Coffee Break 17:15 – 17:45 Micromanagement of the metaproteome: taking control of your data

Lennart Martens (Belgium) 17:45 Closing of the Workshop

MONDAY 12 JUNE Conference hall of the Rectorate 12:00 – 19:00 Congress Registration 18:00 – 18:15 Opening ceremony 18:30 – 19:15 Opening Lecture

Next-generation proteomics: building a precision toolkit for life science Albert Sickmann (Germany)

19:15 – 21:00 Welcome Cocktail

TUESDAY 13 JUNE Conference hall of the Rectorate SESSION I: NON HUMAN PROTEOMICS 09:15 – 10:00 Keynote Lecture

Proteomics- and Immuno-proteomics lessons learned from S. aureus host-pathogen interactions Frank Schmidt (Germany)

10:00 – 10:45 Distinguished Lecture Integrated proteomic technologies for the characterization of milk products Andrea Scaloni (Italy)

10:45 – 11:15 Coffee Break 11:15 – 12:20 Oral presentations selected from abstracts


12:20 – 13:30 Lunch 13:20 – 14:20 Poster session SESSION II: SYSTEMS BIOLOGY 14:30 – 15:15 Keynote Lecture

neXtProt as a tool to explore the human proteome Lydie Lane (Switzerland)

15:15 – 16:00 Distinguished Lecture Mapping Human Mitochondrial Protein Interactions Linked to Neurodegeneration Mohan Babu (Canada)

16:00 – 16:30 Coffee Break 16:30 – 17:15 Plenary Lecture

Generation of large scale SWATH-MS proteomic datasets and their use Ruedi Aebersold (Switzerland)

17:15 – 17:45 Oral presentations selected from abstracts 17:45 – 18:00 Award ceremony: honorary president

Prof. Paolo Mocarelli Direttore scientifico della Fondazione Don Gnocchi, Milano

WEDNESDAY 14 JUNE Conference hall of the Rectorate SESSION III: TECHNOLOGIES and BIOINFORMATICS 09:00 – 09:45 Keynote Lecture

Shedding new light on Spinal Cord injury Michel Salzet (France)

09:45 – 10:30 Distinguished Lecture Clinical MALDI MS Imaging: From Bench to Surgery room Isabelle Fournier (France)

10:30 – 11:00 Coffee break 11:00 – 12:00 Oral presentations selected from abstracts 12:00 – 13:30 Lunch 13:30 – 14:30 Poster session SESSION IV: HUMAN PROTEOMICS 14:30 – 15:15 Keynote Lecture

From protein biomarker discovery to the development of mass spectrometry based diagnostic tests of clinical utility Stephen R Pennington (Ireland)

15:15 – 16:00 Distinguished Speaker Proteomic strategies for genetic and oncological diseases Piero Pucci (Italy)

16:00 – 16:30 Coffee break 16:30 – 17:30 Oral presentations selected from abstracts 17:45 – 18:45 ItPA assembly 21:00 Social Dinner – Chiostro degli Olivetani

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THURSDAY 15 JUNE Conference hall of the Rectorate 9:30 – 10:00 Poster Prizes Satellite Session Bioengineering 10:00 – 10:40 Building 3D human tissues in vitro for tissueand organ on chip applications

Paolo Netti (Italy) 10:40 – 11:20 Implantable and wearable technologies for lifescience at the Center for

Biomolecular Nanotechnologies Massimo De Vittorio (Italy)

11:20 – 11:50 Coffee Break 11:50 – 12:30 A composite scaffold for bone-cartilage defects bridging: an in vivo study

Alessandro Sannino (Italy) End of the Satellite Session 12:30 – 13:15 Closing Lecture

Neuronal interfaces: innovative therapies and neuroprosthetics Fabio Benfenati (Italy)

13:15 Conference Closing


ItPA XII Annual Congress

Conference hall of the Rectorate Via Tancredi, 7 - Lecce

9

Food & Nutrition (FaN) Proteomics Special Focus on One Health Microbiome

Conference hall of the Rectorate of the University of Salento

Monday June 12th 2017 14:00 – 14:15 Opening

Andrea Urbani Università Cattolica del Sacro Cuore and Fondazione Santa Lucia – IRCCS, Rome

Session chairs: George Tsangaris (Greece), Adriano Brandelli (Brasil)

14:15 – 15:00 The central role of microbiota: from environment to food

production Paola Roncada Istituto Sperimentale Italiano Lazzaro Spallanzani, Milano, Italy

15:00 – 15:30 Ex vivo screening of individual human gut microbiomes

Daniel Figeys University of Ottawa, Ottawa, Canada

15.30 – 16:00 Role of the microbiome in food allergy and asthma

Liam O’ Mahony Swiss Institute of Allergy and Asthma, University of Zürich, Davos, Switzerland

16.00 – 16:30 Microbiota from childhood to adulthood in health and disease

Lorenza Putignani IRCCS Ospedale Pediatrico Bambino Gesu, Human Microbiome Unit, Rome, Italy

16:30 – 17:15 Coffee Break 17:15 – 17:45 Micromanagement of the metaproteome: taking control of

your data Lennart Martens VIB-UGent Center for Medical Biotechnology, VIB, Ghent, Belgium

17:45 Closing of the Workshop


SCIENTIFIC PROGRAM Monday June 12th 2017 12:00 – 19:00 Congress Registration 14:00 – 18:00 Food & Nutrition (FaN) Proteomics Special Focus on One Health Microbiome 18:00 – 18:15 Opening Ceremony

Chair: Luigi Bonizzi, Michele Maffia

18:30 – 19:15 Opening Lecture Next-generation proteomics: building a precision toolkit for life science Albert Sickmann Institute for Analytical Sciences, Dortmund, Dortmund, Germany

19:15 – 21:00 Welcome Cocktail Conference hall of the Rectorate of the University of Salento

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Tuesday June 13th 2017 SESSION I: NON HUMAN PROTEOMICS Session Chairs Paola Roncada, Vincenzo Cunsolo 09:15 – 10:00 Keynote Lecture

Proteomics- and Immuno-proteomics lessons learned from S. aureus host-pathogen interactions Frank Schmidt EMA University Medicine Greifswald, Department of Functional Genomics, ZIK-FunGene Group Applied Proteomics, Greifswald, Germany

10:00 – 10:45 Distinguished Lecture Integrated proteomic technologies for the characterization of milk products Andrea Scaloni Consiglio Nazionale delle Ricerche, Proteomics and Mass Spectrometry Laboratory, Roma, Italy

10:45 – 11:15 Coffee break Session Chairs Frank Schmidt, Paola Roncada Oral presentations selected from abstracts: 11:15 – 11:35 Proteomic analysis of the mode of action of the disinfectants

based on pyridoxal oxime derivatives against food borne pathogens Djuro Josic Department of Biotechnology, University of Rijeka, Rijeka, Croatia

11:35 – 11:55 Peptidomic analysis of bioactive ovine milk

caseinate hydrolysates Adriano Brandelli Universidade Federal do Rio Grande do Sul, Laboratório de Bioquímica e Microbiologia Aplicada, Porto Alegre, Brazil

11:55 – 12:10 BacteriaMS: an open source tool for bacterial whole cell typing by

mass spectra pattern matching with in silico hypothesis validation Zhuoxin Chen Department of Chemistry and Institute of Biomedical Sciences, Fudan University, Shanghai,China

12:10 – 12:25 Proteomics to improve serodiagnosis of Brucellosis

Alessio Soggiu Dipartimento di Medicina Veterinaria, Università degli studi di Milano, Milano, Italy

12 XII ItPA National Congress Lecce, June 12th-15th, 2017 12:25 – 13:30 Lunch 13:30 – 14:30 Poster Session (I-II) SESSION II: SYSTEMS BIOLOGY Session Chairs Laura Giusti, Mauro Fasano 14.30 – 15.15 Keynote Lecture

neXtProt as a tool to explore the human proteome Lydie Lane Swiss Institute of Bioinformatics, Geneve, Switzerland

15:15 – 16:00 Distinguished Lecture Mapping Human Mitochondrial Protein Interactions Linked to Neurodegeneration Mohan Babu Department of Biochemistry, University of Regina, Regina, Canada

16:00 – 16:30 Coffee break Session Chairs Andrea Urbani, Mohan Babu 16:30 – 17:15 Plenary Lecture

Generation of large scale SWATH-MS proteomic datasets and their use Ruedi Aebersold Department of Biology, Institute of Molecular Systems Biology, ETH Zurich and Faculty of Science, University of Zurich, Switzerland

Oral presentations selected from abstracts:

17:15 – 17:30 Activation of Nrf2-element signalling pathway in response to

H2S related oxidative stress Viviana Greco Laboratorio di Proteomica e Metabonomica, Fondazione Santa Lucia, Roma, Italy

17:30– 17:45 Proteomic and interactomic characterization of PARK2-mutated Parkinson’s disease patients Mara Zilocchi Department of Science and High Technology, Center of Neuroscience, University of Insubria, Busto Arsizio, Italy

17:45 – 18:00 Award ceremony: honorary president Paolo Mocarelli

Direttore scientifico della Fondazione Don Gnocchi, Milano Chair Luigi Bonizzi

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Wednesday June 14th 2017 SESSION III: TECHNOLOGIES and BIOINFORMATICS Session Chairs Michele Maffia, Maurizio Ronci 09:00 – 09:45 Keynote Lecture

Shedding new light on Spinal Cord injury Michel Salzet Laboratoire de Protéomique, Réponse Inflammatoire, Spectrométrie de Masse (PRISM), Université Lille, Villeneuve d’Ascq, France

09:45 – 10:30 Distinguished Lecture Clinical MALDI MS Imaging: From Bench to Surgery room Isabelle Fournier Laboratoire de Protéomique, Réponse Inflammatoire, Spectrométrie de Masse (PRISM), Université Lille, Villeneuve d’Ascq, France

10:30 – 11:00 Coffee break Session Chairs Michel Salzet, Michele Maffia Oral presentations selected from abstracts: 11:00 – 11:15 Nanotechnological approaches for epithelial–mesenchymal

transition biomarkers screening Paola Priore CNR-NANOTEC, Institute of Nanotechnology c/o Campus Ecotekne, University of Salento, Lecce, Italy

11:15 – 11:30 Clinical applications of universal S-Trap sample processing

John Wilson Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

11:30 – 11:45 Terminal Amine Isotopic Labeling of Substrates (TAILS): a positional proteomics approach to study mitochondrial proteases in Parkinson's disease Marta Lualdi Department of Science and High Technology, University of Insubria, Busto Arsizio, Italy

11:45 – 12:00 Diagnostic “rhinomic” profile of Allergic and non-Allergic Rhinits Subject by Mesoporous Silica Particles and MALDI-TOF Mass Spectrometry Chiara Villella Department of Health Sciences, University Magna Graecia of Catanzaro, Catanzaro

12:00 – 13:30 Lunch 13:30 – 14:30 Poster Session (III-IV)


SESSION IV: HUMAN PROTEOMICS Session Chairs Margherita Ruoppolo, Tiziana Cabras 14:30 – 15:15 Keynote Lecture

From protein biomarker discovery to the development of mass spectrometry based diagnostic tests of clinical utility

Stephen R Pennington School of Medicine, UCD Conway Institute, University College Dublin, Dublin, Ireland

15:15 – 16:00 Distinguished Lecture Proteomic strategies for genetic and oncological diseases Piero Pucci Università degli Studi di Napoli Federico II, Dipartimento di Scienze Chimiche and Ceinge Biotecnologie Avanzate, Naples, Italy

16:00 – 16:30 Coffee break Session Chairs Stephen R Pennington, Margherita Ruoppolo

Oral presentations selected from abstracts: 16:30 – 16:45 Urinary proteomics in Bardet-Biedl syndrome

Michele Costanzo Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli ‘‘Federico II’’, Naples, Italy

16:45 – 17:00 Proteomic of exosome’s cargo and membrane proteins from

Neuroblastoma Victor Corasolla Carregari Fondazione Santa Lucia, IRCCS, Rome-Italy

17:00 – 17:15 Proteomics analysis to assess the role of mitochondria in BRCA1-mediated breast tumorigenesis Domenica Scumaci Laboratory of Proteomics, Dpt. of Experimental and Clinical Medicine; Magna Græcia University of Catanzaro, Catanzaro, Italy.

17:15 – 17:30 Peptidomics analysis of simulated mouth, gastric and

intestinal digestion pattern of allergenic tropomyosin Cristian Piras Department of Veterinary Medicine (DIMEVET), University of Milan, Italy

17:45 – 18:45 ItPA General Assembly

21:00 Social Dinner – Chiostro degli Olivetani

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Thursday June 15th 2017 CLOSING SESSION Session Chairs Michele Maffia, Piero Pucci 09:30 – 10:00 Poster Prizes 10:40 – 12:30 Bioengineering: Satellite Session 12:30 – 13:15 Closing Lecture

Neuronal interfaces: innovative therapies and neuroprosthetics Fabio Benfenati Center for Synaptic Neuroscience and Technology, Fondazione Istituto Italiano di Tecnologia, Genova, Italy

13:15 – 13:30 Conference Closing


Bioengineering: Satellite Session

Conference hall of the Rectorate of the University of Salento

THURSDAY June 15th 2017 Session Chairs Michele Maffia, Piero Pucci 10:00 - 10:40 Building 3D human tissues in vitro for tissue and organ on

chip applications Paolo Netti Istituto Italiano di Tecnologia, Center for Advanced Biomaterials for Health Care, Genoa, Italy

10:40 - 11:20 Implantable and wearable technologies for lifescience at the

Center for Biomolecular Nanotechnologies Massimo De Vittorio Dipartimento di Ingegneria dell’Innovazione, Università del Salento, Lecce, Italy

11.20 – 11:50 Coffee Break Session Chairs Michele Maffia, Daniele Vergara 11.50 – 12:30 A composite scaffold for bone-cartilage defects bridging: an in

vivo study Alessandro Sannino Università del Salento, Department of Engineering for Innovation, Lecce, Italy

ORAL COMMUNICATIONS: Keynote Lectures 17

KEY NOTE LECTURES


K-01

Next-generation proteomics: building a precision toolkit for life science

Albert Sickmanna*

aLeibniz-Institut für Analytische Wissenschaften – ISAS – e.V., Dortmund, Germany. Mass-spectrometry-based proteomics is a rapidly developing field during the last twenty years. It benefits strongly from major inventions in mass spectrometry, separation science, sample preparation, bioinformatics, and novel tools for life science research. After this period of technology development and continuous improvements, proteomics is starting to mature and deliver solutions to complex biological challenges in research areas focusing on for example biodiversity, food, and health. Complied with the central dogma of biology introduced by Francis H. C. Crick in 1958 [1,2], proteomics is the method to explore the building blocks of cellular function encoded by the genome. Moreover, the proteome is extremely dynamic owing to splicing, posttranslational modification, protein targeting, and higher level organization in protein complexes and functional pathways. Today, the next generation of proteomics is ready to provide precision tools able to analyze protein structures, crosstalk between posttranslational modification, activity of decision-making pathways, single-cell organisms, cell clusters, and tissues at once, precision medicine in the near future. Recent achievements and strategies will be discussed in the presentation. * Corresponding author: Albert Sickmann. Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund, Germany Phone.: +49 231 1392 100 Fax: +49 231 1392 200 E-mail address: [email protected] (A. Sickmann) References: [1] Crick, F.H.C. (1958). "On Protein Synthesis". In F.K. Sanders. Symposia of the Society for Experimental Biology, Number XII: The

Biological Replication of Macromolecules. Cambridge University Press. pp. 138–163. [2] Crick, F (August 1970). "Central dogma of molecular biology." (PDF). Nature. 227 (5258): 561–3. doi:10.1038/227561a0.

mailto:[email protected]


K-02

Proteomics and Immunoproteomics lessons learned from S. aureus host-pathogen interactions

Frank Schmidta*

a EMA University Medicine Greifswald, Department of Functional Genomics, ZIK-FunGene Group

Applied Proteomics, Friedrich-Ludwig-Jahnstr. 15a, D-17487 Greifswald, Germany.

S. aureus related diseases range from mild to severe infections and proteomics is able to identify key components important for such diseases. However, general proteome analysis approaches using data dependent acquisition (DDA) are known to provide lower reproducibility and comprehensiveness when compared to data-independent acquisition (DIA). A spectral library, which is suitable for the analysis of DIA data, was generated, benchmarked with a well characterized biological standard data set, and used for an in-depth analysis of in vitro S9 cell and in vivo murine infection experiments. Protein samples from S. aureus grown under different conditions were extensively fractionated to obtain a comprehensive proteome map for routine proteomics approaches. The DDA data (N=152) were collected on a Q Exactive and integrated to provide a spectral library suitable for DIA analysis. The comprehensive S. aureus DIA library was then used to perform DIA-based proteome studies in vitro and in vivo. Benchmarking of the S. aureus DIA library, which covers approx. 72% of the S. aureus proteome, reveals that this library provides very high reproducibility (majority of CVs < 10%) and cross-MS compatibility. When analyzing 47 Staphylococcus strains, peptides of each strain covered > 80% of our library. Thus the library can be used as a global staphylococcal DIA library. DIA analysis of murine infection samples revealed up-regulation of proteins involved in oxidative stress and downregulation of proteins involved in dNTP synthesis and protein biosynthetic activity, which was confirmed by the S9 cell infection experiments. DIA data allowed a deeper insight in the pathogen adaption to the host during infection. We further overexpressed infection related S. aureus proteins as recombinant proteins and investigated the antibody response of patients suffering on sepsis and their controls and profiled the blood proteome by DIA in order to find new candidates for diagnosis and prognosis. * Corresponding author: Dr. Dipl.-Ing Frank Schmidt EMA University Medicine Greifswald, Department of Functional Genomics, ZIK-FunGene Group Applied Proteomics, Friedrich-Ludwig-Jahnstr. 15a, D-17487 Greifswald, Germany, E-mail address: [email protected]



K-03

neXtProt as a tool to explore the human proteome

Lydie Lanea*

a) CALIPHO group, SIB Swiss Institute of Bioinformatics and University of Geneva, Switzerland; Around 20,300 protein-coding genes have been predicted from the analysis of the human genome, and more than half of them give rise to alternative splicing isoforms. These genes are expressed in a large dynamic range in the human body. During or after translation, numerous chemical modifications of the protein products occur, resulting in a huge range of proteoforms. One can estimate that one million proteoforms coexist in a single individual. This variability does not take into account the inter-individual variations due to frequent polymorphisms or rare mutations. The aim of neXtProt (www.nextprot.org) is to document the inter- and intra-individual diversity of human proteins at genomic, transcriptomic and proteomic levels, and to support applications specifically relevant to human proteins [1]. It has been chosen as the reference knowledgebase for the HUPO Human Protein Project [2]. neXtProt integrates a wide range of quality-filtered data converted into precise annotations with fully traceable data provenance. In the current neXtProt release there are 20,179 entries, encompassing 42,164 splicing isoforms, 5,317,610 single amino acid variations and 183,666 PTMs. In addition to a web interface, neXtProt provides an application programming interface that allows programmatic access to the data in the resource, and an advanced search capacity based on the SPARQL language, one of the key technologies of the semantic web. Using a series of examples, I will show how this language can be used to explore the complexity of the human proteome under various angles, using data from neXtProt and semantically compatible resources. * Corresponding author: Lydie Lane. CALIPHO group, SIB Swiss Institute of Bioinformatics and University of Geneva, Switzerland. Phone.: +41 22 379 58 41; E-mail address: [email protected] References: [1] Gaudet P, Michel PA, Zahn-Zabal M, Britan A, Cusin I, Domagalski M, Duek PD, Gateau A, Gleizes A, Hinard V, Rech de Laval V, Lin J, Nikitin F, Schaeffer M, Teixeira D, Lane L, Bairoch A. (2017) The neXtProt knowledgebase on human proteins: 2017 update. Nucleic Acids Res. 45(D1):D177-D182. [2] Omenn GS, Lane L, Lundberg EK, Beavis RC, Overall CM, Deutsch EW. (2016) Metrics for the Human Proteome Project 2016: Progress on Identifying and Characterizing the Human Proteome, Including Post-Translational Modifications. J Proteome Res. 15(11):3951-3960.

http://www.nextprot.org/

https://en.wikipedia.org/wiki/SPARQL

https://en.wikipedia.org/wiki/Semantic_web


https://www.ncbi.nlm.nih.gov/pubmed/27899619




K-04 Generation of large scale SWATH-MS proteomic datasets and their use

Ruedi Aebersolda

a Department of Biology, Institute of Molecular Systems Biology, ETH Zurich and Faculty of Science,

University of Zurich

The original draft of the human genome sequence in 2000 was a monumental achievement. However, genomics only became a mainstream technology when next generation, massively parallel (in space) sequencing made the analysis of large sample cohorts feasible. Similarly, we expect that the ability to perform protein measurements of large sample cohorts will make proteomics a mainstream life science technology. So far, the precise and reproducible quantification of the proteome in biological or clinical sample cohorts by conventional mass spectrometric methods has been technically challenging. In this presentation, we will discuss SWATH-MS, a mass spectrometry based, massively parallel (in time) protein quantification technique that has the capacity to accurately and reproducibly quantify thousands of proteins across samples. In SWATH-MS a biological sample is converted in a single sample injection into a digital file that can be perpetually searched in silico, using a targeted data analysis strategy, to detect and quantify proteins. We will discuss the principles of SWATH-MS and the computational challenges it poses. Using data from cross-lab benchmarking studies, we will demonstrate that SWATH-MS is presently the most extensively benchmarked large scale proteomic technique. Finally, we will illustrate the present performance of the method with selected applications.


K-05

Shedding new light on Spinal Cord injury

Michel Salzeta*

a Inserm U-1192, Laboratoire de Protéomique, Réponse Inflammatoire, Spectrométrie de Masse (PRISM), Université Lille 1, Cité Scientifique, 59655 Villeneuve D’Ascq, France

Spinal cord injury (SCI) belongs to currently incurable disorders of the CNS and is accompanied by permanent health consequences-disability. In order to mimic a SCI, a balloon-compressive technique was used at thoracic Th8-9 spinal level in adult rat. Shot-gun proteomic was used to identify proteins in each spinal cord segment-derived conditioned medium along the rostral-caudal axis after SCI with time course. 3D MALDI Imaging, tissue microproteomics were undertaken combined with confocal imaging [1,2]. In-vitro and in-vivo tests were realized. We established the spatial and temporal events occurring in acute phase after SCI. Caudal segment has clearly been detected as the therapeutic target1,2]. We then assessed in a rat SCI model the in vivo impact of a sustained RhoA inhibitor administered in situ via functionalized-alginate scaffold [3,4] . In order to decipher the underlying molecular mechanisms involved in such a process, an in vitro neuroproteomic-systems biology platform was developed in which the pan-proteomic profile of the dorsal root ganglia (DRG) cell line ND7/23 DRG was assessed in a large array of culture conditions using RhoAi and/or conditioned media obtained from SCI ex-vivo derived spinal cord slices. A fine mapping of the spatio-temporal molecular events of the RhoAi treatment in SCI was performed. The data obtained allow a better understanding of regeneration induced above and below the lesion site. Results notably showed a time-dependent alteration of the transcription factors profile along with the synthesis of growth cone-related factors (receptors, ligands, and signaling pathways) in RhoAi treated DRG cells. Furthermore, we demonstrate the inflammatory response process involvement via immunoglobulins by binding to their C16/CD32 receptors on the DRG cells upon neurite outgrowth initiation and thus modulating the neurite outgrowth process. We then validate our results by an in vivo proteomic studies along the spinal cord segments. Taken together, we shed new light on Spinal Cord injury. * Corresponding author: Michel Salzet Inserm U-1192, Laboratoire de Protéomique, Réponse Inflammatoire, Spectrométrie de Masse (PRISM), Université Lille 1, Cité Scientifique, 59655 Villeneuve D’Ascq, France Phone.: +33 3 2043 4194 Fax: +33 3 2043 4054 E-mail address: [email protected] References: [1] Cizkova, D., Le Marrec-Croq, F., Franck, J., Slovinska, L.,Grulova, I., Devaux, S., Lefebvre, C. Fournier, I. Salzet, M. Microglial response along the rostro-caudal axis after spinal cord injury: in vivo, in vitro and proteomic analyses. Frontiers in Cell. Neurosc. 2014,;8:105 [2] Devaux S., Cizkova D., Qunaico J., Franck J., Ntaf S., Pays L., Hauberg-Lotte L., Mass P., Hobart J.H., Kobeissy F., Merieux, C., Wisztorski M., Slovinska L., Blaska J., Cifankova V., Fournier I., Salzet M. Proteomic analysis of the spatio-temporal based molecular kinetics of acute spinal cord injury identifies a time- and segment-specific window for effective tissue repair Mol Cell Proteomics. (2016) 15(8):2641-70 [3] Cizkova, D., Devaux, S., Le Marrec-Croq, F.,Franck, J., Slovinska, L., Rosocha, J, Spakova, T., Lefebvre, C. Fournier, I. Salzet, M.Modulatory properties of mesenchymal stem cells on BV2 microglia after spinal cord injury secretome activation: Proteomic and physiological studies. Sci. Report. (2014) 4:7514. [4 ] Grulova I, Slovinska L, Blaško J, Devaux S, Wisztorski M, Salzet M, Fournier I, Kryukov O, Cohen S, Cizkova D. Delivery of Alginate Scaffold Releasing Two Trophic Factors for Spinal Cord Injury Repair.Sci Rep. (2015);5:13702



K-06

From protein biomarker discovery to the development of mass spectrometry based diagnostic tests of clinical utility

Stephen R Penningtona*

a School of Medicine, UCD Conway Institute, University College Dublin, Dublin, Ireland

Despite a few decades of proteomics research and the apparent discovery of multiple protein biomarkers to support the diagnosis and treatment of patients, the number of biomarkers that have been developed to the stage of being used routinely is disappointingly low. So, it's clear that whilst there’s huge interest in the development of new biomarkers and major drivers for this to happen, including the pharmaceutical industry’s need to better stratify patients for effective treatment, as yet biomarker development and delivery is proving very challenging. This presentation will cover: 1.The problem: For many diseases, developing biomarkers to support the decisions of which patients to treat, when to treat them and with what treatment, remains challenging. 2.Biomarkers of Utility: The use of proteomics for new protein biomarker discovery has not yielded many clinically used biomarker tests - why is this? 3.A strategy: A pragmatic, 'real world' and patient-centric approach for the discovery development and delivery of biomarkers of potential clinical utility will be introduced. 4.Solutions: The application of this strategy for the development of biomarker tests to support clinical decision in prostate cancer and psoriatic arthritis will be described. 5.Delivery: Potential strategies for the delivery and implementation of multiplexed protein biomarker tests will be explored. * Corresponding author: Stephen R Pennington School of Medicine, UCD Conway Institute, University College Dublin, Belfield, Dublin, D4, Ireland Phone: 00353 1 716 6783 E-mail address: [email protected] References: Leacy E, Finn S, Pennington SR. Need for biomarkers in active surveillance of prostate cancer. Oncology News. 2016. In Press. Includes ‘View from the Clinic – A Patient’s Perspective’ Tonry CL, Leacy E, Raso C, Finn SP, Armstrong J, Pennington SR. The Role of Proteomics in Biomarker Development for Improved Patient Diagnosis and Clinical Decision Making in Prostate Cancer. Diagnostics. 2016, 6(3), 27; doi:10.3390/diagnostics6030027. Percy AJ, Byrns S, Pennington SR, Holmes DT, Anderson NL, Agreste T, Duffy M. Clinical Translation of MS-based Quantitative Plasma Proteomics: Status, Challenges, Requirements, and Potential. Expert Rev Proteomics. doi: 10.1080/14789450.2016.1205950. Epub 2016 Jul 8. PubMed PMID: 27341553. McArdle A, Butt AQ, Szentpetery A, de Jager W, de Roock S, FitzGerald O, Pennington SR. Developing Clinically Relevant Biomarkers in Inflammatory Arthritis: A Multi-Platform Approach for Serum Candidate Protein Discovery. Proteomics Clin Appl. 2015 Sep 2. doi: 10.1002/prca.201500046. Hernández B, Pennington SR, Parnell AC. Bayesian methods for proteomic biomarker development. EuPA Open Proteomics. 2015 (9) 54-64. Mc Ardle A, Flatley B, Pennington SR, FitzGerald O. Early biomarkers of joint damage in rheumatoid and psoriatic arthritis. Arthritis Res Ther. 2015 Jun 1;17:141. Tonry CL, Doherty D, O'Shea C, Morrissey B, Staunton L, Flatley B, Shannon A, Armstrong J, Pennington SR. Discovery and Longitudinal Evaluation of Candidate Protein Biomarkers for Disease Recurrence in Prostate Cancer. J Proteome Res. 2015 Jul 2;14(7):2769-83. Ademowo OS, Hernandez B, Collins E, Rooney C, Fearon U, van Kuijk AW, Tak PP, Gerlag DM, FitzGerald O, Pennington SR. Discovery and confirmation of a protein biomarker panel with potential to predict response to biological therapy in psoriatic arthritis. Ann Rheum Dis. 2014 Sep 3. pii: annrheumdis-2014-205417. doi: 10.1136/annrheumdis-2014-205417 Hernández B, Parnell A, Pennington SR. Why have so few proteomic biomarkers "survived" validation? (Sample size and independent validation considerations). Proteomics. 2014 Jul;14(13-14):1587-92.



