bio-energetics, metabolism and nutrition: from molecules ... · a proteomic approach to study the...

MeetingannualedeiGruppiMembrane,NutrizioneeBiologia

computazionaleedeisistemidellaSIB

25-26GiugnoSalaUlissedell'AccademiadelleScienze

viaZamboni,31,Bologna

Bio-energetics,MetabolismandNutrition:frommoleculestosystems

ProgramandAbstractbook

ComitatoScientifico:VitoDePinto(CT),SilvanaHrelia(BO),PierLuigiMartelli(BO),AlessandraBaracca(BO),RitaCasadio(BO),Angela

Messina(CT),GiancarloSolaini(BO)

ComitatoOrganizzatore:VitoDePinto(CT),SilvanaHrelia(BO),PierLuigiMartelli(BO),AlessandraBaracca(BO),AndreaMagrì(CT),

SalvatoreNesci(BO),CastreseSavoiardo(BO),GiancarloSolaini(BO)

SIBGROUPSMEETING,Bologna25-26Giugno:Bio-energetics,MetabolismandNutrition:frommoleculestosystems

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Program

Monday25June20189,30Registration10,30OpeningAddress10,45PlenaryLectureChair:RitaCasadioLiliaAlberghina(MilanoBicocca)Mitochondriaandmetabolism:theirtimehascomeagain!Session1:Structure,function,systembiologyofmembraneproteinsChair:PierLuigiMartelli11,30DanielaGaglio(CNR-Roma)Building systems metabolomics 1: Biological challenges which will advance by using GC/LC mass spectrometrymetabolomeanalysis11,45ChiaraDamiani(MilanoBicocca)Buildingsystemmetabolomics2:constraint-basedmodelingtoidentifypatternsandrulesofmetabolicswirings12,00FedericaZinghirino(Catania)AnalysisofNRF1andHIFSroleintrascriptionalregulationofVDACisoformsundermetabolicstressconditions12,15SalvatoreNesci(Bologna)TheATPSynthasemembrane-embeddeddomain:structuralimplicationsinhealthanddisease12,30MicheleGalluccio(Cosenza)StructuremodelingoftheOCTN1transporter:validationbysite-directmutagenesis12,45MariaGaetanaG.Pittalà(Catania)Characterizationofpost-translationalmodificationsofVDACisoformsfromrat-livermitochondriabyhigh-resolutionmassspectrometry13,00Lunchbreak14,30PlenaryLectureChair:SilvanaHreliaPaolaAntoniaCorsetto(Milano)NutritionandfastinginthepreventionandtreatmentofcancerSession2:Naturalsubstancesandnutrition:frombiochemistrytopathology


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Chair:SilvanaHrelia15,15EleonoraDaPozzo(Pisa)Anti-oxidantandanti-senescenceeffectsofBergamotjuice15,30SofiaPugnaloni(Ancona)Nutritionalqualityofwholegrains:polyphenolcontentandglycemicloadofSenatoreCappellidurumweathpasta15,45EneaFelrizza(Bologna)BiochemicalcharacterizationofArthrospirasppusedasnutritionalsupplement16,00Coffeebreak16,30LauraGiusti(Pisa)AproteomicapproachtostudytheneuroprotectiveeffectofoleocanthalinSH-SY5Ycells16,45SimonaDaniele(Pisa)TumorNecrosisFactoraregulatesGRK2turnoverthroughtheE3ubiquitinligaseMdm2andsupportsosteogenesis17,00TatianaCarrozzini(Milano-Bicocca)Toinvestigatetheprotectiveeffectofphyto-complexesagainstoxidativestreesinacellularmodelofstroke17,15DeborahPietrobono(Pisa)HumanglioblastomacellapoptosisisinducedbyRosemaryofficinalisthroughthep53functionalreactivation17,30MarcoNecci(Padova)PhytoTypeDB,adatabaseforplantproteinfunctionandvariability

Tuesday26June201810,00PlenaryLectureChair:VitoDePintoCesareIndiveri(Calabria)Theintriguingaminoacidtransporter,LAT1:relevancetohumanhealthanddrugdiscoverySession3:MembraneproteinsinactionChair:GiancarloSolaini10,45AnnaCostanzini(Bologna)RoleoftheF1F0-ATPaseInhibitorIF1inosteosarcomacellsunderanoxicconditions11,00CeciliaPrata(Bologna)SulforaphaneinfluencesAQP8-linkedredoxsignalinginaleukemiccellline11,15Coffeebreak11,30LorenaPochini(Cosenza)RegulatoryaspectsofthehumanorganiccationtransporterOCTN1(SLC22A4)


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12,00MariaTolomeo(Bari)AlteredexpressionofSLC52Amembersinhumancancer12,15MauraSamarani(Milano)ALyposome-plasmamembrane-sphingolipidaxixlinkinglysosomalstoragetocellgrowtharrest12,30StefanoContiNibali(Catania)Proteinpurification'smethodaffectstheelectrophysiologicalfeaturesofyeastVDAC212,45LunchBreak14,15PlenaryLectureChair:AlessandraBaraccaSabinaPassamonti(Trieste)Membranetransportofbilirubinandflavonoids:fromkineticstodietSession4:NutritionandpathologiesChair:CristinaAngeloni15,00LiviaCabitta(Milano)IdentificationoftheantigenrecognizedbyRHIGM22,aremyelination-ptomotinghumanmonoclonalantibodyandhiseffectongliacells15,15ChiaraGiacomelli(Pisa)Humangingivalmesenchymalstemcelltrophismismodulatedbyinflammatorymicroenvironment:effectsofRibesnigrumbudextract15,30SonilaAlia(Marche)Tastesensitivityandbodyweigth:istherealink?15,45ElisaBoschetti(Bologna)Intestinalepithelialbarrierabnormalitiesinpatientswithchronicintestinalpseudo-obstruction16,00GiuliaFrisco(Bologna)LactobacilluscrispatusinterfereswithChlamydiatrachomatisinfectivitythroughmodulationofintegrinexposureincervicalcells16,15NataliaCalonghi(Bologna)Effectof9-Hydroxy-stearicacidonglucosemetabolisminahumancoloncancercellline16,30AntoninaOrlando(Milano-Bicocca)Evaluation of endothelial dysfunction markers in children with cardiovascular risk factors: obesity and/orHypertension16,45CristianaCaliceti(Bologna)IsNOTCHinvolvedinproteomeeffectsofestrogenonendothelialfunctionandangiogenesis?Endofthemeeting


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MeetingannualedeiGruppiMembrane,NutrizioneeBiologia

computazionaleedeisistemidellaSIB

Bologna25-26Giugno,AccademiadelleScienze

Bio-energetics,MetabolismandNutrition:frommoleculestosystems

ABSTRACTBOOK


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PLENARYLECTUREMitochondriaandmetabolism:theirtimehascomeagain!LiliaAlberghinaUniversitàdiMilano-Bicocca,Milano,[email protected]

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Session1:Structure,function,systembiologyofmembraneproteinsBuildingsystemsmetabolomics1:BiologicalchallengeswhichwilladvancebyusingGC/LCmassspectrometrymetabolomeanalysisDanielaGaglio1,2,MarcellaBonanomi1,3,GloriaCampioni1,3,GiuseppinaVotta1,2,MarcoVanoni1,3,LiliaAlbergina1,31 SYSBIO.IT, Centre of Systems Biology, Milano, Italy; 2 Institute of Molecular Bioimaging andPhysiology,NationalResearchCouncil (IBFM-CNR),Segrate, Italy;3DepartmentofBiotechnologyandBiosciences,UniversityofMilano-Bicocca,Milano,Italydaniela.gaglio@ibfm.cnr.itMetabolomicshasbeenestablishedasarobusttooldescribingcomplexbiologicalnetworksandaccuratelythefunctionalandphysiologicalstatesofanorganism.Themetabolomeisthesetofallsmallmolecules,suchasaminoacids,sugarsandlipids,inabiologicalsystem.Itisconsideredtobe an endpoint of biological processes and carries an imprint of all genetic, epigenetic andenvironmental factors.Aimingat in-depthcharacterizationofcomplexmetabolitemixtures, therecenttechnologicaldevelopmentsinthefieldofmetabolomicshaveopenedupawiderangeofresearchfields: inbiological,biomedical,environmentalandnutritionalresearch. Inbiomedicalresearch,metabolomicsisakeytechniqueforsystemsbiology,diseasediagnosticsandbiomarkersespeciallyforcancerstaging,predictionofrecurrence,prognosisandtreatmentselection.Infactis a powerful tool able to identify themain cancermetabolic alterations in tumors, aiming attargetingthecancermetabolicrewiring[1]asnovelandpersonalizedtherapeuticapproachesasopposite tocurrentstandardtreatments.Moreover,metabolomicsdatasetcanbeused insilicolinkingthemetabolicfluxes[2]andpredictivemetabolicmodels[3]abletoinvestigatemetabolicalterationsofcomplexhumandiseases.[1]Gaglioetal,Oncotarget.2016Aug9;7(32):52017-52031;[2]Gaglioetal,MolSystBiol.2011Aug16;7:523;[3]Damianietal,PLoSComputBiol.2017Sep28;13(9):e1005758.

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Buildingsystemmetabolomics2:constraint-basedmodelingtoidentifypatternsandrulesofmetabolicwiringsChiaraDamiani1,2,DavideMaspero3,MarziaDiFilippo1,3,DarioPescini1,4,GiancarloMauri1,3,HansV.Westerhoff5,6,MarcoVanoni1,3,LiliaAlberghina1,31SYSBIOCentreofSystemsBiology,Milano,Italy;2DeptofInformatics,SystemsandCommunication,UniversityMilano-Bicocca,Milano,Italy;3DeptofBiotechnologyandBiosciences,UniversityMilano-Bicocca, Milano, Italy; 4 Dept of Statistics and Quantitative Methods, University Milano-Bicocca,Milano Italy; 5Dept ofMolecular Cell Physiology, VUUniversity, Amsterdam, TheNetherlands; 6Manchester Centre for Integrative SystemsBiology,University ofManchester,Manchester,[email protected],whichprovidesafunctionalreadoutofcellularbiochemistry,isanticipatedto clarify themechanismsof complexdiseases, leading to better diagnosis and treatment thangenomicsorproteomicsalone.Regrettably,currentmetabolomicstechnologiesoftenportraytheaverage behavior of intermixed heterogeneous subpopulations, overlooking the internalinteractionsanddifferenceswithinacellpopulation,takenforexamplefromcancerbiopsiesortumors-on-chips, that may be crucial for facilitating the disease progression. Along withheterogeneous genetic and epigenetic factors, variations in the tumor microenvironmentcontribute indeed to intra-tumor heterogeneity, leading to a complex cancer populationarchitecture in which differently specialized cells exchange nutrients and cooperate to massgrowth, by activating complex networks of interactions. Despitemajor advances in single-cellsequencing,single-cellmetabolicanalysesarelaggingbehind.Tobridgethisgap,wepresentacomputationalframeworktocharacterizemetabolismatthesinglecelllevel,byintegratingbulkmetabolomics and single-cell transcriptomics data, representative of different levels of a cellpopulation.Weexploitconstraint-basedmodeling tosimulateasetof replicatesofametabolicnetworkofhumancentralcarbonmetabolism,correspondingtodifferentcells,whichmayinteractbyexchangingmetabolites, givennutritional constraintson the totalpopulation.By simulatingscenariosinwhichlactatecannotbefullyreleasedinplasmabutcanbeexchangedinthetumormicro-environment,ourmethodologyprovedabletoreproducetheexistenceofaphenomenonknownas“reverseWarburgeffect”,accordingtowhichtumorstromalcellsaerobicallyproducelactate,whichisusedascarbonsourcebyadjacenthighproliferativecancercells.Otherscenariosmay be simulated with our approach, by exploiting single-cell experimental information tospecificallyconstraintheboundariesforthefluxesofthesinglecellsinthepopulation,insteadoflettingthemfreetoadjusttheirconsumptionratesaccordingtolinearprogrammingoptimization.Single-cellfluxesarecomputedasafunctionofthetranscriptomeatthecelllevel,whileassuringbiomassformationatthepopulationlevel.Weintegratedthegeneexpressionprofilesofdozensofindividualtumorcellsisolatedfrommousexenograftsderivedfromlungadenocarcinomapatients.We were able to characterize the existence of a more aggressive subpopulation in terms ofmetabolicgrowthrate.

