vol 83(1) (2018) communications in agricultural and … · 2018. 12. 11. · vol 83(1) (2018)...
TRANSCRIPT
VOL83(1)(2018)
COMMUNICATIONSINAGRICULTURALANDAPPLIEDBIOLOGICALSCIENCES
Publishers
PROF.GUYSMAGGHEPROF.STEVENSLEUTELPROF.JANBAETENS
DR.CHRISCALLEWAERT
Editorialaddress
Coupurelinks6539000Gent(Belgium)
ISSN1379-1176
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Theresultspublishedinthisbookofabstractsareunderthefullresponsibilityoftheauthors.Theorganizingcommitteecannotbeheldresponsibleforanyerrorsinthispublicationandpotentialconsequencesthereof.
PROCEEDINGS
23RDNATIONALSYMPOSIUMONAPPLIEDBIOLOGICALSCIENCES
FACULTYOFSCIENCE
ANDBIOSCIENCEENGINEERINGVUB
FEBRUARY8TH,2018
Websitehttp://www.vub.ac.be/events/nsabs2018-conference#nsabs-2018
ORGANIZINGCOMMITTEEWimDeMalsche(VUB),JulienCousin-Saint-Remi(VUB),KrisPauwels(VUB),ChristianHermans(ULB),BenoitScheid(ULB)VrijeUniversiteitBrussel–hostinstitute2018:WimDeMalsche,JulienCousin-Saint-Remi,KrisPauwelsUniversitéLibredeBruxelles–hostinstitute2018:ChristianHermans,BenoitScheid,DavidCannella,JulienLouvieuxUniversiteitGent:JanBaetens,ChrisCallewaert,StevenSleutelKULeuvenBarbaraDeConinck,BramVandenBergh,ElineVanuytrecht,LiesbethFrançois,MariyaPetrovaUniversiteitAntwerpen:JelleHofman,DieterVandenheuvelUniversitédeLiège:JérômeBindelle,HélèneSoyeurtUniversitéCatholiquedeLouvainValentinCouvreur,XavierDraye,GauthierLequeueUniversitédeMonsSylvainGabrieleREVIEWERCOMMITTEEKULeuven:BarbaraDeConinck,BramVandenBergh,ElineVanuytrecht,LiesbethFrançois,MariyaPetrova,TreesDeBaerdemaeker,NielsDeBrierUniversiteitAntwerpen:JelleHofman,TomTytgat,DieterVandenheuvel,ErikVanEynde,SammyVerbruggen,KarenWuytsUniversiteitGent:JanArends,JanBaetens,ChrisCallewaert,AislingDaly,TimDepraetere,NicoDeStorme,JoachimSchouteten,MajaSimpraga,StevenSleutel,MichielStock.UniversitéCatholiquedeLouvain:XavierDraye,ValentinCouvreur,SixtinePassot,GauthierLequeueUniversitédeLiège:HélèneSoyeurt,JérômeBindelle,AngéliqueLéonard,Jean-MicelRomuee,BenoitMercatoris,MagaliDeleu,GeorgesC.LognayUniversitéLibredeBruxelles:DavidCannella,FredericDebaste,BenoitHaut,ChristianHermans,BenoitScheid,GuillaumeSmitVrijeUniversiteitBrusselGeertAngenon,JulienCousin-Saint-Remi,WimDeMalsche,PierreGelin,FrederickLeroy,FrederickMatheuse,KrisPauwels,YannSterckx,IngeVanMolle,WimVranken
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ACADEMICSUPPORTTheNationalSymposiumonAppliedBiologicalSciences(NSABS2018)isjointlyorganizedbythefollowinginstitutes:
- FacultyofScienceandBioscienceEgineering,VrijeUniversiteitBrussel- UniversitéLibredeBruxelles- FacultyofBioscienceEngineering,KULeuven- DepartmentofBioscienceEngineering,UniversityofAntwerp- FacultyofBioscienceEngineering,GentUniversity- FacultyofBioscienceEngineering,UniversitéCatholiquedeLouvain- EarthandLifeInstitute,UniversitéCatholiquedeLouvain- UniversitédeLiège
WEBSITEhttp://www.vub.ac.be/events/nsabs2018-conference#nsabs-2018ACADEMICANDGOVERNMENTALFINANCIALSUPPORTTheorganizingcommitteeofNSABS2018isgratefulforthefinancialandlogisticsupportbythefollowinginstitutesandtheirdoctoralschools:
- KULeuven- UAntwerp- VUB- UGent- ULB- UCL- ULg- UMons- UGentDoctoralSchools(Bioscience)Engineering- YouReCa- FNRSGraduateSchoolonEnvironmentalSciences,TechnologiesandManagement
(ENVITAM)- FNRSGraduateSchoolonAgriculturalSciencesandBiologicalEngineering(EDT-SCAIB)- FNRSGraduateSchoolinPlantSciences- Vlaamseoverheid- VlaandereninActie- RichtingMorgen(Vlaamseoverheid)
ENVITAM
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INDUSTRIALSPONSORSNSABS2018receivesthegeneroussupportoftheErnestSolvayFund(KingBaudouinFoundation)andoursponsors:
Gold sponsor
Silver+ sponsor
Silver sponsors
Bronze+ sponsor
Bronze sponsors
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PREFACE
With great pleasure the organizing committee presents you the proceedings of the 23rdNationalSymposiumonAppliedBiologicalSciences(NSABS2018).
ThissymposiumhasitsrootsinGhent,wherethefirstconferencewasorganizedin1995byGhentUniversity.Inthefollowingyears,otherBelgianuniversitieshavejoinedincontributingtotheconference.CurrentlyKULeuven,UAntwerp,UGent,ULg,UCL,UMONS,ULBandVUBare involved in reviewing, programming, promoting and financially supporting theconference.Themainpurposeoftheconferencehasbeenpreservedthroughouttheyears:to explore and to bring together the Belgian expertise in the field of Applied BiologicalSciences,focusingmainlyoncontributionsofPhDstudents.
Thisyears’NSABSisorganizedforthefirsttimeinBrussels,hostedbyboththeFlemishandFrenchspeakingFreeUniversityofBrussels,VUBandULB.Inthecurrenteditiontheemphasisis put on ‘crossing borders’ and a number of incentives have been taken to endorse thistheme.Next to best poster and presentation prizes, also thematic prizes are awarded tocontributions that specifically focus on and excel in multidisciplinarity, sustainability andvalorization.Thesetopicswillalsobeaddressedbyseveralrenownedspeakerswithinspiringstories,whilecareerperspectivesforyoungscientistswillbeputinthespotlightaswell.
Thecoreoftheconferenceremainsinthehandsofyoungresearchers,thefloorisgiventoPhDstudentsfor30oraland106posterpresentations.
We hope that you will enjoy NSABS 2018 and we look forward to a fruitful, interactive,informal,enjoyable,open-mindedandmemorableexperience.
Prof.Dr.ir.WimDeMalsche
Onbehalfoftheorganizingcommittee
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PROGRAMNSABS2018
23rdNationalSymposiumforAppliedBiologicalSciences(organizedbyVUBandULB)
08:30 Registrationandwelcomingcoffee(N.MandelaRoom)09:10 Openingconference(AulaA)
09:25PlenaryI(AulaA)
AlbertVandenBerg(UTwente,NL)“FromLabonaChiptoOrgansonChip”
Parallelsessions1AulaB AulaC AulaD
ChemistryandChemicalEngineering Biomathematics EnvironmentI
10:10 O1 O11 O2110:30 O2 O12 O2210:50 O3 O13 O2311:10 Coffeebreak(N.MandelaRoom)
Parallelsessions2AulaB AulaC AulaD
AgricultureandFoodTechnologyI HumanHealthI Key-enabling
Technologies
11:35KeynoteI
TimStakenborg(IMEC)
KeynoteIIStéphanieDescroix(InstitutCurie,FR)
KeynoteIIIDavidCannella(ULB)
12:05 O4 O14 O2412:25 O5 O15 O2512:45 O6 O16 O2613:05 Lunchandpostersession1(N.MandelaRoom)
14:05PlenaryII(AulaA)
LuciaSmit(Braingain)"HowtomanageyourcareerasaPhD:challengesandopportunities"
Parallelsessions3AulaB AulaC AulaD
AgricultureandFoodTechnologyII HumanHealthII EnvironmentII
14:40 O7 O17 O2715:00 O8 O18 O2815:20 O9 O19 O2915:40 O10 O20 O3016:00 Coffeebreakandpostersession2(N.MandelaRoom)17:00 PlenaryIII(AulaA)
FrédéricvanGansberghe(Galacticsa/nv)“Andnow,what’snext?”
17:30 Closingremarksandawards(AulaA)17:45 Networkingreception(N.MandelaRoom)
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ORALPRESENTATIONS
ChemistryandChemicalEngineering
O1.PierreGelin(VUB).UseofacousticsinmicrofluidicReactors.O2. Sander Stroobants (VUB).Anoveldevice for studyingnucleationof crystals andaggregates incontrolledshearflow.O3.HennieValkenier (ULB).Crossing theborder:organicmolecules transport chlorideacross lipidbilayers
AgricultureandFoodTechnologyI
Keynote I. Tim Stakenborg (IMEC). New Developments in Silicon Nanotechnology for HealthcareApplications.O4. Julie Vanderstraeten (UGent). Designer cellulosomics for a customized conversion oflignocellulosicbiomasstovaluablebulkandfinechemicals.O5. Kamal Belhaj (UniversityMohammed 1st,Morocco). The effect of tow traditionalMoroccan'scookingmethodsonOmega-6/omega-3andfattyacidshypo/hypercholesterolemicratiosofBeni-GuillambmeatbreedinginnortheastofMorocco.O6.IlseVanhoutte(UGent).Microbialdetoxificationofthemycotoxindeoxynivalenol.
AgricultureandFoodTechnologyII
O7.ElienLemmens(KUL).Theimpactofcontrolledsteepingandgerminationofwheatonhydrolysisofphytateandarabinoxylanandonmineralbio-accessibility.O8. Inga Tarasenko (UCL). The immediate response of tomato and rice to tephra deposition: anexperimentalstudy.O9.MarieVandermies(ULg).CatabolicselectablemarkereasesgenomeeditinginYarrowialipolytica.
Biomathematics
O11.MauricioMacossayCastillo(VUB).Proteinintrinsicdisorder:adouble-edgedsword?O12.ThomasSchalck (KUL). Identificationofcausalmutationsconferringhigh-ethanol tolerance inEscherichiacoli.O13.AuroreWoller (ULB).Metabolicsyndromeduetoconflictsbetween lightandfood:astoryoftwistedclocks.
HumanHealthI
KeynoteII.StéphanieDescroix(InstitutCurie,FR).Microfluidicsforbiomimetism:from3Dco-culturetogutonchip.O14.XeniaGeeraerts(VUB).Theimpactoflactateonthemetabolicphenotypeoftumor-associatedmacrophages.O15. Gaétan Kalala (ULg). Effect of a diet rich in prebiotic fibers of inulin type and behavior onintestinalhealthinobesepatientsinanhumaninvitrofermentationpatternsO16. Pieter De Bruyn (VUB). Biophysical characterization of a three-component toxin-antitoxinmodulefromthepathogenicbacteriumEscherichiacoliO157.
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HumanHealthII
O17. Hans Gerstmans (UGent). High throughput generation of designer enzybiotics againstAcinetobacterbaumannii.O18.RobertAdamuShey(ULB).PredictionandvalidationofthestructuralfeaturesofOv-58GPCRasanimmunodominantantigenofOnchocercavolvulus:implicationsinonchocerciasiscontrolefforts.O19.FrédéricGoormaghtigh(ULB).Single-Cellimagingofbacterialpersisters.O20.AndreyKossarev(KUL).Shuttlevectorsystemforimprovedpestiviralreversegenetics.
EnvironmentI
O21.SteveHuysman(UGent).UHPLC-HRMSbasedtargetedanduntargetedscreeningofplasticizersinthemarineenvironment.O22.RosanneReitsema(UA).Effectsofclimatechangeonmacrophytegrowth,biomassallocation,physiologyandnutrientstoichiometry.O23.HeleenDeroo(UGent).ProcessesofCH4emission inpaddysoilunderwater-saving irrigationmanagement.
EnvironmentII
O27.ValérieVanEesbeeck (SCK-CEN/UGent).Assessing themicrobiologicaldiversity in thecoolingwatersofanuclearresearchreactor.O28.VereLeybaert(KUL).Quantifyingprecipitationerrorsviasoilmoisturedataassimilation.O29.FrancisVanryckeghem(UGent).ScreeningoftheBelgianPartoftheNorthSeatowardsemergingorganicmicropollutants–comparisonoftwoSPE-techniquespriortoUHPLC-Orbitrap-HRMSanalysis.O30. Myrsini Sakarika (UA). Bridging resource recovery to reuse: microbes as next-generationfertilizer.
Key-enablingTechnologies
Keynote III. David Cannella (ULB). Light powered enzymatic degradation of lignocellulose: a newphoto-bio-catalysisforfuelsandchemicals.O24.PaulineHerpels(KUL).PhotoreactivecrosslinkingrevealsnoveldirectinteractionpartnersofthepersisterregulatorOBG.O25.ShuntaFutagami(VUB).TheEffectoftheLayerThicknessonthePerformanceofPorousLayeredRadiallyElongatedPillarAllayColumns.O26. Marie Versaevel (UMons). MatrXcell: improving cell culture assays with tunable hydrogelrigidities.
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PLENARYTALKS
ALBERTVANDENBERG(UTWENTE,NL)"FROMLABONACHIPTOORGANSONCHIP"
AlbertvandenBergobtainedhisPhD inappliedphysics in1988at theUniversityofTwente
(UTwente,Netherlands).Afterwards,heworkedonminiaturizedchemicalsensorsattheCSEMcenter
andIMTinstituteinNeuchatel(Switzerland).From1993until1999hewasresearchdirectoratMESA
(UTwente). After a part-time professor position “Biochemical Analysis Systems”, in 2000 he was
appointed as full professor on Miniaturized Systems for (Bio)Chemical Analysis in the faculty of
ElectricalEngineeringandpartoftheMESA+InstituteforNanotechnology(UTwente).Heisco-author
ofmore than 400 peer reviewed publications (H=51) and obtained several prestigous awards and
honorssuchastwoERCAdvanced(2008,2015)andERCProofofConcept(2011,2013)grants,Spinoza
prize(2009)andboardmemberoftheRoyalDutchAcademyofSciences(KNAW,2011).