K-07

Neuronal Interfaces: Innovative Therapies and Neuroprosthetics

Fabio Benfenati a*

a) Center for Synaptic Neuroscience and Technology, Fondazione Istituto Italiano di Tecnologia, Genova, Italy

The brain is characterized by highly complex organization, parallel computation, integration of afferent information, emergent properties and functional/structural adaptation. Recent research in the field of neuroscience have stimulated the creation of biomimetic hybrid devices, in which neurons are interfaced with electronic chips, organic electronics or have been genetically modified to generate opto-neural interfaces. By creating these interfaces, one can monitor and change the altered neuronal activity in experimental models of brain diseases and create hybrid devices capable of regulating the excitability and plasticity of neural networks. We recently engineered an optogenetic probe capable, upon illumination, to stimulate transcription in pathological neurons in which the expression of neuronal genes is depressed. Another interesting field is the direct reprogramming of neurons from skin cells and their subsequent grafing to compensate for the neuronal degeneration that accompanies many diseases of the nervous system. We were able to reprogram dopaminergic and GABAergic neurons and studied their incorporation in the host neuronal networks after transplantation in experimental models of Parkinson's disease and epilepsy. Finally, we successfully interfaced organic electronics with nerve tissue. Using photovoltaic polymers, we were able to stimulate or inhibit the activity of neurons with light, mimicking the process that occurs in retinal phototransduction. We have also shown that this bioorganic interface restores light sensitivity in the retina affected by photoreceptor degeneration, suggesting an important future in the field of retinal prostheses. Optogenetics, cellular reprogramming and hybrid interfaces therefore represent concrete and promising therapeutic approaches to diseases of the nervous system. * Corresponding author: Fabio Benfenati E-mail address: [email protected]



K-08

Ex vivo screening of individual human gut microbiomes

Daniel Figeysa*

a) University of Ottawa, Ottawa, Canada; Host–microbiota interactions have been associated to a growing list of diseases, including inflammatory bowel diseases (IBD), obesity, diabetes, cardiovascular diseases. We are interested in studying the molecular changes that occurs in host and microbiota during gut dysbiosis and to develop screening techniques for microbiome. Our understanding of the microbiome has been primarily driven by Next-generation sequencing (NGS). Particularly, large-scale cohort studies through either 16S rRNA gene or shotgun metagenomic sequencing have provided fine taxonomic characterizations of the human microbiome. While NGS-based techniques have been widely applied, the application of metaproteomics, which measures the expressed proteins in the microbiome, is far less common. Metaproteomics was limited by the insufficient sensitivity and resolution of mass spectrometers to measure low abundant proteins from complex microbial communities such as intestinal microbiota. In addition, the lack of easy-to-use metaproteomic computational platforms that can handle the huge reference protein database also limited the application of metaproteomics. Here we will report on the development of a metaproteomics protein identification platform called MetaPro-IQ[1] (universal workflow for gut MetaProteome Identification and Quantification). Eventhough omic approaches can be used to study dysbiosis, our abiltiy to intervene to correct the dysbiosis is limited. This is primarily due the lack of tools for screening compounds against microbiome. Currently, compounds are either screened against individual bacteria or using animal models. Instead, here we report on the development of a rapid assay for individual’s microbiome (called RapidAIM). RapidAIM is an ex vivo assays to study the effects of drugs on the human gut microbiome. In this assay, series of compounds can be screen against a panel of individual microbiomes allowing the characterisation of the microbiome responses and the pathways activated by the compounds. Overall, these metaproteomics technologies make it possible to measure changes in individual microbiome during dysbiosis and to screen compounds against individual microbiomes. * Corresponding author: Daniel Figeys University of Ottawa, Department of Biochemistry, Microbiology, and immunology, Ottawa, Canada. Phone: +1 562 5800 x 8674; E-mail address: [email protected] (D. Figeys) References: [1] Zhang X, Ning Z, Mayne J, Moore JI, Li J, Butcher J, Deeke SA, Chen R, Chiang CK, Wen M, Mack D, Stintzi A, Figeys D., Microbiome.

2016 Jun 24;4(1):31. doi: 10.1186/s40168-016-0176-z. [2] Zhang X, Ning Z, Mayne J, Deeke SA, Li J, Starr AE, Chen R, Singleton R, Butcher J, Mack DR, Stintzi A, Figeys D., Anal Chem. 2016

Jun 21;88(12):6120-5. doi: 10.1021/acs.analchem.6b01412.



K-09

Role of the microbiome in food allergy and asthma

Liam O’Mahonya*

a) Swiss Institute of Allergy and Asthma, University of Zürich, Davos, Switzerland; The mucosal immune system is intimately connected with the vast diversity of microbes present within the gut and on mucosal surfaces. The discovery of novel molecular mechanisms, which mediate host-microbe-nutrient communication, have highlighted the important roles played by microbes and dietary factors in influencing mucosal immune responses. Dendritic cells, ILCs, epithelial cells, T regulatory cells, effector lymphocytes, NKT cells and B cells can all be influenced by the microbiome. Many of the mechanisms being described are bacterial strain or metabolite-specific. The balance between immune tolerance and inflammation is regulated in part by the crosstalk between innate and adaptive immune cells and the microbiota. Many human studies now clearly provide strong connections between the composition and metabolic activity of the bacterial microbiota and the development of allergic disease. In murine studies, germ-free mice display an exaggerated anaphylactic response to challenge with a food allergen, while transfer of the microbiota from food allergy-prone mice (with a gain-of-function mutation in the IL-4 receptor-α chain) to wild type germ-free animals transfers the food allergy phenotype. The deliberate administration of specific bacterial strains, such as Bifidobacteria or Clostridia, to mice can protect against allergen sensitization due to the induction of Tregs within the mucosa, while certain Bifidobacteria strains have also been shown to induce Tregs in humans. Dynamic interactions between a wide range of host immune cells and the microbiota determine whether allergy or tolerance develops. However, significant gaps in our knowledge on the natural induction of tolerance have hampered the development of microbial-based immunoregulatory protocols that fully replicate this process, for both the prevention and treatment of allergic disorders. Significant research is still required to fully appreciate and understand the complexities of tolerance development to the microbiota and its associated importance for tolerance induction to potential allergens. * Corresponding author: Liam O’Mahony Swiss Institute of Allergy and Asthma, University of Zürich, Davos, Switzerland E-mail address: [email protected]



K-10

Micromanagement of the metaproteome: taking control of your data

Lennart Martensa,b *, Thilo Muthc, Tim Van Den Bosschea,b, Bart Mesuerea,b,d, Peter Dawyndtd

a) VIB-UGent Center for Medical Biotechnology, VIB, Ghent, Belgium

b) Department of Biochemistry, Ghent University, Ghent, Belgium c) Robert Koch Institute, Berlin, Germany

d) Department of Applied mathematics, computer science and statistics, Ghent University, Ghent, Belgium

A key issue in metaproteomics is the identification of acquired fragmentation mass spectra [1]. Indeed, identification rates in metaproteomics analyses are small compared to single species proteomics experiments. This is caused by the increased search space in multi-species searchers, and the corresponding growth in decoy sequence diversity [2]. While this also puts a burden on memory requirements and processing speed of search algorithms, these issues are technical in nature, and will be quite easy to resolve [3]. Several approaches are typically used to reduce the search space in metaproteomics identifications, most notably two-pass search strategies, an dthe wholesale removal of organisms from a compound database. Unfortunately, the former tends to silently increase the peptide false discovery rate to unacceptable levels [4], while the latter can cause good spectra to match with a sufficiently high score against incorrect peptides [5]. We are therefore investigating a completely novel way of reducing the database by eliminating a priori those peptides that are unlikely to yield identifiable spectra. Built on our CP-DT tool for tryptic peptide cleavage prediction [6], we have so far been able to show that this intelligent database size reduction strategy indeed looks very promising for the large databases encountered in metaproteomics and proteogenomics studies. Moreover, we have also recently shown the promise of alternative search engine strategies in the specific context of metaproteomics searches [7]. Overall, it is clear that the field of metaproteomics is urgently in need of novel, customized bioinformatics solutions for its identification issues, and we are hard at work to develop and deploy several innovative ways to address these issues. Moreover, we will endeavour to implement these innovations in a user-friendly way through the continued evolution of our MetaproteomeAnalyzer software, which is the first integrated solution for metaproteomics data processing, management and interpretation [8]. * Corresponding author: Lennart Martens. Ghent University and VIB, Albert Baertsoenkaai 3, Gent, Belgium. Phone: +32 264 93 58; E-mail address: [email protected] (L. Martens) References: [1] Muth T et al. Expert Rev Proteomics (2016) 13: pp. 757-769. [2] Colaert N et al. J. Proteome Res. (2011) 10: pp. 5555-5561. [3] Chatterjee S et al. BMC Genomics (2016) 17: p. 642. [4] Muth T et al. Proteomics (2015) 15: pp. 3439-3453. [5] Sticker A et al. Nature Methods (2017) in press. [6] Fannes T et al. J. Proteome Res. (2013) 12: pp. 2253-2259. [7] Peters JS et al. Front Microbiol (2016) 7: p. 813. [8] Muth T et al. J. Proteome Res. (2015) 14: pp. 1557-1565.



DISTINGUISHED SPEAKERS

ORAL COMMUNICATIONS: Distinguished Lectures 29

D-01

Integrated proteomic technologies for the characterization of milk products

Andrea Scalonia* a Proteomics & Mass Spectrometry Laboratory, ISPAAM-CNR, Naples, Italy

Farm animal milk and its products (e.g. cheese, yoghurt and other transformed foods) are an important part of the human diet, with beneficial effects for all ages. Milk is a colloidal dispersion of fat globules within a water-based fluid mainly containing dissolved carbohydrates and protein aggregates with minerals, vitamins and other nutrients. In this context, proteins are key functional milk molecules that, depending on their nature, act as a source of amino acids, nutrient carriers, fat micelle stabilizers, cell proliferation and organ development stimulators, innate/acquired immune defense components or enzymes reflecting mammary gland cell secretory pathways [1,2]. Furthermore, bioactive polypeptides encrypted within primary structure of milk proteins occur naturally in dairy products and/or are released following gastrointestinal digestion. Milk proteomic description may represent a major challenge in analytical terms based on single protein relative abundance, solubility in water/fat globules, susceptibility to hydrolytic processing, post-translational modifications and non-enzymatic chemical adductions resulting from heating- and fermentation-associated technological treatments; thus integrated separation, trapping and mass spectrometric procedures have often been used for a comprehensive characterization of dairy products. This presentation provides an updated picture of the dedicated bottom-up methods developed so far for the comparative proteomic characterization of variably-heated (native, pasteurized, UHT and sterilized for infant nutrition) milk products [3-6]. These studies provided the largest inventory of non-enzymatic glycation modifications occurring in milk products and of corresponding protein targets; the first ones mostly derived from a network of concomitant heating-dependent chemical processes also known as the Maillard reaction [7,8]. Possible effects on above-mentioned milk protein nutritional and functional properties are discussed. Dedicated proteomic procedures were then set up for the rapid assessment of milk quality that, together with those developed for raw material speciation and freezing detection, represent an important tool for evaluating the occurrence of dairy product adulterations [9,10]. * Corresponding author: Andrea Scaloni ISPAAM-National Research Council, via Argine 1085, 80147 Naples, Italy. Phone.: +39 081 5966006; Fax: +39 081 5965291. E-mail address: [email protected] References: [1] D'Alessandro et al. Human milk proteins: an interactomics and updated functional overview. J Proteome Res. 2010;9:3339-3373. [2] D'Alessandro et al. The bovine milk proteome: cherishing, nourishing and fostering molecular complexity: an interactomics and

functional overview. Mol Biosyst 2011;7:579-597. [3] Scaloni et al. Characterization of heat-induced lactosylation products in caseins by immunoenzymatic and mass spectrometric

methodologies. Biochim Biophys Acta 2002;1598:30-39. [4] Arena et al. Modern proteomic methodologies for the characterization of lactosylation protein targets in milk. Proteomics 2010;10:3414-

3434. [5] Arena et al. Redox proteomics of fat globules unveils broad protein lactosylation and compositional changes in milk samples subjected

to various technological procedures. J Proteomics 2011;74:2453-2475. [6] Renzone et al. Proteomic characterization of intermediate and advanced glycation end-products in commercial milk samples. J

Proteomics 2015;117:12-23. [7] Siciliano et al. Mass spectrometry for the analysis of protein lactosylation in milk products. Food Res Int 2013;54:988-1000. [8] Arena et al. Dairy products and the Maillard reaction: a promising future for extensive food characterization by integrated proteomics

studies. Food Chem 2017;219:477-489. [9] Sassi et al. MALDI-TOF-MS platform for integrated proteomic and peptidomic profiling of milk samples allows rapid detection of food

adulterations. J Agric Food Chem 2015;63:6157-6171. [10] Arena et al. Identification of protein markers for the occurrence of defrosted material in milk through a MALDI-TOF-MS profiling

approach. J Proteomics 2016;147:56-65.



D-02

Mapping Human Mitochondrial Protein Interactions Linked to Neurodegeneration

Ramy Malty1, Hiroyuki Aoki1, and Mohan Babu1*

1Department of Biochemistry, University of Regina, 3737 Wascana Parkway, Regina, Canada

Mitochondrial protein (MP) dysfunction has been linked to neurodegenerative disorders (NDs), however, the discovery of molecular mechanisms underlying NDs has been impeded by the limited characterization of interactions governing MP function. Using affinity-purified mitochondrial fractions isolated from 27 epitope-tagged human ND-linked MPs in HEK293 cells coupled with mass spectrometry (MS), we report a high-confidence MP network involving 1,971 interactions among 774 proteins (>90% previously unreported). Nearly half of these interactions were confirmed in differentiated neuronal cells by primary antibody immunoprecipitation and MS, with many linked to NDs. I will also discuss, how by taking this systematic, integrated look at inter- and extra-mitochondrial protein function, we were able to establish a new functional role for ND-linked factors coupled with cell signaling in Parkinson Disease patients. Our study identify new mechanisms for MPs with direct consequences for human disease, and expands the interaction landscape of human mitochondria * Corresponding author: Mohan Babu Department of Biochemistry, University of Regina, 3737 Wascana Parkway, Regina, Canada E-mail address: [email protected]



D-03

Clinical MALDI MS Imaging: From Bench to Surgery room Marie Duhamela, Philippe Saudemonta, Benoit Fatoua,b, Emilie Le Rhuna, Michael Ziskindb,

Michel Salzeta, Isabelle Fourniera*

a Université de Lille, Laboratoire Protéomique, Répnse Inflammatoire et Spectrométrie de Masse (PRISM), Inserm U1192, Villeneuve d’Ascq, France

b Université de Lille Physique des Lasers Atomes et Molécules (PhLAM), CNRS UMR 8523,Villeneuve d’Ascq, France

More than 15 years after its introduction, Mass Spectrometry Imaging (MSI) has now gained, its letters of mobility demonstrating to be a robust technology for studying the distribution of endogenous and exogenous biomolecules. It has opened a new dimension by allowing the correlation between molecules identification to their distribution and their function. Within clinics MSI reseals an important potential for the understanding of physiopathological processes. In particular coupling MSI with Spatially-resolved Large Scale proteomics allow to better understand the changes associated to a specific local microenvironment. For example such strategies can be evaluated for studying the classification of glioma brain tumors using fully unsupervised classification approach. For Glioma Grade III, based on such strategy we identified 3 groups of patients sharing different molecular patterns. We then evaluated the concordance with the WHO 2016 classification [1]. For Traumatic Brain Injury (TBI) Spatiotemporal MSI studies combined to microproteomics allow to unreal new interesting features of the pathology. If MSI becomes a real bench tool for clinics there is a growing interest to be able to move forward analyzing in the real-time in-vivo context as make MS become a tool for guided surgery. To tackle this challenge a new instrument based on Remote Laser Ablation Microsampling coupled to MS analysis was developed [2]. This system permits in-vivo analysis with minimal invasiveness and painless and the discrimination of cancer tissues and cancer typing as demonstrated on dogs sarcoma. * Corresponding author: Isabelle Fournier PRISM Inserm U 1192, University of Lille, Villeneuve d’Ascq, France Phone.: +33 3 20 43 41 94; Fax: +33 3 20 43 40 54. E-mail address: [email protected] References: [1] Le Rhun E, Duhamel M, Wisztorski M, Gimeno JP, Zairi F, Escande F, Reyns N, Kobeissy F, Maurage CA, Salzet M, Fournier I.; Biochim

Biophys Acta. 2016 Nov 24, Evaluation of non-supervised MALDI mass spectrometry imaging combined with microproteomics for glioma grade III classification.

[2] Fatou B, Saudemont P, Leblanc E, Vinatier D, Mesdag V, Wisztorski M, Focsa C, Salzet M, Ziskind M, Fournier I.; Sci Rep. 2016 May 18;6:25919; In vivo Real-Time Mass Spectrometry for Guided Surgery Application.



D-04

Proteomic approaches to genetic and oncological diseases

Flora Cozzolinoa, Diana Canettia, Flavia Mocciaa, Maria Montia, Piero Puccia*

a Department of Chemical Sciences and CEINGE Biotecnologie Avanzate, University Federico II of Napoli, Napoli, Italy

A genetic disorder is caused by one or more abnormalities in the genome, especially a condition that is present from birth (congenital). These disorders may be either hereditary, passed down from the parents' genes, or caused by new DNA mutations. Pure genetic diseases are caused by single point mutations occurring in a single gene and, though relatively rare, they affect millions of people worldwide. Nowadays, several single point mutations have been associated to specific pathologies making the diagnosis of genetic diseases relatively straightforward. However, the molecular mechanisms originating the disease remain very often elusive preventing the development of effective therapeutic treatments. A mutated gene gives rise to a variant protein carrying an amino acid substitution that affects its biological activity. It is then the aberrant protein that prevents the cell from functioning properly generating the pathological state. Protein defects can cause diseases in a variety of ways, through "loss-of-function", when the mutation prevents the protein from functioning or makes the protein unstable leading to its degradation, or "gain-of-function" when the protein takes on new functions harmful to the cell, by interfering with cell functions or being no longer controlled by its normal regulatory partners. A further mechanism for causing disease is associated with defects in protein traffic that prevent a functional protein to reach its normal localization within the cell. This lecture will focus on the application of proteomic strategies to the elucidation of alteredmolecular mechanisms occurring in two genetic diseases, the Wilson Syndrome and the Pompe Disease. * Corresponding author: Prof. Piero Pucci Department of Chemical Sciences and CEINGE Biotecnologie Avanzate University Federico II of Napoli Via Cinthia 6, 80126 Napoli, Italy. Phone.: +39 081-674318; 3737896; E-mail address: [email protected]



D-05

The central role of microbiota: from environment to food production

Paola Roncadaa *, Alessio Soggiub, Cristian Pirasb, Luigi Bonizzib

a) Istituto Sperimentale Italiano L. Spallanzani, Milano, Italy b) Dipartimento di Medicina Veterinaria, Università degli Studi di Milano, Milano, Italy

A better understanding of the links between biodiversity, health and disease presents major opportunities for policy development, and can enhance our understanding of how health-focused measures affect biodiversity, and conservation measures affect health. Loss of biodiversity, habitat fragmentation and the loss of natural environments threaten the full range of life-supporting services provided by ecosystems at all levels of biodiversity, including species, genetic and ecosystem diversity. The disruption of ecosystem services has direct and indirect implications for public health, which are likely to exacerbate existing health inequities, whether through exposure to environmental hazards or through the loss of livelihoods. Moreover, the global composition of diet, from microbiome to nutrient, including life style, can affect every step from gene expression to protein synthesis until degradation, leading to profound modulation of metabolic functions. Proteomics can help the development of a novel sustainable personalized medicine through nutritional intervention. These studies will play an important role in solving major nutrition problem in human and animals, on the verge of one health approach,(www.onehealthinitiative.com) including obesity, metabolic and cardiovascular disease, cancer, ageing, allergy and foetal health and development. Profiling food, microbiome, and biomarkers of nutritional status from a proteomics point of view will potentially lead to a new pillar of personalized medicine. This include also a special focus to food safety, security and quality issues, providing new insights and technologies to ensure safety, starting from the study of microbiome and functional composition of microbial consortia. The definition of microbiota in animal species is useful also to increase food safety, and to counteract antibiotic resistance. * Corresponding author: Paola Roncada. Istituto Sperimentale Italiano L. Spallanzani, Milano, Italy Phone: +39 0250318138; E-mail address: [email protected] [1] Roncada P, One medicine--one health--one biology and many proteins: proteomics on the verge of the One Health approach. Mol Biosyst. 2014 Jun;10(6):1226-7. doi: 10.1039/c4mb90011a



D-06

Microbiota from childhood to adulthood in health and disease

Lorenza Putignani* *Ospedale Pediatrico e Centro di Ricerche Bambino Gesù, Piazza Sant’Onofrio 4, 00165 Rome, Italy The microbiota "organ" is the central bioreactor of the gastrointestinal tract, populated by a total of 1014 bacteria and characterized by a genomic content (microbiome), which represents more than 100 times the human genome. The microbiota plays an important role in child health by acting as a barrier against pathogens and their invasion with a highly dynamic modality, exerting metabolic multistep functions and stimulating the development of the host immune system, through well-organized programming, which influences all of the growth and aging processes. The advent of "omics" technologies (genomics, proteomics, metabolomics), characterized by complex technological platforms and advanced analytical and computational procedures, has opened new avenues to the knowledge of the gut microbiota ecosystem, clarifying some aspects on the establishment of microbial communities that constitute it, their modulation and active interaction with external stimuli as well as food, within the host genetic variability. With a huge interdisciplinary effort and an interface work between basic, translational, and clinical research, microbiologists, specialists in "-omics" disciplines, and clinicians are now clarifying the role of the microbiota in the programming process of several gut-related diseases, from the physiological symbiosis to the microbial dysbiosis stage, through an integrated systems biology approach, to generate the genotype-phenotype "trajectories" under physiological and disease constraints. * Corresponding author: Lorenza Putignani UOS of Parasitology, UOC Microbiology, Parasitology, Virology; UdR Human Microbime, Genetic and Rare Diseases, Ospedale Pediatrico e Centro di Ricerche Bambino Gesù, Piazza Sant’Onofrio 4, 00165 Rome, Italy E-mail address: [email protected] [email protected]




D-07

Building 3D human tissues in vitro for tissue- and organ on chip applications

Paolo A. Nettia,b,*

a) Center for Advanced Biomaterials for HealthCare@CRIB Istituto Italiano di Tecnologia, Napoli,

Italy b) Department of Chemical Materials and Industrial Production, University of Naples Federico II,

Napoli, Italy Tissue and organ on chip (TOC) have been developed to permit the study of human physiology in a tissue-specific context, to enable development of novel in vitro disease models, and to potentially serve as replacements of animals in drug development and toxics testing. For the TOC technology to meet the expectation to replace in part the animal model, it is mandatory to proceed towards the use of tissue and organs that correctly reproduce in composition and organization the extracellular space. Indeed, while sophisticated microdevices have been designed, the engineered tissues still remain surrogates of the native counterparts. On this basis, we established a bottom-up tissue engineering strategy to build-up functional tissue in vitro. The basic idea driving our strategy is based on the concept that ECM is not a merely “passive” matrix holding cells and tissues in place, but it has a functional importance as dynamic repository for morphogens, cytokines and growth factors, which in vivo regulate diverse cellular processes [1]. Starting from this awareness we produced 3D tissue equivalent in which cells are embedded in their own ECM. We succeeded in obtaining a library of tissues and organs histologically and functionally competent and cultured them into opportunely designed microdevices under continuous flow and mechanical forces, thereby recreating key factors known to influence cell and tissues functions in vivo. Due to their somewhat unique properties such TOCs well mimic the organ-specific context, representing a biological environment that is much more effective at predicting human response than today’s cell cultures or animal testing. * Corresponding author: Paolo Netti. Center for Advanced Biomaterials for HealthCare@CRIB Istituto Italiano di Tecnologia, Largo Barsanti e Matteucci 53, 80125 Napoli, Italy Phone: +39 08119933100; Fax: +39 08119933140. E-mail address: [email protected] (P. Netti) References: [1] Endogenous human skin equivalent promotes in vitro morphogenesis of follicle-like structures. Biomaterials 101 (2016) 86-95



D-08

Implantable and wearable technologies for lifescience at the Center for Biomolecular Nanotechnologies

Massimo De Vittorioa,b *

a) Center for Biomolecular Nanotechnologies, Istituto Italiano di Tecnologia, Arnesano (LE) - Italy

b) Dipartimento di Ingegneria dell’Innovazione, Università del Salento, Lecce - Italy

Micro and nanotechnologies and functional materials are being more and more applied to lifescience and medicine by virtue of their ability to produce compact, efficient and fast tools for diagnostics and therapeutics. The combination of genetics, photonics, electronics and micromechanics is producing completely new technological approaches for physical and chemical biosensors and bioactuators which can be disposable, wearable, implantable or tattooable. These new approaches are opening the way to closed loop theranostics, i.e. device integrating diagnostic capabilities and therapeutic response. The integration of on-board intelligence in these device and their communication with portable smart electronics is enabling real time monitoring of health, wellness and sport performance, relating physiological and biochemical parameters to nutrition and lifestyle. In this context, the center for biomolecular nanotecnologies (CBN) is exploiting nanophotonic, nanoelectronic and micromechanical technological approaches to produce innovative in-vivo implantable/wearable devices and in-vitro assays. Recent results on implantable optrodes for optogenetics, on piezoelectric microelectromechanical systems (MEMS) for wearable sensing and for energy harvesting from the human body will be presented and discussed. * Corresponding author: Massimo De Vittorio. E-mail address: [email protected]



D-09

A composite scaffold for bone-cartilage defects bridging: an in vivo study Alessandro Sanninoa, Francesca Gervasoa*, Francesca Scaleraa, Antonio Crovaceb, Giuseppe

Perettic, d

a) Department of Engineering for Innovation, Università del Salento, Lecce, Italy;

b) Department of Emergency and Organ transplantation, University of Bari "Aldo Moro", Bari, Italy; c) Laboratory of Tissue Engineering and Biomaterials, IRCCS Istituto Ortopedico Galeazzi, Milan,

Italy; d) Department of Biomedical Sciences for Health, University of Milan, Milan, Italy.

Several scaffolds have been developed for osteochondral lesions, which usually repair forming fibrocartilage, with poor biomechanical properties. The aim of this new project is to develop and in vivo test a novel configuration of a cell-free biphasic scaffold for osteochondral lesions [1]. A critical osteochondral defect was generated in the medial femoral condyle of 36 skeletally mature sheep. Six defects were left untreated (CNTRL), thirty lesions were divided into three groups: ten lesions treated with a biphasic scaffold made of collagen type I and small cylinders of Magnesium hydroxyapatite embedded in collagen type I (HMG); ten lesions treated with a biphasic substituted formed by collagen type I and Wallastonite (BWS); ten lesions treated with a scaffold made of collagen type I and small cylinders of Wallastonite embedded in collagen type I (HWS). The animals were sacrificed after 3 and 6 months and the samples were analyzed by CT and MRI, macroscopic and histological evaluation. At 3 months, the ICRS macroscopic assessment showed a significant difference between HMG and CTRL. The CT and MRI scans displayed a reparative tissue especially in the HMG group. Histological evaluation at 3 months displayed a cartilaginous tissue in the HMG group. The significant difference between HMG and CTRL was only partially confirmed after 6 months by CT, MRI and histology. In conclusion, our study demonstrated that these novel biphasic scaffolds can be applied for one-stage procedures for osteochondral defects. Moreover, we proved the superior reparative potential of the HMG scaffold at the earlier time point.

