Napoli,17maggio2019,ore9:00-16:30

AulaMagnadelComplessodelleBiotecnologie

ViaTommasodeAmicis,95,Napoli

2°WorkshopBIO/10

diDocentieRicercatoridiBiochimicadellaCampania

Segreteriascientifica/organizzativa:

AngelaArciello,FabioCattaneo,ConcettaValeriaGiosafatto,DaniloSwannMatassa,DariaMariaMonti,EmiliaPedone,AnnapinaRusso,LoredanaMariniello(loredana.mariniello@unina.it),RosarioAmmendola(rosario.ammendola@unina.it)

PartecipantiUniversitàdegliStudidiNapoliFedericoII;UniversitàdegliStudidellaCampaniaLuigiVanvitelli;

UniversitàdiSalerno;UniversitàdelSannio;UniversitàParthenope;ConsiglioNazionaledelleRicerche

La seconda edizione del Workshop dei docenti e ricercatori di Biochimica della Campania ha lo scopo di rafforzare la comunità scientifica campana e di stimolare la partecipazione di giovani aspiranti ricercatori. In ossequio a questo spirito, il Workshop non prevede quote di partecipazione, riserva spazio a brevi comunicazioni orali al maggior numero possibile di giovani non strutturati al di sotto dei 35 anni, mette in palio piccoli premi e iscrizioni alla Società Italiana di Biochimica (SIB), che patrocina l’evento. Il presente libro degli Abstract riporta i contributi suddivisi nelle rispettive categorie SIB: 1. Biochimica e Farmacologia (BIOFAR) 2. Biotecnologie (BIOTEC) 3. Differenziamento e Trasformazione Neoplastica (DTN) 4. Invecchiamento e Varie Patologie (IVP) 5. Proteomica e Metabolomica (PROMET) La lingua ufficiale del workshop è l’italiano; sono comunque ben accette le comunicazioni in inglese.

SEGRETERIA SCIENTIFICA / ORGANIZZATIVA

Rosario Ammendola (rosario.ammendola@unina.it) Angela Arciello (angela.arciello@unina.it) Fabio Cattaneo (fabio.cattaneo@unina.it) C. Valeria L. Giosafatto (giosafat@unina.it) Loredana Mariniello (loredana.mariniello@unina.it) Danilo Swann Matassa (daniloswann.matassa@unina.it) Daria Maria Monti (mdmonti@unina.it) Emilia Pedone (empedone@unina.it) Annapina Russo (annapina.russo@unina.it)

SITO INTERNET https://workshopbio10campania.wordpress.com/notizie-utili/

PAGINA FACEBOOK https://www.facebook.com/Workshop-BIO10-Campania-607379806397129/?modal=admin_todo_tour #WorkshopBIO10

SEDE CONGRESSUALE Il Workshop si svolge a Napoli presso il Complesso delle Biotecnologie in via Tommaso de Amicis 95, in Aula Magna.

Come raggiungerci Con i mezzi pubblici Dalla stazione Ferroviaria (P.zza Garibaldi) Metropolitana Linea 2 fino a p.zza Cavour e poi Metropolitana Linea 1, fermata “Policlinico” Autobus R2 fino a via De Pretis poi R4 fino al Policlinico Dall’Aeroporto di Capodichino Alibus fino alla stazione centrale di p.zza Garibaldi In auto Chi proviene dall’A1, dalla A3, e dalla A14, può immettersi sul raccordo per la tangenziale di Napoli (A56), uscendo allo svincolo n. 7 “Zona Ospedaliera”. Per parcheggiare: è disponibile un ampio parcheggio a pagamento alle spalle della stazione della metropolitana, a pochi metri dall’ingresso di Via Tommaso de Amicis.

P

PROGRAMMA

09:00 – 09:30 Registrazione e affissione poster

09:30 – 09:45 Apertura dei lavori

09:45 – 11:05 1a Sessione – Flash presentations categorie BIOFAR-DTN (elenco completo nella pagina successiva)

11:05 - 11:15 “Innovative Western and Protein Analysis Solutions” – Giuseppe Tortoriello, PhD, Technical Sales Specialist, Life Sciences Solutions

11:15 – 11:45 Coffee break

11:45 – 12:50 2a Sessione – Flash presentations categorie BIOTEC-IVP-PROMET (elenco completo nella pagina successiva)

12:50 – 13:00 “Microgem: coperazione tra Azienda e Università” - Loreta D’Amico, PhD

13:00 – 14:00 Lunch break

14:00 – 15:30 Visita ai Poster

15:30 – 16:00 Discussione generale

16:00 – 16:30 Premiazioni e chiusura dei lavori

1a Sessione – Flash presentations categorie BIOFAR-DTN (ore 9:45 – 11:05) 1. Anna Virginia Adriana Pirozzi - Active components of mediterranean herbs (carvacrol,

thymol and eugenol) are effective on fat deposit in in vitro nafld model 2. Laura Mosca - Ruolo antiproliferativo della S-adenosilmetionina in cellule di

carcinoma della laringe 3. Marialuisa Piccolo - Preclinical development of original cationic Ru-based

nanosystems in human models of breast cancer 4. Daniela Cristina Vuoso - L'estratto polifenolico di mela annurca induce selettivamente

la morte di cellule di carcinoma mammario triplo negativo attraverso la generazione di livelli tossici di specie reattive dell'ossigeno

5. Alberto Zullo - Varianti nel canale RYR1 associate a miopatie: caratterizzazione molecolare e funzionale in due famiglie

6. Prathyush Pothukuchi - Golgi organization determines the channelling of glycosphingolipid biosynthetic flux

7. Federica De Lise - Structural and functional insight into a novel bacterial α-L-rhamnosidase from the marine microorganism Novosphingobium sp. Pp1y

8. Andrea Bosso - GVF27 and IMY47: two unsuspected host defense peptides hidden in human enzymes

9. Roberta Iacono - Pharmacological enhancement of the human acid alpha-glucosidase by allosteric chaperones

10. Annalisa Pecoraro - Ribosomal protein rpL3 targets E2F1 through PARP-1: a new pathway linking ribosomal stress to cell proliferation

11. Martina Castaldo - Transattivazione del recettore tirosina kinasi TRKA mediata dal recettore GPCR FPR1 in cellule di neuroblastoma umano

12. Arianna Pastore - Potenziale oncogenico della proteina ZNF224 nel melanoma 13. Rosarita Nasso - Effetti dei polifenoli estratti da buccia di limone sull’invasività e

sull’espressione delle metalloproteasi indotte da Interleuchina-6 in cellule di carcinoma gastrico

14. Daniela Criscuolo - The molecular chaperone TRAP1 drives inflammation-induced platinum resistance via oxidative stress in human ovarian cancer

15. Silvia Trombetti - Quercetin is able to revert the apoptotic resistance triggered by the short isoform of GATA-1 in myeloid leukemia cells

2a Sessione – Flash presentations categorie BIOTEC-IVP-PROMET (ore 11:45-12:50) 1. Eliana dell’Olmo - Human apolipoprotein B: an unpredictable and promising source

of novel food preservatives 2. Paola Imbimbo - A green cascade approach for the downstream of high value

bioproducts from Galdieria sulphuraria 3. Manar Abdalrazeq - Effect of alkaline pH and heat treatment on the formation of

bioplastics from milk whey proteins in the presence of different concentrations of glycerol

4. Asmaa al-Asmar - Edible coatings and films to reduce acrylamide formation in fried foods and to extend strawberry shelf-life

5. Gennaro Sanità - Nanoparticelle ibride con proprietà fotoacustiche per applicazioni teranostiche

6. Rosa Merlo - A new biotechnological tool for an in vivo enzyme labelling and immobilization

7. Giovanni Gallo - TtSmtB; a thermophilic member of the ArsR/SmtB transcription factor family: insights into stability and metal binding properties

8. Silvia Sposito - The interplay between type 2 transglutaminase and gliadin peptides in celiac disease

9. Nunzia d’Onofrio - Attività antiossidante ed antinfiammatoria dei metaboliti bioattivi del latte di bufala

10. Emanuela Marchese - Metabolomics: an emerging powerful tool for dissecting biological aberrations in Bardet Biedl renal dysfunction

11. Vittoria Monaco - The role of cyclin D3 in differentiation and proliferation processes 12. Rosita Russo - Reliable identification of lactic acid bacteria by targeted and

untargeted high-resolution tandem mass spectrometry

BIOFAR.1

ACTIVE COMPONENTS OF MEDITERRANEAN HERBS (CARVACROL,

THYMOL AND EUGENOL) ARE EFFECTIVE ON FAT DEPOSIT IN IN VITRO NAFLD MODEL

ANNA VIRGINIA ADRIANA PIROZZI1* (adripirozzi@gmail.com; SIB), ROSARIO FINAMORE1 (rosario.finamore@unicampania.it), ANTONIETTA STELLAVATO1 (antonietta.stellavato@unicampania.it),

VALENTINA VASSALLO1 (valentina.vassallo92@gmail.com), CHIARA SCHIRALDI1 (chiara.schiraldi@unicampania.it; SIB)

1 Dipartimento di Medicina Sperimentale, sezione di Biotecnologie, Istologia e Biologia Molecolare,

Università della Campania L. Vanvitelli, Napoli, Italia. *Corresponding Author

Pediatric abdominal obesity has emerged as a significant global health problem that is closely associated to cardiovascular disease, diabetes, and fatty liver disease. The important message is that childhood obesity poses important health problems including, but not limited to, potentially severe chronic liver disease. Within liver pathologies, Non Alcoholic Fatty Liver Disease (NAFLD) is spreading more and more in childhood population. NAFLD is characterized by an accumulation of intracellular fatty acids due to a diets too rich in edible fats and sedentary life style. This condition leads to liver inflammation, cell necrosis, up to cirrhosis and liver failure. Nowadays there is no drug treatment for NAFLD; thus, only nutritional guidelines are indicated to reduce the progression of this pathological process. Gradual weight loss through increased regular exercise and a low-fat diet appears to be effective. Typical mediterranean diet appears to be effective in adult people, but for pediatric population more troubles are noticed related to doses administration and taste. Therefore our attention has focused on the use of essential oils that when added to a food such as tomato sauce, making it functional. Specifically, carvacrol, thymol and eugenol, were tested in vitro on a steatosis model. To mimic NAFLD in vitro, human hepatic cancer cells (HepG2) have been treated with a mix of fatty acids oleic (18:2) and linoleic (18:1) acid ratio 1:1 w/w at 6 mM concentration for 24h. Then the cells have been incubated with the substances alone and in mixture for 24h. After treatment the amount of intracellular fatty acids produced is evaluated by Oil Red staining and the steatosis markers as PPARs were analysed by western blotting. Our data show that this in vitro model may provide preliminary important informations for new therapeutic strategies based on nutraceutical approaches in prevention and reduction of fat deposits. References Stellavato A, Pirozzi AVA, de Novellis F, Scognamiglio I, Vassallo V, Giori AM, De Rosa M, Schiraldi C. In vitro assessment of nutraceutical compounds and novel nutraceutical formulations in a liver-steatosis-based model. Lipids Health Dis. 2018 Feb 5;17(1):24. doi: 10.1186/s12944-018-0663-2. Stellavato A, Lamberti M, Pirozzi AVA, de Novellis F, Schiraldi C. Myclobutanil worsens nonalcoholic fatty liver disease: An in vitro study of toxicity and apoptosis on HepG2 cells. Toxicol Lett. 2016 Nov 16;262:100-104. doi: 10.1016/j.toxlet.2016.09.013. Pirozzi AV, Stellavato A, La Gatta A, Lamberti M, Schiraldi C. Mancozeb, a fungicide routinely used in agriculture, worsens nonalcoholic fatty liver disease in the human HepG2 cell model. Toxicol Lett. 2016 May 13;249:1-4. doi: 10.1016/j.toxlet.2016.03.004.

BIOFAR.2

RUOLO ANTIPROLIFERATIVO DELLA S-ADENOSILMETIONINA IN CELLULE DI CARCINOMA DELLA LARINGE

MARTINA PAGANO1 (martina.pagano@unicampania.it; SIB), LAURA MOSCA1 (laura.mosca@unicampania.it; SIB), ALESSANDRA COPPOLA1 (alessandra.coppola@unicampania.it; SIB), FRANCESCA VITIELLO1 (francesca.vitiello4@studenti.unicampania.it; SIB), CONCETTA PAOLA ILISSO1

(concettapaola.ilisso@unicampania.it; SIB), GIOVANNA CACCIAPUOTI1 (giovanna.cacciapuoti@unicampania.it; SIB), MARINA PORCELLI1* (marina.porcelli@unicampania.it; SIB)

1Dipartimento di Medicina di Precisione, Università della Campania “Luigi Vanvitelli”, Napoli, Italia

*Autore corrispondente

La S-adenosil-L-metionina (AdoMet) è un nucleoside solforato ubiquitario che esercita una varietà di funzioni biologiche (1). Nell’ultimo decennio è stato evidenziato un coinvolgimento della molecola in diversi processi cellulari quali proliferazione, differenziamento, regolazione del ciclo cellulare e apoptosi (2,3). Al fine di valutare gli effetti esercitati dall’AdoMet sui tumori testa-collo, abbiamo selezionato la linea cellulare JHU-SCC-011 di carcinoma della laringe. Su tale linea cellulare sono stati condotti inizialmente esperimenti di vitalità cellulare in seguito al trattamento con AdoMet, dimostrando inibizione della proliferazione cellulare dose- e tempo dipendente. Per valutare l’effetto biologico esercitato dal composto di solfonio sono stati effettuati esperimenti di analisi del processo apoptotico, del processo autofagico e del meccanismo di stress del reticolo endoplasmatico (ER-stress) a vari tempi in seguito al trattamento con 200 e 300 µM AdoMet. L’analisi citofluorimetrica ha evidenziato circa il 20% di apoptosi dopo 72 ore dal trattamento, dato confermato dall’analisi mediante Western blotting dei principali mediatori del processo. L’analisi al FACS e la microscopia a fluorescenza, hanno indicato che l’autofagia è precocemente attivata a 24 e 48 ore di trattamento con AdoMet. L’attivazione dell’ER-stress risultava invece particolarmente evidente dopo 48 e 72 ore di trattamento con il composto di solfonio, come evidenziato dalle immagini di microscopia a fluorescenza. Tale dato è stato confermato dall’attivazione trascrizionale di CHOP, uXBP1 e sXBP1 e dall'attivazione traduzionale di CHOP. Lo studio delle vie di segnalazione attivate dall’AdoMet ha mostrato un incremento del rapporto pJNK/JNK e pERK/ERK1/2, MAPK coinvolte nei processi di morte attivati dal cisplatino, un farmaco chemioterapico utilizzato nella cura dei tumori testa-collo. La combinazione del cisplatino con l'AdoMet ha evidenziato un forte effetto sinergico. Nel complesso, i dati ottenuti rafforzano le evidenze sperimentali già presenti in letteratura sulle potenzialità pro-apoptotiche dell’AdoMet, e suggeriscono nuove strategie terapeutiche per il trattamento del carcinoma testa-collo. References

1. Mato JM, Martínez-Chantar ML, Lu SC. Ann Hepatol 12(2):183-9, 2013. 2. Cave DD, Desiderio V, Mosca L, Ilisso CP, Mele L, Caraglia M, Cacciapuoti G, Porcelli M. Journal

of Cellular Physiology, 233, 1370–1383, 2018. 3. Mosca L, Pagano M, Ilisso CP, Cave DD, Desiderio V, Mele L, Caraglia M, Cacciapuoti G, Porcelli

M. J Cell Physiol., 18:197, 2018.

BIOFAR.3

PRECLINICAL DEVELOPMENT OF ORIGINAL CATIONIC RU-BASED NANOSYSTEMS IN HUMAN MODELS OF BREAST CANCER MARIALUISA PICCOLOI (marialuisa.piccolo@unina.it; SIB), MARIA GRAZIA FERRAROI

(mgferraro8@gmail.com), CHIARA TAMMAROI (chiara.tammaro92@gmail.com), CLAUDIA RICCARDIIII (claudia.riccardi@unina.it), FRANCESCO MAIONEI (francesco.maione@unina.it), GABRIELLA MISSOII

(gabriella.misso@unicampania.it) MICHELE CARAGLIAII (michele.caraglia@unicampania.it; SIB), MARCO TRIFUOGGIIII (marco.trifuoggi@unina.it), DANIELA MONTESARCHIOIII (daniela.montesarchio@unina.it),

CARLO IRACEI* (carlo.irace@unina.it), RITA SANTAMARIAI* (rita.santamaria@unina.it; SIB)

IUniversità “Federico II”, Dipartimento di Farmacia, Napoli IIUniversità della Campania Luigi Vanvitelli, Dipartimento di Medicina di Precisione, Napoli

IIIUniversità “Federico II”, Dipartimento di Scienze Chimiche, Napoli *Autore Corrispondente

Il carcinoma mammario è il secondo tumore più comune al mondo, nonchè la principale causa di morte per cancro nelle donne. Secondo l'OMS, la sua incidenza è in costante aumento, motivo per cui lo sviluppo di nuovi promettenti farmaci antitumorali con specifici meccanismi di azione è considerato una priorità dalla comunità scientifica per sconfiggere specifici tipi di neoplasie, per limitare effetti collaterali tossici e sviluppo di chemioresistenza. Nella ricerca di nuovi farmaci antitumorali a base di metalli di transizione, negli ultimi anni numerosi complessi di rutenio sono stati proposti e testati come farmaci alternativi al cisplatino e congeneri, in quanto dotati di una spiccata attività antiproliferativa ed antimetastatica, e soprattutto di una minore tossicità. Nell'ambito di un progetto di ricerca multidisciplinare, abbiamo sviluppato un nuovo approccio per il delivery in vivo di complessi di Ru (III), basato sull'impiego di nanoaggregati nucleolipidici biocompatibili capaci di veicolare efficacemente complessi metallici in cellule cancerose1. Studi preclinici in vitro condotti su un selezionato pannello di cellule umane di carcinoma mammario - tra cui cellule di adenocarcinoma endocrino-responsivo (MCF-7) e cellule di adenocarcinoma mammario triplo negativo (MDA-MB-231) - hanno evidenziato che i nostri nanosistemi sono in grado di inibire significativamente la proliferazione cellulare mediante attivazione di pathways multipli di morte cellulare2. Successivi esperimenti in vivo hanno confermato l’azione antitumorale delle formulazioni più promettenti a base di rutenio. In particolare, risultati ottenuti mediante allestimento di un modello xenograft di cellule umane di carcinoma mammario, hanno provato l’efficacia di un nanosistema cationico a base di rutenio (HoThyRu/DOTAP) nel bloccare la crescita tumorale in topi atimici3. Sono in corso esperimenti ex-vivo di biologia molecolare al fine di migliorare la comprensione dei meccanismi alla base delle risposte cellulari osservate, essenziale per contrastare eventuali fenomeni di chemioresistenza nella ricerca di nuove opportunità terapeutiche per la lotta al cancro. References

1. Riccardi et al., EurJOC. 7: 1099-1119, 2017. 2. Irace et al., SREP. 7:45236, 2017. 3. Piccolo et al., SREP. In press. 2019.

BIOFAR.4

L'ESTRATTO POLIFENOLICO DI MELA ANNURCA INDUCE SELETTIVAMENTE LA MORTE DI CELLULE DI CARCINOMA MAMMARIO

TRIPLO NEGATIVO ATTRAVERSO LA GENERAZIONE DI LIVELLI TOSSICI DI SPECIE REATTIVE DELL'OSSIGENO

ELISA MARTINO+1 (elisa.martino@unicampania.it; SIB), DANIELA CRISTINA VUOSO+1 (danielacristina.vuoso@unicampania.it; SIB), STEFANIA D'ANGELO2 (sdangelo@uniparthenope.it), NUNZIA D'ONOFRIO1 (nunzia.donofrio@unicampania.it; SIB), MARINA PORCELLI1 (marina.porcelli@unicampania.it;

SIB) and GIOVANNA CACCIAPUOTI1* (giovanna.cacciapuoti@unicampania.it; SIB)

1 Dipartimento di Medicina di Precisione, Università della Campania "Luigi Vanvitelli" 2Dipartimento di Scienze Motorie e del Benessere, Università degli Studi di Napoli “Parthenope”

+uguale contributo *Autore corrispondente

Molti composti naturali possono cooperare nel potenziare l'efficacia terapeutica dei farmaci chemioterapici, superare la farmacoresistenza e ridurre gli effetti collaterali della chemioterapia (1). Tra i nutraceutici, i polifenoli costituiscono una delle classi di composti più interessanti e studiati. La maggior parte degli effetti benefici dei polifenoli naturali è stata attribuita alla capacità di agire da scavengers delle specie reattive dell’ossigeno (ROS). Tuttavia, recenti evidenze sperimentali indicano che in cellule neoplastiche i polifenoli possono comportarsi come agenti citotossici che portano alla morte cellulare attraverso la generazione di livelli tossici di ROS (2). La mela “Annurca” è caratterizzata da un contenuto estremamente alto in polifenoli, che conferiscono una più forte attività antiossidante, comparata con altre varietà. Studi pregressi del nostro gruppo di ricerca hanno dimostrato l'effetto antiproliferativo dell'estratto polifenolico di mela Annurca (APE) in cellule MCF-7 di carcinoma mammario (3). Il presente lavoro di ricerca ha riguardato lo studio dell'attività antitumorale di APE e del relativo meccanismo di azione in cellule MDA-MB-231 di carcinoma mammario triplo negativo (TNBC), un fenotipo di tumore mammario particolarmente resistente alle convenzionali terapie. In tali cellule APE inibisce con modalità dose- e tempo-dipendente la crescita cellulare, causa l'arresto del ciclo cellulare in fase G2/M, e induce apoptosi e autofagia. Le evidenze sperimentali indicano che l'azione antitumorale di APE è mediata dalla generazione di ROS e dall'attivazione della chinasi JNK, coinvolta nella via di segnalazione JNK/c-Jun che in condizioni di stress ossidativo è in grado di indurre efficacemente morte cellulare. APE, inoltre, inibisce alcune oncoproteine coinvolte nei processi di crescita e sopravvivenza cellulare. L'effetto citotossico di APE è selettivo per le cellule tumorali e risparmia cellule non tumorigeniche dove, al contrario APE riduce lo stress ossidativo indotto da H2O2. I risultati ottenuti suggeriscono che APE potrebbe rappresentare un potenziale candidato per il potenziamento delle terapie contro il TNBC.

References

1. Wang, P., Yang, H.L., Yang, Y.J., Wang, L. & Lee, S.C. Overcome Cancer Cell Drug Resistance Using Natural Products. Evid Based Complement Alternat Med. 2015, 767136 (2015).

2. León-González, A.J., Auger, C., Schini-Kerth, V.B. Pro-oxidant activity of polyphenols and its implication on cancer chemoprevention and chemotherapy. Biochem Pharmacol. 98, 371-380 (2015).

3. D'Angelo S, Martino E, Ilisso CP, Bagarolo ML, Porcelli M and Cacciapuoti G. Int. J Oncol. 51, 939-948 (2017).

BIOFAR.5

VARIANTI NEL CANALE RYR1 ASSOCIATE A MIOPATIE:

CARATTERIZZAZIONE MOLECOLARE E FUNZIONALE IN DUE FAMIGLIE

ALBERTO ZULLO,1,2 GIUSEPPA PERROTTA,1 LUCIA RUGGIERO,3 LUCIO SANTORO,3 FRANCESCO SALVATORE,1,4 AND ANTONELLA CARSANA,1,4,*

1 CEINGE-Biotecnologie Avanzate, Via Gaetano Salvatore 486, 80145 Napoli, Italia

2 Dipartimento di Scienze e Tecnologie, Università del Sannio, Via Port’Arsa 11, 82100 Benevento, Italia 3 Dipartimento di Neuroscienze, Scienze della Riproduzione e Ortodonzia, Università di Napoli “Federico II”,

Via Pansini 5, 80131 Naploli, Italia 4 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli “Federico II”, Via

Pansini 5, 80131 Naploli, Italia *Autore corrispondente

I recettori rianodinici di tipo 1 (RyR1) e diidropiridinici (DHPR) sono i principali canali del calcio coinvolti nell'accoppiamento eccitazione-contrazione nel muscolo scheletrico. RyR1 e DHPR sono localizzati nelle cellule muscolari striate rispettivamente nel reticolo sarcoplasmatico (SR) e sulla membrana del tubulo T. Mutazioni nel canale RyR1 sono associate sia a patologie della muscolatura scheletrica con trasmissione ereditaria autosomica dominante, come la suscettibilità all’ipertermia maligna e la miopatia da Central Cores (CCD), sia a miopatie autosomiche recessive con eterogeneità fenotipica e istopatologica. In questo studio sono state identificate quattro varianti nel canale RyR1 in soggetti appartenenti a due famiglie italiane: una affetta da miopatia e portatrice delle varianti c.4003C>T (p.R1335C) e c.7035C>A (p.S2345R); una famiglia affetta da CCD e con le varianti c.9293G>T (p.S3098I) e c.14771_14772insTAGACAGGGTGTTGCTCTGTTGCCCTTCTT (p.F4924 V4925insRQGVALLPFF). Mediante studi di espressione in vitro in cellule linfoblastoidi dei pazienti abbiamo dimostrato che la variante c.4003C>T non è espressa e che la variante con l’ inserzione in frame di 30-nucleotidi è espressa a bassi livelli. Inoltre, con esperimenti di fluorimetria, è stato misurato il rilascio di Ca2+ in risposta a un agonista specifico di RyR1 (4-cloro-m-cresolo) e alla tapsigargina, una molecola che induce lo svuotamento passivo dei depositi di Ca2+ del SR. I risultati degli esperimenti hanno mostrato che la variante c.7035C> A (p.S2345R) causa una riduzione delle riserve di Ca2+ del SR e che la condizione di eterozigosità composta per la variante c.9293G>T (p.S3098I) e l’inserzione in frame di 30-nucleotidi aumenta il rilascio di Ca2+ mediato da RyR1 e non modifica le riserve di Ca2+ del SR. In conclusione, in questo studio abbiamo caratterizzato i difetti molecolari dei canali RyR1 in due famiglie con patologie della muscolatura scheletrica.

BIOFAR.6

GOLGI ORGANIZATION DETERMINES THE CHANNELLING OF GLYCOSPHINGOLIPID BIOSYNTHETIC FLUX

PRATHYUSH POTHUKUCHI1 (p.roy@ibp.cnr.it), ILENIA AGLIARULO1 (i.agliarulo@ibp.cnr.it), MARINELLA PIROZZI1 (m.pirozzi@ibp.cnr.it); GABRIELE TURACCHIO1 (g.turacchio@ibp.cnr.it);

RICCARDO RIZZO1 (r.rizzo@ibp.cnr.it); NINA DATHAN1 (n.dathan@ibp.cnr.it); DANIELA SPANO1 (d.spano@ibp.cnr.it); LAURA CAPOLUPO1 (l.capolupo@ibp.cnr.it); ADVAIT SUBRAMANIAN1

(a.subramanian@ibp.cnr.it); PETRA HENKLEIN2 (petra.henklein@charite.de); GAELLE BONCOMPAIN3 (Gaelle.Boncompain@curie.fr); FRANCK PEREZ3 (Franck.Perez@curie.fr); LINA OBEID4

(Lina.Obeid@stonybrookmedicine.edu); ALBERTO LUINI1 (a.luini@ibp.cnr.it); GIOVANNI D’ANGELO5 (giovanni.dangelo@epfl.ch); SEETHARAMAN PARASHURAMAN1* (r.parashuraman@ibp.cnr.it)

1Institute of Protein Biochemistry, National Research Council, Via P. Castellino 111, Italy

2Universitätsmedizin Berlin Institut für Biochemie Charité, Germany; 3 Institute Curie – CNRS UMR1 44, Research Center, France

4 Stony Brook University Medical Center, United States 5 EPFL-SV-IBI, Switzerland

*Corresponding Author

Glycans are one of the four biopolymers that cells are built of. The importance of polymers in human physiology is highlighted by numerous disorders that are associated with its impaired production. The production of the biopolymers like proteins or DNA depends on large molecular machines that copy from a template to produce a single defined product and thus are deterministic. On the contrary, production and defined cell type specific distribution of glycans depends on the action of enzymes which are sequentially positioned and localized to Golgi apparatus. Thus, the production chain for glycan polymers resembles more a metabolic pathway/network. The channelling of the metabolic flux of glycan production along the network can be influenced by several factors including the levels and Km of the enzymes like every other metabolic network. In the current study, we explored the role of a rather unique feature of the glycan metabolic pathway - the spatial organization of the enzymes in the Golgi in determining the channelling of the metabolic flux. Here, by using glycosphingolipid biosynthesis as a model system we demonstrate spatial organization Golgi enzymes is indeed an important factor determining the metabolic channelling and identified a dedicated molecular machinery that plays an active role in restricting the localization of biosynthetic enzymes to appropriate sub-compartments of the Golgi complex. Specifically, our results demonstrate that Grasp55, influences the proper intra-Golgi localization of key glycosphingolipid biosynthetic enzymes, lactosylceramide synthase and glucosylceramide synthase, by regulating the entry of enzymes into retrograde transport vesicles. Deletion of Grasp55 function leads to a mislocalization of these enzymes, and change glycosphingolipid output of the Golgi. These findings highlight how the proper configuration of the Golgi apparatus in terms of both composition and spatial/sequential organization influence the channelling of the glycan metabolic flux to determine the glycan production by the cell.

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BIOFAR.7

STRUCTURAL AND FUNCTIONAL INSIGHT INTO A NOVEL BACTERIAL Α-L-RHAMNOSIDASE FROM THE MARINE

MICROORGANISM NOVOSPHINGOBIUM SP. PP1Y FEDERICA DE LISE1 (federica.delise@unina.it); FRANCESCA MENSITIERI1

(francesca.mensitieri@unina.it); STRAZZULLI ANDREA1,2 (andrea.strazzulli@unina.it), MORACCI MARCO1,2 (marco.moracci@unina.it); NOTOMISTA EUGENIO1 (notomist@unina.it); CAFARO VALERIA1 (valeria.cafaro@unina.it), DI DONATO ALBERTO1 (didonato@unina.it), IZZO VIVIANA3* (vizzo@unisa.it)

1 Department of Biology, University Federico II of Naples, Via Cinthia 26, 80127, Naples, Italy. 2 Institute of Biosciences and Bioresources, National Research Council of Italy, Via P. Castellino 111,

80131, Naples, Italy. 3 Department of Medicine, Surgery and Dentistry "Scuola Medica Salernitana", University of Salerno, Via

S. Allende 2, 84131, Salerno, Italy. *Corresponding Author

Keywords: Rhamnosidase,Flavonoid,Homology modeling, Novosphingobiumsp. PP1Y. α-L-Rhamnosidases (α-RHAs, EC 3.2.1.40) are glycosyl hydrolases (GHs) hydrolyzing terminal α-L-rhamnose residues from different substrates such as heteropolysaccharides, glycosylated proteins and natural flavonoids. Although the possibility to hydrolyze rham-nose from natural flavonoids has boosted the use of these enzymes in industry, to date only few bacterial rhamnosidases have been fully characterized and only one crystal structure of a α-RHA of the GH106 family has been solved [1]. A novel α-RHA (RHA-P) activity was identified in the crude extract of Novosphingobium sp. PP1Y [2]; this enzyme is an inverting GH, for which an initial biochemical characterization has been performed [3]. In this work, we show that the enzyme, whose recombinant expression in E.coli and purification was optimized, has a good stability in various conditions of temperature and pH. An initial homology modeling study of RHA-P, in combination with a site directed mutagenesis analysis, confirmed a pivotal role of residues D503, E506, E644, likely located at the catalytic site. The definition of the 3D structure through X-ray crystallography is currently underway and will give important details concerning the the catalytic mechanism of this protein. RHA-P showed activity on natural flavonoids such as naringin, rutin, hesperidin and quercitrin, with a catalytic efficiency comparable or even higher than other bacterial α-RHAs described in literature. These results make this enzyme appealing for the bioconversion and de-rhamnosylation of natural flavonoids. In this framework, the possibility to use as biocatalyst, according to the needs, either the purified enzyme or the recombinant whole cell of E. coli expressing the protein, render RHA-P a versatile tool for biotechnological purposes.