LUCIASMIT"HOWTOMANAGEYOURCAREERASAPHD:CHALLENGESANDOPPORTUNITIES"
Dr. Lucia Smit is founder of Braingain, organization sociologist and certified professional co-
active coach (CPCC). She started her career as management consultant for companies and
governm4ents. Afterwards, as a member of the OESO work group she studied the careers of
researchers. From2007, Lucia guides professionals at developping their ambitions into theirwork
environmentandjobmarket,withastartingpointbeingtheenhancementoftheirself-knowledge.
(Braingain,https://www.braingain.be/)
FRÉDÉRICVANGANSBERGHE“ANDNOW,WHAT’SNEXT?”
Mr.FrédéricvanGansbergheisCEOatGalacticSA/NV,ChairmanatCompagnieduBoisSauvage
SAandPresidentatGalactic,Inc.Hegraduatedaselectro-mechanicalengineerfromECAMandgota
post-graduatedegreeinbiotechnologyatUCL.
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Lecturesession
CHEMISTRYANDCHEMICALENGINEERING
2
USEOFACOUSTICSINMICROFLUIDICREACTORS
PierreGelin1,DominiqueMaes1,WimDeMalssche1
1VrijeUniversiteitBrussel,Belgium
Microfluidicsisanupcomingtechnologyinawidevarietyoffieldsincludingbiology,chemistry
andmedicine.InthesesmallchannelstheReynoldsnumberisverylow(Re<1).AttheselowRevalues,
viscositydominatesover inertiaensuringpredictable laminar flows,whichprovidesanexceptional
controloverthehandledfluidstreams.Thismakesitpossibletocontinuouslyproducehighlyuniform
droplets and particles. Continuous and high throughput production of micron size particles is
becomingmoreandmorerelevantevenintheindustry.Particlematurationisoftenoneofthelimiting
factor.Fortheproductionofpoly(lactic-co-glycolicacid)(PLGA)microparticlesforexample,therate
limitingstepisthesolventextractionfromtheproduceddroplettoyieldthefinalparticle.
Theuseofstandingacousticwavesinmicrofluidicdevicesgivesthepossibilitytoenhancethe
masstransferratefarbeyondtherateofdiffusion.Acousticstreamingisaflowphenomenonoccurring
whenanacousticwavepropagatesthroughanenclosedfluid.Thesestreamingflowstaketheformof
vorticestransversetothemicrofluidicchannel.However,whenparticlesaresuspendedinaliquida
secondphenomenonoccurs,namelyacousticradiation.Acousticradiationarisesfromthescattering
of the soundwave on the particle, this pushes it towards the pressure node. Both phenomena,
radiationandstreaming,havebeeninvestigatedinthiscontribution.Forlargeparticles(5µm)radiation
dominates,whileforsmallparticles(0.5µm)streamingdominates.
CFDsimulationshavebeenperformedtogetinsightinthestreamingvelocityfieldsinsideahalf
wavelengthchannelforachanneldepthof100µm.Dependingontheappliedenergytothechannel,
velocitiesofapproximately50µm/scouldbereached,showingaveryfastmixingoftwostreams.This
has also been shown experimentally with fluorescence experiments. Tracking of small particles
revealed that thevortex flowsbehaveaspredictedandconform that theboundaryconditionsare
realistic. Large particle displacement distances and velocities have also been investigated
experimentallyasafunctionoftheappliedenergyandfrequency.Veryfastfocussingofparticleson
thepressurenodehasbeenshown.Theuseofboththesephenomena isofgreatrelevance inthe
productiononmicroparticles.Streamingcanbeusedtoenhancetheformationoftheparticleswhile
radiationenablestofocusandfurtherconcentratethedesiredparticles.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
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ANOVELDEVICEFORSTUDYINGNUCLEATIONOFCRYSTALSANDAGGREGATESINCONTROLLEDSHEARFLOW
SanderStroobants1,ManlyCallewaert1,SudhaChinnu1,
WimDeMalsche1,DominiqueMaes1
1VrijeUniversiteitBrussel,Belgium
Nucleation, the first step in any first-order phase transition, is one of the most secretive
processes in chemistry, physics, materials science, biophysics and biotechnology; predictions of
nucleation rates and yields often deviate from reality by several orders ofmagnitude. It iswidelyacceptedthatthenucleationofproteincrystalsandorderedaggregatesfollowatwo-stepmechanism:
inafirststep,clustersofdenseproteinliquidareformed,withinwhich,inthesecondstep,ordered
nucleiform[1,2].Becauseitisthefirstphaseofthecrystallization/aggregationprocess,nucleation
controlsnumerouspropertiesoftheemergingphase.NucleationalsounderliestheformationoffibrilsandaggregatesobservedindiseasessuchasAlzheimerandcataract.
Boththeory[3]andexperiments [4]have indicatedthatshear flowduringcrystallizationwill
affect the nucleation rate, the size of the emerging phase and even polymorphism. To further
investigatethisinfluence,wehavedevelopedanewsetupthatwillenableustocreateacontrolledconstantshearflowprofileinasolution.Theequipmentconsistsofastationaryglassplate;afused
silica wafer with microfluidics channels connected to a rotation stage and Peltier elements for
temperature control. Tomonitor the crystallization an invertedmicroscope setup is usedwith theoptionofaddingfiltersintheopticalpathway,enablingfluorescentmicroscopy.
Thewaferwillrotatewithrespecttothestationaryglassplate,thiswillresultinaconstantshear
flow in the channels. Simulation performed with COMSOL Multiphysics 5.0 [5] confirm these
assumptions and characterize the deviations from the ideal profile at the walls and inlets. Thenucleationandgrowthcanbecontrolledbychangingtemperature,assuchthenucleationphasecan
beseparatedfromthegrowthphase,enablingthestudyoftheeffectofshearflowduringbothphases.
References
[1]D.Maesetal.,“Doproteincrystalsnucleatewithindenseliquidclusters?,”ActaCrystallogr.Sect.FStruct.Biol.Commun.,vol.71,no.Pt7,pp.815–822,Jul.2015.
[2]M.A.Vorontsova,D.Maes,andP.G.Vekilov,“Recentadvancesintheunderstandingoftwo-stepnucleationofproteincrystals,”FaradayDiscuss.,vol.179,no.0,pp.27–40,Jun.2015.
[3]F.MuraandA.Zaccone,“Effectsofshearflowonphasenucleationandcrystallization,”Phys.Rev.E,vol.93,no.4,p.042803,Apr.2016.
[4]A.Penkova,W.Pan,F.Hodjaoglu,andP.G.Vekilov,“NucleationofProteinCrystalsundertheInfluenceofSolutionShearFlow,”Ann.N.Y.Acad.Sci.,vol.1077,no.1,pp.214–231,Sep.2006.
[5]COMSOLMultiphysicsReferenceManual,version5.0.www.comsol.com:COMSOL,Inc.
4
CROSSINGTHEBORDER:ORGANICMOLECULESTRANSPORTCHLORIDEACROSSLIPIDBILAYERS
HennieValkenier1,GlennGrauwels1,AnthonyDavis2,KristinBartik1
1UniversitélibredeBruxelles,Belgium2UniversityofBristol,UnitedKingdom
Absenceormalfunctionofmembraneproteinswhich formanion channels are the causeof
several diseases. Cystic fibrosis is for instance caused by a deficiency in transport of chloride and
restoringthistransport isan importantchallenge.Syntheticanioncarriers,whichareembedded inlipidbilayers,havethepotential todothis.Thesemoleculescanbindananion,extract it fromthe
aqueousphase,transportitthroughthelipophilicinteriorofalipidbilayer,tothenreleaseitonthe
othersideofthemembrane[1].Chemicalpotentialenergy,intheformofaconcentrationgradient,is
thedrivingforceforthistransportprocess.
Wewillshowhowthegeometryofthebindingsiteoftransportersplaysanimportantrole.The
typesofinteractionsanddegreeofencapsulationoftheaniondeterminetheselectivity[2],whilethe
binding site canbeeitherpreorganised,orundergo largeconformational changesuponbindingofchloride[3].
The potential of synthetic receptors as anion carriers to restore chloride transport will be
demonstrated:ourmostactivecarrierreachesratesoftransportupto800Cl-/sbyasinglecarrier,as
measuredinliposomes[4]andgiantunilamellarvesicles(GUVs)[5].Anothercompoundwasfoundtorestoresignificantchloridetransportinepithelialcellswithoutshowingtoxicity,makingitapromising
candidatefortherapeuticalapplicationsandbiomedicalresearch[6].
References
[1]P.A.Gale,J.T.Davis,R.Quesada,Chem.Soc.Rev.2017,46,2497.
[2]M.Lisbjerg,H.Valkenier,B.M.Jessen,H.Al-Kerdi,A.P.Davis,M.Pittelkow,J.Am.Chem.Soc.2015,137,4948.
[3]H.Valkenier,C.M.Dias,C.P.Butts,A.P.Davis,Tetrahedron2017,73,4955.
[4]H.Valkenier,L.W.Judd,H.Li,S.Hussain,D.N.SheppardandA.P.Davis,J.Am.Chem.Soc.,2014,136,12507.
[5]H.Valkenier,N.LópezMora,A.KrosandA.P.Davis,Angew.Chem.Int.Ed.,2015,54,2137.
[6]H.Li,H.Valkenier,L.W.Judd,P.R.Brotherhood,S.Hussain,J.A.Cooper,O.Jurček,H.A.Sparkes,D.N.SheppardandA.P.Davis,NatChem,2016,8,24.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
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Lecturesession
AGRICULTUREANDFOODTECHNOLOGY
6
KEYNOTEI
NEWDEVELOPMENTSINSILICONNANOTECHNOLOGYFORHEALTHCAREAPPLICATIONS
TimStakenborg
IMEC,Leuven,Belgium
TimStakenborggraduated in1998at theUniversityof Leuvenasamaster inengineering in
chemistryandbiochemistry.AfterbeingaprojectengineeratAlcon-NovartisandhisPhDinmolecular
biology,hejoinedIMEC.
AtIMEC,Timiscurrentlyaprincipalmemberofthetechnicalstaffandgroupleaderinthelife
sciencetechnologydepartment.Hehasbeenaprojectleadinseveralfundedandindustrialresearch
projectsespeciallyfocusingonmergingbiologywithtechnology.Inhiscurrentroleofgroupleader,he
is heavily involved in the transfer and integration of key technical components in fully functionaldevices.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
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DESIGNERCELLULOSOMICSFORACUSTOMIZEDCONVERSIONOFLIGNOCELLULOSICBIOMASSTOVALUABLEBULKANDFINECHEMICALS
JulieVanderstraeten1,M.Fonseca1,A.Thibau1,F.Hooghe1,Y.Briers1
1LaboratoryforAppliedbiotechnology,DepartmentofBiotechnology,GhentUniversity,
ValentinVaerwyckweg1,9000,Gent,Belgium
INTRODUCTION
Lignocellulose isamajorcomponentofplantcellwallsand ispresent ina largeamountofwasteandsidestreams.Efficientuseofthiscomponentasaresourceisoneofthemostimportantchallengesinthefieldofindustrialbiotechnology.
Inlignocellulosicbiomass,celluloseissurroundedandtightlyconnectedtohemicelluloseandthe hemicellulose itself forms a compact matrix with lignin. The crystallinity of cellulose, thehydrophobicity of lignin, the encapsulation of cellulose and the general non-uniform three-dimensionallignocellulosicstructureexplainsthehighresistancetowardsenzymaticdegradationandassuchtofurtherconversion.
Cellulosomesaremulti-enzymecomplexesproducedbyspecialistmicro-organismsthatfeedon plant cell wall carbohydrates. These ‘nanomachines’ consist of two complementary structuralmodules,alargescaffoldin,whichcomprisesdifferentcohesinmodulesanddockingenzymes,whichcomprise a dockerin module and a catalytic enzyme. A specific interaction between the cohesindomainsofthescaffoldinandthedockerindomainsofthecatalyticenzymesensuresthearrangementofdifferentcatalyticactivitiesonasinglebackbone.Assuch,allcatalyticmoduleswithcomplementaryfunctionsareclosetoeachother,enhancingtheirsynergism(Artzietal.,2016).
Becausecellulosomesarehighlyefficientindegradinglignocellulosicbiomass,theseenzymecomplexeshaveattractedtheinteresttoadaptandusetheminthelignocellulosicconversionprocess.Thegoalistousetheknowledgeaboutnaturallyoccurringenzymecomplexestocreateengineereddesigner cellulosomeswith thepotential todegradeall lignocellulosic componentsand to convertthese degradation products into valuable end products (Vazana et al., 2012). Unlike nativecellulosomes, the use of designer cellulosomes enables control over the composition andarrangementoftheselectedenzymes(Figure1).
Figure 1: Designer cellulosomes in the conversion process of lignocellulose. Designer cellulosomescompriseascaffoldinbearingdifferentdockingenzymes.Celluloseandhemicellulosecomponentsoflignocellulose are degraded to C5 and C6 monomers, which can be converted into valuable end-products.
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Severalresearchgroupsaroundtheworldhavesuccessfullycreateddesignercellulosomes.However,theuseofstandardcloningtechniquesmakestheconstructionprocesstediousandallowsthecreationandanalysisofonlyoneorafewdesignercellulosome(s)atatime.OurlabhasdevelopedaDNAassemblymethod,whichenablesustocreatemodularproteinsatahighrate.