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Analysis of NRF1 and HIFs role in transcriptional regulation of VDAC isoforms undermetabolicstressconditionsFedericaZinghirino,VitoDePinto,LiaMela,FrancescaGuarinoDepartmentofBiomedicalandBiotechnologicalSciences,UniversityofCatania,Catania,[email protected](VDAC)area familyofpore-formingproteins thatplay a crucial role in transport of ions and smallmetabolites through themitochondrial outermembrane.InmammalstherearethreeisoformsofVDAC(VDAC1,VDAC2andVDAC3),encodedby three different genes [1]. To investigate on the role of VDAC isoforms in mitochondrialdysfunctionandadaptationduringmetabolicstress,weanalyzedtheirexpressionandregulationby inducinghypoxiaandnutrientdeprivation thatarecommon features inpathologiessuchascancer and neurodegenerative diseases. Interestingly, VDAC1 and VDAC2 expression levelsincreasedinatimerelatedmannerwhileVDAC3transcriptlevelsremainedunchangedorwereslightlydown-regulated.Usingabioinformaticapproach,weperformedapredictionanalysisoftranscriptionfactorbindingsitesonVDACpromoters.Wefocusedourattentiononbindingsitesspecifically recognized by NRF1 and HIFs factors, respectively involved in mitochondrialbiogenesisinducedbynutrientdepletion[2]andincellresponsetolowlevelofO2(hypoxia)[3].WefoundthatonallthreepromotersanalyzedthereareNRF1putativebindingsitesbutwithamajornumericaldistributionandstatisticalsignificanceonVDAC3promotersequence.Instead,theHIFsfactorbindingsiteshavebeenfoundonlyonVDAC1andVDAC2promoters.TherearealsoseveralregulativemodulesformedbyotherfactorssuchasCREB,ETS,SP1thatareinvolvedinmitochondrial biogenesis and in response to oxidative stress. Furthermore, we found that onVDAC3promotertherearebindingsitesfortranscriptionalrepressorsadjacenttoNRF1bindingsites.Thus,wehypothesizedadifferenttranscriptionalregulationofVDAC3probablycorrelatedwithotherfactorsthatcouldrepresstheNRF1activationorcompetewithitsoverlappingbindingsitesidentifiedonthesequence.Inconclusion,withthispredictiveanalysisweidentified,onVDACpromoters,thebindingsitesforNRF1andHIFsthatcouldbetheregulatorsofVDACsexpressionin response to metabolic stress conditions. This suggests that VDAC proteins could have animportantroleinmitochondrialdysfunctionandadaptation.[1]Messinaetal,BiochimBiophysActa(2012)1818,1466-1476;[2]Scarpulla,BiochimBiophysActa(2012)1819,1088-97;[3]Iommarinietal,FrontOncol(2017)7:286.

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The ATP Synthase membrane-embedded domain: structural implication in health anddiseaseSalvatoreNesci,FabianaTrombetti,VittoriaVentrella,CristinaAlgieri,AlessandraPagliaraniDepartmentofVeterinaryMedicalSciences,UniversityofBologna,OzzanoEmilia,[email protected]/ATPaseisabifunctionalenzymecomplexarrangedintwomain domains: the hydrophilic F1, protruding in the matrix and capable of ATPsynthesis/hydrolysis,andthemembrane-embeddedFOwhichtranslocatesH+throughtheinnermitochondrial membrane (IMM). The H+ translocation coupled to ATP catalysis involves twoasymmetricanddiscontinuoushalf-channels,locatedonhorizontalα-helicesofasubunit[1]andopeningoneitherIMMsides.TheequallyspacedarrangementofcrucialaminoacidsinvolvedintheH+uptake/releaseinasubunit[2]guaranteestheH+flowacrosstheIMM,whosedirectionisdrivenbythec-ringrotation[3]andallowstheaccommodationofdifferently-sizedc-rings.TheholoenzymeformsFOdimers,whichbendtheIMMtoyielditscharacteristiccurvatureattheapexofcristae[4]andensuremitochondrialbioenergetics.Conversely,thedimerdissociationimpairsoxidativephosphorylationandformsthemitochondrialpermeabilitytransitionporebetweenthedetachedmonomers [5], thus initiatingregulatedcelldeath.FO is targetedbyendogenousandexogenousmodulators[6],whichthroughpost-translationalmodificationsofcrucialaminoacids[7],changetheenzymefeatures.Indeed,anystructuralchangewhichaffectstheH+translocationmechanismand/orthedimerassemblyimpactsonmitochondrialefficiencyandconstituteariskfactor not only for mitochondrial diseases but also for a variety of pathologies in whichmitochondrial dysfunctions are involved. So, FO emerges as molecular link between the(micro)environmentandmitochondrialbioenergeticsandapromisingtargetforinnovativedrugs[6].[1]Allegrettietal,2015,Nature521(7551):237–240;[2]Srivastavaetal,2018,Science360(6389);[3]Nescietal,2015,JMembrBiol248(2):163–169;[4]Guoetal,2017,Science358(6365):936–940;[5]Nesci,2018,TrendsBiochemSci43(5):311–313;[6]Pagliaranietal,2016,MiniRevMedChem16(10):815–824.[7]Nescieta,2017,BiochimBiophysActa1861(11PtA):2902–2912.

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StructuremodelingoftheOCTN1transporter:validationbysite-directmutagenesisMicheleGalluccio,LorenaPochiniandCesareIndiveriUnit of Biochemistry and Molecular Biotechnology, Department BEST, University of Calabria,ArcavacatadiRende,Italymichele.galluccio@unical.itTheOrganicCationTransporterNovel1(OCTN1) isamemberof theSLC22 family involved incationtransportatplasmamembranelevel.Eventhoughitsphysiologicalsubstrateisstillunderdebate,itsinvolvementininflammatorypathwaysrelatedtonon-neuronalacetylcholinesystemisacknowledged[1].Todate,usingdifferentapproachesandtemplates,homologymodelshavebeenbuilt by different research groups to gain insights in the structure/function relationships.However,thepresenceintheOCTN1sequenceofalargeextracellularhydrophilicloopbetweenthefirstandthesecondtransmembranedomains,impairsthemodelingduetotheabsenceofacorrespondingloopinanyofthepossibletemplatespresentinthePDBdatabase.Modeller9.19software,I-TASSERandPhyre2serverhavebeenusedtomodelOCTN1usingasdifferenttemplatetheglycerol-3-phosphatetransporterfromE.coli(1PW4),thehigh-affinityphosphatetransporter(PiPT)fromPiriformosporaindica(4J05)orthehumanGLUT3transporter(4ZW9).Tovalidatethemodels,site-directedmutagenesisstudieshavebeenperformedbyexploitingthesevenCysresidues of the protein and their reactivity toward hydrophobic or hydrophilic thiol specificreagents.IndeedfouroftheCysteinesarepresentinthelargehydrophilicloopandthreeinthetransmembrane domains. cDNA encoding for the OCTN1 protein has been codon optimizedaccordingtoE.coligenomeandclonedunder thecontrolof the tacpromoter into thepH6EX3plasmid.E.coliLemo21(DE3)strainhasbeenculturedat28°Cfor6hoursinpresenceof0.4mMIPTG in order to over-express the protein of interest that has been purified by affinitychromatography.Site-directedmutagenesishasbeenperformedsubstitutingeachofthesevenCysresidues toAla.Theeffectof–SHreagents, suchasMTSEA,havebeen tested for the reactivitytowardsOCTN1wt andCys/Alamutants.Thedataobtainedare consistentwith thehomologymodelobtainedbyPhyre2server.[1]Pochinietal,BiochimBiophysActa,1818(2012)559-565.

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Characterization of post-translational modifications of VDAC isoforms from rat livermitochondriabyhigh-resolutionmassspectrometryMariaGaetanaG.Pittalà1,RosariaSaletti2,PierpaoloRisiglione1,SalvatoreAntonioM.Cubisino1,SalvatoreFoti2,VitoDePinto11DepartmentofBiomedicalandBiotechnologicalSciences,UniversityofCatania,Catania,Italy;2DepartmentofChemicalSciences,UniversityofCatania,Catania,[email protected] anion selective channels (VDAC1, VDAC2, VDAC3) are integral membraneproteins found in themitochondrial outermembranewhoseprimary function is topermit thecommunication and exchange of molecules related to the mitochondrial functions. The outermembranecancollectsignalsfromandtomitochondriarelatedtobioenergeticsmetabolism,tothe dynamics of the organelle with its fusion/fission processes, to the quality control of theorganelleitself.Howeverthereareonlyrareandlimitedinformationaboutthemoleculesinvolvedinthisroleofswitchbetweentheinandoutofmitochondria.ThepresentworkispartofaresearchlineconcerningthestructuralcharacterizationoftheVDACproteins.Wehaverecentlyreportedaboutthepeculiarover-oxidationofcysteinesfromVDAC3ratlivermitochondria[1]andnowwehaveextendedtheanalysistotheothertwoisoformsVDAC1andVDAC2[2].Inparticular,wehavefocusedonthesequenceanalysisandsomeoftheirpost-translationalmodifications(PTM).Wehave found also in these proteins, as for VDAC3, over-oxidation of cysteines. In fact, since theintermembranespaceisastronglyoxidizingareaofthecell,wewantedtoseeifthiscouldmodifytheredoxstateoftheseproteins.Theattentionwasthereforefocusedonthestudyoftheoxidationstateofthemethionineandcysteineresidueswhich,amongalltheaminoacids,arethemosteasilysusceptible to oxidation. Furthermore, serine, threonine and tyrosine phosphorylation and thecysteine succinations were studied. Interestingly, cysteine over-oxidation appears to be anexclusivefeatureofVDACs,sinceitisnotpresentinothertransmembranemitochondrialproteinseluted by hydroxyapatite [2]. The analyses were performed by tryptic and chymotrypticproteolysisandhigh-resolutionnanoUHPLC/nanoESI-MS/MSandtheresultsweresubmittedtobioinformaticsresearch.Wespeculatethatsuchmodificationscouldhaveasignalingmeaning,inthesensethattheycouldshowamodifiedinterfacebetweentheexternalsurfaceoftheorganelleandcytosol resident systems [3].Theassignmentof a functional role to thesemodificationsofVDACswillbeafurthersteptowardsthefullunderstandingoftherolesoftheseproteinsinthecell.[1]Salettietal,BiochimBiophysActa.2016,1859,301-311.[2]Salettietal,BiochimBiophysActa.2018,inpress.[3]Reinaetal,Oncotarget.2016,Jan19;7(3):2249-68.