BIOFAR.8

DEFINIZIONE DELLE BASI MOLECOLARI DELL’AMILOIDOSI AL: CARDIOTOSSICITA’ DELLE CATENE LEGGERE

IMMUNOGLOBULINICHE ESTHER IMPERLINI (imperlini@ceinge.unina.it) (SIB)a,b,#, FRANCESCA LAVATELLIc,#

(francesca.lavatelli@unipv.it), DANIELA SARNATARO (daniela.sarnataro@unina.it)d, PAOLA ROGNONI (paola.rognoni@unipv.it)c, GIAMPAOLO MERLINI (gmerlini@unipv.it)c,*, FRANCESCO SALVATORE

(salvator@unina.it) (SIB)b,d,*

aIRCCS SDN, Napoli, Italia bCEINGE-Biotecnologie Avanzate, Napoli, Italia

cCentro per lo Studio e la Cura delle Amiloidosi Sistemiche, Fondazione IRCCS Policlinico San Matteo di Pavia e Dipartimento di Medicina Molecolare, Università degli studi di Pavia, Pavia, Italia

dDipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli studi di Napoli “Federico II”, Napoli, Italia

#Equal contribution *Corresponding Author

Le amiloidosi sistemiche sono patologie da misfolding proteico caratterizzate da modificazioni conformazionali di proteine che si aggregano e si depositano nei tessuti sotto forma di fibrille di amiloide [1]. Nell’amiloidosi AL, le fibrille sono formate dalla porzione N-terminale di catene leggere (CL) immunoglobuliniche monoclonali, prodotte in eccesso da un clone plasmacellulare midollare. L’amiloidosi AL è una patologia progressiva da deposito, con conseguente disfunzione degli organi interessati dal processo amiloidogenico; il coinvolgimento cardiaco interessa l’80% dei pazienti, determinandone anche la diminuzione della sopravvivenza. Esistono evidenze cliniche/sperimentali a supporto della proteotossicità delle CL solubili amiloidogeniche e cardiotossiche. Il razionale di tale studio è basato sull’ipotesi che tali CL stabiliscono interazioni non fisiologiche con altre proteine alterandone la loro normale funzione e/o l’espressione in cellule cardiache. A supporto di tale ipotesi, abbiamo dimostrato che le CL interagiscono con specifiche proteine mitocondriali [2] e modulano l’espressione di proteine ad attività mitocondriale, coinvolte nei processi di riorganizzazione del citoscheletro e del controllo-qualità della degradazione proteica [3]. Tuttavia, la cascata degli eventi molecolari alla base della cardiotossicità delle CL non è ancora nota. A tale scopo, abbiamo analizzato l’internalizzazione cellulare delle CL cardiotossiche mediante esperimenti di immunofluorescenza. Tali CL patologiche sono state purificate da pazienti con amiloidosi AL cardiaca. Le CL di controllo sono state isolate da pazienti affetti da mieloma multiplo. Entrambe le CL sono state coniugate con il fluoroforo Oregon Green (OG) 488 ed incubate a diversi tempi (30min, 4h e 24h) con fibroblasti cardiaci umani. I nostri dati hanno evidenziato differenze di internalizzazione tra i due tipi di CL: a 30min, l’internalizzazione della CL cardiotossica è minore rispetto a quella di controllo, ma maggiore a 24h di esposizione con le cellule cardiache. La comprensione dei meccanismi di internalizzazione delle CL permetterà di identificare le possibili associazioni molecolari che ne determinano il destino intracellulare. References 1. Merlini G, Dispenzieri A, Sanchorawala V, Schönland SO, Palladini G, Hawkins PN, Gertz MA. Systemic

immunoglobulin light chain amyloidosis. Nat Rev Dis Primers. 2018;4(1):38. 2. Lavatelli F, Imperlini E, Orrù S, Rognoni P, Sarnataro D, Palladini G, Malpasso G, Soriano ME, Di Fonzo

A, Valentini V, Gnecchi M, Perlini S, Salvatore F, Merlini G. Novel mitochondrial protein interactors of immunoglobulin light chains causing heart amyloidosis. FASEB J. 2015;29(11):4614-28.

3. Imperlini E, Gnecchi M, Rognoni P, Sabidò E, Ciuffreda MC, Palladini G, Espadas G, Mancuso FM, Bozzola M, Malpasso G, Valentini V, Palladini G, Orrù S, Ferraro G, Milani P, Perlini S, Salvatore F, Merlini G, Lavatelli F. Proteotoxicity in cardiac amyloidosis: amyloidogenic light chains affect the levels of intracellular proteins in human heart cells. Sci Rep. 2017;7(1):15661.

BIOFAR.9

MERCURY TOXICITY IN HUMAN ERYTHROCYTES AND PROTECTIVE ROLE OF HYDROXYTYROSOL

FABIANA TORTORA1 (fabiana.tortora1989@libero.it; SIB); CATERINA MANNA1* (caterina.manna@unicampania.it; SIB)

1University of Campania “Luigi Vanvitelli”, Naples, Italy.

*Corresponding Author

The possibility of reducing heavy metal toxicity through diet has attracted great interest recently and the use of phytochemicals able to significantly counteract oxidative alterations as an attractive tool for the reduction of mercury (Hg) toxicity has been proposed. We therefore investigated the possible protective effects of hydroxytyrosol (HT), an olive oil phenolic antioxidant, on the programmed cell death induced by Hg treatment in red blood cells (RBC). Similar to apoptosis of nucleated cells, RBC may undergo suicidal death (eryptosis), in which the most important hallmark is cell membrane scrambling with PS translocation to the cell surface (1). In recent papers, we offer experimental evidence that HT has the potential to modulate cytotoxicity and to counteract the oxidative stress induced in RBC by Hg treatment. We also report that HT treatment reduces morphological alterations, which are associated with phosphatidylserine (PS) exposure (2). Intact human RBC has been incubated in vitro in the presence of mercuric chloride (HgCl2) and the protective effect of HT against eryptosis has been evaluated by measuring several hallmarks including PS exposure, cell shrinkage, increase in intracellular calcium and ATP and glutathione depletion. Cell preconditioning with HT (1-5 µM), prior to exposure to 2,5 µM HgCl2, causes a noteworthy decrease in PS-exposing RBC, almost restoring ATP and glutathione content. Conversely, HT shows no effect against decrease in cell volume nor against influx of extracellular calcium. Interestingly, no increase in oxidative stress was observed at the HgCl2 concentration utilized. Our data provide evidence of the efficacy of HT in modulating the programmed suicidal death in an anucleated cell, devoid of mitochondria and thus lacking any mitochondria-mediated apoptotic pathways (3). Taken together, the data on HT reveal that the prevention of metal toxicity should be regarded as an additional mechanism responsible for the health-promoting potential of this dietary phenol. References 1. Lang E, Qadri SM, Lang F (2012). Killing me softly-suicidal erythrocyte death. Int J Biochem Cell Biol 44: 1236-1243. Doi: 10.1016/j.biocel.2012.04.019. 2. Tagliafierro L, Officioso A, Sorbo S, Basile A, Manna C (2015). The protective role of olive oil hydroxytyrosol against oxidative alterations induced by mercury in human erythrocytes. Food Chem Toxicol 82:59-63. Doi: 10.1016/j.fct.2015.04.029. 3. Officioso A, Alzoubi K, Lang F, Manna C (2016). Hydroxytyrosol inhibits phosphatidylserine exposure and suicidal death induced by mercury in human erythrocytes: Possible involvement of the glutathione pathway. Food Chem Toxicol 89: 47-53. Doi: 10.1016/j.fct.2016.01.003.

BIOFAR.10

NUOVI INIBITORI DELLA FOSFATASI CDC25B: STUDIO DEI PARAMETRI CINETICI E STRUTTURALI E DEGI EFFETTI BIOLOGICI SU

LINEE DI MELANOMA FEDERICA ALIOTTA1 (federica.aliotta@unina.it; SIB), ROSARITA NASSO1,2

(rosarita.nasso@uniparthenope.it; SIB), CARMEN CERCHIA5 (carmen.cerchia@unina.it), MARTINA SIMONETTI1 (marti.simonetti@studenti.unina.it), ROSARIO RULLO3 (rosario.rullo@cnr.it), ALESSANDRO

ARCUCCI4 (alessandro.arcucci2@unina.it), MARIOROSARIO MASULLO2 (mario.masullo@uniparthenope.it; SIB), ANTONIO LAVECCHIA5 (antonio.lavecchia@unina.it), EMMANUELE DE VENDITTIS1*

(emmanuele.devendittis@unina.it; SIB), MARIA ROSARIA RUOCCO1* (mariarosaria.ruocco@unina.it)

1 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II 2 Dipartimento di Scienze Motorie e del Benessere, Università di Napoli “Parthenope”

3 Istituto per il Sistema Produzione Animale in Ambiente Mediterraneo, Consiglio Nazionale delle Ricerche, Napoli

4 Dipartimento di Sanità Pubblica, Università degli Studi di Napoli Federico II 5 Dipartimento di Farmacia, Università degli Studi di Napoli Federico II

*Autore Corrispondente La fosforilazione/defosforilazione delle proteine è un evento che regola importanti processi cellulari, come la crescita, il differenziamento, la migrazione, la sopravvivenza e l’apoptosi. In particolare, gli enzimi “cell division cycle 25” (Cdc25) sono fosfatasi coinvolte nella regolazione del ciclo cellulare. L’alterazione dell’espressione di queste proteine è stata osservata in diversi tipi di tumori e quindi gli enzimi Cdc25 rappresentano promettenti “targets” per la terapia del cancro. In un precedente lavoro svolto dal mio gruppo di ricerca è stato dimostrato che il composto NSC28620 agisce come inibitore reversibile di Cdc25B (Ki 5.3 µM) e a 200 µM esercita un effetto antiproliferativo su alcune linee di cellule tumorali [J.Med.Chem. 55 (2012) 4142-4158]. Al fine di migliorare il potere inibitorio su Cdc25, è stato condotto un programma di ottimizzazione del “lead compound” NSC28620 che ha portato alla sintesi di trentuno derivati. Le proprietà di queste nuove molecule sono state evidenziate attraverso la misura dell’attività fosfatasica della forma ricombinate di Cdc25B contenente solo il dominio catalitico, in assenza o in presenza di questi inibitori. È stato osservato che alcune molecole sono dotate di un potere inibitorio maggiore rispetto a quello del lead compound; in particolare, il più attivo (cpd 7j) ha un valore di Ki pari a 0.8 μM. L’analisi dei grafici di Lineweaver-Burk ha suggerito che questi nuovi composti agiscono come inibitori reversibili, essenzialmente appartenenti a due tipologie (non competitivi e incompetitivi). La presenza in Cdc25B di un unico residuo di triptofano ha permesso poi, tramite l’analisi della fluorescenza intrinseca, di avere informazioni sulla regione della proteina coinvolta nell’interazione con i vari inibitori. In fine, l’effetto biologico dei composti più attivi è stato valutato mediante saggi di proliferazione su linee di melanoma umano. A bassa concentrazione, 2.5 μM, un solo composto (cpd 4a) riduce significativamente la crescita di cellule di melanoma, in maniera tempo-dipendente.

BIOFAR.11

NOVEL SYNTHETIC DERIVATIVES COMPOUNDS FEATURED 5-LIPOXYGENASE INHIBITOR: STUDY OF ANTI-TUMOR AND ANTI-

INFLAMMATORY EFFECT COSSU AM1,3,5* (alessiamaria.cossu@biogem.it; SIB), Luce A1(amalia.luce@unicampania.it) ,RUSSO M1

(margherita.russo@unicampania.it), TAKEUCHI T1 (takashi19900706@gmail.com), SCIAMMARELLA C1, BOCCHETTI M1 (marco.bocchetti@unicampania.it), RAMIREZ AC2, PACE S3 (simona.pace@uni-jena.de),

WERZ O3 (oliver.werz@uni-jena.de), FILOSA R4 (rfilosa@hotmail.it) , ZAPPAVIGNA S1 (silvia.zappavigna@unicampania.it), CARAGLIA M1,5 (michele.caraglia@unicampania.it)

1Dep. of Precision Medicine, University of Campania “Luigi Vanvitelli”, via L. De Crecchio 7, Naples, 80138,

Italy. 2Dep. of Environmental Sciences, Faculty of Chemistry and Biology, University of Santiago de Chile,

8320000, Santiago, Chile. 3Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University

Jena, 07743, Jena, Germany. 4Dep. of Experimental Medicine, Second University of Naples, Via L.De Crecchio, 7, Naples, 80138, Italy.

5Biogem scarl, Institute of Genetic Research, Laboratory of Molecular and Precision Oncology, 83031, Ariano Irpino (AV), Italy. *Corresponding Author

5-lipoxygenase (5-LO), is involved in the proliferation and survival of tumour cells. The inhibition of 5-LO representing a valid drug-target for pharmacotherapy of inflammatory disorders and neoplasms. We investigated the biological and antitumor activity of new benzoquinonic derivatives: RF-Id, Ea100C and Ea100Cred, potent 5-LO inhibitors, and their possible use as future candidates for Glioblastoma treatment. Finally, we evaluated, the ability of new catechol derivatives CI, CS, AS and AI, to inhibit 5-LO activity. In details we found that: v RF-Id induced growth inhibition, apoptosis and an increase of cell in G2/S phase together

with high level of p21 and p27 expression. Moreover, it induced a significant cleavage of caspases 8, 9, 3 and 7, blocked the interaction of cIAP2 / XIAP by the degradation of XIAP and inhibited NF-kB pathway.

v Ea100C induced apoptosis, cell cycle modulation, and autophagy on U87-MG. Ea100c red induced apoptosis mediated by ER stress associated with autophagy and cell cycle modulation on LN229. The ER stress induced the activation of NF-kB and JNK and consequently of the caspase cascade., through CHOP up-regulation

v CI and CS induced a potential inhibition of 5-LO in both cell-free and cell-based tests. CI blocked p38 pathway required for 5-LO activation and prevented the interaction between 5-LO/ FLAP protein. AI and AS didn’t induce significant effects.

In conclusion, these new benzoquinonic derivatives could represent useful candidates for GBM treatment. References

1. Zappavigna S, Scuotto M, Cossu AM, Ingrosso D, De Rosa M, Schiraldi C, Filosa R, Caraglia M. The 1,4 benzoquinone-featured 5-lipoxygenase inhibitor RF-Id induces apoptotic death through downregulation of IAPs in human glioblastoma cells. J Exp Clin Cancer Res. 2016 Oct 22;35(1):167.

2. Filosa R, Peduto A, Aparoy P, Schaible AM, Luderer S, Krauth V, Petronzi C, Massa A, de Rosa M, Reddanna P, Werz O. Discovery and biological evaluation of novel 1,4-benzoquinone and related resorcinol derivatives that inhibit 5-lipoxygenase. Eur J Med Chem. 2013 Sep; 67:269-79.

BIOFAR.12

EXPRESSION, PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF A NOVEL BETA-GALACTOSIDASE FROM

THERMOPHILIC BACILLUS COAGULANS STRAIN MA-13 FERDINANDO SANSONE (ferdinando.sansone@unina.it); MARTINA AULITTO (MAulitto@lbl.gov);

ANDREA STRAZZULLI (andrea.strazzulli@unina.it; SIB); SALVATORE FUSCO (salvatore.fusco@unina.it; SIB); MARCO MORACCI (marco.moracci@unina.it; SIB); SIMONETTA

BARTOLUCCI (simonetta.bartolucci@unina.it) (SIB); PATRIZIA CONTURSI* (patrizia.contursi@unina.it; SIB)

Università degli studi di Napoli Federico II, Dipartimento di Biologia, Italia.

*Corresponding Author Industrial processes of lignocellulose’s degradation exploit microbial hydrolytic enzymes for the treatment of biomass [1]. In particular, synergistic effects of enzymatic mixtures for hydrolysis of hemicelluloses have already been described [2]. This study has been focused on enzymes specifically required for xyloglucans breakdown. A putative beta-galactosidase (EC number 3.2.1.23) from the thermophilic Bacillus coagulans strain MA-13 [3], named Bcb-Gal, has been characterized. Bcb-Gal belongs to the glycoside hydrolase family GH42 which catalyzes the hydrolysis of b-D-galactosides from non-reducing terminal of di- and oligosaccharides through a retaining mechanism. The beta-galactosidase gene (BC_022) has been cloned and expressed recombinantly in E. coli Rosetta (DE3). Subsequently, the enzyme has been purified at homogeneity by affinity chromatography and his molecular weight analyzed on SDS-PAGE. According to the sequence analysis, the protein showed a molecular mass of about 76 kDa. The recombinant enzyme displayed an optimal activity at 60 °C and pH 5.0 in citrate buffer using 2-Nitrophenyl-b-D-galactopyranoside as substrate. In addition to this, the stability at different pH values has been studied within the range pH 4.0-8.0. In particular, the enzyme retained more than 40% of the enzymatic activity at pH 5.0 after incubation up to 6 hours. Moreover, Bcb-Gal displayed a low thermostability, retaining less than 40% of residual activity after incubation at 40°C for 2 hours. Finally, the effects of metal ions on the enzyme activity were investigated. Bcb-Gal was strongly inhibited by Cu2+ while Mn2+, Mg2+, Ca2+, and Zn+ turned out to have an enhancing effect on the enzyme activity. Further biochemical characterization of this enzyme is under way in order to fully exploit its potential in biotechnological applications. References

1. Chundawat SP, Beckham GT, Himmel ME, Dale BE. Deconstruction of lignocellulosic biomass to fuels and chemicals. Ann Rev Chem Biomol Eng. 2011;2:121–145. doi: 10.1146/annurev-chembioeng-061010-114205.

2. Van Dyk JS, Pletschke BI. A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes-Factors affecting enzymes, conversion and synergy. Biotechnol Adv. 2012;30:1458–80.

3. Aulitto, Martina, et al. "Bacillus coagulans MA-13: a promising thermophilic and cellulolytic strain for the production of lactic acid from lignocellulosic hydrolysate." Biotechnology for biofuels 10.1 (2017): 210.

BIOFAR.13

CHITOSAN-BASED POLYELECTROLYTE COMPLEXES FOR DOXORUBICIN AND ZOLEDRONIC ACID COMBINED THERAPY TO

OVERCOME MULTIDRUG RESISTANCE MARIANNA ABATE1*, ALESSIA MARIA COSSU4, AMALIA LUCE1, AGOSTINO FESTA1, TAKASHI

TAKEUCHI1, FLORIANA LASALVIA1, SIMONA GIARRA2, VIRGINIA CAMPANI2, CARLO LEONETTI3, MANUELA PORRU3, LAURA MAYOL3, GIUSEPPE DE ROSA2, SILVIA ZAPPAVIGNA1, MICHELE

CARAGLIA1.

1Department of Precision Medicine University of Campania “Luigi Vanvitelli”, Naples, Italy. 2Department of Pharmacy, University of Naples “Federico II”, Naples, Italy. 3UOSD SAFU, IRCCS Regina Elena National Cancer Institute, Rome, Italy.

4BIOGEM Istituto di Ricerche Genetiche “Gaetano Salvatore”, Ariano Irpino, Italy. *Corresponding Author

The present work aimed to develop nanovectors co-encapsulating doxorubicin (Doxo) and zoledronic acid (Zol) for combined therapy against Doxo-resistant tumors. Chitosan-based polyelectrolyte complexes (PECs) prepared by ionotropic gelation technique were proposed. The influence of some experimental parameters was evaluated in order to optimize the PECs in terms of size and polydispersity index. PECs stability was studied by monitoring size and zeta potential over the time. In vitro studies were carried out on wild type and Doxo-resistant cell lines, to assess both the synergism between Doxo and Zol, as well as the restoring of Doxo sensitivity. Polymer concentration, incubation time and use of a surfactant were found to be crucial to achieve small size and monodisperse PECs. Doxo and Zol, only when encapsulated in PECs, showed a synergistic antiproliferative effect in all the tested cell lines. Importantly, the incubation of Doxo-resistant cell lines with Doxo/Zol co-encapsulating PECs resulted in restoration of Doxo sensitivity.

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BIOFAR.14

IDENTIFYING THE DNA-TARGET SITE OF MUCR FROM BRUCELLA ABORTUS AND THE PROTEIN REGION RESPONSIBLE FOR HIGHER-ORDER OLIGOMER FORMATION. EXAMINING THE ROLE OF MUCR

OLIGOMERIZATION IN GENE REGULATION ILARIA BAGLIVO1* (ilaria.baglivo@unicampania.it; SIB), LUCIANO PIRONE2 (luciano.pirone@unina.it; SIB),

JOSHUA EDISON PITZER3 (PITZERJ@ecu.edu), ROY MARTIN ROOP II3 (ROOPR@ecu.edu), EMILIA MARIA PEDONE2 (empedone@unina.it; SIB), PAOLO V. PEDONE1* (paolov.pedone@unicampania.it; SIB)

1Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of

Campania “Luigi Vanvitelli”, Caserta, 81100, Italy. 2Institute of Biostructures and Bioimaging, C.N.R, Naples, 80134, Italy

3Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, NC, USA.

*Corresponding Author MucR from Brucella abortus and Ml proteins (Mls) from Mesorhizobium loti are members of the Ros/MucR family of prokaryotic zinc-finger proteins found in the α-proteobacteria, which regulate the expression of genes required for the successful pathogenic and symbiotic interactions of these bacteria with the eukaryotic hosts. The structure and function of their distinctive zinc-finger domain have been extensively studied in the last years. The aim of our study is to disclose the DNA-target site of MucR and Mls and to analyse the possible quaternary structure of these proteins. The results of our experiments demonstrate that the DNA-target site of the proteins studied is an AT-rich region containing a T-A step and that the full-length proteins are able to form higher-order oligomers (1). Finally, we demonstrate that a conserved hydrophobic region at the N-terminus of MucR is responsible for higher-order oligomer formation and that oligomerization is essential for MucR regulatory function in Brucella (2). All these features of MucR are shared by the histone-like nucleoid structuring protein, (H-NS) (3), leading us to propose that the prokaryotic zinc-finger proteins in the MucR/Ros family control gene expression employing a mechanism similar to that used by the H-NS proteins, rather than working as classical transcriptional regulators. References

1. Ilaria Baglivo*, Luciano Pirone, Emilia Maria Pedone, Joshua Edison Pitzer, Lidia Muscariello, Maria Michela Marino, Gaetano Malgieri, Andrea Freschi, Angela Chambery, Roy-Martin Roop II & Paolo Vincenzo Pedone*. “Ml proteins from Mesorhizobium loti and MucR from Brucella abortus: an AT-rich core DNA-target site and oligomerization ability.” Sci Rep.(2017), 7(1):15805.

2. Luciano Pirone, Joshua Edison Pitzer, Gianluca D’Abrosca, Roberto Fattorusso, Gaetano Malgieri, Emilia Maria Pedone, PaoloVincenzo Pedone*, Roy Martin Roop II* & Ilaria Baglivo*. “Identifying the region responsible for Brucella abortus MucR higher-order oligomer formation and examining its role in gene regulation”. Sci Rep. (2018) 8:17238

3. Dorman, C. J. “H-NS: A Universal Regulator For a Dynamic genome.” Nature Rev Microbiol. (2004) 2, 391–399

BIOFAR.15

GOLPH3 PROMOTES ONCOGENESIS BY CONTROLLING THE INTRA-GOLGI TRAFFICKING OF GLYCOSPHINGOLIPID SYNTHASES

RICCARDO RIZZO1 (r.rizzo@ibp.cnr.it; SIB); DOMENICO RUSSO1 (d.russo@ibp.cnr.it; SIB); KAZUO KUROKAWA2 (kkurokawa@riken.jp); DOMENICO SUPINO1 (d.supino@ibp.cnr.it); PRANOY SAHU1

(p.sahu@ibp.cnr.it); BERNADETTE LOMBARDI1 (b.lombardi@ibp.cnr.it); FRANCESCO RUSSO1 f.russo@ibp.cnr.it, MIKHAIL ZHUKOVSKY1 (m.zhukovsky@ibp.cnr.it); PRATHYUSH POTHUKUCHI1

(p.roy@ibp.cnr.it); LUCIA STICCO1 (l.sticco@ibp.cnr.it); SERENA CAPASSO1 (s.capasso@ibp.cnr.it,);LAURA CAPOLUPO1 (l.capolupo@ibp.cnr.it); GAELLE BONCOMPAIN3

(Gaelle.Boncompain@curie.fr); NINA DATHAN1 (n.dathan@ibp.cnr.it), GABRIELE TURACCHIO1(g.turacchio@ibp.cnr.it); FEDERICA ZITO MARINO4 (zitomarino@istitutotumori.na.it);

GABRIELLA ACQUINO4 (g.aquino@istitutotumori.na.it); CARLO VITAGLIANO4 (carlo.vitagliano@istitutotumori.na.it); PETRA HENKLEIN5 (petra.henklein@charite.de); HENRIK

CLAUSEN6 (hclau@sund.ku.dk); ULLA MANDEL6 (ulma@sund.ku.dk); TOSHIYUKI YAMAJI7 (tyamaji@nih.go.jp), KENTARO HANADA7 (hanak@nih.go.jp); ALFREDO BUDILLON4

a.budillon@istitutotumori.na.it); SEETHARAMAN PARASHURAMAN1 (r.parashuraman@ibp.cnr.it); FRANCK PEREZ3 (Franck.Perez@curie.fr); LINA M. OBEID8 (Lina.Obeid@stonybrookmedicine.edu); AKI

NAKANO2 (nakano@riken.jp); YUSUF A HANNUN8 (Yusuf.Hannun@stonybrookmedicine.edu); GIOVANNI D’ANGELO9* (giovanni.dangelo@epfl.ch); ALBERTO LUINI1* (a.luini@ibp.cnr.it).

1Institute of Protein Biochemistry, CNR, Italy

2Live Cell Super-Resolution Imaging Research Team, Japan 3 Institute Curie – CNRS UMR1 44, Research Center, France

4Istituto Nazionale Tumori G. Pascale, Italy 5Universitätsmedizin Berlin Institut für Biochemie Charité, Germany

6University of Copenhagen, Faculty of Health Sciences, Denmark 7Department of Biochemistry & Cell Biology National Institute of Infectious Diseases, JAPAN

8Stony Brook University Medical Center, United States 9EPFL-SV-IBI, Switzerland

*Corresponding Author The GOLPH3 oncogene is located on a human chromosome region, which is frequently amplified in solid tumours. GOLPH3 encodes a Golgi complex associated protein involved in membrane trafficking and Golgi structure maintenance. While its role in the stimulation of cell proliferation pathways is widely accepted, how GOLPH3 gain of function mediates oncogenesis is not completely understood. Here we show that GOLPH3 regulates proliferation by promoting sphingolipid glycosylation. Specifically, we found that GOLPH3 interacts with sphingolipid glycosylating enzymes at the Golgi complex and controls their intra-Golgi recycling during cisternal progression. Through this mechanism GOLPH3 counteracts sphingolipid glycosylating enzymes leakage to lysosomes and consequent degradation resulting in increased enzymes levels. Alterations in glycosylating enzymes proteostasis and sphingolipid metabolism induced by increased GOLPH3 levels impact on growth signalling pathways, thus promoting uncontrolled proliferation. Importantly, GOLPH3 overexpression/ amplification tightly correlates with tumour cells sensitivity to pharmacological inhibition of sphingolipid glycosylation suggesting that inhibition of sphingolipid metabolism represents a valuable therapeutic option for patients bearing GOLPH3 dependent tumours.

BIOFAR.16

CRYPTIDES IDENTIFIED IN HUMAN APOLIPOPROTEIN B: NEW WEAPONS TO FIGHT ANTIBIOTIC-RESISTANCE

ROSA GAGLIONE1 (rosa.gaglione@unina.it; SIB), ANGELA CESARO1 (angela.cesaro@unina.it), ELIANA DELL’OLMO1 (eliana.dellolmo@unina.it), EUGENIO NOTOMISTA2 (notomist@unina.it), ELIO PIZZO2

(elipizzo@unina.it; SIB), *ANGELA ARCIELLO1 (anarciel@unina.it; SIB)

1Department of Chemical Sciences, University of Naples Federico II, 80126 Naples, Italy; 2Department of Biology, University of Naples Federico II, 80126 Naples, Italy.

*Corresponding Author Chronic respiratory infection is the main cause of morbidity and mortality in cystic fibrosis (CF) patients. One of the hallmarks of these infections led by opportunistic pathogens is their long-term persistence despite intensive antimicrobial therapy. This inevitably determines the development in CF of antimicrobial resistance [1]. Hence, novel strategies to counteract biofilm growth and to combat multidrug-resistant strains are urgently needed. Several peptides produced upon maturation processes of precursor proteins play central roles in innate defense systems. We recently reported the characterization of two novel cryptic HDPs, identified in human Apolipoprotein B by using a bioinformatic method recently developed by our research group [2]. These two novel HDPs, here named ApoBL and ApoBS, have been found to be endowed with a broad-spectrum anti-microbial and anti-biofilm activity, being active against both Gram-negative and Gram-positive bacterial strains [3]. Interestingly, both HDPs were found to be able to act in synergy with either commonly used antibiotics or EDTA, also against multi-drug resistant bacteria. Moreover, ApoB derived cryptides were found to be neither toxic nor haemolytic towards eukaryotic cells [3]. Altogether, these findings open interesting perspectives to peptides therapeutic applications. We also analyzed ApoB-derived peptides antimicrobial activity against bacterial pathogens clinically isolated from CF patients. We found that both peptides are effective against several strains, such as Burkholderia multivorans LMG 17582, P. aeruginosa RP 73 and P. aeruginosa KK 27. Even more interestingly, ApoB-derived peptides were also found to be endowed with significant anti-biofilm activity against pathogens isolated from CF patients. Considering the world burden of Anti-Microbial Resistance, these findings represent a promising starting point for the future development of novel effective therapies useful to counteract bacterial infections. References

1. López-Causapé C., et al. Expert Rev Respir Med. (2015) 9, (1):73-88. 2. Pane, K., et al. J Theor Biol (2017) 419, 254-265 3. Gaglione, R., et al. Biochem Pharmacol (2017) 130, 34-505.

BIOFAR.17

ANGIOGENIN: A RIBONUCLEASE INVOLVED IN THE RECRUITMENT OF STRESS GRANULES IN KERATINOCYTES

ROSANNA CULURCIELLOa (rosanna.culurciello@unina.it); ANDREA BOSSOa (andrea.bosso@unina.it); MARIAGRAZIA SILVESTROa (mariagrazia.silvestr@libero.it); MICHELA FERRAROa

(michela.ferraro@libero.it); MARIA CARMEN VALOROSOa ( mariacarmen.valoroso@unina.it); SERENA ACETOa ( serena.aceto@unina.it); ANGELA ARCIELLOb (anarciell@unina.it; SIB); ELIO PIZZOa*

(elipizzo@unina.it; SIB)

a Department of Biology, Federico II University of Naples, 80126 Naples, Italy. b Department of Chemical Sciences, Federico II University of Naples, 80126 Naples, Italy.

*Corresponding Author Angiogenin is an atypical ribonuclease, also known as RNase 5, widespread expressed in several organs and tissues and involved in physiological and pathological processes. The finding that angiogenin is able to directly stimulate both cancer and endothelial cell proliferation marked the first functional expansion of this enzyme [1]. Recent experimental evidences show that angiogenin is involved in the cellular response to stress through its ability to hydrolyse tRNA, and thus contribute to translational inhibition and recruitment of Stress Granules. These data further expanded the role of angiogenin from cell growth to cell survival [2]. In the wake of this inspiration, several authors have appropriately focused on the study of the additional properties of angiogenin in cells subjected to different growth conditions [3]. However, there is no particular experimental evidence relating to angiogenin in the epidermis, the outermost layer of human skin, continually exposed to external stressors. Therefore, taking advantage of the scarce knowledge of the role of angiogenin in this layer, our study is mainly aimed at clarifying the possible role of this enzyme in human keratinocytes HaCaT. Our results clearly indicate that angiogenin is constitutively expressed in HaCaT cells and its expression is not influenced following stimuli that alter cellular homeostasis. Further analyses revealed that angiogenin, in HaCaT subjected to oxidative or thermal stresses, dramatically changes its intracellular localization moving largely towards transient cytoplasmic ribonucleoprotein complexes, also noted as Stress Granules (SGs). As SGs represent a useful cell survivor index during a stress response, a further strategy has been made up with the aim to verify if a larger amount of angiogenin was able to modulate number and size of Stress Granules. Interestingly, preliminary results indicate that recombinant angiogenin is able to attenuate Stress Granules assembly induced by treatment with oxidative agents. All these results suggest that modulation of angiogenin expression could represent a potential protectant tool against skin damage. References 1. Li S, Hu GF. Angiogenin-mediated rRNA transcription in cancer and neurodegeneration. Int J Biochem

Mol Biol. 2010;1(1):26-35. 2. Emara MM, Ivanov P, Hickman T, Dawra N, Tisdale S, Kedersha N, Hu GF, Anderson P. Angiogenin-

induced tRNA-derived stress-induced RNAs promote stress-induced stress granule assembly. J Biol Chem. 2010 Apr 2;285(14):10959-68.