Below,wedescribethefirsthigh-throughputconstructionofdockingenzymesandscaffoldinsandtheinitialactivityanalysisoftheassembleddockingenzymes.Specifically,wehavefocusedonthe construction of the first galactomannan degrading designer cellulosome, termed themannanosome.Currentlydesignedcellulosomeshavemainlyfocussedonthedegradationofcellulose(cellulosome)andthepredominanthemicelluloseofhardwoods,xylan(xylanosome).However,thelargesthemicellulosefractioninsoftwood,mannan,hasnotbeencoveredyet.Mannansarestoredinendospermwallsandvacuolesofseedsandvegetativetissues(e.g.ivorynut,coconut,coffeebean).Astheseplantsarewidelyusedinthefoodindustry,severalmannan-richmaterialsarediscardedaswasteinthefoodsupplychain(Yamabhaietal.,2016).
MATERIALANDMETHODS
Cloning
The DNA encoding enzymatically active domains, dockerins, cohesins and carbohydratebindingmodule(CBM)wasamplifiedfromgenomicDNAusingsequencespecificprimersandclonedinentryvectorstocreatethebuildingblocks.Inasecondassemblyreaction,thebuildingblockswerecombinedinasinglereactiontubeandassembledinapre-definedorderinadestinationvector.
Expressionandpurification
Toexpressandpurify the recombinantproteins,E. coliBL21(DE3)RIL cells, containing thecorrectvector,weregrownat37°CinLB,supplementedwiththeappropriateantibioticsand2mMCaCl2.WhenA600reached0.6,isopropylthio-β-galactosidewasaddedtoafinalconcentrationof0.5mM. After overnight incubation at 16°C and 180 rpm, cells were harvested by centrifugation (5minutes,20000g)and resuspended inTris-buffered saline (TBS)pH7,4, supplementedwith2mMCaCl2.Cellswerethendisruptedbyaddinglysozymetoafinalconcentrationof1mg/ml,performingthreefreeze-thawcyclesandsonication.Proteinpurificationwasperformedbyimmobilizedmetal-ionaffinitychromatographyonaHisGraviTrap™column(GEHealthcare).
Enzymaticactivityassays
Mannanase and galactosidase activity was determined quantitatively by measuring thereducingsugarsreleasedfrommannanorgalactomannanbythedinitrosalicylicacid(DNS)method.An assay mixture consisted of 1 ml 0.5 % (w/v) mannan or galactomannan in 0.2 M Sørensen’sphosphate buffer (pH 7.3) and 1 µl docking enzyme (equimolar concentrations). At specific timepoints, 100 µl sampleswere taken,mixedwith 100 µl DNS reagent and incubated at 100°C for 5minutes. After cooling down to room temperature, the degree of enzymatic hydrolysis wasdeterminedspectrophotometricallybymeasuringtheabsorbanceat540nm.Thereleasedreducingsugarsweredeterminedagainstastandardcurveobtainedwithmannose.
RESULTS
Asour in-housedevelopedmodular assemblymethodallowsus to createDNA sequencesencodingmodularproteinsinhigh-throughput,wewereabletosystematicallyanalyseseveraldockingenzymeandscaffoldinparameters.TheworkflowshowninFigure2wasimplemented.
Thefirststepwastoconvertmodulesthatareusedtobuildupcellulosomesintocompatiblebuilding blocks (Figure 2, Step I). Four cohesin-dockerin pairs and one CBM were selected fromnaturallyoccurringcellulosome-producingorganismstoserveasthenon-catalyticmodules.Thethree
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selectedcatalyticactivemodulesareall(galacto)mannanactiveenzymes.ß-1-4-mannanasecatalysesthe randomhydrolysis of ß-1,4 glycosidic bonds of themannanbackbone andß-1,4mannosidasereleasesmannoseresiduesfromß-1,4-linkedmannosides.BothenzymesoriginatefromThermobifidafusca, a natural plant cellwall degrader. α-(1,6)-linkedD-galactose side chains are released by α-galactosidase,originatingfromClostridiumcellullolyticum,anaturalcellulosomeproducer.
Thesecondstepwastoassemblethesemodulesintospecificscaffoldinanddockingenzymesequences(Figure2,StepII)).Weaimedfortheconstructionof24rationallydesignedscaffoldinsand19rationallydesigneddockingenzymesinonerun.ThescaffoldinsallcontainedonetofouroftheselectedcohesinsandoneornoCBM.Forthedockingenzymeswecombinedoneortwoofthethreedifferent catalyticmodules acting on galactomannanwith one out of the four different dockerinsavailableinourcollection.Cloneanalysisprovedthattheusedmethodhasa96%efficiency.
Afterassembly,allconstructswereexpressedinE.coliBL21(DE3)RILcellsandpurifiedbyHis-tagpurification.
Figure2:Adoptedworkflow.Westartfromaselectionofcellulosomebuildingblocks.Thesebuildingblocks are assembled into the coding sequences for docking enzymes and scaffoldins in high-throughput. Systematic parameter analysis then leads us to the determination of optimal dockingenzymeandscaffoldincombination.
Basedonourhigh flexibility in theconstructionofdifferentmodulardockingenzymes,wewereabletoinvestigatetheeffectofdifferentparametersontheperformanceofdockingenzymes(Figure2,StepIII).TheenzymaticactivitywasmeasuredbyDNSanalysis.Specifically,weanalysedtheeffectof thedockerinoriginand the specificorderof themodules. In addition,we compared the
10
activity of a bicatalytic docking enzyme to a mixture of two monocatalytic enzymes with thecorrespondingcatalyticmodules.Figure3showsaselectionofobtainedactivitypatterns.
Thetypeofdockerinthatischosentoconstructadockingenzymehasnosignificanteffectontheactivityofthedockingenzyme(p=0.1).Allvariantsreleasethesameamountofreducingsugarsovertime(Figure3A).
Figure3Bshowstheimportanceofthepositionofdifferentmodulesinsideafusionprotein.For thebicatalyticdockingenzymecombiningtheusedgalactosidaseandmannanaseenzyme, theoptimalconfigurationconsistsofgalactosidaseattheN-terminusandmannanaseattheC-terminus,linkedwithacentraldockerindomain(p<0.05).
Figure3Cindicatesthattwomonocatalyticdockingenzymeshaveahigheractivitythenonebicatalyticdockingenzymecomposedofthesameenzymaticallyactivedomains.
Figure3:Dockingenzymeactivityanalysis.Thecurvesshowdegradationbytheconstructeddockingenzymesrepresentedschematicallynexttoeachgraph.KeytotheusedsymbolscanbefoundinFigure2.A:Degradationpatternsforthreedockingenzymescomposedofthesamecatalyticmodules,butdifferentdockerins.B:Degradationpatternsfortwodockingenzymescomposedofthesamecatalyticmodulesanddockerin,butassembledindifferentorders.C:Activitypatternofabicatalyticdockingenzymecomparedtoamixtureoftwomonocatalyticdockingenzymeswiththecorrespondingcatalyticmodules.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
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DISCUSSION
Our first results indicate that preliminary research is crucial prior to the constructionof adesigner cellulosome. There is a high number of variables that can influence the performance ofdesignercellulosomes.Foreachspecificcollectionofenzymes,optimization isrequired inordertoassemblethemostoptimalsetofdockingenzymesontheidealscaffoldin.
Forexample,weobservedthatchangingthepositionofdifferentmoduleshasalargeeffectontheactivityofthefulldockingenzyme.Arrangementofdockingenzymesmustguaranteeoptimalfoldingofeachmoduletoensurethatdockerinsretainspecificbindingtotheirmatchingcohesinsandcatalytic modules preserve their hydrolytic properties. On top of that, earlier research hasdemonstrated the importance of the relative position of different docking enzymes within thescaffoldin(Sternetal,2015).
Theseessentialpreliminaryassays, inwhichanextensivenumberofdockingenzymes(andscaffoldins)arecompared,call foraquickandeasyassemblymethod.Ourmodularapproachwasproventobe96%efficient,thuslendingitselfperfectlyfortheconstructionofdesignercellulosomes,themostadvancedmodularproteinspresentinnature.
CONCLUSION
We have efficiently implemented a straight-forward workflow in which high-throughputassemblywasextendedtosystematicparameteranalysis.Assuch,wewereabletoanalyseseveraldockingenzymevariables.Thisisanimportantstepincellulosomeresearch,ascurrentlyusedcloningtechniquesallowtheconstructionandanalysisofonlyafewdockingenzymesorscaffoldinsatthesametime.
ACKNOWLEDGEMENTS
J. V. has received a doctoral scholarship of the Bijzonder Onderzoeksfonds of Ghent University(BOF17/DOC/086).
REFERENCES
ArtziL,BayerEA,MoraïsS.Cellulosomes:bacterialnanomachinesfordismantlingplantpolysaccharides.NatRevMicrobiol.2017;15:83-95
Stern J,KahnA,VazanaY,ShamshoumM,MoraïsS, LamedR,BayerEA.Significanceof relativepositionofcellulases indesignercellulosomesforoptimizedcellulolysis.PLoSONE.2015;10(5):e0127326
VazanaY,MoraïsS,BarakY, LamedR,BayerEA.Designercellulosomes forenhancedhydrolysisof cellulosic substrates.MethodsEnzymol.2012;510:429-52.
Yamabhay M, Sak-Ubol S, Srila W, Haltrich D. Mannan biotechnology: from biofuels to health. Crit Rev Biotechnol.2016;36:32-42
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THEEFFECTOFTOWTRADITIONALMOROCCAN'SCOOKINGMETHODSONOMEGA-6/OMEGA-3ANDFATTYACIDSHYPO/HYPERCHOLESTEROLEMICRATIOSOFBENI-GUIL
LAMBMEATBREEDINGINNORTHEASTOFMOROCCO
BelhajK1,2,MansouriF1,SindicM2,FauconnierM-L3,BoukhartaM4,SerghiniCaidH.1,ElamraniA.1
1LaboratoiredeBiologiedesplantesetdesmicro-organismes,FacultédesSciences,
UniversitéMohamedIer,Oujda;Maroc.2LaboratoireQualitéetSécuritédesProduitsAlimentaires,GemblouxAgro-BioTech,
UniversitédeLiège;Belgique.3LaboratoiredeChimiegénéraleetOrganique,GemblouxAgroBio-Tech,
UniversitédeLiége;Belgique.4InstitutSupérieurIndustrielagronomique,Huy,Belgique.
INTRODUCTION
Meatcookingisessentialtoimprovemeatconsumer’spalatability.Infact,thermaltreatmentsimprovehygienicqualityofmeat(deactivationandeliminationpathogenicmicroorganisms),increaseitsshelflife,enhanceitsorganolepticqualityflavor,tendernessandcolor(Modzelewska-Kapitułaetal., 2012). However,meat cookingmodifies chemical composition (fatty acids, amino acids, …) bylosseswithaconsequentchangeofthemeatnutritionalvalue.Fatandfattyacidscompositionofmeatareaveryimportantcretariaforagoodhealth(Woodetal.,2004).Also,it'sveryimporanttoknowthatanimbalancedpolyunsaturatedfattyacids(PUFA)/saturatedfattyacids(SFA)andahighn-6/n-3ratiosmaycauseserioushealthproblemssuchascardiovasculardisease,obesityandcertaincancers(Simopoulos,2002,Simopoulos,2016).Moreover,thoseparametersareveryinfluencedbycookingmethods(Flakemoreetal.,2017).Theobjectiveofthisstudyistocompareanddeterminetheeffectof twoMoroccancookingmethods (BarbecueandTajine)onnutritionalqualityofBéni-guil's lambmeatespeciallytheomega-6/omega-3andhypo/hypercholesterolemicratios.
MATERIALANDMETHODS
ThetrialswereconductedontheLongissimusLumborum(LL)ofBéni-guilslambmeatobtained24hpostmortem(onemuscleforeachcarcass).ThepHultimewasanalyzedtoavoidanomalycarcass,byusingaportablepHmeter(pH/Cond340iWTW,Weilheim,Germany),equippedwithapenetrationelectrode.Then,wedividedthesamplesintothreegroupsaccordingtocookingmethods(30samplesforeachgroup).A totalof90 sampleswereobtained includingonegroupusedas control.Meats,denudedof external fatswere cooked, to a core temperatureof 70 °C, using twoMoroccanmeatcookingmethods:grilledusinganelectricalgriddle(drycooking)andTajineusing20mlofwater(moistcooking). After cooking the samples were freeized, lyophilised, crushed and stored at -20 C° forsubsuquentfattyacidsanalysis.Fattyacidsprofile
The intramusculare fat was extracted according to Bligh and Dyer (1959) using achloroform/methanol/watermixture(2/1/1;v/v/v).Thelipidextractwasmethylatedandconvertedtofattyacidmethylesters(FAME)accordingtothemethoddescribedby(BenMoumenetal.,2015).SeparationoftheFAMEwasperformedonanAgilentgaschromatograph(GC)(HP6890series,AgilentTechnologies,USA),equippedwithanOmegawaxcapillarycolumn(30m×0.25mm×0.25μmfilmthickness)fromSupelco(Bellefonte,PA,USA)andaflameionisationdetector(FID).Helium(99.999%,
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AirLiquide,Liège,Belgium)wasusedasthecarriergas,ataflowrateof1.7mlmin-1.Thetemperatureoftheinjectoranddetectorweresetat150and250°C,respectively,andtheoventemperaturewasset at 210 °C. The injection volume was 1 µl, in splitless mode. A FAME standard, containing 37components(Supelco,Bellefonte,PA,USA),wasusedtoidentifytheindividualpeaks.Statisticalanalysis
Theanalysisofeffect'scookingmethods(BarbecueandTajine)onnutritionalvaluewascarriedouton triplicate foreachsampleandeachparameter.All statisticalanalysiswereconductedusingStatisticalPackagefortheSocialSciences(SPSSforWindows,version20,SPSSInc.,Chicago,IL,USA).ThenormaldistributionwasverifiedaccordingtoShapiroWilktest.One-wayANOVAstatisticalanalysisandDuncan'spost-hoctestwasusedformeanscomparison,thedifferencewasconsideredsignificant(atP<0.05).RESULTS
ThemeanvalueofpH24ofLLusedinthisstudywas5.66.Theresultsshowedthat100gofrawmeatcontains6.14%(table1)ofintramuscularFat(IMF).TheeffectofcookingmethodsshowedthatBarbecuecookingmethodpreserveditsIMFthanTajinecookingmethodwithacontentof5.31%and4.83%(table1)offreshmeat,respectively.TheeffectofcookingmethodsonfattyacidsratiosandsumsofBéni-guil'slambmeatproducedineasternMoroccoaresummarizedinTable1.Table1:Cookingmethodseffectoncooking loss, IMFandfattyacidsratiosandsumsofBéni-guil'slambmeat
Fattyacids(%) Rawmeat CookingmethodBarbecue Tajine
SFA 46,67a 42,21b 44,23cPUFA 9,47a 21,93b 16,36cUFA 53,32a 57,78b 55,76cUFA/SFA 1,14a 1,39b 1,27cPUFA/SFA 0,20a 0,54b 0,38cn-6 7,79a 18,70b 14,15cn-3 1,46a 2,69b 1,81an-6/n-3 5,70a 7,92a 11,45bh/H 1,62a 2,17b 1,98bCookingloss --- 21.14a 27.06b
IMF 6.14a 5.31b 4.83c
pH 5.66 --- ---Significantdifferencesareshownbydifferentletters(a-c)SFA:Saturatedfattyacids;PUFA:Polyunsaturatedfattyacids;UFA:Unsaturatedfattyacids;n-6:n-6PUFA;n-3:n-3PUFAh/H=(18:1n9c+18:2ω6+20:4ω6+18:3ω3+20:5ω3+22:5ω3+22:6ω3)/(14:0+16:0).