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PLENARYLECTURENutritionandfastinginthepreventionandtreatmentofcancerPaolaAntoniaCorsetto,G.Montorfano,S.Zava,I.Pastori,I.Colombo,A.M.RizzoDepartmentofPharmacologicalandBiomolecularSciences,UniversitàdegliStudidiMilano,[email protected],butalsodietaryhabits,playfundamentalroleincanceronset,progression and prognosis. Nutrient starvation is a promising approach to reduce metaboliteavailabilitytotumourscells,impairingtherapidsynthesisofnewintracellularcomponents,suchas lipidmembranes and enzymatic or structural proteins. Several studies have suggested thatcycles of prolonged fasting or of fasting-mimicking diets (FMDs) enhance the activity and thetoleranceofchemo-andradio-therapyinpreclinicalcancermodels.Nutrientstarvationstronglyincreases the antitumor activity of tyrosine kinase inhibitors inmice carrying human tumourxenograftstoblocksignallingviathepro-tumorigenicmitogen-activatedproteinkinasecascade.Fastingorshort-termstarvationdecreasescirculatinginsulin-likegrowthfactor1(IGF-1),thatiscrucialfortheestablishmentofthedifferentialstresssensitization,aconditionthatmakescancercells,butnotnormalcells,sensibletocytotoxicdrugs.Moreover,fastingalsodown-regulatesthemechanistic target of rapamycin (mTOR). mTOR is a metabolic regulator on which signallingpathways dependent on IGF-1, glucose, and amino acids converge to regulate growth andautophagy and which has a central role in cancer proliferation FMDs may enhance thechemotherapy efficacy by tumour immunogenicity increase, in part by an HO-1-dependentmechanism,whichinducestherecruitmentofcytotoxicCD8+Tcellstothetumour,andreducestumour-associatedTregs.Breastcancerisveryintricatediseaseduetoitsheterogeneousnatureandthereisapositiveassociationbetweenobesityandbreastcancermortality.OnesubtypeisTripleNegativeBreastCancer(TNBC),whichisdeficientintheestrogenreceptorα,progesteronereceptor,andhumanepidermalgrowthfactorreceptorexpression.TNBChasanaggressiveclinicalbehavior and is extremely metastatic. Since there is a complex network between exogenousnutrientsandcancerabnormalmetabolism,wehaveevaluated,aspreclinicaldata,theeffectsofnutrientdeprivationoncellmigrationandlipidmetabolisminMDA-MB-231celllinederivedfromTNBC;weassessedtheconsequencesofmediumglucose,glutamineandserumreductionsoncellviability,migrationandEpithelialMesenchymalTransition.Moreover,weevaluatedtheeffectonlipidphenotypeby lipidomic approach.The resultsobtained indicate thatnutrient restrictionsreducecell viabilityand tumorcellmigrationgreatly influencing the lipidpatternofMDA-MB-231cells.Wemeasuredtotal,phospholipidandneutrallipidfattyacids,especiallyarachidonic(AA)andeicosapentaenoic (EPA)acids that areprecursorsof eicosanoids involved in the cross-talkbetweencancercellsandimmunecells.Thedatasuggestsignificantchangesinlipidcomposition,especiallyinomega-6/omega-3.Moreover,wehaveobservedalterationsoftriglycerideandsterolcontent.Inconclusion,nutrientdeprivationinfluenceslipidphenotypeandinvasivenessofTNBCcells suggesting a possible future clinical approach for the prevention and treatment of breastadenocarcinoma,althoughfurtherstudiesandclinicaltrialsareneededtodemonstrateitsefficacy.


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Session2:Naturalsubstancesandnutrition:frombiochemistrytopathologyAnti-oxidantandanti-senescenceeffectsofBergamotjuiceEleonoraDaPozzo1,2,MarinellaDeLeo1,2,ImmacolataFaraone3,LuigiMilella3,ChiaraCavallini1,2,EugeniaPiragine1,2,LaraTestai1,2,VincenzoCalderone1,2,LuisaPistelli1,2,AlessandraBraca1,2,andClaudiaMartini1,21DepartmentofPharmacy,UniversityofPisa,Pisa,Italy;2ResearchCentreforNutraceuticalandHealthyFoods“NUTRAFOOD”,UniversityofPisa,Pisa,Italy;3DepartmentofScience,UniversityofBasilicata,Potenza,Italyeleonora.dapozzo@unipi.itAgingisoneofthemainriskfactorfortheonsetofcardiovasculardiseases;oneofthepossibleexplanationscouldbelinkedtotheage-associatedover-productionoffreeradicals.Thisincreaseof oxidative stress can be overcomewith a high intake of food antioxidants. In this context, anumberofstudieshavebeenaddressedtoassesstheanti-agingpotentialofnaturalantioxidantcompounds. Recently, it has been shown that the juice of bergamot (Citrus bergamia Risso etPoiteau), a fruitmostly produced in the Ionian coastal areas of Southern Italy (Calabria), is avaluable sourceofhealthpromotingconstituentswith, amongother, antioxidantproperties. Inorder to investigate the potential anti-aging effects of this Mediterranean natural antioxidantsource,bergamotjuicesofthreedifferentcultivars('Fantastico', 'Femminello'and'Castagnaro')werehereincharacterizedbythemeanofhighperformanceliquidchromatography-photodiodearray-electrosprayionization-tandemmassspectrometry.Then,juiceswereinvestigatedfortheevaluationoftotalpolyphenolicandflavonoidcontents,cellfreemodelantioxidantactivitiesandinvitroanti-agingpropertiesontwodifferentcellularmodelsofinducedmyocardialsenescence.Thebestperformingjuicewasalsoassessedinvivo.Thephytochemicalprofilesconfirmedthatjuiceswererich inflavonoids,bothflavoneandflavanoneglycosides. Inaddition, twolimonoidglycosides were also identified in all cultivars. Each cultivar showed different phenolic andflavonoidcontents. In tube results showed the juice robustantioxidantactivities that correlatewith their phenolic and flavonoid contents.Moreover, for the first time, the ability of juice tocounteract the chemical-induced senescence was here demonstrated in both cellular models.Lastly,theinvivodataobtainedfrommiceheartsevidencedanincreaseintranscriptionofgenesinvolvedinanti-agingandanti-oxidantresponses.TheoverallresultssuggestthatBergamotjuiceexertsanti-oxidantandanti-senescenceeffects,makingitusefulfornutraceuticalpurposes.

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Nutritionalqualityofthewholegrains:polyphenolcontentandglycemicloadofSenatoreCappellidurumwheatpastaSofiaPugnaloni1,SonilaAlia1,AriannaVignini1,TizianaBacchetti2,MarcelloGabrielli3,EleonoraSalvolini1,GiannaFerretti1,LauraMazzanti11Dipartimentodi ScienzeClinicheSpecialistiche edOdontostomatologiche,FacoltàdiMedicinaeChirurgia,UniversitàPolitecnicadelleMarche;2DipartimentodiScienzedellaVitaedell’Ambiente,Facoltà di Scienze, Università Politecnica delle Marche; 3 Scuola di Specializzazione in Scienzedell’alimentazione,FacoltàdiMedicinaeChirurgia,UniversityofBologna,Bologna,Italypugnalonisofia@gmail.comPastaisoneofthebestpartsoftheMediterraneandiet.UNESCOrecognizedtheMediterraneandiet as an Intangible Cultural Heritage of Humanity in November 2010. As a result, researchregarding its potential health benefits has received considerable attention in the last decades.Pastaandbread,whileoftenpresentinthedietaryItalianhabits,areconsumedmainlyintheirrefined forms.Severalstudiesencouragedtoconsumewholegrainbecauseof itshigh levelsofvitamins, dietary fiber and bioactive compounds (polyphenols) with antioxidant and anti-inflammatoryproperties.Evidences fromdifferentstudiesshowapositiveassociationbetweenrefined carbohydrates and insulin resistance. The Glycaemic Index (GI) has been extensivelystudiedasanindicatorofthephysiologicaleffectsofacarbohydratemealwithapplicationsinthemanagementandpreventionofdiabetesandobesity.Theaimofthepresentstudywastoevaluatetheproperties(suchasglycemicindexandpolyphenolcontent)ofapastaobtainedusingSenatoreCappelliDurumWheat.ItisanAutumnalcultivarofdurumwheat(Triticumdurum),obtainedatthebeginningofthelastcenturybythegeneticistNazarenoStrampelli.Weobtainedthefollowinginformationrelatedtothetotalpolyphenolandflavonoidscontentsinthestudiedpastasampleusing the spectrophotometric method: total polyphenol=113.5 mg/100 g, flavonoids=52.96mg/100 g. A standard assay was performed to measure the GI of two significant sources ofcarbohydrates following the World Health Organization (WHO) recommended methodology,determiningtheincrementalareaunderthebloodglucoseresponsecurveofa50gcarbohydrateportionofthetestfoodcomparedtothesameamountofcarbohydratefromaglucosesolutionbythesamesubjectmeasuredincapillarywholebloodbeforeand15,30,45,60,90and120minutesafteringestioninatotalof14healthyadultvolunteers(6malesand8females).Twoformatsofthesamepasta(providing50gavailablecarbohydrate)weretestedinordertoenablecomparisonoftheresultingGIvalues,andtodeterminewhetherthedifferentformatsgavethesameresult.Thefollowingresultswereobtained:longformatpasta(spaghetti)=47.9,shortformatpasta=68.5.Ourstudyconfirmsthelowglycemicindexofpastamadefromdurumwheat,andithasalsohighlightedone of the multiple factors affecting the GI of a food, its physical form, which illustrates theimportanceofmeasuringtheGIvaluesoffoodsratherthanapplyingvaluesfromfoodsofsimilardescription.Suchinformationisusefultoresearchers interestedincalculatingtheGI indietarysurveystostudydiet-diseaserelationships,andintheplanningofdietaryinterventionstudies,inorder tohaveaclear ideaof theGIof the interventiondiets. It alsoprovidesvaluabledata forpractitionerswhohaveresponsibilityforadvisingindividualsontheirdiet.


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* * * BiochemicalcharacterizationofArthrospirasppusedasnutritionalsupplementEneaFerlizza1,GiuliaAndreani2,MartinaBertocchi2,GiorgioFedrizzi1,GloriaIsani21IstitutoZooprofilatticoSperimentaledellaLombardiaedell’EmiliaRomagna;2DipartimentodiScienzeMedicheVeterinarie,UniversitàdiBologna,Bologna,[email protected] centuries, native populations from Mexico used the cyanobacteria Arthrospira as food.Nowadays, thismicroorganismisgloballyrecognizedasasourceofchemicalconstituentswithwideapplicationsindifferentfields,includinghumanandanimalnutritionaswellasnutraceutical,cosmeceutical,andpharmaceutical industries.Infact,Arthrospirashowshighconcentrationsofproteins, vitamins andminerals, in particular essential trace elements. In the last decade, theinteresttowardscyanobacteriaaspossiblenutraceuticalsandfunctionalfoodgrewexponentially,leadingtomassivecommercializationofArthrospira-basedfoodsupplementsunderthenameofSpirulina.Inconsiderationoftheincreasingpopularityoftheseproductsandtheassociatedqualityproblems,thepurposeofthispreliminaryresearchwastoanalysetracemetalconcentrationsandtoisolateproteinsandpigmentsincommercialsamplesofArthrospirasppusedforhumanandanimal nutritional supplementation. Samples of Arthrospiramaxima andArthrospira platensiswereobtainedfromthemarket.Nineteenessentialandnon-essentialtraceelements(Mn,Fe,Co,Cu,Zn,Se,Mo,Cr,V,Pb,Cd,Hg,As,Al,Ag,Ni,TI,U,Sb)wereanalysedbyICP-MS.CytosolicproteinswereextractedandamolecularexclusionchromatographyonSephadexG-75wassubsequentlyperformed to separate different protein fractions. In the resulting fractions total proteins andpigments were determined, as well as Fe, Zn and Cu. The analyses by ICP-MS revealed highconcentrations of essential trace elements in the examined samples; in particular, Feconcentrationsrangedfrom432to576µg/gdryweightresultingoneorderofmagnitudehigherthanthosegenerallyfoundinfoodsofanimalandplantorigin.Hgconcentrationswerelowerthan0.05µg/gdryweight,whileAlconcentrationsshowedwidevariations(4-175µg/gdryweight)andraisedconcernduetohighvaluesfoundinsomesamples.Regardingcytosolicproteins,allthesamplesshowedapeakcontainingtheblueproteinphycocyanin;amongthedifferentapplicationofthisprotein,thepossibleuseinmedicineandbiologyhasrecentlyattractedincreasingattentionduetoitsantioxidant[1]andanti-inflammatory[2]properties.Asecondpeakwaspresentatthelowmolecularweightsfractions,relatedtothepresenceofsmallpeptidesandfreeaminoacids,including the so-calledmycosporin-likeaminoacids [3]. In conclusion,due to theabundanceofinteresting bioactive molecules and essential trace elements, Arthrospira-based supplementsdeservemoreattentioninfuturestudies.[1]Fernández-Rojasetal,2014,JFunct.Food11:375-392;[2]Qianetal,2016,E-CAM,ID7803846;[3]Chrapustaetal,2017,Mar.Drugs15:1-29.