3. Pizzo E, Sarcinelli C, Sheng J, Fusco S, Formiggini F, Netti P, Yu W, D'Alessio G, Hu GF. Ribonuclease/angiogenin inhibitor 1 regulates stress-induced subcellular localization of angiogenin to control growth and survival. J Cell Sci. 2013 Sep 15;126(Pt 18):4308-19.

BIOFAR.18

GVF27 AND IMY47: TWO UNSUSPECTED HOST DEFENSE PEPTIDES HIDDEN IN HUMAN ENZYMES

ANDREA BOSSO1 (andrea.bosso@unina.it); ROSA GAGLIONE2 (rosa.gaglione@unina.it; SIB); ROSA AMBROSIO1 (rosaambrosio.991@gmail.com); FRANCESCA GRIMALDI1

(francesca.grimaldi92@gmail.com); MICHELA FERRARO1 (michela.ferraro93@libero.it) ROSANNA CULURCIELLO1 (rosanna.culurciello@unina.it); ANGELA ARCIELLO2 (anarciel@unina.it; SIB); EUGENIO

NOTOMISTA1 (notomist@unina.it); ELIO PIZZO1* (elipizzo@unina.it; SIB)

1. Department of Biology, University Federico II, 80126, Naples, Italy 2. Department of Chemical Sciences, Federico II University, Naples, 80126, Italy

*Corresponding Author The term cryptome refers to a large group of bioactive peptides generated by the proteolysis of precursor proteins. Several human proteins, including those apparently not involved in immunity, can behave as sources of Host Defence Peptides (HDPs), hidden in their primary structures and released by the action of appropriate proteases. The use of bioinformatic approaches has increased the possibility to highlight the presence of putative HDPs in any proteins, known as HDP-releasing proteins. It is extremely suggestive to image that these proteins would release biologically active peptides upon proteolytic cleavage by bacterial and/or host proteases. We recently developed a bioinformatic strategy for the identification of potential HDP-releasing proteins and the accurate localization of the fragment(s) hidden in their amino acidic sequences [1]. Analyzing different protein libraries by using this approach, we identified novel intriguing cryptic HDPs, never described so far, in proteins including cytokines and cytokine receptors, apolipoproteins, proteins involved in coagulation cascade and enzymes. Some of these HDPs have been successfully produced in recombinant form and extensively characterized [2,3]. Here, we report the characterization of novel intriguing cryptic HDPs identified in two different cationic enzymes, describing their bactericidal properties against planktonic and sessile bacteria and their anti-inflammatory effects. Remarkably, GVF27 efficiently inhibits the release of proinflammatory cytokines induced by LPS on both human and mouse cells whereas IMY47 promotes the release of chemokines making us imagine its hypothetical role in the recruitment of macrophages to the site of infection. Similar indications were also obtained in experiments in vivo, where we found that IMY47 mitigates recruitment of neutrophils and macrophages in lung infected by Pseudomonas aeruginosa. Collected data not only highlight the effectiveness of the computational strategy to identify HDPs included inside the structure of larger proteins, but also draw attention on a new potential role for enzymes as reservoirs of multi-functional host defense molecules.

References

1. Pane K. et al, Antimicrobial potency of cationic antimicrobial peptides can be predicted from their amino acid composition: Application to the detection of "cryptic" antimicrobial peptides. J Theor Biol. 2017 Apr 21;419:254-265. doi: 10.1016/j.jtbi.2017.02.012.

2. Bosso A. et al, A new cryptic host defense peptide identified in human 11-hydroxysteroid dehydrogenase-1 β-like: from in silico identification to experimental evidence. Biochim Biophys Acta Gen Subj. 2017 Sep;1861(9):2342-2353. doi: 10.1016/j.bbagen.2017.04.009.

3. Pane K. et al, Identification of Novel Cryptic Multifunctional Antimicrobial Peptides from the Human Stomach Enabled by a Computational-Experimental Platform. ACS Synth Biol. 2018 Sep 21;7(9):2105-2115. doi: 10.1021/acssynbio.8b00084.

BIOFAR.19

THE NITRODI SPRING WATER PROMOTES CELL MIGRATION AND VITALITY IN VITRO AND EXERTS ANTIINFLAMMATORY EFFECTS

THROUGH DOWN-REGULATION OF PROTEIN S-NITROSYLATIONS ANTONIETTA AVERSANO1 (antonietta.aversano10@gmail.com); FRANCESCA CAMMAROTA1

(francesca.cammarota88@gmail.com), FRANCESCA DURATURO1 (francesca.duraturo@unina.it; SIB); FRANCESCA WANDA ROSSI2 (frawrossi@yahoo.it); AMATO DE PAULIS2 (depaulis@unina.it); PAOLA

IZZO1 (paola.izzo@unina.it; SIB); MARINA DE ROSA1* (marina.derosa@unina.it; SIB).

1University of Naples Federico II, Department of Molecular Medicine and Medical Biotechnology, Naples, Italy;

2University of Naples Federico II, Department of Translational Medical Sciences, Naples, Italy; *Corresponding Author

The Nitrodi spring in the island of Ischia is classified as a mineral, hypothermal, sulphate alkaline one and both its internal use (i.e. for drinking cures) and the external use, are able to determine different benefits on many diseases and conditions, such as psoriasis, arthropathy, gastritis and gastroduodenitis. It has been reported that its name derives from the word ‘nitro’, namely ‘soda’ since it was believed the springs’ waters were rich in (1-3). The mechanism by which thermal water therapy from Nitrodi spring water works is still unknown. However several cases report that the effects of thermal water continues for months after completion of treatment. In this work we investigated the molecular basis of therapeutic effects of the Nitrodi spring water. We performed an in vitro model in wich the colorectal adenocarcinoma cell line RKO was alternatively treated with PBS or PBS prepared with the Nitrodi water, for 4 hours per day, 5 days for week, for 6 weeks and analysed by using MTT assay, transwell migration assay, western blot assay, fluorometric detection of protein S-nitrosothiols and S-Nitrosylation Western Blot specific assay. This stategy allowed us to demonstrate that the Nitrodi spring water promotes cell migration and cell vitality together with down-regulation of nitrosylated active form of Cox2 protein throught a more general mechanism which involves down-regulation of protein nithrosylations and, in this way, playing an antiinflammatory role. This results are in agreement with all reported therapeutic property of the Nitrodi spring water, conferring strengthness to this important natural resource available as alternative care for health. References

1. Mancioli M. Le proprietà terapeutiche delle acque di Nitrodi e Olmitello. Anallisi Editore 1984; 2. Forti L.: Rilievi dedicati alle Ninfe di Nitrodi. Estratto dal VoI. XXVI dei Rend. Accademia di Archeol.

Lettere e Belle Arti di Napoli. L'arte Tipograf., Napoli, 1951. 3. Filippelli A, Costantino M Relazione clinica per il riconoscimento delle proprietà terapeutiche dell’acqua

minerale naturale nitroli – del complesso “Sorgente Nitrodi – Fonte delle Ninfe”. Università degli Studi di Salerno – Scuola di medicina salernitana. Marzo 2017.

BIOFAR.20

ANTIPROLIFERATIVE TANSHINONES FROM SALVIA MILTIORRHIZA BUNGE: EVALUATION ON HUMAN GLIOBLASTOMA MODELS IN VITRO

MARIALUISA PICCOLOI (marialuisa.piccolo@unina.it; SIB), MARIA GRAZIA FERRAROI (mgferraro8@gmail.com), CHIARA TAMMAROI (chiara.tammaro92@gmail.com), GABRIELLA CONTINOI (gcontino@studenti.unina.it), FILOMENA IOVINOI (iovinofilomena94@libero.it), FRANCESCO MAIONEI

(francesco.maione@unina.it), CARLO IRACEI* (carlo.irace@unina.it), RITA SANTAMARIAI* (rita.santamaria@unina.it; SIB)

IUniversità “Federico II”, Dipartimento di Farmacia, Napoli

*Autore corrispondente

Le piante medicinali e gli estratti di erbe della medicina tradizionale cinese sono sempre più comunemente usati in tutto il mondo come integratori alimentari e nutraceutici per le loro proprietà benefiche. Tra questi, la radice di Salvia miltiorrhiza – da secoli rimedio naturale per il trattamento di diverse patologie – viene tradizionalmente usata nei paesi asiatici come agente antiossidante, antitumorale ed antinfiammatorio. I suoi effetti farmacologici sono principalmente attribuiti alla presenza di composti simili a diterpenoidi lipofili, quali diidrotanshinone (DTA), tanshinone IA (TIA), tanshinone IIA (TIIA) e criptotanshinone (CRY), che di per sé sono in grado di superare la barriera ematoencefalica e inibire l'aggregazione di peptidi Aβ, proteggendo i neuroni da condizioni infiammatorie1. Inoltre, diverse evidenze supportano l'ipotesi che i tanshinoni estratti dalle radici di Salvia miltiorrhiza (note come Danshen) esercitino una significativa attività citotossica e/o antiproliferativa in vitro. Pertanto, scopo del presente studio è stato quello di indagare il profilo di bioattività di Danshen e dei suoi costituenti attivi (TIIA e CRY) in modelli umani di patologie tumorali, quali glioblastoma e carcinoma del colon-retto. In questo modo, l'attività antitumorale dei composti selezionati è stata valutata in diverse linee cellulari, dimostrando che Danshen ed i suoi costituenti attivi sono dotati di spiccata attività antiproliferativa, selettiva verso cellule di glioblastoma umano (linee LN-229 e U-87 MG)2. Sebbene siano stati fatti molti progressi nella terapia antineoplastica, i gliomi hanno finora mostrato una scarsa risposta a farmaci antiproliferativi. Pertanto, sulla base dei risultati ottenuti, sono in corso ulteriori esperimenti per approfondire i meccanismi molecolari d’azione dei tanshinoni coinvolti nell'attivazione di pathways di morte cellulare. Oltre ad essere utilizzate come integratori alimentari, i nostri risultati suggeriscono che le radici di Salvia miltiorrhiza possono essere considerate come una nuova potenziale fonte di composti bioattivi per lo sviluppo di nuove strategie terapeutiche antitumorali. References

1. Maione et al., Pharmacological Research. 129:482-490. 2018. 2. Piccolo et al., Biomedicine and Pharmacotherapy. 105:1042-1049. 2018.

BIOFAR.21

IN VITRO ANTI-TUMOR ACTIVITY OF A NOVEL LOCKED NUCLEIC ACID (LNA)-INHIBITOR-MIR-221 IN HEPATOCELLULAR CARCINOMA

AGOSTINO FESTA1 (agostino.festa@unicampania.it), MAYRA RACHELE ZARONE1

(mayra.zarone@virgilio.it) MARIANNA ABATE1 (marianna.abate@unicampania.it), MARGHERITA RUSSO1 (margherita.russo@unicampania.it), MICHELA FALCO1 (michela.falco1@studenti.unicampania.it), MICHELE

CARAGLIA1 (michele.caraglia@unicampania.it), GABRIELLA MISSO1* (gabriella.misso@unicampania.it)

1 University of Campania “Luigi Vanvitelli”, Department of Precision Medicine, Naples, Italy. *Corresponding Author

miR-221 and miR-222 (miR-221/222) are two highly homologous microRNAs (miRNAs), whose upregulation has been described in several types of human diseases. miR-221/222 have been considered to act as oncogenes in advanced malignancies, including hepatocellular carcinoma (HCC), by controlling the expression of target proteins involved in cell cycle, differentiation and apoptosis. Silencing miR-221/222 could represent a promising therapeutic strategy for the treatment of HCC [1]. Here, we investigated the in vitro therapeutic activity of a novel phosphorothioate (PS) modified backbone 13-mer locked nucleic acid (LNA)-Inhibitor-miR-221 (LNA/PS 13-mer LNA-i-miR-221) against PLC/PRF/5 HCC cells. Our data demonstrate that LNA-i-miR-221 specifically recognizes the miR-221 complementary sequence and effectively knocks down miR-221 levels in HCC cells. We found that enforced expression of LNA-i-miR-221 increases G0/G1-phase and induces up-regulation of the p27Kip1, which is a well- established miR-221 target. Moreover, LNA-i-miR-221 reduces cell viability, colony formation, migration and invasive behavior of PLC/PRF/5 cells. Silencing miR-221 increases the expression of PTEN, another well-known canonical target of miR-221, and promotes autophagic flux by antagonizing PI3K/AKT/mTOR pathway. We also revealed that LNA-i-miR-221 reduces protein expression of SIRT1, which negatively regulates the tumor suppressor p53 through deacetylation. Based on this observation, we proposed a pivotal role of LNA-i-miR-221 in the modulation of miR-34a/SIRT1/p53 loop, wherein p53 induces the tumor suppressor miR-34a, that in turn activates p53 by inhibiting SIRT1, whose expression is further silenced by LNA-i-miR- 221, thus promoting miR-34a up-regulation [2]. Taken together, these data provide the rationale for in vivo studies for the treatment of HCC. References

1. Song J, Ouyang Y, Che J, et al. Potential Value of miR-221/222 as Diagnostic, Prognostic, and Therapeutic Biomarkers for Diseases. Front Immunol. 2017 Feb 16;8:56.

2. Misso G, Di Martino MT, De Rosa G, et al. Mir-34: a new weapon against cancer? Mol Ther Nucleic Acids. 2014 Sep 23;3:e194.

BIOFAR.22

NUTRITIONAL VALUES AND AMINO ACID CONTENT OF CHICKPEA SEEDS GROWN IN VALLE AGRICOLA DISTRICT, ITALY

NICOLA LANDI (nicola.landi@unicampania.it; SIB); SARA RAGUCCI (sara.ragucci@unicampania.it; SIB); PAOLO V. PEDONE (paolov.pedone@unicampania.it; SIB); ANTIMO DI MARO*

(antimo.dimaro@unicampania.it; SIB)

Dept. of Environmental, Biological and Pharmaceutical Sciences and Technologies (DiSTABiF), University of Campania ‘Luigi Vanvitelli’, Caserta, Italy

*Corresponding Author The local cultivars are tied up to a tradition of transformation for “homemade” products. Therefore, it is interesting to consider possible intervention strategies in order to preserve genetic variability of local cultivars, according to the traditional approach of biochemical and nutritional valorisation (1). Chickpea (Cicer arietinum L.) cultivation has a positive impact on agriculture and environment. In this framework, we have investigated nutritional values and amino acid profile of chickpea seeds grown in ‘Valle Agricola’ (traditional product surveyed by the Campania region; Gazzetta Ufficiale n. 60 del 12.03.2019) because there are no nutritional data available on them. The analysis is focused on the content of moisture, ash, total protein, carbohydrates, lipids, total and free amino acid composition. The content of total proteins, lipids and carbohydrates in the chickpea seeds is 19.70 g/100g, 6.41 g/100g, 59.90 g/100g of flour respectively, while ashes 3.02 g/100g and moisture 10.97 g/100g of flour. The total amino acid analysis shows a high content of essential amino acids (about 37%); in particular, the most abundant were lysine, leucine and phenylalanine, whereas methionine and cysteine are limiting amino acids as reported for other legumes (2). Considering free amino acids, the total amount in ‘Valle Agricola’ chickpea was 107.82 mg per 100 g of flour. The analysis evidenced the presence of several non-protein amino acids, such as Aaba, Ethan, Pea, Phser and Taur (about 8% total content of free amino acids), while asparagine was by far the most abundant among free amino acids (about 17%). Furthermore, the amount of free essential amino acids, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val, in ‘Valle Agricola’ chickpea was 30.6 mg per 100 g of flour (not significant as a contribution in a human diet). Future studies will be aimed at the characterization of fatty acids, antioxidant power and protease inhibitors content with and without heat treatment (boiling) (3). References 1. Guarrera and Savo (2016). Wild food plants used in traditional vegetable mixtures in Italy. J

Ethnopharmacol., 185, 202-234. 2. Wood and Grusak (2007). Nutritional value of chickpea. In: Yadav, S.S., Redden, B., Chen, W. and Sharma,

B., Eds., Chickpea Breeding and Management, CAB International, Wallingford, 101-142. 3. Landi et al. (2017). Nutritional values and metabolic profile with and without boiled treatment of 'Gallo

Matese' beans (Phaseolus vulgaris L.), a landrace from Southern Italy. Acta Sci Pol Technol Aliment., 16(3), 331-344.

BIOFAR.23

OSTREATIN, A NOVEL RIBOTOXIN-LIKE PROTEIN FROM FRUITING BODIES OF PLEUROTUS OSTREATUS

SARA RAGUCCI (sara.ragucci@unicampania.it; SIB); NICOLA LANDI (nicola.landi@unicampania.it; SIB); ANTIMO DI MARO* (antimo.dimaro@unicampania.it; SIB)

Dept. of Environmental, Biological and Pharmaceutical Sciences and Technologies (DiSTABiF), University of Campania ‘Luigi Vanvitelli’, Caserta, Italy

*Corresponding Author

Ribotoxins are specific extracellular ribonucleases (RNases), produced by several

ascomycetes fungal species, such as Aspergillus giganteus, from which has been found the prototype a-sarcin (1). Ribotoxins cleave a single phosphodiester bond in a universally conserved sequence of large rRNA in the ribosomes, known as Sarcin-Ricin Loop (SRL). This cleave produces a small rRNA fragment of 300-400 nucleotides (depending on the ribosome origin), known as a-fragment. These toxins are involved likely in fungal survival or colonization purposes, while are promising candidates for biotechnological applications such as in medicine (e.i.: immunotoxins in cancer therapy) and agriculture (e.i.: biopesticides as pest control agents). Recently, our group has isolated and characterized a homologous enzyme, named Ageritin, from the basidiomycete Agrocybe aegerita, an edible mushroom (2). Ageritin is an RNase that shows the same target for enzymatic action, but is different in primary structure and chemical-physical properties (3). Thus, Ageritin is the prototype of a new class of ribotoxin-like proteins in basidiomycetes fungi.

In this framework, we report the purification and partial characterization of a novel member of this class, named Ostreatin, from Pleurotus ostreatus (known as ‘orecchione’ in Italy), another basidiomycete edible mushroom. Ostreatin, purified at homogeneity by using the same purification protocol of Ageritin (2), shows a molecular weight of 15 kDa, a basic pI (> 9) and a single free cysteinyl residue. Enzymatically, this enzyme releases the a-fragment when incubated with both yeast and rabbit ribosomes, while unspecific RNase activity has not been detected, thus Ostreatin is a novel specific ribotoxin-like protein as Ageritin. Overall, our findings suggest that this novel class of specific RNases or ribotoxin-like proteins is well represented in some basidiomycetes and, in particular, in edible mushrooms (3). References 1. Olombrada et al., (2017). Fungal Ribotoxins: A Review of Potential Biotechnological Applications. Toxins (Basel), 9(2), pii: E71. 2. Landi et al., (2017). Purification, characterization and cytotoxicity assessment of Ageritin: The first ribotoxin from the basidiomycete mushroom Agrocybe aegerita. BBA GS, 1861(5 Pt A):1113-1121. 3. Landi et al. (2018). Structural insights into nucleotide and protein sequence of Ageritin: a novel prototype of fungal ribotoxin. J. Biochem. doi: 10.1093/jb/mvy113.

BIOFAR.24

HYBRID COOPERATIVE COMPLEXES BASED ON HIGH AND LOW MOLECULAR WEIGHT HYALURONIC ACID IMPROVE PROLIFERATION

AND REDUCE INFLAMMATION IN AN OSTEOARTHRITIS IN VITRO MODEL BASED ON HUMAN SYNOVIOCYTES AND CHONDROCYTES ANTONIETTA STELLAVATO1#* (antonietta.stellavato@unicampania.it; SIB), VALENTINA VASSALLO1# (valentina.vassallo92@gmail.com; SIB), ANNA VIRGINIA ADRIANA PIROZZI1 (adripirozzi@gmail.com;

SIB), ANNALISA LA GATTA1(annalisa.lagatta@unicampania.it; SIB), MARIO DE ROSA1

(mario.derosa@unicampania.it), GIOVANNI BALATO2 (giovannibalato@gmail.com), ALESSIO D’ADDONA2

(alessio.daddona@gmail.com), VIRGINIA TIRINO1 (virginia.tirino@unicampania.it), CARLO RUOSI2

(caruosi@unina.it) and CHIARA SCHIRALDI1* (chiara.schiraldi@unicampania.it; SIB)

1. Department of Experimental Medicine, Section of Biotechnology, Medical Histology and Molecular Biology, University of Campania “Luigi Vanvitelli”, Naples, Italy.

2. School of Medicine and Surgery “Federico II” of Naples, Department of Public Health, A.O.U. Federico II of Naples, Via S. Pansini, 80131, Naples, Italy.

*Corresponding Author

Hyaluronan has a pivotal role in the maintenance of normal functions of synovial fluid and structure of the articular joint, but it has been reported that its concentration is reduced in patients affected by degenerative cartilage diseases, such as osteoarthritis (OA). The aim of this study was to investigate the anti-inflammatory effects and properties of hybrid cooperative complexes based on high and low molecular weight hyaluronan (HCC) compared to H-HA on human primary cells derived by pathological joints. Rheological behavior of HCC was evaluated in order to define their potential as viscosupplement gel in degenerated joints. Also, experimental characterization was performed using an in vitro model of OA based on human chondrocytes and synoviocytes isolated from affected patients joints undergone to surgical replacement. In addition, in order to investigate the anti-inflammatory effects of HCC, NF-kB, COMP-2, IL-6, and IL-8 were assayed as specific biomarkers at the transcriptional and protein level. Moreover, the proliferative properties of HCC were evaluated using time lapse video microscopy. Results showed that chondrocytes and synoviocytes clearly presented an altered cytokine profile compatible with a severe ongoing inflammation status. H-HA and, above all, HCC significantly reduced the specific biomarkers level tested and improved cartilage healing. The rheological profile indicated HCC suitability for intra-articular injection in joint diseases. HCC viscoelastic properties and the protective/anti-inflammatory effect on human chondrocytes and synoviocytes suggest the novel HCC-based gels as a valid support for OA control. References 1. Stellavato A, de Novellis F, Reale S, de Rosa M and Schiraldi C. “Hybrid Complexes of High and Low

Molecular Weigh: Evaluation using an in vitro model of osteoarthritis”. Journal of Biological Regulators & Homeostatic Agent, vol. 30, pp. 7-16, 2016.

2. La Gatta A, De Rosa M, Frezza MA, Catalano C, Meloni M, Schiraldi C. “Biophysical and biological characterization of a new line of hyaluronan-based dermal fillers: A scientific rationale to specific clinical indications”. Mater Sci Eng C Mater Biol Appl., vol. 68, pp. 565-572. 2016.

3. Calamia V, Fernàndez-Puente P, Mateos J, Lourido L, Rocha B, Montell E, Vergés J, Ruiz-Romero C and Blanco FJ. “Pharmacoproteomic Study of Three Different Chondroitin Sulfate Compounds on Intracellular and Extracellular Human Choncrocyte Proteomes”. Molecular & Cellular Proteomics 11(6):M111.013417, 2012.

BIOFAR.25

ANALISI DEGLI EFFETTI STRUTTURALI E FUNZIONALI NELL’INTERAZIONE TRA L’ENZIMA GALT NATIVO E MUTATO E IL

POTENZIALE CHAPERONE ARGININA ANNA VERDINO (averdino@unisa.it; SIB), GAETANO D'URSO (g.durso8@studenti.unisa.it), ANNA

MARABOTTI* (amarabotti@unisa.it; SIB)

Università degli Studi di Salerno, Fisciano (SA) *Autore corrispondente

La “Galattosemia classica” o di tipo I è una malattia metabolica rara causata dal malfunzionamento dell’enzima galattosio-1-fosfato uridiltransferasi (GALT), associato alla presenza di mutazioni nel gene codificante per tale enzima. Attualmente, l’unica terapia è una dieta priva di galattosio, che deve essere continuata per tutta la vita, ma che non è in grado di evitare effetti negativi sullo sviluppo neuromotorio dei pazienti. La mutazione missenso più inabilitante e diffusa è p.Q188R, che ha una ripercussione sia da un punto di vista funzionale, provocando una riduzione nell'attività enzimatica, sia da un punto di vista strutturale, con riduzione del numero di legami di idrogeno all’interfaccia delle due subunità dell’omodimero GALT e conseguente destabilizzazione dell'enzima. Un approccio tentato, negli ultimi anni, per contrastare gli effetti destabilizzanti nelle mutazioni nelle malattie genetiche è stato l’utilizzo di chaperoni molecolari, che interagendo con la struttura soggetta ad aberrazione, tendono a compattarla e a ripristinarne la funzione. Un potenziale chaperone molecolare, l'arginina, è stato testato anche su questo enzima, per verificare la sua efficacia nel recuperarne la funzionalità. Alla luce di queste evidenze sperimentali, nel presente lavoro, sono state effettuate analisi di dinamica molecolare sia dell'enzima GALT nativo che del mutante p.Q188R in assenza e in presenza dello chaperone arginina, per studiarne gli effetti su questo enzima da un punto di vista energetico e strutturale a tre differenti temperature. Le suddette analisi hanno mostrato che l’arginina, pur interagendo con l’enzima, non sembra avere un ruolo di compattazione sulla forma mutata dell'enzima. Particolarmente rilevanti sono state le analisi dei legami idrogeno in assenza e in presenza dello chaperone, per comprendere l'effettiva capacità della molecola di arginina nel permettere la stabilizzazione dell'enzima. Tali indagini sono inoltre il punto di partenza per la ricerca di nuove molecole potenzialmente in grado di stabilizzare GALT tramite approcci computazionali.

BIOFAR.26

NEW INSIGHTS ON THE PROTEOGLYCAN LIKE DOMAIN OF THE HUMAN CARBONIC ANHYDRASE IX REVEAL ITS DISORDERED

NATURE MARTINA BUONANNO1 (martinabuonanno@gmail.com; SIB), EMMA LANGELLA1 (emma.langella@cnr.it),

GIUSEPPINA DE SIMONE1 (gdesimon@unina.it), SIMONA MARIA MONTI1* (marmonti@unina.it; SIB)

1 Institute of Biostructures and Bioimaging, IBB - CNR, 80134 – Napoli, Italia *Corresponding Author

The human Carbonic Anhydrase IX (hCA IX) is a zinc-containing metalloenzyme which catalyzes the reversible hydration of CO2 to bicarbonate and proton. [1] It is a multi-domain transmembrane enzyme consisting of a N-terminal proteoglycan (PG)-like region, a catalytic domain, a transmembrane segment (TM) and an intracellular tail (IC). Since hCA IX is overexpressed in numerous solid tumours, this enzyme has been widely studied. Indeed, structural information are available on the CA IX catalytic domain, whose structure was solved by our group and on the C-terminal region. [2] On the contrary, only few data were available for the N-terminal PG-like domain which has a key role in tumour progression being able to enhance the catalytic activity of CAIX and mediate its binding to the extracellular matrix. [3] Here we report for the first time the full characterization of the PG domain, providing insights into its structural and functional features. The obtaining of a great yield of this recombinant domain allowed us to characterize it with a multidisciplinary approach by means of biochemical, biophysical, and molecular dynamics studies. Results showed that PG domain can be classified as an Intrinsically Disordered Protein (IDP) globally unfolded with only some local residual polyproline II secondary structure. The observed conformational flexibility is in agreement with the hypothesis that this domain could be responsible for the interactions of hCA IX with several partners assisting survival and metastatic spread of tumours. References

1. Supuran, C.T., Di Fiore, A., Alterio, V., Monti, S.M., De Simone, G., Curr. Pharm. Des., (2010) 16, 3246-3255.

2. Buonanno M, Langella E, Zambrano N, Succoio M, Sasso E, Alterio V, Di Fiore A, Sandomenico A, Supuran CT, Scaloni A, Monti SM, De Simone G., ACS Chem Biol., (2017) 12,1460-1465.

3. Csaderova L., Debreova M., Radvak P., Stano M., Vrestiakova M., Kopacek J., Pastorekova S. and Svastova E. Front. Physiol. (2013), 4, 271

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DEFINITION OF MICRORNA SIGNATURES AS DIAGNOSTIC AND PROGNOSTIC BIOMARKERS AND THERAPEUTIC TOOLS IN

LARYNGEAL CANCER PATIENTS MICHELA FALCO1* (michela.falco1@studenti.unicampania.it), TAKASHI TAKEUCHI1,2*

(takashi.takeuchi@unicampania.it), HIROMICHI KAWASAKI1,3, ANGELA LOMBARDI1 (angela.lombardi@unicampania.it), MARIANNA ABATE1 (marianna.abate@unicampania.it), AGOSTINO

FESTA1 (agostino.festa@unicampania.it), MARCO BOCCHETTI1 (marco.bocchetti@unicampania.it), TERESA ABATE4 (abate.teresa@yahoo.it), GIOVANNI MOTTA1 (giovannimotta95@yahoo.com), FILIPPO

RICCIARDIELLO4 (filippo.ricciardiello@aocardarelli.it), GAETANO MOTTA5 (gaetano.motta@unicampania.it), GABRIELLA MISSO1 (gabriella.misso@unicampania.it), MICHELE

CARAGLIA1 (michele.caraglia@unicampania.it)

1 University of Campania “Luigi Vanvitelli”, Department of Precision Medicine, Italy 2 Wakunaga Pharmaceutical Co., Ltd, Molecular Diagnostics Division, Japan

3 Wakunaga Pharmaceutical Co., Ltd, Drug Discovery Laboratory, Japan 4 Cardarelli Hospital, Department of Ear Nose and Throat Unit, Italy

5 University of Campania “Luigi Vanvitelli”, Department of Ear Nose and Throat Unit, Italy *Corresponding Author

Laryngeal cancer (LCa) is the second most common cancer in head and neck region. Surgery, chemotherapy, radiation and combination therapy are often selected as the treatment options. Despite the development of the diagnosis and the treatments, disease morbidity and mortality has not been remarkably declined. One of the reasons is metastatic cancer which can lead to progression and recurrence of LCa. However, accurate diagnosis of small metastases is still a challenge. MicroRNAs (miRNAs) are a subset of small non-coding RNAs and post-transcriptionally regulate the expression of multiple genes. miRNAs act as either oncogenes or tumor-suppressors in the pathogenesis of cancers. The relation between aberrant miRNAs and lymph node metastasis has not been well understood in LCa. Our recent studies shown that miR-449a is significantly down-regulated in LCa tissues with lymph node metastasis compared to the cases without metastasis. To investigate the biological functions, we transfected either miR-449a mimic or negative scramble control into Hep-2 laryngeal squamous carcinoma cell line. The transient expression of miR-449a significantly suppressed both cell growth and migration of Hep-2 cells. Furthermore, based on in silico analysis, we focused on Notch1 and 2 genes as direct target candidates of miR-449a. Notch family is known as a regulator of various cellular processes such as differentiation, proliferation, apoptosis and migration. Both mRNA and protein expression of Notch1 and 2 were significantly suppressed by ectopic expression of miR-449a in Hep2 cells. Our findings suggest that miR-449a acts as a tumor-suppressor and inhibits metastasis factors such as cell proliferation and motility through the targeting of Notch1 and 2 genes.