DISCUSSION
Theresult forcooking lossshowedthatthermaltreatments inducedan internal juice loss inmeat.Thesmallest internal juicelossvaluewasrecordedinbarbecue(21.14%)thanTajinecookingmethod (27.06%). So, the first cookingmethod holds back its internal juice than the second one.Similarresultwasreportedbyotherauthors(Lorenzoetal.,2015).Also,thebarbecuecookingmethodallowshighpreservationofIMFthanTajinewith6.14%,5.31%and4.83%forfreshmeat,barbecue
14
and Tajine respectively. Consequently, Barbecuemethod guaranteed a better juiciness than Tajinemethod.GC-FIDanalysis revealedasignificanteffectofcookingmethodson fattyacidsprofileandratios(Table1).Thesaturatedfattyacids(SFA),polyunsaturatedfattyacids(PUFA),andunsaturatedfattyacids(UFA)percentageswereveryinfluencedbycookingmethods.BarbecuecookingmethodsimprovesmeatnutritionalvaluebyincreasingPUFAfraction.So,theincreaseofPUFA/SFAratiorangedbetween0.20,0.38and0.54forRawmeat,Tajineandbarbecuerespectively.Similaroutcomeswerefind by other authors (Ramamurti, 1986, Domínguez et al., 2015). This implies an increase, ofhypocholesterolemicfattyacidsandthereforeanincreaseoftheh/Hcholesterolemicratiowhichhelpsreducecardiovasculardisease.Theh/HratiofoundforBarbecuecookingmethod(2.17)washigherthan those reported inSpanish (Campoetal., 2013))andAustralian (Flakemoreetal., 2017) lambmeats.
Thisresultcanbeexplainedbycookinganddriploss,becausetheproportionalchangeinfattyacidscompositionandthisisconsistentwiththeresultsreportedbyRamamurti(1986).Additionally,then-6/n-3ratiowasaffectedbycookingmethods.Theresultsofn-6/n-3ratioinourstudyishigherthanthosereportedinbeef(Alfaiaetal.,2010)andinfoal(Domínguezetal.,2015)meats.
CONCLUSION
ForthefirsttimewestudiedtheeffectofdifferentMoroccan'scookingmethodsonnutritionalquality of the Béni-Guil Protected Geographical Indication meat from eastern Morocco and weconcludethat froma taste,nutritionalanddietaryqualitypointofviewbarbecuecookingmethodallowsbetterpreservationofmeatnutritionalqualitythanTajineanditisthecookingmethodtoberecommended.REFERENCESALFAIA,C.M.,ALVES,S.P.,LOPES,A.F.,FERNANDES,M.J.,COSTA,A.S.,FONTES,C.M.,CASTRO,M.L.,BESSA,R.J.&PRATES,
J.A.2010.Effectofcookingmethodsonfattyacids,conjugatedisomersoflinoleicacidandnutritionalqualityofbeefintramuscularfat.MeatScience,84,769-777.
BENMOUMEN,A.,MANSOURI,F.,RICHARD,G.,ABID,M.,FAUCONNIER,M.L.,SINDIC,M.,ELAMRANI,A.&SERGHINICAID,H.2015.Biochemicalcharacterisationoftheseedoilsoffoursafflower(Carthamustinctorius)varietiesgrowninnorth-easternofMorocco.InternationalJournalofFoodScience&Technology,50,804-810.
BLIGH,E.G.&DYER,W.J.1959.Arapidmethodoftotallipidextractionandpurification.Canadianjournalofbiochemistryandphysiology,37,911-917.
CAMPO,M.,MUELA,E.,OLLETA,J.,MORENO,L.,SANTALIESTRA-PASÍAS,A.,MESANA,M.&SAÑUDO,C.2013.InfluenceofcookingmethodonthenutrientcompositionofSpanishlightlamb.Journaloffoodcompositionandanalysis,31,185-190.
DOMÍNGUEZ,R.,BORRAJO,P.&LORENZO, J.M.2015.Theeffectofcookingmethodsonnutritionalvalueof foalmeat.JournaloffoodCompositionandAnalysis,43,61-67.
FLAKEMORE, A. R.,MALAU-ADULI, B. S., NICHOLS, P. D. &MALAU-ADULI, A. E. O. 2017. Omega-3 fatty acids, nutrientretentionvalues,andsensorymeateatingqualityincookedandrawAustralianlamb.Meatscience,123,79-87.
LORENZO,J.M.,CITTADINI,A.,MUNEKATA,P.E.&DOMÍNGUEZ,R.2015.Physicochemicalpropertiesoffoalmeatasaffectedbycookingmethods.Meatscience,108,50-54.
MODZELEWSKA-KAPITUŁA,M.,DĄBROWSKA,E., JANKOWSKA,B.,KWIATKOWSKA,A.&CIERACH,M.2012.Theeffectofmuscle,cookingmethodandfinalinternaltemperatureonqualityparametersofbeefroast.Meatscience,91,195-202.
RAMAMURTI,K.1986.Effectsofcooking,freezingandfrozenstorageonthefattyacidPatternofgroundbeef.LWTFoodSci.Technol.,19,481.
SIMOPOULOS, A. P. 2002. The importance of the ratio of omega-6/omega-3 essential fatty acids. Biomedicine &pharmacotherapy,56,365-379.
SIMOPOULOS,A.P.2016.Anincreaseintheomega-6/omega-3fattyacidratioincreasestheriskforobesity.Nutrients,8,128.
WOOD,J.,RICHARDSON,R.,NUTE,G.,FISHER,A.,CAMPO,M.,KASAPIDOU,E.,SHEARD,P.&ENSER,M.2004.Effectsoffattyacidsonmeatquality:areview.Meatscience,66,21-32.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
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MICROBIALDETOXIFICATIONOFTHEMYCOTOXINDEOXYNIVALENOL
IlseVanhoutte1,KrisAudernaert1,LeenDeGelder1
1GhentUniversity,Belgium.
Mycotoxins are secondarymetabolites producedby fungi.When these fungi arepresent on
agricultural commodities, they canposeahigh risk toanimalandhumanhealth.As theEuropean
Commissionimposesstrictregulationsonmaximumlevelsofseveralmycotoxins,itisaprerequisitefor farmers tomeet these regulations toavoidcomplete lossofacontaminatedbatch. Inanagro-
ecosystem, farmers try to avoid fungal infection and the occurrence of mycotoxins in their
commoditiesviapreventionandinterventionapproaches,butoftenfailincompletelyeliminatingthe
risk. Therefore, detoxification strategies are needed such as the application of binders or theapplicationofmicroorganismsabletodetoxifythesemycotoxins.
Our project focuses on the microbial detoxification of deoxynivalenol (DON), a worldwide
prevalent mycotoxin. Microorganisms were screened for their biotransformation capacity of DONthroughnon-targetedLC-MS/MS.Furthermore,abioassayusingtheaquaticplantLemnaminorwas
developedtoassesstheresidualtoxicityofDONmetabolitesafterbiotransformation.Pursuingthis
approach,twomixedcultureswereobtained,originatingfromsoilandactivatedsludge,capableof
biotransformingDONintotheepimerofDONandtheepimerofDOM-1,bothconveyingnoresidualtoxicityfortheLemnaminorplant.
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THEIMPACTOFCONTROLLEDSTEEPINGANDGERMINATIONOFWHEATONHYDROLYSISOFPHYTATEANDARABINOXYLANANDONMINERALBIO-ACCESSIBILITY
ElienLemmens1,NielsDeBrier1,PeterGoos2,ErikSmolders3,JanDelcour1
1Laboratoryofchemistryandbiochemistry,KULeuven.
2DivisionofMechatronics,BiostatisticsandSensors,KULeuven.3DivisionofSoilandWaterManagement,KULeuven.
Wheatwholegrain foodproductsdeserve tobean importantpartofahealthydietas they
reducetheriskofdevelopingdiet-relateddisorderssuchasobesity,typeIIdiabetes,cardiovascular
diseaseandcancer.Theirhealthbenefitsaremainlyascribedto thehighconcentrationsofdietaryfiberandotherbio-activessuchasBvitaminsandminerals inbran.However,thesebio-activesare
entrappedincellswithrigidwallswhichwithstandconventionalmillinganddigestionbythehuman
enzymearsenal.Moreover,wheatphosphorusismainlystoredasphyticacidwhichchelatesdivalentcationssuchasironandzinc.Asaconsequence,thebio-accessibilityofwheatmineralsonlyamounts
to5to25%.Hydrolyticenzymessuchasxylanaseandphytaseareactivatedanddenovosynthesized
inwheataleuronecellstofuelseedlinggrowth.Xylanaseshydrolyzeandsolubilizearabinoxylan(AX),
themainaleuronecellwalldietaryfibre(DF)component.Atthesametime,theincreaseinphytaseactivityduringgerminationmakesphosphate,mineralelementsandmyo-inositolavailableforplant
growthanddevelopment.Inafoodsystemcontext,thisisimportantsinceopeningwheataleurone
cellwallsandbreakingdownphytatestructuresby(partial)germinationmaywellresultinagreater
wheatmineralaccessibility inthehumangastro-intestinaltract.WeherestudiedhowsteepingandgerminationaffectthewheatDFcomplexandincreasethebio-accessibilityofminerals.
Duringsteeping(29h)andgermination(120h)ofwheatgrainsat15°Cthexylanaseandphytase
activity levels increasedabout five times.As a result, the contentofwater extractableAX (WEAX)
doubledduringsteepingandgermination[from0.5%to1.0%ofinitialdrymatter(idm)].However,thephytatecontentdecreasedonlyfrom0.96%to0.81%ofidm.Theironbio-accessibilityincreasedfrom
7% in regularwheat to 15% in germinatedwheat. In a next step,we used an I-optimal design of
experimentstomaximizetheimpactofcontrolledsteeping(3-36h;15-50°C)andgermination(6-120h;15-30°C)onAXandphytatehydrolysisinwheatand,hence,improvethelevelsofsolubleDFand
elemental bio-accessibility. The WEAX content was mainly determined by the main effect of
germinationtimeandthequadraticeffectofsteepingtemperature.Whenwheatgrainsweresteeped
at24°Cfor36handgerminatedat22°Cfor120h,theWEAXcontentinwheatincreasedfrom0.5%to1.3%of idmandthephytatecontentdecreasedfrom0.96%to0.66%of idm.Weconcludethat
controlled steeping and germination of wheat offers novel raw materials which can be used for
manufacturing cereal products with different DF properties and an improved bio-accessibility of
minerals.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
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THEIMMEDIATERESPONSEOFTOMATOANDRICETOTEPHRADEPOSITION:ANEXPERIMENTALSTUDY
IngaTarasenko1,StanleyLutts2,PierreDelmelle1
1EnvironmentalSciences,EarthandLifeInstitute,UniversitéCatholiquedeLouvain,
Louvain-la-Neuve,Belgium.2Agronomy,EarthandLifeInstitute,UniversitéCatholiquedeLouvain,Louvain-la-Neuve,Belgium.
During a volcanic explosive eruption, tephra particlesmay interact with terrestrial plants in
variousways,possiblyresultingindetrimentaleffectssuchasalterationofleafphysiologyandreducedplantgrowth.Inagriculturalareas,tephradepositioncanleadtoseverecropdamagesandsignificant
economic losses. Themagnitude of tephra impacts on vegetation ismodulated by several tephra
propertiesactingincombination;theseincludethickness,porosity,grainsizedistribution,colourand
composition.Whilemanystudieshighlightthemechanicalimpactofheavytephrafallonvegetation,littleisknownaboutthephysiologicalresponseofplantleavestoathintephradeposit.
Here, we present the results of an experimental study aimed at better understanding the
physiologicalconsequencesoftephra-leaf interaction.Tephrafromthe1991Mt.Pinatuboeruption
wasappliedtoriceandtomatoplantsgrownincontrolledconditionsinaphytotron.Twoscenariosoftephradepositionwereconsidered:aone-eventscenario(S1)inwhichagivenamountoftephrawas
appliedtoplantsonce,andamultiple-eventscenario(S2) inwhichtephrawasappliedthreetimes
overaperiodoftwoweeks. Inbothcases,thetotalamountoftephrareceivedbytomatoandrice
plants was ~90 g and ~180 g, respectively. Each experiment lasted two weeks and involved sixreplicates.