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AproteomicapproachtostudytheneuroprotectiveeffectofoleocanthalinSH-SY5YcellsLauraGiusti1,CristinaAngeloni2,SerenaLacerenza3,FedericaCiregia4,MariaCristinaBarbalace5,AndreaUrbani6,MaurizioRonci7,ClementinaManera3,MariaDigiacomo3,MarcoMacchia3,MariaRosaMazzoni3,AntonioLucacchini7,SilvanaHrelia51Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy; 2 School ofPharmacy,UniversityofCamerino,Camerino,Italy;3DepartmentofPharmacy,UniversityofPisa,Pisa,Italy;4DepartmentofRheumatology,GIGAResearch,CentreHospitalierUniversitairedeLiège,University of Liège, Liège, Belgium; 5Department for Life Quality Studies, University of Bologna,Rimini, Italy; 6 Istituto di Biochimica e Biochimica Clinica, Università Cattolica, Roma, Italy; 7Department of Medical, Oral and Biotechnological Sciences, University G. d’Annunzio of Chieti-Pescara,Italylaura.giusti@unipi.itOliveleavesandvirginoliveoilcontainmanyphenolicseffectiveagainstagingandseverallifestyle-relateddiseases,includingneurodegeneration,bothinanimalmodelsandinhumans.Oleocanthalisasecoiridoid,oneofthemostrepresentedclassofphenolsinoliveoil,anditisresponsibleofthe stinging effect at pharynx level perceived after extra virgin olive oil ingestion. Recently,different studies demonstrated that oleocanthal possesses anti-aggregation activities on tauproteinandIbuprofen-likeactivitythankstoitsabilitytoinhibitsCOX-1andCOX-2.Theaimofthisworkistoinvestigatetheneuroprotectiveeffectofoleocanthalinneuron-likeSH-SY5YcellsbeforeandafteroxidativestressinducedbyH2O2.Using2DEcoupledtomassspectrometrytheproteinmapsfordifferentconditionsoftreatmenthavebeenobtainedandanalyzedbySamespots(TotalLab).PCRanalyseswereperformedtovalidateproteomicresults.Seventeenspotsresultedsignificantly differentially expressed with respect to control after treatment with hydrogenperoxide,twenty-sevenaftertreatmentwitholeocanthal(10µM)followedbyhydrogenperoxide,whiletwospotsfordirecteffectofoleocanthal.SpotsofinterestwereexcisedandidentifiedbyLC/MS/MS.Oleocanthalsignificantlyrevertedthedown-regulationinducedbyhydrogenperoxideof26Sproteasomenon-ATPaseregulatorysubunit1,proteasomesubunitbetatype-4,Ubiquitincarboxyl-terminalhydrolaseandPyruvatekinase,moreoveritincreasedtheexpressionofHeatshockproteinHSP90-betaandProteinDJ1.Moreover,10µMoleocanthalwasabletocounteractoxidativestressinducedbyH2O2inSH-SY5YasmeasuredbyMTTviabilityassayandtoincreasereduced-GSH level both in the absence and in the presence of H2O2 as measured bymonochlorobimaneassay.Ourfindingssuggestthatoleocanthalmayhavebeneficialhealtheffectincounteractingneurodegeneration.

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TumorNecrosisFactorαregulatesGRK2turnoverthroughtheE3ubiquitinligaseMdm2andsupportsosteogenesisSimonaDaniele,LetiziaNatali,ChiaraGiacomelli,DeborahPietrobono,RebeccaPiccarducci,MariaLetiziaTrincavelli,ClaudiaMartini.DepartmentofPharmacy,UniversityofPisa,Pisa,[email protected](TNF-α)isinvolvedinbonehealingbyaffectingmesenchymalstemcell (MSC)proliferationanddifferentiation inadose-and time-dependentmanner [1,2]. In thebonecell,TNF-αaffectstheexpressionandfunctionalityofdifferentGprotein-coupledreceptors(GPCRs), expressed on MSC membranes, and of their intracellular regulatory proteins, GPCR-regulated kinases (GRKs) [3], thus dictating their final biological outcome in controlling boneanabolicprocesses.AmongtheseGPCRs,aprimaryroleinosteogenesishasbeenemergingfortheA2B adenosine receptor (A2BAR), a Gs-coupled receptor that triggersMSCdifferentiation intoosteoblasts [4,5]. Herein, the effects of TNF-α were investigated in particular on theexpression/responsivenessoftheA2BAR,inordertoinvestigatethefunctionalconsequencesofthereceptormodulationonMSCdifferentiation.Tothispurpose,MSCswereincubatedwithTNF-αandcellulardifferentiationwasmonitoredbybothReal-timePCRandfluorescenceanalysesofosteogenic markers. Low TNF-α concentrations showed a pro-differentiating effect on MSCs,promotingtheosteoblastphenotype.Inparticular,thecytokinereducedA2BARdesensitization,forcingthereceptor-mediatedosteoblastdifferentiation,throughtoaregulationinGRK2turnoverandexpression.TheseeffectsonGRK2levelsdidnotinvolveatranscriptionalmechanism,buttheyweremediated,atleastpartially,bytheproteasomesystem.Inparticular,thecytokineinducedasignificant GRK2 association with the E3 ubiquitin ligase Mdm2 and promoted the kinaseubiquitination.LowlevelsofthekinasesGRK2reducedA2BARdesensitizationcausinganincreaseof functional receptors. Overall, these data indicate that the release of cytokines in theinflammatoryenvironmentdirectsMSCdifferentiationandrepresentsausefultargettoenhancebone formation.Moreover, the pivotal role of A2BAR availability/functionality in osteogenesisappearstobestrictlylinkedtoadecreaseofreceptordesensitizationmediatedbyGRK2,whoselevelsarecontrolledbytheubiquitin-ligaseMdm2binding.[1]FrontImmunol2014;5:1-9;[2]JCellPhysiol2010;223:168-177;[3]MolPharmacol2006;69:1311–1319;[4]BiochimBiphysActa2014;1843:2957-66;[5]MolCellBiol2017;37(8):E00442-16.

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To investigate the protective effects of phyto-complexes against oxidative stress in acellularmodelofstrokeTatianaCarrozzini1,ElenaLonati1,LauraBotto1,EmanuelaCazzaniga1,FrancescaFarina1,ChiaraMagoni2,MassimoLabra2,PaolaPalestini1,AlessandraBulbarelli11DepartmentofMedicineandSurgery,UniversityofMilano-Bicocca,Milano,Italy;2DepartmentofBiotechnologiesandBiosciences,UniversityofMilano-Bicocca,Milano,[email protected] oxidative stress, a pathological condition caused by the breakdown of the physiologicalequilibrium between the production and the elimination of reactive oxygen species (ROS) isinvolvedinthepathogenesisofdifferenthumandisease. IndeedthepresenceofROScaneasilyactivate innate immune responses, neuroinflammation, microglial activation, cerebrovasculardysfunction, and alterations in the blood-brain barrier contributing to CNS pathology such asAlzheimer’sandotherneurodevelopmentaldisorders.Epidemiologicalandobservationalstudieshavefocusedtheattentionontheresearchofanti-oxidantsubstancesabletoreducetheeffectofROSonthenervoussystem.Ahealthydietwithadequateintakeofessentialmicronutrientsmaybecrucialtopreventthedevelopmentofchronicdiseases.Increasedintakeofantioxidants,aswellas other anti-inflammatory nutrients, may attenuate the oxidative stress and inflammation,therebyprovidingausefuladditiontocurrentdiseasemanagementstrategies.Furthermore,theidentificationofnaturalphyto-compoundswithantioxidantactivity,couldbeusedtoenrichfood,strengthening protective properties. Moreover, the potential health and economic benefits ofestablishing non-pharmacological approaches (e.g., dietary supplementation) to diseasemanagementcouldbeenormous.Forthereasonsabovementioned,ourresearchaimstoidentifynewnaturalsubstanceswithantioxidantproperties.Thenaturalphyto-extractsderivingfromtheprocessingofthecoffeebeanwaste,wereusedonlivercellstoexaminetheirtoxicitysincetheliveristheorganresponsibleofdetoxification.Aftertheresultsindicatedthatthesubstanceswerenottoxic,theantioxidantpropertiesofthesephyto-extractswereevaluatedinpresenceofTert-butyl hydroperoxide aspro-oxidant. The results obtained show that ourphyto-extracts have aprotectiveeffectagainstoxidativestress.ConsideringthatthereoxygenationoftissueafterstrokedeterminestheformationofROS,wearetestingtheantioxidanteffectofthecoffeebeanwasteontheneuronalcellsandthehematoencephalicbarriercells,wheretheoxidativestresswasinducedwithOGD,aninvitromodelofstroke(oxygenglucosedeprivationandreoxygenation).Inparallel,wearealsocarryingstudieswithcoffee-derivedsubstancemodifiedbyintestinalmicrobiomeonthesamecellularmodel.

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Human glioblastoma cell apoptosis is induced by Rosemary officinalis through the p53functionalreactivationDeborah Pietrobono1, Chiara Giacomelli1, Marinella De Leo1,2, Simona Daniele1, AlessandraBertoli1,AlessandraBraca1,2,M.LetiziaTrincavelli1,2,ClaudiaMartini1,21DepartmentofPharmacy,UniversityofPisa,Pisa, Italy;2CentroInterdipartimentalediRicerca"NutraceuticaeAlimentazioneperlaSalute",UniversityofPisa,Pisa,[email protected],naturalproductshavegainedpopularityaseffectiveandlowtoxicityco-adjuvantstocanonicaltherapyinreducingcancercellgrowth(Oncotarget.2018Apr24;9(31):22194–22219).Amongtheseproducts,Carnosol(CAR)hasattractedagreatinterestforitsanti-proliferativeeffectsonGlioblastomaMultiforme(GBM)cells.GBMisahighaggressiveglialtumorwithahighproliferationrateandresistancetochemotherapy.Adysregulationofp53signalingpathwayhasbeenimplicatedinGBMcellresistancetostandardtherapy,highlightingtheneedofnewagentsabletoreactivatethispathwayandtriggeringapoptoticprocessesinthesecancercells.Herein,theabilityofaRosemaryofficinalisextract(RMO)tomodulatetheproliferationofdifferenthumanGBMcelllineswasdemonstrated.Furthermore,itsabilitytopromotethep53reactivationwasmeasured,incomparisontothesinglemoleculeCAR,evaluatingthep53proteinlevelsandtheexpressionofp53targetgenes(e.g.p21,MDM2,PUMAandBAX).RMOcausedasignificantanti-proliferativeeffectonGBM,especiallyincellsexpressingwild-typep53.ThiseffectappearedtobesignificantalsoinGBMstaminalcells(CSCs),whicharethemostresistantcellularcomponentinthetumorbulkandareresponsiblefortumorresistanceandrecurrence.RMOanti-proliferativeeffectsweremediatedbythefunctionalreactivationofp53,asdemonstratedbytheincreaseofapoptosisandbytheactivetranscriptionofp53targetgenes.TheRMOactivitywasdemonstratedto be higher respect to CAR alone demonstrating that the overall composition of the extractspotentiatestheantiproliferativeeffectswithrespecttoasinglecomponent.Inconclusion,thesedatahighlightedtheabilityofRMOtorestoreandreactivatethep53functionalityinglioblastomacells.Furthermore,theaugmentedactivityofRMOrespecttoCARshedlightonthepossibilitytodevelopcombinatorytherapytopotentiatetheconventionalGBMtreatment.