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ENEA: IDENTIFICAZIONE E CARATTERIZZAZIONE DI UN NUOVO ALLERGENE DELLA PESCA E DELL’ALBICOCCA

TERESA RICCIARDIa,b (teresa.ricciardi@ibbr.cnr.it), IVANA GIANGRIECOa,b (ivana.giangrieco@ibbr.cnr.it), CLAUDIA ALESSANDRIb,c,d (claudia.alessandri@caam-allergy.com), LISA TUPPOa,b,

lisa.tuppo@ibbr.cnr.it), ANNA FILOMENA DIGILIOa (anna.digilio@ibbr.cnr.it), BEATRICE COBUCCI-PONZANOa (beatrice.cobucciponzano@ibbr.cnr.it; SIB), MAURIZIO TAMBURRINIa

(maurizio.tamburrini@ibbr.cnr.it; SIB), ADRIANO MARIb,c,d (adriano.mari@caam-allergy.com), MARIA ANTONIETTA CIARDIELLOa* (mariaantonietta.ciardiello@ibbr.cnr.it)

aInstitute of Biosciences and BioResources (IBBR), CNR, Naples, Italy

bAllergy Data Laboratories (ADL), Latina, Italy

cAssociated Centers for Molecular Allergology (CAAM), Rome, Italy

dCenter for Molecular Allergology, IDI-IRCCS, Rome, Italy *Corresponding Author

La pesca può causare reazioni allergiche con sintomi lievi oppure gravi come lo shock anafilattico1.Finora nella pesca sono state isolate cinque proteine allergeniche (www.allergome.org), ma molti dati clinici indicano che questo frutto contiene allergeni ancora sconosciuti. Gli obiettivi di questo studio sono stati la caratterizzazione, e la produzione della forma ricombinante, di ENEA, una nuova proteina della pesca che mostrava somiglianza strutturale con un allergene maggiore del lattice, Hev b 5, e con un allergene della manioca, Man e 5. ENEA è stata isolata dalla polpa di pesca mediante separazioni cromatografiche e identificata tramite sequenziamento diretto dell’estremità N-terminale. Essa è presente in quantità molto variabili nella pesca, e talvolta risulta degradata. E’ stata, quindi, prodotta la forma ricombinante utilizzando la sequenza della proteina omologa da albicocca, allora disponibile in banca dati. La sequenza di ENEA di pesca, ora disponibile in banca dati, mostra una identità di sequenza del 97% con ENEA di albicocca. Esperimenti di dicroismo circolare hanno evidenziato che ENEA è una proteina non strutturata, appartenente al gruppo delle “intrinsecally disordered proteins”. La caratterizzazione immunologica, effettuata mediante dot blot, ABA2 e FABER3 test, ha mostrato che ENEA è riconosciuta in maniera specifica da IgE di pazienti allergici alla pesca e/o albicocca. Esperimenti di inibizione della interazione IgE specifiche-allergene hanno dimostrato che sia ENEA naturale, sia la sua forma ricombinante, cross-reagiscono con l’allergene del lattice Hev b 5. Sulla base dei dati immunologici ottenuti, ENEA di albicocca è stata registrata dalla WHO/IUIS nella banca dati internazionale degli allergeni con il nome di Pru ar 5. In conclusione, è stata identificata e caratterizzata una nuova proteina allergenica, ENEA, presente nella pesca e nell’albicocca. Le caratteristiche immunologiche della forma ricombinante di ENEA sono state verificate e presto questa proteina sarà introdotta nel sistema FABER e contribuirà alla diagnosi delle allergie alimentari.

References 1. Alessandri, C., Ferrara, R., Bernardi, M.L., Zennaro, D., Tuppo, L., Giangrieco, I., Tamburrini, M., Mari, A., Ciardiello, M.A., 2017. Diagnosing allergic sensitizations in the third millennium: why clinicians should know allergen molecule structures. Clin Transl Allergy 7, 21. 2. Pomponi, D., Bernardi, M.L., Liso, M., Palazzo, P., Tuppo, L., Rafaiani, C., Santoro, M., Labrada, A., Ciardiello, M.A., Mari, A., Scala, E., 2012. Allergen micro-bead array for IgE detection: a feasibility study using allergenic molecules tested on a flexible multiplex flow cytometric immunoassay. PLoS One 7, e35697. 3. Tuppo, L., Giangrieco, I., Alessandri, C., Ricciardi, T., Rafaiani, C., Ciancamerla, M., Ferrara, R., Zennaro, D., Bernardi, M.L., Tamburrini, M., Mari, A., Ciardiello, M.A., 2018. Pomegranate chitinase III: Identification of a new allergen and analysis of sensitization patterns to chitinases. Mol Immunol 103, 89-95.

BIOFAR.29

CHITINASI DI CLASSE III DEL MELOGRANO: IDENTIFICAZIONE E CARATTERIZZAZIONE DI UN NUOVO ALLERGENE ALIMENTARE

LISA TUPPOa,b (lisa.tuppo@ibbr.cnr.it), IVANA GIANGRIECOa,b (ivana.giangrieco@ibbr.cnr.it), CLAUDIA ALESSANDRIb,c, (claudia.alessandri@caam-allergy.com), TERESA RICCIARDIa,b

(teresa.ricciardi@ibbr.cnr.it), MAURIZIO TAMBURRINIa (maurizio.tamburrini@ibbr.cnr.it; SIB), ADRIANO MARIb,c (adriano.mari@caam-allergy.com), MARIA ANTONIETTA CIARDIELLOa*

(mariaantonietta.ciardiello@ibbr.cnr.it)

aInstitute of Biosciences and BioResources (IBBR), CNR, Naples, Italy. bAllergy Data Laboratories (ADL), Latina, Italy

cAssociated Centers for Molecular Allergology (CAAM), Rome, Italy. *Corresponding Author

Negli ultimi anni il consumo di melograno è particolarmente aumentato perché questo frutto è considerato un “functional food” con effetti benefici nella prevenzione di diverse patologie. Tuttavia, il melograno è anche una fonte di proteine capaci di scatenare reazioni allergiche, anche molto gravi, nei soggetti sensibili. Finora in questo frutto sono state descritte due proteine allergeniche1: una 9k-LTP, Pun g 1, e la pommacleina, Pun g 7 (www.allergome.org). Gli obiettivi di questo studio sono stati la caratterizzazione di un nuovo allergene dal frutto di melograno, la chitinasi di classe III,2 e il confronto della sua reattività verso le IgE con quella delle chitinasi di altre classi disponibili sul FABER® test.3 La chitinasi III è stata isolata dalla polpa del melograno mediante separazioni cromatografiche e identificata tramite sequenziamento diretto della regione N-terminale e spettrometria di massa. È una proteina di 29 kDa che mostra il 69% di identità di sequenza con un allergene maggiore del lattice, la hevamina. In esperimenti di dot blotting, immunoblotting e FABER® test, la chitinasi III di melograno è stata riconosciuta da anticorpi IgE di pazienti allergici. In una popolazione di 357 soggetti sensibilizzati ad uno o più allergeni di melograno presenti su FABER®, 69 (19,3%) avevano IgE specifiche verso la chitinasi III. Di questi pazienti, il 34,8% e il 7.2% erano sensibilizzati anche alla chitinasi di classe IV di kiwi e classe I del lattice, rispettivamente. In conclusione, nel frutto di melograno è stata identificata una nuova molecola allergenica che è stata sottomessa alla WHO/IUIS affinché venga considerata la sua registrazione con il nome Pun g 14. La sua inclusione nei sistemi diagnostici contribuirà al miglioramento della diagnosi di allergia alimentare. Questo studio ha, inoltre, evidenziato l’importante ruolo delle chitinasi III e IV come molecole capaci di produrre sensibilizzazione allergica in un numero significativo di pazienti. References 1. Tuppo L., Alessandri C, Pasquariello M.S., Petriccione M., Giangrieco I., Tamburrini M., Mari A., Ciardiello M.A. (2017) Pomegranate Cultivars: Identification of the New IgE-Binding Protein Pommaclein and Analysis of Antioxidant Variability. Journal of Agricultural and Food Chemistry, 65 (13), 2702-2710 2. Tuppo L., Giangrieco I., Alessandri C., Ricciardi T., Rafaiani C., Ciancamerla M., Ferrara R., Zennaro D., Bernardi M.L., Tamburrini M., Mari A., Ciardiello M.A. (2018) Pomegranate chitinase III: Identification of a new allergen and analysis of sensitization patterns to chitinases. Molecular Immunology 103, 89–95 3. Alessandri C., Ferrara R., Bernardi M. L., Zennaro D., Tuppo L., Giangrieco I., Tamburrini M., Mari A. and Ciardiello M. A. (2017) Diagnosing allergic sensitizations in the third millennium: why clinicians should know allergen molecule structures. Clinical and Translational Allergy 7:21, DOI 10.1186/s13601-017-0158-7

BIOFAR.30

IN VITRO PROTEIN DIGESTION FOLLOWING THE COST ACTION PROTOCOL “INFOGEST”

C. VALERIA L. GIOSAFATTO (giosafat@unina.it; SIB) and LOREDANA MARINIELLO* (loredana.mariniello@unina.it; SIB)

University of Naples “Federico II”, Department of Chemical Sciences, 80126 Naples, Italy.

* Corresponding author In this work we show some data obtained by using an in vitro digestion protocol set up within INFOGEST, an international network that aims at “Improving Health Properties of Food by Sharing our Knowledge on the Digestive Process”. In particular, the specific objectives of the network are focussed mainly in:

Ø comparing the existing digestion models, harmonize the methodologies; Ø in validating them towards in vivo data and propose guidelines for performing new

experiments. Recently a review reporting an adaptation of the sequential static digestion model suitable for foods, which was used in order to mimic the oral, gastric and duodenal digestion conditions in respect to elderly, adult and infant has been published (1). In our laboratories we have assessed the digestibility of several food proteins both as purified forms (such as egg proteins, like ovalbumin, and legume proteins, such as phaseolin) and as part of the food matrix represented by common bean and grasspea flour. As the structure of the food plays an important role in food digestion, some protein sources have been modified by means of the enzyme microbial transglutaminase (TG), widely used as food matrix structuring agent. The enzymatic modification regarded both the flour from common bean legumes and a bio-tofu (2), a novel food obtained by soy and lactic acid bacteria. Finally the digestibility of protein based-edible films, the matrix of which was modified or not by TG, was evaluated as well (3). References 1. Levi et al. Trends in Food Science & Technology 60: 52-63, 2017. 2. Xing et al. Journal of Functional Foods 53, 292-298, 2019. 3. Giosafatto et al. Coatings, 245, 191-198, 2019.

BIOFAR.31

PHARMACOLOGICAL ENHANCEMENT OF THE HUMAN ACID ALPHA-GLUCOSIDASE BY ALLOSTERIC CHAPERONES

ROBERTA IACONO1,2 (roberta.iacono@unina.it; SIB); BEATRICE COBUCCI-PONZANO2 (beatrice.cobucciponzano@ibbr.cnr.it; SIB); MARIA CARMINA FERRARA2 (mc.ferrara14@gmail.com);

CATERINA PORTO3 (porto@tigem.it); BARBARA ROSSI3 (brossi@tigem.it); VÉRONIQUE ROIG-ZAMBONI4 (veronique.zamboni@afmb.univ-mrs.fr); STANLEY GERMANY4

(stanley.germany@genoscreen.com); YVES BOURNE4 (bourne@afmb.univ-mrs.fr); GIANCARLO PARENTI3,5 (parenti@tigem.it); GERLIND SULZENBACHER4 (gerlind.Sulzenbacher@afmb.univ-mrs.fr);

MARCO MORACCI*1,2 (marco.moracci@unina.it; SIB)

1Department of Biology, Federico II University, Via Cupa Nuova Cinthia 21, 80126 Naples, Italy 2Institute of Biosciences and Bioresources CNR, Via P.Castellino 111, 80131 Naples, Italy.

3Telethon Institute of Genetics and Medicine, Via Campi Flegrei 34, 80078, Pozzuoli, Naples, Italy. 4Centre National de la Recherche Scientifique (CNRS), Aix-Marseille Univ, AFMB, 163 Avenue de

Luminy, 13288 Marseille, France. 5Department of Translational Medical Sciences, Federico II University, Via Pansini 5, 80131 Naples, Italy

*Corresponding Author Pompe disease (also known as glycogen storage disease type 2) is a rare lysosomal storage disease caused by deficiency of the lysosomal acid α-glucosidase (GAA). The disease is characterized by glycogen accumulation in lysosomes triggering severe secondary cellular damage and resulting in progressive motor handicap and premature death. To date enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA) is the only approved treatment for Pompe disease, but therapeutic efficacy is limited by the insufficient bioavailability and delivery of the drug. An alternative is the use of small-molecule pharmacological chaperones (PC) that enhance activity levels of both endogenous and recombinant enzymes by facilitating their folding and trafficking to the lysosomes. However, the chaperones identified so far are also active site-directed molecules and potential inhibitors of target enzymes. We demonstrated that N-acetylcysteine (NAC) is a novel allosteric chaperone for GAA. NAC improved the stability of rhGAA as a function of pH and temperature without disrupting its catalytic activity. NAC enhanced the residual activity of mutated GAA in cultured PD fibroblasts and in COS7 cells overexpressing mutated GAA. In a PD mouse model the combination of NAC and rhGAA resulted in better correction of enzyme activity in liver, heart, diaphragm and gastrocnemia, compared to rhGAA alone. We obtained the high-resolution crystal structures of rhGAA in the unbound form and in complex with the NAC. This provide the molecular framework for the rationalization of the deleterious effects of disease-causing mutations, offering guidance for optimization of the in silico screening for more efficient small molecule chaperones serving for future therapies.

BIOFAR.32

FABER: UN NUOVO TEST DIAGNOSTICO PER L’ALLERGIA, REALIZZATO IN NANOTECNOLOGIA MULTIPLEX

IVANA GIANGRIECOa,b (ivana.giangrieco@ibbr.cnr.it), CLAUDIA ALESSANDRIb,c (claudia.alessandri@caam-allergy.com), LISA TUPPOa,b (lisa.tuppo@ibbr.cnr.it); TERESA RICCIARDIa,b (teresa.ricciardi@ibbr.cnr.it), CHIARA RAFAIANIc (pedrigna@gmail.com), MICHELA CIANCAMERLAc

(michelaciancamerla@gmail.com), ROSETTA FERRARAb,c (rosetta.ferrara@caam-allergy.com), MARIA LIVIA BERNARDIb,c (maria.livia.bernardi@caam-allergy.com), DANILA ZENNAROb,c

(danila.zennaro@caam-allergy.com), MAURIZIO TAMBURRINIa (maurizio.tamburrini@ibbr.cnr.it; SIB), ADRIANO MARIb,c (adriano.mari@caam-allergy.com), MARIA ANTONIETTA CIARDIELLOa*

(mariaantonietta.ciardiello@ibbr.cnr.it)

aInstitute of Biosciences and BioResources (IBBR), CNR, Napoli, Italy

bAllergy Data Laboratories (ADL), Latina, Italy

cAssociated Centers for Molecular Allergology (CAAM), Rome, Italy * Autore Corrispondente

L’allergologia molecolare e la sua applicazione in campo diagnostico hanno fatto registrare un salto di qualità nella individuazione e gestione della malattia allergica.1 I test molecolari permettono di conoscere con precisione la proteina a cui una persona è allergica consentendo la valutazione del livello di rischio a cui il paziente è esposto. In questo contesto, in collaborazione con gli allergologi del CAAM, abbiamo realizzato il sistema diagnostico per l’allergia FABER®2,3 FABER® è realizzato in nanotecnologia multiplex e, per la prima volta, combina i vantaggi della allergologia molecolare con quelli dei metodi tradizionali che usano estratti proteici totali. Il biochip di FABER® contiene 244 allergeni (da alimenti, pollini, acari, epiteli, muffe, lattice, etc.), 122 molecole e 122 estratti, analizzati tutti contemporaneamente con un solo test. Gli allergeni, coniugati a specifiche nano-particelle attivate chimicamente, sono immobilizzati su un supporto solido per costituire un microarray proteomico. Alcuni di questi allergeni sono esclusivi, cioè sono presenti soltanto su FABER®. E’ il caso di allergeni importanti come chitinasi e kirola da kiwi, peamacleina di pesca e melograno, LTP di piselli, etc. FABER® è un sistema dinamico, continuamente aggiornato sulla base delle nuove conoscenze scientifiche, ed è l’unico ad esecuzione centralizzata, cioè i test si effettuano in un unico laboratorio che riceve i campioni di siero provenienti da tutto il mondo. I risultati ottenuti, raccolti e conservati in una banca dati, costituiscono una enorme fonte di dati scientifici che permettono analisi di tipo epidemiologico a livello globale. Abbiamo osservato, ad esempio, che la sensibilizzazione alla LTP di arachide ha una prevalenza maggiore negli italiani rispetto agli statunitensi e altri paesi non mediterranei. In conclusione, FABER® rappresenta un sistema all’avanguardia che (i) permette una diagnosi personalizzata consentendo l’applicazione della medicina di precisione e dell’information technology in allergologia e (ii) contribuisce all’aumento delle conoscenze in campo allergologico. Bibliografia 1. Giangrieco I., Rafaiani C., Liso M, Palazzo P., Pomponi D., Tuppo L., Crescenzo R., Tamburrini M., Mari A., Ciardiello M.A. (2012) Allergens in allergy diagnosis: a glimpse at emerging new concepts and methodologies. Translational Medicine @ UniSa, 4(3), 27-33 2. Alessandri C., Ferrara R., Bernardi M. L., Zennaro D., Tuppo L., Giangrieco I., Tamburrini M., Mari A. and Ciardiello M.A. (2017) Diagnosing allergic sensitizations in the third millennium: why clinicians should know allergen molecule structures. Clinical and Translational Allergy 7:21, DOI 10.1186/s13601-017-0158-7 3. Tuppo, L., Giangrieco, I., Alessandri, C., Ricciardi, T., Rafaiani, C., Ciancamerla, M., Ferrara, R., Zennaro, D., Bernardi, M.L., Tamburrini, M., Mari, A., Ciardiello, M.A., 2018. Pomegranate chitinase III: Identification of a new allergen and analysis of sensitization patterns to chitinases. Molecular Immunology 103, 89-95.

BIOFAR.33

ANTI-CANCER ACTIVITY OF STEROIDS FROM HELLEBORUS CAUCASICUS

STEFANIA MARTUCCIELLO (smartucciello@unisa.it), GAETANA PAOLELLA (gpaolella@unisa.it; SIB), SONIA PIACENTE (piacente@unisa.it), IVANA CAPUTO* (icaputo@unisa.it; SIB)

University of Salerno, Italy

*Corresponding Author Helleborus caucasicus (Ranunculaceae) is an endemic plant of the Caucasian flora, widely distributed in West Georgia. Biological activities for the extracts of some Helleborus species including H. caucasicus have been reported. We found that butanolic extract of the underground parts of H. caucasicus and some isolated compounds decreased cell viability in vitro of a cancer cell line of lung origin (Calu-1) in a concentration-dependent manner, compared to the normal cell line. In particular, we identified that furostanol derivative (25S)-22α,25-epoxyfurost-5-ene-3β,11β,26-triol 26-O-β-d-glucopyranoside (compound 5), 20-hydroxyecdysone ( compound 6), and 3β,5β,14β-trihydroxy-19-oxo-bufa-20,22-dienolide 3-O-α-l-rhamnopyranoside, known as deglucohellebrin (compound 7) exerted a strong cytotoxic effect on Calu-1 cells and on other cancer cell lines (HepG2 and Caco-2). In addition they reduced the cell cycle progression and induced apoptosis, with a concomitant decrease of GRP78 expression, a pro-survival protein. Our findings suggest that selected compounds from H. caucasicus are potential interesting agents in anti-cancer and chemopreventive strategies.

BIOFAR.34

A STEP TOWARDS THE COMPREHENSION OF THE INSURGENCE OF THE CONGENITAL CENTRAL HYPOVENTILATION SYNDROME

LUCIANO PIRONE1 (luciano.pirone@unina.it; SIB), LAURA CALDINELLI2 (laura.caldinelli@uninsubria.it) (SIB); SIMONA DI LASCIO3 (s.dilascio@in.cnr.it), ROCCO DI GIROLAMO4 (rocco.digirolamo@unina.it),

SONIA DI GAETANO1 (digaetan@unina.it; SIB), LOREDANO POLLEGGIONI2 (Loredano.Pollegioni@uninsubria.it; SIB), ROBERTA BENFANTE3 (r.benfante@in.cnr.it), EMILIA

PEDONE1* (empedone@unina.it; SIB)

1 Institute of Biostructures and Bioimaging, CNR, Napoli, Italy. 2Department of Biotechnology and Life Sciences, Università degli Studi dell'Insubria, Varese Italy, and The Protein Factory Research Center, Università degli studi dell'Insubria and Politecnico di Milano, Milano, Italy.

3Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Milan, Italy.

4Dipartimento di Scienze Chimiche, Università di Napoli Federico II , Complesso Monte S. Angelo, Napoli, Italy

5CNR- Neuroscience Institute, Milan, Italy *Corresponding Author

About 90% of Congenital Central Hypoventilation Syndrome (CCHS) patients show poly-alanine triplet expansions in the coding region of transcription factor PHOX2B, which renders this protein an intriguing target to understand the insurgence of this syndrome and for the design of a novel therapeutical approach1. Consistently with the role of PHOX2B as a transcriptional regulator, it is reasonable that a general transcriptional dysregulation caused by the poly-alanine expansion might represent an important mechanism underlying CCHS pathogenesis2. Therefore, this study focused on the biochemical characterization of different PHOX2B variants3, such as a variant containing the correct C-terminal (20-alanines) stretch, one of the most frequent poly-alanine expansions (+7-alanines), and a variant lacking the complete alanine stretch (0-alanines). Comparison of the different variants by a multidisciplinary approach based on different methodologies (including circular dichroism, spectrofluorimetry, light scattering and Atomic Force Microscopy studies) highlighted the propensity to aggregate for the PHOX2B variant containing the poly-alanine expansion (+7-alanines), especially in the presence of DNA, while the 0-alanines variant resembled the protein with the correct poly-alanine length. Moreover, and unexpectedly, the formation of fibrils was revealed only for the pathological variant, suggesting a plausible role of such fibrils in the insurgence of CCHS. References

1. Di Lascio S, et al. (2013) Neurobiol Dis. 50:187-200. 2. Di Lascio S, et al. (2016). J Biol Chem. 291:13375-93. 3. Pirone et al. (2019) FEBS J. 2019 Apr 7. doi: 10.1111/febs.14841.

BIOFAR.35

NUOVE STRATEGIE EPIGENETICHE PER IL TRATTAMENTO DEL CARCINOMA MAMMARIO BASATE SU COMBINAZIONI TRA IL DONATORE DI METILI S-ADENOSILMETIONINA ED RNA NON

CODIFICANTI ALESSANDRA COPPOLA1 (alessandra.coppola@unicampania.it; SIB), CONCETTA PAOLA ILISSO1

(concettapaola.ilisso@unicampania.it; SIB), LAURA MOSCA1 (laura.mosca@unicampania.it; SIB), MARTINA PAGANO1 (martina.pagano@unicampania.it; SIB), FRANCESCA VITIELLO1

(francesca.vitiello4@studenti.unicampania.it; SIB), GIOVANNA CACCIAPUOTI1 (giovanna.cacciapuoti@unicampania.it; SIB), MARINA PORCELLI1* (marina.porcelli@unicampania.it; SIB)

1Dipartimento di Medicina di Precisione, Università della Campania “Luigi Vanvitelli”, Napoli, Italia

*Autore Corrispondente

Nell’ultimo decennio molti studi sia in vitro che in vivo hanno evidenziato che la S-adenosilmetionina (AdoMet) è in grado di contrastare la progressione di molti tumori umani sebbene con differenti meccanismi molecolari (1,2). Recentemente una nuova classe di molecole di RNA non codificanti, noti come microRNA (miRNA), è stata associata a diverse malattie umane incluso il tumore della mammella (3). Al fine di valutare gli effetti antiproliferativi esercitati dall’AdoMet nella linea cellulare MDA-MB 231di carcinoma mammario triplo negativo, abbiamo analizzato il profilo d’espressione dei miRNA miR-34a, miR-34c e miR-486-5p, dei quali era già nota la modulazione esercitata dall’AdoMet in cellule di carcinoma mammario MCF-7. In tale linea cellulare i livelli di espressione dei miRNA analizzati risultavano incrementati dal trattamento con AdoMet. Le cellule MDA-MB 231 sono state trasfettate con miR-34a e miR-34c, mimico e inibitore in presenza o meno di 500 μM AdoMet per 72 ore. I risultati ottenuti mostravano che la combinazione sia del miR-34a che del miR-34c mimico con AdoMet potenziava fortemente l’effetto pro-apoptotico del composto di solfonio attraverso la diminuzione dei livelli della pro-caspasi iniziatrice 8, della pro-caspasi effettrice 6 e della poli (ADP ribosio) polimerasi (PARP). Questi dati confermano che l’AdoMet può regolare l'espressione di miRNA nelle cellule di carcinoma mammario aumentando le conoscenze sulle basi molecolari dell'effetto antitumorale dell’AdoMet e suggerendo l'uso del composto di solfonio per lo sviluppo di nuove strategie terapeutiche miRNA-mediate finalizzate ad interventi terapeutici su pazienti affetti da carcinoma mammario. Bibliografia

1. Fontecave M, Atta M, Mulliez E. Trends in Biochemical Sciences, 29: 243-249, 2004. 2. Frau, M., Feo, F., Pascale, R.M. J. Hepatol., 59 (4): 830-841, 2013. 3. Ilisso, C.P., Delle Cave D, Mosca L, Pagano M, Coppola A, Mele L, Caraglia M, Cacciapuoti G, Porcelli

M. Cancer Cell Int., 4;18:197, 2018.

BIOTEC.1

HUMAN APOLIPOPROTEIN B: AN UNPREDICTABLE AND PROMISING SOURCE OF NOVEL FOOD PRESERVATIVES

ELIANA DELL’OLMO1 (eliana.dellolmo@unina.it), ROSA GAGLIONE (rosa.gaglione@unina.it; SIB), CONCETTA VALERIA LUCIA GIOSAFATTO (giosafat@unina.it; SIB), ROCCO DI GIROLAMO1 (rocco.digirolamo@unina.it), ANGELA CESARO1 (angela.cesaro@unina.it), RENATA PICCOLI1

(renata.piccoli@unina.it; SIB), EUGENIO NOTOMISTA2 (notomist@unina.it), EDWIN J.A VELDHUIZEN3 (E.J.A.Veldhuizen@uu.nl), RAFFAELE PORTA1 (raffaele.porta@unina.it; SIB), ANGELA ARCIELLO1,*

(anarciel@unina.it; SIB)

1Department of Chemical Sciences, University of Naples Federico II, 80126 Naples, Italy; 2Department of Biology, University of Naples Federico II, 80126 Naples, Italy;

3Department of Infectious Diseases and Immunology, Division Molecular Host Defence, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands;

*Corresponding author Nowadays, consumers are particularly aware of the health concerns regarding food additives, and the health benefits of “natural” and “traditional” foods, without chemical preservatives, are becoming more and more attractive (1). In this scenario, Host Defence Peptides (HDPs) have stimulated great interest because of their wide range of activities, the absence of toxic effects and the ability to preserve foods without altering their quality (2). Here, we report the characterization of two novel HDPs, identified in human Apolipoprotein B (ApoB) by using a bioinformatic tool, and named r(P)ApoBLPro and r(P)ApoBSPro. ApoB-derived peptides showed a wide range of activities, such as antimicrobial, anti-biofilm and immunomodulatory properties (3). Interestingly, ApoB-derived peptides were found to exert significant antimicrobial effects also towards main food-borne pathogens, such as Salmonella strains. Scanning electron microscopy analyses highlighted that both peptides are able to induce significant bacterial surface morphological changes, whereas stability analyses revealed that they maintain antimicrobial activity at high temperature and at extreme pH values. Besides, ApoB-derived peptides were found to exert significant fungicidal activity against Candida albicans cells and Aspergillus niger spores. Peptides were also found to be endowed with anti-biofilm properties, as demonstrated by crystal violet assays and confocal microscopy analyses, revealing their ability to disrupt bacterial biofilm matrix at sub-minimum inhibitory concentration values. Finally, to evaluate the applicability of both peptides in food bio-preservation, edible bitter vetch protein (BVP) films were prepared (4) by incorporating them into the BVP matrix. The obtained materials were found to retain a strong antimicrobial activity, as indicated by agar diffusion assays, opening interesting perspectives to their applicability in food industry. References

1. Settanni & Corsetti (2008), Int J Food Microbiol. 121(2):123-38. 2. Wanget al. (2016), Int J Mol Sci 17(603):1–13. 3. Gaglione et al. (2017), Biochem Pharmacol. 130:34-50. 4. Arabestani et al. (2016), Food Hydrocolloid. 60:232-242.

BIOTEC.2

A GREEN CASCADE APPROACH FOR THE DOWNSTREAM OF HIGH VALUE BIOPRODUCTS FROM GALDIERIA SULPHURARIA

1PAOLA IMBIMBO (paola.imbimbo@unina.it); 2 MONICA BUENO (monica.bueno@cial.uam-csic.es); 1LUIGI D’ELIA (luigi.delia@unina.it); 3GIUSEPPE OLIVIERI (giuseppe.olivieri@wur.nl), 4ANTONINO

POLLIO (antonino.pollio@unina.it), 2ELENA IBANEZ (elena.ibanez@csic.es), 1DARIA MARIA MONTI* (mdmonti@unina.it; SIB)

1 University of Naples Federico II, Department of Chemical Sciences,,Italy

2 Universidad Autónoma de Madrid, CIAL (CSIC-UAM), Spain. 3 Wageningen University, Bioprocess Engineering, AlgaePARC, The Netherlands.

4 University of Naples Federico II, Department of Biology, Italy *Corresponding author

In the last years, microalgae have attracted great interest, since they may serve as a continuous and reliable source of safe natural products, including pigments, polyunsaturated fatty acids (PUFA) and proteins of industrial interest. To date, microalgae production is currently limited to a few small industries, mainly for feed, nutrition and cosmetic sectors. However, obtaining an economic and sustainable method to increase the production of commercial products from microalgae, still remains a challenge. In this scenario, a new biorefinery process is urgently needed to fully exploit microalgae. We set up a cascade approach to optimize the recovery of high valuable bioproducts starting from the wet biomass of Galdieria sulphuraria, a unicellular thermo-acidophilic red alga, capable of accumulating several bioactive compounds. Extractions were performed in two steps, coupling a conventional high pressure procedure to recover phycocyanin with a pressurized liquid extraction for carotenoids recovery. These innovative, green and cost-effective procedures are able to improve the yields without affecting the quality of compounds, avoiding the use of organic solvents and the drying processes.

BIOTEC.3

EFFECT OF ALKALINE PH AND HEAT TREATMENT ON THE FORMATION OF BIOPLASTICS FROM MILK WHEY PROTEINS IN THE

PRESENCE OF DIFFERENT CONCENTRATIONS OF GLYCEROL MANAR ABDALRAZEQ (manar.abdalrazeq@unina.it; SIB), C. VALERIA L. GIOSAFATTO*

(giosafat@unina.it; SIB) , and RAFFAELE PORTA (raffaele.porta@unina.it; SIB)

University of Naples “Federico II”, Department of Chemical Sciences, 80126 Naples, Italy. *Corresponding author

Milk whey (MW) represents the major by-product of cheese industry. One possibility to recycle the MW wastes is the use of their globular proteins (MWPs) as a polymer source for the production of biodegradable plastic materials. MWP-based films are usually obtained by protein heat treatment in the presence of glycerol (GLY) as plasticizer at pH 7 [1], a method which would require commercially high costing process. Since it is known that denaturation and aggregation of MWPs are pH dependent, with strong alkalis producing rod-like microstructures able to forms fine-stranded fiber-like matrices, it was exploited the possibility to produce manageable MW-derived materials without any heat-treatment but under alkaline conditions. Our results demonstrated that the casting at pH 12 of the unheated MWP film forming solutions, containing either 40 or 50% GLY, led to produce more resistant and flexible films than the ones obtained at pH 7. Film opacity was observed significantly increased, being higher in the samples obtained at alkaline pH without MWP heating and with higher GLY concentrations. The developed experimental conditions allowed to produce hydrocolloid films with improved properties probably because MWPs denatured under alkaline conditions form small primary aggregates able to combine into large clusters [2]. Finally, also moisture content was observed to decrease with the reduction of GLY content, both in heated and unheated MWP-based films, whereas water uptake of the different films prepared at pH 12 did not significantly change. References

1. Galus, S.; Kadzinska, J. Moisture sensitivity optical mechanical and structural properties of whey protein-based edible films incorporated with rapeseed oil. Food Technol. Biotechnol. 2016, 54, 78-89, Doi: 10.17113/ft b.54.01.16.3889.

2. Bryant, C.M., McClements, D.J. Optimizing preparation conditions for heat-denatured whey protein solutions to be used as cold-gelling ingredients. Afr. J. Food Sci. 2000, 65, 259–263, Doi: 10.1111/j.1365-2621.2000.tb15990.x.