*Corresponding author: Francesca Gervaso, PhD Università del Salento, Via Monteroni, Lecce, Italy. Phone: +39 0832 297238; Fax: +39 0832 297240. E-mail address: [email protected] (F.Gervaso) [1] Sosio, C., A. Di Giancamillo, D. Deponti, F. Gervaso, F. Scalera, M. Melato, M. Campagnol, F. Boschetti, A. Nonis, C. Domeneghini, A. Sannino and G. M. Peretti Tissue Eng Part A 2015; 21: 704-715



SELECTED ORAL COMMUNICATIONS

ORAL COMMUNICATIONS: Session I non-human proteomics 39

SESSION I

NON-HUMAN PROTEOMICS

40 XII ItPA National Congress Lecce, June 12th-15th, 2017 O-I-01

Proteomic analysis of the mode of action of the disinfectants based on pyridoxal oxime derivatives against food borne pathogens

Martina Šrajer Gajdošik1, Uroš Andjelković2, Dajana Gašo Sokač3, Hrvoje Pavlović3, Tamara

Martinovicć2, Djuro Josic2,4*

1 Department of Chemistry, J. J. Strossmayer University, HR-31000 Osijek, Croatia 2 Department of Biotechnology, University of Rijeka, HR-51000 Rijeka, Croatia

3 Faculty of Food Technology, J. J. Strssmayer University, HR-31000 Osijek, Croatia 4 Department of Medicine, Warren Alpert Medical School, Brown University, Providence, RI, USA

Comprehensive proteomic data and changes in proteomes of food borne pathogens after treatment with the disinfectants based on ammonium salts of pyridinium oxime were investigated. The Gram-positive bacterium Bacillus subtilis and the Gram-negative one, Escherichia coli were evaluated. Up and down-regulated proteins in these bacteria after growth under the inhibition with four different disinfectants based on chloride and bromide salts of pyridinium oxime were identified and their cellular localizations and functions were determined by gene ontology searching. Proteome changes presented here demonstrate different mechanism of action of these disinfectants. In Gram-positive food pathogen Bacillus subtilis, the inhibitory substances seem to act mainly at the cell surface and caused most changes in the integral membrane components. It results in significant alternations in membrane and cell surface proteins, while in the Gram-negative pathogen Escherichia coli intracellular proteins were more affected. These investigations are a contribution for investigation of the virulence and pathogenicity of food borne bacteria and their survival under stress conditions, and can also lead the way for further development of new inhibitors of microbial growth and studies of mechanism of their actions. *Corresponding Author: Djuro Josić Department of Biotechnology, University of Rijeka, HR-51000 Rijeka, Croatia, Department of Medicine, Warren Alpert Medical School, Brown University, Providence, RI, USA E-mail address: [email protected]


ORAL COMMUNICATIONS: Session I non-human proteomics 41 O-I-02

Peptidomic analysis of bioactive ovine milk caseinate hydrolysates Adriano Brandelli a, Roberta Fontoura a, Ana Paula Folmer Corrêa a, Ana Carolina Ritter a,

Lucélia Santi b, Walter Orlando Beys da Silva b, John Yates III b

a Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

b The Scripps Research Institute, La Jolla, CA, USA

Bioactive peptides have been defined as specific protein fragments that have a positive impact on body functions or conditions and may ultimately influence health. Upon oral administration, bioactive peptides, may affect the major body systems - namely, the cardiovascular, digestive, immune and nervous systems, depending on their amino acid sequence. The objective of this study was to obtain ovine caseinate hydrolysates with a novel microbial protease derived from Chryseobacterium sp. kr6 for evaluation of their biological activities and identification of their peptide composition. The hydrolysates were evaluated for antimicrobial, angiotensin I-converting enzyme (ACE)-inhibitory and dipeptidyl peptidase IV (DPP-IV)-inhibitory activities. Antibacterial activity was observed for ovine caseinate hydrolyzed for 1 and 2 h. ACE-inhibitory activity was increased for up to 6 h of hydrolysis (86.6% of inhibition), although 60% inhibition was already observed after 30 min of hydrolysis. DPP-IV-inhibitory activity was maximum with 1 h hydrolysates (>90%), and thereafter decrease to reach no inhibition after 6 h. Hydrolysates were analyzed using a LTQ Orbitrap XL mass spectrometer and peptide identification and quantification was carried out in an Integrated Proteomics Pipeline IP2. Peptidomic analysis of the hydrolysates revealed that most abundant peptides were derived from αS1-casein and β-casein, whereas relative abundance of αS2-casein derived peptides increased at 6 h hydrolysis. The peptides RPKHPIKHQGLSPEVLNENLL, RFVVAPFPEVFR and RFVVAPFPEVF from αS1-casein were the most abundant at 30 min, 1 h and 6 h hydrolysis, respectively. The β-casein-derived peptide LYQEPVLGPVRGPFPILV was one of the most abundant at 1 h hydrolysis. *Corresponding Author: Adriano Brandelli Universidade Federal do Rio Grande do Sul Av. Bento Gonçalves 9500 – ICTA, 91501-970 Porto Alegre, Brazil Phone/Fax: +5551 3308 6249 / +5551 3308 7048 E-mail address: [email protected]


42 XII ItPA National Congress Lecce, June 12th-15th, 2017 O-I-03

BacteriaMS: an open source tool for bacterial whole cell typing by mass

spectra pattern matching with in silico hypothesis validation

Zhuoxin Chen a,#* Yi Yang a,#, Tianqi Gong a, Yu Lin b, Yingdi Zhu c, Hubert H. Girault c, Baohong Liu a,d,, Liang Qiao a,d,*, and Pengyuan Yang a,*

a Department of Chemistry and Institute of Biomedical Sciences, Fudan University, Shanghai,China

b Research School of Computer Science, The Australian National University, Canberra, Australia c Laboratoire d’Electrochimie Physique et Analytique, Ecole Polytechnique Federale de Lausanne,

Sion, Switzerland d Shanghai Stomatological Hospital, Fudan University, Shanghai, China

Bacterial cell typing is of great importance in clinical diagnosis, environmental monitoring, food safety analysis and bioanalytical research. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is now widely used to analyze bacterial samples. However, the corresponding algorithms for spectra analysis are closed source commercial software, considering only the similarity of sample spectra to a spectra bank and suffering from accurate identification at the strain and species level for samples with low cell copies. Herein, we have developed a new open source algorithm, BacteriaMS, for MALDI-MS based bacteria typing. A distinctive feature of the algorithm is that in silico fake mass spectra and in silico sample mass spectra are generated via a random spectra model (RSM) and a similar spectra model (SSM), respectively, for hypothesis validation. With RSM, the species or genus of a bacterial sample can be predicted. With SSM, the inherent drawback of MALDI-MS in reproducibility can be overcome and identification at the strain level is guaranteed. The new algorithm is superior for identification of bacterial samples with low cell copies, and can provide reliable hierarchical clustering based-bacteria taxonomy. Source code of the algorithm is freely available at https://github.com/lmsac/BacteriaMS. The algorithm has also been developed as a web-based application (http://bacteriams.fudan.edu.cn) that is free for public usage with a spectra bank of more than 400 strains. *Corresponding author: Zhuoxin Chen Department of Chemistry and Institute of Biomedical Sciences, Fudan University, Handan Road 220, Shanghai 200433, China

ORAL COMMUNICATIONS: Session I non-human proteomics 43 O-I-04

Proteomics to improve serodiagnosis of Brucellosis

Paola Roncadaa*, Cristian Pirasb, Isabella Alloggiob, Viviana Grecoc, Tiziana Di Febod, Ivanka Krastevad, Andrea Urbanic,e, Giuliano Garofolod, Manuela Tittarellid, Luigi Bonizzib

Alessio Soggiub

a) Istituto Sperimentale Italiano Lazzaro Spallanzani, Milano, Italy b) Dipartimento di Medicina Veterinaria, Università degli Studi di Milano, Milano, Italy

c) Fondazione Santa Lucia, Roma, Italy d) Istituto Zooprofilattico Sperimentale dell' Abruzzo e del Molise "G. Caporale", Teramo, Italy

e) Università Cattolica del Sacro Cuore, Roma, Italy

Brucellosis still causes a public health impact and economic losses in animal husbandry in Mediterranean area also if it is eradicated in most of developed Countries. The design of adequate strategies for preventing animal brucellosis and consequently human disease requires in-depth knowledge of different strains that are circulating. Moreover, the occurrence of false positive reactions in the serological tests available currently reduces their specificity [1]. Furthermore, the mass vaccination policy using Rev.1 vaccine, in many endemic areas of the Mediterranean with the aim of controlling/reducing disease prevalence in small ruminants, interfere with serological diagnosis of the disease. Since the 'perfect antigen' has not been developed yet, it is useful to make comparative proteomics characterization of of Brucella spp to set to obtained proteins that could permit to develop rapid diagnostic test. We analyze three strains of Brucella melitensis coming from library of National Reference Laboratory of Brucellosis (IZSAM- Teramo ITALY) in three technical replicates for each strain used a combined methods of bead beating and solubilization [2]. After 2D electrophoresis, image analysis was performed with Progenesis same spots and proteins were analyzed with MALDI-TOF/MS. Combined methods of extraction physical plus chemical were able to resolve huge numbers of proteins that can be used to set up immune-proteomics analysis to improve and complete serodiagnosis of brucellosis Work supported by BrucMedNet- ARIMNet2 - Coordination of Agricultural Research in the Mediterranean-Grant agreement no. 618127. * Corresponding author: Paola Roncada, PhD. Istituto Sperimentale Italiano Lazzaro Spallanzani, Milano Phone.: +39 0250318138 E-mail address: [email protected] References: [1] Di Febo T, Luciani M, Portanti O, Bonfini B, Lelli R, Tittarelli M Development and evaluation of diagnostic tests for the serological diagnosis of brucellosis in swine. Vet Ital. 2012 Apr-Jun;48(2):133-56. [2] Piras C, Soggiu A, Bonizzi L, Greco V, Ricchi M, Arrigoni N, Bassols A, Urbani A, Roncada P.Identification of immunoreactive proteins of Mycobacterium avium subsp. paratuberculosis. Proteomics. 2015 Feb;15(4):813-23. doi: 10.1002/pmic.201400276



SESSION II

SYSTEMS BIOLOGY

ORAL COMMUNICATIONS: Session II Systems Biology 45

O-II-01

Activation of Nrf2-element signalling pathway in response to H2S related oxidative stress

Viviana Grecoa*, Alida Spallonib, Luisa Pieronia, Patrizia Longoneb, Andrea

Urbania,c

a) Laboratorio di Proteomica e Metabonomica, Fondazione Santa Lucia, Roma, Italy b) Laboratorio di Neurobiologia Molecolare, Fondazione Santa Lucia, Roma, Italy

c) Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Roma, Italy

Hydrogen sulphide (H2S) is an essential body product recognized as an endogenous neuromodulator in the central nervous system. In our previous work, we have highlighted a close connection of H2S to Amyotrophic Lateral Sclerosis (ALS) pathogenesis [1]. H2S was reported to be primarily toxic for ALS motorneurons increasing intracellular Ca2+ concentrations. Although this compelling evidence points to a role of H2S in ALS pathology, much needs to be done to unravel these mechanisms at a molecular level. Therefore, the aim of this study is to elucidate the mechanisms and pathways through which H2S contributes to trigger and amplify motorneuron damage. Mixed spinal cord cultures prepared from C57BL/6J were treated with Necrostatin (inhibitor of necroptosis) and V5 (Bax-Inhibiting Peptide), alone and together, exposed or not to H2S donor NaHS. Differential protein expression of this dataset was carried out by shotgun proteomics analysis based on nLC-MSE. Bioinformatics analysis, using QIAGEN’s Ingenuitys Pathway Analysis, showed higher expression of peroxiredoxins and molecules Type Citokine in H2S treated protein extracts compared to the untreated condition. Moreover, Nrf2-element signaling pathway is activated in response to H2S-related oxidative stress. It has been shown that activation of this signaling pathway is one of major mechanism in the cellular defense against oxidative or electrophilic stress [2]. We retain that H2S contributes notably to reinforce oxidative stress inducing the motorneuron to protect itself. In ALS, where motorneurons are already compromised and vulnerable and H2S levels are poisonous, these defense mechanisms could be not enough to counteract motorneuron death. * Corresponding author: Viviana Greco Laboratorio di Proteomica e Metabonomica, Fondazione Santa Lucia, Roma Italy Phone.: +3906501703220 E-mail address: [email protected] References: [1] Davoli A., Greco V. Spalloni A., et al "Evidence of Hydrogen Sulphide involvement in Amyotrophic Lateral Sclerosis” Ann Neurol. 2015 Jan 27. doi: 10.1002/ana.24372. [2] Obuobi S, Karatayev S, Chai CL, Ee PL, Mátyus P. The role of modulation of antioxidant enzyme systems in the treatment of neurodegenerative diseases. J Enzyme Inhib Med Chem. 2016;31(sup3):194-204. Epub 2016 Jul 7.


46 XII ItPA National Congress Lecce, June 12th-15th, 2017 O-II-02

Proteomic and interactomic characterization of PARK2-mutated Parkinson’s disease patients.

Mara Zilocchia*, Ilaria Colugnata, Luisa Pieronib, Barbara Garavagliac, Zoran Minicd, Mauro

Fasanoa, Mohan Babud, Tiziana Alberioa

a) Department of Science and High Technology, Center of Neuroscience, University of Insubria,

Busto Arsizio, Italy. b) Santa Lucia IRCCS Foundation Rome, Italy.

c) Molecular Neurogenetics Unit, "Carlo Besta" Neurological Institute IRCCS Foundation, Milan, Italy.

d) Department of Biochemistry, Research and Innovation Centre, University of Regina, Canada.

Mitochondrial dysfunction and mitophagy impairment are important hallmarks of Parkinson’s disease (PD) [1; 2]. Mutations in the PARK2 gene cause the development of an autosomal recessive form of PD [3]: indeed, this gene encodes for the protein Parkin, an E3 ubiquitin ligase involved in the removal of damaged mitochondria [4]. Little is known about mitochondrial alterations that occur in PARK2-mutated patients. For this reason, the aim of our research was to characterize the mitochondrial proteome and interactome of these PD patients. To this purpose, we isolated mitochondria from primary skin fibroblasts cell lines of three PARK2-mutated patients and three control subjects using a commercial kit based on surfactants. Mitochondrial protein extracts were then analyzed by quantitative shotgun proteomics (Synapt G2, Waters). For the interactome analysis, a size-exclusion chromatography was performed in order to isolate clusters of interacting proteins. All fractions thus obtained were then analyzed using a LTQ Orbitrap Velos Tandem Mass Spectrometer. We found several mitochondrial proteins that quantitatively and qualitatively change in PARK2-mutated patients in comparison with control subjects. Also the protein-protein interactions were affected in the presence of PARK2 mutations that cause a loss of function of Parkin. These findings were followed by an in-depth study of the mitochondrial network in terms of shape parameters in order to combine the proteome/interactome data with the morphological study. * Corresponding author: Mara Zilocchi. Title: PhD student. Institution: Department of Science and High Technology, Center of Neuroscience, University of Insubria, Busto Arsizio, Italy. Address: via Manara 7, I-21052 Busto Arsizio (VA), Italy. Phone.: +39-0331-339414; E-mail address: [email protected] (M. Zilocchi) References: [1] Subramaniam SR, Chesselet MF. Mitochondrial dysfunction and oxidative stress in Parkinson's disease. Prog Neurobiol, 2013. [2] Fernández-Moriano C., González-Burgos E., Gómez-Serranillos M. P. Mitochondria-Targeted Protective Compounds in Parkinson’s and

Alzheimer’s Diseases. Oxidative Medicine and Cellular Longevity, 2015. [3] Sun .M, Latourelle J.C., Wooten G.F., Lew M.F., Klein C., et al. Influence of heterozygosity for parkin mutation on onset age in familial

Parkinson disease: the GenePD study. Arch Neurol, 2006. [4] Chen Y., and Dorn G. W. PINK1-phosphorylated mitofusin 2 is a Parkin receptor for culling damaged mitochondria. Science, 2013.


ORAL COMMUNICATIONS: Session IV Human Proteomics 47

SESSION III

TECHNOLOGIES AND BIOINFORMATICS


O-III-01

Nanotechnological approaches for epithelial–mesenchymal transition biomarkers screening

Paola Priorea, Monica Biancoa*, Daniele Vergarab, Flora Guerrab, Simona Bettinic, Rosanna

Paganob, Cecilia Buccib, Michele Maffiab, Valentina Arimaa, Antonio Gaballoa*,

a) CNR-NANOTEC, Institute of Nanotechnology c/o Campus Ecotekne, University of Salento, Lecce, Italy

b) Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy

c) Department of Innovation Engineering c/o Campus Ecotekne, University of Salento, Lecce, Italy.

The epithelial–mesenchymal transition (EMT) program has emerged as a central driver of tumor malignancy, thus representing a potential source of cancer clinical markers that may improve the sensitivity and specificity of most traditional ones [1]. Here, we used proteomic and molecular approaches to identify a dataset of proteins whose expression varied significantly between two different breast cancer cell lines with epithelial and mesenchymal features: MCF-7 and MDA-MB-231, respectively. Several putative extracellular biomarkers were identified. Western blot, mass spectrometry, and bioinformatics analyses of proteins released into cell culture media (secretomes) led to identification of two classical EMT markers: E-cadherin a transmembrane protein implicated in cell–cell adhesion (epithelial phenotype), and Vimentin, an intermediate filament protein (mesenchymal phenotype). In fact, as tumor-associated proteolysis of transmembrane proteins with the release of ectodomains is a hallmark of invasive tumors it has been argued that detection of the soluble ectodomain of E-cadherin, may serve as a serum diagnostic biomarker. To confirm this hypothesis novel nanotechnological approaches based on gravimetric biosensors and high resolutions surface science techniques have been used. In this view, we exploited the Quartz Crystal Microbalance with simultaneous frequency and dissipation monitoring (QCM-D) as high sensitive method to monitor human serum interactions with specific antibodies [2]. QCM-D allowed us to study in real time the interactions between anti-E-cadherin, immobilized on the gold surface of the sensor via a self-assembled alkanethiol monolayer, and secretomes of different breast cancer cell lines. Overall, this approach can be used to detect with high sensitivity EMT markers. * Corresponding authors: Dr. Gaballo Antonio, Dr. Bianco Monica CNR-NANOTEC, Institute of Nanotechnology c/o Campus Ecotekne, University of Salento, Via Monteroni, 73100, Lecce, Italy. Phone.: +39 0832-319823; +39 0832-319306 E-mail addresses: [email protected] (A. Gaballo), [email protected] (M. Bianco) We gratefully acknowledge funding from the Apulia Regional Cluster project “SISTEMA” project code T7WGSJ3 References: [1] Vergara Daniele et al. Translating epithelial mesenchymal transition markers into the clinic: Novel insights from proteomics. EuPA Open

Proteomics 10 (2016) 31–41. [2] Bianco Monica et al. Quartz crystal microbalance with dissipation (QCM-D) as tool to exploit antigen–antibody interactions in pancreati

ductal adenocarcinoma detection. Biosensors and Bioelectronics 42 (2013) 646–652.



ORAL COMMUNICATIONS: Session III Technologies and Bioinformatics 49

O-III-02

Clinical applications of universal S-Trap sample processing

John P. Wilson1*, Nikita Saha Turna1, Rosamonde Banks2, Darryl J.C. Pappin1, Alexandre Zougman2

1Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; 2University of Leeds, Leeds, England

Bottom-up proteomics has been long hampered by limitations in sample preparation. The vastly different solubilities of proteins and myriad protocols to dissolve them lead to very different answers depending on what proteins are (or are not) present in the analyte solution. S-Trap sample processing solves this problem by integrating: 1) strong SDS-based protein solubilization (5% SDS) and complete denaturation (pH < 1 and > 70% organic); 2) simultaneous sample concentration and cleanup; and 3) rapid reactor-type protein digestion. The universal S-Trap protocol has been successfully applied without alteration on many kinds of samples (biological fluids, tissues, bacteria, yeast, cell lines, etc.). It enables reproducible sample processing using standard lab equipment from protein to peptides in around 1 – 2 hrs. It also allows sample preparation in the case of unknown sample composition, for example in submissions to core labs. Here, we apply S-Traps to clinical applications. First, we explore the application of S-Traps to dried blood spots beginning with protein extraction via 5% SDS. Second, we apply S-Trap sample processing to biological fluids and assay the reproducibility and efficiency in a 96-well plate format. Third and finally, we explore the effect of the harsh protein denaturation in S-Trap sample processing on antibody-antigen interactions. Especially when endogenous autoantibodies are present, these antibody-antigen interactions have been reported to alter susceptibility to digestion and thus change the results of mass spec based clinical assays * Corresponding author: John P. Wilson. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Phone.: +001 650.207.3796 E-mail address: [email protected] References: [1]Zougman A1, Selby PJ, Banks RE. Suspension trapping (STrap) sample preparation method for bottom-up proteomics analysis. Proteomics.

2014 May;14(9):1006-0.



O-III-03

Terminal Amine Isotopic Labeling of Substrates (TAILS): a positional proteomics approach to study mitochondrial proteases in Parkinson's

disease

Marta Lualdia, Federica Cornoa, Maurizio Roncib, Tiziana Alberioa and Mauro Fasanoa

a Dept. of Science and High Technology, University of Insubria, Busto Arsizio, Italy

b Dept. of Medical, Oral and Biotechnological Sciences, University “G. D’Annunzio” of Chieti-Pescara, Chieti, Italy

Mitochondrial dysfunction appears to have a major pathogenetic role in Parkinson’s disease (PD). The surveillance of protein quality control within mitochondria is orchestrated by a highly conserved proteolytic system, whose impairment may contribute to the onset of neurological disorders. Using a cellular model of impaired dopamine (DA) homeostasis (i.e., the human neuroblastoma SH-SY5Y cell line treated with DA) it has been demonstrated that dopamine aberrantly activates mitochondrial proteases1,2. However, a detailed characterization of mitochondrial proteases and their substrates in both physiological and pathological conditions is still lacking. The aim of the present work is the identification of candidate dopamine-activated mitochondrial proteases involved in PD pathogenesis. To this end, a newly developed proteomics approach has been employed, named TAILS (Terminal Amine Isotopic Labeling of Substrates)3. Using this procedure, a detailed analysis of the N-terminome of dopamine-treated SH-SY5Y cells has been performed, thus leading to the identification of dopamine-induced proteolytic cleavages in mitochondrial proteins. The sequence of the proteolytic fragments will be instrumental for the identification of candidate DA-activated mitochondrial proteases, which could represent novel drug targets or diagnostic biomarkers. * Corresponding authors: Marta Lualdi, PhD Department of Science and High Technology – University of Insubria via Manara 7, 21052 - Busto Arsizio (VA) Phone: +39 0331 339414 Fax: +39 0332 395599 e-mail: [email protected] References: (1) Alberio, T.; Bondi, H.; Colombo, F.; Alloggio, I.; Pieroni, L.; Urbani, A.; Fasano, M. Mitochondrial proteomics investigation of a cellular model of impaired dopamine homeostasis, an early step in Parkinson’s disease pathogenesis. Mol. Biosyst. 2014, 10 (6), 1332–1344 DOI: 10.1039/c3mb70611g. (2) Di Pierro, A.; Bondi, H.; Monti, C.; Pieroni, L.; Cilio, E.; Urbani, A.; Alberio, T.; Fasano, M.; Ronci, M. Experimental setup for the identification of mitochondrial protease substrates by shotgun and top-down proteomics. EuPA Open Proteomics 2016, 11, 1–3 DOI: 10.1016/j.euprot.2016.02.002. (3) Kleifeld, O.; Doucet, A.; Prudova, A.; auf dem Keller, U.; Gioia, M.; Kizhakkedathu, J. N.; Overall, C. M. Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates. Nat. Protoc. 2011, 6 (10), 1578–1611 DOI: 10.1038/nprot.2011.382.


ORAL COMMUNICATIONS: Session III Technologies and Bioinformatics 51

O-III-04 Diagnostic “rhinomic” profile of Allergic and non-Allergic Rhinits Subject

by Mesoporous Silica Particles and MALDI-TOF Mass Spectrometry

Chiara Villellaa*, Maria Stella Murfunia, Mariaimmacolata Preianòa, Giuseppina Maggisanoa, Nadia Lobelloa, Nicola Lombardob, Rocco Savinoa, Rosa Terraccianoa

aDepartment of Health Sciences, University Magna Graecia of Catanzaro, Catanzaro

bDepartment of Medical and Surgical Sciences, University Magna Graecia of Catanzaro, Catanzaro

The nasal fluid (NF) highly reflects the pathophysiology of inflammatory diseases. Discriminating different rhinitis can sometimes be difficult, as the diagnostic criteria used to identify the various subgroups are not always unambigous1. In the present study we propose a biomarker discovery strategy based on NF collected from nasal swab, a non-invasive diagnostic procedure compared to nasal lavage fluid (NFL) collection. We demonstrate that the combined use of mesoporous silica (MPS) with MALDI-TOF MS allows the rapid detection of differential nasal peptide profiles from nasal swabs of healthy (H), allergic rhinitis (AR) and non-allergic rhinitis (NAR) subjects. Depending on the pore diameter and on the chemical groups of the mesoporous surface, complex peptide mixtures are extracted from NF and converted into reproducible MALDI-TOF fingerprints2. As a proof-of-principle, we also explored the ability of our platform to discriminate between nasal swabs of patient with AR and NAR, and between these groups and H controls. Four peaks resulted differentially expressed between NAR and AR, two peaks discriminated AR from H while one peak segregated NAR from H group. Therefore, peptides selected and enriched by our platform could form part of a diagnostic “rhinomic” profile of allergic and non-allergic patients. Finally, we also forecast that the rhinomic fingerprints extrapolated by our tool may be able to reveal a distinctive pattern of molecular features correlated by specific relative expression in each analyzed subject and may provide a more practical route to personalized treatment3. * Corresponding Author: Chiara Villella, Phd Student University of Catanzaro “Magna Graecia” Europa Avenue, Germaneto, 88100, Catanzaro, Italy. Phone.: +3909613694204 Fax: +3909613694090 E-mail address: [email protected] (C. Villella) References: [1] Toppila-Salmi, S., van Drunen, C. M., Fokkens, W. J., Goleb- ski, K. et al., Molecular mechanisms of nasal epithelium in rhinitis and rhinosinusitis. Curr. Allergy Asthma Rep. 2015, 15, 495. [2] Savino, R., Terracciano, R., Mesopore-assisted profiling strategies in clinical proteomics for drug/target discovery. Drug Discov. Today 2012, 17, 143–152. [3] Lombardo, N., Preianò, M., Maggisano, G., Murfuni M.S., Messina, L., Pelaia, G., Savino, R., Terracciano, R. A rapid differential display analysis of nasal swab fingerprints to distinguish Allergic from non-Allergic subjects by mesoporous silica particles and MALDI-TOFMass Spectrometry. Proteomics. 2017 Mar;17(6). 10.1002.



SESSION IV

HUMAN PROTEOMICS


O-IV-01

Urinary proteomics in Bardet-Biedl syndrome

Michele Costanzo1, Giuliana Bruno2, Marianna Caterino1,3,4, Rosa A. Siciliano5, Maria F.

Mazzeo5, Miriam Zacchia6, Giovambattista Capasso6, Margherita Ruoppolo1,3,4

1 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli ‘‘Federico II’’, Naples, Italy

2 IRGS Biogem, Ariano Irpino, Avellino, Italy 3 CEINGE - Biotecnologie Avanzate scarl, Naples, Italy

4 Associazione culturale DiSciMuS RCF 80026, Casoria, Naples, Italy 5 Istituto di Scienze dell’Alimentazione – CNR, Avellino, Italy

6 Prima divisione di Nefrologia, Dipartimento di Scienze Cardio-toraciche e Respiratorie, Università della Campania "Luigi Vanvitelli", Naples, Italy

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive genetic disorder. This ciliopathy has genetic heterogeneity and at least 21 genes have been identified. Clinical manifestations include retinal degeneration, polydactyly/syndactyly, learning disabilities, obesity, and kidney dysfunction. The management of BBS patients is actually symptomatic. Many efforts have pointed at the topical delivery of missing BBS genes by adenoviruses. Nevertheless, much about molecular pathogenesis of BBS is still unknown. From a population of 44 Italian BBS patients, 7 males and 7 females were chosen, based on age, sex, body size and glomerular filtration rate. 10 male and 10 female age/gender-matched healthy volunteers were included as controls. The second urine in the morning was collected, and suitably prepared for proteomic experiments. Controls were pooled in order to reduce the biologic variability. Proteins were fractionated by 1D-SDS-PAGE. Each lane underwent in-gel tryptic digestion. Mixtures of peptides were analysed by tandem mass spectrometry. The relative abundance of each protein was determined by the Spectral Counting approach, using the normalized spectral abundance factor (NSAF). 12 and 71 proteins were identified as up-regulated and down-regulated in this patients’ cohort. Renal abnormalities appear to have high frequency in BBS. Dysregulated proteins could be directly involved in the pathogenesis or in the progression of the renal phenotype, or could be markers of kidney dysfunction. According to our knowledge about urinary proteomics and renal diseases, the quantitative proteomic approach is able to refine our view, thus providing more insights about the mechanisms underlying renal and extra-renal disease in this setting. * Corresponding author: Michele Costanzo Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli ‘‘Federico II’’, Via S. Pansini 5, 80131, Naples, Italy Phone.: +39-081-3737807; Fax: +39-081-3737808 E-mail address: [email protected] .



O-IV-02

Proteomic of exosome’s cargo and membrane proteins from Neuroblastoma

Victor Corasolla Carregari1, OlesyaChayka2, Lene Jakobsen3, Luisa Pieroni1, Martin R.

Larsen3, Andrea Urbani1, Arturo Sala2 and Giuseppe Palmisano1,4**

1-Fondazione Santa Lucia, IRCCS, Rome-Italy 2- Brunel Institute of Cancer Genetics and Pharmacogenomics, London, UK

3- Department of Biochemistry and Molecular biology, Odense, DK 4- Glyco. Prot. Laboratory, Department of Parasitology, University of Sao Paulo, Brazil

Neuroblastoma is the most common extracranial solid tumour in childhood comprising around 10% of all childhood cancers (1). The prognosis of patients with disseminated neuroblastoma is grim, with a 5-year survival rate of approximately 30% (2). As well know, the MYC-N expression is strongly related with the high aggressiveness and treatment resistance of the Neuroblastoma (3). We isolated extracellular vesicles from the SH-EPs with/without MYC-N expression. Through the common antibodies for exosome’s membrane proteins CD63, CD81, CD56 and CD9 attached with the beads, our flow cytometry analysis allowed us to identify the size and the distribution of these vesicles. After the morphological characterization of the exosomes, we performed a quantitative proteomic analysis identifying 890 proteins, merging the results of two different mass spectrometers (LTQ-Orbitrap Velos and Synapt G2 respectively). MYC-N expression induced the regulation of 152 proteins involved in several molecular networks such as Protein synthesis, Glycolysis and Extra Cellular Matrix-Interactions. Aiming to understand better the interaction of the exosomes with the recipient cell membrane, we evaluated the membrane proteins profile from cells with/without MYC-N expression. We identified 417 proteins in the cells with the MYC-N and 228 without the MYC-N. 185 proteins were present in both of them and 228 only in the cells with MYC-N+ expressed and 39 in the cells with MYC-N- non-expressed. Moreover, the expression analysis showed that from the 185 proteins shared between both cells, 157 are statistically regulated. Indeed, all results are suggesting a strong relationship between MYC-N activation and exosome protein cargo modulation. *Corresponding author: Victor Corasolla Carregari Fondazione Santa Lucia: Via Del Fosso di Fiorano 64, 00143– Roma – Italy Phone: +39 06501703220; E-mail: [email protected]. References 1-Brodeur GM, Bagatell R. Mechanisms of neuroblastoma regression. Nat Rev Clin Oncol. 2014;11:704-713. 2-Aydin GB, Kutluk MT, et al. Neuroblastoma in Turkish children: experience of a single center. Journal of Pediatric Hematology and Oncology 2009;31:471–80. 3-Cady Ploessl, PharmD, et al. Dinutuximab: An Anti-GD2 Monoclonal Antibody for High-Risk Neuroblastoma. Annals of pharmacotherapy 2016, 1-7.



O-IV-03 Proteomics analysis to assess the role of mitochondria in BRCA1-mediated

breast tumorigenesis

Antonio Concolino, Angela M. Perri, Claudia V. Fiumara, Laura Tammè, Erika Olivo,

Giovanni Cuda, Domenica Scumaci*

Laboratory of Proteomics, Dpt. of Experimental and Clinical Medicine; Magna Græcia University of Catanzaro, Catanzaro, Italy.