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PhytoTypeDB,adatabaseforplantproteinfunctionandvariabilityMarcoNecci1,2,3,DamianoPiovesan1,AlessandroCestaro3,SilvioTosatto1,41 Department of Biomedical Sciences, University of Padua, Padova, Italy; 2 Department ofAgricultural Sciences, University of Udine, Udine, Italy; 3 Fondazione Edmund Mach, S. Micheleall'Adige,Italy;4CNRInstituteofNeuroscience,Padova,[email protected] important plant species are poorly studied at the point thatmore than half A. thalianaproteinscompletelylackfunctionalannotationbyGeneOntlogy.WithafocusoncultivatedcropswestartedourworkfromMalusdomestica.Malusdomestica(DomesticatedApple)it’sthemostintensivelygrownfruitcropintheworld,fromwhichitsimportance.Despiteitsdomestication(aprocess that usually shrinks down diversity), it retained a large amount of genetic diversitythroughoutevolution,originatingmanydifferentcultivarsthatdisplaydifferentphenotypes,likefruit features or resitance to pathogens. Despite the great importance,M. domestica is poorlystudied.TostudyM.domesticagenefunctionsandvariabilityamongdifferentcultivarswestartedfrom an experiment where more than 500,000 high quality SNPs were identified from theresequencingof78M.domesticacultivars.Anaccurategenepredictionidentifiedaround46,000genesforthereferencegenome,whosecodingsequencingwerethenanalyzedinordertoidentifytheirfunction.Wedevelopedandgatheredasetoftoolsadresourcesfortheanalysisofproteinsequences, includingwidelyusedbioinformatics tools likeBLASTor InterProScan.AnnotationsproducedbystandardtoolsarecomplementedbyafunctionpredictionfromanimprovedversionofINGA(amongthewinnersofCAFA2),namelyINGA2.ItscoredamongthebestinCAFA3andexcelledinpredictionofplantproteinlocalization,representingtheperfecttooltoachieveafinerpredictionofproteinfunction.Furthermore,mobiDB-lite,atoolwedevelopedforproteindisorderannotationandwaslaterincludedinInterProScan5,extendsthecoverageofdomainannotationtoregionsofproteindisorder,whichhostthemostvariability.Thissetoftoolwasorganizedinapipelinethatisusedtoannotateproteomesforfunction.Tobroadenourscopewearegoingtoannotate ‘orphan genomes’, results ofwhole genome sequencing experiments thatwereneverfurtheranalyzed(e.g.Theobromacacao).Theannotationswereorganizedinadatabase(NOSQLMongoDBdatabase)andawebappwasdevelopedwithcuttingedgetechnologiestoquerythedatabaseandvisualizeandfiltertheannotations.


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PLENARYLECTURETheintriguingaminoacidtransporter,LAT1:relevancetohumanhealthanddrugdiscoveryMariafrancescaScalise,MicheleGalluccio,LaraConsole,CesareIndiveriDepartmentDiBEST(Biologia,Ecologia,ScienzedellaTerra),UnitofBiochemistryandMolecularBiotechnology,UniversityofCalabria,ArcavacatadiRende,[email protected](SLC7A5) isanHeterodimericAminoAcidTransporter interactingwiththeglycoproteinCD98 (SLC3A2) through a conserved disulfide. LAT1 mediates an antiport of branched andaromaticneutralaminoacids.Conversely,itwasrecentlyshownthatHisisapreferredsubstrate.LAT1 is over-expressed inmany tumors being a potential pharmacological target. It has beenstudied by bioinformatics, intact cell and proteoliposomes using the native or recombinantproteins. CD98 is not required for transport since [3H]His transport was detected either inproteoliposomesharboringtheLAT1/CD98heterodimerorLAT1alone.ThehomologymodelofLAT1 predicted four crucial residues for substrate binding, then confirmed by site-directedmutagenesis:F252whichhasagatefunction;S342andC335whichareresponsibleforsubstratedocking;C407whichplaysaminorrole in the intracellularside.ThepresenceofCyshasbeenexploited fordesigning inhibitors,basedondithiazolering,abletoreactwiththiols.Among50compounds, 8 have been identified as best inhibitors that interact with C407. Two of thecompoundsarethemostpotentinhibitorsofLAT1sofaridentified,andwereabletoinducecancercells death. An intriguing aspect of LAT1 biology is its expression in Blood Brain Barrier.Interestingly,some inheritedmutationsofLAT1areresponsibleofAutismSpectrumDisordersduetofunctionimpairment.

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Session3:Membraneproteinsinaction

RoleoftheF1F0-ATPaseinhibitorIF1inosteosarcomacellsunderanoxicconditionsAnnaCostanzini,GianlucaSgarbi,FrancescaLiuzzi,AlessandraBaracca,GiancarloSolainiDepartmentofBiomedicalandNeuromotorSciences,LaboratoryofBiochemistryandMitochondrialPathophysiology,UniversityofBologna,Bologna,[email protected] provide most of the ATP necessary to the cell via oxidative phosphorylation(OXPHOS), process in which the F1F0-ATPase catalyzes the ATP synthesis driven by theelectrochemicalgradient(ΔµH+).Asshowninheartandliver,ischemialeadstoaΔµH+collapsethatinducestheF1F0-ATPasetoreverseitsactivityandtohydrolyzeATP.Inthiscondition,theendogenousATPaseinhibitoryfactor1(IF1),caninhibithydrolysis,preventingATPdissipationtoensurecellsviability.Innormoxia,IF1cooperatesinthemitochondrialstructuredeterminationandpromotesOXPHOS [1,2].Theoverexpressionof IF1 in severalhumancancer suggested itsinvolvementintumorsdevelopmentandgrowth.Foritsfeatures,IF1isproposedasatargetforcancer therapy, but its mechanisms of action are still unclear. We recently investigated thebioenergeticsofcancercellsinoxygendeprivation,provingthattheATPhydrolysisoccursonlyinanoxiaconditions,butnotinhypoxia.Inanoxia-mimickingconditions,wealsodemonstratedthatIF1expressionfavorscancercellsgrowthandcellularATPlevelspreservation,byinhibitingtheATPase[3].Asa further investigation,hereweexploredincancercellsuponanoxia-mimickingconditions, the role of IF1 expression on the mitochondria content and composition. For thispurpose,weexposedIF1-expressingandIF1-silencedosteosarcomacellstotheuncouplerFCCPandweevaluatedthemitochondrialmass.Surprisingly,wefoundthatthemitochondriamasswaspreservedequallyinbothcelllinemodels,comparedtocontrols.Nevertheless,thecitratesynthaseactivityandtheOXPHOScomplexesexpressionshowedadecrease,butinpartsustainedinIF1-silenced clones.Thesedata suggested that IF1 couldmodulatemitochondrial functionality andcomposition through the mitochondria turnover. Indeed, the analysis of mitophagy andmitochondrialbiogenesismarkersunderlinedtheactivationofbothprocessesinIF1-expressingcells, conversely sloweddown in IF1-silenced clones.Taken together, our findings support thehypothesis that, in temporary anoxia, IF1 promotes cell survival by preserving ATP and bypromotingthequalitycontrolofmitochondria.Thisoccurswiththeactivationofbothmitophagyand mitochondrial biogenesis, in order to eliminate and to replace damaged mitochondria,providingfunctionalorganellesavailableincaseofoxygenlevelsrestoration.Conversely,intheabsenceofIF1,thesustainmentofΔψmbyATPhydrolysismayactsasasignaloffunctionality,leading to OXPHOS complexes preservation and mitochondria turnover inhibition, fictitiousadvantagesforthecellsbecauseofthedetrimentalimpactoftheenergydissipationoncellsurvival.[1]Barbatoetal,JBiolChem.2015Mar6;290(10):6338-48.[2]Faccendaetal,CellRep.2017Feb21;18(8):1869-1883.[3]Sgarbietal,BBABioenergetics,1859(2018)99-109.

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SulforaphaneinfluencesAQP8–linkedredoxsignalinginaleukemiccelllineCecilia Prata1, Carlotta Facchini1, Emanuela Leoncini2, Monia Lenzi1, Tullia Maraldi3, LauraZambonin1,CristinaAngeloni4,SilvanaHrelia2,DianaFiorentini11DepartmentofPharmacyandBiotechnology,UniversityofBologna,Bologna,Italy;2DepartmentforLifeQualityStudies,UniversityofBologna,Rimini,Italy;3DepartmentofSurgical,Medical,DentalandMorphological Sciences, University ofModena and Reggio Emilia,Modena, Italy; 4 School ofPharmacy,UniversityofCamerino,Camerino,[email protected](SFN),anisothiocyanatecompoundpresentinabundanceinCruciferousvegetables,exertspotentialbenefitsforpreventionandco-treatmentofseveralhealthdisorders,asreportedin both experimental and epidemiological studies [1]. In cancer cells, signalling pathways thatpromotecellproliferation,survival,angiogenesisandmetastasisarehyper-activatedbecauseofan increase in localized reactive oxygen species (ROS) production and of an altered redoxenvironment, compared tonormalcells. Inparticular,hydrogenperoxide (H2O2)derived fromNOXfamilyisinvolvedinvariousredoxsignaltransductionpathwaysandtheaquaporin8(AQP8)hasbeenidentifiedasaH2O2transportfacilitatoracrosstheplasmamembrane.Recentevidencedemonstrated that many tumor cell types express elevated level of aquaporin isoforms andhighlightedapositivecorrelationbetweenhistologicaltumorgradeandtheAQPexpression[2].WepreviouslydemonstratedthatAQP8funnelsNOX-derivedH2O2, triggeredbyendogenouslygenerated VEGF, which, in turn, provokes VEGFR-2 phosphorylation and the consequentmodulationofmanycellularactivities, resulting incell survivalandproliferation inamodelofacutemyeloidleukemia[3].Therefore,thisstudyaimedattheevaluationofthepotentialeffectofSFNonthemodulationofredoxsignalinginvolvingAQP8,NOX2andp-VEGFR-2expression.TheelucidationofAQP8roleincancerredoxsignallingandtheeffectexertedbySFNonitsmodulationcanoffernewpotential target foranti-cancertherapy,suggestingthe importanceofanti-tumoreffectexertedbydietarycompounds.[1]Račkauskasetal,OncolRep.(2017)37,3660-3666;[2]Ribattietal,Biochim.BiophysActa(2014)1840,1550-1553;[3]VieceliDallaSegaetal,Biochim.BiophysActa(2014)843,806-814.

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RegulatoryaspectsofthehumanorganiccationtransporterOCTN1(SLC22A4)LorenaPochini,MariafrancescaScalise,MicheleGalluccio,CesareIndiveriDepartment DiBEST, Unit of Biochemistry and Molecular Biotechnology, University of Calabria,ArcavacatadiRende,[email protected](SLC22A4)belongstotheOCTNsubfamily(OrganicCation Transporter Novel) which includes three members. Two of these, namely OCTN2 andOCTN3 are well acknowledged as carnitine transporters. Differently, OCTN1 displays lowefficiency in transporting carnitine. In intact cells studies OCTN1 was found to transport theprototype organic cation tetraethylammonium (TEA) and the mushroom metaboliteergothioneine;since the twosubstratesarenotphysiological inhumanmetabolism, theroleofOCTN1 is still unclear. To get further insights into the possible role of OCTN1 in humans, thetransporter has been overexpressed in E. coli and functionally assayed in proteoliposomes bystudyinguptakeandeffluxofpossiblesubstrates.Inthissystemacetylcholine(Ach)wasidentifiedasasubstrate.Achuptake,butnotefflux, is inhibitedbyextraliposomal (extracellular)sodiumindicatingthattheeffluxprocessmaybethephysiologicalone.ThistransportfunctionindicatesapossibleinvolvementofOCTN1inthe“NonNeuronalCholinergicSystem”thatisubiquitousandrequiresnonquantalAchreleasefromcellsofmanytissues.RegulationofAchtransporthasbeeninvestigatedbystudyingtheeffectontransportactivityofROSinducingcompounds,pHchangesandlipidcompositionofthemembrane.HydrogenperoxideincreasesAchtransportmediatedbyOCTN1.Thiseffectisprobablymediatedbydisulfideformationwhichisinducedbythecompound.The hypothesis is confirmed by the finding that the C-less mutant of OCTN1 is insensitive tohydrogen peroxide. A pH gradient (acidic inside) imposed in proteoliposomes increases Achuptake. This indicates the possible involvement of a proton in the transport cycle. Finally,cholesterolincludedintheproteoliposomalmembranestimulatesOCTN1increasingitstransportactivity.Thesiteofactionofthislipidhasbeenpredictedbybioinformatics.