BIOTEC.4

EDIBLE COATINGS AND FILMS TO REDUCE ACRYLAMIDE FORMATION IN FRIED FOODS AND TO EXTEND STRAWBERRY

SHELF-LIFE ASMAA AL-ASMAR 1,2 (asmaa.alasmar@unina.it; SIB), CONCETTA VALERIA L. GIOSAFATTO1

(giosafat@unina.it; SIB), LOREDANA MARINIELLO1 * (loredana.mariniello@unina.it; SIB)

1 Department of Chemical Sciences, University of Naples “Federico II,” 80126 Naples, Italy. 2 Analysis, Poison control and Calibration Center (APCC), An-Najah National University, P.O. Box 7

Nablus, Palestine. *Corresponding author

According to European Food Safety Authority (EFSA), acrylamide is produced during cooking in numerous foods (e.g. french fries, breads). EFSA scientists concluded that acrylamide is a health concern. Higher temperature and low moisture content are very convenient conditions to produce acrylamide. Edible coating is a thin layer that covers the food surface that can be applied by dipping or spraying methods. Moreover, the edible coating or film can be consumed without causing any adversely health problem for consumers. Hydrocolloid materials are the most common materials used to prepare edible coatings and films. French fries are popular products worldwide with an acrylamide content that can reach 2089 µg/kg [1]. Recently, we ended up that pectin-based solutions are able to reduce acrylamide content in French fries of about 48%. Moreover, grass pea flour solutions treated with microbial transglutaminase also reduced acrylamide content of about 37%, while coating solutions containing grass pea flour not crosslinked by the enzyme could reduce acrylamide of about 31% [1]. In this work we checked the effectiveness of edible films in reducing acrylamide also in falafel, a traditionally fried and street food in Middle Eastern, made of grinded chickpeas with parsley and spices. Our studies have assessed that, after frying, the acrylamide content reached 7229 µg/kg. After adding transglutaminase (20 U/g chickpea proteins) to the falafel dough, the acrylamide content was reduced of about 34.4%, while the reduction was of about 84.5% when transglutaminase-containing falafels were dipped in the pectin solution [2]. We have also studied the effectiveness of mesoporous silica nanoparticles (MSN) in reinforcing pectin films and verified MSN ability to reduce the permeability to CO2, O2 and water vapor. Thus, cast pectin–based films containing MSN have been used to wrap strawberries. Results have indicated the ability of our films to increase strawberry shelf-life [3]. References

1. Al-Asmar A., Naviglio D., Giosafatto C.V. L., Mariniello L. (2018). Hydrocolloid-Based Coatings are Effective at Reducing Acrylamide and Oil Content of French Fries. Coatings, 8: 147-159. doi:10.3390/coatings8040147

2. Al-Asmar A., Giosafatto C.V. L., Panzella L., Mariniello L. Improving the health quality of fried falafel (Middle Eastern food) by using transglutaminase and/or pectin coating. Submitted to Coatings.

3. Al-Asmar A., Giosafatto C.V.L., Sabbah M., Sanchez A., Villalonga Santana R., Mariniello L. Effect of mesoporous silica nanoparticles on physicochemical properties of pectin packaging material for strawberry wrapping. Submitted to Nanomaterials.

BIOTEC.5

NANOPARTICELLE IBRIDE CON PROPRIETÀ FOTOACUSTICHE PER APPLICAZIONI TERANOSTICHE

GENNARO SANITÀ*1 (gennaro.sanita@unina.it; SIB), BARBARA CARRESE1 (b.carrese@studenti.unina.it), BRIGIDA SILVESTRI2 (brigida.silvestri@unina.it), GIUSEPPINA LUCIANI2 (luciani@unina.it), NUNZIA

MIGLIACCIO1 (nunzia.migliaccio@unina.it), GAETANO CALÌ3 (g.cali@ieos.cnr.it), ANNALISA LAMBERTI1 (annalisa.lamberti@unina.it)

1 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli “Federico II”, Napoli,

Italia 2 Dipartimento di Ingegneria chimica, dei Materiali e della Produzione industriale, Università di Napoli

“Federico II”, Napoli, Italia 3 Istituto per l’endocrinologia e l’oncologia sperimentale “G. Salvatore”, Consiglio Nazionale delle

Ricerche, Napoli, Italia *Corresponding author

Uno dei limiti della diagnostica per immagini è quello di non poter identificare i tumori nelle primissime fasi della malattia, portando spesso ad una diagnosi tardiva con esito infausto. Le nuove metodiche di diagnostica per immagini basate sull’utilizzo di nanoparticelle (NPs) vantano, invece, una maggiore sensibilità e selettività in ambito diagnostico1. In particolare, l’uso di NPs con proprietà fotoacustiche (PAI) attira l’interesse della comunità scientifica grazie alla semplicità d’utilizzo ed alla precisione. Inoltre, il PAI può essere efficacemente combinato con strategie terapeutiche, come l’effetto fototermico (PTT), così da mettere in atto una strategia teranostica2. In questo lavoro saranno presentate NPs ibride (MelaSil_Ag) progettate per applicazioni teranostiche. Le MelaSil_Ag sono costituite da un nucleo d’argento metallico, circondato da un guscio di silicio e un composto simile alla melanina (acido 5,6-diidrossiindol-2-carbossilico, DHICA). Il DHICA e il silicio sono stati scelti per la loro elevata biocompatibilità, il basso costo e le loro proprietà fisiche, mentre l’argento è in grado di amplificare i segnali PAI e PTT prodotti dal DHICA. La valutazione biologica preliminare è stata eseguita su due linee cellulari pancreatiche (PANC-1 e BxPC-3). I risultati hanno mostrato un buon livello d’internalizzazione e una lieve diminuzione di vitalità nelle cellule incubate con le NPs. Inoltre, per valutare l’emotossicità delle MelaSil_Ag, è stata stimata la percentuale di globuli rossi lisati dopo incubazione con le NPs. I dati hanno mostrato un’emotossicità del 100% già dopo 60 minuti d’incubazione, ma l’aggiunta di albumina umana (HSA) sulla superficie delle NPs, in presenza di EDC e NHS, aboliva totalmente tale emotossicità. Inoltre, le NPs modificate con HSA sono meno citotossiche di quelle non modificate. Infine, l’analisi delle proteine “corona” interagenti con le NPs, con e senza HSA, hanno indicato come la presenza dell’albumina diminuisca di molto l’interazione di tali proteine con la superficie delle nanoparticelle. References

1. Yu MK, Park J, Jon S. Targeting strategies for multifunctional nanoparticles in cancer imaging and therapy. Theranostics. 2012;2(1):3-44. doi:10.7150/thno.3463

2. Jaque D, Martínez Maestro L, Del Rosal B, et al. Nanoparticles for photothermal therapies. Nanoscale. 2014;6(16):9494-9530. doi:10.1039/c4nr00708e

BIOTEC.6

A NEW BIOTECHNOLOGICAL TOOL FOR AN IN VIVO ENZYME LABELLING AND IMMOBILIZATION

ROSA MERLO1 (rosa.merlo@ibbr.cnr.it), SONIA DEL PRETE1 (sonia.delprete@ibbr.cnr.it), ANNA VALENTI1 (anna.valenti@ibbr.cnr.it) ,ROSANNA MATTOSSOVICH1 (rosanna.mattossovich@ibbr.cnr.it),

VINCENZO CARGINALE1 (vincenzo.carginale@ibbr.cnr.it), CLAUDIU T. SUPURAN2 (claudiu.supuran@unifi.it), CLEMENTE CAPASSO1 (clemente.capasso@ibbr.cnr.it), GIUSEPPE

PERUGINO1, * (giuseppe.perugino@ibbr.cnr.it)

1Institute of Biosciences and Bioresources, National Research Council of Italy – Naples, Italy 2University of Florence, Neurofarba Department, Polo Scientifico – Sesto Fiorentino Firenze, Italy

*Corresponding author Chemical, physical or natural methods have been proved very advantageous strategies for enzymes immobilization. In particular, the use of natural systems unifies in a one-step the production and the in vivo immobilization of proteins, avoiding problems related to high costs of purification. Starting from this knowledge, we developed a novel Anchoring-and-Self-Labelling-protein-tag system (hereinafter ASLtag) [1]. The ASLtag consists of two moieties: the transmembrane N-terminal domain of the ice nucleation protein (INP) from Pseudomonas syringae, which allows the anchoring of proteins to the outer membrane of Gram-negative bacteria, and the thermostable version of the SNAP-tag (H5), an engineered O6-alkylguanine-DNA-alkyl-transferase (AGT or OGT, EC 2.1.1.63) from Sulfolobus solfataricus [2]. This enzyme covalently bounds O6-benzylguanine (O6-BG) derivatives with an irreversible one-shot trans-alkylation reaction, which allows the transfer of the chemical group conjugated with the O6-BG to a cysteine residue in the active site. Enzymes of interest fused to the ASLtag can be simultaneously in vivo immobilized on the outer membrane of Escherichia coli and indirectly labelled with the SNAP-tag reaction. Furthermore, this new biotechnological tool led to enhance both the expression and the thermal stability of the neo-synthesized enzymes, without affecting their folding or activity [3, submitted]. Finally, the possibility to conjugate an unlimited number of chemical groups to the substrate for the activity of the H5 moiety significantly increases its biotechnological potential. References

1. Merlo R, Del Prete S, Valenti A, Mattossovich R, Carginale V, Supuran C.T, Capasso C, Perugino G (2019) An AGT-based protein-tag system for the labelling and surface immobilization of enzymes on E. coli outer membrane, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 490-499, doi: 10.1080/14756366.2018.1559161

2. Vettone A, Serpe M, Hidalgo A, Berenguer J, del Monaco G, Valenti A, Rossi M, Ciaramella M, Perugino G (2016) A novel thermostable protein-tag: optimization of the Sulfolobus solfataricus DNA alkyl-transferase by protein engineering, Extremophiles, 20(1):1-13. doi: 10.1007/s00792-015-0791-9

3. Del Prete S, Merlo R, Valenti A, Mattossovich R, Rossi M, Carginale V, Supuran C.T, Perugino G, Capasso C, (2019) Thermostability enhancement of the 𝛼-carbonic anhydrase from Sulfurihydrogenibium yellowstonense by using the anchoring-and-self-labelling-protein-tag system (ASLtag), Journal of Enzyme Inhibition and Medicinal Chemistry, submitted.

BIOTEC.7

IDENTIFICATION, ISOLATION AND CHARACTERIZATION OF THERMO-RESISTANT ANTIOXIDANTS FROM EXTREMOPHILE MICROALGAE

LUIGI D’ELIA (luigi.delia@unina.it)1; PAOLA IMBIMBO (paola.imbimbo@unina.it)1; GIUSEPPE OLIVIERI (giuseppe.olivieri@wur.nl)2; ANTONINO POLLIO (anpollio@unina.it)3; DARIA MARIA MONTI

(mdmonti@unina.it; SIB)1*

1University of Naples Federico II, Department of Chemical Sciences, Naples, Italy;

2Department of Agrotechnology and Food Sciences, Bioprocess Engineering, Wageningen, Netherlands; 3 University of Naples Federico II, Department of Biology, Naples, Italy

*Corresponding author In the last few years, the demand for natural antioxidants to be used in the food industry is increased. This is mainly due to the toxicity of synthetic antioxidants and to their high production costs. Microalgae are rich in proteins, fatty acids and pigments endowed with antioxidant activity1. However, the concept of algal biorefinery needs to be applied for the production and subsequent commercialization of these molecules, in order to improve the economic sustainability of microalgae production1. Food processing and preservation need high temperature during the sterilization processes2. A short cooking time may be insufficient to kill microorganisms and to inactivate some of the enzymes involved in food browning reactions or in the degradation of antioxidants; on the contrary, an excessive cooking time reduces the nutritive properties of bioactive molecules. To overcome the problem of thermal degradation, the use of antioxidants isolated from extremophile microalgae would be interesting. In this study, different extremophile microalgal strains, able to grow at high temperature, were investigated as antioxidants producers. The cyanobacteria Synechococcus bigranulatus, was selected as it is able to grow at different pH. The microalga showed a productivity of 0.14 g/L at pH 11. In vitro antioxidant activity of the algal extract was performed using 2,2′- azino-bis (ethylbenzthiazoline-6-sulfonic acid, ABTS.+) radical cation assay, and the biocompatibility was performed on human eukaryotic cells. HPLC analyses showed that S. bigranulatus is an excellent producer of Zeaxanthin an β-carotene. References

1. Ruiz J, Olivieri G, de Vree J, et al. Towards industrial products from microalgae. Energy Environ Sci. 2016;9(10):3036-3043. doi:10.1039/C6EE01493C

2. ISEKI-Food (Network) ID, Jagtap DD, Mohapatra D, Joshi DC, Sutar RF, Kapdi SS. International Journal of Food Studies. Vol 2.; 2013.

BIOTEC.8

TtSmtB; A THERMOPHILIC MEMBER OF THE ArsR/SmtB TRANSCRIPTION FACTOR FAMILY: INSIGHTS INTO STABILITY AND

METAL BINDING PROPERTIES GIOVANNI GALLOa (giovanni.gallo2@unina.it; SIB), IMMACOLATA ANTONUCCIa

(immacolata.antonucci@unina.it), LUCIANO PIRONEb (luciano.pirone@unina.it; SIB), ANGELA AMORESANOc (angela.amoresano@unina.it), PATRIZIA CONTURSIa (contursi@unina.it; SIB), DANILA

LIMAUROA (limauro@unina.it; SIB), EMILIA PEDONEb (empedone@unina.it; SIB), SIMONETTA BARTOLUCCIa (bartoluc@unina.it; SIB) and GABRIELLA FIORENTINO*a (gabriella.fiorentino@unina.it; SIB)

a. Department of Biology, University of Naples Federico II, Napoli, Italy

b. Institute of Biostructure and Bioimaging, CNR, Napoli, Italy c. Department of Chemical Sciences, University of Naples Federico II, Napoli, Italy

*Corresponding author

The abundance of heavy metals in the environment has guided the evolution of multiple defense strategies in all microorganisms which sense the metals and regulate the transcription of genes coding for resistance proteins. Knowledge on the molecular components involved in heavy metals metabolism or detoxification represents a prerequisite to develop bio-systems for monitoring heavy metals in the environment. In this context, the metalloproteins of the ArsR/SmtB family are homodimeric transcriptional repressors widespread within the bacterial and archaeal kingdoms. They are able to sense a variety of metals, undergo allosteric changes and derepress genes involved in detoxification. Besides sharing many common features, they display great diversity in metal-sensing motifs and in the number of metals binding sites with a big variation in metal selectivity and metal coordination geometry [1]. Even though a large number of metal-responsive transcriptional regulatory proteins has been described so far [2], the molecular determinants of specificity, selectivity, and metal binding mechanism have been scarcely investigated in thermophilic microorganisms. In the genome of Thermus thermophilus HB27 TtSmtB is the unique ArsR/SmtB representative [3] chosen as a model to study molecular metal binding mechanisms at high temperature. Our study shows that TtSmtB has two different metal binding sites per monomer and interacts with di-tri-penta-valent metal ions with different affinity; the metal ions characterized prevent association in vitro of TtSmtB /DNA complexes in a way that reflects their binding capability. Conversely, binding of Zn(II) to TtSmtB stabilizes protein /DNA complexes pointing to a structural rather than a regulatory role for this ion. References

1. Saha RP, Samanta S, Patra S, Sarkar D, Saha A, Singh MK: Metal homeostasis in bacteria: the role of ArsR-SmtB family of transcriptional repressors in combating varying metal concentrations in the environment. Biometals 2017, 30:459-503.

2. Osman D, Cavet JS: Bacterial metal-sensing proteins exemplified by ArsR–SmtB family repressors. Natural product reports 2010, 27:668-680.

3. Antonucci I, Gallo G, Limauro D, Contursi P, Ribeiro AL, Blesa A, Berenguer J, Bartolucci S, Fiorentino G: An ArsR/SmtB family member regulates arsenic resistance genes unusually arranged in Thermus thermophilus HB27. Microbial biotechnology 2017, 10:1690-1701.

BIOTEC.9

CHEMICALLY-INDUCED DIRECT CARDIAC REPROGRAMMING AS AN INNOVATIVE STRATEGY FOR HEART REPAIR AND REGENERATION Giorgia Di Benedetto 1, Gianluca Testa 2,3, Flora Pirozzi 4, Matteo Barbato1, Luigi Ambrosone 2,3, Carlo

Gabriele Tocchetti 4, Pasquale Abete 4, Domenico Bonaduce 4, Tommaso Russo 1, Fabiana Passaro 1,*.

1 Department of Molecular Medicine and Medical Biotechnologies; University of Naples “Federico II”, apoli, taly

3 Interdepartmental Centre for Nanotechnology Research - NanoBem, University of Molise, Campobasso, Italy

4 Department of Translational Medical Sciences; University of Naples “Federico II”, Napoli Italy *Corresponding author

Cardiovascular diseases represent the first cause of morbidity in western countries, and, although in recent years substantial strides have been made in treatment strategies, mortality still remains high. Due to the limited regenerative capacity of the adult human heart, following myocardial infarction (MI) a large number of cardiomyocytes (CM) are necrotized, lost and replaced by scar tissue, leading to cardiac dysfunction. By now, the only available strategy to restore cardiac function is heart transplantation. A regenerative approach based on cardiomyocyte replacement seems to be a promising alternative. Indeed, direct cardiac reprogramming of non-contractile cells into cardiomyocytes represents an intriguing treatment option and different laboratories are searching for factors that could drive the trans-differentiation of the abundant population of cardiac fibroblasts (CFs) found in the scar of MI zones, into therapeutically suitable cells, such as CMs. However, the use of integrative viruses, frequently adopted in many direct reprogramming approaches, generate concerns, mostly related to their association with the risk of gene damage and oncogenesis. Recently, the use of small molecules able to induce direct cardiac reprogramming via non-genetic strategies in mice and in human, provided substantial foundation for pharmacological interventions to be translated into the clinic. Nevertheless, protocols derived from this very thriving research activity, while proving effective in confirming the overall concept of direct reprogramming and its achievements, still face the limitation of a poor yield of cardiomyocytes. In this picture, we set up a chemical compound-based direct cardiac reprogramming strategy. Starting from a defined cocktail of small molecules, which have been previously identified, we have generated a new protocol which, modifying CFs epigenetic state, increases cardiac precursor-like cells and improves the yield of induced cardiomyocyte (iCM).

BIOTEC.10

AN ARSENATE REDUCTASE-HYDROPHOBIN CHIMERA FOR ARSENIC BIO-SENSING

Rosanna Puopolo (rosanna.puopolo@unina.it; SIB), Alessandra Piscitelli (alessandra.piscitelli@unina.it; SIB), Paola Giardina (paola.giardina@unina.it; SIB), Simonetta Bartolucci (simonetta.bartolucci@unina.it;

SIB), Gabriella Fiorentino* (gabriella.fiorentino@unina.it; SIB)

Università degli Studi di Napoli Federico II, Italia *Corresponding author

Arsenic (As) is a toxic metalloid widespread in soil, water and air, harmful to humans and the environment; due to its toxicity, one of the biotechnological challenges is centred on the development of biosensors to monitoring its concentration in the environment. Microbial activities play important roles in the mobilization of arsenic; in particular, the arsenate reductase of Thermus thermophilus HB27 (TtArsC) is an enzyme capable of reducing As(V) in As(III). This enzyme, thanks to its thermophilic nature, has high resistance to changes in pH, temperature and ionic strength, characteristics that make it a good candidate as component of a biosensor [1], [2]. In the last few years it has also been demonstrated that biosensors based on self-assembling amyloid proteins display improved sensitivity compared to other systems. In this context, we aimed at developing a miniaturized biosensor which combined the recognition properties of TtArsC with the self-assembling properties of the hydrophobin Vmh2 of Plerotus ostreatus [3].Two chimeric genes codifying for two alternative fusion proteins (Vmh2-ArsC and ArsC-Vmh2) were designed and cloned in the pEt28(b+) expression vector. The recombinant proteins were expressed in Escherichia coli BL21(DE3)RIL, purified and characterised. The results obtained show that the chimeras can be firmly anchored on a surface with a precise orientation, pointing to them as a new platform for the development of arsenic biosensors. References

1. I. Del Giudice, D. Limauro, E. Pedone, S. Bartolucci, and G. Fiorentino, “A novel arsenate reductase from the bacterium Thermus thermophilus HB27: Its role in arsenic detoxification,” Biochim. Biophys. Acta - Proteins Proteomics, vol. 1834, no. 10, pp. 2071–2079, 2013.

2. J. Politi, J. Spadavecchia, G. Fiorentino, I. Antonucci, and L. De Stefano, “Arsenate reductase from Thermus thermophilus conjugated to polyethylene glycol-stabilized gold nanospheres allow trace sensing and speciation of arsenic ions,” J. R. Soc. Interface, vol. 13, no. 123, p. 20160629, 2016.

3. A. Piscitelli, A. Pennacchio, S. Longobardi, R. Velotta, and P. Giardina, “Vmh2 hydrophobin as a tool for the development of ‘self-immobilizing’ enzymes for biosensing,” Biotechnol. Bioeng., vol. 114, no. 1, pp. 46–52, 2017.

BIOTEC.11

DEVELOPMENT OF A INNOVATIVE BIO-SENSORS CHIMERA FOR THE ANALYTICAL DETERMINATION OF ENVIRONMENTAL O6-ALKYL-GUANINE CONTAINING AND DNA PRODUCTION OF A NEW AGT

SUBSTRATE BASED ON A DNA TRIPLEX Rosanna Mattossovich1 (rosanna.mattossovich@ibbr.cnr.it); Rosa Merlo1 (rosa.merlo@ibbr.cnr.it); Anna

Valenti1 (anna.valenti@ibbr.cnr.it); Giuliana d’Ippolito2 (gdippolito@icb.cnr.it); Angelo Fontana2 (afontana@icb.cnr.it); Alessandro Porchetta3 (prclsn01@uniroma2.it), Francesco Ricci3 (francesco.ricci@uniroma2.it) and Giuseppe Perugino*1 (giuseppe.perugino@ibbr.cnr.it)

1Institute of Biosciences and BioResources – National Research Council of Italy, Via Castellino 111, 80131

Naples, ITALY. 2Institute of Biomolecular Chemistry – National Research Council of Italy, Via Campi Flegrei 34, 80078

Pozzuoli (NA), ITALY. 3Chemical science and technology department, University of Rome, Tor Vergata, Via della Ricerca

Scientifica, 00133, Rome, Italy *Corresponding author

Cellular DNA is subjected to covalent modifications by intracellular chemical compounds and coming from the external environment. Alkylating agents are reactive molecules transferring chemical groups in nuclebases causing alterations in their functions. AGTs are ezymes which remove alkyl adducts from the O6- and O4- position of guanine and thymine on DNA, respectively, by a peculiar irreversible reaction [1]. We are currently focussing our attention on the development of analytical methods for the measure of O6-AG containing DNA, based on optical biosensors. These methods are useful for: i) the determination of DNA damages in the environment from the food and, in general, industrial productions; ii), the identification and optimization of protective molecules against the action of alkylating agents on DNA, useful in the cosmetic industry. One approach is based on the use of a protein chimera, which is based on a AGT and a Halotag moieties: the former recognizes and repairs O6-AGs in the DNA sample, whereas the latter part of the chimera is used for the direct and specific immobilization on the bio-chip support, conjugated with its relative specific substrates, where an optical signal will allow the determination of the AGT activity. An alternative method is based on the switching DNA Triplexes, which have been further introduced into DNA nanotechnology as sequence-specific targeting agents or nanodevices. The conformational switches between the duplex and the triplex form could be monitored through a FRET-pair fluorescent probes, located in an internal position and at the 5’ end, respectively [2]. In particular, we demonstrated that the presence of O6-methylated guanines affects the Triplex formation at neutral pHs. I performed a well-characterised assay on the AGT from the hyperthermophilic archea Sulfolobus solfataricus (SsOGT) [2] by using a fluorescent AGT substrate in competition with these oligonucleotides. References

1. Perugino et al., 2015 2. Amodio et al., 2014 3. Perugino et al., 2012

BIOTEC.12

CHARACTERIZATION OF A NEW ANTIOXIDANT ENZYME FROM THERMUS THERMOPHILUS

MARIO DE SIMONE, GABRIELLA FIORENTINO (fiogabri@unina.it; SIB), PATRIZIA CONTURSI (contursi@unina.it; SIB), SIMONETTA BARTOLUCCI (bartoluc@unina.it; SIB), DANILA LIMAURO*

(limauro@unina.it; SIB)

Dept. of Biology, University of Naples Federico II, Campus Monte S. Angelo, Via Cinthia, 80126, Naples, Italy

*Corresponding author

To fight oxidative damage due to reactive oxygen species (ROS), cells are equipped of different enzymes, among which Peroxiredoxins (Prxs) play a key role (1). Prxs are thiol- based enzymes in which one (1-Cys Prxs) or two cysteine residues (2-cys Prxs) are involved in the catalysis. The cysteine residues in 2-Cys Prx form a disulfide bridge following reduction of peroxide and generally the enzyme is recycled by disufide reductase system: Thioredoxin reductase (Tr) /Thioredoxin (Trx). Thermus thermophilus is a thermophilic bacterium whose antioxidant system was partially investigated; nowadays only a catalase and a superoxide dismutase were deeply studied (2). We characterized a Prx of T. thermophilus HB27, TtPrx, that could represent a key enzyme in the defence to oxidative stress. Based on sequence of T. thermophilus, TT_C0933, encoding a putative Prx (TtPrx), a synthetic gene was produced and expressed in Escherichia coli. The recombinant protein was purified and biochemically characterized. TtPrx is active on both organic and inorganic peroxides using dithiothreitol as thiol reductant, and it showed optimal temperature at 80°C. To go insight into physiological recycling system of TtPrx, a thermophilic heterologous regeneration system composed by SsTr of Sulfolobus solfataricus and Protein Disulphide Oxidoreductase of T. thermophilus (TtPDO) (3) was used. Interestingly in this system TtPDO, was able to reduce TtPrx in presence of H2O2. Analysis of secondary structure and thermal stability of TtPrx were also performed by Circular Dichroism analysis. Furthermore, TtPrx can protect supercoiled DNA from oxidative damage. Our results showed that TtPrx can prevent ROS damage, not only scavenging peroxides, but also defending integrity of DNA. Finally, the thermostability and high optimal temperature of TtPrx make this enzyme a good candidate for possible biotechnological applications. References

1. Rhee SG. Mol Cells. 2016 ;39(1):1-5. 2. Ebihara A, et al. Extremophiles. 2015 ;19(4):775-85 3. Pedone E, et.al. Extremophiles. 2014;18(4):723-31

BIOTEC.13

INVESTIGATING MOLECULAR WEIGHT DIFFERENCES OF HEPAROSAN- AND CHONDROITIN-LIKE CAPSULAR POLYSACCHARIDES FROM WILD

TYPE AND ENGINEERED ESCHERICHIA COLI STRAINS Odile Francesca Restaino1* (odilefrancesca.restaino@unicampania.it; SIB); Sergio D’Ambrosio1

(sergio.dambrosio@unicampania.it; SIB); Elisabetta Cassese1 (eli.cassese@gmail.com); Simona Barbuto Ferraiuolo1 (simona.barbutoferraiuolo@unicampania.it); Alberto Alfano1 (alberto.alfano@unicampania.it);

Riccardo Ventriglia1 (riccardo_ventriglia@hotmail.it); Adelaide Marrazzo1 (adelaide.marrazzo@gmail.com); Chiara Schiraldi1 (chiara.schiraldi@unicampania.it; SIB); Donatella Cimini1 (donatella.cimini@unicampania.it)

1Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of

Campania “Luigi Vanvitelli”, Via De Crecchio 7, 80138, Naples, Italy *Corresponding author

Heparin and chondroitin sulfate are world-wide used as anti-thrombic and anti-osteoarthritis drugs, respectively. In the last years new biotechnological approaches were successful in producing heparin and chondroitin sulfate starting from the heparosan and chondroitin-like capsular polysaccharides of wild type and engineered E. coli K5 and K4 strains. In order to obtain natural-like homologues, the molecular weights of the microbial capsular polysaccharides might be precisely determined, but so far they have never been reported. In this study, for the first time, a new protocol was developed to purify the capsular polysaccharides of three wild type and three engineered E. coli K5 and K4 strains and to determine their molecular weights. A small scale purification train was designed by coupling ultra-filtration and anion exchange chromatography to purify, directly from the fermentation broth supernatants, the microbial capsular polysaccharides. High recovery (>85.0%) and the removal of the main contaminant, the lipopolysaccharide, were obtained. Analyses by size exclusion chromatography with triple detector array determined that the wild type capsular polysaccharides ranged from 51.3 to 90.9 kDa, while the engineered strains produced polysaccharides with higher molecular weights, ranging from 68.4 to 130.6 kDa, but with similar polydispersity values, between 1.1 and 1.5.

BIOTEC.14

THE HIPPO PATHWAY EFFECTOR YAP1 AND ITS POSSIBLE ROLE IN THE DNA DAMAGE RESPONSE OF MOUSE EMBRYONIC STEM CELL

Matteo Barbato1, Giorgia Di Benedetto 1, Tommaso Russo 1, Fabiana Passaro 1,*

1 Department of Molecular Medicine and Medical Biotechnologies; University of Naples “Federico II”, Napoli,

Italy *Corresponding author

Embryonic stem cells (ESCs) exhibit unlimited self-renewal capacity and differentiate into multiple cell types representative of the three germ layers. Their high proliferative capacity is coupled to rapid G1-S transition and elevated levels of CDKs and other cell cycle regulators. DNA replication is a central cellular process that allows duplication of the genetic material before its proper segregation during cell division. The DNA damage response (DDR) protects cells from deleterious mutations during replication and helps maintaining genome stability in face of exogenous genotoxic stress. Such pathways must be particularly robust in ESCs, since they are highly expose to different sources of stress replication, such as oxidative stress, nucleotide deficiency, interference between replication and transcription and hypoxia. Hippo pathway is emerging as an important constituent of DDR. Its components are responsive to stress signals and are also involved in preserving genome integrity from DNA lesions. Several components of the Hippo pathway are targeted by the two major kinase cascades that coordinate cellular responses after the induction DNA damage, the ATM/CHK2 and ATR/CHK1 pathways. Yes-associated protein (YAP) and its paralog WW domain containing transcription regulator 1 (TAZ) are key elements of the Hippo pathway kinase cascade. We collected evidences suggesting that YAP1 might be involved in preserving mESC integrity upon physiological stresses, including replicative stress. As such, our data may contribute to assign a new role to YAP1 in mESCs.