Mitochondria are the organelles deputed to energy production, but they are also involved in carcinogenesis, cancer progression, and metastasis, playing a role in altered energy metabolism in cancer cells. Mitochondrial metabolism is connected with several mitochondrial pathways such as ROS signaling, Ca2+ homeostasis, mitophagy, and mitochondrial biogenesis. These pathways are merged in an interactive super-network that seems to play a crucial role in cancer [1,2]. Germline mutations of the BRCA1 gene account for 5–10% of breast cancers and confer a risk of developing the disease 10- to 20-fold much higher in BRCA1 mutation carriers than in non-carriers [3]. By considering metabolic networks that could reconcile both genetic and non-genetic causal mechanisms in BRCA1 driven tumorigenesis, we herein based our study on the hypothesis that BRCA1 haploinsufficiency might drive metabolic rewiring in breast epithelial cells, acting as a push toward malignant transformation. Using 2D-DIGE we analyzed and compared the mitochondrial proteomic profile of sporadic breast cancer cell lines (MCF7) and BRCA1 mutated breast cancer cell lines (HCC1937). Image analysis was carried out with Decider Software, and proteins differentially expressed were identified by LC-MS/MS on a quadrupole-orbitrap mass spectrometer Q-Exactive. Ingenuity pathways analysis software was used to analyze the fifty mitochondrial proteins whose expression resulted significantly altered in response to BRCA1 mutation status. Mitochondrial Dysfunction and oxidative phosphorylation, Energy production and nucleic acid metabolism were, respectively the canonical pathway and the molecular function mainly affected. Western blotting analysis was done to validate the expression and the peculiar compartmentalization of specific proteins such us HSP60. Data obtained lead us to hypothesize an interesting connection between BRCA1 and mitochondria pathways, capable to trigger metabolic changes which, in turn, sustain the high energetic and anabolic requirements of the malignant phenotype. *Corresponding author: Domenica Scumaci Laboratory of Proteomics, Dpt. of Experimental and Clinical Medicine; Magna Græcia University of Catanzaro, Catanzaro, Italy Phone.: +39 0961 369 4224 Cell.: +39 329 347 22 47 E-mail address: [email protected] References: [1] Taylor RW, Turnbull DM. Mitochondrial DNA mutations in human disease. Nat Rev Genet (2005) 6(5):389–402. doi:10.1038/nrg1606 2. [2] Holmuhamedov E, Lewis L, Bienengraeber M, Holmuhamedova M, Jahangir A, Terzic A. Suppression of human tumor cell proliferation through mitochondrial targeting. FASEB J (2002) 16(9):1010–6. doi:10.1096/fj.01-0996com [3] Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, Liu Q, Cochran C, Bennett LM, Ding W, Bell R, Rosenthal J, Hussey C, et al. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science. 1994; 266:66–71. 2.



O-IV-04

Peptidomics analysis of simulated mouth, gastric and intestinal digestion pattern of allergenic tropomyosin

Cristian Pirasa, Alessio Soggiua, Viviana Grecob, Takács Krisztinac, Gelencsér Évac,

Thomas Holzhauserd, Annette Kuehne, Karin Hoffmann-Sommergruberf, Andrea Urbanib,g, Luigi Bonizzia, Paola Roncadah

a) Department of Veterinary Medicine (DIMEVET), University of Milan, Italy; b) Santa Lucia Foundation, Rome, Italy:

c) NARIC Food Science Research Institute, Department of Biology, Budapest, Hungary; d) Recombinant Allergen Therapeutics, Division of Allergology, Paul-Ehrlich-Institut, Federal

Institute for Vaccines and Biomedicines, Langen, Germany; e) Allergology - Immunology - Inflammation Research Unit, Department of Infection and Immunity,

Luxembourg Institute of Health, Luxembourg; f) Dept. of Pathophysiology and Allergy Research, Medical University of Vienna, Austria

g) Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Rome, Italy; h) Istituto Sperimentale Italiano “Lazzaro Spallanzani” Milano, Italy;

The digestion patterns of allergenic proteins play a key role in allergenicity. In order to completely remove allergenic properties of a protein it is necessary a complete enzymatic digestion. Whether the enzymatic digestion is not efficient, the persistence of bigger peptides can occur and put the basis for the development of the sensitization process. The hypothesis of the current work is based on the probability that shrimp tropomyosin (TM) is not fully digested or presents a digestion pattern that generates some peptides that can be immunogenic. Therefore, the work plan designed aims to study the cleavage pattern of respectively purified chicken TM, recombinant chicken TM, purified TM of Penaeus monodon (Pen m 1), recombinant TM of Penaeus monodon (rPen m 1) and recombinant TM of Crangon crangon (rCrac c 1). One mg of each orthologue has been processed through simulated mouth, gastric and intestinal digestion. The sample was frozen after this step, concentrated and cleaned through protein precipitation. Afterwards, the protein pellet was analyzed for peptidomic analysis through 1D tricine gel electrophoresis, 2D tricine gel electrophoresis and mass spectrometry. Shrimp TM digestion pattern highlighted the presence of a resistant band at an average MW of 25 kDa that could be involved in the immunogenic process. This approach (peptidomics through 1D-2D Tricine/MS) could represent a milestone for the study of digestion patterns of allergenic proteins or for the study of allergenic potential of novel foods. Authors are grateful to COST Action FA1402 entitled: Improving Allergy Risk Assessment Strategy for New Food Proteins (ImpARAS) * Corresponding author: Cristian Piras University of Milan, Department of Veterinary Medicine, Via Celoria 10, 20133, Milan, Italy. Phone.: +39 0250318138; E-mail address: [email protected] (Cristian Piras)


POSTERS: Session II systems biology 57

POSTERS


P-I-01

Characterization of Arabian camel milk proteome over lactation

Isabella Alloggioa*, Alexandra Contreras-Jodarb, Cristian Pirasa, Alessio Soggiua, Viviana Grecoc, Andrea Urbanid, Luigi Bonizzia, Moez Ayadie,f, Rihyad S. Aljumaahe, Mohammed A.

Alshaikhe, Elena Díaz-Medinab, Gerardo Cajab, Paola Roncadag

a) Università degli Studi di Milano, Milano, Italy b) Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain

c) Fondazione Santa Lucia, Roma, Italy d) Università Cattolica del Sacro Cuore, Roma, Italy

e) King Saud University, Riyadh, Saudi Arabia f) Université de Jendouba, Tunisia

g) Istituto Sperimentale Italiano Lazzaro Spallanzani, Milano, Italy

The World Health Organization recommends that infants be exclusively breastfed for the first 6 months of life for their optimal growth and health. However, less than 35% of infants worldwide even complete breastfeeding for more than 4 months [1]. Infant formula is intended as a substitute to breast milk and it is formulated to mimic nutritional composition of breast milk using bovine milk as protein source [2]. However, hypersensitivity to bovine milk proteins (mainly to αs1-casein and β-lactoglobulin) is one of the major food allergy affecting between 2% and 3% of the general child population [3]. In recent years, camel milk is getting attention due to its reported lower αs1-casein content compared with cow milk and the absence of β-lactoglobulin that may be a promising new protein source for children allergic to bovine milk proteins [4]. This work aimed to characterize the proteomic profile of camel milk at two different stage of lactation. Three multiparous she-camels of the herd of Oasis Park Fuerteventura (La Lajita, Las Palmas, Spain) were milked at early (42 ± 11 DIM) and late (252 ± 15 DIM) stage of lactation for sampling collection. Milk samples protein characterization was performed through 1D and 2D-electrophoresis. Differentially expressed proteins between the two groups (early and late lactation) were identified by MALDI TOF mass spectrometry. Multivariate data analyses, including PCA, PLS-DA were used to generate an integrated vision of changes of the protein profile of camel milk over lactation. Results highlighted the presence of 7 differentially expressed proteins between two groups. These findings could be useful for a better understanding of the protein composition dynamics of camel milk. * Corresponding author: Isabella Alloggio Università degli Studi di Milano Phone.: +39 0250318138 E-mail address: [email protected] References: [1] World Health Organization. 2003. Global Strategy for infant and young child feeding. [2] Martin, C.R.; Ling, P.-R. and Blackburn, G.L. 2016. Review of infant feeding: key features of breast milk and infant formula. Nutrients 8, 279 [3] Luyt, D.; Ball, H.; Makwana, N.; Green, M.R.; Bravin, K.; Nasser, S.M. and Clark, A.T. 2014. BSACI guideline for the diagnosis and management of cow’s milk allergy. Clin Exp Allergy. 44:642-72. [4] Hinz, K.; O’Connor, P.M; Huppertz, T., Ross, R.P. and Kelly, A.L. 2012. Comparison of the principal proteins in bovine, caprine, buffalo, equine and camel milk. J. Dairy Res. 79:185:191.



P-I-02

Comparative proteomic analyses of Salmonella Enteritidis SE86 exposed to cold plasma

Ana Carolina Rittera, Giorgia Gozzib, Lucelia Santic, Walter O. Beys-da-Silvac, John R.

Yatesd, III, Luigi Ragnib, Adriano Brandellia, Lucia Vanninib

a Universidade Federal do Rio Grande do Sul, Rio Grande do Sul, Brasil

b Università di Bologna, Bologna, Italy c Hospital de Clínicas de Porto Alegre, Rio Grane do Sul, Brasil

d The Scripps Research Institute, California, United States In response to increasing demand for high quality and food safety, important technological innovations are being developed for the food industry, once the sensory and nutritional changes are mainly attributed to decontamination processes where eradication of pathogenic microorganisms is achieved by heat and chemical processes. The non-thermal plasmas (cold plasma) is an ionized gas containing free electrons and neutral reactive species such as atoms, molecules and radicals, at room pressure and temperature. Gas plasma has demonstrated efficiency in the removal of microbial contamination from fresh and minimally processed food, especially pathogenic bacteria, such as Listeria spp. and Salmonella spp. Therefore, the aim of this study was to investigate the protein expression obtained from S. Enteritidis SE86 treated with cold plasma at different times, in order to observe the mechanisms of action of gas plasma in control of this microorganism. The number of viable S. Enteritidis SE86 cells was analyzed at different time intervals upon exposure to gas plasma for up to 90 min by plate and approximately 100 μg of S. Enteritidis SE86 protein extracts were analyzed by Multidimensional Protein Identification Technology (MudPIT). The results demonstrated that no significant changes in cell counts were detected up to 30 min exposure to gas plasma. However, after 60 min, 2 log reduction was achieved. Overall, 1096 proteins were identified, with 249 more abundant in plasma-treated samples, and 9 more abundant in non-treated control samples. In plasma treated cells, proteins associated to ion binding showed higher abundance. * Corresponding author: Ana Carolina Ritter. Universidade Federal do Rio Grande do Sul Av. Bento Gonçalves 9500 – ICTA, 91501-970 Porto Alegre, Brazil Phone/Fax: +5551 3308 6249 / +5551 3308 7048 E-mail address: [email protected]



P-I-03

Ancient and modern durum wheat varieties: a comparative proteomic analysis of the metabolic fractions

Antonella Di Francesco, a* R. Saletti,a V. Cunsolo,a V. Muccilli,a, P. De Vita,c S. Gallinab

and S. Fotia

a) Department of Chemical Sciences, University of Catania, Catania b) B.R.I.T. (Bio-Nanotech Research and Innovation Tower), University of Catania, Catania

c) CREA Cereal Research Centre (CREA-CER), Foggia

Wheat, due to its adaptability and for the unique functional properties of its flour, represents the most widely grown, processed, and consumed cereal by humankind. Today most of the wheat species grown are hybrids which have been created from ancient wheat over the last 100 to 50 years [1]. These "new" wheats have beneficial properties in terms of yield compared with the original ancient ones. It is important to note that wheat is also the causing factors of many adverse reactions, such as celiac disease, allergies, and non-celiac wheat sensitivity (NCWS) in susceptible people. This leads to an increasing interest for the gluten-free products and for the ancient wheat genotypes which are generally considered better tolerated than the modern ones, but without any scientific evidence. The aim of the present work is the comparison by proteomic approaches of the salt-soluble protein fractions extracted from the mature kernel of ancient durum wheats, and Simeto, an improved durum wheat variety, widely spread in Italy and other Mediterranean countries. Preliminary results revealed a remarkable similarity in the metabolic protein composition of these varieties, but also evidenced some peculiar differences, such as the presence of some wheat allergens only in the modern cultivar Simeto. * Corresponding author: Antonella Di Francesco PhD Department of Chemical Sciences, University of Catania Viale A. Doria, 6 – Catania, Italy +39 0957385028. E-mail address: [email protected] References: [1] Shewry, P.R., J Exp Bot, 2009, 60,1537



P-I-04 Identification of Trypanosoma equiperdum low molecular weight proteins

recognized by antibodies from dourine infected horses

Mirella Luciania*, Tiziana Di Feboa, Ivanka Krastevaa, Angela Cattaneob, Massimiliano Orsinia, Chiara Di Pancrazioa, Angela Bachib, Manuela Tittarellia

a) Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Teramo, Italy;

b) Istituto FIRC di Oncologia Molecolare (IFOM), Milano, Italy

Trypanosoma equiperdum is the causal agent of dourine, a disease of equids transmitted directly from animal to animal during coitus. Diagnosis of dourine depends on the recognition of clinical signs, identification of the parasite and serological tests (CFT, IFAT, ELISA). T. equiperdum is closely related to other Trypanosoma species, as T. brucei and T. evansi. and cross-reactions among them are possible [1]. In those cases, history, clinical and pathological findings are necessary to confirm T. equiperdum infection [2]. Improvement in dourine serodiagnosis will require more T. equiperdum specific subunit antigens and monoclonal antibodies to them. In this work, six electrophoretic bands with molecular weight ranging between 37 and 10 kDa, recognised by antibodies from infected horses [3], were subjected to standard in-gel destaining, reduction, alkylation and trypsin digestion; peptides were analyzed using an UPLC EASY-nLC 1000 system coupled to a Q Exactive-HF mass spectrometer, in order to identify immunogenic proteins that could be used as biomarkers in the diagnosis of dourine. A total of 167 proteins were identified; functional features for proteins were derived from the correspondent Uniprot entries. They were further annotated in terms of topological and immunological features. Twenty-five proteins resulted surface proteins and all of them contained B-cell linear epitopes exposed to the solvent. Among them, 8 proteins were found unique for T.equiperdum. Those proteins could be potential candidates for development of diagnostic assays specific for dourine disease. * Corresponding author: Mirella Luciani Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Via Campo Boario, Teramo, Italy Phone.: +39 0861332448; Fax: +39 0861332251. E-mail address: [email protected] References: [1] World Organisation for Animal Health (2013). Chapter 2.5.3. Dourine. In: Manual of diagnostic tests and vaccines for terrestrial animals.

OIE, Paris, 1-10 [2] Calistri P, et al (2013). Dourine Reemergence in Italy. J Equine Vet Sci, 33, 83-89 [3] Luciani M, et al (2013). IgG antibodies from dourine infected horses identify a distinctive Trypanosoma equiperdum antigenic pattern of

low molecular weight molecules. Vet Immunol Immunopathol, 151, 140-146



P-I-05

Proteome analysis in dystrophic mdx mouse muscle reveals a drastic alteration of Key Metabolic and Contractile Proteins after chronic exercise

and the potential modulation by anti-oxidant compounds

Tania Gamberia, Tania Fiaschia, Elisa Valocchiaa, Alessandra Modestia, Paola Mantuano b, Jean-Francois Rollandb, Francesca Sanarica b, Annamaria De Lucab* and Francesca

Magherinia*

a) Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence,

Italy b) Section of Pharmacology, Department of Pharmacy & Drug Sciences, University of Bari “Aldo

Moro”, Bari, Italy

Weakness and fatigability are typical features of Duchenne muscular dystrophy patients and are aggravated in dystrophic mdx mice by chronic treadmill exercise. In the present study, we describe for the first time, the pattern of protein expression variation that is associated to the worsening of dystrophy phenotype induced by chronic exercise. Our proteomic analysis pointed out 34 proteins differentially expressed between sedentary and exercised mdx mice. These proteins belong mostly to glucose metabolism, energy production and sarcomere structure categories. Interestingly exercise induced an increase of typical fast twitch fiber proteins (Troponin T fast skeletal muscle, Troponin I fast skeletal muscle and Myozenin-1) combined with an increase of several glycolytic enzymes. Concerning energy transfer, Adenylate kinase, showed a marked decrease when compared with non exercised mdx. The decline of this enzyme correlates with increased Creatin kinase enzyme, suggesting that a compensatory energy metabolism mechanism could be activated in mdx mouse skeletal muscle following exercise, as already proven for Ak1 knockout mice. In addition, we also analysed muscles from exercised mdx mice treated with two natural anti-oxidant compounds, apocynin and taurine, that in our previous study, were proved to be beneficial on some pathology related parameters, and we showed that these compounds can counteract exercise-induced expression changes of several proteins. * Corresponding authors: Francesca Magherini University of Florence, Florence, Italy. Phone.: +39 0552751237; E-mail address: [email protected] Annamaria de Luca University of Bari “Aldo Moro”, Bari, Italy Phone.: 39 0805442245 E-mail address: [email protected]




P-I-06

Immunomodulatory Properties of L. gasseri Investigated by Proteomics

Maria Fiorella Mazzeo, Diomira Luongo, Mauro Rossi, Rosa Anna Siciliano*

Institute of Food Sciences, CNR, Avellino, Italy

The interaction with the immune system and its modulation are key mechanisms underlying the health-promoting effects of probiotic bacteria, which could be mediated by molecules on the microorganism surface or secreted probiotic-derived factors, recently defined as "postbiotics” [1]. The immunomodulatory properties of Lactobacillus gasseri, a well known probiotic bacterium [2], were assessed investigating its effects in vitro on murine bone-marrow dendritic cells (DCs). DCs play a pivotal role in both innate and adaptive immunity. In this cell model, L. gasseri OLL2809 was able to modify the cytokine profile, dramatically inducing the production of IL-12, TNF-α and IL-10 [3]. In order to get a deeper insight on the immunomodulatory properties of this strain, a comparative proteomic study was performed on secretomes of both immature and mature (LPS-induced) DCs, cultured in serum-free medium and challenged with L. gasseri OLL2809, collected in log growth phase and subjected to γ-irradiation, to prevent further bacteria proliferation. Quantitative proteomics strategies based on the spectral counting method [4] led to identify proteins involved in the maturation process and, most importantly, forty seven proteins whose production was modulated by co-incubation with L. gasseri. The functional classification confirmed the extracellular localization of most of these proteins, and clearly highlighted their involvement in the regulation of immune processes. Therefore, a detailed DC molecular fingerprint was associated with the immunomodulatory properties of this probiotic strain. This study could give a contribution to better design functional applications of probiotics for the amelioration of specific immunological conditions. *Corresponding author: Rosa Anna Siciliano Institute of Food Sciences, CNR, via Roma 64, Avellino, Italy. Phone.: +39 0825 299363; Fax: +39 0825 781585. E-mail address: [email protected] (R.A. Siciliano) References: [1] Tsilingiri K, Rescigno M. Postbiotics: what else? Benef Microbes. 2013, 4:101-107. [1] Selle K, Klaenhammer TR. Genomic and phenotypic evidence for probiotic influences of Lactobacillus gasseri on human health. FEMS Microbiol Rev. 2013, 37:915-935. [2] Luongo D, Miyamoto J, Bergamo P, Nazzaro F, Baruzzi F, Sashihara T, Tanabe S, Rossi M. Differential modulation of innate immunity in vitro by probiotic strains of Lactobacillus gasseri. BMC Microbiol. 2013, 13:298. [3] Zybailov B, Mosley AL, Sardiu ME, Coleman MK, Florens L, Washburn MP. Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae. J Proteome Res. 2006, 5:2339-2347.



P-I-07

Vegetal Coal: Proteomic Study Of Tomato Fruits

Maria Tartaglia, Simona Arena, Andrea Scaloni, Mariapina Rocco*

a) 1 Department of Science and Technology, University of Sannio, Benevento, Italy b) 2 Proteomics and Mass Spectrometry Laboratory, ISPAAM, National Research Council, Naples,

Italy

The biochar is a rich-carbon coal obtained by pyrolysis of different biomasses. This coal can be used for soil carbon long-term storage, reducing the atmosphere CO2 amount and furthermore to improve the chemical and physical characteristics of the soil. There are several biochars produced by several combustion techniques and input materials. Despite this heterogeneity, biochar can improve soil fertility and therefore the efficiency of plants. Tomato's cultivation was choosen because of its great economic relevance and the its wide diffusion in Mediterranean basin. Using tomato plants San Marzano variety, it was observed an increase of plant growth if a small amount of biochar was added to the soils. Several studies focused their interest on biochar effects in plants, demonstrating higher yield and productivity. In this work we evaluated the power of biochar like soil amendment and its effects on tomato plants treated with different carbon and nitrogen concentrations in culture media. 2-DIGE proteomic analysis was performed in tomato fruits, identifying 37 differentially expressed proteins, clustered according to their biochemical function. Notably, most important identified pathways were involved in stress/defense response, indicating a still unclear role of biochar not only on growth, development and ripening processes in tomatoes, but also in protection.

*Corresponding author: Mariapina Rocco University of Sannio, Via Port’Arsa 11, Benevento, Italy Phone.: +390824305168 Fax.: + 390824305120 Email address: [email protected]

References: 1) M. Yamato, Y. Okimori, I. F. Wibowo, S. Anshori, M. Ogawa (2006) Soil Sci. Plant Nutr., 52: 489–495 2) J. Lehmann, Jr. J.P. Da Silva, C. Steiner, et al. (2003) Plant Soil, 249: 343–57 3) M. K. Hossain, V. Strezova, K. Y. Chanb, P. F. Nelsona (2010) Science, 78: 1167–1171 4) M. Viger, R. D. Hancock, F. Miglietta and G. Taylor (2014) GCB Bioenergy, 7, 658–672



P-I-08

Proteome modulation in microencapsulated Lactobacillus reuteri DSM 17938 during simulated gastrointestinal conditions (SGC)

Alessio Soggiu a*, Annachiara De Priscob, Cristian Pirasa, Isabella Alloggioa, Viviana Grecoc,

Andrea Urbanic,d ,Luigi Bonizzia, Gianluigi Mauriellob, Paola Roncadae.

a) Dipartimento di Medicina Veterinaria, Università degli studi di Milano, Milano, Italy; b) Dept. of Agricultural Sciences, Division of Microbiology, University of Naples Federico II,

Portici (NA), Italy ; c) Fondazione S. Lucia, Roma, Italy ;

d) Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Rome, Italy ; e) Ist. Sper. Italiano « l. Spallanzani », Milano, Italy.

Probiotics have known in recent years an important rediscovery and re-evaluation. Many studies that have been conducted by numerous research groups have highlighted their fundamental role in maintaining our health and well-being and the prevention of certain diseases. However, as living microorganisms they can also be partially destroyed during production, storage and even from the stomach acid environment. Several technological approaches have been proposed to overcome this problem and microencapsulation has gained a great attention because of its potentiality in enhancing their capability to resist to several environmental stress factors[1]. However at this moment are almost lacking experimental evidences that describe the effects of microencapsulation on protein modulation of probiotic bacteria as well as the effect of this protection during SGC. In this work, our aim is to investigate through a gel-based proteomic approach proteome dynamics of chitosan-alginate encapsulated Lactobacillus reuteri DSM 17938 during SGC. Overnight cultures of L. reuteri DSM17938 were encapsulated in core-shell microcapsules[2]. Free and encapsulated bacteria were incubated in ratio 1:4 in gastric simulated solution for 2h at 37 °C and subsequently for 3h at 37 °C in intestinal simulated solution. After incubation bacterial protein have been extracted as described[3] and separated by 1D and 2D electrophoresis. Differentially expressed spot between free and encapsulated bacteria after SGC (p<0.05) have been detected using Progenesis Same Spot 4.6 and will be identified by MALDI-TOF/TOF MS. Several significant differences have been highlighted between the different experimental conditions. The identification of the proteins modulated during SGC in free and encapsulated bacteria will shed light on metabolic response of probiotic bacteria to physiological and technological stress factors. * Corresponding author: Alessio Soggiu. Dipartimento di Medicina Veterinaria, Università degli studi di Milano, Milano, Italy; Phone.: +39 0250318138; Fax: +39 0250318104 E-mail address: [email protected] References: 1. De Prisco, A. and G. Mauriello, Probiotication of foods: A focus on microencapsulation tool. Trends in Food Science & Technology, 2016.

48: p. 27-39. 2. De Prisco, A., et al., Microencapsulation by vibrating technology of the probiotic strain Lactobacillus reuteri DSM 17938 to enhance its

survival in foods and in gastrointestinal environment. LWT - Food Science and Technology, 2015. 61(2): p. 452-462. 3. Piras, C., et al., Identification of immunoreactive proteins of Mycobacterium avium subsp. paratuberculosis. Proteomics, 2015. 15(4): p.

813-823.



P-I-9 Proteomic analysis of mitochondria from AMPK/Snf1- depleted yeast cells

grown in presence or absence of methionine

Elisa Maffiolia,Francesca Grassi Scalvinia, Silvia Morellid, Fabiana Santagataa, Armando Negria, Gennaro Agrimib, Paola Coccettic, Gabriella Tedeschia,d*

a) Dipartimento di Medicina Veterinaria, Università degli Studi di Milano, Milano, Italy;

b) Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di BARI ALDO MORO, Bari, Italy;

c) Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di MILANO-BICOCCA, Milano, Italy;

d) Fondazione Filarete, Milano, Italy;

The AMPK is an evolutionary conserved regulator of cellular energy homeostasis in eukaryotes, which orthologue in S. cerevisiae, Snf1, is of primary importance in yeast for the growth in presence of low glucose concentration and in the utilization of alternative carbon sources. Another evolutionary conserved complex in cellular metabolism is MCP, a key regulator of pyruvate uptake into mitochondria. Cancer cell lines with an inactivation of AMPK or MCP show the so-called Warburg effect: loss of Snf1/AMPK activity determines a reliance on amino acid utilization for growth in yeast, a typical hallmark of cancer cells. On the other hand, the restoration of both activities causes the suppression of the cancerous phenotype. To deeply characterize the role of Snf1/AMPK activity, mitochondria were isolated from the yeast Saccharomyces cerevisiae according to the protocol described in (1). We performed a LC-ESI MS/MS analysis to compare the proteome of mitochondria from WT/ snf1D yeast cells grown in presence or absence of methionine. Mitochondrial proteins were derivatized, digested and analyzed by LC-nanoESI-LTQ Orbitrap Velos. Raw data files were processed using MaxQuant software and the proteins were functionally profiled using bioinformatics annotation tools and pathway databases. This proteomic approach allowed to elucidate the role of Snf1/AMPK activity and to investigate the connection between AMPK and MPC in the regulation of yeast metabolic network as well as in models of human cancer cell lines, also enlightening the role of AMPK in regulating MPC activity through specific chemical modulators of AMPK activity. * Corresponding author: Gabriella Tedeschi. Dipartimento di Medicina Veterinaria, Università degli Studi di Milano, Milan, Italy. Phone.: +390250318124; Fax: +39 02 50318123. E-mail address: [email protected] (G. Tedeschi) References: [1] Chris Meisinger,Thomas Sommer,and Nikolaus Pfanner. Purification of Saccharomcyes cerevisiae mitochondria devoid of microsomal and cytosolic contaminations. Analytical Biochemistry 287(2), 339-342 (2000).



P-II-01

Proteomic profiling of fractionated Head and Neck cancer cell secretome capable of activating the immune response in vitro

Ada Consalvoa, b *, Maurizio Roncia,b,c, Verena Damiania,b, Mirco Zucchellia,b, Domenico

Ciavardellib,d, Vincenzo De Laurenzia,b .

a Department of Medical, Oral and Biotechnological Sciences, University G. D’Annunzio, Chieti, Italy

b Centro Scienze dell’Invecchiamento e Medicina Traslazionale- CeSI-Met , Chieti, Italy c Fondazione S.Lucia, IRCCS, Rome, Italy

d School of Human and Social Science, “Kore” University of Enna, Enna, Italy

Head and neck (H&N) cancers account for 5 to 10% of all cancers worldwide. The annual incidence of H&N cancers worldwide is more than 550,000 cases with around 300,000 deaths each year. Recently the role of tumor microenvironment (TME) has gained attention. In fact it has been shown that TME undergoes numerous changes during tumor progression including the development of immune-escaping features influencing migration and proliferation of cancer cells [1]. In a previous study we have demonstrated that specific fractions of H&N cancer cells secretome are capable of activating THP1 monocytes in-vitro. We also demonstrated the presence of Bcl-2-associated athanogene 3 (BAG3) in the secretome of different H&N cancer cells. BAG3 is a cytoplasmatic protein often found upregulated in many types of tumors and has been recently associated to the modulation of TME [2]. The aim of this study was to characterize the protein profile of the fractionated conditioned medium in order to better understand the involvement of BAG3 in activating the immune system. We first tested the activating properties of fractions from H&N cancer cells conditioned medium obtained after size exclusion chromatography. Then we performed a proteomic characterization of these fractions by one-dimensional SDS gel electrophoresis coupled to mass spectrometry. We found that fractions in different mass regions have different activation properties with the most active being in the range 40-150 KDa. More than 20 proteins were identified in this range. Among them, many known interactors of BAG3 were detected, including 2 heat shock proteins (HSPA8 and HSP90AB1), suggesting a potential role of these proteins in the BAG3-mediated activation of immune response. *Corresponding author: Ada Consalvo, PhD Analytical Biochemistry and Proteomics Unit, Research Centre on Aging and Translational Medicine (Ce.S.I-MeT), “G. d’Annunzio” University, Via Luigi Polacchi 66100 Chieti, Italy Phone: +39 0871 541596 Fax: +39 0871 541598 E-mail address: [email protected] Reference: [1] Oncogene (2008) 27, 5904–5912. [2] Nature Communication (2015) 6, 1-11.