Applicationoftephraontomatoandriceplantsresultedinleafwilting,twistingandfolding.A
colourchangewasalsonoticedandplantgrowthwasaffected.Inbothtephradepositionscenarios,theshootbiomassoftomatoandriceattheendoftheexperimentwasreducedbyupto~30and
~50%,respectively,whencomparedtocontrolplantsthatdidnotreceivetephra.InS2,thetephra-
coveredriceplantsshoweda~30%lowerrateofphotosyntheticactivitythanthecontrolplantsthat
didnotreceivetephraduringthetwoweeksofexperiment.InS1,therateofphotosyntheticactivityinricedecreasedby~40%withinonedayoftephraapplication;itremainedsignificantlyloweruntil
the firstweek of the experiment. The rate of photosynthetic activitymeasured for tomato leaves
coveredbytephrawasalwayslower(byafactorofupto2)thanthatmeasuredforcontrolplants.Leaf
temperature in both the rice and tomato experiments increasedby ~10%.Our preliminary resultsalreadysuggestvariousphysiologicaleffectsintomatoandriceplantsaffectedbyatephradeposit.
Additionalmeasurementsonleafchlorophyllandsugarcontents,plantwateruseefficiency,stomatal
conductance,internalCO2concentrationandtranspirationratewillbepresented.
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CATABOLICSELECTABLEMARKEREASESGENOMEEDITINGINYARROWIALIPOLYTICA
MarieVandermies1,OliviaDenies1,Jean-MarcNicaud2,PatrickFickers1
1UniversitédeLiège2INRA-AgroParisTech
Selectable markers are a central component of genome edition technologies. In the yeast
Yarrowia lipolytica, thesemarkersaretraditionallybasedonantibioticresistance(hygromycinB)orauxotrophy (e.g., leucine, uracil). However, the use of the former is impaired by a high level of
spontaneous resistance, and the use of the latter by continuous complementation of the culture
mediumorrestorationofprototrophytothestrains.Asanalternative,genesrelatedtothecatabolism
ofcarbonsources,or“catabolicselectablemarkers”,presenttheadvantageofnotbeinginvolvedinessentialmetabolicpathways.TherecentlyidentifiedEYK1,encodingerythrulosekinase,canserveas
anefficientcatabolicselectablemarkerforgenomeeditinginY.lipolytica.Comparedtoauxotrophic
markerssuchasURA3andLEU2,EYK1increasesthegrowthrateoftransformantsonselectivemedium
andtheefficiencyofgenomeedition.TheutilityofthemarkerEYK1inareplicativevectorwasalsodemonstrated. Besides, the cloning-free strategy developed here simplifies the construction of
disruptioncassettesforgenomeeditinginY.lipolytica.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
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Lecturesession
BIOMATHEMATICS
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PROTEININTRINSICDISORDER:ADOUBLE-EDGEDSWORD?
MauricioMacossayCastillo1,MainakGuharoy1,GiulioMarvelli1,PeterTompa1,2,ShoshanaJ.Wodak1
1VIB-VUBCenterforStructuralBiology,VrijeUniversiteitBrussel
2InstituteofEnzymology,HungarianAcademyofSciences
Intrinsicallydisorderedproteins and regions thereof (IDPs/IDRs) adopthighlyheterogeneous
anddynamicconformationalstates.Thesepropertiesenablethemtoperformimportantregulatory
andsignalingrolesinthecell.Moreover,IDPs/IDRswereshowntoengageinweakinteractionswithotherproteins.Onemaythereforewondertheextenttowhichevolutionoperatestolimitthenon-
functionalinteractionsthatIDP/IDRswouldforminthecell.Sinceaccordingtothelawofmassaction,
such interactions should increase with the protein abundance level, we investigated how various
intrinsic properties of S. cerevisiae proteins, in particular those of IDPs/IDRs, vary with proteinabundance.Inaddition,weanalyzetherelationsbetweentheintrinsicpropertiesoftheproteins,their
abundancelevelsandthepatternsofphysicalandfunctional interactionstheyform,asdetectedin
oneoftherecenthighconfidenceprotein-proteininteractionnetworkinvolvingyeastsolubleproteins.
ResultsshowthatIDRsofmoreabundantproteinstendtobemorepolar,lesshydrophobicand
lesspronetobindtootherproteins,thanthoseoflowabundanceproteins,justasforsurfaceresidues
inglobularproteins.Incontrast,theseproteinsfeaturealargernumberofdistinctstructured(Pfam)
domains.ThissuggeststhatevolutionreducesthelikelihoodofhighabundanceproteinstoengageinspuriousinteractionsbyincreasingthesolubilityofIDPs/IDRs,whilelikelyallowingthemtoengagein
morefunctionallydiverseinteractionsthroughtheirstructureddomains.Analysisoftheyeastprotein-
interaction network then confirmed earlier findings that the number of interacting partners per
protein,increaseswithitsabundancelevel.Furthermore,usingasemanticsimilaritymeasureofthegeneontology(GO)annotations,andgeneco-expressionlevels,wefindthathigh-abundanceproteins
tendtohavemorefunctionallydiverseinteractionpartnersthatarealsolesscoherentlyexpressedbut
that IDPs/IDRs were notmore prone to having such partners than globular proteins. Our current
interpretationofthesefindingsisthatevolutionstrikesadelicatebalancebetweenenablingdiversefunctionalroleswhileminimizingspuriousinteractions.Itdoessonotonlybyregulatingtheprotein
abundance levels and their cellular localization, but also by modulating the intrinsic molecular
propertiesoftheproteins,inbothglobularandintrinsicallydisorderedregions.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
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IDENTIFICATIONOFCAUSALMUTATIONSCONFERRINGHIGH-ETHANOLTOLERANCEINESCHERICHIACOLI
ThomasSchalck1,2,T.Swings1,2,C.Bonte1,B.VandenBergh1,2&J.Michiels1,2
1CentreofMicrobialandPlantGenetics,K.U.Leuven,KasteelparkArenberg20,Heverlee,Belgium2VIB-KULeuvenCenterforMicrobiology
INTRODUCTION&RESEARCHSCOPE
Fordecades,fossiloilwasdrivingthedevelopmentofmanyindustries.Asaresult,petroleumoveruse not only depleted our oil reserves to alarmingly low levels but also lumbers us withenvironmentalproblems.Fuelingvehiclesandsupplyingtheindustrywithessential,chemical‘buildingblocks’canhowever,alsobedonebyrelyingonrenewableenergysourceswhichwouldavoidtheadverseeffectsofcrudeoil.Oneofthesefossiloilalternativesthatisalreadywidelyintegratedinoursociety is bioethanol. Mainly, because this alcohol has extraordinary properties that meet therequirementsforusageasafuelandasabuildingblock inpetrochemical industry.Theproductionprocess of bioethanol involves the fermentation of sugar-rich plant-or algae-derived biomass byethanologenic microorganisms, including the yeast Saccharomyces cerevisiae or bacteria, such asEscherichiacoliandZymomonasmobilis.Inbrief,thisconversionofsugarstoethanolhasanetzerocarbonfootprintasthealgaeorcrops, includingmaize,wheatandswitchgrass,photosyntheticallycapture the carbon dioxide released during combustion of bioethanol in vehicles and duringproductionofplasticsinthepolymerindustry.
Traditionally,microbial fermentationprocessesarewellestablished inthefood industrytoproducealcoholicbeveragessuchasbeerorwine.Incontrast,industrialbioethanolcanbeconsideredasabulkproductanddiffersfromdrinkingalcoholintermsofprice(around70UScents/liter),volumeandgrade(sometimesupto99%foranhydrousbioethanol)(Fairley,2011).Therefore,highethanoltitersjustaftermicrobialfermentationarepreferredasenergyconsumptionduringdistillationcouldbe limited to keep the overall bioethanol production process profitable. However, themaximumethanol content in the fermentationbatch is inevitably determinedby themicrobe’s tolerance toethanol. Indeed,ethanolandotheralcoholsexert toxiceffectson themicrobial cell envelopeandothercellularprocesses.Therefore,strategiestoprotectmicroorganismsagainstlethalethanolstresscouldholdthecluetoimprovebioethanolyields(Dunlop,2011andLingetal.2014).Althoughthisrelationship between productivity and tolerance may seem trivial at first glance, the geneticbackground underlying the tolerance phenotype is extremely complex and remains incompletelyunderstood.
ANEVOLUTION-DRIVEN INSILICOMODELTOUNDERSTANDTHEMECHANISMSBEHINDETHANOLTOLERANCE
Inpreviouswork,ourlabusedexperimentalevolutiontoincreaseethanoltoleranceinE.coli.The long-term evolution experiment under gradually increasing ethanol concentrations yieldedseveral highly ethanol tolerant strain. Subsequently, these evolved strains were sequenced andrevealedaglimpseofthemajor,cellularmechanismsresponsiblefortheincreaseinethanoltolerance.Aidedby thesedata,wenow start tounderstandhow themodel bacterium,E. coli, adapts to anextremelystressfulenvironment(Swingsetal.2017a).Indepthstudyofthesehypertolerantstrainsrevealedastrongcorrelationbetweenmutation frequencyandthepropensity to increaseethanoltoleranceinawaythatonlystrainsthataccumulatedamutatormutationwereabletoadapttohighethanollevels.Asaresultofahypermutationstate,hundredsofmutationsaccumulatedthroughoutthe entire genome (some evolved strains harbor even up to thousandmutations). Someof those
22
mutations,classifiedas‘passenger’,settledinthepopulationbyhitchhikingwithanotherbeneficialmutation and tend tohave aneutral or evendeleterious effects.Others, called ‘drivers’, improvefitnessandcontributetothetolerancephenotype,butoftenremainedhiddenunderthenoiseofthepassenger mutations (Bozic et al. 2010 and Lenski, 2017). Thus, retaining the ethanol tolerance-associateddrivermutationsanddiscardingthepassengersisasuitablestrategytodefineaframeworkforstrainengineeringinthecontextofethanolproduction.Ideally,thisindustrialstrainshouldharbortheminimumsetofdrivermutationstoprovideprotectionagainstacertainethanolconcentrationwithout affecting critical industry-related, microbial parameters like growth rate and productionproperties.
Todistinguishbetweendriverandpassengermutations,weidentifiedcorepathwayswhicharedirectlylinkedtoimprovedethanoltoleranceinE.colithroughabioinformaticsnetworkanalysistool, called Identification of Adaptive Mutations in Evolutionary Experiments or shortly IAMBEE(Swingsetal.2017b).Thismethodappliesacombinationofwhole-genomesequencinginformationfrom individually, evolved E. coli populations together with preexisting network databases.Morespecifically, the algorithm focuses on genomic data at selective sweeps, corresponding to ethanolthresholds at which favorable adaptive mutations take the upper hand thereby purging geneticdiversitywithinabacterialpopulation(Cohan,2016).Asaconsequence,mutationswhichoccurredduringtheseparticularevolutionarytimepointsarelikelytobeadaptiveunderethanolconditionsandthus provide thebackbone for subsequent network analysis. Finally, the in silico study revealed avariety of subnetworks involved in different cellular processes, ranging from cell envelopehomeostasis to transcription. Some identified pathways, such as a pathway involved in plasmamembranefattyacidcomposition,werealreadyreportedinliterature,whereasothers,suchastheAcrAB-TolCeffluxpumpcomplex,werenotlinkedtoethanoltoleranceyet.
FROMANINSILICOTOWARDSANINVIVOCONTEXTHere, we sought to verify IAMBEEs outcome and estimate the relative contribution of
common point mutations within the key IAMBEE-identified subnetworks in vivo. To study thesepolymorphisms individually, it is clear that the mutation of interest has to be isolated from thecorresponding,evolvedcellpopulationandtransferredtotheoriginal,ancestralbackground.Forthispurpose,wedevelopedamodifiedtwo-plasmidCRISPR/Cas9approachwhichenablesto introduceanypointmutationinanygeneofinterestinastraightforwardandconvenientworkflow(Swingsetal. 2018).Moreover, this genome editing technique allows to combinemultiple single nucleotidepolymorphisms(SNPs)inonesinglecelltoinvestigatetheeffectofepistasisintheethanoltolerancecontext.Aftergenomemodification,thefitnessoftheCRISPR-mutantsarecomparedtowild-typeE.coliintermsofgrowthdynamicsandcellsurvivalinethanol-richmediumusingopticaldensitydevicesandclassicalplatingtechniques.Importantly,thesemicrobiologicaldatacanprovideaninvivorealitychecktoeitherprovetheevolutionaryrelevanceofinsilicomodelsor,incaseofinconsistencies,tofine-tune the prediction power of computational tools.Moreover, besides being a research tool,CRISPR/Cas9 can also serve as a useful method for (tolerance) engineering purposes in order todevelopsuperiorethanol-producingstrains.
Inasecondpart,weareplanningtoset-upaninvivophagetransduction-basedscreeningset-upfortheinvivoidentificationofethanoltolerance-improvingmutations(Nehringetal.2015).Inanutshell,thisconceptwillallowustoexchangegenomiclocifromthewild-typeancestortoanevolvedstrainortheotherwayaround.Inthefirstcase,ethanoltoleranceisexpectedtodropwhendrivermutationsareremovedbyphagetransductionwhereas,inthesecondsituation,theintroductionofadvantageous,adaptivemutationsinthewild-typebackgroundshouldimprovegrowthcharacteristicsandsurvivalunderethanolstress.Bothtransductionapproachesshouldprovideagenome-widescan,indicatingthetrueadaptivemutations,whichshouldincludethegenesandpathwaysidentifiedbyIAMBEEandlikelysupplementthelistofcausalmutationswithnewethanoltolerance-associatedloci.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
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The previous paragraphs described a pipeline for in silico and in vivo identification ofmutations conferring ethanol tolerance as well as a methodology to integrate individual or acombinationofSNPs intoanyE. colibackground.Obviously, creating superiorE. coli fermentationstrains using the obtained list of ethanol-associated driver mutations in combination with theCRISPR/Cas9editingtoolshouldbeinreachatthispoint.Subsequently,weareplanningtoevaluatethemicrobialperformanceoftheseengineeredstrainsintermsofethanolproductiontoestablishaclearlinkbetweentoleranceandproductivityinafollow-upstudy.Inaddition,theseeditedstrainswillbesuitableresearchsubjectstounderstandhowmutationscanalterthefunctionofaspecific,cellularpathwaytopushthecell’sethanoltolerancelimit.