BIOTEC.15

IDENTIFICATION AND CHARACTERIZATION OF THE FIRST ARCHAEAL GLYCOSIDE HYDROLASE 109 FROM METAGENOMIC DATASET OF

PISCIARELLI SOLFATARA NICOLA CURCI1,2 (nicola.curci@unina.it); ANDREA STRAZZULLI1 (andrea.strazzulli@unina.it; SIB);

ROBERTA IACONO1,2 (roberta.iacono@unina.it); FEDERICA DE LISE1 (federica.delise@unina.it); LUISA MAURELLI2 (luisa.maurelli@ibbr.cnr.it); BEATRICE COBUCCI-PONZANO2

(beatrice.cobucciponzano@ibbr.cnr.it; SIB); MARCO MORACCI1,2* (marco.moracci@unina.it; SIB)

1 Department of Biology University of Naples ‘Federico II’ Naples, Italy 2 Institute of Biosciences and BioResources, National Research Council of Italy, Naples, Italy

*Corresponding author

Carbohydrate active enzymes (Cazymes) are of considerable interest in many fields such as human health and biotechnology. Currently, CAZy database (www.cazy.org) groups Cazymes in classes, families, and subfamilies based on amino acid sequence revealing common 3D structures and reaction mechanisms among the biochemically characterized members[1]. The discovery of new Cazymes, in particular Glycoside hydrolases (GH) from hyperthermophilic microorganisms, gained interest for their great industrial potential. These biocatalysts, functioning under conditions in which their mesophilic counterparts would quickly denature, are used in several biotechnological applications. However, the majority of hyperthermophilic microorganisms are unculturable and the metagenomic study of extreme environments is a key approach to exploit the enzymes from these complex microbial populations, providing the access to far more microbial diversity than genomic approaches[2]. Here, we report the identification and characterization of the first archaeal GH belonging to family 109, in which only an α-N-acetylgalactosaminidase activity, operating by unusual NAD+ dependent mechanism, has been characterized so far[3]. The sequence was identified from metagenomic dataset of the hot spring Pisciarelli Solfatara. The recombinant enzyme was characterized in detail, showing activity at 85°C and pH 8.0, and the highest substrate specificity on pNP-�-N-acetylglucosaminide. References

1. G. J. Davies and S. J. Williams (2016) Carbohydrate-active enzymes: sequences, shapes, contortions and cells. Biochem. Soc. Trans. 44, 79–87; doi:10.1042/BST20150186

2. Strazzulli A, Iacono R, Giglio R, Moracci M and Cobucci-Ponzano (2017). Metagenomics of hyperthermophilic environments: biodiversity and biotechnology. In: “Microbial ecology of extreme environment book” Spinger, London (GBR)

3. Liu QP, Sulzenbacher G, Yuan H, Bennett EP, Pietz G, Saunders K, Spence J, Nudelman E, Levery SB, White T, Neveu JM, Lane WS, Bourne Y, Olsson ML, Henrissat B, and Clausen H. (2007) Bacterial glycosidases for the production of universal red blood cells. Nat Biotechnol. 25, 454-64. DOI:10.1038/nbt1298

BIOTEC.16

NOVEL ESTERASE FROM THE THERMOPHILIC BACTERIUM GEOBACILLUS THERMODENITRIFICANS

NICOLA CURCI1,2, (nicola.curci@unina.it); ANDREA STRAZZULLI1, (andrea.strazzulli@unina.it; SIB); ROBERTA IACONO1,2, (roberta.iacono@unina.it); FEDERICA DE LISE1, (federica.delise@unina.it); LUISA

MAURELLI2, (luisa.maurelli@ibbr.cnr.it); BEATRICE COBUCCI-PONZANO2, (beatrice.cobucciponzano@ibbr.cnr.it; SIB); MARCO MORACCI1,2*, (marco.moracci@unina.it; SIB)

1Department of Biology University of Naples ‘Federico II’ Naples, Italy

2Institute of Biosciences and BioResources, National Research Council of Italy, Naples, Italy *Corresponding author

Lipolytic enzymes, represented by esterases and lipases, catalyse the hydrolysis and synthesis of ester bond. These groups of enzymes differ in their biochemical properties and share little primary sequence similarity but present a conserved catalytic triad of amminoacid arranged in the same order along the sequence: serine (Ser), aspartate (Asp) or glutamate (Glu), and histidine (His), with the catalytic Ser embedded in the consensus motif Gly-X-Ser-X-Gly. Lipolytic enzymes do not require cofactors, are usually rather stable, active in organic solvent and exhibit high regio- and stereo-specificity. These proprieties make them attractive biocatalysts for biotechnological applications and in particular those isolated from (hyper)thermophiles have received great attention due to their stability at elevated temperature and a wide pH range in organic solvents [1]. Here we report on the identification of a new thermostable esterase from the thermophilic bacterium Geobacillus thermodenitrificans and its biochemical characterization. Based on its sequence and on the preserved consensus pentapeptide motif, the enzyme is not attributable to any of the microbial esterase families classified. The gene GTNG0744 was cloned and expressed in E. coli. The recombinant enzyme is a monomer of 31.5 KDa and showed high selectivity for the synthetic substrate 4-NP-O-Octanoate. It is also active on the 4-NP-O-Acetate and 4-NP-O-butirrate, but no activity detected on the 4-NP-dodecanoate, suggesting its preference for esters with short acyl chains. This esterase showed thermal stability and activity in a wide pH range with promising potential for biotechnological and industrial applications. References

1. López-López O, Cerdán M and González Siso M. (2014) Curr Protein Pept Sci 15:445–455

BIOTEC.17

IBISBA 1.0 IS A PAN-EUROPEAN RESEARCH INFRASTRUCTURE PROVIDING TRANSLATIONAL RESEARCH SERVICES TO INDUSTRIAL

BIOTECHNOLOGY Mauro Di Fenza1* (mauro.difenza@ibbr.cnr.it), Beatrice Cobucci-Ponzano1

(beatrice.cobucciponzano@ibbr.cnr.it; SIB), Marco Moracci 1,2 (marco.moracci@unina.it; SIB), Michael O’Donohue3 (Michael.Odonohue@inra.fr), and the IBISBA 1.0 consortium4

1Institute of Biosciences and BioResources-CNR, Italy

2Dept. of Biology, University of Naples ‘Federico II’, Italy 4CEPIA, Division of Science Food and Bioproducts Engineering-INRA, France

4www.ibisba.eu. *Corresponding author

Industrial Biotechnology is at a crucial point in its development, because it has been increasingly empowered by progress in life sciences research and is now benefitting from synthetic biology. To move industrial biotechnology further along the road to industrial maturity, progress in Research & Innovation is required to better translate new knowledge into innovative preindustrial processes that can be taken up by industry. This is vital to support the development of the European industrial biotechnology sector and Europe's circular bioeconomy transition. Europe possesses a lot of research infrastructures that can be used to accelerate the development of innovative bioprocesses. Currently, these are mostly disconnected and thus unable to host the development of efficient R&I project pipelines. However, networking of individual infrastructure facilities is a viable way to overcome this problem. IBISBA 1.0 (www.ibisba.eu) is a research infrastructure project aiming to create a Pan-European research infrastructure network to provide innovation services to industrial biotechnology. These services include the hosting of bioprocess development projects, helping to translate results into preindustrial innovation, the development of experimental workflows and standards to improve interoperability and reproducibility, a web-based repository for knowledge asset management and first-rate training for early career stage researchers. As a step towards reaching these ambitions, IBISBA 1.0 is operating a transnational access (TNA) programme, which provides subsidized access to a set of research facilities. The TNA programme is open to all eligible researchers wishing to translate their research results into pre-industrial innovation.

BIOTEC.18

HYALURONAN-BASED HYDROGELS AS DERMAL FILLERS: THE BIOPHYSICAL PROPERTIES FOR SPECIFIC CLINICAL USES

ANNALISA LA GATTA*(annalisa.lagatta@unicampania.it; SIB), ROSANNA SALZILLO

(ros.salzillo@libero.it), CLAUDIA CATALANO (claudiacatalano92@libero.it), ANTONELLA D’AGOSTINO (antonella.dagostino@unicampania.it), ANNA VIRGINIA ADRIANA PIROZZI (adripirozzi@gmail.com; SIB),

CHIARA SCHIRALDI* (chiara.schiraldi@unicampania.it; SIB)

Università degli Studi della Campania “Luigi Vanvitelli”, Dipartimento di Medicina sperimentale, Naples, Italy. *Corresponding author

Hyaluronan (HA)-based hydrogels, obtained by crosslinking the biopolymer with 1,4 butandiolediglycidylether (BDDE), are widely used as dermal fillers. Lines of HA-fillers including “volumetric”, “global performance” and “skinbooster” gels, with specific clinical indications and recommended planes of injection are currently marketed [1]. The biophysical characterization of these products strongly supports clinicians when selecting the product for a specific need and, combined with clinical studies, is key to further improve gel design [1-2]. Here, we aimed to ascertain the biophysical properties of a filler that translate into a “volumetric”, “global action” or “skinbooster” effect. At least three commercial gels for each class of fillers were evaluated for composition (soluble/insoluble-HA), hydration capacity, rheological behavior, and cohesivity [1-3]. Water-soluble HA (up to 50wt%) was detected in all the samples. The soluble HA consists of chemically modified, short (Mw<500kDa) HA chains, however, a relevant fraction of unmodified, longer chains was found in some samples (SEC-TDA analyses). Gel hydration and cohesivity varied extensively even within the same class of gels. The same was found for rigidity (G’:40-600Pa). For the volumetric gels, 1H-NMR analyses revealed a wide range of HA-chemical modification extent also suggesting diversities in the crosslinking efficiency. The gels proved diversely sensitive to hyaluronidases and ROS. Studies using Human Dermal Fibroblasts demonstrated gel capacity to prompt collagen I, elastin and aquaporin3 synthesis supporting a positive effect of HA fillers on skin restoration and hydration. The findings represent a wide assessment of properties that well characterize the three classes of HA-fillers. Differences in behavior were highlighted among the products with the same clinical indications, thus providing practitioners with useful information to drive the selection toward one product or another depending on the specific case. Finally, the work valuably contributes to our knowledge of HA-fillers and to the optimization of their use and manufacture. References

1. La Gatta A, De Rosa M, Frezza M A. Catalano C, Meloni M, Schiraldi C. Biophysical and biological characterization of a new line of hyaluronan-based dermal fillers: a scientific rationale to specific clinical indications. Mater Sci Eng C Mater Biol Appl. 2016; 68:565-72

2. Kablik J, Monheit GD, Yu LP, Chang G, Gershkovich J. Comparative physical properties of hyaluronic acid dermal fillers. Dermatol Surg. 2009; 35: 302–12.

3. Sundaram H, Rohrich RJ, Liew S, Sattler G, Talarico S, Trévidic P et al. Cohesivity of Hyaluronic Acid Fillers: Development and Clinical Implications of a Novel Assay, Pilot Validation with a Five-Point Grading Scale, and Evaluation of Six U.S. Food and Drug Administration-Approved Fillers. Plast Reconstr Surg. 2015; 136: 678-86.

BIOTEC.19

THE HUMAN GENOME STABILITY MAINTENANCE DNA HELICASE FANCJ IS RECRUITED TO THE DNA REPLICATION FORKS BY DIRECT

INTERACTION WITH THE AND-1/CTF4 FACTOR ETTORE NAPOLITANO (e.napolitano@ibp.cnr.it), ALIDA IAGROSSI (a.iagrossi@ibp.cnr.it) and

FRANCESCA M. PISANI* (fm.pisani@ibp.cnr.it)

Istituto di Biochimica delle Proteine, CNR, Via P. Castellino, 111. 80131 - Naples. Italy *Corresponding author

Human FANCJ is an ATP-dependent DEAH-box helicase that unwinds DNA with a 5'-to 3' directionality. FANCJ shares sequence similarity with the Super-Family 2 Fe-S cluster-containing DNA helicases DDX11, XPD and RTEL1 [1]. All of these helicases play important roles in genome stability maintenance and are implicated in rare genetic syndromes and cancer development [2]. FANCJ is mutated in hereditary breast and ovarian cancer as well as in Fanconi anemia (FA). Here we report that, in unperturbed conditions, FANCJ is associated to the ongoing DNA replication forks in mammalian cells. This association is mediated by a direct interaction with the replication factor AND-1 (acidic and nucleoplasmic DNA-binding protein), the human ortholog of yeast Ctf4 (chromosome transmission fidelity 4). This is a homo-trimeric protein, which links the Cdc45-MCM-GINS (CMG) replicative DNA helicase with DNA polymerase a/primase operating on the leading and lagging strand, respectively [3]. Moreover, Ctf4/AND-1 acts as an interaction platform recruiting and coordinating the activities of many additional replication factors and enzymes at the fork either in yeast or in mammalian cells. Using a combination of structural predictions, site-specific mutagenesis and co-pull down experiments carried out on cell extracts and on purified recombinant proteins, we have been able to identify the FANCJ AND-1-interacting site, an evolutionarily conserved sequence motif, located between the helicase box IV and V. Furthermore, we have precisely mapped the FANCJ:AND-1 interaction surfaces using protein truncated forms and site-specific mutants. Results of this study will allow us to unveil the role played by the FANCJ DNA helicase in assisting replisomes to deal with difficult-to-replicate DNA templates, that represent endogenous sources of replication stress. References

1. Brosh RM Jr (2013) DNA helicases involved in DNA repair and their roles in cancer. Nat Rev Cancer 13, 542-558.

2. Brosh RM Jr, Cantor SB (2014) Molecular and cellular functions of the FANCJ DNA helicase defective in cancer and in Fanconi anemia. Front Genet 5, 372.

3. Simon AC et al. (2014) A Ctf4 trimer couples the CMG helicase to DNA polymerase alpha in the eukaryotic replisome. Nature 510, 293–297.

BIOTEC.20

FUNGAL PROTEIC BIOSURFACTANTS FOR THE DEVELOPMENT OF BIOSENSING AND BIOMEDICAL PLATFORMS

Ilaria Stanzione* (ilaria.stanzione@unina.it), Paola Cicatiello (p.cicatiello@gmail.com), Rossana Pitocchi (rosspitocchi@gmail.com), Alessandra Piscitelli (apiscite@unina.it; SIB), Paola Giardina (giardina@unina.it;

SIB)

Dipartimento di Scienze Chimiche, Università Federico II, Napoli. *Corresponding author

Biosurfactants are amphiphilic molecules mainly produced by microorganisms (including bacteria, yeast and fungi) and occur in nature as diverse groups comprising glycolipids, lipopeptides and lipoproteins, fatty acids, phospholipids and proteins. Some proteic biosurfactants are able both to act as emulsion stabilizers and to modify surface properties. Among the proteic biosurfactants, the hydrophobins (HFBs) are self-assembling proteins typical of filamentous fungi, described as the most powerful surface-active proteins known. Vmh2 from Pleurotus ostreatus is the HFB more studied by our research group. Furthermore, using a bioprospecting approach, some marine fungi were selected for their ability to produce foam in shaken cultures, and two new HFBs, Pac2 and Pac3, were isolated and characterized (Cicatiello et al. 2017). Vmh2 and Pac3 exhibit anti-biofilm activities, inhibiting the adhesion on the functionalized surfaces of Staphyloccocus epidermidis, a nosocomial pathogen, involved in infections of medical devices such as catheters (Artini et al, 2017). HFB layers on different surfaces can be useful for easy immobilization of biomolecules with specific activities to develop innovative biosensors. Using genetic engineering techniques, Vmh2 was fused to other biotechnologically relevant proteins as a tool for the development of self-immobilizing proteins. A homogeneous bio-layer was formed by these chimerae, characterized both by the adhesive property of Vmh2 and the catalytic/recognition activities of target proteins and used for high throughput analyses to detect different analytes in real matrices (Sorrentino et al, 2019). Moreover, Vmh2 was used to exfoliate and functionalize 2D nanomaterials, such as graphene and MoS2, making these dispersions stable in water solution and suitable for nanotechnological applications.

References

1. Artini et al, 2017, Hydrophobin coating prevents Staphylococcus epidermidis biofilm formation on different surfaces; Biofouling.; 33(7):601-611.

2. Cicatiello et al, 2017, Self-assembly of two hydrophobins from marine fungi affected by interaction with surfaces; Biotechnol Bioeng.;114(10):2173-2186.

3. Sorrentino et al, 2019, Development of a biosensing platform based on a laccase-hydrophobin chimera. Appl Microbiol Biotechnol; doi: 10.1007/s00253-019-09678-2.

BIOTEC.21

CAZYMES DISCOVERY FROM GEOTHERMAL ENVIRONMENTS: A METAGENOMIC APPROACH

ANDREA STRAZZULLI1,2 (andrea.strazzulli@unina.it; SIB), BEATRICE COBUCCI-PONZANO3 (beatrice.cobucciponzano@ibbr.cnr.it; SIB), ROBERTA IACONO1,3 (roberta.iacono@unina.it), ROSA

GIGLIO3 (rosagiglio@hotmail.it), NICOLA CURCI1,3 (nicola.curci@unina.it), LUISA MAURELLI3

(luisa.maurelli@ibbr.cnr.it), BERNARD HENRISSAT4,5 (Bernard.Henrissat@afmb.univ-mrs.fr), FEDERICO M. LAURO6,7 (flauro@ntu.edu.sg), CARLOS M.G.A. FONTES8 (cafontes@fmv.ulisboa.pt), MARCO

MORACCI1,2,3,* (marco.moracci@unina.it; SIB)

1Department of Biology, University of Naples "Federico II", Naples, Italy. 2Task Force on Microbiome Studies, University of Naples Federico II, Naples, Italy

3Institute of Biosciences and BioResources, National Research Council of Italy, Naples. 4Centre National de la Recherche Scientifique, AFMB, USC 1408, Marseille, France.

5Department Biological Sciences, King Abdulaziz University, Jeddah, Saudi Arabia 6Asian School of the Environment, Nanyang Technological University, Singapore

7Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore

8NzyTech LDA, Lisbon, Portugal *Corresponding author

Novel thermophilic glycosidases, showing uncommon intrinsic stability to pH extremes and temperatures >80°C[1], are very promising candidates for the biotransformations and biotechnological application requiring extreme reaction condition as for lignocellulosic materials in second-generation biorefineries. We report here a metagenomic approach aimed to search novel Cazymes[2] within the hyperthermophilic microbial communities populating geothermal sites. The metagenomic analysis of the microbial consortia in two neighboring mud/water pools in the solfataric field of Pisciarelli (Naples, Italy) that differ in temperature and pH (Pool1 T=85°C and pH 5.5; Pool2 T=94°C and pH 1.5) was performed. Moreover, to identify enzymes to be exploited in the conversion of lignocellulosic biomasses for second-generation biofuels, we enriched in-lab Pool1 community to select microorganisms able to grow on different plant biomasses. The analysis of metagenomic data revealed a high abundance of cazymes in the solfataric samples. In particular, within the cazymes present in Pool2, we identified and characterized a novel hyperthermostable GH5 b-mannosidase and the first archaeal GH109 showing activity on glucosides and N-acetyl-glucosides. In addition, among the sample enriched in-lab on plant biomasses, a remarkable selection of specific cazyme families was observed and the characterization of a set of these enzymes revealed novel b-glucosidase and glucanase activities. We show here that a combined approach of metagenomic of extreme environments, in-lab enrichments, and detailed enzymatic characterization is a powerful tool to exploit natural biodiversity and obtain novel biocatalysts for industrial applications. References

1. Cobucci-Ponzano B, Aurilia V, Riccio G, Henrissat B, Coutinho PM, Strazzulli A, Padula A, Corsaro MM, Pieretti G, Pocsfalvi G, Fiume I, Cannio R, Rossi M, Moracci M. J Biol Chem 2010, 285, 20691-20703.

2. Menzel P, Gudbergsdóttir SR, Rike AG, Lin L, Zhang Q, Contursi P, Moracci M, Kristjansson JK, Bolduc B, Gavrilov S, Ravin N, Mardanov A, Bonch-Osmolovskaya E, Young M, Krogh A, Peng X. Microb Ecol 2015, 70 411-24

BIOTEC.22

TRANSGLUTAMINASE-CROSSLINKED PROTEIN FILMS REINFORCED BY MESOPOROUS SILICA NANOPARTICLES

PROSPERO DI PIERRO1 (prospero.dipierro@unina.it; SIB), INIGO FERNANDEZ1* (iferbats@gmail.com), CARLOS REGALADO-GONZALEZ2 (regcarlos@gmail.com), BLANCA E. GARCIA-ALMENDAREZ2

(blancag31@gmail.com), RAFFAELE PORTA1 (portaraf@unina.it; SIB)

1Department of Chemical Sciences, University of Naples “Federico II”, Naples, Italy; 2Facultad de Química, Universidad Autónoma de Querétaro, Querétaro, Mexico

*Corresponding Author

The environmental impact of plastic wastes is escalating rising widespread global concern since disposal systems are inadequate. Therefore, it is crucial to find enduring plastic alternatives, especially in short-term food packaging and disposable applications. One possible solution is the production of bio-based (polysaccharide and/or protein-derived) biodegradable/edible materials. The major limit of hydrocolloid films in food packaging is their relatively poor mechanical and barrier properties which currently hinder their industrial use. The advancement of nanotechnology has boosted interest to new types of composites in which the filler has at least one dimension smaller than 100 nm (nanocomposites). These innovative biomaterials exhibit generally increased mechanical and barrier properties, as well as improved heat resistance compared to their neat polymers and conventional composites. We suggest here a new strategy to produce nano-reinforced biomaterials by using as polymer matrix protein mixture extracted from bitter vetch (BV) seeds and as filler the mesoporous silica nanoparticles (MSN) functionalized or not with (3-aminopropyl)-triethoxysilane (APTES). To improve the structural network, the nanoparticle containing film forming solution was incubated in the presence of transglutaminase (TG), a protein crosslinking enzyme. The obtained results showed that all the BV protein films reinforced with MSN or MSN-APTES showed improved mechanical and barrier properties to both gases and water vapor, and that TG addition further reduced film permeability values to the ones of the well known and widely commercialized starch-based MaterBi bioplastics.

DTN.1

RIBOSOMAL PROTEIN RPL3 TARGETS E2F1 THROUGH PARP-1: A

NEW PATHWAY LINKING RIBOSOMAL STRESS TO CELL PROLIFERATION

A. PECORARO (annalisa.pecoraro@unina.it; SIB), R. DI DOMENICO (rossel.didomenico@studenti.unina.it), A. PELLEGRINO (anna.pellegrino2@studenti.unina.it), M. LOPATRIELLO (m.lopatriello@studenti.unina.it), F. DI DONATO (francesca.didonato2@studenti.unina.it), G. RUSSO (giulrus@unina.it; SIB) and A. RUSSO*

(annapina.russo@unina.it; SIB)

Department of Pharmacy, University of Naples “Federico II”, Via Domenico Montesano 49, 80131 Naples, Italy

*Corresponding Author Manipulating cell cycle regulatory pathways represents a good strategy in the treatment of cancer. Our previous data demonstrated that nucleolar stress induced by anticancer drugs in colon cancer cells devoid of p53 leads to the activation of ribosomal protein L3 (rpL3). In this condition, rpL3 exerts additional ribosomal functions including DNA repair, cell cycle arrest and apoptosis. In the present study, we demonstrate that upon drug-induced nucleolar stress, rpL3 enriches in the nucleolus and localizes in the nucleus acting as a regulator of cell cycle progression. In particular, we demonstrate that rpL3 is required for the E2F-1 mediated transcriptional activation of G1/S transition genes. Luciferase assays establish that rpL3 negatively regulates the activity of E2F-1 promoter. Induced ribosome free rpL3 reduces Cyclin D1 amount at mRNA and protein levels. Using protein/protein immunoprecipitation studies, we demonstrate that rpL3 physically interacts with PARP-1 contributing negatively to PARP-1 stability. rpL3/PARP-1 complex affects E2F1 transcriptional activity. Our findings led to the identification of a new pathway mediated by rpL3 involving E2F-1 and PARP-1 and contributing to regulate cell cycle progression. References 1. Russo A. et al., IJMS (2017) 18,140. 2. Russo A. et al., Cell Cycle (2016) 15, 41.

DTN.2

TRANSATTIVAZIONE DEL RECETTORE TIROSINA KINASI TrkA MEDIATA DAL RECETTORE GPCR FPR1 IN CELLULE

DI NEUROBLASTOMA UMANO MARTINA CASTALDO1 (martina.castaldo@unina.it) (SIB), CRISTIANA ZOLLO1 (cristianazollo@gmail.com),

ROSARIO AMMENDOLA1 (rosario.ammendola@unina.it) (SIB), FABIO CATTANEO1* (fabio.cattaneo@unina.it) (SIB)

1 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II,

Italia *Autore corrispondente

Il recettore per formil peptidi FPR1 appartiene alla famiglia dei recettori GPCR accoppiati a proteine Gi sensibili alla tossina della pertosse (PTX). FPR1, in seguito al legame con il suo ligando ad alta affinità, N-formil-Metionil-Leucil-Fenilalanina (N-fMLP), induce la mobilizzazione di calcio intracellulare, la generazione di specie reattive dell’ossigeno (ROS) mediata dalla NADPH ossidasi, la migrazione e la proliferazione cellulare (1). Diversi studi hanno evidenziato un’elevata espressione di FPR1 in patologie tumorali e neurodegenerative (2). In questo studio, abbiamo dimostrato che FPR1 è funzionalmente espresso in cellule di neuroblastoma umano SH-SY5Y. Infatti, la stimolazione con N-fMLP di cellule SH-SY5Y deprivate di siero per 24h, induce l’attivazione del complesso della NADPH ossidasi. Questa risulta inibita dalla preincubazione con PTX o con la Ciclosporina H (CSH), un antagonista di FPR1. Le cascate di segnalazione innescate dai GPCR possono promuovere la transattivazione dei recettori tirosina chinasi attraverso diversi meccanismi molecolari (3). Le cellule SH-SY5Y esprimono il recettore tirosina kinasi TrkA che lega, ad alta affinità, il fattore di crescita delle cellule nervose (NGF), una neurotrofina. che influenza la sopravvivenza delle cellule neuronali, la crescita assonale e la plasticità sinaptica. Esperimenti di western blotting dimostrano che il trattamento con N-fMLP di cellule SH-SY5Y induce la transattivazione di TrkA e la fosforilazione di Erk1/2 e Akt in assenza di stimolazione con NGF. Tali fosforilazioni sono prevenute dalla preincubazione con PTX, CSH o con GW441756, un inibitore di TrkA. Inoltre, il pretrattamento con l’ apocinina, un inibitore selettivo della NADPH ossidasi, previene la trans-fosforilazione di TrkA, Erk1/2 e Akt, suggerendo che la generazione di ROS mediata dalla NADPH ossidasi gioca un ruolo chiave nella transattivazione di TrkA indotta da FPR1. Tali risultati possono contribuire all’identificazione di nuovi potenziali bersagli terapeutici in grado di modulare la proliferazione e la migrazione cellulare nelle malattie neurodegenerative. References: 1. Migeotte I., Communi D., Parmentier M., Formyl peptide receptors: a promiscuous subfamily of G protein-coupled receptors controlling immune responses. Cytokine Growth Factor Rev. 2006 Dec;17(6):501-19. 2. Russo R., Cattaneo F., Lippiello P., et al. Motor coordination and synaptic plasticity deficits are associated with increased cerebellar activity of NADPH oxidase, CAMKII, and PKC at preplaque stage in the TgCRND8 mouse model of Alzheimer’s disease. Neurobiol Aging. 2018 Aug;68:123-133. 3. Cattaneo F., Guerra G., Parisi M., et al. Cell-surface receptors transactivation mediated by g protein-coupled receptors. Int J Mol Sci. 2014 Oct 29;15(11):19700-28.

DTN.3

POTENZIALE ONCOGENICO DELLA PROTEINA ZNF224 NEL

MELANOMA ARIANNA PASTORE (arianna.pastore@unina.it), MARIANNA TORIELLO (mar.toriello@studenti.unina.it),

MARCO PALLANTE (marco.pallante@icloud.com), ALESSIA POLVERINO (alessia.polverino@studenti.unina.it), LORENZO MANNA (lo.manna@studenti.unina.it), PAOLA

COSTANZO (paola.costanzo@unina.it; SIB), ELENA CESARO* (elena.cesaro@unina.it) Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli studi di Napoli “Federico II”,

Napoli. *Autore Corrispondente

Il melanoma è uno dei tumori più aggressivi che colpisce gran parte della popolazione Occidentale. Nonostante i numerosi progressi terapeutici degli ultimi anni, l’eterogeneità e la plasticità della patologia rendono il melanoma ancora incurabile. Pertanto, si rende necessario l’approfondimento dei meccanismi molecolari che sono alla base della resistenza alla terapia. Recenti studi hanno mostrato l’importanza del TGF-β nel promuovere la progressione tumorale del melanoma, inducendo cambiamenti fisiologici e morfologici cellulari tipici della Transizione Epitelio-Mesenchimale (EMT)1. ZNF224 è una proteina “KRAB-zing finger”, in grado di funzionare sia da oncosoppressore2, che da promotore tumorale3, regolando l’espressione di geni coinvolti nell’apoptosi e nella proliferazione, in modo dipendente dal contesto cellulare e dai suoi interattori. In questo studio analizziamo il ruolo di ZNF224 nel melanoma. Abbiamo dimostrato che i livelli di espressione di ZNF224 sono indotti a seguito del trattamento con TGF-β. Inoltre, l’oversepressione di ZNF224 in linee cellulari di melanoma causa una prolungata fosforilazione delle proteine Smad, suggerendo un ruolo di ZNF224 come mediatore del pathway del TGF-β. Ancora, l’iper-espressione di ZNF224 si accompagna all’attivazione trascrizionale di geni associati con l’EMT e indotti dal TGF-β, quali Slug, Snail e TGF-β. Inoltre, abbiamo osservato che la modulazione dei livelli di ZNF224 in linee cellulari di melanoma influisce positivamente su aspetti legati alla definizione di un fenotipo tumorale aggressivo quali la proliferazione, la migrazione e l’adesione cellulare. I dati ottenuti indicano un ruolo di ZNF224 come fattore pro-tumorigenico nel melanoma attraverso la regolazione di elementi chiave associati al pathway del TGF-β. L’identificazione di ZNF224 come regolatore della via di segnalazione del TGF-β potrà contribuire alla comprensione dei meccanismi molecolari alla base del pathways del TGF-β necessaria allo sviluppo di terapie innovative nel trattamento del melanoma. Referenze

1. Roesch, A. Tumor heterogeneity and plasticity as elusive drivers for resistance to MAPK pathway inhibition in melanoma. Oncogene. 2015 Jun 4;34(23):2951-7

2. Sodaro G, Blasio G, Fiorentino F, Auberger P, Costanzo P, Cesaro E. ZNF224 is a transcriptional repressor of AXL in chronic myeloid leukemia cells. Biochimie. 2018 Nov;154:127-131.

3. Busiello T, Ciano M, Romano S, Sodaro G, Garofalo O, Bruzzese D, Simeone L, Chiurazzi F, Romano MF, Costanzo P, Cesaro E. Role of ZNF224 in cell growth and chemoresistance of chronic lymphocitic leukemia. Hum Mol Genet. 2017 Jan 15;26(2):344-353.

DTN.4

EFFETTI DEI POLIFENOLI ESTRATTI DA BUCCIA DI LIMONE

SULL’INVASIVITÀ E SULL’ESPRESSIONE DELLE METALLOPROTEASI INDOTTE DA INTERLEUCHINA-6 IN CELLULE DI CARCINOMA

GASTRICO ROSARITA NASSO1 (rosarita.nasso@uniparthenope.it; SIB); VALENTINA PAGLIARA1

(valentina.pagliara83@gmail.com; SIB); ILARIA FINORE2 (ilaria.finore@icb.cnr.it); ANNARITA POLI2

(annarita.poli@icb.cnr.it); PAOLA DI DONATO3 (paola.didonato@uniparthenope.it); MARIOROSARIO MASULLO1 (mario.masullo@uniparthenope.it; SIB); ROSARIA ARCONE1*

(rosaria.arcone@uniparthenope.it; SIB)

1 Dipartimento di Scienze Motorie e del Benessere, Università degli studi di Napoli “Parthenope”, Napoli, Italia

2 Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Napoli, Italia 3 Dipartimento di Scienze e tecnologie, Università degli studi di Napoli “Parthenope”, Napoli, Italia

*Autore corrispondente I polifenoli sono composti organici di origine vegetale molto abbondanti nella dieta mediterranea. Negli ultimi anni, i polifenoli hanno attratto un grande interesse scientifico, non solo come agenti anti-ossidanti, ma anche per le loro proprietà protettive nei processi infiammatori correlati all’insorgenza di patologie come quelle cardiovascolari, metaboliche, neurodegenerative e nel cancro [1]. In questo studio, abbiamo indagato gli effetti dei polifenoli estratti dalla buccia di limone (LPE) sull’invasività cellulare e sull’espressione delle metalloproteasi (MMP)-9/2, in un contesto infiammatorio, utilizzando le linee umane di adenocarcinoma gastrico, MKN28 e AGS. Abbiamo valutato l’effetto indotto dal pretrattamento con LPE sull’espressione delle MMP-9 e MMP-2 e sulla invasività cellulare nelle cellule MKN-28 ed AGS esposte ad Interleuchina-6 (IL-6) [2]. Saggi di migrazione ed invasione cellulare hanno dimostrato che il pre-trattamento con LPE (0.5 o 1 µg/ml di equivalente di acido gallico) riduce l’invasività cellulare indotta da IL-6. Inoltre, LPE inibisce i livelli d’espressione (mRNA e proteina) delle MMP-9 e MMP-2 indotti da IL-6. I nostri risultati indicano che il trattamento con LPE è in grado anche di ridurre l’attività gelatinasica delle MMP-9 e MMP-2, che sono enzimi chiave coinvolti nella degradazione della matrice extracellulare nel processo di formazione di metastasi. Infine, è stato analizzato anche l’effetto di LPE sullo stato di attivazione di STAT3 in seguito al trattamento da IL-6. Questi risultati suggeriscono che LPE riduce l’invasività delle MKN28 e AGS attraverso la diminuzione dei livelli di espressione di MMP-9 e MMP-2 dopo trattamento con IL-6. In conclusione, questi dati indicano un possibile meccanismo molecolare che media il ruolo protettivo esercitato da LPE contro i processi metastatici del cancro gastrico. Bibliografia

1. [1] Di Donato P., Taurisano V., Tommonaro G., Pasquale V., Silvàn Jimènez J.M., de Pascual-Teresa S., Poli A., Nicolaus B. Biological properties of polyphenols extract from Agro Industry’s wastes Waste Biomass Valor (2017) doi: 10.1007/s12649-017-9939-4

2. [2] Arcone R., Palma M., Pagliara V., Graziani G., Masullo M., Nardone G. Green tea polyphenols affect invasiveness of human gastric MKN28 cells by inhibition of LPS or TNF-α induced Matrix Metalloproteinase-9/2 Biochimie Open 3 (2016) doi: 10.1016/j.biopen.2016.10.002

DTN.5

THE MOLECULAR CHAPERONE TRAP1 DRIVES INFLAMMATION-

INDUCED PLATINUM RESISTANCE VIA OXIDATIVE STRESS IN HUMAN OVARIAN CANCER

DANIELA CRISCUOLO (daniela.criscuolo@unina.it), DANILO SWANN MATASSA (daniloswann.matassa@unina.it; SIB), FRANCA ESPOSITO* (franca.esposito@unina.it; SIB)

Università di Napoli “Federico II”, Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Italy *corresponding author Ovarian cancer, the most lethal gynecological tumor, is an initially chemosensitive neoplasm with frequent relapse and development of chemoresistance (1). Contrarily to conventional wisdom, aggressive subtypes of ovarian cancer cells have been proven to be particularly dependent on oxidative phosphorylation rather than glycolysis. In the context of metabolic remodelling, we investigated the role of TRAP1, a molecular chaperone involved in the regulation of respiratory complex activity and ROS production in cancer cells. We previously demonstrated that TRAP1 silencing in ovarian cancer cells increases oxidative metabolism and induces the expression of proinflammatory cytokines and members of the ABC transporter family, which mediate drug resistance, suggesting that inflammation may mediate cisplatin resistance induced by increased respiration (2). In order to identify the molecular mechanisms linking the metabolic remodeling induced by TRAP1 downregulation with inflammation and chemoresistance, we performed a differential gene expression analysis in chemosensitive ovarian cancer cells PEA1 upon siRNA-mediated TRAP1 silencing. Results showed a significant deregulation of inflammatory and oxidative stress pathways. In parallel, to identify genes whose expression correlates with that of TRAP1 in patients, we performed a coexpression analysis in a large database of high grade serous ovarian cancer samples. The overlap between the resulting list and the gene expression analysis allowed us to identify a small subset of genes involved in metabolism (GLS), inflammation (CSF2, IL6) and chemoresistance (CYP1B1). To confirm the hypothesis that inflammatory response is triggered by oxidative stress induced by increased respiration, we treated the PEA1 cells with H2O2, finding significant increase of proinflammatory factors IL8 and CSF2. Accordingly, longer time treatments resulted in upregulation of the drug resistance gene CYP1B1. These results highlight TRAP1 as a potential prognostic and predictive biomarker for ovarian cancer patient selection and the opportunity to develop metabolism-directed therapeutic strategies to improve the effectiveness of platinum-based regimens. References

1. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin 2014; 64: 364. 2. Matassa DS, Amoroso MR, Lu H, Avolio R, Arzeni D, Procaccini C, Faicchia D, Maddalena F,

Simeon V, Agliarulo I, Zanini E, Mazzoccoli C, Recchi C, Stronach E, Marone G, Gabra H, Matarese G, Landriscina M, Esposito F. Oxidative metabolism drives inflammation-induced platinum resistance in human ovarian cancer. Cell Death Differ. 2016;23(9):1542-54.