P-II-02

System biology analysis for dissecting the epithelial mesenchymal transition metabolic program

Daniele Vergaraa*, Anna Giudettia, Eleonora Stancaa, Stefania De Domenicob, Pasquale

Simeonec, Franck Juliend, Paola Lunettia, Loredana Capobiancoa, Giuseppe Nicolardia, Isabelle Fournierd, Michel Salzetd, Michele Maffiaa

a) Department of Biological and Environmental Sciences and Technologies, University of Salento,

Lecce, Italy; b) Institute of Food Production Sciences, C.N.R. Unit of Lecce, Lecce, Italy

c) Unit of Cytomorphology, CeSI-MeT and Department of Medicine and Aging Sciences, School of Medicine and Health Sciences, University “G. d’Annunzio”, Chieti, Italy

d) Laboratoire PRISM - U1192 INSERM, Université de Lille 1, Villeneuve d'Ascq, France The activation of the epithelial mesenchymal transition (EMT) program is accompanied by proteomic modifications including alterations in the expression of metabolic enzymes. Deciphering the functional implications of this dysregulated metabolism requires the analysis of multiple molecules including mRNAs, proteins, and lipids at omics level. Here, we applied a LC-MS/MS approach to define proteomic alterations in breast cancer models with epithelial and mesenchymal features. Significant changes in metabolic pathways promoting cell growth were identified. Overall, the metabolic protein expression program characterised mesenchymal models for a limited metabolic flexibility with a functional consequent dependence on specific metabolic substrates such as lipids. This metabolic phenotype reflects changes in lipid-signaling pathways and enzymes that satisfy the increasing uptake of lipids from the microenvironment. This is in difference with epithelial cells that metabolizes glucose in the mitochondria to produce fatty acids. We also demonstrated how these proteomic changes impact on lipid content profile by in situ lipidomic analysis of our models. Moreover, metabolic alterations identified from MS profiling were integrated with genomic and transcriptomic data revealing a comprehensive and complex picture of regulation. In the contest of EMT metabolism, this omics approach improved our understanding of the genetic and molecular events associated with the metabolic phenotype of epithelial and mesenchymal cells. We gratefully acknowledge funding from the Apulia Regional Cluster project “SISTEMA” project code

T7WGSJ3

* Corresponding author: Daniele Vergara. University of Salento, Lecce, Italy. E-mail address: [email protected] (D. Vergara)


POSTERS: Session III technologies and bioinformatics 69

P-III-01 An integrated procedure for the simultaneous analysis of mt-HPP shotgun

data

Mauro Fasanoa*

a) Center of Bioinformatics and Dept. Of Science and High Technology, University of Insubria, Busto Arsizio, Italy;

The mitochondrial B/D-HPP consortium has been committed in a standardization project, where mitochondria were isolated from ten cell lines by several procedures and analyzed by shotgun proteomics. Samples were produced in three biological replicates. MS data were processed by Peaks-Studio, thus yielding 63 datasets to be analyzed. Tables were in the comma separated values (.csv) format. The file names were written according to the following syntax: Cellname Operator Method Instrument_PREPn.csv. The tabulated information contains, among the others, the following columns: Column 3: UniProtKB Entry Name; Column 4: Significance (-10LogP); Column 9: Molecular Mass; Column 10: Description (e.g., Filamin-A OS=Homo sapiens GN=FLNA PE=1 SV=4). All csv files in the working directory were iteratively opened three at a time for the three biological replicates of the same preparation. Attributes (i.e. Cell line, isolation method) were obtained by parsing the file name. Gene names were obtained from the field “Description” using “GN=” and “ “ as text separators. After removal of duplicated gene names, amounts are computed as the ratio between Significance and Molecular Mass and normalized with respect to the total amount in each experiment. Normalized amounts lower than 5×10-4 were removed to reduce the number of less-represented identifications. Eventually, only proteins observed in at least two out three biological replicates were considered in the following procedures. A summary table reporting identities and computed amounts for the three biological replicates of each preparation was written as a .csv file. Gene name lists were then translated into non-redundant UniProtKB accession numbers and saved as .txt files in the same directory. Gene names were then mapped to Mitocarta and IMPI databases to extract identities associated to mitochondria. Eventually, subcellular location for all identified proteins was obtained from UniProtKB. The most represented categories were “Mitochondria”, “Cytoplasm”, “Nucleus” and “Endoplasmic Reticulum”. For each category, association with the extraction method was obtained by non-parametric Kruskal-Wallis test followed by the post-hoc Dunn test. The procedure runs in the R environment and will be available for demonstration during the Poster Session.

* Corresponding author: Mauro Fasano. Insubria University, via Manara 7, Busto Arsizio, Italy. Phone.: +39 0331 339450; Fax: +39 0332 395599. E-mail address: [email protected] (M. Fasano)



P-III-02

MARNORA: a tool to perform Meta-Analysis, Reaction Network, Over Representation Analysis.

Chiara Monti a,b,*, Mauro Fasano a,b , Simone Tini a,b, Tiziana Alberio a,b

a) Department of Science and High Technology, University of Insubria, Busto Arsizio/Como, Italy;

b) Center of Bioinformatics, University of Insubria, Italy;

The omics techniques, including proteomics, can be used to investigate pathogenetic mechanisms of multifactorial disorders such as Parkinson’s Disease (PD). To get a picture as complete as possible, it is possible to analyze at the same time results coming from all the proteomics studies about the subject of interest (meta-analysis) [1,2]. In this project, an automated process to execute the meta-analysis semi-automatically was developed. To this purpose, we wrote a routine in R [3], which searched in PubMed original proteomics articles focusing on PD. We filtered out papers with data obtained by the analysis of peripheral fluids. To select papers using a shotgun proteomic approach, we searched in the full text of the publication the keyword “shotgun”. Eventually, the publications were manually inspected. Studies were included only if they were case–control studies. Altogether, 538 literature hits were identified through the PubMed database. The automatic filter allowed us to eliminate biomarker studies or papers not dealing with the shotgun proteomics approach. Eventually, 30 full-text articles were included in the meta-analysis. The list of all proteins found to be altered in PD included 2961 proteins. The proteins list extracted can be analyzed with MARNORA to generate protein-protein interaction and/or functional networks and to perform an over representation analysis using several reference databases. This should be an interesting strategy both for the proteomics community, to better interpret experimental data, and for scientists outside the community, to exploit big data set and generate new hypotheses in their field of interest. * Corresponding author: Chiara Monti PhD Student Department of Science and High Technology Laboratory of Biochemistry and Functional Proteomics University of Insubria, via Manara 7, I-21052 Busto Arsizio (VA) Phone +39-0331-339423 Reference: [1] Monti C, Bondi H, Urbani A, Fasano M, Alberio T. Systems biology analysis of the proteomic alterations induced by MPP(+), a Parkinson's disease-related mitochondrial toxin. Front Cell Neurosci. 2015. doi: 10.3389/fncel.2015.00014. [2] Monti C, Colugnat I, Lopiano L, Chiò A, Alberio T. Network Analysis Identifies Disease-Specific Pathways for Parkinson's Disease. Mol Neurobiol. 2016. doi: 10.1007/s12035-016-0326-0. [3]RStudio Team (2015). RStudio: Integrated Development for R. RStudio, Inc., Boston.

POSTERS: Session III technologies and bioinformatics 71

P-III-03

New innovations implemented on the Q Exactive HF mass spectrometer

Tabiwang N. Array1; Sega Ndiaye2; Eugen Damoc1; Erik Couzijn1; Jens Grote1; Oliver Lange1; Christian Thoeing1; Kerstin Strupat1; Catherina Crone1; Anastassios Giannakopulos1,

Thomas Moehring1 and Alexander Harder1

1Thermo Fisher Scientific, Bremen, GERMANY 2Thermo Fisher Scientific, Paris, FRANCE

We made both hardware and software changes to enhance the performance of the Q Exactive HF instrument. A brighter ion source interface was realized by replacing the heated capillary with a modified design with higher throughput, and replacing the S-lens with an ion funnel for improved ion transmission. On the modified Q Exactive HF instrument, a brighter ion source was implemented with improved ion transmission. Using our standard positive calibration mixture at a flow rate of 5 uL/min, we observed up to fourfold improvement in ion transmission; injection times were approximately 2.5 times shorter and electrometer currents correspondingly higher relative to the unmodified Q Exactive HF instrument. The modified instrument also showed reduced solvent cluster formation in the bent flatapole relative to the unmodified version. Through optimization of the electronics switching times and the measurement strategy, combined with a resolution setting of 7,500 @ m/z 200, a maximum scan speed of over 40 Hz can be attained. The productivity of this resolution setting was verified using a complex HeLa digest sample. We could perform a full scan (6,'000 resolution @ m/z 200) and 40 MS/MS (7,500 resolution @ m/z 200) in one second on the modified instrument. On average, 25,000 unique peptides were identified in 30 min (58 min total run time); the same number of identified peptides could only be achieved on the unmodified Q Exactive HF using a 60 min gradient. A direct comparison with a 60 min gradient on the modified instrument afforded approximately 39% more identified peptides. * Corresponding author: Sega NDIAYE. Thermo Fisher Scientific Les Ulis Paris France E-mail address: [email protected] (S. Ndiaye)



P-III-04

Enrichment strategies for improvement of mass spec analysis of chemical cross-linked peptides

Rosa Viner1; RYAN BOMGARDEN2; Kratika Singhal2; Sergei Snovida2; Craig Gutierrez3;

Lan Huang3

1Thermo Fisher Scientific, San Jose, CA; 2Thermo Fisher Scientific, Rockford, IL;

3UC Irvine Department of Physiology & Biophysics, Irvine, CA Chemical cross-linking in combination with mass spectrometry is a powerful method to determine protein-protein interactions. This method has been applied to recombinant and native protein complexes and, more recently, to whole cell lysates or intact unicellular organisms in efforts to identify protein-protein interactions on a global scale. However, this method suffers from low identification rates without enrichment/fractionation, as the typical yield of cross-linked peptides is less than 1 % of total identified peptides. In this study, we evaluated multiple, widely used enrichment/fractionation techniques andbenchmarked newly developed SCX spin columns for cross-linked peptide analysis using an Orbitrap Fusion Lumos mass spectrometer. Methods Different amine-reactive, homobifuctional crosslinkers including DSS, DSSO and BuUrBu were used to crosslink protein and protein complex standards. Cross-linked samples were reduced, alkylated and digested with trypsin for MS analysis. Protein and peptide concentrations were determined using the Pierce™ BCA Protein Assay Kit and the Pierce™ Quantitative Colorimetric Peptide Assay, respectively. Cross-linked peptides were pre-fractionated on SCX stage tips, SCX spin columns and SEC Superdex Peptide PC column (GE Health). Enriched samples were separated using a 50cm Thermo Scientific™ EASY-Spray™ column and an EASY-nLC™ 1200 UPLC system in 60min gradient, followed by detection on the Thermo Scientific™ Orbitrap™ Fusion Lumos™ mass spectrometer. Data were analyzed using Thermo Scientific™ Proteome Discoverer™2.2 software and XlinkX node. * Corresponding author: Sega NDIAYE. Thermo Fisher Scientific Les Ulis Paris France E-mail address: [email protected] (S. Ndiaye)


POSTERS: Session IV human proteomics 73

P-IV-01

Iron depletion alters mitochondrial signaling and metabolism in breast cancer cells

Maida De Bortoli1, Elena Taverna1, Elisa Maffioli2, Patrizia Casalini1, Gabriella Tedeschi2

and Italia Bongarzone1

1Laboratorio di Proteomica, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano 2 Dipartimento di Medicina Veterinaria – Università degli Studi di Milano

Iron is an essential element for cellular metabolism and an adequate iron supply is critical for various cellular processes including DNA synthesis and cell cycle progression. Iron-depletion results in cell cycle arrest and also apoptosis although their mechanism(s) of action have only recently been more comprehensively elucidated. Conclusive evidence, however, has not yet been adduced to prove the effects of iron depletion agents in cancer therapy. We combined biochemistry, microscopy, flow cytometry and liquid chromatography tandem-mass spectrometry (LC-MS/MS) analyses to investigate cellular and molecular events induced by desferrioxamine (DFO) or thiosemicarbazone (Dp44mT), on two human breast cancer cell lines, MDA-MB-231 and MDA-MB-157. We observed a persistent cellular stress and organelle dysfunctions because of disruption of cellular homeostasis leading to cell death. Quantitative global proteomic analysis of the stressed cells before onset of cell death, showed deregulation of a number of proteins involved in mitochondria homeostasis; protein network analyses identified upregulated metabolic pathways that may be associated with stress tolerance and pro-survival mechanism. Iron depletion produced a cascade of reactions were initiated by conditions stimulated mitochondria to release reactive oxygen species (ROS) and by the impairment of iron prolyl hydroxylase (PHD) enzymes resulting in the stabilization of hypoxia inducible factors (HIFs) and lipid accumulation. This study suggests that there is an intricate connection between iron depletion, hypoxia, stress tolerance and lipid metabolism. However, more studies are needed to understand the correlation between molecular mechanisms of action that have been identified for iron chelators and their clinical implications. * Corresponding author: Italia Bongarzone Laboratorio di Proteomica, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano Phone.: +393455364726 E-mail address: [email protected]



P-IV-02

MAPPING OF TRANSGLUTAMINASE-2 REACTIVE GLUTAMINE RESIDUES IN SEVERAL HUMAN SALIVARY PROLINE-RICH

PEPTIDES BY MS/MS.

Mozhgan Boroumanda, Federica Iavaroneb, Federica Vincenzonib, Alessandra Padigliaa, Massimo Castagnolab,c, Irene Messanac, Tiziana Cabrasa.

aDipartimento di Scienze della Vita e dell’Ambiente – Università di Cagliari

bIstituto di Biochimica e Biochimica Clinica- Facoltà di Medicina-Università Cattolica, Roma cIstituto di Chimica del Riconoscimento Molecolare CNR- Roma, Italy

Transglutaminase-2 (TG-2) is an ubiquitous enzyme responsible for the generation of a cross-link between a lysine (the lone-pair donor) and a glutamine residue (the lone-pair acceptor) with the loss of an ammonia molecule. TG-2 is a Ca++-dependent enzyme, negatively modulated by GTP and by the reversible formation of an internal Cys-Cys bridge. TG-2 is the principal transglutaminase present in oral cavity, released by the epithelial cells, and plays a principal role in the formation of the “protein pellicle” covering the oral epithelia. Due to the distinctive features of the oral epithelia in wound repair, determination of the proteins involved and the mechanism of its formation is demanding. Previous studies demonstrated participation of acidic proline-rich proteins and statherin, proteins secreted by salivary glands, to the “oral pellicle formation” [1]. We present in this study preliminary data supporting the potential involvement of basic proline-rich proteins (bPRPs). bPRPs are the most complex and polymorphic class of salivary proteins and, due to great structural similarities, their purification is challenging. We were able to purify several members of this family by different steps of preparative gel filtration and RP-HPLC. Some purified bPRPs (P-C, P-H, P-D) were incubated in vitro with TG-2 in the presence of monodansyl-cadaverine to map potential reactive glutamine sites. HPLC- High resolution MS and MS/MS analyses of the reaction products showed that these bPRPs are active substrates for TG-2 and that only specific glutamine residues are able to react with monodansyl-cadaverine. * Corresponding author: Mozhgan Boroumand. Dipartimento di Scienze della Vita e dell’Ambiente- Università di Cagliari, Cagliari, Italy. Phone.: +39-070-675-4500; Fax: +39- 070-675-4523 E-mail address: [email protected] References: [1] Yao, Y., Lamkin, M. S., Oppenheim, F. G., J. Dent. Res. 2000, 79, 930–938..



P-IV-03

Characterization of lipid metabolism in the differentiation of oligodendrocytes in myelinating forms

Ilaria Cicalinia*, Daniele Vergarab, Damiana Pieragostinoc, Lorenza Falconea, Patrizia

Ballerinid, Nicola Petragnanid, Stefania De Domenicoe ,Michele Maffiab, Piero Del Boccioa

aDep. of Pharmacy, “G. d’Annunzio” University of Chieti-Pescara, Chieti, Italy. b Dep. of Biological and Environmental Sciences and Technologies, University of Salento, Lecce,

Italy cDep. of Medical, Oral and Biotechnological Sciences, “G. d’Annunzio” University of Chieti-

Pescara, Chieti, Italy dDep. of Psychological Sciences, Health and Territory, “G. d’Annunzio” University of Chieti-Pescara,

Chieti, Italy eInstitute of Food Production Sciences, C.N.R. Unit of Lecce, Lecce, Italy

Multiple Sclerosis (MuS) is a neurodegenerative disease linked to an autoimmune response to the oligodendrocytes-produced myelin. Since axonal damage is an early event, the study of an oligodendrocytes cell model would provide crucial information about the aetiology of disorder. Starting from the known involvement of lipid metabolism in the pathology, our purpose was to characterize lipid metabolism in the maturation process of the oligodendrocytes in the differentiate form. Oligodendrocytes cell line (MO3.13) was used to characterize the metabolic profiling of the precursor form, focusing to key enzymes involved in lipid uptake and triacylglycerols (TAGs) synthesis. WB experiments show that MO3.13 is characterized by low levels of fatty acids synthesis, high lipid uptake and TAG synthesis. The cells were then subjected to a differentiation protocol by adding phorbol myristate acetate (PMA) to the serum-free medium. Differentiation marker (GFAP), lipid metabolism markers (FASN and MGAL), and metabolic regulator (SIRT1) levels were evaluated by WB experiments and confirmed by qPCR. Data show that PMA positively influences the migration and lysis of TAGs, instead, the absence of serum is sufficient to modulate SIRT1 expression and differentiate the cell line by escaping the astrocytic phenotype. Finally, MO3.13 were analysed through MALDI-TOF analysis in order to characterized in situ lipidomic profiling of precursor cells comparing to the differentiated form. In conclusion, we suppose the involvement of lipid metabolism in oligodendrocytes differentiation. These experimental evidences may contribute to better understand the biological mechanisms cause and effect of MuS, contributing both to diagnostic and pathogenetic purposes. - This study is supported by FISM, Fondazione Italiana Sclerosi Multipla cod 2015/R/12 and by ItPA Foundation (award for young researchers) *Corresponding author: Ilaria Cicalini, PhD student Analytical Biochemistry and Proteomics Unit, Research Centre on Aging and Translational Medicine (Ce.S.I-MeT), “G. d’Annunzio” University, Via Luigi Polacchi 66100 Chieti, Italy Phone: +39 0871 541586 E-mail address: [email protected]



P-IV-04

Comparative proteomic approach of mitochondrial proteins in a mouse model for creatine transporter deficiency

Federica Ciregiaa*, Claudia Boldrinia, Angelo Molinarob, Laura Barocellib, Tommaso

Pizzorussob, Maurizio Roncic, Andrea Urbanid , Maria Rosa Mazzonia, Antonio Lucacchinia, Laura Giustia

aDepartment of Pharmacy, University of Pisa, Pisa, Italy

bInstitute of Neuroscience, National Research Council, Pisa, Italy c Department of Medical, Oral and Biotechnological Sciences, University G. d'Annunzio of Chieti-

Pescara, Chieti, Italy dIstituto di Biochimica e Biochimica Clinica, Università Cattolica, Roma, Italy

Mutations in the creatine (Cr) transporter (CrT) gene lead to cerebral Cr deficiency syndrome-1 (CCDS1), an X-linked metabolic disorder characterized by cerebral Cr deficiency causing intellectual disability, seizures, movement and autistic like behavioral disturbances, language and speech impairment. The lack of knowledge about the effects of Cr deficiency on neuronal circuits derives at least partially from the paucity of studies on animal models. Evidence of mitochondrial dysfunction in animal models of Cr deficiency suggests that mitochondrial function may also be abnormal. In this study for the first time we investigate the mitochondrial proteome in a CCDS1 mouse model with the aim to show the potential protein alterations induced by CCDS1. Mitochondria were obtained by differential centrifugation from brain of wild type (CrT +/y, n=3) and knock-out (CrT -/y, n=3) mice sacrificed at 30 days-old. Mitochondrial proteins of CrT +/y and CrT -/y samples were separated by 2DE and proteomic profiles compared by Same spot software. Fifty-five spots resulted differentially expressed in significant manner in CrT -/y with respect to CrT +/y , in particular 14 spots showed fold > 2. All these spots were increased in CrT-/y deficiency mice. Spots of interest were cut and analyzed by mass spectrometry. Identified proteins appertained essentially to mitochondrial respiratory chain, and to oxidative stress. Finally, Ingenuity Pathways analysis was also performed and the network generated involves developmental and hereditary disorder and metabolic disease. Overall our results suggest that the lack of uptake of creatine actives the processes of ATP production and as a consequence the increase of systems involved in controlling oxidative stress. * Corresponding Author: Federica Ciregia, Phd University of Pisa Via Bonanno 6, 56126 Pisa, Italy. Phone.: +390502219535 E-mail address: [email protected]



P-IV-05

The proteomic map of porcine claustrum: what differences in the protein expression in insula and putamen tell us about the claustrum ontogenetic

origin?

Federica Ciregiaa*, Laura Giustia, Andrea Pironeb, Vincenzo Miragliottab, Bruno Cozzic, Maurizio Roncid, Maria Rosa Mazzonia, Antonio Lucacchinia

aDepartment of Pharmacy, University of Pisa, Italy bDepartment of Veterinary Sciences, University of Pisa, Italy

cDepartment of Comparative Biomedicine and Food Science, University of Padova, Italy d Department of Medical, Oral and Biotechnological Sciences, University G. d'Annunzio of Chieti-

Pescara, Chieti, Italy The claustrum (CL) is a subcortical nucleus present in all mammalian species examined so far, including man. Its structure, function and origin are still a matter of debate. Over the past decades, the studies employing the pig brain as a model for neurochemical studies has dramatically increased. The key translational features of the pig brain are its size, and the similarities with the cortical and subcortical structures of the human brain. The aim of our study is to define, for the first time, the proteomic map of porcine CL and to compare it with those of putamen and insula to identify proteins able to characterize this brain area. Moreover, another goal is to obtain information on the CL ontogenesis. After the sacrifice of pigs, tissue from the three different areas were immediately excised and stored at -80°C until the use. Tissues were resuspended in rehydration solution and protein extracts separated by two-dimensional electrophoresis. Images were acquired by Image Quant LAS4010 and a comparative analysis was carried out using SameSpots software. Principal component analysis was performed for different comparisons to obtain a list of spots able to characterize the different areas. Ten spots resulted over-expressed in significant manner with fold > 2 after comparison of CL vs insula and 23 after comparison of CL vs putamen. Spots of interest were cut and analyzed by mass spectrometry. The identification of proteins typical for the three different areas are in progress. Our preliminary results identify differences between protein expression in the CL and surrounding insula and putamen. The more significant dissimilarities with the last one suggest a clear different ontogenetic origin between the CL and putamen. * Corresponding Author: Federica Ciregia, Phd University of Pisa Via Bonanno 6, 56126 Pisa, Italy. Phone.: +390502219535 E-mail address: [email protected]



P-IV-06

ProLyPALS: Proteomics of Lymphocytes from Parkinson’s Disease and Amyotrophic Lateral Sclerosis patients

Ilaria Colugnata*, Chiara Montia, Chiara Sironia, Leonardo Lopianob, Adriano Chiòb, Alice

Di Pierroc, Cristoforo Comid, Mauro Fasanoa, Tiziana Alberioa

a) Department of Science and High Technology, Center of Neuroscience, University of Insubria,

Busto Arsizio, Italy. b) “Rita Levi Montalcini” Department of Neuroscience, University of Torino, Torino, Italy.

c) Department of Health Sciences, University of Piemonte Orientale, Novara, Italy. d) Movement Disorders Centre, Neurology Unit, Department of Translational Medicine, Univeristy

of Piemonte Orientale, Novara, Italy.

Parkinson’s Disease (PD) and Amyotrophic Lateral Sclerosis (ALS) are neurodegenerative disorders characterized by the progressive loss of specific neurons in selected regions of the central nervous system whose etiology is mostly unknown [1]. Nevertheless, several studies have shown that PD and ALS share common pathogenic mechanisms. Indeed, the frequency of extra-pyramidal symptoms in ALS patients is higher than in the general populations [2]. The aim of the present research was to characterize the proteome of Peripheral Blood Mononuclear Cells (PBMC) derived from 20 riluzole-treated ALS patients, 20 de-novo patients with ALS, 20 de-novo PD patients and 20 ALS patients with PD symptoms (ALS-PD). Total proteins (500 µg) were separated by two-dimensional electrophoresis (2-DE) using 18 cm IPG DryStrips with nonlinear 3-10 pH gradient followed by 12.5% SDS-PAGE. Maps were stained with Ru(II)-tris-bathophenanthroline disulfonate. Significant spots were excised and submitted to trypsin digestion. Peptides were separated by nanoflow-HPLC Chip system and the resulting proteins were identified by MS/MS and database search. To this purpose, as a proof of principle, we achieved 2-DE analysis of PBMC collected by 10 PD patients, 10 ALS patients treated with riluzole, 10 ALS patients and 5 ALS-PD. We identified different proteins altered in ALS patients treated or not with riluzole and we decided to exclude them because these proteins were therapy-sensitive. We also identified various proteins significantly altered in PD, ALS and ALS-PD patients that allowed us to contradistinguish the different groups of patients by principal component analysis and hierarchical cluster analysis. * Corresponding author: Ilaria Colugnat. PhD student. Department of Science and High Technology, Center of Neuroscience, University of Insubria. Via Manara 7, I-21052 Busto Arsizio (VA), Italy. +39-0331-339414; E-mail address: [email protected] References: [1] Agrawal M, Biswas A (2015) Molecular diagnostics of neurodegenerative disorders. Front Mol Biosci 2:54. [2] Körner S, Kollewe K, Ilsemann J, Müller-Heine A, Dengler R, Krampfl K, Petri S (2013) Prevalence and prognostic impact of comorbidities in amyotrophic lateral sclerosis. Eur J Neurol 20: 647–654.



P-IV-07

Potential new role for PRUNE during microtubule polymerization and cytoskeletal rearrangements

Flora Cozzolino1,§, Maria Monti1,§, Veronica Ferrucci1,2,3, Angela di Somma§, ,Francesco

Pennino1,2, Fatemeh Asadzadeh1, Iolanda Scognamiglio2, Angela Duilio§, Massimo Zollo1,2,3, Pucci Piero1,§

1-CEINGE Biotecnologie Avanzate, Naples, Italy §- Department of Chemical Sciences, University “Federico II” of Naples, Italy

2- DMMBM Department of Molecular Medicine and Medical Biotechnology, Naples, Italy 3- SEMM European School of Molecular Medicine, University of Milan, Italy

Genetic studies found biallelic mutations in PRUNE-1 genes in several families with tubulinopathies and neurodevelopmental disorders showing primary Microcephaly and clinical features of PEHO/PEHO-like syndrome, including cerebellar and optic nerve atrophy (1; 2). Neurodevelopmental disorders with intellectual disabilities and brain size malformations are known to be caused by perturbations in neuronal progenitor cells proliferation, migration and differentiation (5). Prune-1 belongs to DHH-phosphoesterase superfamily with an exopolyphosphatase (PPase/PPX) activity (3) strongly expressed in developing brain and cerebellum (4) with a role in proliferation and motility. To gain further insight into the molecular mechanism by which PRUNE-1 regulate neurogenesis, we performed pull-down experiments followed by mass-spectrometry analyses using a breast carcinoma cellular model (MDA-MB231T) overexpressing FLAG-tagged-PRUNE-1 wild type protein. Novel protein interactors of PRUNE-1were identify. Among these we found different proteins with a role in cytoskeleton organization during cell division as potential PRUNE-1 binding partners. We focused our effort on the potential interaction between PRUNE-1 and β-Tubulin. In order to validate the interaction between PRUNE-1 and β-Tubulin, co-immunoprecipitation assays were performed using Neuroblastoma cell clones (SHSY5Y) previously engineered to induce both Wild Type or mutated (D30N and R297W) PRUNE-1 proteins in presence of Doxycycline (1). The immunoprecipitation assays showed that both Wild Type and mutated PRUNE-1 proteins have the ability to bind both β- and α-Tubulin (1). Furthermore, in vitro polymerization assays demonstrated that Wild Type PRUNE-1 significantly enhances the microtubule polymerization, while the presence of both mutants caused a delay in microtubule assembly processes. Consequently, PRUNE-1 may have a role during cell division thus regulating neuronal progenitor proliferation processes during brain/cerebellum development. Altogether these data show that PRUNE-1 syndrome as a novel tubulinopathy with both neurodevelopmental and degenerative components. Corresponding author: Prof. Piero Pucci. Department of Chemical Sciences, University “Federico II” of Naples, Via Cintia 4, 80125, Naples Phone.: +39 081674318; +39 0813737896 Fax: +39 0813737808. E-mail address: [email protected] (P. Pucci) References:

1. Zollo et al. Brain. 2017 Apr 1;140(4):940-952 2. Karaca E et al., Volume 88, Issue 3, 4 November 2015, pp. 499–513 3. A. D’Angelo et al. Cancer Cell, 5 (2004), pp. 137–149 4. Carotenuto P. et al., 2006 Aug;38 (3-4); pp. 233-46 5. Barbelanne M et al. 2014:547986. doi: 10.1155/2014/547986. Epub 2014 Dec 8


http://www.sciencedirect.com/science/journal/08966273/88/3

http://www.ncbi.nlm.nih.gov/pubmed/?term=Barbelanne%20M%5BAuthor%5D&cauthor=true&cauthor_uid=25548773


P-IV-08 Study of tears metabolome for the research of new molecular biomarkers

in multiple sclerosis

Damiana Pieragostinoa, Ilaria Cicalinib, Claudia Rossia, Maria Di Ioiac, Giovanna De Lucac, Mirco Zucchellia, Alessandra Lugaresid, Giuseppina Bolognae, Marco Marchisioe, Paola

Lanutie, Piero Del Bocciob*

aDep. of Medical, Oral and Biotechnological Sciences, “G. d’Annunzio” University of Chieti-

Pescara, bDep. of Pharmacy, “G. d’Annunzio” University of Chieti-Pescara, Chieti, Italy.

cDep. of Neuroscience, Imaging and Clinical Sciences, “G. d’Annunzio” University of Chieti-Pescara, Chieti, Italy

d Dep. of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italia eDep. of Medicine and Aging Sciences, “G. d’Annunzio” University of Chieti-Pescara, Chieti, Italy

To date the most important biomarkers for Multiple Sclerosis (MuS) diagnosis are the oligoclonal bands (OCBs) in CSF and Link Index. CSF is the body fluid that might better provide information about the pathological processes occurring in the CNS but it is obtained through an invasive procedure that cannot be easily repeated. By preliminary metabolomics study in CSF we demonstrated low levels of several sphingomyelins in MuS patients correlated to an up regulation of acid sphingomielinase (ASMase), highlighting an unbalance of the pathway sphingomyelin/ceramide in CSF. Tears could be considered as an intermediate fluid between the CSF and the serum. Tears of 12 MuS patients and 12 controls were obtained by Schirmer’s test, subjected to a methanol extraction that allowed to obtain a sample containing amino acids, acyl-carnitines and phospholipids which was subjected to LC-MS/MS screening analysis. Furthermore, tears exosomes and micro-vesicles (EMVs) were extracted by phosphate buffer solution and analyzed by polychromatic flow cytometry to characterize the amount and phenotype of tears EMVs. Metabolomics approach revealed a perturbation of aminoacids and carnitines metabolism and confirmed low levels of sphingomyelins in tears probably correlated to an unbalance of sphingomyelinase pathways already found in CSF. Moreover we explored and characterized the EMVs population in tears fluid, finding interesting aspects in term of number, subclasses and ASMase exposure in MuS. -This study was supported by FISM, Fondazione Italiana Sclerosi Multipla cod 2015/R/12 *Corresponding author: Prof. Piero Del Boccio, Department of Pharmacy, University “G. d’Annunzio” Chieti-Pescara, Chieti, Italy. Phone: +39 0871 541596 Fax: +39 0871 541598 E-mail address: [email protected]



P-IV-09 14-3-3 Proteins and their isoforms in breast cancer tissues

Gianluca Di Caraa*, Rosa Mussoa, Patrizia Cancemia,b, Nadia N. Albanesea, Salvatore Minafraa and Ida Pucci-Minafraa

a) Centro di Oncobiologia Sperimentale, University of Palermo and La Maddalena Hospital,

Palermo; b) Department of Biological, Chemical, and Pharmaceutical Sciences and Technologies

(STEBICEF), University of Palermo, Palermo, Italy. The oncological research is still mainly focused on the identification of the mechanisms which characterize the malignant phenotype, in order to improve the clinical approach to the patients. In this context, the proteomic approach is a powerful tool to identify the putative protagonists of biochemical pathways involved, as a cause or as a consequence, in the tumor progression. Here we report the results of a large-scale proteomic study on one hundred surgical fragments of breast cancer, collected at the La Maddalena Hospital from informed consenting patients and characterized for the clinical and molecular parameters. The tissue samples were carefully washed and homogenized for the protein extraction [1]. The protein concentration was determined by Bradford assay and aliquots of samples were subjected to the analytical and preparative IPG-2D electrophoresis. The gels were stained by ammoniacal silver staining and the interesting spots identified by MALDI-TOF (AbSciex) mass spectrometer. To date we identified 454 proteins (encoded by 271 genes) subdivided into two major groups on the basis of their occurrence in the proteomic maps and furtherly subclassified into functional clusters, according to the major gene ontology databases (NextProt, Panther, David). Among the identified proteins the members of the 14-3-3 protein family deserves a particular attention. The 14-3-3 human protein family includes 7 specific phospho-serine and -threonine binding proteins (14-3-3B, -E, -F, -G, -S, -T, -Z), involved in many biological processes related to the regulation of the cell cycle, the cell proliferation, the metabolic pathways, the protein trafficking, the cell migration and the cell survival, both in physiological and pathological conditions [2]. In our proteomic maps we have identified all the seven 14-3-3 proteins and have evaluated their different occurrence in the proteomes of the patients. Among these proteins, six (B, E, G, S, T, Z) resulted ubiquitously expressed in all the patients, while the expression of the 1433F resulted sporadic. The relationship between the expression of each 14-3-3 and other identified proteins were evaluated by applying a biostatistic (Pearson’s r) approach. The proteins whose espression showed a significant linear correlation with each 14-3-3 protein were selected for the bioinformatic analysis (String). The results provided a twofold evidence: firstly, all the 14-3-3 proteins seem to be involved in the pro-survival pathways in the tumor samples; secondly, each 14-3-3 protein seems to play a central role in different molecular mechanisms, crucial for the tumor progression. In conclusion, we believe that the results here reported strenghten the candidacy of the 14-3-3 proteins as usefull biomarkers for clinical applications to the breast cancer. *Corresponding author: Dr. Gianluca Di Cara Centro di Oncobiologia Sperimentale, Palermo, Italy Phone.: +390916806418 E-mail address: [email protected] References: [1] Pucci-Minafra I et al. Ann N Y Acad Sci 2002, 963:122-39. [2] Freeman AK et al. Semin Cell Dev Biol 2011, 22:681-7.