CONCLUSIONTo summarize, this project covers the entire research flow from the identification of
tolerance-associatedmutationstowardsapplicationofelementaryknowledgeinstrainengineering.In addition, fundamental insights into the ethanol tolerance mechanism could offer a deeperunderstandingonhowlivingorganismsareabletocopewithextremestressesthroughoutevolution.Finally,duetotheapplicablenatureofthisresearchtopic,strainimprovementofindustriallymorerelevant production strains, such as Z. mobilis or S. cerevisiae, could be tackled by focusing onhomologouspathwayswhichappearedpredominantforethanoltoleranceinourmodelorganismE.coli.
REFERENCESBozic,I.etal.Accumulationofdriverandpassengermutationsduringtumorprogression.ProcNatlAcadSci107,18545–
18550(2010).Cohan,F.M.DispatchesBacterialSpeciation:GeneticSweepsinBacterialSpecies.Curr.Biol.26,R112–R115(2016).Dunlop,M.J.Engineeringmicrobesfortolerancetonext-generationbiofuels.Biotechnol.Biofuels4,32(2011).Fairley,P.Nextgenerationbiofuels.Nature474,S2–S5(2011).Lenski,R.E.Experimentalevolutionandthedynamicsofadaptationandgenomeevolutioninmicrobialpopulations.Int.Soc.
Microb.Ecol.11,2181–2194(2017).Ling,H.,Teo,W.,Chen,B.,Leong,S.S.J.&Chang,M.W.Microbialtoleranceengineeringtowardbiochemicalproduction:
Fromlignocellulosetoproducts.CurrentOpinioninBiotechnology(2014).Nehring,R.B.etal.Anultra-denselibraryresourceforrapiddeconvolutionofmutationsthatcausephenotypesinEscherichia
coli.NucleicAcidsRes.(2015).Swings,T.etal.AdaptivetuningofmutationratesallowsfastresponsetolethalstressinEscherichiacoli.Elife6,(2017).Swings,T.etal.Network-basedidentificationofadaptivepathwaysinevolvedethanol-tolerantbacterialpopulations.Mol.
Biol.Evol.34,2927–2943(2017).Swings,T.,MarcianoD.etal.CRISPR-FRT:Targetingsharedsitesinaknock-outcollectionforoff-the-shelfgenomeediting.
NatureCommunications(inreview).
24
METABOLICSYNDROMEDUETOCONFLICTSBETWEENLIGHTANDFOOD:ASTORYOFTWISTEDCLOCKS
AuroreWoller1,DidierGonze1
1UniversiteLibredeBruxelles,Belgium
Circadianrhythmsarebiologicalprocessesthatoscillatewithaperiodofabout24hours.These
rhythmsareubiquitous,endogenousandcanbeentrainedbyexternalperiodicevents(suchaslight-
darkcycleandfeeding-fastingcycle).Themammaliancircadiansystemiscomposedofmultipleclocksthroughoutthebody.Theselocalclocksareentrainedbynutrient,neuralandendocrinecuesanddrive
diversephysiologicalfunctionsincludingmetabolism.Inparticular,theclockofthepancreaticbetacell
rhythmically regulates the transcription of genes involved in glucose-stimulated insulin secretion.
Perturbationsof this fine-tunedoscillatorynetwork increase the susceptibility todiseases.Besideschronicjetlagandshiftwork,commonperturbationsareill-timedeatingpat-ternswhichcanleadto
metabolictroubles(suchhypoinsulinemia).Wehavebuiltamathematicalmodeldescribingtheclock-
dependentpancreaticregula-tionofglucosehomeostasisinrodents.Aftercalibratingthemodel,wehaveinvestigatedtheeffectofrestrictingfoodaccesstothenormalrestphase.Oursimulationshave
shownthattheresultingconflictbetweenthe light-darkcycleandthefeeding-fastingcyclecreates
differential phase shift in the expression of core clock genes (consistent with experimental
observations).Ourmodelfurthershowsthatthisinducesanon-concomittancebetweennutrientcuesand clock-controlled cues controlling metabolic outputs: this results in hypoinsulinemia,
hyperglycemiaaswellasinalossoffoodanticipation.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
25
Lecturesession
HUMANHEALTH
26
KEYNOTEII
MICROFLUIDICSFORBIOMIMETISM:FROM3DCO-CULTURETOGUTONCHIP
StéphanieDescroix
MacromoleculesandMicrosystemsinBiologyandMedicine,InstitutCurie(MMBM),France
Stéphanie Descroix has an initial background in biochemistry and she obtained her PhD in
AnalyticalChemistryfromtheUniversityPierreandMarieCurie in2002.ShewashiredbyCNRSas
researcheratESPCI(Paris)in2004todevelopmicrofluidicdeviceforbioanalyticalapplication.In2011,
she joined the lab PhysicoChemistry Curie at Institut Curie, a unique interdisciplinary and clinicalenvironment.Her research focuseson thedevelopmentofmicrofluidicdevices forbiophysicaland
biomedicalapplications.Shehasauthoredmorethan70papersand5patents.Shehasbeenawarded
in 2013 by the French Chemical Society. Since 2013, she is head of the CNRS French Micro andNanofluidicNetwork.Sheisalsoco-founderofINOREVIAlaunchedin2016.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
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THEIMPACTOFLACTATEONTHEMETABOLICPHENOTYPEOFTUMOR-ASSOCIATEDMACROPHAGES
XeniaGeeraerts1,JoVanGinderachter1,SarahMariaFendt2
1LaboratoryofCellularandMolecularImmunology,VrijeUniversiteitBrussel,Brussels,Belgium
2LaboratoryofCellularMetabolismandMetabolicRegulation,VIBCenterforCancerBiology,VIB,Leuven,Belgium
One of the outstanding challenges in macrophage biology is to understand how particular
microenvironmental cues translate into macrophage functions and ultimately in the outcome of
macrophage-regulatedpathophysiologicalprocesses.Averyrelevantdiseaseinthisrespectiscancer.Tumor-associatedmacrophages(TAM)areamongstthemostabundantinflammatorycellsintumors
andasignificantcorrelationwas foundbetweenhighTAMdensityandaworseprognosis formost
cancers.Theco-existenceofdistinct,specializedTAMsubpopulationswithinthesametumorhasbeendefinedbeforeandindicatedthepresenceofprotumoralM2-likeMHC-II(lo)TAMandantitumoralM1-
likeMHC-II(hi)TAM.Therefore,strategiesthatreprogramprotumoralM2TAMintoanantitumoralM1
phenotype,arecurrentlyhighlyinvestigated.
Inthiscontext,ourworkhasfocusedonunravellingthelargelyunknownmetabolicphenotypeof TAM subsets in a murine lung carcinoma model. Interestingly, we found that the metabolic
phenotypeofMHC-II(hi)TAMandMHC-II(lo)TAMshowssimilaritieswithinvitrostimulatedM1and
M2macrophages,characterizedbyaerobicglycolysisfollowedbyatruncatedTCAcycleandoxidative
metabolism,respectively.13Ctraceranalysisrevealedlactate,whichisknowntohighlycontributetotumoracidosis,asacarbonsourceforMHC-II(lo)TAMwhichcanbefueledintotheTCAcyclecoupled
toOXPHOS.Ourdatasuggestthatlactatestronglyaffectsmacrophagemetabolism,asMHC-II(hi)TAM
metabolismisdisturbedandforcedtowardsanintactTCAcycleinthepresenceoflactate.
Overall,ourfindingssuggestthatlactatemightbeanimportantregulatorofTAMmetabolism.Sincemetabolism is shown to be inextricably connected to immune cell function, interferingwith
(lactate)metabolismcouldbeanelegantwaytometabolicallyrepolarizeTAMtowardsanantitumoral
phenotypeandsubsequentlyaffecttumorgrowthandmetastasis.
28
EFFECTOFADIETRICHINPREBIOTICFIBERSOFINULINTYPEANDBEHAVIORONINTESTINALHEALTHINOBESEPATIENTSINANHUMANINVITROFERMENTATION
PATTERNS
GaétanKalala1,2,4,B.Kambashi2,N.Everaert2,Y.Beckers2,A.Richel2,B.Pachikian3,A.M.Neyrinck3,N.M.Delzenne3,J.Bindelle2
1UniversitédeKinshasa,DepartmentofAnimalProduction,Kinshasa-X,DRCongo
2UniversitédeLiège,GemblouxAgro-BioTech,B-5030,Belgium3UniversitécatholiquedeLouvain,MetabolismandNutrition,B-1200,Brussels,Belgium
4WallonieBruxellesInternational,B-1080,Brussels-Belgium.
Obesityanditsassociatedpathologieshaveseriousconsequencesonpatients’lives,leadingto
high costs for society. Consumption of dietary fiber (DF) and prebiotics remains essential for the
modulationofthegutmicrobiotatopreventthesediseases(Delzenneetal.2013).Inthisregard,theFood4Gutmultidisciplinaryresearchprojectisinvestigatingtheuseoffiber-richvegetables,especially
fructans, in order to observe their positive effects on intestinal health. In this framework, obese
patients are subjected for 3months tooneout of twopossible dietary treatments: onebasedon
fructan-richvegetablesandonebasedonvegetablespoorinfructans(placebo).Fecesweresampledand used as inoculum in a dual in vitro model of the gastro-intestinal to study the changes in
fermentationpatternsofthefibrefractionofvegetablesaccordingtothetreatmentthepatienthad
received.Sixvegetablesweresampledintriplicates(N=3)andsteamedfor20to30min:Jerusalemartichoke, salsify, asparagus, pumpkin, fennel and swede (Kalala et al.2017). They were chosen
becausetheydisplayavarietyofcontentsinfructans,soluble(SDF)andinsolubledietaryfiber(IDF).
Steamedvegetable sampleswerehydrolyzed invitrowithporcinepepsinandpancreatin tomimic
digestionintheuppergut.Undigestedfiberresidueswererecoverusinga6kDadialysismembraneand fermented in vitro with the fecal inoculums collected before and after the treatment.
Fermentationkineticsover24haswellasshort-chainfattyacidproductionandprofileswerecompared
accordingtotheindividualdonorandthevegetablespecies.Theextentandtherateoffermentation
were strongly related to the content and fiber profile of vegetables,with high variability betweenindividuals. This variability included a significant difference in the SCFA profile due to the dietary
treatmentwithahigherproportionofbutyrateaftertheinterventionwithfructan-baseddiets.Itcan
be concluded that fermentation potential of fecesmicrobiota of obese patients is highly variable
between individual and that a dietary interventionwith fructans for 3months positively shift thefermentationpatternofvegetablesfibre.
References
DelzenneNetal.2013.Gutmicrobiotaandmetabolicdisoder.BJN
KalalaGetal.2017.Characterizationoffructansanddietaryfiberprofilesinrawandsteamedvegetables.InpressIJFSN
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
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BIOPHYSICALCHARACTERIZATIONOFATHREE-COMPONENTTOXIN-ANTITOXINMODULEFROMTHEPATHOGENICBACTERIUMESCHERICHIACOLIO157
PieterDeBruyn1,RemyLoris1
1VrijeUniversiteitBrussel,Belgium
Bacteriahaveextraordinarywaystodealwithstressingeneralandantibioticsinparticular.The
use and over-use of antibiotics in Europe and the USA has resulted in the rapid development of
resistancetomultipletypesofantibioticsinpathogenic.Asidefrominheritableantibioticresistance,multidrugtolerancecanalsobelinkedtoanon-inheritabletraitcalledpersistence,thesurvivalofa
certain percentage of bacteria in a stressful environment by entering a dormant state. Because
antibiotics preferably work on metabolically active cells, these bacteria are capable to withstand
differentantibioticsduringthetimeoftreatment.
Toxin-antitoxinmoduleshavebeenlinkedtoinducingpersistenceviaamechanismthatinvolves
theactivityofthetoxin.ThepaaR2-paaA2-parE2operonisauniquethree-componenttoxin-antitoxin
module present in pathogenic Escherichia coli O157. Aside from the toxin (ParE2) and antitoxin
(PaaA2),thereisanadditionalregulator(PaaR2)thatisinvolvedintherepressionofthetranscriptionoftheoperon.TheobjectiveistounderstandhowPaaR2autoregulatesthetranscriptionoftheoperon
andhowthisregulationislinkedtotheonsetofpersistence.Viaacombinationofstructuralbiology,
biochemistryandbiophysicalexperiments,amechanisticmodelfortheautoregulationofthepaaR2-paaA2-parE2operonwasconstructedtounravelitsroleinpersistence.
30
HIGHTHROUGHPUTGENERATIONOFDESIGNERENZYBIOTICSAGAINSTACINETOBACTERBAUMANNII
HansGerstmans1,2,RobLavigne2,YvesBries1
1UniveristeitGent,Belgium
2KatholiekeUniversiteitLeuven,Belgium
Overthepastfewyears,antimicrobialresistancehasevolvedfromarareeventtoaneverydayoccurringproblem.Managingthesebacteriainhealthcareiscomplicatedasthelackofnewantibioticsandtheongoingspreadofmulti-andevenpandrug-resistantstrainsleaveuswithareducednumberofpotentialantibiotics,especiallyagainstGram-negativepathogens1.Fallingshortofnewtreatmentswould lead to thedeathof10millionpeopleeachyearworldwide 2, anda cumulativeworldwideeconomiclossupto$124trillionby20503.Moreover,carbapenemresistantAcinetobacterbaumanniiisrecentlycategorizedasoneofthemostdangerouspathogensbytheWHO4.
Enzyme-basedantimicrobialsorenzybioticsexploitthenaturalantibacterialactivityofenzymes.Specifically,weusedendolysinsthatarefoundinbacterialviruses,alsoknownasbacteriophages5.Byprotein engineering we proved in the past that we can customize the antibacterial properties ofendolysinsandwewerethefirsttomakethemhighlyfunctionalagainstGram-negativebacteria(i.e.Artilysins)forwhichmostantibioticsdonotwork6–8.
In2016,pipelineportfolioreviewaboutalternativestoantibioticshighlightedenzybioticsasthealternative class of antibacterials with the greatest potential, based on their clinical impact andtechnicalfeasibility9.Enzybioticshaveseveraluniqueattributes:(1)theirfast(secondstominutes)6,mode-of-actionthatisindependentofanactivehostmetabolism(e.g.bacterialpersisters)6,7(2)theirspecificity against both Gram-positive and Gram-negative pathogens (including antibiotic resistantstrains)5,8(3)thelimitedresistancedevelopmentreportedsofar6.Recently,wehavedevelopedandgradually optimized designer enzybiotics against the four major epidemiological strains ofAcinetobacterbaumannii.