DTN.6

QUERCETIN IS ABLE TO REVERT THE APOPTOTIC RESISTANCE TRIGGERED BY THE SHORT ISOFORM OF GATA-1 IN MYELOID

LEUKEMIA CELLS S. TROMBETTI1 (silvia.trombetti@unina.it), R. SESSA1 (raffaele.sessa@unina.it), R. CATAPANO1

(rosacatapano92@gmail.com), M. MIRANDA1 (mirandamariag@outlook.com), A. LO BIANCO1

(al.lobianco@studenti.unina.it), M. CABALLERO CAVALLÈ1,3 (marta.caballero@estudiants.urv.cat), P. IZZO1,2 (paola.izzo@unina.it; SIB), M. GROSSO1,2 *(michela.grosso@unina.it; SIB)

1Dept. Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy;

2Ceinge- Biotecnologie Avanzate, Naples, Italy; 3Dept. Chemistry and Organic Chemistry, University Rovira I Virgili, Tarragona, Spain

*Corresponding Author Aberrant expression of GATA-1, a master regulator of several hematopoietic genes has been reported in myeloid leukemias. Maintenance of a balanced expression of the two isoforms of this transcription factor, the full-length protein (GATA-1FL) and a shorter isoform (GATA-1S), contributes to control hematopoiesis, whereas their dysregulation can alter the differentiation/proliferation potential of hematopoietic precursors (WY Lee et al. 2018; JD Crispino et al. 2017). Given the role played by ROS in myeloid leukemogenesis, and on the basis of our previous observations, we investigated the role of GATA1 isoforms in oxidative stress and resistance to pro-apoptotic stimuli in the erithroleukemia K562 cell line. Cytoplasmatic ROS resulted to be significantly increased in cells over-expressing GATA-1FL (GATA-1FL cells) compared to cells over-expressing the shorter isoform (GATA-1S cells). Accordingly, total GSH levels as well as the GSH/GSSG ratio were found reduced in GATA-1FL cells and conversely increased in GATA-1S cells.We also found a significant decrease in the MnSOD protein levels in GATA1S cells compared to GATA-1FL cells. As a whole, these findings reinforce the evidence of an enhanced antioxidant capacity in GATA1S cells and a pro-oxidant redox status in GATA-1FL cells. Furthermore, in contrast with the apoptotic resistance shown by GATA-1S cells, GATA-1FL cells were more sensitive to cis-platin exposure. Changes in proliferation and apoptosis related to cell redox states were also demonstrated by analyzing the antioxidant effects of quercetin on GATA-1 cells. After 24 h quercetin treatment, GATA-1S cells showed an enhanced apoptotic rate with respect to the GATA-1FL counterpart. Finally, co-tratment with quercetin and cis-platin demonstrated an enhanced apoptosis susceptibility in both cell types and, more intriguingly quercetin’s ability to revert the apoptosis resistance shown by GATA-1S cells when treated with cis-platin alone. In conclusion our study suggests a mechanism to bypass the apoptotic resistance triggered by GATA-1s expression and to design more effective ROS-based therapies in myeloid leukemias.

DTN.7

AN INTRONIC MUTATION C. 589-9_589-6DELGTTT IN THE MLH1 GENE

CAUSES THE LOSS OF MLH1 PROTEIN EXPRESSION IN COLON CARCINOMA FROM A YOUNG PATIENT WITH LYNCH SYNDROME

VALENTINA D’AMORE (vale.damore@studenti.unina.it); RAFFAELLA LICCARDO (raffaella.liccardo@unina.it); MATILDE LAMBIASE (matilde.lambiase@libero.it); MARINA DE ROSA (marina.derosa@unina.it; SIB); PAOLA IZZO (paola.izzo@unina.it SIB); FRANCESCA DURATURO*

(francesca.duraturo@unina.it; SIB)

Dipartimento di Medicina Molecolare e Biotecnologie Mediche Università degli Studi di Napoli Federico II, Via Pansini, 5 80131 Napoli.

*Corresponding Author

Lynch syndrome (LS) is an autosomal dominant disorder characterized by an increased risk of colorectal and extracolonic cancers showing early age of onset. It is associated with germline mutations in one of the mismatch repair (MMR) genes, including MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MSH6, PMS1 homolog 2, mismatch repair system component (PMS2), MLH3 and MSH3. The mutations identified in MMR genes are point mutations or large rearrangements. The point mutations are certainly pathogenetic whether they determine formation of truncated protein. This study aimed to investigate the effect of an intronic mutation, c. 589-9_589-6delGTTT, identified in a young boy with colon adecarcinoma, on mRNA splicing of the MLH1 gene. This mutation is reported as uncertain pathogenetic significance variant on international database of MMR variants (www.insight-database.org). Functional analysis was performed to investigate the effect of this intronic mutation at the transcriptional level. The Human Splicing Finder was used to predict its effect. The in-silico tool predicted that the mutation induces altered mRNA splicing by using a cryptic acceptor site. cDNA sequencing confirmed that this mutation causes an alteration of the canonical splicing site and at level of mRNA determines the skipping of the entire exon 8 (c.589_c.677). The skipping of exon 8 determines the shift of the mRNA reading frame with consequent formation of a premature stop codon that could determine a truncated protein, likely not functional (p.Gln197Argfs*8). We confirmed these data on colon tumoral tissue paraffin-embedded of our patient. Indeed, this mutation is associated with the lack of expression of the MLH1 protein (revealed by immunohistochemistry) and a high status of Microsatellite Instability (MSI) in colon tumour tissue. Therefore, our findings suggest that this intronic variant plays a pathogenic role and it is responsible of early onset of colon cancer and, thus of Lynch syndrome phenotype. References

1. Duraturo F, Liccardo R, Cavallo A, De Rosa M, Rossi GB, Izzo P. Multivariate analysis as a method for evaluating the pathogenicity of novel genetic MLH1 variants in patients with colorectal cancer and microsatellite instability. Int J Mol Med. 2015 Aug;36(2):511-7. Epub 2015 Jun 19.

2. Duraturo F, Liccardo R, De Rosa M, Izzo P. Genetics, diagnosis and treatment of Lynch syndrome: Old lessons and current challenges. Oncol Lett. 2019 Mar;17(3):3048-3054. doi: 10.3892/ol.2019.9945. Epub 2019 Jan 18.

3. Liccardo R, De Rosa M, Izzo P, Duraturo F. Novel Implications in Molecular Diagnosis of Lynch Syndrome. Gastroenterol Res Pract. 2017;2017:2595098. doi: 10.1155/2017/2595098. Epub 2017 Jan 29.

DTN.8

GPR55 TARGETING WITH PEPTIDES: A NOVEL THERAPEUTIC STRATEGY FOR THE TREATMENT OF OSTEOCLAST-RELATED

PATHOLOGIES JOLE FONDERICO §1 (j.fonderico@ibp.cnr.it; SIB), MARIA MANGINI#1 (m.mangini@ibp.cnr.it), ROSSELLA

D'ANGELO# (rosa.dangel88@gmail.com), STEFANIA FULLE§ (sfulle@unich.it), MARIA GIOVANNA MOSCA# (mariagiovanna.mosca@libero.it), STEFANIA MARIGGIO#* (s.mariggio@ibp.cnr.it)

#National Research Council, Department of Biomedical Sciences, Institute of Protein Biochemistry, Naples, Italy

§Department of Neurosciences, Imaging and Clinical Sciences –‘G. d’Annunzio’ University of Chieti-Pescara, Italy

1These authors equally contributed *Corresponding Author

G-protein-coupled receptors (GPCRs) are associated with several physiopathological conditions and, in recent years, have emerged as crucial players in tumour growth and metastatisation. GPR55, recently identified as the lysophosphatidylinositol receptor, is involved in different physiological and pathological processes, including bone regulation, endothelial function, inflammation and pain. Together with its ligand, GPR55 was reported to promote tumour cell proliferation, invasion and metastatic spread. Indeed, GPR55 is overexpressed in different kinds of tumours and in vitro studies have also shown high levels of lysophosphatidylinositol in transformed and tumour cells. This study is aimed to investigate GPR55/lysophosphatidylinositol function in the development of osteoclast precursors toward bone-resorbing osteoclast syncytia and to define the physiological role of GPR55 in bone resorption as well as in bone metastasis formation. We have identified an active role of the GPR55/lysophosphatidylinositol axis in osteoclast maturation and fusion. Indeed, RANKL-induced differentiation of RAW264.7 cells into mature osteoclasts resulted in a strong up-regulation of GPR55 mRNA levels. Moreover, GPR55 pharmacological inhibition, with different antagonists, or protein downregulation, with RNA interference, affected the completion of the osteoclastogenesis process by inhibiting the osteoclast marker appearance, while the lysophosphatidylinositol stimulated the process. By taking advantage of a phage-displayed peptide library, we have identified peptidic binders of GPR55, able to specifically recognise the receptor and to regulate its downstream signalling and internalisation rate. Also the effect of these peptides in the osteoclastogenesis process was evaluated. Since bone-metastasis development can be blocked through inhibition of bone resorption, osteoclasts are reasonable targets for anti-metastatic treatments and GPR55 peptides could represent useful tools for novel therapeutic approaches of bone diseases related to GPR55 dysregulation.

DTN.9

DUAL ROLE OF FKBP5 GENE ISOFORMS IN THE ACTIVATION OF AKT ONCOGENIC PATHWAY AND IN THE PROLIFERATION OF MELANOMA

CANCER CELLS MARTINA TUFANO1 (martina.tufano@unina.it), SIMONA ROMANO1 (simona.romano@unina.it), PAOLO D’ARRIGO1 (paolo.darrigo@unina.it), VINCENZA VIGORITO1 (v.vicky@hotmail.it), SALVATORE RUSSO1

(salvatore.russo36@studenti.unina.it) and MARIA FIAMMETTA ROMANO1* (mariafiammetta.romano@unina.it)

1Università degli Studi di Napoli “Federico II”, Dipartimento di Medicina Molecolare e Biotecnologie Mediche,

Napoli, Italia *Corresponding Author

Akt upregulation is a major mechanism sustaining cancer survival and progression. FKBP51 is an immunophilin, whose relevant role in sustaining cancer cell growth and aggressiveness is widely documented3. A previous paper has proposed a role for FKBP51 as a tumor suppressor because, by interacting with Akt and PHLPP, it functions as a scaffold in Akt dephosphorylation1. This result is not in agreement with results from our group and others2 that found increased pAkt levels following FKBP51 overexpression. Recently, we identified for the first time that FKBP5 gene generates a spliced isoform lacking TPR domain (FKBP51s), involved in protein protein interaction. Aim of this work was to address whether a unique gene could, at the same time, regulate opposite mechanisms, as Akt phosphorylation/dephosphorylation. Immunoblot results were in line with our hypothesis that pAkt levels resulted increased and decreased by FKBP51- and FKBP51s-overexpression, respectively. Using TPR-mutants, we clarified that the TPR domain mediates Akt phosphorylation. In line with increased p-Akt levels, FKBP51-overexpression resulted in high levels of Akt targets p-70S6K and cyclin D1, whereas FKBP51s-overexpression clearly counteracted this increase. Accordingly, cell cycle analysis showed FKBP51s-overexpression produced a significant increase of cells in G0/G1 to the detriment of S- and G2/M-phase (50% vs 70% G0/G1; 24% vs 18% S; 21% vs 10% G2/M). We also demonstrated that the TPR domain is essential in promoting the Akt-K63-ubiquitination. Such process required also Hsp90, a known interactor of FKBP51. FKBP51-KO melanoma cells, obtained by Crispr/cas9 technology, showed impaired Akt K63-Ub, in line with results obtained in melanoma cells transiently transfected. Collectively, these results suggest that FKBP51 promotes Akt activation by serving as a scaffold to build the macrocomplex deputed to Akt ubiquitination and phosphorylation. By converse, the spliced FKBP51s was unable to support ubiquitination and reasonably functions as a decoy that subtracts Akt to phosphorylation machinery. References

1. Pei H, Li L, Fridley BL, Jenkins GD, Kalari KR, Lingle W, Petersen G, Lou Z, Wang L. FKBP51 affects cancer cell response to chemotherapy by negatively regulating Akt. Cancer Cell. 2009 Sep 8;16(3):259-66. doi: 10.1016/j.ccr.2009.07.016.

2. Fabian AK, März A, Neimanis S, Biondi RM, Kozany C, Hausch F. InterAktions with FKBPs--mutational and pharmacological exploration. PLoS One. 2013;8(2):e57508.

2. Romano S, Sorrentino A, Di Pace AL, Nappo G, Mercogliano C, Romano MF. The emerging role of large immunophilin FK506 binding protein 51 in cancer. Curr Med Chem. 2011;18(35):5424-9.

DTN.10

VALUATION OF THE MUTATIONAL STATUS OF GENES INVOLVED IN CARCINOGENESIS AND PREDICTION OF RESPONSE TO DRUGS OF

HUMAN NEOPLASIA BY NEXT GENERATION SEQUENCING

Carcinogenesis is a multiphase process that drives the progressive transformation of a normal cell into a tumor cell: decades of research proved that cancer results from accumulation of several genomic aberrations that control tumor initiation and progression. The difference for researchers was the "possibility to query the genes": molecular photography of a tumor allows to learn which mechanisms are altered and to draw specific therapies capable to interact only with cells which have these molecular targets, to decrease side effects on healthy cells. This is the revolution: from a "one size fits all medicine" to a personalized and specific vision where therapy is established on the molecular profile of the single tumor in the single patient. The main purposes are to maximize the care potential, to minimize the toxicity and to identify the patients who will be able to benefit from the therapy. Our molecular diagnostic service “Cytometric and Mutational Diagnostics” of University of Campania “Luigi Vanvitelli” provides the possibility to perform molecular characterization of Non Small Cell Lung Cancer (NSCLC), Metastatic Colorectal Cancer (mCRC) and Melanoma patients. For this molecular characterization we use three different sequencing methods: Ion Torrent Personal Genome Machine (PGM), Therascreen Rotor Gene Q and Pyromark. Simultaneously with the production of the medical reports, we created a database which is updated weekly with all the mutations found in patients. The intent is to correlate the molecular data with the follow-up and clinical data of patients in order to identify new molecular targets useful for the identification of new targeted therapies.

RUSSO MARGHERITA1(margherita.russo@unicampania.it), GRIMALDI ANNA1

(anna.grimaldi@unicampania.it), FERRI CARMELA1 (carmela.ferri@icloud.com ),FESTA AGOSTINO1

(agostino.festa@unicampania.it), SABETTA ROSALAURA2 (rosalaurasabetta@libero.it), LUCE AMALIA1(amalia.luce@unicampania.it), COSSU ALESSIA MARIA1( alessiamaria.cossu@biogem.it),

PAPACCIO GIANPAOLO3 (gianpaolo.papaccio@unicampania.it),FRANCO RRANCO2

(renato.franco@unicampania.it), CARAGLIA MICHELE1, (michele.caraglia@unicampania.it), LOMBARDI ANGELA (angelalombardi@hotmail.it)1*

1Department of Precision Medicine, University of Campania “Luigi Vanvitelli”.

2Pathological Anatomy Unit, Department of Psychic and Physic health and preventive medicine, University of Campania “Luigi Vanvitelli”

3Department of Experimental Medicine, University of Campania “Luigi Vanvitelli” *Corresponding Author

IVP.1

THE INTERPLAY BETWEEN TYPE 2 TRANSGLUTAMINASE AND GLIADIN PEPTIDES IN CELIAC DISEASE

GAETANA PAOLELLA (gpaolella@unisa.it; SIB), SILVIA SPOSITO (ssposito@unisa.it), MARILENA LEPRETTI (mlepretti@unisa.it), CARLA ESPOSITO (cesposito@unisa.it),

STEFANIA MARTUCCIELLO (smartucciello@unisa.it), IVANA CAPUTO* (icaputo@unisa.it; SIB)

UNIVERSITY OF SALERNO, ITALY

*Corresponding author Celiac disease (CD) is an intestinal inflammatory disorder triggered by the ingestion of cereals containing gliadins. Type 2 transglutaminase (TG2) has been strongly implied in CD pathogenesis. In intestinal Caco-2 cells, TG2 expression and activity were increased by exposure to a subset of toxic gliadin peptides, of which peptide 31-43 (p31-43) is the prototype. On the other hand, antibodies against TG2 were able to reduce p31-43 uptake by Caco-2 cells. Our aim has been to investigate the interplay between p31-43 and TG2 in a model of primary skin-derived CD fibroblasts. We observed that anti-TG2 antibodies were unable to reduce p31-43 uptake by CD fibroblasts while they protected normal fibroblast from p31-43 entrance. Moreover, p31-43 induced an increase of TG2 expression only in CD fibroblasts, whereas TG2 activity was increased more in normal cells than in CD fibroblasts. The different way that CD and normal cells handle p31-43 in the presence on anti-TG2 antibodies and the different effect that the toxic peptide exerts on TG2 activation into the two groups of cells reinforce the idea that the interplay between p31-43 and TG2 could have an important role in CD pathogenesis.

IVP.2

ATTIVITÀ ANTIOSSIDANTE ED ANTINFIAMMATORIA DEI METABOLITI BIOATTIVI DEL LATTE DI BUFALA

Nunzia D’Onofrio*1 (nunzia.donofrio@unicampania.it; SIB); Angelo Capasso1 (angelocapasso27@libero.it); Rosario Casale1 (rosario.casale@unicampania.it); Giuseppe Campanile2 (giuseppe.campanile@unina.it);

Maria Luisa Balestrieri1 (marialuisa.balestrieri@unicampania.it; SIB)

1Dipartmento di Medicina di Precisione, Università degli Studi della Campania “L. Vanvitelli”, 80138 Napoli, Italia.

2Dipartimento di Medicina Veterinaria e Produzioni Animali, Università degli Studi di Napoli Federico II, 80137 Napoli, Italia

*Corresponding author Il latte di bufala (Bubalus bubalis), il secondo latte più consumato nel mondo, è una buona fonte di sostanze ad alto valore nutritivo, necessarie per preservare e migliorare lo stato di salute e di benessere, e ridurre il rischio di diverse patologie. Le proprietà funzionali dei metaboliti bioattivi del latte di bufala sono state recentemente ampliate. Infatti, i risultati ottenuti hanno mostrato un peculiare profilo di betaine e acilcarnitine a catena corta nel latte di bufala, con particolare riferimento alla δ-valerobetaina (δVB), γ-butirrobetaina (γBB), acetilcarnitina e propionilcarnitina. Il latte di bufala mostra attività antiossidanti ed antinfiammatorie in grado di contrastare il danno endoteliale indotto dalle alte concentrazioni di glucosio. Questi effetti risultano potenziati dall'arricchimento con la δVB, con conseguente diminuzione delle specie di ossigeno reattivo, della perossidazione lipidica e del rilascio di citochine (P <0,05). Di interesse, la δVB si oppone all’attivazione del processo infiammatorio indotto dalle alte concentrazioni di glucosio, modulando i livelli di espressione di SIRT1, SIRT6 e NF-κB (P <0,05). In conclusione, questo studio evidenzia le proprietà del latte di bufala nel ridurre, a livello endoteliale, lo stress ossidativo e l'infiammazione, suggerendo la δVB quale nuovo composto di origine alimentare con proprietà funzionali. Bibliografia D'Onofrio N, Balestrieri A, Neglia G, Monaco A, Tatullo M, Casale R, Limone A, Balestrieri ML, Campanile G. Antioxidant and Anti-Inflammatory Activities of Buffalo Milk δ Valerobetaine. J Agric Food Chem. 2019. 13;67(6):1702-1710.

IVP.3

UCHL3 INTERACTS WITH KEAP1 TO FINE-TUNE THE NRF2-DEPENDENT STRESS RESPONSE

Antonia Marcone (antonia.marcone@hotmail.it), Rossella Izzo (rossellaizzo95@gmail.com), Fabio Martinelli (fabiomartinelli@alice.it); SIB), Giuseppina Minopoli (giuseppina.minopoli@unina.it),

Raffaella Faraonio*(raffaella.faraonio@unina.it; SIB)

Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, Napoli, Italy *Corresponding author

Introduction. Activation of the NRF2 [(erythroid-derived 2)-like 2] antioxidant program during oxidative/electrophilic stresses is an essential pathway to fight deleterious consequences of reactive oxygen species (ROS) accumulation. Controlled NRF2 activity is necessary to preserve physiological functions of organisms: alterations of the NRF2 cytoprotective pathway, indeed, has been linked to natural aging as well as to the pathogenesis of numerous degenerative and immunological disorders, including cancer (1). NRF2 activity is mainly regulated by KEAP1, a cysteine-rich adaptor for Cullin3-based E3 ubiquitin ligase that in basal conditions targets NRF2 for ubiquitination and subsequent degradation. However, the mechanisms by which KEAP1 can be regulated need further investigation. Deubiquitinating enzymes (DUBs) catalyze the removal of ubiquitin, thus reversing the action of the E3 ligases. In particular, the Ubiquitin C-Terminal Hydrolase L3 (UCHL3) has emerged as a new player in cellular surveillance to DNA damage, and its loss has been associated to induction of stress response markers (2). These observations led us to test the involvement of UCHL3 in the NRF2/KEAP1 signaling. Results. The expression level (mRNA and protein) of UCHL3 was analyzed in different cellular systems with gain NRF2 function (cells resistant to oxidative stress). The results obtained highlight increased levels of UCHL3 protein and demonstrated a positive correlation between UCHL3 and NRF2 across a panel of human cancer cell lines. Furthermore, TCID (4,5,6,7-tetrachloroindan-1,3-dione), a selective UCHL3 inhibitor, counteracted the induction of NRF2-dependent genes, due to a concurrent decrease of NRF2 protein. In agreement with the above results, UCHL3 manipulations affected NRF2-dependent gene expression. Additional studies showed that UCHL3 directly interacts with KEAP1 under basal and stress-conditions, with the two proteins forming a tight complex. Preliminary experiments indicate a possible role of UCHL3 on KEAP1 stability and/or degradation. Conclusion. Our results suggest a new deubiquitylation-independent function of UCHL3 on the NRF2-KEAP1 signaling. References

1. Cuadrado A. et al. Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases Nat Rev Drug Discov. 2019 Apr;18(4):295-317.

2. Liao C. et al. UCHL3 Regulates Topoisomerase-Induced Chromosomal Break Repair by Controlling TDP1 Proteostasis. Cell Rep. 2018 Jun 12;23(11):3352-3365.

IVP.4

TARGETING HEPARAN SULFATE PROTEOGLYCANS AS A NOVEL STRATEGY TO REVERT CELL SIGNALING ALTERATIONS IN

MUCOPOLYSACCHARIDOSES VALERIA DE PASQUALE1 (valeria.depasquale@unina.it; SIB), VALERIA PISTORIO1

(valeria.pistorio@unina.it), MELANIA SCARCELLA (melania_scarcella@libero.it), LUIGI MICHELE PAVONE1* (luigimichele.pavone@unina.it; SIB)

1Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, Italia

*Corresponding author Mucopolysaccharidoses (MPSs) are inherited metabolic diseases caused by the deficiency of lysosomal enzymes needed to catabolize glycosaminoglycans (GAGs). Four alternative therapies are currently considered: enzyme replacement therapy, substrate reduction therapy, gene therapy, and hematopoietic stem cells transplantation. However, while some of them exhibit limited clinical efficacy and require high costs, others are still under development. Therefore, innovative alternative treatments for MPSs need to be explored. Here, we describe a novel approach based on the use of a recombinant protein that is able to bind the excess of extracellular accumulated heparan sulfate (HS). We demonstrate that this protein is able to reduce GAGs content and lysosomal defects in primary fibroblasts from MPS patients and to reduce some clinical symptoms of the disease in the MPS IIIB mouse model. We also show that by masking the excess of extracellular accumulated HS in MPS fibroblasts, FGF signaling transduction can be positively modulated. We, therefore, suggest the use of a binding molecule for HS in MPSs as an alternative strategy to prevent the detrimental extracellular substrate storage.

IVP.5

POSSIBLE ATTENUATOR EFFECT OF INTRONIC STK11 VARIANT IN A PEUTZ-JEGHERS FAMILY WITH GERMLINE PATHOGENIC SPLICING

MUTATION SHOWING HIGH PHENOTYPIC VARIABILITY FRANCESCA CAMMAROTA1(francesca.cammarota88@gmail.com), ANDREA CERASUOLO2

(andrea.cerasuolo91@gmail.com); ANTONIETTA AVERSANO1 (antonietta.aversano10@gmail.com); FRANCESCA DURATURO1 (francesca.duraturo@unina.it; SIB); RAFFAELLA LICCARDO1

(raffaella.liccardo@unina.it); ERASMO MIELE3 (erasmo.miele@unina.it); ANNAMARIA STAIANO3 (staiano@unina.it); PAOLA IZZO1 (paola.izzo@unina.it, SIB); MARINA DE ROSA1*

(marina.derosa@unina.it, SIB).

1 Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Italy; 2 Molecular Biology and Viral Oncology Unit, Istituto Nazionale per lo studio e la cura dei tumori,

“Fondazione Giovanni Pascale” IRCCS-Naples, Italy; 3 Department of Translational Medical Science, Section of Pediatrics, University of Naples

"Federico II," Naples, Italy *Corresponding author

Peutz-Jeghers syndrome (PJS) is a rare autosomal-dominant inherited precancerous condition characterized by multiple polyps in the gastrointestinal tract and distinctive mucocutaneous pigmentation. Germline mutations in the serine/threonine kinase 11 gene (STK11/LKB1) (OMIM*602216) are documented in up to 70%–80% of the PJS patients (1-3). We have analyzed the disease-causing mutation in 2 italian young sisters in which PJS was suspected. They showed classical cafe au lait spots associated with the disease, in absence of any other symtoms or familiarity. Sequencing all 9 exons of STK11 gene led to the identification, in both probands, of a novel, unique germline mutation, named c.597G→A; this is a splicing mutation hitting the last nucleotide of exon 4. Interestingly, their unaffected father was carrier of this mutation. He also showed a second intronic substitution, the c.465-51T→C (rs2075606), which was not inherited by his affected daughters. An alterated splicing messenger, consisting in exon 4 skipping, was identified in all subjects carrier of the c.597G→A mutation. In silico analysis, performed by using ESEFinder software, showed that the c.465-51T→C intronic mutation probably activates a silent Enhancer Splicing Element. Finally, we determined the quantitative expression of the STK11 wilde type messenger by using Real-Time qRT-PCR, observing that the expression of the wild-type STK11 mRNA in both probands appeared about the half when compared to that observed in their healthy mother, while the unaffected father, also carrier of the pathogenic mutation, showed only a small down-expression. In light of these findings, we hypotesize that the c.465-51T→C intronic variant, which segregates with the wild type allele and not inherited together with the pathogenetic mutation, could compensate for the deleterious effect of the splicing mutation, attenuating the disease aggressiveness and giving rise to the phenotypic variability observed in this interesting family. References

1. De Rosa M, Galatola M, Quaglietta L, Miele E, De Palma G, Rossi GB, Staiano A, Izzo P. Alu-mediated genomic deletion of the serine/threonine protein kinase 11 (STK11) gene in Peutz-Jeghers syndrome. Gastroenterology. 2010 Jun;138(7):2558-60

2. De Rosa M, Pace U, Rega D, Costabile V, Duraturo F, Izzo P, Delrio P. Genetics, diagnosis and management of colorectal cancer (Review). Oncol Rep. 2015,34(3):1087-96.

3. Lucci-Cordisco E, Risio M, Venesio T, Genuardi M. The growing complexity of the intestinal polyposis syndromes. Am J Med Genet A. 2013,161A(11):2777-87.

IVP.6

THERAPEUTIC POTENTIAL OF 5-THIOHISTIDINES IN HUMAN INFLAMMATORY DISEASES

MARIARITA BRANCACCIO1 (mariarita.brancaccio@szn.it; SIB), ALFONSINA MILITO1

(alfonsina.milito@szn.it; SIB), CARLA VIEGAS2,3 (caviegas@ualg.pt), DINA SIMES2,3 (dsimes@ualg.pt), IMMACOLATA CASTELLANO1* (immacolata.castellano@szn.it; SIB)

1Stazione Zoologica Anton Dohrn, Department of Biology and Evolution of Marine Organisms, Naples, Italy

2University of Algarve, Centre of Marine Sciences (CCMAR), Faro, Portugal. 3University of Algarve, GenoGla Diagnostics, Centre of Marine Sciences (CCMAR), Faro, Portugal.

*Corresponding author Introduction 5-thiohistidines are histidine-derived thiols first isolated from marine invertebrates eggs where they play a key role in the protection of cells towards the oxidative burst associated with fertilization. Ovothiols are methyl-5-thiohistidines from marine invertebrates, bacteria, and microalgae, which protect cells from environmental stressors. Recently, we have demonstrated that ovothiol, purified from sea urchin P. lividus eggs, exhibits anti-inflammatory activity, in an in vitro model of endothelial dysfunction [1], and in an in vivo model of liver fibrosis [2]. Moreover, we have previously shown that the same molecule induces autophagy in a human liver carcinoma cell line, HepG2 [3]. Thanks to their chemical properties, they represent promising bioactive molecules to use as anti-inflammatory compounds in human diseases. Material and Methods human keratinocytes (HaCat) and human embryonic kidney 293 cells (HEK293) were used to test the cytotoxicity of these molecules by resazurin-based assay. An ex vivo human skin model was used to investigate the anti-inflammatory properties of these molecules. Results and Discussion In this preliminary study we showed that two types of 5-thiohistidines are not cytotoxic in epithelial cells, indeed the vitality is even greater than in untreated cells. In addition, using specific ELISA assays, we observed that the pre-treatment of ex vivo human skin tissue with ovothiol A and the desmethylated form 5-thiohistidine at the concentration of 5 μM before the induction of inflammation with IL-β1 10 ng/ml, led to a significant decrease in IL-6 and IL- 8 production, indicating a stronger anti-inflammatory effect compared to the pre-treatment of dexamethasone, a type of corticosteroid medication, used as an anti-inflammatory in the treatment of many skin diseases or during the Cushing's syndrome. Conclusions These findings indicate that marine 5-thiohistidines have significant anti-inflammatory properties and can be considered as new marine drugs or dietary supplements for the treatment of human inflammatory diseases. References

1. Castellano I, Di Tomo P, Di Pietro N, Mandatori D, Pipino C, Formoso G, Napolitano A, Palumbo A, Pandolfi A. “Anti-Inflammatory Activity of Marine Ovothiol A in an In Vitro Model of Endothelial Dysfunction Induced by Hyperglycemia “. Oxid Med Cell Longev. 2018.