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AlteredexpressionofSLC52AmembersinhumancancerMariaTolomeo,MariannaLoredanaDefrancesco,GiordanoEsperti,MariaBarileDepartmentofBiosciences,Biotechnology,andBiopharmaceutics,UniversityofBariAldoMoro,Bari,[email protected],otherwiseknownasvitaminB2,isanessentialdietarycomponentandrepresentstheprecursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which areimportantenzymaticcofactorsrequiredforcarbohydrate,aminoacidandlipidmetabolism,andother cellular regulatory roles [1]. In different cells, riboflavin uptake occurs via specializedcarrier-mediatedprocessessupportedbythreespecificmembersofthesolutecarrierfamily52(SLC52A), identifiedandnamedriboflavintransporter1(RFVT1;SLC52A1),RFVT2(SLC52A2),andRFVT3(SLC52A3),respectively[2].Insidethecells,riboflavinisphosphorylatedtoFMNbyriboflavinkinaseanditissubsequentlymetabolizedbyFADsynthasetoFAD,theflavincofactormainlylocatedinmitochondria[1].Alterationsofsomeoftheseproteinshavebeencorrelatedwithrareinheritedneuromusculardiseases[3].Herewepointourattentiononthepossibleinvolvementofflavincofactorhomeostasisinhumancancer.WepresentevidencesinfavorofaprofoundalterationofflavincofactorlevelinsometypesofhumancanceraccompaniedbydysregulationofRFVTexpression[4].[1]Barileetal,JInheritMetabDis(2016)39:545-557;[2]Yonezawa&Inui,MolAspectsMed(2013)34:693-701;[3]Jaeger&Bosch,InheritMetabDis(2016)39:559-564;[4]Tutinoetal,AnticancerRes(2018)38:2659-2667.

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Alysosome-plasmamembranesphingolipidaxislinkinglysosomalstoragetocellgrowtharrestMauraSamarani1,NicolettaLoberto1,GiuliaSoldà2,LetiziaStraniero2,RosannaAsselta2,StefanoDuga2,GiuliaLunghi1,FabioA.Zucca3,LauraMauri1,MariaGraziaCiampa1,DomitillaSchiumarini1,RosariaBassi1,PaolaGiussani1,ElenaChiricozzi1,AlessandroPrinetti1,MassimoAureli1,SandroSonnino11Department ofMedical Biotechnology and TranslationalMedicine, University ofMilan,Milano,Italy;2DepartmentofBiomedicalSciences,HumanitasUniversity,PieveEmanuele,andHumanitasClinical and Research Center, Rozzano, Italy; 3 Institute of Biomedical Technologies, NationalResearchCouncilofItaly,Segrate,Italymaura.samarani@unimiIncreasingevidenceimplicates lysosomaldysfunctionintheetiopathologyof lysosomalstoragediseases, neurodegenerative disorders, and the aging process. Nevertheless, the molecularmechanisms by which the perturbation of lysosomal homeostasis induced by the storage ofundegradedmetabolitesmayaffectthecellfunctionandviabilityarestillunknown.Inthiscontext,alteredmetabolismofsphingolipidscouldplayanactiveroleintheonsetofcelldamage.Toexplorethis issue, we used human fibroblasts loaded with sucrose as a simple model of lysosomalaccumulation. In sucrose-loaded fibroblasts, we observed a significant increase in lysosomalbiogenesis followed by arrested cell proliferation. Lysosome-related genes were the mostsignificantly enriched among the upregulated transcripts, mainly activated by the nucleartranslocation of TFEB. However, despite induced lysosomal biogenesis we found reducedlysosomalcatabolismandautophagyblockage.Theseconditionsareresponsiblefortheincreasedcontent of several sphingolipid species (i.e. sphingomyelin, glucosylceramide, ceramide, andgangliosidesGM3andGD3)bothintracellularlyandattheplasmamembrane(PM)level.Moreover,weobservedanincreaseinthelysosomalmembraneproteinLamp-1onthePMofsucrose-loadedfibroblastsandagreaterreleaseofthesolublelysosomalproteincathepsinDintheirextracellularmediumcomparedwithcontrols.TheseresultsindicateincreasedfusionbetweenlysosomesandthePM,asalsosuggestedbytheincreasedactivityoflysosomalglycosphingolipidhydrolasesonthePMofsucrose-loadedfibroblasts.Theinhibitionofβ-glucocerebrosidaseandnonlysosomalglucosylceramidase, both involved in ceramide production resulting from glycosphingolipidcatabolismonthePM,partiallyrestoredcellproliferation.Ourfindingsindicatetheexistenceofanewmolecularmechanismunderlyingcelldamagetriggeredbylysosomalimpairment.

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Proteinpurification’smethodaffectstheelectrophysiologicalfeaturesofyeastVDAC2StefanoContiNibali1,AndreaMagrì1,2,MariaCarmelaDiRosa1,ValentinaI.CastruccioCastracani1,LucianoS.Cavallaro1,VitoDePinto11DepartmentofBiomedicalandBiotechnologicalSciences,UniversityofCatania,Catania,Italy;2DepartmentBiological,GeologicalandEnvironmentalSciences,UniversityofCatania,Catania,[email protected] Voltage-Dependent Anion selective Channel (VDAC) represents themost important pore-forming protein family of the Mitochondrial Outer Membrane. VDACs allow the exchanges ofmetabolites(ATP,ADP)andions(Mg,K,Cl)betweencytosolandinnerofmitochondria,playinganimportantroleincellmetabolismandintheregulationofapoptosis[1].TheyeastSaccharomycescerevisiaehastwodistinctgenesencodingfortwoVDACisoforms.yVDAC1,themainandmostabundant porin, shares with human VDAC1 about 70% of sequence homology and the mainelectrophysiologicalproperties (i.e.: channel activityat thePlanarLipidBilayer (PLB), voltage-dependentchannelsof4nSin1MKCl).Duetoitsimportant,cellslackingyVDAC1displaysastronginhibitionofcellgrowthinnon-fermentableconditions,asresultofablockingofmitochondrialmetabolism[2].Onthecontrary,theroleofyVDAC2wasunclearsincefromitsdiscovery.Infact,theabsenceofyVDAC2hasnoeffectoncellgrowth,indicatingthattheproteinisnotinvolvedinmitochondrialmetabolismand,possibly,itisdevoidofchannelactivity.Onlyrecently,yVDAC2wasextracted from yeast mitochondria in native condition and its electrophysiology was deeplyanalyzedat thePLB, revealingaclearpore-formingactivity (i.e.: channelsof3.6nS in1MKClcharacterizedbyareducedvoltagesensitivity)[3].AnalternativestrategyforyVDAC2purification,consisting in the heterologous expression in bacterial system, is presented in this work. Theencoding sequence of yVDAC2 was cloned in expression vector in frame with a 6xHis-tag,expressedinE.coliandpurifiedbyaffinitychromatography.Then,theproteinwasrefoldedinvitroand characterized at the PLB. Our results displayed that the recombinant yVDAC2 maintainssimilarfeaturestothatofnativeonebut,atthesametime,severalimportantdifferencesbetweenthe twoproteins in ionselectivityandvoltagesensitivityhavebeen found.Overall, our resultsconfirmthatyVDAC2isanothermemberofVDACfamilyandthatthemethodsusedforproteinpreparationisdeterminantfortheelectrophysiologicalproperties.[1]DePintoetal,BiochimBiophysActa(1989)987:1-7;[2]Blachly-Dysonetal,MolCellBiol(1997)5727-5738;[3]Guardianietal.,BiochimBiophysActa(2018)270–279.


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PLENARYLECTUREMembranetransportofbilirubinandflavonoids.FromkineticstodietSabinaPassamonti1,FedericaTramer1,LovroZiberna2,MarjanaNovic31UniversitàdiTrieste,DipartimentodiScienzedellaVita,Trieste, Italy;2UniversityofLjubljana,Faculty of Medicine, Department of Pharmacology. Ljubljana, Slovenia; 3 National Institute ofChemistry,LaboratoryofCheminformatics,Ljubljana,Sloveniaspassamonti@units.itBilirubinistheendproductofhemecatabolism,withadailyproductionofabout200–300mginanormaladult.Inthecirculation,bilirubinistransportedbyalbuminasareversiblecomplex,andthenistakenupbytheliver,whichexcretesitasadiglucuronidederivative.Bilirubinuptakeintotheliveristransporter-mediated,asshownbykineticdata.Despitedecade-longeffortstoidentifythehepaticbilirubintransporter,nonehaveyetconfirmedbymulti-levelcharacterisationtofulfilthisfunction.Amongthestudiedentitiesisthebilirubintransporternamedbilitranslocase(BTL;TCDB2.A.65.1.1).TheinvitroBTLtransportassayusesthepH-indicatordyesulfobromophthalein(BSP)as transport substrate.Dietaryanthocyanins (AC), glycosidated flavonoidmolecules thathavephenolic-quinoidaltautomerismasBSP,arestrongcompetitiveinhibitorsofBTLtransport.ACaretransportedintovariouscelltypesexpressingBTL.Similarlytotheextremelyfastuptakeofbilirubinintheliver,ACalsodistributefromthecirculationtothemainorgans,includingthebrain,atanextraordinaryfastrate.Thisremainsoneofthemostcompellingpiecesofevidenceabout theprincipal roleofmembrane transporters indetermining thepatternofbioactivityofdietarycompounds.Thepresentationwillcoverthefollowing:1)overviewofbilirubinmembranetransporters[1];2)StructuraldetailsofBTL[2];3)QSARofBTLsubstrates[3];4)Absorption,distributionandmetabolismofACintherat:focusonkinetics[4];5)FromACkineticstodietandhealth [5]; 6) The current work in our lab and network of collaborators to develop newtechnologiestostudythebilirubin-dietinterplay.[1]Čvorovićetal,FrontPharmacol2017,8,887;[2]ChoudhuryetalPLoSOne.2015;10(8):e0135455;[3]Zuperletal,AnalChimActa2011705(1–2):322–33;[4]Fornasaroetal,SciRep.2016;6:22815;[5]Zibernaetal,FreeRadicBiolMed.2012;52(9):1750–9.


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Session4:NutritionandpathologiesIdentificationof theantigenrecognizedbyRHIGM22,a remyelination-promotinghumanmonoclonalantibody,andhiseffectonglialcellsLivia Cabitta1, SaraGrassi1, SimonaPrioni1, LauraMauri1,MariaGrazia Ciampa1, Yana Zorina2,SandroSonnino1,AlessandroPrinetti11DepartmentofMedicalBiotechnologyandTranslationalMedicine,UniversityofMilano,Milano,Italy;2AcordaTherapeutics,Inc.,Ardsley,[email protected](rHIgM22)bindstomyelinandoligodendrocytes(OLs)andpromotesremyelination in mouse models of Multiple Sclerosis. rHIgM22 preferentially reacts withsulfatidepositive(O4+)OLs[1],andbindingofrHIgM22isabolishedinCNStissueslicesfromCst(-/-) mice [2], suggesting that its binding requires the presence of a product of cerebrosidesulfotransferase,possiblysulfatide,highlyexpressedinOLsandmyelin.Howevertheidentityofthe antigen recognized by this antibody remains to be elucidated. We tested the binding ofrHIgM22topurifiedlipidsandlipidextractsfrommousebrain,CNSmyelin,mixedglialcells,andO4+OLsusingTLCimmunostaining.OurpreliminaryresultsshowthatIgM22bindstosulfatideinvitro,while it doesnot bind to othermyelin sphingolipids suggesting that sulfatide at theOLssurfacemightbe important for thebindingof rHIgM22 to these cells and tomyelin.However,IgM22doesnotbind structures expressing sulfatideoutside thenervous system, so additionalfactorsarelikelyrelevantfortheimmunoreactivityofIgM22inCNS.Indeed,inlipidextractsfromdifferentsourceswefoundanotherlipidantigenselectivelyrecognizedbyrHIgM22.Toattemptthe identificationof the antigen, sampleswerepurifiedusing columnchromatographyand thesecond rHIgM22-immunoreactive band enriched fractions were analyzed by ESI MassSpectrometry. The results obtained led to hypothesize that the unknown antigens could bePhosphatidylinositol(16:0/18:1-PI)andtwodifferentPhosphatidylserinespecies,18:0/22:6-PSand18:0/18:1-PS.Thislipidisalsopresentintheextractsfrommixedglialcultures,whichdonotcontainmatureO4+OLs,suggestingthatotherglialcellsinadditiontoOLsmightbeimportantinthe response to rHIgM22. Furthermore, literature suggests that rHIgM22 biological activity ismediatedbythereorganizationofLyn, integrinαvβ3andPDGFRαat thecellsurface to formasignalingcomplextriggeringLynactivationwhich,inturn,promotesoligodendrocyteprecursorcells (OPCs) survival and proliferation [3]. We assessed the effect of a 24 hours, single dosetreatmentwithrHIgM22onOPC,OLandmixedglialcell(MGC).NosignificantdifferenceinthelipidpatternofMGCtreatedcellswasobserved,whileinOPCandOLtreatedwithrHIgM22thereis an increase inGD3andGM3, supporting thehypothesis that thebindingof rHIgM22on thesurface of OL could elicit biological responses mediated by alterations of lipid-dependentmembraneorganizationand/orsignaling.Wealsoobservedan increasedexpressionofseveralproteins,includingPDGFRα,Lyn,activatedLynandintegrinαVinbothOPCandOL.[1]Howeetal,NeurobiolDis,2004;[2]Wrightetal,ArchNeurol,2009;[3]Watzlawiketal,Glia,2010.