References
1.WHO,WHO’sfirstglobalreportonantibioticresistancerevealsserious,worldwidethreattopublichealth.2014.2.Neill,J.O.,AntimicrobialResistance:Tacklingacrisisforthehealthandwealthofnations.2014.3.Taylor,J.etal.Estimatingtheeconomiccostsofantimicrobialresistance.2014.4.Kahlmeter,G.etal.GlobalPriorityListOfAntibiotic-ResistantBacteriaToGuideResearch,DiscoveryAndDevelopmentOf
NewAntibiotics.2017.5.Schmelcher,M.etal.Bacteriophageendolysinsasnovelantimicrobials.FutureMicrobiol.2012.6.Defraine,V.etal.EfficacyofArtilysinArt-175againstResistantandPersistentAcinetobacterbaumannii.AntimicrobAgents
Ch.2016.7.Briers,Y.etal.Art-175IsaHighlyEfficientAntibacterialagainstMultidrug-ResistantStrainsandPersistersofPseudomonas
aeruginosa.AntimicrobAgentsCh.2014.8.Briers, Y. et al. Engineeredendolysin-based ‘Artilysins’ to combatmultidrug-resistant gram-negativepathogens.MBio
2014.9.Czaplewski,L.etal.Alternativestoantibiotics—apipelineportfolioreview.LancetInfect.Dis.2016.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
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PREDICTIONANDVALIDATIONOFTHESTRUCTURALFEATURESOFOV-58GPCRASANIMMUNODOMINANTANTIGENOFONCHOCERCAVOLVULUS:IMPLICATIONSIN
ONCHOCERCIASISCONTROLEFFORTS
RobertAdamuShey1,2*,FerdinandNgaleNjume1,2,LeaOliveTchouateGainkam1,PhilippePoelvoorde1,LeonMutesa4,AnnieRobert5,PerrineHumblette2,Jean-Pierre
Munyampundu4,JosephKamgno3,LucVanHamme1,StephenGhogomu2,JacobSouopgui1
1UniversiteLibredeBruxelles,Belgium
2UniversityofBuea,Cameroon3CRFilMT,Cameroon
4UniversityofRwanda,Rwanda5UniversitéCatholiquedeLouvain,Belgium
Onchocerciasisisadevastatingyetneglectedtropicaldiseasethatcreatesaremarkablestigma,generates and perpetuates poverty, and is a major impediment to socioeconomicdevelopment/progressofaffectedcommunities.Itisthesecondleadinginfectiouscauseofblindnessworldwide,affectingabout15.5millionpeopleincluding12.2millionwithskindisease,1.025millionwithvisionloss[1]andanadditional172millionpeopleinneedpreventivechemotherapy.Currently,Ivermectinistheonlydrugusedformasstreatmentagainstthedisease[2].Ivermectin,howeverhaslimited effects on adult parasites, so treatments must be expanded over at least 12-15 years,correspondingtothereproductivelifespanoftheadultwormwhenexposedtodrugpressure,hopingnofurtherinfectionoccurswithinthistime.
TheWHO’sambitiousgoalofeliminatingthediseaseby2025willnotbeachievedwithoutthedevelopmentofnewtools [3].Measures tocertifydiseaseeliminationandsurveillanceareofdireneedforhealthsystemsincountrieswhereonchocerciasisisapublichealthproblem.
Here,wereportthat(i)O.volvulusantigenOv58isaG-proteincoupledreceptorconservedinnematodes, (ii) synthetic peptides predicted in the extracellular domain (ECD) of Ov58GPCR elicitpositiveresponsestoserafromonchocerciasispatients,(iii)syntheticpeptidecocktailsdiscriminateserafrominfected,treatedpatientsandnormalAfricancontrols,(iv)polyclonalantibodiesagainsttherecombinantECDrevealedasinglebandonblotsofO.volvulustotalextracts,correspondingtotheexpectedsizeoftheendogenousnativeantigen,(v)Ov58GPCRistranscriptionallyactivatedinboththe larvaeandadult parasite, (vi) humoral immune responsesdepictdeclineswith IVM treatmentcompliance.Hence,weconcludethattheECDofOv58GPCRharborsantigenicfeaturesofinterestinthecontextofonchocerciasisdiagnosisandcontrolefforts.
References1.Lustigman,S.,etal.,Onchocercavolvulus:TheRoadfromBasicBiologytoaVaccine.TrendsParasitol,2017.2.Kuesel,A.C.,ResearchfornewdrugsforeliminationofonchocerciasisinAfrica.IntJParasitolDrugsDrugResist,2016.
6(3):p.272-286.3.Hotez,P.J.,etal.,EliminatingtheNeglectedTropicalDiseases:TranslationalScienceandNewTechnologies.PLoSNeglTrop
Dis,2016.10(3):p.e0003895.
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SINGLE-CELLIMAGINGOFBACTERIALPERSISTERS
FrédéricGoormaghtigh1,LaurenceVanMelderen1,TatjanaSchlechtweg1
1UniversitéLibredeBruxelles,Belgium
Bacterialpersistersarerarephenotypicvariantsthataretoleranttolethaldosesofantibiotics.
Onthecontrarytoresistance,persistenceisatransientphysiologicalstatethatdoesnotrelyongenetic
mutationsandisthereforenotheritable.Persistercellsarethoughttoenteradormantstateinwhichthey are protected against the lethal effect of antibiotics. Two types of persister cells have been
identifiedsofar:typeIpersisters,originatingfromthepreviousstationaryphaseandtypeIIpersisters,
whichareformedstochasticallyduringexponentialgrowth.Uponremovaloftheantibiotic,persister
cellsareabletoresumegrowthandgiverisetoanewbacterialpopulation,whichwillbeassensitiveto the antibiotic as the initial population. Their low frequency (± 1/10,000) associated with their
transientnaturemakethephenomenonparticularlydifficulttocharacterizeusingclassicalpopulation
approaches.Asaresult,thephysiologyofthesephenotypicvariantsremainsmostlyuncovered.
Inthiswork,bycombiningmicrofluidicsandfluorescencemicroscopy,wewereabletoidentify
and track E. coli persisters to ofloxacin (a fluoroquinolone). For the first time, persister cellswere
observedthroughouttheirlifecycle:exponentialgrowth,antibiotictreatmentandgrowthresumption
afterofloxacinremoval.Morphology,growthrate,nucleoidslocalizationandSOSresponseinductionweremonitoredforbothpersisterandsensitivecells.Interestingly,wefoundthatwhiletypeIpersister
cellsarenon-dividingduringthegrowthperiod,typeIIpersistersdodividenormallywithinthesame
period. Interestingly,persistersdosustainDNAdamageasshownbythecompactionof theirDNA,
induction of SOS response and appearance of membrane perturbations. Upon removal of theantibiotic,persistercellsstarttoelongate,formingverylongfilaments(44±23µm)andinducethe
SOS response, allowing them to repair DNA damages caused by the ofloxacin treatment. DNA
replicationthenoccurs,resultingintheformationofmulti-nucleatedfilaments.Finally,divisionevents
takeplaceatmultiplelocationswithinthefilamentandanewbacterialpopulationisgenerated.
Asaconclusion,thisisthefirsttimetypeIIpersistercellswereobservedandcharacterizedat
the single-cell level before, during and after the antibiotic treatment. Such an analysis provides a
powerful tool for studying persister cells physiology and would be valuable to most populationanalysesperformedpreviouslyinthefield.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
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SHUTTLEVECTORSYSTEMFORIMPROVEDPESTIVIRALREVERSEGENETICS
AndreyKossarev1,JanPaeshuyse1
1LaboratoryofHost-PathogenInteractions,DepartmentofBiosystems,KULeuvenUniversity
PestivirusesareubiquitousRNAviruses,generaoftheFlaviviridaefamily,whichpredominately
affectlivestockandresultinseriouseconomiclosses.Someprominentmembersincludebovineviral
diarrhoea (BVD) and classical swine fever virus. On the level of pathogenicity, pestiviruses arecharacterizedbyhighgeneticvariabilityandagradientinvirulence.DuetotheinherentnatureofRNA
viruses,theirinstabilitypresentsachallengeasgeneticengineeringiscumbersometasktoachieve.
Contemporary methods in reverse genetics circumvent this problem through generation of
complementaryDNA (cDNA),whichallows fordirectmanipulationsandpropagationasabacterialcDNAplasmidclone.Furthermore,suitableRNApolymerase(RNAP)promotorspermitgenerationof
infectious genomic viral RNA by in vitro transcription.Modern strategies combine stable bacterial
artificialchromosomes(BAC)witheukaryoticorviralpromotersforinvivo/invitrotranscriptions.
Our prime objective is to establish an improved reverse genetics system with simplified
workflow and provide rigorous tools for molecular studies of pestiviruses. For this purpose we
engineered a BAC shuttle vector system, partially inspired by the KU Leuven PLLAV system
(WO2014174078A1),withasetofregulatoryelementsasafirststeptowardsthisgoal.Currentshuttlevectorversioniscomposedofcopycontrolregulatoryelementsandyeastoriginofreplication(ori).
Thefirstelementfurtherincreasestheoverallvectorstabilitybyreducingtheplasmidcopynumberto
aminimum.A switch from single copyori to high copyori is achieved through arabinose induced
activationoftransactingtrfAelement,increasingthecopynumberupto50times.Yeastorielementallows for propagation and maintenance of the shuttle vector in various yeast strains while
simultaneouslyexploitingyeast’shighlyefficienthomologousrecombinationgeneticmanipulationsof
characterized and uncharacterized pestiviral strains. This step is performed in combination with
CRISPR-Cas9 technology,providingmoreefficientgeneticengineeringmethods.Furthermore,eachviral cDNAclone ispositionedunder the controlofT7promoterand terminator.However, in vitro
transcriptionstepisomittedthroughutilizationofstableT7RNAPexpressingMDBKcell lines.Such
strategyeliminatestheneedforexpensiveT7RNAPkits,significantly increasestheoverallreactionyieldsandmaximizesthechancesforsuccessfulinitiationofviralinfectiouscycle.Lastly,generationof
genuine3’endsandeliminationofpotentialrun-offeffectisrealisedbyflankingviral3’UTRdirectly
withaHepatitisDeltavirusribozyme.Ongoingeffortsareaimedatenhancingcurrentshuttlevector
withstableeukaryoticepisomalretentioncapabilitiesandaninduciblebiotypeswitchforBVDstrains.Finalizedsystemwillbepresentedandpublishedforscientificuseinreversegeneticsofpestiviruses
andRNAvirusesingeneral.
34
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
35
Lecturesession
ENVIRONMENT
36
UHPLC-HRMSBASEDTARGETEDANDUNTARGETEDSCREENINGOFPLASTICIZERSINTHEMARINEENVIRONMENT
SteveHuysman1,LievenVanMeulebroek1,FrancisVanryckeghem2,HermanVan
Langenhove2,KristofDemeestere2,LynnVanhaecke1
1GhentUniversity,DepartmentofVeterinaryPublicHealthandFoodSafety,LaboratoryofChemicalAnalysis(LCA)
2GhentUniversity,DepartmentofSustainableOrganicChemistryandTechnology,ENVOC
Thecumulativeamountofplasticsenteringouraquaticenvironmentispredictedtoincreaseby
an order of magnitude by 2025, resulting in the extensive entree of plasticizers. Plasticizers, i.e.
phthalateacidestersandalkylphenols,areeasilyreleasedintotheaquaticenvironmentasaresultoftheirweakphysicalbondingtoplasticpolymers.As ithasbeenshownthatcontinuousexposureto
plasticizers results inaplethoraofadverseecologicaleffects,monitoringphthalateacidestersand
alkylphenolsinthemarineenvironmentisofgreatimportancetoprovideinsightinthewidespreadoccurenceofthesecompoundsinouraquaticecosystems.
Therefore, this study engaged on the analysis of the abovementioned compound groups in
seawater samples obtained from different locations in the Belgian Part of the North Sea during
differentseasons(2016-2017).Theseawatersampleswereanalyzedusinganin-housedevelopedandvalidatedultra-high-performanceliquidchromatographichigh-resolutionmassspectrometric(UHPLC-
HRMS) method. This novel analytical method was specifically designed to minimize false-positive
detectionsresultingfromlaboratorycontaminationofplasticizers.Performingthetargetedanalysisof
27plasticizersrevealedtheprevalenceof16uniqueplasticizersintheseawatersamples.Alkylphenolsandphthalateswererespectivelydetectedatconcentrationsrangingfrom24to8000ngL-1and20to
2580ngL-1.Moreover,multivariatestatisticsdisplayedthehighestconcentrationsofethylphenoland
dibutyl phthalate in the harbor of Ostend. Next to the native plasticizers, also primary plasticizer
metabolites(mono-phthalates)wereubiquitouslydetectedwithconcentrationsrangingbetween25and2550ng L-1. Subsequent to the targeted screening,untargetedanalysisof the sameseawater
samples revealed 3688 different unknown compounds. Further data analysis for identification
purposesoccurredthroughonlineenvironmentaldatabasesearch,includingMarineLit,MarineDrugs,EPA, drugsbank, ACTor and EAWAG biodegradation. In conclusion, the ubiquitous prevalence of
plasticizers inourmarinewaterssuggeststhatplasticizerswillhavereacheddifferenttrophic levels
throughoutouraquaticecosystemandmaythuscontributetoadverseecologicaleffectsthreatening
thequalityofourmarineenvironment.