P-IV-10

An integrated cytologic and proteomic study on breast cancer cell lines with low and high motility properties

Rosa Musso, Gianluca Di Cara, Adriana Celesia and Ida Pucci-Minafra*

Centro di Oncobiologia Sperimentale, University of Palermo and La Maddelena Hospital, Palermo,

Italy. Breast cancer is the most common malignant tumor in women. The different types of breast cancer can be further classified as invasive and non-invasive forms. Among the invasive types, the ductal infiltrating carcinoma (DIC) is the most common and aggressive one. Using an in vitro model consisting of two cell lines derived from mammary cancers, the 8701-BC [1] with high aggressive potentialities, and the SKBR3 [2] very poorly tumorigenic, we investigated on differential cell behaviours, using integrated cytological and proteomics experimental approaches. The microscopic and immune-cytochemical observations highlighted significant phenotypic differences between the two cell lines, being the SKBR3 more regular in shape, more adhesive and stationary, with respect to the 8701-BC which display pleomorphic cell shapes and the presence of long cell surface protrusions, typical of very motile and potentially invasive neoplastic cells. To test the motility attitude of these cells, we performed the “scratch assay” on the individual cell lines: the results showed a high mobility response for the 8701-BC cells and a very low for the SKBR3. Subsequently, this assay was performed on the SKBR3 cultures grown in the presence 8701-BC conditioned medium. The experimental data showed an increase of the SKBR3 cell motility, due probably to the release of secretory molecules, either soluble or within secretory vesicles [3] by the 870-BC cells. The proteomic analysis highlighted several functional clusters involved in the cytoskeleton organization and cell motility, related to putative invasive phenotypes. Experiments are in progress to identify the nature of the signalling between the 8701-BC and the SKBR3 cell types. * Corresponding author: Prof. Ida Pucci-Minafra Centro di Oncobiologia Sperimentale, University of Palermo, Italy Phone/Fax.: +39 0916806418 E-mail address: [email protected] References: [1] Pucci Minafra I, et al. Cell Biol Int Rep. 1985, 9(3):291-6. [2] Lacroix M, et al. Breast Cancer Res and Treat. 2004, 83:249–289. [3] Palazzolo, G, et al. Anticancer Res. 2012, 32:847-60.



P-IV-11

Proteomic Approaches to Understand the cytotoxic, apoptotic and antitumoral effects of new N-heterocyclic carbene–gold(I) complexes

Francesca Magherinia, Tania Fiaschia, Alessandra Modestia, Chiara Gabbianib, Alessandro

Pratesic, Tiziano Marzoc, Ida Landinid, Stefania Nobilie, Enrico Minid, Luigi Messoric, Tania Gamberia

a) Dipartimento Scienze Biomediche, Sperimentali e Cliniche “Mario Serio” Università degli Studi di

Firenze, Italia b) Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Italia

c) Dipartimento di Chimica “Ugo Schiff”, Università degli studi di Firenze, Italia d) Dipartimento di Medicina Sperimentale e Clinica, Università degli Studi di Firenze, Italia

e) Dipartimento di Scienze della Salute, Università degli Studi di Firenze, Italia

Au(NHC) and Au(NHC)2 are two novel gold(I) carbene compounds endowed with potent cytotoxic properties in vitro toward various cancer cell lines [1]. Both complexes produce strong cytotoxic effects in A2780 human ovarian cancer cells resulting in apoptotic cell death. Comparative proteomic analysis pointed out that the number of modulated proteins was far greater in Au(NHC)2-treated cells (50 proteins) than in Au(NHC)-treated cells (20 proteins). Both carbene complexes affected proteins belonging to protein synthesis, metabolism, cytoskeleton and stress response and chaperones. Notably, only Au(NHC)2 elicited a conspicuous down-regulation of some glycolytic enzymes. To obtain a more detailed view of these metabolic differences we evaluated the ability of these gold(I) carbenes to affect glycolytic and mitochondrial activity. It was found that only Au(NHC)2 caused an impairment of respiration and a metabolic shift towards glycolysis probably as a compensatory mechanism, suggesting mitochondria as the possible cellular target. Mitochondria had already been identified as primary cellular target for the gold(I) compound Auranofin most probably through inhibition of the seleno-enzyme thioredoxin reductase (TrxR) [2]. We demonstrated that Au(NHC)2 was the most effective inhibitor of TrxR. In conclusion, in terms of structure–activity relationships, we can state that the introduction of the second carbene ligand in Au(NHC)2 greatly enhances the antiproliferative effects of this gold(I) carbene complex. We also demonstrated that Au(NHC)2 inhibits TRXR activity leading to a metabolically stressed phenotype. Corresponding Author: Tania Gamberi, Dr. Dipartimento Scienze Biomediche, Sperimentali e Cliniche “Mario Serio”, Università degli Studi di Firenze. Viale GB Morgagni n.50, 50134 Firenze, Italia Phone. +390552751234 Fax. +390552751201 E-mail address: [email protected] References: [1] Messori L, Marchetti L, Massai L, Scaletti F, Guerri A, et al. Chemistry and biology of two novel gold(I) carbene complexes as prospective anticancer agents. Inorg Chem. 2014; 53:2396-403. [2] Rigobello MP, Messori L, Marcon G, Agostina Cinellu M, el al. Gold complexes inhibit mitochondrial thioredoxin reductase: consequences on mitochondrial functions. J Inorg Biochem. 2004; 98:1634-41.





P-IV-12

Dissecting the TRIM8 role in the pathogenesis of glioblastoma

Ilaria Iacobucci1, Diana Canetti1, Flora Cozzolino1, Pietro Pucci1, Santina Venuto2, Giuseppe Merla2, Lucia Micale2, Maria Monti1

1Department of Chemical Sciences and CEINGE Advanced Biotecnologies, University of Naples

"Federico II", Naples, Italy 2Medical Genetics Unit, I.R.C.C.S. "Casa Sollievo della Sofferenza" Hospital, San Giovanni

Rotondo, 71013, Italy Tripartite motif (TRIM) proteins constitute a superfamily that share a conserved motif architecture known as RBCC. Some TRIM proteins possess E3-ubiquitin ligase activity and are involved in many biological processes and changes in their abundance or activity are associated with several pathological conditions. In particular, TRIM8 controls multiple physiological functions, including inflammation, cell survival, differentiation and exerts central roles in cancer. TRIM8 is aberrantly expressed in glioblastoma, a lethal brain tumor that arises from glial cells, and its expression inversely correlates with tumor grade. In order to find novel approaches for glioblastoma treatment, molecular mechanisms responsible of the glioma onset and involving TRIM8 were investigated by a functional proteomic approach. Immunoprecipitation experiments and LC/MSMS analyses were performed on mouse embryonic neurospheres cells expressing FLAG-Trim8 in order to purify and identify protein complexes involving FLAG-Trim8. Cells expressing an empty vector were used as negative control. Co-immunoprecipitation experiments were carried out and interaction analysed by western blotting. Almost 50 TRIM8 putative interacting proteins were identified and clustered in biological processes and pathways according to UniProt annotation. Eight mitosis-related protein (22%) were identified; four of them are members of the kinesin-like protein family. Co-immunoprecipitation assays showed that Trim8 physically bound to previously identified Kif11, Kif2c, Kifc1, and Haus. Hence, functional relationships between Trim8 and mitotic motor proteins were also explored. Our data show that TRIM8 is involved in regulation of mitotic spindle machinery: during mitosis, different dynamic morphological transitions are coordinated in a temporal and spatial manner through ubiquitination of key mitotic factors, providing directionality and fidelity to this process. TRIM8 aberrations lead to mitotic checkpoint defects, common also in human glioma cancers. Corresponding author: Prof.ssa Maria Monti. Department of Chemical Sciences, University “Federico II” of Naples, Via Cintia 4, 80125, Naples Phone.: +39 081674414; +39 0813737919 Fax: +39 0813737808. E-mail address: [email protected] (M. Monti)



P-IV-13

Top-Down proteomic analysis of Gingival crevicular fluid in deciduous, permanent and exfoliating teeth in children

Federica Marinia, Romeo Patinib, Laura Di Tonnob, Tiziana Cabrasc, Maria Teresa Sannac,

Alessandra Olianasc, Barbara Manconic, Massimo Cordarob, Irene Messanad, Massimo Castagnolaad, Federica Iavaronea*

aIstituto di Biochimica e Bioch. Clinica-Fac. di Medicina-Università Cattolica, Roma

bIstituto di Clinica Odontostomatologica-Fac. di Medicina-Università Cattolica, Roma

cDip. di Scienze della Vita e dell’Ambiente, Università di Cagliari

dIstituto di Chimica del Riconoscimento Molecolare CNR-Roma. Gingival crevicular fluid (GCF) arises from the gingival plexus of blood vessels in the gingival corium subjacent to the epithelium lining of the dento-gingival space. GCF basically contains local breakdown products such as tissues, inflammatory mediators, host inflammatory mediators, serum transudate found in gingival sulcus, subgingival microbial plaque, extracellular proteins, and cells. Composition of GCF varies between periodontium in healthy and diseased conditions [1]. Analysis by Top-down platforms have evidenced that this fluid contains high concentrations of α-defensins [2], Tβ4 and Tβ10 [3] in adults. α-defensins probably originates from different leukocyte families transmigrating in the gingival pocket, while the origin of β-thymosins is currently unknown. Both peptides could modify their concentrations in several pathologies, offering new opportunities for early diagnosis of local and systemic diseases by this fluid. In the present study GCF was collected from deciduous, exfoliating and permanent teeth of a sample of 20 children (8M 12F mean age:9, min:8 yr.o.; max:10 yr.o.) with the aim to characterize and quantify by a Top-down approach the peptidome of GCF and verify possible variations occurring during the exfoliating process. HPLC-ESI- high-resolution MS-MS experiments were carried out by an Ultimate 3000 Micro HPLC (Dionex) coupled to an Orbitrap Elite (ThermoFisher). High resolution MS and MS2 data were elaborated both manually and by means of Protein Discoverer 2.1 software (2016, ThermoFisher Scientific), based on SEQUEST cluster as search engine against the Swiss-Prot human database. The results obtained confirmed the presence of α-defensins, Tβ4 and Tβ10 and its fragments and revealed the presence of other interesting peptides such as specific fragments of alpha-1-antitrypsin, alpha-1-antichymotrypsin, fibrinopeptide A and B, LVV Hemorphin-7, S100, as well as some other bioactive peptides deriving from α and β subunits of hemoglobin. These peptides showed a statistically significant different level in GCF deriving from deciduous, exfoliating and permanent teeth suggesting a biological role in processes like inflammation and dentition. *Corresponding author: Dr. Federica Iavarone, Ist. di Biochimica e Biochimica Clinica, Facoltà di Medicina, Università Cattolica, Largo F. Vito, 00168, Roma, Italy. Phone and/or Fax: ++39-06-3053598; E-mail address: [email protected] References: [1] Barros, S.P.;Williams, R.; Offenbacher, S.; Morelli, T. Gingival crevicular fluid as a source of biomarkers for periodontitis. Periodontol. 2000 2016, 70, 53–64. [2] Pisano E., Cabras T., Montaldo C., Piras V., Inzitari R., Olmi C. Castagnola M., Messana I. Peptides of human gingival crevicular fluid determined by HPLC-ESI-MS, Eur. J. Oral Sci., 2005; 113, 462-468. [3] Inzitari R., Cabras T., Pisano E., Fanali C., Manconi B., Scarano E., Fiorita A, Paludetti G, Manni A, Nemolato S, Faa G, Castagnola M, Messana I. HPLC-ESI-MS analysis of oral human fluids reveals that gingival crevicular fluid is the main source of oral thymosins beta(4) and beta(10), J. Se.p Sci., 2009; 32, 57-63.


http://www.ncbi.nlm.nih.gov/pubmed?term=Fiorita%20A%5BAuthor%5D&cauthor=true&cauthor_uid=19035385

http://www.ncbi.nlm.nih.gov/pubmed?term=Paludetti%20G%5BAuthor%5D&cauthor=true&cauthor_uid=19035385

http://www.ncbi.nlm.nih.gov/pubmed?term=Manni%20A%5BAuthor%5D&cauthor=true&cauthor_uid=19035385

http://www.ncbi.nlm.nih.gov/pubmed?term=Nemolato%20S%5BAuthor%5D&cauthor=true&cauthor_uid=19035385

http://www.ncbi.nlm.nih.gov/pubmed?term=Faa%20G%5BAuthor%5D&cauthor=true&cauthor_uid=19035385

http://www.ncbi.nlm.nih.gov/pubmed?term=Castagnola%20M%5BAuthor%5D&cauthor=true&cauthor_uid=19035385

http://www.ncbi.nlm.nih.gov/pubmed?term=Messana%20I%5BAuthor%5D&cauthor=true&cauthor_uid=19035385


P-IV-14

Zoledronic acid boosts γδ-T-cell activity in children given hematopoietic transplantation

Alice Bertaina1, Alessia. Zorzoli2, Andrea Petretto3, Giulia Barbarito2, Elvira Inglese3, Pietro Merli1, Chiara Lavarello3*, Letizia P. Brescia1, Biagio De Angelis1, Gino Tripodi4, Lorenzo

Moretta5, Franco Locatelli1,6 and Irma Airoldi2

1 Department of Pediatric Hematology and Oncology, IRCCS Ospedale Bambino Gesù, Rome, Italy 2 Laboratorio di Oncologia, Istituto Giannina Gaslini, Genova, Italy

3 Core Facilities, Istituto Giannina Gaslini, Genova, Italy 4 Dipartimento Ricerca Traslazionale, Medicina di Laboratorio, Diagnostica e Servizi, Istituto

Giannina Gaslini, Genova, Italy 5 Area di Ricerca Immunologica, IRCCS Ospedale Bambino Gesù, Rome, Italy

6 Department of Pediatric Science, Università di Pavia, Italy. A new method of graft manipulation based on physical removal of αβ+ T cells and CD19+ B cells, leaving mature NK and γδ T cells in the graft, has been recently developed for HLA-haploidentical hematopoietic stem cell transplantation (HSCT). We recently demonstrated that γδ T cells collected from transplanted patients are endowed with capacity of killing leukemia cells after ex vivo treatment with zoledronic acid (ZOL). Thus, we hypothesized that infusion of ZOL in patients may enhance γδ T-cell cytotoxic activity against leukemia cells. Patients given 3 or more ZOL infusions had a better probability of survival in comparison to those given 1 or 2 treatments (86% vs 54%, respectively, p=0.008). Proteomic analysis of γδ T cells purified from patients showed that ZOL caused both up-regulation of proteins involved in activation processes and immune response and down-regulation of proteins involved in proliferation. These findings are consistent with an induction of Vδ2 cell differentiation and increased cytotoxicity of both Vδ1 and Vδ2 cells against primary leukemia blasts. Finally, a proteomic signature was identified for each ZOL treatment. * Corresponding author: Chiara Lavarello. Core Facilities, Istituto Giannina Gaslini, Genova, Italy. Phone.: 0039 010 56362911; E-mail address: [email protected] (C. Lavarello)



P-IV-15

Gut microbiota profiling in Autism Spectrum Disorders patients: a metaproteomics approach

Stefano Levi Morteraa*, Pamela Vernocchia, Ilaria Basadonneb, Alessandro Zandonàc,

Marco Chiericic, Andrea Quagliarielloa, Antonio Gasbarrinid, Andrea Urbanid, Paola Venutib, Cesare Furlanelloc, Lorenza Putignania

a) Human Microbiome Unit, Bambino Gesù Children’s Hospital, Rome, Italy,

b) Department of Psychology and Cognitive Sciences, University of Trento, Rovereto, Italy; c) Bruno Kessler Foundation, Trento, Italy,

d) Catholic University of the Sacred Heart, Rome, Italy

The existence of a strong link between gastrointestinal disorders and the gut microbiota (GM) is now considered a fact, with particular evidence for inflammatory bowel diseases. A further possible relationship with Autism Spectrum Disorders (ASD) has been recently supposed but, given the complex assessment of the different cognitive-behavioural phenotypes for ASD, is very hard to define how the gastrointestinal tract and the gut microbiota could be correlated with the central nervous system and some states of the pathology. In this study, we collected fecal samples from ten families in which two parents, an ASD child, and a possible sibling were present. We performed a GM profiling by LC-MS/MS shotgun proteomics experiments on isolated bacterial proteins, taking into consideration some important factors for the microbial population development, such as nutritional history, antibiotics assumption, current diet, and the presence of gastrointestinal disorders. A series of bioinformatics tools were employed to identify Operational Taxonomic Units (OTUs) and their distribution, to perform functional analysis (COG and KEGG classification) and for statistics. Co-abundance networks were inferred, modelling COGs as nodes and Pearson correlation among COGs abundance profiles as edges. Wilcoxon and Kruskal-Wallis tests were performed to identify differentially expressed COGs between ASD and non-autistic subjects. A preliminary analysis based on 30 samples from seven families highlighted interesting results in the OTUs distribution of some ASD patients. Sutterellaceae and Ruminococcaceae seem to be possible candidates as microbial biomarkers, as already reported in the literature [1] and according to other metagenomics studies carried on by our group. * Corresponding author: Stefano Levi Mortera. Bambino Gesù Children’s Hospital, Viale di San Paolo 15, 00146,Roma, Italy. Phone.: +39 0668594372 E-mail address: [email protected] (S. Levi Mortera) References: [1] L. Wang, C.T. Christophersen, M.J. Sorich, J.P. Gerber, M.T. Angley, M.A. Conlon, Increased Abundance of Sutterella spp. and Ruminococcus torques in feces of children with autism spectrum disorder, Mol. Autism. 4 (2013) 42.


P-IV-16 Altered profile of salivary proteins in patients with Autoimmune Hepatitis

Barbara Lioria*, Barbara Manconia, Tiziana Cabrasa, Simona Onalib, Francesco Figorillib, Luchino Chessab, Federica Iavaronec, Massimo Castagnolacd, Irene Messanad, Alessandra

Olianasa.

a) Dipartimento di Scienze della Vita e dell’Ambiente, Università di Cagliari, Cagliari, Italy b) Centro per lo studio delle malattie del fegato, Dipartimento di Scienze mediche e Sanità

pubblica, Università di Cagliari, Cagliari, Italy c) Istituto di Biochimica e Biochimica Clinica, Università Cattolica, Roma, Italy

d) Istituto di Chimica del Riconoscimento Molecolare CNR, Roma, Italy

Autoimmune hepatitis (AIH) is a rare chronic liver disorder of unknown etiology characterized by the loss of immunological tolerance to autologous liver tissue. In this study we investigated the levels of salivary proteins to evaluate potential changes in the salivary proteome of AIH patients compared to control subjects. Indeed, human saliva is an attractive bodily fluid, due to the easiness and non-invasivity of the sample collection, in studies finalized to characterize novel biomarkers useful for diagnosis of diseases of the oral cavity and systemic. The acidic soluble fractions of unstimulated whole saliva of 45 AIH patients and 41 control subjects were analyzed by RP-HPLC-ESI-MS and salivary peptides/proteins were quantified by eXtracted Ion Current procedure. During this preliminary study we evaluated the levels of the following salivary peptides/proteins: statherin, histatin 1, histatin 3, 5 and 6, cystatin S, S1, S2, SN and SA. Significant increased levels in AIH patients with respect to control subjects were observed for monophosphorylated statherin (p = 0.02), statherin (p = 0.04), histatin 1 (p = 0.04), histatin 6 (p = 0.02), cystatin S1 (p = 0.006) and cystatin S2 (p = 0.01). On the contrary the levels of the non-phosphorylated statherin, non-phosphoryilated histatin 1, histatin 3, histatin 5, cystatin S, SN and SA did not show any significant differences between patients and controls. Further studies are needed to assess whether the level modifications of these specific salivary proteins/peptides could reflect a different oral microenvironment and represent a potential non-invasive biomarker of the disease.

* Corresponding Author: Dott. Barbara Liori Università di Cagliari Cittadella Universitaria di Monserrato Cagliari, Italy Phone.: +390706754507 E-mail address: [email protected]



P-IV-17 Protein composition of human blastocoel fluid isolated from women aged <

and ≥ 37 years

Elisa Maffiolia*, Francesca Grassi Scalvinia, Silvia Morellid, Fabiana Santagataa, Armando Negria, Elena Albanib, Elena Monica Borronic, Gabriella Tedeschia,d

a) Dipartimento di Medicina Veterinaria, Università degli Studi di Milano, Milano, Italy; b) Istituto Clinico Humanitas, Rozzano, Milano, Italy;

c) Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milano, Italy;

d) Filarete Foundation, Milan, Italy;

Maternal ageing represents one of the major causes of altered embryo-endometrial communication during implantation. The molecular mechanisms have been poorly investigated in humans due to technical difficulties and ethical limitations; thus, no clinically useful markers have been identified to assess embryo quality and positive pregnancy outcome. To this purpose, proteomics is an emerging powerful technology in the identification of both biomarkers and disease targets in the field of embryology. We examined the protein composition of human blastocoel fluid isolated from blastocysts of women aged <37 years (group A) and women aged ≥ 37 years (group B) by LC-ESI-MS/MS using a shotgun proteomic approach and label free quantification. Mass spectra analysis allowed to identifying 148 proteins, out of which 109 are differentially expressed between the two groups. These proteins were functionally profiled using bioinformatics annotation tools and pathway databases. The main perturbed networks refer to fertilization and embryo implantation, as well as to signaling and nucleic acid interactions. Many of the proteins differentially expressed are related to processes involving ubiquitin and endocannabinoids: the main molecular functions of these proteins consist of histone turnover and transcription factors, crucially required for cell cycle progression and proliferation. The results provide valuable information on the molecular player involved in biological processes related to blastocyst features in aged women which can be used as a starting point towards the identification of non-invasive tools to investigate and ameliorate blastocyst viability, and improving clinical outcome of IVF cycles. These data have been recently published in (1). * Corresponding author: Elisa Maffioli. Department of Veterinary Medicine, University of Milan, Milan, Italy. Phone.: +390250318124; Fax: +39 02 50318123. E-mail address: [email protected] (E. Maffioli) References: [1] Tedeschi G, Albani E, Borroni EM, Parini V, Brucculeri AM, Maffioli E, Negri A, Nonnis S, Maccarrone M, Levi-Setti PE. Proteomic profile of maternal-aged blastocoel fluid suggests a novel role for ubiquitin system in blastocyst quality. J Assist Reprod Genet. 2017;34(2):225-238.



P-IV-18 Interactors of human salivary Cystatin D explored by immunoprecipitation

coupled to Mass Spectrometry

Barbara Lioria, Alessandra Olianasa, Tiziana Cabrasa, Federica Vincenzonib, Simone Serraoa, Irene Messanac, Massimo Castagnolab,c, Barbara Manconia *

a) Dipartimento di Scienze della Vita e dell’Ambiente, Università di Cagliari, Cagliari, Italy

b) Istituto di Biochimica e Biochimica Clinica, Università Cattolica, Roma, Italy c) Istituto di Chimica del Riconoscimento Molecolare CNR, Roma, Italy

Cystatin D belongs to the cystatins superfamily that includes a large number of related proteins widely distributed in the higher organisms. It is encoded by CST5, a polymorphic gene that produces two proteoforms with either Cys or Arg at position 26. The main biological role of cystatin D is related to its inhibitory activity against cysteine proteases, and recently other interesting activities unrelated to protease inhibition have been described, such as tumor suppressor effect on colon carcinoma cells [1]. In order to gain new insights into human cystatin D structure and interactors, a sample of unstimulated whole saliva was submitted to a co-immunoprecipitation assay with an antibody recognizing cystatin D-Cys26 and the co-immunoprecipitated proteins were separated by SDS-PAGE. In the absence of reducing agent two main bands with apparent MW higher than 200 kDa were evidenced, while under reducing conditions several bands, ranging between 12-160 kDa, were obtained. These results highlighted that cystatin D-Cys26 forms copolymers with other protein partners present in human saliva. Co-immunoprecipitated proteins were submitted to in-gel and on-beads trypsin digestion and fragments characterized by LC- high resolution MS/MS. MS/MS data confirmed the presence of cystatin D-Cys26 and evidenced the following in vivo interactors of cystatin D-Cys26: pIgR, IgA1, IgA2, amilase 1, lysozime C, S100A9, S100A8 and PIP. Collectively, these results demonstrated that cystatin D-Cys26 associated with a large set of protein partners, which are all involved in the innate and/or acquired immune system.

* Corresponding Author: Barbara Manconi, PhD Università di Cagliari Cittadella Universitaria di Monserrato Cagliari, Italy Phone.: +390706754508 E-mail address: [email protected] Reference: [1] Alvarez-Díaz S. et al. J Clin Invest. 2009;119:2343-58.



P-IV-19

Top-down molecular characterization of pediatric craniopharyngioma

Claudia Martellia*, Riccardo Serrab, Ilaria Inserraa, Federica Iavaronea, Federica Vincenzonia, Diana Valeria Rossettia, Gianpiero Tamburrinib, Massimo Caldarellib, Luca

Massimib; Massimo Castagnolaa,c, Claudia Desiderioc

a) Istituto di Biochimica e Biochimica Clinica, Università Cattolica, Rome; Italy b) Unità Operativa Complessa di Neurochirurgia Infantile, Fondazione Policlinico Universitario A.

Gemelli, Università Cattolica del Sacro Cuore, Rome, Italy c) Istituto di Chimica del Riconoscimento Molecolare, CNR, Rome, Italy

Adamantinomatous Craniopharyngioma (AC) is a brain tumor which develops in sellar/suprasellar region and presenting, although its benign nature, an aggressive behavior. Generally in children it is associated to a invasive cystic mass which destructs neighboring eloquent or neurovascular structures [1]. Since AC pathogenesis is still not defined, its molecular characterization by proteomic tools could provide new insights into mechanisms involved in its development and progression. The present work represents the first comprehensive proteomic investigation on AC intracystic fluid and tissue. Thirteen intracystic fluids [2, 3] and seven solid tumor from different AC patients were analyzed by liquid chromatography coupled with high resolution LTQ-Orbitrap tandem mass spectrometry applying a top-down approach. Among the common proteins/peptides identified in solid and cystic parts, α-defensins 1-3, β-thymosins, ubiquitin and α-hemoglobin chain fragments were characterized, confirming the role of inflammation in AC pathogenesis. The intracystic fluid was mainly constituted by extracellular proteins such as apolipoproteins (Apo A-I, Apo A-II and Apo C-I) that could be involved in the accumulation of lipids within the cystic fluid. In tissue extracts β-thymosins and ubiquitin and glial fibrillary acidic protein fragments, α-thymosins, α-defensin 4, calmodulin, cysteinylated and glutathionylated S100-A6, S100-A10 and S100A-11 were identified. S100A proteins could be either related to the aggressive behavior of AC tumor and/or to the role of calcium inside the disease, since the identification of calmodulin and the presence of calcium flaks within the intracystic fluid. These results could be relevant in the future for the development of novel diagnostic and therapeutic strategies. * Corresponding author: Claudia Martelli. Istituto di Biochimica e Biochimica Clinica, Università Cattolica, L.go F. Vito 1, 00168 Rome, Italy. Phone.: +39 0630154215 Fax: +39 063053598 E-mail address: [email protected] References: [1] Apps JR, Martinez-Barbera, JP. Molecular pathology of adamantinomatous craniopharyngioma: review and opportunities for practice.