2. Brancaccio M, D’Argenio G, Lembo V, Palumbo and Castellano I. “Antifibrotic Effect of Marine Ovothiol in an In Vivo Model of Liver Fibrosis”. Oxid Med Cell Longev. 2018.

3. Russo G, Russo M, Castellano I, Napolitano A, and Palumbo A. “Ovothiol isolated from sea urchin oocytes induces autophagy in the Hep-G2 cell line.” Marine Drugs. 2014.

PROMET.1

METABOLOMICS: AN EMERGING POWERFUL TOOL FOR DISSECTING BIOLOGICAL ABERRATIONS IN BARDET BIEDL RENAL DYSFUNCTION

EMANUELA MARCHESE1,2 (marchese@ceinge.unina.it, SIB), MIRIAM ZACCHIA1

(Miriam.ZACCHIA@unicampania.it), MARIANNA CATERINO2,3 (marianna.caterino@unina.it, SIB), GIOVAMBATTISTA CAPASSO1,4 (gb.capasso@unicampania.it), MARGHERITA RUOPPOLO* 2,3

(margherita.ruoppolo@unina.it, SIB)

1. Università degli Studi della Campania ‘L. Vanvitelli’, Napoli 2. CEINGE Biotecnologie Avanzate, Napoli

3. Università degli Studi di Napoli, “Federico II”, Naples 4. IRGS Biogem, Via Camporeale, Ariano Irpino, Avellino, Italy

*Corresponding author Bardet Biedl Syndrome (BBS) is a rare genetic disorder which is included in the group of disease named Ciliopathies. Interestingly, BBS and related disorders patients show a wide range of clinical phenotypes, even in the presence of the same primary genetic defect. So, large observational studies are required to get insight into clinical variability among patients and in understanding possible differences derived from the same genetic conditions. In this setting proteomics and metabolomics are potential complementary tools contributing to the better understanding of molecular mechanisms of disease. Our previous urinary proteomic investigations in BBS patients suggested possible aberrations in fibrosis, cell adhesion and extracellular matrix organization (1). In this regard, a well-designed metabolomic study would enable the identification of the downstream effects caused by the action of deregulated genes and proteins. The present study aims to compare urinary metabolomic profile of BBS patients with that of different control groups including aged-gender-matched healthy volunteers, aged-gender-matched chronic kidney disease patients by other causes and obese individuals. The differentially represented metabolites were identified by an untargeted strategy. This approach could help us to discover specific biomarkers for BBS and to elucidate biological/metabolic aberrations underlying renal disease that represents the major cause of mortality in BBS patients. The results were then linked to some of clinical data (Glomerular Filtration Rate, the annual eGFR decline and the Body Mass Index) using bioinformatics tools in order to identify predictor factors of renal outcome and disease progression. Overall, the obtained results suggest a possible deregulation of cellular metabolism, including changes in fuel source, but also lipid digestion, mobilization and transport. Reference

1. Caterino M. et al. Kidney Blood Press Res. 2018, 43:389-405

PROMET.2

THE ROLE OF CYCLIN D3 IN DIFFERENTIATION AND PROLIFERATION PROCESSES

VITTORIA MONACOB, SIMONA CELENTANOb, AGNESE BONATOc, MAURIZIA CARUSOc, PIERO PUCCI a,b, MARIA MONTI*a,b

a. Dipartimento di Scienze Chimiche Università di Napoli Federico II, Via Cinthia, 21, 80126 Napoli

b. CEINGE Biotecnologie Avanzate S.c.a.r.l., Via Gaetano Salvatore 486, 80145 Napoli c. National Research Council (CNR), Institute of Cell Biology and Neurobiology, Campus Adriano Buzzati-

Traverso, Via Ramarini, 32, 00015 Monterotondo Scalo, Rome, Italy *Corresponding author

D-type cyclins (D1, D2, and D3) are a family of proteins involved in cell cycle control. These play a direct role in proliferation by activating cyclin-dependent kinases, CDK4 and CDK6, in the G1 phase. Cyclin D3/CDK4 complex phosphorylates the retinoblastoma protein (Rb) that when is hyper-phosphorylated activates the transcription factor E2F, which induces the expression of the genes necessary for G1/S transition [1]. Furthermore, molecular biology studies using D3-/- knock-out mice show a role of cyclin D3 in myogenic differentiation [2]. The strict dualism existing between proliferation and differentiation makes difficult to affirm the existence of a direct mechanism of cyclin D3 on the regulation of muscle-specific gene expression. Functional proteomics approach has been carried out allowing purification and identification of cyclin D3 protein complexes in order to define molecular mechanisms in proliferation and differentiation processes in which it is involved. For this purpose, myoblasts in proliferating (t0h) and differentiating (t72h) conditions were lysed and protein extracts were immunoprecipitated. The isolated protein complexes were identified by nano-LC-MS/MS experiments and the D3 interactors[3], under the two conditions, were compared in order to identify specific interactors. Finally, each identified protein was attributed to a specific functional complex by employing protein-protein data bases. Our results confirmed the canonical role of cyclin D3 in the regulation of the G1/S transition in t0h and t72h conditions. In proliferating myoblasts we found several proteins belonging to the anaphase-promoting complex or cyclosome (APC/C), which shows a role of cyclin D3 also in G2/M transition. Furthermore, the identification of RNF20/40 E3 ubiquitin-protein ligase complex in differentiating cells suggests a pivotal role of cyclin D3 in epigenetic modification regulation since this complex mediates monoubiquitination of 'Lys-120' of histone H2B.

References

1. Sherr CJ, McCormick F. (2002); The RB and p53 pathways in cancer. Cancer Cell; 2(2): 103-12. 2. De Luca, G., Ferretti, R., Bruschi, M., Mezzaroma, E., and Caruso, M. (2013). Cyclin D3 critically

regulates the balance between self-renewal and differentiation in skeletal muscle stem cells. Stem Cells, 31: 2478–2491.

3. Monti M, Cozzolino M, Cozzolino F, Vitiello G, Tedesco R, Flagiello A Pucci P. (2009); Puzzle of protein complexes in vivo: a present and future challenge for functional proteomics; Expert Rev Proteomics.; 6(2):159-69.

PROMET.3

RELIABLE IDENTIFICATION OF LACTIC ACID BACTERIA BY TARGETED AND UNTARGETED HIGH-RESOLUTION TANDEM MASS

SPECTROMETRY ROSITA RUSSO1 (rosita.russo@unicampania.it; SIB), MARIANGELA VALLETTA1

(mariangela.valletta@unicampania.it), CAMILLA REGA (camilla.rega@gmail.com), ROSANGELA MARASCO (rosangela.marasco@unicampania.it), LIDIA MUSCARIELLO

(lidia.muscariello@unicampania.it), PAOLO VINCENZO PEDONE1 (paolovincenzo.pedone@unicampania.it), MARGHERITA SACCO

(margherita.sacco@unicampania.it), ANGELA CHAMBERY1 (angela.chambery@unicampania.it; SIB)

1 Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania “Luigi Vanvitelli”, 81100 Caserta, Italy

Probiotic lactic acid bacteria (LAB) are generally employed in food industry because they contribute to nutritional value of fermented foods. Although knowledge of LAB composition is of high relevance for various industrial and biotechnological applications, the comprehensive identification of LAB species is sometimes technically challenging. Recently, MALDI-TOF MS-based methodologies for bacteria detection/identification in clinical diagnostics and agri-food proved to be an attractive strategy, complementary to traditional techniques for their sensitivity and specificity. In this study, we propose, for the first time, a novel methodology based on high resolution nano-LC-ESI-MS/ MS for LAB identification at genus, species and sub-species level by using the sequence regions 33–52 and 72–82 of the S16 ribosomal protein as proteotypic peptide markers. The developed methodology was then applied to the analyses of buffalo and bovine whey starter cultures, thus assessing the applicability of the approach for the detection of LAB also in complex matrices.

PROMET.4

EPIGENETIC ANALYSIS OF HISTONE MODIFICATIONS BY MASS SPECTROMETRY

FLORA COZZOLINOa,b, ILARIA IACOBUCCIa,b, CARMELA RICCIOb, VITTORIA MONACOa,b, TIZIANA ANGRISANOc, PIERO PUCCI a,b, MARIA MONTIa,b*

a. Dipartimento di Scienze Chimiche Università di Napoli Federico II, Via Cinthia, 21, 80126 Napoli

b. CEINGE Biotecnologie Avanzate S.c.a.r.l., Via Gaetano Salvatore 486, 80145 Napoli c. University of Naples “Federico II”, Biology Department, Naples, Italy

*Corresponding author

The DNA in eukaryotic cells is packed in chromatin with nucleosomes as basic unit. Nucleosomes are composed by octamer of four histones, and the global chromatin structure is altered by histone post-translational covalent modifications. Several types of histone modifications are well known such as acetylation, methylation, phosphorylation, and ubiquitination that play a role in the regulation of transcription activity. Histone modifications dysregulation as well as disruption of chromatin remodeling machinery play a fundamental role in many pathologies and cellular mechanisms. The analysis of histone modifications with standard mass mapping procedures is complicated by the highest occurrence of basic residues mainly in the regions interested by the modifications (N-terminal regions of H3 and H4 histones). We developed a methodology based on limited proteolysis coupled to MALDI-MS to achieve a complete sequence coverage; then we focused on H4 lysine acetylation and by LC-MSMS and ion extract procedures, we got a relative quantification of the modification. Once optimized the procedure on standard chicken core histones, the methodology was applied to the investigation of H4 acetylation state in mouse embryonal stem cells treated with DMSO and Trichostatin A (TSA), respectively. TSA inhibits histone deacetylases inducing cell differentiation. By employing the present procedure, we were able to identify the acetylated lysines and quantify the variation of the levels of acetylation occurring in the two different conditions.

PROMET.5

STUDY OF THE REACTIVITY OF CYSTEINE RESIDUES IN OXIDATIVE FOLDING PROCESS

ORNELLA DI FUSCOb, FLORA COZZOLINOa,b, ALESSIO BOCEDIc, GIADA CATTANic, GIORGIA GAMBARDELLAc, SILVIA TICCONIc, GIORGIO RICCIc, PIERO PUCCI a,b

a. Department of Chemical Science, University of Naples “Federico II”, Naples, Italy b. CEINGE Biotecnologie Avanzate, University of Naples “Federico II”, Naples, Italy

c. Department of Chemical Sciences and Technologies, University of Rome “Tor Vergata”, Rome, Italy *Corresponding author

Investigations on lysozyme (Lyz) and ribonuclease A (RNase A) reoxidation in the presence of 0.4 mM GSSG revealed that one single cysteine in each protein acquire a never observed and specific hyper-reactivity toward GSSG, with the two residues displaying about 3000-3500 times higher reactivity than an unperturbed protein cysteine. Moreover, in the case of lysozyme, the fast glutathionylation of this hyper-reactive cysteine causes a complete inhibition of the spontaneous protein aggregation. The aim of this study was the identification of hyper reactive cysteine residue in each model protein. Samples of reduced Lyz and RNase A, treated with GSSG and alkylated with bromopyruvic acid on a time-course basis, have been subjected to controlled pepsin hydrolysis, followed by mass spectrometry analysis, in order to find the glutathionylated residues. Using MALDI-MS and nanoLC-MS/MS approaches, we identified these unusual residues as Cys94 for lysozyme and Cys95 for ribonuclease A. Overall these results support the hypothesis that the incipit of the oxidative folding of these two proteins may be the very fast glutathionylation of these two cysteines without any enzymatic assistance by PDI. This phenomenon is partially explained by a lower pKa value of these two residues in comparison with free cysteine. However, the occurrence of a reversible GSSG-protein complex has been demonstrated to be reasonably responsible for the observed hyper-reactivity. In conclusion, all data collected support the hypothesis that the occurrence of a hyper-reactive cysteine in an oxidative protein folding process might be a general process selected by the evolution to contrast collateral folding events (i.e. protein aggregation) [1, 2]. References

1. The extreme hyper-reactivity of Cys94 in lysozyme avoids its amorphous aggregation – Bocedi A, Cattani G, Martelli C, Cozzolino F, Castagnola M, Pucci P, Ricci G – Sci Rep. 2018 Oct 30; 8(1): 16050

2. Hyper-reactivity of Cys95 in the reduced Ribonuclease A reveals the incipit of its oxidative folding - Bocedi A, Cattani G, Gambardella G, Ticconi S, Cozzolino F, Di Fusco O, Pucci P, Ricci G - Manuscript Submitted (5th March 19)

PROMET.6

A COMPARATIVE STUDY OF THE INTERNALIZATION OF TWO RECOMBINANT Α-GALACTOSIDASE A, EMPLOYED IN THE

TREATMENT OF FABRY-ANDERSON DISEASE ILARIA IACOBUCCI1,2, CHIARA VOLLARO 2, FLORA COZZOLINO1,2, VALENTINA CITRO3, MARIA

VITTORIA CUBELLIS 3, PIERO PUCCI 1,2, MARIA MONTI 1,2

1University of Naples "Federico II", Chemical Sciences Department, Naples, Italy 2CEINGE-Advanced Biotechnology, Naples, Italy

3University of Naples “Federico II”, Biology Department, Naples, Italy *Corresponding author

Fabry-Anderson disease (FD) is an X-linked lysosomal storage disease caused by mutations on the GLA gene encoding for α-galactosidase A, a lysosomal hydrolase involved in the catabolism of α-galactosides. Certain mutations in GLA lead to the accumulation of globotriaosylceramide in tissues, damaging organs like heart and kidneys. Enzymatic Replacement Therapy (ERT) has been approved for FD treatment; it aims to reduce the accumulation of substrates through the periodic administration of the recombinant protein. Up to date two drugs for ERT are available: the agalsidase alfa known as Replagal® produced by Shire Human Genetic Therapies, Inc. and the agalsidase beta, known as Fabrazyme® produced by Genzyme Corp. Surprisingly, the two therapeutic principles don’t show the same outcome; so we tried to define the internalization molecular mechanisms thus to obtain information about the pathways in which the two recombinant proteins are involved. FD patient fibroblasts, not expressing GLA, were incubated with Replagal® or, alternatively, with Fabrazyme®. A functional proteomic approach, based on immunoprecipitation experiments and nano-LC-MS/MS analyses was performed to purify and identify α-Gal A partners, using patient fibroblasts not treated as negative control. Previously endogenous α-Gal A interactome in WT fibroblasts was also investigated to deeply understand its route towards the lysosomes in physiological conditions. 184 proteins were identified in the experiments with recombinant proteins: 50 of which shared by the two conditions. Common proteins mainly belong to endocytosis and vesicle traffic processes. Data confirmed that both enzymes are internalized through clathrin dependent and caveolae endocytosis. Additionally in Fabrazyme® treated cells, we found proteins associated with exocytosis, suggesting a partial disposal of the protein that might explain the different efficacy of the therapy.

PROMET.7

SCREENING NEONATALE ESTESO: UNA LEZIONE DAI FALSI POSITIVI VILLANI GRD*1,2 (guglielmorosariodomeni.villani@unina.it; SIB), GALLO G2 (giovannag80@gmail.com),

ALBANO L2 (lucyalb@live.it), CRISCI D2 (crisci@ceinge.unina.it), DI TOMMASO S2 (silviagm@hotmail.it), FISCO M2 (mariagraziafisco@gmail.com), PECCE R1,2 (pecce@dbbm.unina.it), TURTURO MG2

(turturomariag@live.it), VALLONE F2 (vallone@ceinge.unina.it), PERFETTO R2 (perfetto@gmail.unina.it), SACCHETTINI R2 (sacchettini@ceinge.unina.it), AURIEMMA F2 (auriemma@ceinge.unina.it), RUOPPOLO

M1,2 (margherita.ruoppolo@unina.it; SIB)

1Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, Napoli, Italia

2Ceinge Biotecnologie Avanzate scarl, Napoli, Italy *Corresponding author

Introduzione: I programmi di screening neonatale esteso (SNE) mediante spettrometria di massa tandem (MS/MS), permettono, sin dalla nascita di identificare numerose malattie: la LC-MS/MS può identificare più di 70 difetti metabolici su un singolo spot di sangue (DBS) preparato a 48-72 ore di vita. In Italia, solo nel 2016 è stata approvata una legge che rende obbligatorio lo SNE; nel 2007 il nostro laboratorio ha iniziato uno studio pilota di screening neonatale (1) ed oggi rappresenta il laboratorio regionale per lo screening neonatale esteso delle malattie metaboliche ereditarie. La flow-chart per l’SNE prevede tests di I livello per la determinazione delle acilcarnitine e degli aminoacidi su DBS e tests di II livello, su urine (acidi organici, AO) o su siero (aa e acilcarnitine) per conferma. Qui riportiamo un caso interessante di un neonato con alterazioni ai tests di I e II livello contrastanti. Risultati: Il neonato allo screening mostrava un aumento di C5OH (3-idrossi-isovaleril- e 2-metil-3-idrossi-butirril-carnitina) (0.66 �mol L-1; v.n.: 0.03-0.4). Questa alterazione è indicativa di un possibile difetto del metabolismo degli aa a catena ramificata (BCAA) oppure della biotina. Il neonato è stato quindi richiamato per i tests di II livello con risultati sorprendenti: al primo controllo risultava ancora aumento di C5OH con un lieve incremento di ornitina, mentre l’analisi degli AO evidenziava, invece, un significativo aumento di acido metilmalonico (MMA) e di acido orotico. Ai controlli successivi l’aumento di C5OH è rientrato mentre MMA e orotico sono rimasti alterati. Conclusioni: i tests di II livello hanno evidenziato che il neonato non è affetto da difetto del metabolismo dei BCAA o della biotina, motivo del richiamo allo screening, ma ha alterazioni indicative contemporaneamente di difetti del metabolismo della vitamina B12, o del propionato, e di un difetto del ciclo dell’urea o del metabolismo delle pirimidine. Bibliografia

1. Scolamiero E et al. Targeted metabolomics in the expanded newborn screening for inborn errors of metabolism. Mol Biosyst 2015;11(6):1525-35.

PROMET.8

MACROPHAGE MIGRATION INHIBITORY FACTOR IS A MOLECULAR DETERMINANT OF THE ANTI-EGFR MONOCLONAL ANTIBODY

CETUXIMAB RESISTENCE IN HUMAN COLORECTAL CANCER CELL

ROSITA RUSSO1 (rosita.russo@unicampania.it; SIB), NUNZIA MATRONE2 (nu.matrone@gmail.com), VALENTINA BELLI2 (valentina.belli@hotmail.com), DAVIDE CIARDIELLO2 (davideciardiello@yahoo.it),

MARIANGELA VALLETTA1 (mariangela.valletta@unicampania.it), SABRINA ESPOSITO1 (sabrina.esposito@unicampania.it; SIB), PAOLO VINCENZO PEDONE1 (paolovincenzo.pedone@unicampania.it), FORTUNATO CIARDIELLO2

(fortunato.ciardiello@unicampania.it), TERESA TROIANI2 (teresa.troiani@unicampania.it), ANGELA CHAMBERY1 (angela.chambery@unicampania.it; SIB)

1 Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania “Luigi Vanvitelli”, 81100 Caserta, Italy 2 Department of Precision Medicine, Università degli studi della Campania "Luigi Vanvitelli", 80131, Naples, Italy. The clinical impact of the monoclonal antibody (mAb) cetuximab targeting the epidermal growth factor receptor (EGFR) in colorectal cancer (CRC) has been widely recognized. Nevertheless, the onset of intrinsic and acquired cetuximab resistance is a serious issue that limits the effectiveness and restrict the use of this drug in targeted therapies. Until now, several mechanisms have been involved in the resistance to anti-EGFR mAbs treatments in CRC, including the activation of substitute pathways and constitutive activation of EGFR effector molecules. Unraveling the molecular players involved in cancer resistance is the first step towards the identification of alternative signaling pathways that can be targeted to circumvent resistance mechanisms and to restore the efficacy of therapeutic treatments in a tailored manner.Here, by applying a nanoLC MS/ MS TMT isobaric labeling-based approach, we have delineated a molecular hallmark of cetuximab-resistance in CRC, identifying macrophage inhibitory factor (MIF) as a molecular determinant capable of triggering cancer resistance in sensitive human colorectal cancer cells. We also demonstrated that blocking the MIF axis in resistant cells by using the 4-IPP selective MIF inhibitor, reestablishes cell sensitivity to cetuximab. Of note, the combined treatment with cetuximab and 4-IPP further enhanced proliferation inhibition in the resistant cell line, with a synergistic effect paralleled by a significant induction of apoptosis. Combination of cetuximab and 4-IPP effectively inhibits key downstream effectors of the MAPK and AKT signaling pathways, supporting the involvement of MIF-induced MAPK and AKT activation in the mechanism of intrinsic resistance to cetuximab. Collectively, our results suggest for the first time the association of MIF signaling and its dysregulation to cetuximab drug resistance, paving the way to the development of new personalized combination therapies targeting the MIF axis.

PROMET.9

ACIDEMIA GLUTARICA DI TIPO I: STUDIO DEI MECCANISMI MOLECOLARI ALLA BASE DELL’ACCUMULO CEREBRALE DI NH4+

MARIANNA CATERINO1,2 (marianna.caterino@unina.it, SIB), MICHELE COSTANZO1,2 (michele.costanzo@unina.it, SIB), EMANUELA MARCHESE2,3 (marchese@ceinge.unina.it, SIB),

MARGHERITA RUOPPOLO1,2,* (margherita.ruoppolo@unina.it, SIB)

1. Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli, “Federico II”, Napoli

2. CEINGE Biotecnologie Avanzate, Napoli 3. Dipartimento di Salute Mentale e Fisica e Medicina Preventiva, Università degli Studi della

Campania ‘L. Vanvitelli’, Napoli *corresponding author

L’acidemia glutarica di tipo I (GA-I, OMIM #231670) è un disordine autosomico recessivo dovuto al deficit enzimatico di glutaril-CoA deidrogenase (GCDH, EC 1.3.99.7). E’ una acidemia organica di tipo celebrale, con una incidenza di 1:100000 nati, caratterizzata dall’accumulo di glutarato (GA), 3-idrossi-glutarato (3-OHGA) e glutaril carnitina in plasma, urine e tessuti (1). I pazienti sono affetti da grave distonia, degenerazione striatale bilaterale, emorragie subdurali e vacuolizzazione spongiforme della materia bianca, con perdita neuronale e astrogliosi nello striato. Le strategie terapeutiche, ad oggi impiegate, fondano essenzialmente su diete a basso contenuto di proteine, trattamenti anticatabolici e trapianti di fegato o reni. Da circa un decennio queste malattie sono incluse nei programmi di screening neonatale in diversi paesi. Tuttavia, l'esito complessivo del trattamento terapeutico dei azienti affetti non è soddisfacente in quanto i danni cerebrali iniziano prima della nascita determinando anomalie neurologiche irreversibili anche dopo la diagnosi presintomatica o il trapianto di fegato. La chiave per lo sviluppo di strategie di trattamento più efficaci è, chiaramente, la completa comprensione dei processi patofisiologici che interessano i tessuti celebrali stessi. Le alterazioni metabolomiche ed i profili di espressione proteica nel tessuto cerebrale del modello murino a 3, 6 e 9 settimane sono stati studiati con un approccio di tipo ‘omico’ (4). Il modello murino della patologia è stato ottenuto introducendo la mutazione R411W nel gene Gcdh di topi Sprague-Dawley mediante la tecnologia del genome engineering CRISPR-CAS9 (Gcdhki/ki). Lo studio dei profili proteomici rivela l’alterazione di proteine coinvolte nella trasmissione del segnale sinaptico e nell'organizzazione della sinapsi. Osserviamo, inoltre, un significativo aumento della glutaminasi (Gls), coinvolta nella produzione di NH4+ cerebrale, tipico della neuropatogenesi di GA-I. Questi risultati potrebbero essere la base di nuove strategie terapeutiche per la prevenzione dell'accumulo di NH4+ cerebrale. Bibliografia

1. Kolker S. et al. Neuropediatrics 2003, 34:253-60 2. Caterino M. et al J Inherit Metab Dis. 2015, 38:969-79 3. Caterino M. et al Sci Rep. 2016, 6:25270 4. Ruoppolo M. et al Sci Rep. 2018, 16:4663

PROMET.10

ACIDEMIA METIL-MALONICA: UN APPROCCIO OMICO MARIANNA CATERINO1,2 (marianna.caterino@unina.it, SIB), MICHELE COSTANZO1,2

(michele.costanzo@unina.it, SIB), ARMANDO CEVENINI1,2 (armando.cevenini@unina.it), EMANUELA MARCHESE2,3 (marchese@ceinge.unina.it, SIB), MARGHERITA RUOPPOLO1,2,*

(margherita.ruoppolo@unina.it, SIB)

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

2 CEINGE Biotecnologie Avanzate, Napoli 3 Dipartimento di Salute Mentale e Fisica e Medicina Preventiva, Università degli Studi della

Campania ‘L. Vanvitelli’, Napoli *corresponding author

Le acidemie metilmaloniche (MMA) sono disordini congeniti del metabolismo degli acidi grassi, dovute al deficit dell’enzima mitocondriale, metil-malonil-CoA mutase (MUT, EC 5.4.99.2). L’enzima catalizza la conversione dell’isomero L-metilmalonil-CoA in succinil-CoA, un intermedio del ciclo di Krebs, in presenza di un cofattore l’adenosil-cobalammina. Il metil malonil CoA è un metabolita del catabolismo del propionato, che a sua volta deriva dalla degradazione degli amminoacidi ramificati, isoleucina e valina, del colesterolo e dall’ossidazione degli acidi grassi a catena dispari. La carenza dell’enzima MUT o del suo cofattore determinano accumulo plasmatico di acido metil malonico, di propionil-carnitina e dei suoi derivati (1). Nonostante gli elevati livelli plasmatici dell’acido metilmalonico consentano una precoce diagnosi della patologia, ad oggi non esiste una terapia. I pazienti mostrano infatti iperammonemia, chetoacidosisi, letargia, problemi respiratori, disturbi cognitivi ed epatomegalia. A lungo termine le maggiori conseguenze si traducono in danno neurologico e renale. Sono stati pertanto investigati i meccanismi molecolari influenzati dalla carenza dell’enzima MUT su un sistema cellulare modello, ottenuto silenziando l’espressione proteica dell’enzima MUT in cellule umane embrionali renali HEK (human embryonic kidney) 293 mediante la tecnologia del genome editing CRISPR-CAS9 (MUT-knock out HEK293).Mediante un approccio di proteomica quantitativa label-free, i profili di espressione proteica influenzati dal deficit enzimatico sono stati caratterizzati e quantificati (2, 3). Le abbondanze relative degli hits presenti nel dataset proteico consentono di osservare la deregolazione di proteine coinvolte nel bilancio dell’omeostasi ossido-riduttiva mitocondriale. Poiché l’analisi condotta consente di osservare alterazioni anche in proteine coinvolte nel metabolismo lipidico e glucidico, sono stati allestiti studi di caratterizzazione metabolomica sul medesimo sistema cellulare (4). I dati raccolti indicano una sensibile alterazione dell’omeostasi mitocondriale in risposta alla carente attività enzimatica dell’enzima MUT, supportando l’idea di un coinvolgimento mitocondriale nei meccanismi molecalari che regolano l’insorgenza e la progressione della patologia stessa. Bibliografia

1. Caterino M. et al. Mol Biosyst. 2016, 12:566-74 2. Caterino M. et al. Kidney Blood Press Res. 2018, 43:389-405 3. Costanzo M et al Int J Mol Sci. 2018, 19 4. Ruoppolo M. et al Sci Rep. 2018, 16:4663

PROMET.11

PROTEOMICS ANALYSIS OF HUMAN SERUM OF NON SMALL CELL LUNG CANCER (NSCLC) PATIENTS

PINTO FEDERICA1 (pinto.federica@unicampania.it) , MARIO COPPOLA1 (mario.coppola@unicampania.it), MARIO SANTINI2 (mario.santini@unicampania.it) , MARINA DI DOMENICO1

(marina.didomenico@unicampania.it), LUCIO QUAGLIUOLO1*, (lucio.quagliuolo@unicampania.it; SIB), MARIAROSARIA BOCCELLINO1 (mariarosaria.boccellino@unicampania.it; SIB)

1 Dipartimento di Medicina di Precisione, Università della Campania “Luigi Vanvitelli”, Napoli, Italy

2 Dipartimento di Malattie Cardio-respiratorie, Università della Campania “Luigi Vanvitelli”, Napoli, Italy *Corresponding author

Non-small-cell lung carcinomas (NSCLC) is the most common type of lung cancer and it has poor prognosis, because overall survival after 5 years is 20–25% for all stages. Thus, it is extremely important to increase the survival rate in the early stages NSCLC by focusing on novel screening tests of cancer identifying specific biomarkers expression associated with a more accurate tumor staging and patient prognosis. In this study we focused our attention on quantitative proteomics of three heavily glycosylated serum proteins: AMBP, alpha2 macroglobulin and SERPINA1. In particular, we analyzed serum samples from 20 NSCLC lung adenocarcinoma cancer patients in early and advanced stages, and 10 healthy donors in order to obtain a relative quantification through the MRM analysis of these proteins that have shown to be markers of cancer development and progression. AMBP, alpha2 macroglobulin and SERPINA1 were chosen because all of them possess endopeptidase inhibitor activity and play key roles in cancer. We observe a variation in the expression of these proteins linked to the stage of the disease. Therefore, we believe that proteins like, alpha2 macroglobulin, alpha microglobulin/bikunin and SERPINA1 could be useful biomarkers for early detection of lung cancer and in monitoring its evolution. References

1. Di Domenico, M., Pozzi, D., Palchetti, S., Digiacomo, L., Iorio, R., Astarita, C., Fiorelli, A., Pierdiluca, M., Santini, M., Barbarino, M., Giordano, A., Di Carlo, A., Frati, L., Mahmoudi, M., & Caracciolo, G. (2018). Nanoparticle-biomolecular corona: A new approach for the early detection of non-small-cell lung cancer. J Cell Physiol.

2. Berggard, T., Oury, T.D., Thogersen, I.B., Akerstrom, B., & Enghild, J.J. (1998). Alpha1-microglobulin is found both in blood and in most tissues. J Histochem Cytochem, 46(8), 887-894.

3. Byers, L.A., Wang, J., Nilsson, M.B., Fujimoto, J., Saintigny, P., Yordy, J., Giri, U., Peyton, M., Fan, Y.H., Diao, L., Masrorpour, F., Shen, L., Liu, W., Duchemann, B., Tumula, P., Bhardwaj, V., Welsh, J., Weber, S., Glisson, B.S., Kalhor, N., Wistuba, II, Girard, L., Lippman, S.M., Mills, G.B., Coombes, K.R., Weinstein, J.N., Minna, J.D., & Heymach, J.V. (2012). Proteomic profiling identifies dysregulated pathways in small cell lung cancer and novel therapeutic targets including PARP1. Cancer Discov, 2(9), 798-811.

Si ringrazia la Società Italiana di Biochimica per il patrocinio.

Si ringraziano per il supporto e la partecipazione i seguenti sponsor:

EuroClone S.p.A

Via Figino, 20/22, 20016 Pero (MI) https://www.euroclonegroup.it

info@euroclone.it

Microtech S.R.L.

Viale Pisciarelli 79, 80078, Pozzuoli (NA)

microtech@microtech.eu https://www.microtech.eu/

M&M Biotech

Via Camillo Tutini, 13, 80136 Napoli (NA)

https://www.mmbiotech.it info@mmbiotech.it

Deltek

via Antiniana, 28 - 80078 Pozzuoli (NA)

https://www.del-tek.it info@del-tek.it

Zanichelli editore S.p.A.

via Irnerio 34, 40126 Bologna https://www.zanichelli.it universita@zanichelli.it

Event Horizon s.r.l.

Via Torquato Tasso, 175, 80127 Napoli (NA)

Discimus RFC

https://www.discimus.it info@discimus.it