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Human gingival mesenchymal stem cell trophism is modulated by inflammatorymicroenvironment:effectsofRibesnigrumbudextract.Chiara Giacomelli1, Letizia Natali1, Deborah Pietrobono1, Marinella De Leo1,2, Simona Daniele1,FilippoGraziani2,3,AlessandraBraca1,2,M.LetiziaTrincavelli1,2,ClaudiaMartini1,21DepartmentofPharmacy,UniversityofPisa,Pisa, Italy;2CentroInterdipartimentalediRicerca"NutraceuticaeAlimentazioneperlaSalute",UniversityofPisa,Pisa,Italy;3DepartmentofSurgical,Medical,MolecularandCriticalAreaPathology,UniversityofPisa,Pisa,[email protected](MSCs)playacrucialroleinthemaintenanceoftissuehomeostasisand in promoting regenerative processes. Among the different MSC types, the gingivalmesenchymalstemcells(GMSCs)havearisenasapromisingtooltopromotetherepairofdamagedtissuessecretingtrophic,regeneration-promotingmediators.TNF-αisoneofthekeymediatorsofinflammationthatcouldaffecttissueregenerativeprocessesandmodifytheMSCpropertiesininvitroapplication.Herein,weinvestigated1)theeffectsofTNF-alphaonGMSCtrophismand2)theability of Ribes Nigrum bud extract (RBE) to modulate the effect of this cytokine on GMSCproperties. GMSCwere isolated and characterized from health subjects. TNF-α affected GMSCproliferationandtheexpressionofinflammatory-relatedprotein(IL-6,IL-10,TGF-β,andCOX-2)independenceonitsconcentration.AhighTNF-αconcentrationdecreasedtheGMSCviabilityandimpairedthetrophiceffectofGMSCsonendothelialcells,likelybyenhancingtheamountofpro-inflammatorymediatorsinGMSCsecretome.GMSCincubationwithRBEchangedsecretomacellcompositionsorestoringtheGMSCbeneficialeffectsonendothelialviabilityandmotility.TheseresultsdemonstratedthatahighTNF-αconcentration,asoccurredunderchronicinflammatoryconditions, decreased the GMSC well-being and alter their trophic activity impairing GMSC-endothelial cell communication. These data highlight that the control of inflammatorymicroenvironmentiscrucialtoguaranteeMSC-drivenreparativeprocesses.Furthermore,theuseof natural anti-inflammatory agents restored theGMSC regenerative properties on endothelialcells opening the way to the use and the development of natural extracts in wound healing,periodontalregenerationandtissueengineeringapplicationthatuseMSCs.

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Tastesensitivityandbodyweight:istherealink?SonilaAlia1,SofiaPugnaloni1,FrancescaBorroni1,MarinaTaus2,LauraMazzanti1,AriannaVignini1.1 Dipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche and Scuola diSpecializzazioneinScienzedell’Alimentazione,UniversitàPolitecnicadelleMarche,Italy;2UOCdiDieteticaeNutrizioneClinica-AOUOspedaliRiunitidiAncona,Ancona,[email protected](OB),definedasaclinicalconditioncharacterizedbyanincreasedBodyMassIndex(BMI),is becoming a global epidemic in both children and adults. OB is fueled by individual factors,nutritiontransitionandincreasinglysedentarylifestylesthatleadtoexcesscaloricintake.Amongindividualfactors,tastesensitivityplaysanimportantroleinfoodpreferences,choices,andthusconsumption. The present study was conducted to evaluate the relationship between tastesensitivityandBMI,bystudyingtheresponsetotheadministrationofdifferenttastantsubstancesindifferentgroupsofsubjects.Thirtyhealthynormal-weightvolunteers(18femalesand12males,BMI21.6±1.7Kg/m2),nineteenhealthyoverweight (11 females and8males,BMI27.9±1.4Kg/m2)andtwenty-twosubjectswithobesity(18femalesand4males,BMI36.9±5.7Kg/m2)wererecruited.Theywereaskedtoavoideatinganddrinkinganythingexceptwaterforonehourprior to testing, not to smoke, andnot tobrush their teeth. For each subject the lateralizationOldfieldscore,bodyweight,height,andbloodpressureweredetermined.Thetastetestisbasedonfilterpaperstripssoakedwith4tastants,presentedatdifferentconcentrations,evokingthe4basictastequalities(salty,acid,sweet,bitter);purerapeseedoilandwaterwereadministered,evoking fat and neutral taste. The stimuli were applied to each side of the protruded tongue.Patientswereaskedtoidentifythetastefromalistofeightdescriptionsaccordingtoamultipleforced-choice.TheresultshaveshownageneraldecreaseoftastesensitivitywiththeincreaseofBMI, except for fat taste. Other variables affecting the taste sensitivity are the age (negativeassociation),gender(womengenerallyshowhighersensitivity),tastant’sconcentration(positiveassociation).Our findingscouldprovide important insights for thedesignofnew therapies forweight lossand long-termweightmaintenance,andforthecompositionofdietscombiningthecorrectcaloricandnutritionalsupplywiththeindividualtastepreferences.

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Intestinal epithelial barrier abnormalities in patients with chronic intestinal pseudo-obstructionElisaBoschetti1,AnnaAccarino2,CarolinaMalagelada2,FernandoAzpiroz2,RosannaCogliandro1,CatiaSternini4,VincenzoStanghellini1,RobertoDeGiorgio31DepartmentofMedicalandSurgical Sciences,UniversityofBologna,Bologna, Italy;2Digestive-System-Research-Unit,University-HospitalValld'Hebron,Barcelona,Spain;3DepartmentofMedicalSciences,UniversityofFerrara,Ferrara,Italy;4Digestive-Disease-Division,UniversityofCalifornia,LosAngeles,[email protected](CIPO)isarareconditionduetosevereimpairmentofgutmotility responsible for recurrent sub-occlusive episodes. Altghough neuro-muscular-glial-ICCabnormalities represent the main pathogenetic mechanism, the pathophysiology of CIPOremains poorly understood. Intestinal epithelial barrier (IEB) abnormalities can contribute toneuro-epithelial changes by allowing passage of harmful substances. This study aimed to testwhetherIEBabnormalitiesoccurinCIPOpatientsbyanalyzingthejejunalproteinexpressionofthemajorcomponentsoftightjunctions(TJs):occludin,claudin-4andzonulaoccludens-1(ZO-1),asmarkersofIEBintegrity.Wealsoexaminedtheexpressionofvasoactiveintestinalpolypeptide(VIP)andglial fibrillaryacidicprotein(GFAP),asneuronalandglialcellsmarkers.28clinicallycharacterized CIPO patients (15F; 16-75 yrs) were studied and subdivided according to guthistopathology: n=7 with an apparently normal (AN) neuro-muscular layer; n=11 withinflammatory (INF) changes throughout the neuro-muscularwall and n=10with degenerativeneuro-muscular alterations (DEG). N=8 (3F, 48-73 yrs) non-CIPO subjects undergoing surgeryserved as controls. Protein expression was evaluated on jejunal full thickness biopsies withWestern Blot. Total occludin was significantly decreased in the intestine of CIPO patients vs.controls (P=0.002), particularly in AN (P=0.007) and INF (P=0.004) subgroups; ZO-1 and VIPexpression was decreased selectively in DEG (P=0.015 and P=0.0305). Occludin/claudinoligomers,anindexofTJsassembly,wereabsentin81%ofCIPO(P<0.0001)patients.Claudin-4wasupregulatedinCIPO(P=0.070),particularlyinINF(P=0.050)andDEG(P=0.044)groups.GFAPresultedubiquitously increased inCIPO(P<0.001).Theabsenceofoccludin-claudinsoligomersindicates IEB abnormalities in CIPO patients and provides the morphological basis for thehypothesis of noxious agents passing through the intestinal wall. It is likely that barrierdysfunction in AN and INF is occludin dependent, while in DEG is ZO-1-dependent. IEBabnormalitiesmightaffectneuronalandglialcells.

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Lactobacillus crispatus interferes with Chlamydia trachomatis infectivity throughmodulationofintegrinexposureincervicalcellsGiulia Frisco1, Giorgio Sartor1, Carola Parolin1, Beatrice Vitali1, Antonella Marangoni2, ClaudioFoschi2,NataliaCalonghi11DepartmentofPharmacyandBiotechnology,UniversityofBologna,Bologna,Italy;2Microbiology,DIMES,UniversityofBologna,Bologna,[email protected] women, urogenital CT infections are often asymptomatic, thus remaining unnoticed anduntreated. This can lead to complications and sequelae including pelvic inflammatory disease,tubal infertility and ectopic pregnancy [1,2]. A normal vaginal microbiota, dominated bylactobacilli,iscrucialforthepreventionofseveralurogenitalandsexuallytransmittedinfections,including Chlamydia [3-5]. This aspect is strengthened by the demonstration that in case ofbacterialvaginosis,aclinicalconditioncharacterizedbythedepletionoflactobacilli,ahigherriskofSTItransmissionandacquisitionisreported[6].Thisstudyaimedtoelucidatethemolecularbases of the interaction among lactobacilli, Chlamydia trachomatis and epithelial cells. Weevaluated the capacity of lactobacilli cells and supernatants to interfere with C. trachomatisinfectivity in HeLa cells, by means of competition, exclusion and displacement mechanisms.Lactobacillicellswerethemostactivefraction,bymeansofanexclusionstrategy.Weinvestigatedthe potential mechanism of protection in Lactobacillus crispatus BC5 (model strain), and wedemonstratedthattheincubationofHeLacelllinewithBC5cellsinducesimportantmodificationsat the level of the epithelial plasmamembrane, by altering lipid composition and α5 integrinsubunit exposure. When α5 integrin subunits were masked by a specific blocking antibody,Chlamydia infection was precluded. α5 integrin subunit is thus crucial for the pathogenpenetration into HeLa cells, and the anti-Chlamydia activity of BC5 can be directly linked tomembrane propertiesmodifications in epithelial cells. In conclusion, we identified a potentialmolecularmechanismatthebasisoftheprotectionexertedbyLactobacillusagainstthesexuallytransmitted pathogen Chlamydia trachomatis, getting insights into the role of the vaginalmicrobiotaforthewoman’shealth.[1]Priceetal,AmJEpidemiol2013;178:484–92;[2]Menonetal,ClinMicrobiolRev2015;28:969–85;[3]Nardinietal,SciRep2016;6:29024;[4]ÑahuiPalominoetal,FrontMicrobiol.2017May19;8:906;[5]Foschietal,FrontCellInfectMicrobiol.2017Dec6;7:502;[6]Wiesenfeldetal,ClinInfectDis2003;36(5):663-8.