Neurosurg. Focus. 2016; 41: E4. [2] Desiderio C, Martelli C, Rossetti DV, et al. Identification of thymosins β4 and β 10 in paediatric craniopharyngioma cystic fluid. Childs

Nerv Syst. 2013;29:951-60. [3] Martelli C, Iavarone F, Vincenzoni F, et al. Proteomic characterization of pediatric craniopharyngioma intracystic fluid by LC-MS top-

down/bottom-up integrated approaches. Electrophoresis 2014; 35: 2172–83.



P-IV-20

Proteomic analysis of malignant pleural mesothelioma cell proteome and secretome

Serena Lacerenzaa, Federica Ciregiaa, Laura Giustia, Viviana Grecob, Andrea Urbani b,c,

Antonio Lucacchinia, Maria Rosa Mazzonia

aDepartment of Pharmacy, University of Pisa, Pisa, Italy bProteomics and Metabonomics Unit, IRCCS-Fondazione Santa Lucia, Rome, Italy

cIstituto di Biochimica e Biochimica Clinica, Università Cattolica, Roma, Italy

Malignant pleural mesothelioma (MPM) is an aggressive cancer, linked to asbestos exposure, with a poor prognosis. An analysis of cell proteome and secretome can shed light on disease pathogenesis while offering the possibility to potential therapeutic targets and biomarkers. In the present study we compared proteome and secretome protein profiles of a MPM cell line (NCI-H2052) with those of a normal mesothelial cell line (Met-5A). Cells were grown to 80% confluence in their complete culture media and then incubated in serum-free RPMI-1640 medium for 24 h. At the end, the culture medium (CM) was collected, centrifuged, and concentrated. The CM proteins were precipitated and subjected to 2D-gel electrophoresis. The remaining cells were lysed and proteins of whole cell lysate (WCL) were also separated by 2D-gel electrophoresis. An analysis of gels showed 342 protein spots differentially expressed in NCI-H2052 WCL compared to Met-5A WCL. Of these proteins, 17 were upregulated in NCI-H2052 cell proteome while 13 were downregulated with fold changes major than 3. The analysis of cell secretome showed 91 protein spots differentially expressed in NCI-H2052 cell CM compared Met-5A cell CM. Among these proteins, 14 were upregulated in NCI-H2052 secretome while 10 were downregulated with fold changes major than 3. Some proteins, highly expressed in NCI-H2052 cell proteome and secretome, were identified by MALDI-TOF/TOF. Most upregulated proteins appeared to be involved in cell responses to oxidative stress such as peroxiredoxin-3, cytochrome P450 1B1, and sirtuin 1 in the proteome and sulfhydryl oxidase 1, and prosaposin in the secretome. * Corresponding Author: Prof. ssa Maria R Mazzoni University of Pisa Via Bonanno 6, 56126 Pisa, Italy. Phone.: +390502219524 E-mail address: [email protected]



P-IV-21

The traffic of GAA uptaken in enzymatic replace treatment of Pompe disease

Orsola F. Moccia1, Diana Canetti1, Caterina Porto2, Marcella Coletta2, Giancarlo Parenti2,

Pietro Pucci1, Maria Monti1

1Department of Chemical Sciences, University “Federico II” of Naples, Italy; CEINGE Advanced Biotechnology, Naples, Italy

2 Department of Pediatrics, University “Federico II”, Naples, Italy; Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy

Pompe disease (PD) is a metabolic myopathy caused by the deficiency of the lysosomal acid α-glucosidase (GAA), as a consequence of mutations in the gene coding for this enzyme whose function is degradation of glycogen to glucose. The absence or reduction in its enzymatic activity involves the onset of the disease due to accumulation of glycogen in various tissues, particularly cardiac and skeletal muscles. Among partially effective therapies for some types of Pompe phenotypes, enzyme replacement therapy (ERT) involves the treatment of patients with recombinant enzyme GAA (rhGAA) expressed in Chinese hamster ovary cells (CHO). We have characterized with a functional proteomics approach the intracellular traffic pathways followed by this enzyme after uptake from medium. Fibroblasts from a PD patient in which the GAA is not produced at all, were treated for two or six hours with rhGAA. The internalized protein and its interactors were immunoprecipited and the interactome was identified. Our results confirm that GAA is up taken by an endocytic clathrin-dependent process and that an amount of protein is directed to endosomes and lysosomes while another amount is degraded by proteasome. This result suggests that the unsatisfied efficacy of ERT in PD therapy might be explained by the partial proteasomal degradation of recombinant enzyme following its internalization.

*Corresponding author: Prof. Maria Monti Department of Chemical Sciences, University “Federico II” of Naples, Via Cinthia 4, 80125, Naples, Italy Phone.: +39 081674474; +39 0813737919 Fax: +39 0813737808. E-mail address: [email protected] (M. Monti)



P-IV-22

Investigation of OFD1 interactome by functional proteomic approach

Flora Cozzolino1, Daniela Iaconis2, Pucci Piero 1,3, Brunella Franco2,4, Maria Monti1,3*

1 CEINGE Biotecnologie Avanzate S.c.a.r.l., Naples, Italy. 2Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy

3Department of Chemical Sciences, University of Naples Federico II, Italy 4Department of Medical Translational Sciences, University of Naples “Federico II”, Italy

Ciliopathies represent an emerging group of human genetic disorders associated to functional and/or structural abnormalities of cilia, specialized, evolutionary conserved organelles that protrude from the cell surface of almost all mammalian cells. Defects in cilia formation and/or function result in a wide spectrum of clinical phenotypes including renal cystic disease and disorders in which the renal involvement is associated to eye, skeletal such as the Oral facial digital type I syndrome (OFD1). This latter syndrome is associated to mutations in the OFD1 gene coding for a centrosome/basal body-associated protein which exerts a critical role in cilia formation. OFD1 interactome was investigated by a functional proteomic approach based on the isolation and identification of OFD1 interacting proteins. A large number of putative OFD-1 interacting proteins participates to protein synthesis process, including several subunits of eukaryotic translation initiation factor 3 and ribosomal proteins. The presence of these proteins belonging to so called translation pre-initiation complex (PIC) suggests for the first time the occurrence of translation at centrosome. Immunofluoscence experiments show that OFD1 colocalizes at the centrosome with several other elements of PIC. Present data demonstrate that OFD1 localizes with PIC at the centrosome where controls the translation of specific targets involved in cilia formation. *Corresponding author: Prof. Maria Monti Department of Chemical Sciences, University “Federico II” of Naples, Via Cinthia 4, 80125, Naples, Italy Phone.: +39 081674474; +39 0813737919 Fax: +39 0813737808. E-mail address: [email protected] (M. Monti)



P-IV-23

Dissection of the mechanism of action of a LSD1-inhibitor in Acute Myeloid Leukemia by quantitative proteomics

Luciano Nicosiaa, Roberto Ravasioa, Saverio Minuccia,b and Tiziana Bonaldia*

aDepartment of Experimental Oncology, European Institute of Oncology, Milan, Italy;

bDepartment of Bioscience, University of Milan, Milan, Italy

Lysine specific-demethylase 1 (LSD1), the major histone H3K4/K9 demethylase, is aberrantly expressed in acute myeloid leukemia (AML) and is emerging as a promising target for the epigenetic therapy of AML subtypes that are not responsive to retinoic acid treatment [1]. The Experimental Therapeutic Unit at our Campus optimized a potent and specific LSD1 inhibitor, which has been characterized in vitro for its selectivity towards LSD1 and in vivo for its anti-proliferative effects on self-renewing AML cells [2]. We are investigating the effects of this compound on both the histone post-translational modification (hPTM) patterns and the interaction network in which LSD1 is embedded [3] in NB4 AML cells, by using a panel of quantitative mass-spectrometry (MS) strategies. The in-depth hPTM analysis by MS [4] discovers that 24 hours treatment with the inhibitor induces the increase of H3K4me2 (known LSD1-substrate), H3K9me2/K14ac, H3K9me3/K14ac, H3K27me2 and H3K27me3, paralleled by a decrease of the H3K27me1 levels. A similar trend is also detected in NB4 LSD1-knock out cells by the CRISPR/Cas9 technology, which suggests that these epigenetic changes may be specifically associated to the cellular response to LSD1 absence/inhibition. The investigation of the basal LSD1-interactome by SILAC-based proteomics [5] allows identifying novel interesting interactors mainly involved in DNA binding and transcription regulation, in addition to the well-known interactors RCOR1, HDAC1 and HDAC2. The subsequent analysis of the dynamic LSD1-interactome upon drug treatment allows shedding lights on the changes in interaction networks of the enzyme, contributing to dissect the molecular effect of the compound in this model. * Corresponding author: Tiziana Bonaldi European Institute of Oncology, Milan, Italy. Phone.: +39-0294375123; Fax: +39-0294375990. E-mail address: [email protected] References: [1] Lokken, A.A., and Zeleznik-Le, N.J. (2012). Breaking the LSD1/KDM1A Addiction: Therapeutic Targeting of the Epigenetic Modifier in AML. Cancer Cell 21, 451-453.

[2] Vianello, P., Botrugno, O.A., Cappa, A. et al. (2016). Discovery of a novel inhibitor of histone lysine-specific demethylase 1A (KDM1A/LSD1) as orally active antitumor agent. J. Med. Chem. 59, 1501-1517.

[3] Amente, S., Lania, L., and Majello, B. (2013). The histone LSD1 demethylase in stemness and cancer transcription programs. Biochim Biophys Acta 1829, 981-986.

[4] Soldi, M., Cuomo, A. and Bonaldi, T. (2014). Improved bottom-up strategy to efficiently separate hypermodified histone peptides through ultra-HPLC separation on a bench top Orbitrap instrument. Proteomics 14, 2212-2225.

[5] Ong, S.E., and Mann, M. (2006). A practical recipe for stable isotope labelling by amino acids in cell culture (SILAC). Nat Protoc. 1, 2650-2660.



P-IV-24

Proteomic insights in extracellular microvesicles from CSF and tears of multiple sclerosis patients

Pieragostino Damianaa,* , Cicalini Ilariab , Lanuti Paola c , Marchisio Marcoc , Fontana Antonellab , Zappacosta Rominab , Simeone Pasqualec , Sacchetta Paoloa , di Ioia Mariad,

Lugaresi Alessandrae , Del Boccio Pb

aDep. of Medical, Oral and Biotechnological Sciences, “G. d’Annunzio” University of Chieti-

Pescara, Chieti, Italy bDep. of Pharmacy, “G. d’Annunzio” University of Chieti-Pescara, Chieti, Italy.

c Dep. of Medicine and Aging Science , “G. d’Annunzio” University of Chieti-Pescara, Chieti, Italy d Department of Neuroscience, Imaging and Clinical Sciences, “G. d’Annunzio” University of Chieti-

Pescara, Chieti, Italy e Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italia

Multiple Sclerosis (MuS) is multifactorial neurological disease characterized by great heterogeneity in clinical presentation. Emerging evidences showed that distinct types of brain cells release high number of Extracellular Vesicles (EVs), functioning as shuttles for the delivery of cargo among different cells within an organism. These data mean that EVs carry receptors, bioactive lipids, proteins, and nucleic acids by which EVs may modify the phenotype and functions of target cells. Widely investigated as biomarkers in oncology little is known about EVs roles in the MuS. We carried out a proteomics characterization of EVs isolated from CSF and tears of MuS patients. EVs were analyzed by a polychromatic flow cytometry method. Exosomes and EVs were sorted by using a FACSAria III cell sorter (BD) were characterized by Dynamic Light Scattering (DLS). Proteomics data of pure microvesicles were obtained by LC-MS/MS system (Bruker) and subjected to web-based Ingenuity Pathway Analysis system to rebuild their functional implications in the phato-physiology of MuS. High number of exosomes and microvesicles, measured in MuS both in CSF and tears, were characterized by proteomics approach after sorting and showing a specific protein cargo. In conclusions, on the basis of our knowledge, this is the first time that extracellular vesicles and exosomes are described and characterized in tears, showing a subpopulation originated from neuron and microglia cells. - This study is supported by FISM, Fondazione Italiana Sclerosi Multipla - cod 2015/R/12 *Corresponding author: Dr. Damiana Pieragostino, PhD Department of Medical Oral and Biotechnological Sciences, University “G. d’Annunzio” Chieti-Pescara, Chieti, Italy. Phone: +39 0871 541593 Fax: +39 0871 541598 E-mail address: [email protected] .



P-IV-25

Plasma n-3 PUFA levels correlate with protein markers in age-related disorders: an observational study in Alzheimer’s disease and MCI

Maurizio Ronci1,2*, Debora Cutuli1,3, Fabrizio Piras1, Federica Marini4, Luisa Pieroni1,

Viviana Greco1, Laura Petrosini1,3 , Gianfranco Spalletta1, and Andrea Urbani1,3

a) Fondazione S. Lucia, IRCCS, Rome b) Department of Medical, Oral and Biotechnological Sciences, University G. D’Annunzio, Chieti

c) Department of Psychology, University “Sapienza” of Rome d) Biochemistry and Clinical Biochemistry Institute, Università Cattolica del Sacro Cuore, Rome

The hypothesis on the protective effect of long-chain n-3 PUFA against cognitive decline linked to Alzheimer’s disease (AD) has been around for long time and today is increasingly being accepted. n-3 PUFA are major components of neuronal membranes and have a wide range of modulating functions, from synaptic plasticity to neuroimmunity and neuroprotection. Experimental evidence indicates that n-3 PUFA deficiency has detrimental effects on brain and cognitive functionality, while n-3 PUFA dietary supplementation has beneficial effects [1]. On the other hand, only a few plasma markers of AD were proposed and successfully replicated [2]. The aim of the present study was to profile n-3 PUFA in plasma of subjects affected by AD and MCI and to correlate them with both the disease status and the levels of proposed protein plasma markers of AD in order to define a multivariate panel able to classify confidently the subjects. 48 subjects were enrolled: 13 AD (age=69.58±7.2 years), 27 MCI (age=70.3±7.3 years) and 8 healthy controls (age=69.3±9 years). Plasma fatty acids were measured as pentafluorobenzyl derivates using negative chemical ionization GC-MS [3]. Relative plasma amounts of annexin A5, IgM, pancreatic polypeptide and apolipoprotein E were measured by LC-MS/MS in pseudo MRM acquisition mode. Proteotypic peptides and relative transitions were taken from peptide Atlas database. According to the preliminary results we can state that a multivariate measurement of plasma n-3 PUFA together with the specific relative amounts of apolipoprotein E may represent a powerful tool for the screening of MCI and AD. This study is supported by the Italian Ministry of Health – Project code GR-2011-02351086. * Corresponding author: Maurizio Ronci. Department of Medical Oral and Biotechnological Sciences, University “G. d’Annunzio” Chieti-Pescara, Chieti, Italy. Phone.: +39 0871 541579; Fax: +39 0871 541598; E-mail address: [email protected] References: [1] Front Aging Neurosci. 2014 Aug 25;6:220. doi: 10.3389/fnagi.2014.00220. [2] Front Neurol. 2015; 6: 236. doi: 10.3389/fneur.2015.00236 [3] Biochim Biophys Acta. 2011 Nov;1811(11):648-56. doi: 10.1016/j.bbalip.2011.07.006.



P-IV-26 Tear Film Steroid Profiling in Dry Eye Disease by Liquid Chromatography

Tandem Mass Spectrometry

Damiana Pieragostinoa, b, Luca Agnifilic, Ilaria Cicalinib,d, Roberta Caliennoc, Mirco Zucchellia,b, Leonardo Mastropasquac, Paolo Sacchettaa,b, Piero Del Bocciob,d and Claudia

Rossia, b*

a Department of Medical Oral and Biotechnological Sciences, University “G. d’Annunzio” of Chieti- Pescara, Chieti, Italy.

b Analytical Biochemistry and Proteomics Laboratory, Research Centre on Aging and Translational Medicine (Ce.S.I-MeT), University “G. d’Annunzio” of Chieti-Pescara, Chieti, Italy.

c Ophtalmic Clinic, Department of Medicine and Aging Science, "G. d'Annunzio" University of Chieti-Pescara, Chieti, Italy

d Department of Pharmacy, University “G. d’Annunzio” of Chieti- Pescara, Chieti, Italy.

Dry eye disease (DED) is a multifactorial disorder of the ocular surface unit resulting in eye discomfort, visual disturbance and ocular surface damage; risk of DED increases with age in both sexes, while its incidence is higher among females caused by an overall hormonal imbalance. The role of androgens has recently investigated and these hormones were considered to have a protective function on the ocular surface. In order to correlate DED to tear steroid levels, a robust, specific and selective method for the simultaneous quantification of cortisol (CORT), corticosterone (CCONE), 11-deoxycortisol (11-DECOL), 4-androstene-3,17-dione (ADIONE), testosterone (TESTO), 17α-hydroxyprogesterone (17-OHP) and progesterone (PROG) was developed and applied for the analysis of tear samples. The method involves a simple extraction procedure of steroids from tears collected on Schirmer strips followed by a high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) analysis. The developed method was applied to a small dry eye female group and, CORT, ADIONE and 17-OHP response levels resulted significantly decreased in dry eye patients in respect to controls. The ROC curve obtained by the combination of these three steroids (AUC= 0.964) evidenced good diagnostic power of the differential tear steroids in discriminating DED. In conclusion, the present method allowed for the first time the study of a steroid profiling directly in tear fluid. *Corresponding author: Claudia Rossi PhD Analytical Biochemistry and Proteomics Unit, Research Centre on Aging and Translational Medicine (Ce.S.I-MeT) “G. d’Annunzio” University of Chieti, Via Luigi Polacchi 66100 Chieti, Italy Phone: +39 0871 541596 Fax: +39 0871 541598 E-mail: [email protected]



P-IV-27

Proteomic analysis of K562 erythroid cells after Sox6 induction Gloria Barbarani1, Antonella Ronchi1, Robert Steinfelder2, Sudharshan Elangovan1, Cristina

Fugazza1, Marianna Caterino3,4, Margherita Ruoppolo3,4,5*.

1 Dipartimento di Biotecnologie e Bioscienze, Università degli studi di Milano-Bicocca, Milan, Italy 2 Source BioScience, Nottingham Business Park, Nottingham, United Kingdom

3Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli, “Federico II”, Naples, Italy

4Associazione culturale DiSciMuS RCF 80026, Casoria, Naples, Italy 5 CEINGE Biotecnologie Avanzate, Naples, Italy

The Sox6 transcription factor is crucial for terminal maturation of definitive red blood cells. Sox6-null mouse fetuses present misshapen and nucleated erythrocytes, due to impaired actin assembly and cytoskeleton stability. These defects are accompanied with a reduced survival of Sox6-/- RBCs, resulting in a compensated anemia. Sox6-overexpression in K562 cells and in human primary ex vivo erythroid cultures enhances erythroid differentiation and leads to hemoglobinization, the hallmark of erythroid maturation (1). In order to unravel pathways and processes downstream to Sox6 induction, including both direct and un-direct targets, we performed a differential proteomic analysis on human erythroid K562 cells overexpressing Sox6. We compared total protein extracts obtained from K562 cells and from the same cells overexpressing Sox6. The experiment was performed in quadruplicate on four pairs of samples from cells transduced with either the Sox6-overexpressing vector (Sox6-K562) or the corresponding Empty Vector (EV-K562) as a control, to ensure statistical replication. Sox6-overexpression induces dysregulation of 64 proteins, involved in cytoskeleton remodeling and in protein synthesis, folding and trafficking, key processes for erythroid maturation. Moreover, 43 out of 64 genes encoding for differentially expressed proteins contain within their proximal regulatory regions sites that are bound by SOX6 according to ENCODE ChIP-seq datasets and are possible direct SOX6 targets. SAR1B, one of the most induced proteins upon Sox6 overexpression, shares a conserved regulatory module, composed by a double SOX6 binding site and a GATA1 consensus, with the adjacent SEC24A gene. Since both genes encode for COPII components, this element could concur to the coordinated expression of these two proteins during erythropoiesis. *Corresponding author: Margherita Ruoppolo Full Professor Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Via S. Pansini 5, 80131 Napoli, Italy. Tel: +39-081-3737807, +39-0817462426, Fax: +39-081-3737808 E-mail address: [email protected] References [1] Dumitriu, B., et al. Sox6 is necessary for efficient erythropoiesis in adult mice under physiological and anemia-induced stress conditions.

PLoS One 5 (8), e12088 (2010).

mailto:Tel:%20+39-081-3737807



P-IV-28

Comparative proteomics of acid-insoluble fraction of saliva in type 1 diabetes-celiac disease subjects, celiac disease or type 1 diabetes subjects

Morena Arbaa, Gianluigi Cabiddua, Elisabetta Pisanoa, Pier Paolo Pusceddud, Federica

Iavaroneb, Federica Vincenzonib, Massimo Castagnolab,c, Tiziana Cabrasa, Irene Messanac, Maria Teresa Sannaa*

a) Dipartimento di Scienze della Vita e dell’Ambiente, Università di Cagliari, Monserrato (CA),

Italy; b) Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Roma, Italy; c) Istituto di Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche, Roma,

Italy; d) Azienda Ospedaliera Brotzu, Dipartimento Medicina Interna, Cagliari, Italy

The comparative proteomic analysis, performed by 2-DE coupled to high-resolution ESI-MS, on the acid-insoluble fraction of whole saliva from 25 subjects affected by type 1 diabetes and celiac disease (T1D-CD), type 1 diabetes (T1D) or celiac disease (CD), allowed to evidence a number of proteins with different expression between the groups. Among them, several proteins are involved in immune response: the polymeric immunoglobulin receptor (pIgR) in the secretory component form appeared under-expressed in T1D with respect to controls (CTR) with two spots differing for their pIs probably for their different content in sialic acid [1]. A significant decrease in pIgR has already been reported in Crohn’s disease [2], a chronic inflammation of the digestive system that may be due to an auto-immune reaction. Several spots of J chain, a polypeptide that regulates IgA and IgM polymer formation and their transport across the mucosa mediated by pIgR, appeared over-expressed in T1D with respect to all other groups. α-enolase, a multi-functional enzyme that has been shown to translocate from cytosol to the surface of macrophages and monocytes under inflammatory stimuli, resulted over-expressed in CD saliva with respect to the other groups. Prolactin-induced protein, probably involved in oral mucosa innate immune response [3], resulted under-expressed in T1D-CD with respect to CTR. Although several proteomics studies on diabetic patients biofluids have been published, only few have been reported on CD and T1D-CD patients, and the present study might contribute to the future identification of potential biomarkers of these pathologies. * Corresponding author: Maria Teresa Sanna Dipartimento di Scienze della Vita e dell’Ambiente, Università di Cagliari Cittadella Universitaria - 09042 MONSERRATO (CA), Italia Tel: 070 675 4509 Fax: 070 675 4523 E-mail address: [email protected] References: [1] Huang, J. et al., 2015. Journal of proteome research, 14(3), pp.1335–1349 [2] Arsenescu, R. et al., 2008. Mucosal immunology, 1(5), pp.399–411 [3] Ghafouri, B., Tagesson, C. & Lindahl, M., 2003. Proteomics. pp. 1003–1015.



P-IV-29 In-depth mapping and quantitative evaluation of urinary N-glycoproteome

of clear cell RCC patients by n-LC ESI MS/MS approach

Lucia Santorellia*, Martina Stellaa, Manuel Gallia, Clizia Chinelloa, Marina Pittoa, Marco Grasso b and Fulvio Magnia

a) Dept. of Medicine and Surgery, University of Milano-Bicocca, Milano, Italy

b) Urology Unit, San Gerardo Hospital, Monza, Italy

Protein N-glycosylation is one of the most complex and frequently occurring post-translational modifications involved in many biological processes, such as cell adhesion, molecular trafficking and clearance [1]. Aberrant changes in N-glycosylation protein pattern are closely associated with several diseases, including cancer; indeed, it is known that glycosylation profiles is modulated consequently during its progression and spreading [2]. Thus, efficient identifications of these patterns and reliable differentiation between levels of aberrant protein glycoforms in healthy and tumour conditions would be useful for understanding the molecular mechanism of this multifactorial disease, developing specific biomarkers and finding novel therapeutic targets. In particular, this approach is likely to be promising for the most common and aggressive neoplasm in kidney, the clear cell Renal Cell Carcinoma (ccRCC). The aim of this study is to identify potential glycomarkers in urine belonging to patients affected by ccRCC at different stage. In particular, we mapped the N-glycosylation sites of urinary proteins obtained from patients with ccRCC classified as pT1a (n=15) and pT3a (n=15), and from healthy subjects (n=15), by an N-glyco-FASP-based method [3]. The proteome enriched in glycoproteins were identified and evaluated via label-free nLC-ESI MS/MS. This approach permits to examine the number and the distribution of the modification sites and simultaneously to quantify stage-related changes of N-glycosylated amino acids sequences. These results expand our knowledge of this important class of post-translational modifications also to urine. The translation of this knowledge into pre-clinical studies could gain a remarkable impact for biomarkers and therapeutic targets discovery in kidney cancer. * Corresponding author: Lucia Santorelli. University of Milano - Bicocca, via Cadore, Monza, Italy. Phone: +39 0264488217; E-mail address: [email protected] (L.Santorelli) References: [1] Ohtsubo K, Marth JD. Glycosylation in cellular mechanisms of health and disease. Cell. 2006; 126:855–867. [2] Leticia Oliveira-Ferrer, Karen Legler, Karin Milde-Langosch Role of protein glycosylation in cancer metastasis. Semin Cancer Biol (2017)

https://doi.org/10.1016/j.semcancer.2017.03.002 [3] Zielinska DF, Gnad F, Wiśniewski JR, Mann M. Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence

constraints. Cell. 2010 May 28;141(5):897-907. doi: 10.1016/j.cell.2010.04.012 Acknowlegements: This work was supported by grants from FAR 2013-2016, Fondo Ateneo Quota Competitiva 2014 and in part by Fondazione Gigi & Pupa Ferrari Onlus



P-IV-30

Top-down analysis of the proteomic profile of human amniotic epithelial cells (hAEC) and human amniotic mesenchymal stromal cells (hAMSC):

preliminary data

Sofia Petrocchia*, Federica Iavaronea, Pietro Romeleb, Antonietta Silinib, Massimo Castagnolaa,c, Ornella Parolinib,d

a) b) Istituto di Biochimica e Biochimica Clinica – Facoltà di Medicina-Università Cattolica, Roma,

c) Centro di Ricerca E.Menni – Fondazione Poliambulanza-Istituto Ospedaliero, Brescia, d) Istituto di Chimica del Riconoscimento Molecolare – CNR, Roma

e) Istituto di Anatomia Umana e Biologia– Facoltà di Medicina-Università Cattolica, Roma The fetal portion of human placenta is composed of amnion and chorion membranes. Human amniotic epithelial cells (hAEC) and human amniotic mesenchymal stromal cells (hAMSC) are isolated from amnion; while human chorionic mesenchymal stromal cells (hCMSC) and human chorionic trophoblastic cells (hCTC) from chorion [1]. hAMSC and hAEC show different plasticity and immunomodulatory properties which could be useful for cell therapy. The proteomic profile of these cell lines is still almost completely unknown; for this reason we used a top down platform to analyze the composition of hAMSC and hAEC derived from term placentas obtained from Fondazione Poliambulanza-Istituto Ospedaliero of Brescia. Cells were vitally thawed, protease inhibitors and formic acid were added and then lysed by sonication and thermal shock. After centrifugation, the acidic soluble fractions were immediately analysed with a top down approach. High-resolution HPLC-ESI-MS-MS experiments were carried out by an Ultimate 3000 Micro HPLC (Dionex) coupled to an Orbitrap Elite (ThermoFisher) with ESI source. High resolution MS and MS2 data were elaborated both manually and with Proteome Discoverer 2.1 software. In both hAEC and hAMSC, thymosin β4 and its fragments 2-39 and 2-41, thymosin β10 and its fragment 2-41, S100A4, S100A6, S100A10, S100A11, ubiquitin, histones (H)3.2, H4, H2A type 2A, and H2A type 2C were detected. In addition, hAMSC also expressed thymosin α11, fragment 1-74 of ubiquitin, and fragments 419-466 and 422-466 of vimentin. On the other hand, S100A10 fragment 2-48, S100A10 with 2 N6-acetyllysine, and the macrophage migration inhibitory factor (MIF) were detected only in hAEC. Future studies will be focused on more detailed studies in order to confirm these interesting preliminary results, and understand the significance/role of these molecules in the immunomodulatory actions of amniotic cells.

* Corresponding author: Sofia Petrocchi. Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, Roma, Italia. Phone: +39 063053598; Fax: +39 063057612. E-mail address: [email protected] References: [1] Parolini O, Alviano F, Bagnara GP, Bilic G, Bühring HJ, Evangelista M, Hennerbichler S, Liu B, Magatti M, Mao N, Miki T, Marongiu

F, Nakajima H, Nikaido T, Portmann-Lanz CB, Sankar V, Soncini M, Stadler G, Surbek D, Takahashi TA, Redl H, Sakarugawa N, Wolbank S, Zeisberger S, Zisch A, Strom SC. Concise review: isolation and characterization of cells from human term placenta: outcome of the first international workshop on placenta derived stem cells. Stem Cells. 2008; 26:300-311.



P-IV-31

Proteomic expression profile of injured rat peripheral nerves revealed biological networks and processes associated to nerve regeneration

Vergara D1,2, Romano A3, Stanca E1,2, La Pesa V3, Aloisi AL3, Franck J4, Cicalini I5, Giudetti

AM1, Storelli E3,6, Pieragostino D5, Fournier I4, Sannino A6, Salzet M4, Cerri F3, Quattrini A3, Maffia M1,2,*

1) Department of Biological and Environmental Sciences and Technologies, University of Salento,

via Monteroni, Lecce, Italy 2) Laboratory of Clinical Proteomic, ‘"Giovanni Paolo II"’ Hospital, ASL-Lecce, Italy 3) Division of Neuroscience and INSPE, San Raffaele Scientific Institute, Milan, Italy

4) Univ. Lille, Inserm, U-1192 - Laboratoire Protéomique, Réponse Inflammatoire et Spectrométrie de Masse-PRISM, F-59000 Lille, France

5) Analitical Biochemistry and Proteomics Unit, Research Centre on Aging (Ce.S.I), University "G. d'Annunzio" of Chieti-Pescara, 66100 Chieti, Italy

6) Department of Innovation Engineering, University of Salento, via Monteroni, Lecce, Italy Peripheral nerve regeneration is regulated through the coordinated spatio-temporal activation of multiple cellular pathways. In this work, an integrated proteomics and bioinformatics approach was employed to identify differentially expressed proteins in the proximal sciatic nerve stumps at 20 days of injury. By a label free liquid chromatography mass-spectrometry (LC-MS/MS) approach, we identified 201 differentially proteins that were assigned to specific canonical and disease and function pathways. These include proteins involved in cytoskeleton signalling and remodelling, acute phase response, and cellular metabolism. Metabolic proteins were significantly modulated after nerve injury to support a specific metabolic demand. In particular, we identified a group of proteins involved in lipid uptake and lipid storage metabolism. Immunofluorescent staining for acyl-CoA diacylglycerol acyltransferase 1 (DGAT1) and DAGT2 expression provided evidence for the expression and localization of these two isoforms in Schwann cells in the sciatic nerve at the injury site. This further supports a specific local regulation of lipid metabolism in peripheral nerve after injury. We gratefully acknowledge funding from the Apulia Regional Cluster project “SISTEMA” project code T7WGSJ3 by ItPA Foundation (award for young researchers) Corresponding author: Michele Maffia DISTEBA - University of Salento E-mail address: [email protected] (M. Maffia)


P-IV-32

β-catenin knockdown affects mitochondrial biogenesis and lipid metabolism in breast cancer cells 

Daniele Vergaraa,b, Eleonora Stancab, Flora Guerraa, Paola Priorec, Antonio Gaballoc, Julien Franckd,

Pasquale Simeonee, Marco Trerotolaf, Stefania De Domenicog, Marta Verri a,b, Daniela Gaetani a,b, Federica Paradiso a,b, Isabelle Fournierd, Cecilia Buccia, Michel Salzetd,  Michele Maffiaa,b, Anna

M. Giudettia*

a) Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy

b) Laboratory of Clinical Proteomic, ‘‘Giovanni Paolo II’’ Hospital, ASL-Lecce, Italy c) CNR NANOTEC - Institute of Nanotechnology, Lecce, Italy

d) Laboratoire PRISM - U1192 INSERM, Université de Lille 1, Villeneuve d'Ascq, France e) Unit of Cytomorphology, CeSI-MeT and Department of Medicine and Aging Sciences, School of

Medicine and Health Sciences, University “G. d’Annunzio”, Chieti, Italy f) Unit of Cancer Pathology, CeSI-MeT and Department of Medical, Oral and Biotechnological

Sciences, University “G. d’Annunzio”, Chieti, Italy g) Institute of Food Production Sciences, C.N.R. Unit of Lecce, Lecce, Italy

β-catenin is a multifunctional protein with a role in the regulation of cell organization and gene expression. Here, we applied a liquid chromatography-mass spectrometry (LC-MS/MS) approach to identify proteins modulated after β-catenin knockdown in the breast cancer cell line MCF-7. After trypsin digestion of CTR (shCTR) and β-catenin knockout cells (shβcat), by using a label free analysis, we identified a total of 98 differentially expressed proteins, including 53 up-regulated and 45 down-regulated. Bioinformatics analysis of this dataset led to the identification of proteins involved in multiple biological processes and modulated after β-catenin knockdown, including a large proportion of metabolic proteins. In detail, proteins involved in carbohydrate metabolism and tricarboxylic acid cycle were down-regulated, whereas proteins associated to lipid metabolism were up-regulated in shβcat compared to shCTR. In addition, in shβcat we measured a decreased mitochondrial mass and membrane potential. This is consistent with the reduced expression of transcriptional factors regulating mitochondrial biogenesis we measured in these cells. β-catenin driven metabolic reprogramming resulted also in a significant modulation of lipogenic enzyme expression and activity. Moreover, increased utilization of [1-14C]acetate and decreased incorporation of [14U]glucose into fatty acids, was also measured in shβcat with respect to shCTR. Our data highlight a role for β-catenin in the regulation of metabolism and energy homeostasis in MCF-7 cells.

We gratefully acknowledge funding from the Apulia Regional Cluster project “SISTEMA” project code T7WGSJ3

Corresponding author: Anna M. Giudetti.

University of Salento, via Prov.le Lecce-Monteroni, Lecce, Italy.

Tel.: +39 832.298679

fax: +39 832.298626

E-mail address: [email protected] (A.M. Giudetti)


P-IV-33

Stage- and grade-related alterations of urinary proteome in ccRCC patients investigated by label-free nLC-ESI-MS/MS

Martina Stellaa, Manuel Gallia, Andrew Smitha, Mariia Ivanovaa, Isabella Pigaa, Italo Zoppisb,

Marco Grassoc, Giorgio Bovod, Fulvio Magnia and Clizia Chinelloa*

a) Dept. of Medicine and Surgery, Proteomics and Metabolomics Unit, Univ. of Milano-Bicocca, Italy

b) Department of Information, Systems and Communication, Univ. of Milan-Bicocca, Italy c) Urology Unit, San Gerardo Hospital, Monza, Italy

d) Pathology Unit, San Gerardo Hospital, Monza, Italy

Renal cell carcinoma (RCC) represents the 9th most common cancer worldwide and its incidence is rapidly increasing. Commonly, symptoms only appear at late stages of cancerous disease, when the life expectancy is 10-15 months. Imaging techniques, commonly used for the detection of renal carcinomas, are not always able to clearly distinguish benign renal masses from malignant ones, masses with different aggressiveness and metastatic potential1. Therefore, possible biomarkers for early detection and tumour progression are needed. In a pilot study based on a cohort of 44 patients affected by the clear cell variant of RCC (ccRCC), we investigated the urinary proteome to pinpoint alterations correlating with clinical features (pT and grade)2. To this end, urine samples were digested following FASP protocol and peptide identification was obtained by nLC-ESI MS/MS. Label-free quantification was performed using PEAKS Studio and R platforms. Considering only the proteins identified and quantified with at least two unique peptides, seven showed a significant variation among samples with different pT (pT1 versus pT3). On the other hand, when comparing patients with different grade (low grade:G1-G2 and high grade:G3-G4), A1AG1 was the only protein with a remarkable modulation of its expression. In addition, 11 proteins were dysregulated accordingly to tumour progression. An investigation based on functional annotation was performed to inspect the biological role of these alterations. In conclusion, these outcomes highlighted a panel of promising proteins that could be a useful starting point for future studies aimed at verifying their possible use in the management of RCC patients. * Corresponding author: Clizia Chinello, Ph.D. Dept. of Medicine and Surgery (DMC)-University of Milano-Bicocca. Via Cadore, 48-20900 Monza (MB) Tel.: +39 02-6448-8105 fax: +39 02-6448-8252 E-mail address: [email protected] References: [1] Delahunt B et al.The International Society of Urological Pathology(ISUP) grading system for renal cell carcinoma and other prognostic

parameters. Am.J.Surg.Pathol. 37(10),1490–1504 (2013) [2] Borje Ljungberg, et al. EAU Guidelines on Renal Cell Carcinoma: 2014 Update. European Urology 67 (5), 913–924 (2015) Acknowledgements This work was supported by grants from FAR 2013–2016, Fondo di Ateneo Quota Competitiva 2014 and from Fondazione Gigi & Pupa Ferrari Onlus.



INDEX

Authors index 107 PROGRAM AT GLANCE………………………………………………………………………pag. 5 FaN WORKSHOP PROGRAM………………………………………………………………...pag. 9 SCIENTIFIC PROGRAM ………………………………………………………………...…..pag. 10 SATELLITE SESSION PROGRAM ……...………………………………………..………..pag. 16 KEYNOTE LECTURES ……………………………………………………..………….........pag. 17 K-01 Next-generation proteomics: building a precision toolkit for life science Albert Sickmann K-02 Proteomics and Immunoproteomics lessons learned from S. aureus host-pathogen interactions Frank Schmidt K-03 neXtProt as a tool to explore the human proteome Lydie Lane K-04 Generation of large scale SWATH-MS proteomic datasets and their use Ruedi Aebersold K-05 Shedding new light on Spinal Cord injury Michel Salzet K-06 From protein biomarker discovery to the development of mass spectrometry based diagnostic tests of clinical utility Stephen R Pennington K-07 Neuronal Interfaces: Innovative Therapies and Neuroprosthetics Fabio Benfenati K-08 Ex vivo screening of individual human gut microbiomes Daniel Figeys K-09 Role of the microbiome in food allergy and asthma Liam O’Mahony K-10 Micromanagement of the metaproteome: taking control of your data Lennart Martens DISTINGUISHED SPEAKERS………………………………………………………………..pag.28 D-01 Integrated proteomic technologies for the characterization of milk products Andrea Scaloni D-02 Mapping Human Mitochondrial Protein Interactions Linked to Neurodegeneration Mohan Babu D-03 Clinical MALDI MS Imaging: From Bench to Surgery room Isabelle Fournier D-04 Proteomic approaches to genetic and oncological diseases Piero Pucci D-05 The central role of microbiota: from environment to food production Paola Roncada D-06 Microbiota from childhood to adulthood in health and disease

108 XII ItPA National Congress Lecce, June 12th-15th, 2017 Lorenza Putignani D-07 Building 3D human tissues in vitro for tissue- and organ on chip applications Paolo A. Netti D-08 Implantable and wearable technologies for lifescience at the Center for Biomolecular Nanotechnologies Massimo De Vittorio D-09 A composite scaffold for bone-cartilage defects bridging: an in vivo study Alessandro Sannino SELECTED ORAL COMMUNICATIONS………………………………………………….pag. 38 SESSION I: NON – HUMAN PROTEOMICS……………………………………………….pag. 39 O-I-01 Proteomic analysis of the mode of action of the disinfectants based on pyridoxal oxime derivatives against food borne pathogens Martina Šrajer Gajdošik, Uroš Andjelković, Dajana Gašo Sokač, Hrvoje Pavlović, Tamara Martinovicć, Djuro Josic

O-I-02 Peptidomic analysis of bioactive ovine milk caseinate hydrolysates Adriano Brandelli, Roberta Fontoura, Ana Paula Folmer Corrêa, Ana Carolina Ritter, Lucélia Santi, Walter Orlando Beys da Silva, John Yates III

O-I-03 BacteriaMS: an open source tool for bacterial whole cell typing by mass spectra pattern matching with in silico hypothesis validation Zhuoxin Chen, Yi Yang, Tianqi Gong, Yu Lin, Yingdi Zhu, Hubert H. Girault, Baohong Liu, Liang Qiao, and Pengyuan Yang O-I-04 Proteomics to improve serodiagnosis of Brucellosis Paola Roncada, Cristian Piras, Isabella Alloggio, Viviana Greco, Tiziana Di Febo, Ivanka Krasteva, Andrea Urbanic, Giuliano Garofolo, Manuela Tittarelli, Luigi Bonizzi, Alessio Soggiu

SESSION II: SYSTEM BIOLOGY……………………………………………………………pag. 44 O-II-01 Activation of Nrf2-element signalling pathway in response to H2S related oxidative stress Viviana Greco, Alida Spalloni, Luisa Pieroni, Patrizia Longone, Andrea Urbani O-II-02 Proteomic and interactomic characterization of PARK2-mutated Parkinson’s disease patients Mara Zilocchi, Ilaria Colugnat, Luisa Pieroni, Barbara Garavaglia, Zoran Minic, Mauro Fasano, Mohan Babu, Tiziana Alberio SESSION III: TECHNOLOGIES and BIOINFORMATICS…………………………….…pag. 47 O-III-01 Nanotechnological approaches for epithelial–mesenchymal transition biomarkers screening Paola Priore, Monica Bianco, Daniele Vergara, Flora Guerra, Simona Bettini, Rosanna Pagano, Cecilia Bucci, Michele Maffia, Valentina Arima, Antonio Gaballo O-III-02 Clinical applications of universal S-Trap sample processing John P. Wilson, Nikita Saha Turna, Rosamonde Banks, Darryl J.C. Pappin, Alexandre Zougman O-III-03 Terminal Amine Isotopic Labeling of Substrates (TAILS): a positional proteomics approach to study mitochondrial proteases in Parkinson's disease Marta Lualdi, Federica Corno, Maurizio Ronci, Tiziana Alberio and Mauro Fasano O-III-04 Diagnostic “rhinomic” profile of Allergic and non-Allergic Rhinits Subject by Mesoporous Silica Particles and MALDI-TOF Mass Spectrometry Chiara Villella, Maria Stella Murfuni, Mariaimmacolata Preianò, Giuseppina Maggisano, Nadia Lobello, Nicola Lombardo, Rocco Savino, Rosa Terracciano

Authors index 109 SESSION IV: HUMAN PROTEOMICS…………………………………………...………....pag. 52 O-IV-01 Urinary proteomics in Bardet-Biedl syndrome Michele Costanzo, Giuliana Bruno, Marianna Caterino, Rosa A. Siciliano, Maria F. Mazzeo, Miriam Zacchia, Giovambattista Capasso, Margherita Ruoppolo O-IV-02 Proteomic of exosome’s cargo and membrane proteins from Neuroblastoma Victor Corasolla Carregari, OlesyaChayka, Lene Jakobsen, Luisa Pieroni, Martin R. Larsen, Andrea Urbani, Arturo Sala, Giuseppe Palmisano O-IV-03 Proteomics analysis to assess the role of mitochondria in BRCA1-mediated breast tumorigenesis Antonio Concolino, Angela M. Perri, Claudia V. Fiumara, Laura Tammè, Erika Olivo, Giovanni Cuda, Domenica Scumaci O-IV-04 Peptidomics analysis of simulated mouth, gastric and intestinal digestion pattern of allergenic tropomyosin Cristian Piras, Alessio Soggiua Viviana Greco, Takács Krisztina, Gelencsér Éva, Thomas Holzhauser, Annette Kuehn, Karin Hoffmann-Sommergruber, Andrea Urbani, Luigi Bonizzi, Paola Roncada POSTERS……………………………..……………………………………………………...…pag. 57 POSTERS: SESSION I - NON HUMAN PROTEOMICS………….………………………..……pag. 58 P-I-01 Characterization of Arabian camel milk proteome over lactation Isabella Alloggio, Alexandra Contreras-Jodar, Cristian Piras, Alessio Soggiu, Viviana Greco, Andrea Urbani, Luigi Bonizzi, Moez Ayadie, Rihyad S. Aljumaah, Mohammed A. Alshaikh, Elena Díaz-Medina, Gerardo Caja, Paola Roncada P-I-02 Comparative proteomic analyses of Salmonella Enteritidis SE86 exposed to cold plasma Ana Carolina Ritter, Giorgia Gozzi, Lucelia Santi, Walter O. Beys-da-Silva, John R. Yates III, Luigi Ragni, Adriano Brandelli, Lucia Vannini P-I-03 Ancient and modern durum wheat varieties: a comparative proteomic analysis of the metabolic fractions Antonella Di Francesco, R. Saletti, V. Cunsolo, V. Muccilli, P. De Vita, S. Gallina and S. Foti P-I-04 Identification of Trypanosoma equiperdum low molecular weight proteins recognized by antibodies from dourine infected horses Mirella Luciani, Tiziana Di Febo, Ivanka Krasteva, Angela Cattaneo, Massimiliano Orsini, Chiara Di Pancrazio, Angela Bachi, Manuela Tittarelli P-I-05 Proteome analysis in dystrophic mdx mouse muscle reveals a drastic alteration of Key Metabolic and Contractile Proteins after chronic exercise and the potential modulation by anti-oxidant compounds Tania Gamberi, Tania Fiaschi, Elisa Valocchia, Alessandra Modesti, Paola Mantuano, Jean-Francois Rolland, Francesca Sanarica, Annamaria De Luca and Francesca Magherini P-I-06 Immunomodulatory Properties of L. gasseri Investigated by Proteomics Maria Fiorella Mazzeo, Diomira Luongo, Mauro Rossi, Rosa Anna Siciliano P-I-07 Vegetal Coal: Proteomic Study Of Tomato Fruits Maria Tartaglia, Simona Arena, Andrea Scaloni, Mariapina Rocco P-I-08 Proteome modulation in microencapsulated Lactobacillus reuteri DSM 17938 during simulated gastrointestinal conditions (SGC) Alessio Soggiu, Annachiara De Prisco, Cristian Piras, Isabella Alloggio, Viviana Greco, Andrea Urbani, Luigi Bonizzi, Gianluigi Mauriello, Paola Roncada P-I-9 Proteomic analysis of mitochondria from AMPK/Snf1- depleted yeast cells grown in presence or absence of methionine Elisa Maffioli, Francesca Grassi Scalvini, Silvia Morelli, Fabiana Santagata, Armando Negri, Gennaro Agrimi, Paola Coccetti, Gabriella Tedeschi

110 XII ItPA National Congress Lecce, June 12th-15th, 2017 POSTERS: SESSION II - SYSTEMS BIOLOGY……………………………………………...…pag. 68 P-II-01 Proteomic profiling of fractionated Head and Neck cancer cell secretome capable of activating the immune response in vitro Ada Consalvo, Maurizio Ronci, Verena Damiani, Mirco Zucchelli, Domenico Ciavardelli, Vincenzo De Laurenzi P-II-02 System biology analysis for dissecting the epithelial mesenchymal transition metabolic program Daniele Vergara, Anna Giudetti, Eleonora Stanca, Stefania De Domenico, Pasquale Simeone, Franck Julien, Paola Lunetti, Loredana Capobianco, Giuseppe Nicolardi, Isabelle Fournier, Michel Salzet, Michele Maffia POSTERS: SESSION III - TECHNOLOGIES and BIOINFORMATICS…………………...……pag. 70 P-III-01 An integrated procedure for the simultaneous analysis of mt-HPP shotgun data Mauro Fasano P-III-02 MARNORA: a tool to perform Meta-Analysis, Reaction Network, Over Representation Analysis Chiara Monti, Mauro Fasano, Simone Tini, Tiziana Alberio P-III-03 New innovations implemented on the Q Exactive HF mass spectrometer Tabiwang N. Array, Sega Ndiaye, Eugen Damoc, Erik Couzijn, Jens Grote, Oliver Lange, Christian Thoeing, Kerstin Strupat, Catherina Crone, Anastassios Giannakopulos, Thomas Moehring and Alexander Harder P-III-04 Enrichment strategies for improvement of mass spec analysis of chemical cross-linked peptides Rosa Viner, Ryan Bomgarden, Kratika Singhal, Sergei Snovida, Craig Gutierrez, Lan Huang POSTERS: SESSION IV - HUMAN PROTEOMICS…………………………………………..…pag. 74 P-IV-01 Iron depletion alters mitochondrial signaling and metabolism in breast cancer cells Maida De Bortoli, Elena Taverna, Elisa Maffioli, Patrizia Casalini, Gabriella Tedeschi and Italia Bongarzone P-IV-02 MAPPING OF TRANSGLUTAMINASE-2 REACTIVE GLUTAMINE RESIDUES IN SEVERAL HUMAN SALIVARY PROLINE-RICH PEPTIDES BY MS/MS Mozhgan Boroumand, Federica Iavarone, Federica Vincenzoni, Alessandra Padiglia, Massimo Castagnola, Irene Messana, Tiziana Cabras P-IV-03 Characterization of lipid metabolism in the differentiation of oligodendrocytes in myelinating forms Ilaria Cicalini, Daniele Vergara, Damiana Pieragostino, Lorenza Falcone, Patrizia Ballerini, Nicola Petragnani, Stefania De Domenico, Michele Maffia, Piero Del Boccio P-IV-04 Comparative proteomic approach of mitochondrial proteins in a mouse model for creatine transporter deficiency Federica Ciregiaa, Claudia Boldrini, Angelo Molinaro, Laura Barocelli, Tommaso Pizzorusso, Maurizio Ronci, Andrea Urbani, Maria Rosa Mazzoni, Antonio Lucacchinia, Laura Giusti P-IV-05 The proteomic map of porcine claustrum: what differences in the protein expression in insula and putamen tell us about the claustrum ontogenetic origin? Federica Ciregia, Laura Giusti, Andrea Pirone, Vincenzo Miragliotta, Bruno Cozzi, Maurizio Ronci, Maria Rosa Mazzoni, Antonio Lucacchini P-IV-06 ProLyPALS: Proteomics of Lymphocytes from Parkinson’s Disease and Amyotrophic Lateral Sclerosis patients Ilaria Colugnat, Chiara Monti, Chiara Sironi, Leonardo Lopiano, Adriano Chiò, Alice Di Pierro, Cristoforo Comi, Mauro Fasano, Tiziana Alberio P-IV-07 Potential new role for PRUNE during microtubule polymerization and cytoskeletal rearrangements Flora Cozzolino, Maria Monti, Veronica Ferrucci, Angela di Somma, Francesco Pennino, Fatemeh Asadzadeh, Iolanda Scognamiglio, Angela Duilio, Massimo Zollo, Pucci Piero

Authors index 111 P-IV-08 Study of tears metabolome for the research of new molecular biomarkers in multiple sclerosis Damiana Pieragostino, Ilaria Cicalini, Claudia Rossi, Maria Di Ioia, Giovanna De Luca, Mirco Zucchelli, Alessandra Lugaresi, Giuseppina Bologna, Marco Marchisio, Paola Lanuti, Piero Del Boccio P-IV-09 14-3-3 Proteins and their isoforms in breast cancer tissues Gianluca Di Cara, Rosa Musso, Patrizia Cancemi, Nadia N. Albanese, Salvatore Minafra and Ida Pucci-Minafra P-IV-10 An integrated cytologic and proteomic study on breast cancer cell lines with low and high motility properties Rosa Musso, Gianluca Di Cara, Adriana Celesia and Ida Pucci-Minafra P-IV-11 Proteomic Approaches to Understand the cytotoxic, apoptotic and antitumoral effects of new N-heterocyclic carbene–gold(I) complexes Francesca Magherini, Tania Fiaschi, Alessandra Modesti, Chiara Gabbiani, Alessandro Pratesi, Tiziano Marzo, Ida Landini, Stefania Nobili, Enrico Mini, Luigi Messori, Tania Gamberi P-IV-12 Dissecting the TRIM8 role in the pathogenesis of glioblastoma Ilaria Iacobucci, Diana Canetti, Flora Cozzolino, Pietro Pucci, Santina Venuto, Giuseppe Merla, Lucia Micale, Maria Monti

P-IV-13 Top-Down proteomic analysis of gingival crevicular fluid in deciduous, permanent and exfoliating teeth in children Federica Marini, Romeo Patini, Laura Di Tonno, Tiziana Cabras, Maria Teresa Sanna, Alessandra Olianas, Barbara Manconi, Massimo Cordaro, Irene Messana, Massimo Castagnola, Federica Iavarone P-IV-14 Zoledronic acid boosts γδ-T-cell activity in children given hematopoietic transplantation Alice Bertaina, Alessia. Zorzoli, Andrea Petretto, Giulia Barbarito, Elvira Inglese, Pietro Merli, Chiara Lavarello, Letizia P. Brescia, Biagio De Angelis, Gino Tripodi, Lorenzo Moretta, Franco Franco Locatelli and Irma Airoldi P-IV-15 Gut microbiota profiling in Autism Spectrum Disorders patients: a metaproteomics approach Stefano Levi Morteraa, Pamela Vernocchi, Ilaria Basadonne, Alessandro Zandonà, Marco Chierici, Andrea Quagliariello, Antonio Gasbarrini, Andrea Urbani, Paola Venuti, Cesare Furlanello, Lorenza Putignani P-IV-16 Altered profile of salivary proteins in patients with Autoimmune Hepatitis Barbara Liori, Barbara Manconi, Tiziana Cabras, Simona Onali, Francesco Figorilli, Luchino Chessa, Federica Iavarone, Massimo Castagnolac, Irene Messana, Alessandra Olianas P-IV-17 Protein composition of human blastocoel fluid isolated from women aged < and ≥ 37 years Elisa Maffioli, Francesca Grassi Scalvini, Silvia Morelli, Fabiana Santagata, Armando Negri, Elena Albani, Elena Monica Borroni, Gabriella Tedeschi P-IV-18 Interactors of human salivary Cystatin D explored by immunoprecipitation coupled to Mass Spectrometry Barbara Liori, Alessandra Olianas, Tiziana Cabras, Federica Vincenzoni, Simone Serrao, Irene Messana, Massimo Castagnola, Barbara Manconi P-IV-19 Top-down molecular characterization of pediatric craniopharyngioma Claudia Martelli, Riccardo Serra, Ilaria Inserra, Federica Iavarone, Federica Vincenzoni, Diana Valeria Rossetti, Gianpiero Tamburrini, Massimo Caldarelli, Luca Massimi, Massimo Castagnola, Claudia Desiderio

P-IV-20 Proteomic analysis of malignant pleural mesothelioma cell proteome and secretome Serena Lacerenza, Federica Ciregia, Laura Giusti, Viviana Greco, Andrea Urbani, Antonio Lucacchini, Maria Rosa Mazzoni P-IV-21 The traffic of GAA uptaken in enzymatic replace treatment of Pompe disease Orsola F. Moccia, Diana Canetti, Caterina Porto, Marcella Coletta, Giancarlo Parenti, Pietro Pucci, Maria Monti

112 XII ItPA National Congress Lecce, June 12th-15th, 2017 P-IV-22 Investigation of OFD1 interactome by functional proteomic approach Flora Cozzolino, Daniela Iaconis, Pucci Piero, Brunella Franco, Maria Monti P-IV-23 Dissection of the mechanism of action of a LSD1-inhibitor in Acute Myeloid Leukemia by quantitative proteomics Luciano Nicosia, Roberto Ravasio, Saverio Minucci and Tiziana Bonaldi P-IV-24 Proteomic insights in extracellular microvesicles from CSF and tears of multiple sclerosis patients Pieragostino Damiana, Cicalini Ilaria, Lanuti Paola, Marchisio Marco, Fontana Antonella, Zappacosta Romina, Simeone Pasquale, Sacchetta Paolo, di Ioia Maria, Lugaresi Alessandra, Del Boccio P

P-IV-25 Plasma n-3 PUFA levels correlate with protein markers in age-related disorders: an observational study in Alzheimer’s disease and MCI Maurizio Ronci, Debora Cutuli, Fabrizio Piras, Federica Marini, Luisa Pieroni, Viviana Greco, Laura Petrosini, Gianfranco Spalletta and Andrea Urbani P-IV-26 Tear Film Steroid Profiling in Dry Eye Disease by Liquid Chromatography Tandem Mass Spectrometry Damiana Pieragostino, Luca Agnifili, Ilaria Cicalini, Roberta Calienno, Mirco Zucchelli, Leonardo Mastropasqua, Paolo Sacchetta, Piero Del Boccio and Claudia Rossi P-IV-27 Proteomic analysis of K562 erythroid cells after Sox6 induction Gloria Barbarani, Antonella Ronchi, Robert Steinfelder, Sudharshan Elangovan, Cristina Fugazza, Marianna Caterino, Margherita Ruoppolo P-IV-28 Comparative proteomics of acid-insoluble fraction of saliva in type 1 diabetes-celiac disease subjects, celiac disease or type 1 diabetes subjects Morena Arba, Gianluigi Cabiddu, Elisabetta Pisano, Pier Paolo Pusceddu, Federica Iavarone, Federica Vincenzoni, Massimo Castagnola, Tiziana Cabras, Irene Messana, Maria Teresa Sanna P-IV-29 In-depth mapping and quantitative evaluation of urinary N-glycoproteome of clear cell RCC patients by n-LC ESI MS/MS approach Lucia Santorellia, Martina Stella, Manuel Galli, Clizia Chinello, Marina Pitto, Marco Grasso and Fulvio Magni P-IV-30 Top-down analysis of the proteomic profile of human amniotic epithelial cells (hAEC) and human amniotic mesenchymal stromal cells (hAMSC): preliminary data Sofia Petrocchia, Federica Iavarone, Pietro Romele, Antonietta Silini, Massimo Castagnolaa, Ornella Parolini P-IV-31 Proteomic expression profile of injured rat peripheral nerves revealed biological networks and processes associated to nerve regeneration Vergara D, Romano A, Stanca E, La Pesa V, Aloisi AL, Franck J, Cicalini I, Giudetti AM, Storelli E, Pieragostino D, Fournier I, Sannino A, Salzet M, Cerri F, Quattrini A, Maffia M P-IV-32 β-catenin knockdown affects mitochondrial biogenesis and lipid metabolism in breast cancer cells Daniele Vergaraa, Eleonora Stanca, Flora Guerra, Paola Priore, Antonio Gaballo, Julien Franck, Pasquale Simeone, Marco Trerotola, Stefania De Domenico, Marta Verri, Daniela Gaetani, Federica Paradiso, Isabelle Fournier, Cecilia Bucci, Michel Salzet, Michele Maffia, Anna M. Giudetti P-IV-33 Stage- and grade-related alterations of urinary proteome in ccRCC patients investigated by label-free nLC-ESI-MS/MS Martina Stella, Manuel Galli, Andrew Smith, Mariia Ivanova, Isabella Piga, Italo Zoppis, Marco Grasso, Giorgio Bovo, Fulvio Magni Clizia Chinello

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