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Effectof9-hydroxy-stearicacidonglucosemetabolisminahumancoloncancercelllineChristianBergamini1,CarlaBoga2,RomanaFato1,DianaFiorentini1,GiuliaFrisco1,LucaMasin1,GiorgioSartor1,LauraZambonin1,NataliaCalonghi11DepartmentofPharmacyandBiotechnology,UniversityofBologna,Bologna,Italy;2DepartmentofIndustrialChemistry,UniversityofBologna,Bologna,Italynatalia.calonghi@unibo.itRecentfindingsidentifiedanewclassofendogenouslipids,branchedFattyAcidestersofHydroxyFattyAcids(FAHFAs),abletobehaveasspecificsignalingmoleculesthatcanregulatethecellularmetabolism [1].AmongFAHFAs, thePalmitic-Acid-9-Hydroxy-StearicAcid (9-PAHSA)seems toexert favorable metabolic effects in obesity-related diseases and type 2 diabetes, causing anincreaseininsulinsensitivityandglucoseuptake.IthasbeenrecentlyreportedthattheFAHFAscontentinhumanserumofbreastcancerpatientswassignificantlydecreasedcomparedtohealthycontrols [2], thus it is very interesting to investigate the possible involvement of these lipidmoleculesalsoincancertransformationandprogression.Previousstudiesofourresearchgrouphaveshownthat theadministrationof9-hydroxy-stearicacid (9-HSA) tocoloncarcinomacells(HT29)inducesstrongantiproliferativeanddifferentiatingeffects,withacellcyclearrestinG0/G1phase[3].Since9-HSAcanbeproducedbythehydrolysisof9-PAHSA,itisconceivablethatalsothis lipid could act as signaling molecule. Consequently, the aim of this research was thecharacterizationoftheglucosemetabolismofHT29cellstreatedwith9-HSA.Asafirststep,theeffectof9-HSAonthe lipidorganizationofHT29cellswasstudied,showingan increaseof thefluidityofplasmamembrane.ThetreatmentofHT29with9-HSAfor1hprovokesasignificantincreaseoftheglucosetransporterGlut1(andinalesserextentofGlut3)ontheplasmamembrane,as a result of a translocation from intracellular stores, since the level of expressionof the twotransporterswereunchanged,asdeterminedbyRT-PCR.Accordingly,thehigheramountofGlut1onthecellsurfacecausedalsoanincreaseinglucoseuptakeintothecells.RT-PCRanalysisshowedalsoasignificantincreaseofMCT1,themonocarboxylatetransporter,inHT29cellstreatedwith9-HSA.MCT1iscommonlyoverexpressedbycancercellstomaintainlactateandpHhomeostasis[4]. Therefore, lactate productionwasmeasured in HT29 cells upon 9-HSA treatment for 1 h.Resultsshowthat lactateproductionwassignificantlyincreased, indicatingacellularmetabolicshifttowardglycolysis.TheacutemetabolicchangesobservedinHT29cellsaretypicalcellularresponsestoasignalmolecule,supportingthehypothesisofasignalingrolefor9-HSAincancercells.[1]Yoreetal,Cell,159:318-332,2014;[2]Zhuetal,J.Chromatogr.B,1061-1062:34-40,2017;[3]Calonghietal,Biochem.Biophys.Res.Commun.,314:138-142,2004;[4]Adijanto&Philp,Curr.Top.Membr.,70:275-311,2012.

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Evaluationofendothelialdysfunctionmarkersinchildrenwithcardiovascularriskfactors:obesityand/orhypertensionAntoninaOrlando1,SimonettaGenovesi1,3,MarcoGiussani2,PaolaPalestini1,EmanuelaCazzaniga11DepartmentofMedicineandSurgery,UniversityofMilanoBicocca, Italy;2FamilyPediatrician,Milano, Italy; 3 Department of Cardiovascular, Neural and Metabolic Sciences, S. Luca Hospital,IRCCS,IstitutoAuxologicoItalianoantonina.orlando@unimib.itCardiovasculardiseases(CVDs),responsibleformorethan30%ofannualdeaths,aretheleadingcauseofdeathworldwide[www.who.int].Thesediseasesarisefromasetofriskfactorsthatcanoccurevenatanearlyage.Inthepediatricpopulationincorrectbehaviorsandeatinghabitsleadtopathologiessuchasobesityandpredisposetocardiovasculardamage.Obesityisassociatedwitha number of complications such as hypertension, hyperuricaemia, dyslipidaemia and insulinresistance,which,ifnottreated,exposethechildtoahigherriskofdevelopingCVDsatayoungage[BridgerT.,2009;DanielsS.,2011].EndothelialdysfunctionisanotherriskfactorforCVDs.Thispathologicalconditionischaracterizedbyareducedbioavailabilityofvasodilators,inparticularnitricoxide(NO),andanincreaseinvasoconstrictionfactorsproducedbytheendothelium(suchas endothelin-1) [Lerman A. 1992]. The cause of endothelial dysfunction resides in theinflammatory state of adipose tissue that is triggered in thepresenceof excessweight; in thiscondition,theadiposetissueundergoesamacrophageinfiltrationwhichinducestheproductionofpro-inflammatorymolecules[TranB.,2012].Atthelevelofthewallofbloodvessels,itseemsthatthisinflammationoriginatesaconditionofoxidativestressthatdamagestissuehomeostasisanddeterminestheonsetofendothelialdysfunction[VanGaalL.,2006].Inparticular,adepletionofNOand/oraconcomitantincreaseinvasoconstrictorssuchasendothelin-1inducesanalterationofthereleasecapacityinthesmoothmusclecellsofthevesselsandfavourstheincreaseinarterialpressure[ShulzE.,2008].Thisconditionofvasoconstrictionisinitiallyreversible,butovertimeitstabilizesanddeterminestheonsetofhypertension[BleakleyC.,2015].Giventhesepremises,theaimofthisworkistostudytheassociationbetweentheconditionofendothelialdysfunctionandthepredisposingfactorscardiovascularrisksuchasweightexcessandhypertensioninapediatricpopulation.WeevaluatetheproductionofNOandthereleaseofendothelin-1inplasmasampleofachildrenpopulationattendingaClinicforCardiovascularRiskAssessment(Milano).Resultsshowthatthemarkersofendothelialdysfunctionarerelatedto'excessweightmorethanhypertensionand correlate with metabolic alterations such as hyperuricemia and insulin resistance. Inconclusion, the cardiovascular risk factors associatedwith an altered endothelial function arealreadypresentinpediatricage,howeversincethechildisagrowingorganismthismechanismcouldbereversible.

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IsNotchinvolvedinprotectiveeffectsofestrogenonendothelialfunctionandangiogenesis?CristianaCaliceti1,FrancescaFortini2,FrancescoVieceli2,GiorgioAquila3,4,DonatoCalabria1,AldoRoda1,AntonioPannuti5,LucioMiele5,RobertoFerrari2,3,,4,PaolaRizzo2,4,61 Department of Chemistry, University of Bologna, Bologna, Italy and National Institute ofBiostructuresandBiosystems(INBB),Roma,Italy;2MariaCeciliaHospital,GVMCareandResearch,E.S.Health ScienceFoundation, Cotignola, Italy; 3Departments ofMedical Sciences,University ofFerrara,Ferrara,Italy;4LaboratoryforTechnologiesofAdvancedTherapies(LTTA),UniversityofFerrara, Ferrara, Italy; 5 Stanley Scott Cancer Center, Louisiana StateUniversityHealth SciencesCenterandLouisianaCancerResearchConsortium,NewOrleans,USA;6DepartmentofMorphology,Surgery,UniversityofFerrara,Ferrara,[email protected] age-matched men, premenopausal women benefit from cardiovascular protection. Themechanismsofactionarestillpoorlyunderstood,althougharoleforestrogensinstimulationofangiogenesisandprotectionagainstapoptosisofendothelialcells(ECs),hallmarkofendothelialdysfunction,havebeensuggested. Inestrogen receptor (ER)αpositivebreast cancer cells,17-estradiol(E2)treatmentinhibitsNotch1activity,howeverlittleisknownregardingtheroleofE2inNotchsignalinginendothelium.Theaimsofthisstudyweretoestablishi)whetherestrogensmodulateNotchactivityinendothelialcellsandii)thepossibleconsequencesofthismodulationonendotheliumfunctions.TreatmentwithE2activatesNotchsignalinginhumanumbilicalveinendothelialcells(HUVECs),effectcounteractedbyERsantagonistICI182.780,suggestingthatE2modulation of Notch1 ismediated by ERs. Our datawere in contrastwith findings of Notch1inhibitionbyE2treatmentinERαpositivebreastcancercells;sinceECsdifferentlyfrombreastcancercellsexpresshighlevelsofERβ,wehypothesizedthattheseoppositeresultscouldbeduetothedifferentactivityofthetwoformsofERsonNotch1.TreatmentwithERβspecificagonist(DPN)butnotwithERαspecificagonist(PPT)inducedactivationofNotch1inHUVECs.Wenextevaluated the role of ER-Notch1 axis in angiogenesis andTNFα-induced endothelial apoptosis.Notch1inhibitionincreasedECssprouting,effectabolishedbyE2,evaluatedbyatubeformationassayon3DMatrigelandinmouseaorticringexplants.Moreover,DPNbutnotPPTcounteractedthe increase in ECs sprouting caused by Notch inhibition, suggesting that ERβ but not ERα isinvolved inNotch1modulation.TNFα reduced the levelsof activeNotch1protein,whichwerepartiallyrestoredbyE2treatment.E2counteractsTNFα-inducedapoptosis,effectabrogatedwhenNotch1 is inhibited, whereas ectopic overexpression of Notch1 diminished TNFα-inducedapoptosis.Moreover,theE2-mediatedregulationofthelevelsofactiveNotch1wasabrogatedaftersilencingER,abolishingtheeffectofE2onapoptosis.Interestingly,DPNtreatmentantagonizedTNFα-induceddecreasedofcleavedNotch1andapoptosis.Insummary,ourresultsindicatethatE2 requires active Notch1 through a mechanism involving ERβ to regulate angiogenesis andprotect the endotheliumagainstTNFα-induced apoptosis. These findings couldbe relevant forassessingtheefficacyandapplicabilityofmenopausalhormonetreatment,becausetheysuggestthatreducedlevelsofNotch1signalingmayinterferewiththeprotectiveactionofhormoneontheendothelium.

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Participants

Alberghina,Lilia [email protected]

Algieri,Cristina [email protected]

Alia,Sonia [email protected]

Angeloni,Cristina [email protected]

Baracca,Alessandra [email protected]

Barbalace,MariaCristina [email protected]

Boschetti,Elisa [email protected],Alessandra [email protected]

Cabitta,Livia [email protected]

Caliceti,Cristiana [email protected]

Calonghi,Natalia [email protected]

Carrozzini,Tiziana [email protected]

Cazzaniga,Emanuela [email protected]

ContiNibali,Stefano [email protected]

Corsetto,PaolaAntonia [email protected]

Costanzini,Anna [email protected]

DaPozzo,Eleonora [email protected]

Damiani,Chiara [email protected]

Daniele,Simona [email protected]

DePinto,Vito [email protected]

Fiorentini,Diana [email protected]

Franceschini,Nicola [email protected]

Frisco,Giulia [email protected]

Gaglio,Daniela [email protected]

Galluccio,Michele [email protected]

Giacomelli,Chiara [email protected]

Giusti,Laura [email protected]

Hreli,Silvana [email protected]

Indiveri,Cesare [email protected]

Isani,Gloria [email protected]

Lonati,Elena [email protected]

Lucacchini,Antonio antonio.lucacchini@gmailcom

Magrì,Andrea [email protected]

Malaguti,Marco [email protected]

Marabotti,Anna [email protected]

Marrazzo,Pasquale [email protected]

Martelli,Pierluigi [email protected]

Martini,Claudia [email protected]

Necci,Marco [email protected]

Nesci,Salvatore [email protected]

Orlando,Antonina [email protected]


39

Pagliarani,Alessandra [email protected]

Passamonti,Sabina [email protected]

Pietrobono,Deborah [email protected]

Pittalà,MariaGiovannaG. [email protected]

Pochini,Lorena [email protected]

Prata,Cecilia [email protected]

Pugnaloni,Sofia [email protected]

Rizzo,AngelaMaria [email protected]

Samarani,Maura maura.samarani@unimi

Sartor,Giorgio [email protected]

Solaini,Giancarlo [email protected]

Tioli,Gaia [email protected]

Tolomeo,Maria [email protected],MariaLetizia [email protected]

Trombetti,Fabiana [email protected]

Turina,PaolaM. [email protected]

Zinghirino,Federica [email protected]

bio-energetics, metabolism and nutrition: from molecules ... · a proteomic approach to study the...

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