Acknowledgments: The authors acknowledge the Belgian Science Policy (BELSPO) for funding the
NewSTHEPSproject(BR/143/A2/NEWSTHEPS;www.newstheps.be)inwhichthisworkfits.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
37
EFFECTSOFCLIMATECHANGEONMACROPHYTEGROWTH,BIOMASSALLOCATION,PHYSIOLOGYANDNUTRIENTSTOICHIOMETRY
RosanneReitsema1,PatrickMeire1,JonasSchoelynck1
1EcosystemManagementResearchGroup(Ecobe),DepartmentofBiology,
UniversityofAntwerp,Belgium
Freshwater macrophytes are faced with the effects of climate change. Increased CO2 andnutrientsinthewaterandperiodsofhigherflowvelocityduetoincreasedprecipitationintensityarethreeimportanteffectsinfreshwaterecosystems(Shortetal.,2016).Itisnotfullyunderstoodhowmacrophytesrespondtothisandwhetherthereisaninteractionbetweentheeffects.InaracetrackflumeexperimentshootsofBerulaerectawereexposedtolow(400ppm)andhigh(1000ppm)CO2concentrations under low (0.04m s-1) and high (0.4m s-1) flow conditions. The experimentwasexecutedtwice:undermesotrophicandundereutrophicconditions.After10weeks,biomassofeachplantwasdetermined(roots, leavesandstemsseparately);morphologywasmeasured(numberofstems,leavesandstolons,lengthanddiameterofthelongeststem,andtotalandaverageleafarea);chlorophyllcontentofasubsetofplantswasmeasured;andfinallynutrientstoichiometry(C:N:P:Siratio)willbeanalysed(theresultswillbeinbeforethepresentation).
TheresultsshowthatplantsgrownunderhighCO2concentrations(HC)comparedtolowCO2concentrations (LC) hadmore and thicker stems,more leaves,more stem- leaf- and belowgroundbiomass,ahigherleaf:stemratioandroot:shootratio,morestolons,ahigherRGRahighertotalleafarea, a lower SLA and a higher chlorophyll content. Flow velocity (FV) also had an effect onplantgrowth:plantsgrewtaller,buthadfewerstems.TherewerealsointeractioneffectsbetweenFVandCO2:HighFV reduced stemand leafbiomassunderhighHC,butenhanced stemand leafbiomassunderLC.PlantsgrownunderlowFVandHCalsohadthehighestaverageleafsurfacearea.Undereutrophicconditions,similarpatternswereseen:morebiomasswasproducedunderHC.However,theplantsweresmallerthaninthefirstexperiment.Clearly,eutrophicationhadanegativeeffectonplantbiomass.Thiswasprobablyduetothegrowthofalgaethatcoveredtheplantsandreducedlightavailability.
It is important to understand howmacrophyte quantity and qualitywill respond to climatechange because living macrophytes play an important role in stream structure and functioning(Schoelyncketal.,2012)andmacrophytedetritusaffectsthedecomposersfoodweb(Polunin,1984)andtheaquaticcarboncycle (Regnieretal.,2013).Thisexperimenthasshownthat increasedCO2concentrations can lead tomoremacrophyte biomass, that this effect can be influenced by flowvelocity,butthattheeffectismuchsmallerundereutrophicconditions.Theseresultshighlyrelevanttohelppredictingandunderstandinghowmacrophyteswilladapttonewconditionsunderclimatechange.
References
Polunin,N.V.C.(1984).InAdvancesinEcologicalResearch.Ac.Press,NewYork,115-166Regnier,P.etal.(2013).NatureGeoscience,6(8),597–607.Schoelynck,J.etal.(2012).Ecography,35,760–768.Short,F.T.etal.(2016).AquaticBotany,135,3–17.
38
PROCESSESOFCH4EMISSIONINPADDYSOILUNDERWATER-SAVINGIRRIGATIONMANAGEMENT
HeleenDeroo1,MasudaAkter1,ElizabethVerhoeven2,DanielSaid-Pullicino3,Marco
Romani4,PascalBoeckx1,StevenSleutel1
1GhentUniversity,Belgium2ETHZurich,Switzerland3UniversityofTurin,Italy4EnteNazionaleRisi,Italy
Owingtothedominantanaerobicconditionsinirrigatedricefields,considerablemethane(CH4)
productioninsoil(witheitheracetateorCO2assubstrate)canresultinsubstantialCH4emissiontothe
atmosphere.Management to limit such CH4 emissions during the rice growing season is possible,providedthatthecontrollingprocessesareunderstood.Inparticular,itisnotcleartowhatextenta
reduction of CH4 emissions under water-saving irrigation management may be the resultant of
enhancedCH4oxidation,duetothedifficultytoreliablyquantifyCH4oxidation.Weassessedrelativecontributions of CH4 production and oxidation to its emission in rice fields under water-saving
Alternate Wetting and Drying irrigation (AWD) in comparison with the conventional Continuous
Flooding(CF)inanItalianricefieldin2015and2016.Therefore,wemeasuredsoilCH4emissionusing
closedfluxchambersinthefieldalongsideinsituassessmentofCH4oxidationbytwodistinctmethods.Firstly,CH4oxidationwasmeasuredthroughselectiveinhibitionofCH4oxidiserswithdifluoromethane
(CH2F2).Secondly,CH4oxidationandcontributionsofbothCH4productionpathwayswereestimated
bymeansofstableCisotopemeasurementsinaerobicandanaerobicincubations,plantaerenchym
andemittedCH4.
As it turnsout,acetatewas themain substrate forCH4production in theendof the season
(theoretical2:1ratio),withthecontributionofthispathwaybeingsignificantlylowerunderAWDin
the beginning of the growing season. Furthermore, lower CH4 emissions in fields under AWD (in
average2.9mgCH4h-1m-2versus14.0underCFin2015;and5.4versus9.3underCFin2016)resultmainlyfromlowerCH4production,andtoalesserextentfromprobablyhigherrelativeCH4oxidation
in thebeginningof thecropping season.Caution is required in the interpretationofCH4oxidation
resultsthough,sincebothmethodswerebiased.Forthefirstmethod,weassumethatCH2F2interactedunintentionallywithCH4production,asoftenCH4emissionswerehigherwithoutthanwithaddition
oftheCH4oxidation inhibitortotheclosedchambers’headspace.Concerningthe isotopemethod,
severalincubationsampleswereprobablytoomuchdisturbed–asindicatedbytheiroftenphysically
impossibleresults.Inconclusion,water-savingirrigationmanagementleadstolowerCH4production,andmaybe–buttoalesserdegree–tohigherCH4oxidationinthebeginningoftheseason.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
39
ASSESSINGTHEMICROBIOLOGICALDIVERSITYINTHECOOLINGWATERSOFANUCLEARRESEARCHREACTOR
ValérieVanEesbeeck1,MohamedAhmed1,RubenProps2,NatalieLeys1,CorinneRivasseau3,
JacquesMahillon4,PieterMonsieurs1
1SCK-CEN,Belgium2GhentUniversity,Belgium
3CEA,France4UniversitéCatholiquedeLouvain,Belgium
TheBR2nuclearresearchreactorcomprisesdifferentwateryenvironments:theprimarycoolingcircuitconsistsofaclosedloopcontainingwaterthatcoolsdownthefuelrodsinthereactorcore.Anopenbasinsurroundsthereactorvessel,andwheneverfuelisloadedontoorunloadedfromthecorematrix, anexchangeoccursbetween thebasinand theprimary circuitwater. Spentnuclear fuel isstored in the contiguous spentnuclear fuelpool (SNFP) inorder to cooldownbeforebeing safelydisposed.Remarkably, despite the low-nutrient environment combinedwith thehighly radioactivecharacterofthewaterandthepresenceofdissolvedradioactivemetals,microbialgrowthisnotfullyprevented. Indeed, several microbes appear to be able to survive and thrive in such conditions.Microorganismsidentifiedinthoseenvironmentsthusprovideauniqueopportunitytoacquirenewinsightsintosurvivalstrategiesandradiation-resistancemechanisms.
The objective of thiswork is to explore the bacterial communities present in the describedenvironmentsoftheBR2nuclearresearchreactor.Inparallel,thisresearchalsofocusesonfollowingupthesecommunitiesovertimeduringandoutsidereactoroperationtomonitorthelong-termeffectofionizingradiation.Finally,thisprojectalsoaimsataphenotypicalcharacterizationoftheprevailingspecies.
Forthecharacterizationandthefollow-upofthebacterialcommunities,a16SrRNAampliconsequencingapproachwasadopted.Resultsfromalong-termfollow-upexperimenthighlightedaclearshiftinthebacterialcommunityprofileduringandoutsidereactoroperation,bothforthebasinandthe primary water. More specifically for the basin water, the profiles observed across multipleshutdownperiodsappearedtobequitesimilar.Incontrasttothis,thecommunityinterestinglyshoweddrasticvariationwhencomparedacrossdifferentreactoroperationcycles.Thiscouldbeduetothechangeinphysico-chemicalparametersthatthesewatersundergowhentransitioningfromonestatetotheother.
Withregardtothephenotypicalcharacterization,strainswereisolatedfromthebasinandtheSNFP and subsequently subjected to ionizing radiation in a gamma irradiation facility to test theirradiationresistance.Thisexperimentshowedthatall12testedstrainstoleratedadoseof300Gy,butonly8%ofthestrainswasabletocopewithadoseof2100Gy,indicatinglargevariabilityinradiationresistancebetweendifferentstrains,andassuchnotnecessarilyahighradiationtolerancetosurviveinthebasinandthespentnuclearfuelpool.
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QUANTIFYINGPRECIPITATIONERRORSVIASOILMOISTUREDATAASSIMILATION
VereLeybaert1
1KatholiekeUniversiteitLeuven,Belgium
The partitioning of the water budget is of key importance to global climate and weather
forecasting,andtothemonitoringofagriculturalandothersocioeconomicphenomena.Soilmoisture
iscrucialinthewaterbudgetattheinterfacebetweenlandandatmosphere.Thebestestimatesofsoilmoisturecanbeobtainedbycombiningsatelliteobservationswithlandsurfacemodelsviadata
assimilation. However, by improving soil moisture estimates, some inexplicable imbalances in the
waterbudgetareintroduced.ThisPhDresearchwill,forthefirsttime,usethemostrecentsatellite
data for soil moisture and precipitation estimation in combination with a quasi-operational landsurfacemodelinordertolinksoilmoisturedataassimilationincrementstoerrorsinprecipitationor
othermodelingerrors,andcontributetomoreaccurateestimatesofthewaterbalancecomponents.
Apreliminaryanalysisof theoperationalSoilMoistureActivePassive (SMAP)Level4SoilMoistureproduct (L4_SM) and in situ precipitationmeasurements over theMurrumbidgee area in Eastern
Australia will be presented. The link between precipitation errors and SMAP L4_SM soilmoisture
incrementswillbeanalyzedusingtemporalandspatialplots.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
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SCREENINGOFTHEBELGIANPARTOFTHENORTHSEATOWARDSEMERGINGORGANICMICROPOLLUTANTS–COMPARISONOFTWOSPE-TECHNIQUESPRIORTOUHPLC-
ORBITRAP-HRMSANALYSIS
FrancisVanryckeghem1,S.Huysman2,H.VanLangenhove1,L.Vanhaecke2&K.Demeestere1
1GhentUniversity,Dept.ofGreenChemistryandTechnology,ResearchGroupEnvironmentalOrganicChemistryandTechnology(EnVOC),CoupureLinks653,9000Gent,Belgium
2GhentUniversity,Dept.ofVeterinaryPublicHealthandFoodSafety,LaboratoryofChemicalAnalysis,Salisburylaan133,9820Merelbeke,Belgium
INTRODUCTION
Alargevarietyoforganicchemicals–includingpharmaceuticals,personalcareproductsandpesticides–areusedineverydaylifeacrosstheglobe.Manyofthesechemicalsaredischargedintothe sewer system, either in their unchanged form or as metabolites, and will eventually enterwastewater treatment plants (WWTP). Yet, WWTP are not designed and most often not able tocompletely remove these so-called micropollutants (Vergeynst et al., 2015). As result of WWTPeffluentdischarge, incorrectusage, improperdisposaland industrialprocesses, these traceorganiccompoundsareemerginginouraquaticenvironment.Asmanyofthechemicalsareknowntohavebiological effects, they areof great environmental concern and their occurrence shouldbe closelymonitored. Presently, knowledge about theprevalence andbehaviour of these compounds iswelldocumented for wastewater, riverine water, groundwater and drinking water (Stuart et al., 2012;Caldasetal.,2013;Battetal.,2017).Marinewaters,onthecontrary,havereceivedmuchlessattentionso-farregardingtheoccurrence,fateandeffectsofemergingorganicmicropollutants.Mainreasonscanbefoundin,a.o.,theanalyticalchallengeswithrespecttosamplingandtheirprevalenceatultra-trace (≤ppb-levels) concentrations. Suitable sample preparation techniques are thus of paramountimportancetobeabletodealwiththeselowconcentrationsprevailinginthemarineenvironment.Next to that, highly sensitive and selective instrumental analytic techniques are needed. In thiscontext, modern high-resolution mass spectrometry (HRMS) enables the quantification of knownmicropollutantsatenvironmentalconcentrationsandthescreeningforavirtuallyunlimitednumberofunknownmicropollutantsinasinglerun.Therefore,thisstudyfocussedonthecomparisonoftwosolid-phase extraction (SPE) techniques followed by a newly developed and validated UHPLC-Q-Orbitrap-HRMSmethodfortargetedscreeningofthemarineenvironmenttowardsalargesetofmulti-classpharmaceuticals,personalcareproductsandpesticides.Next,untargetedscreeningfollowedbymulti-variateanalysiswasperformedasaproofofconceptandtoenablethediscriminationbetweenthetwoSPE-methods.MATERIALANDMETHODSSamplinglocations
Triplicategrabsampleswerecollectedduringtwosamplingcampaignsi.e.inwinter2016andspring2017atthreedifferent locations intheBelgianPartoftheNorthSea(BPNS); i.e.harbourofZeebrugge(51.340467°,3.203364°),atopenSeainfrontofZeebrugge(51.360494°,3.113614°),andtheharbourofOostende(51.226300°,2.935556°)(Figure1).Grabsamplesweretakenat2mdepthusing5LNiskinbottlesandwerestoredinpre-rinsedamberbottles.
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Figure1.DetailedmapofthegrabsamplesthatweretakenintheBelgianPartoftheNorthSea,i.e.harbourofZeebruggeⓐ,openseainfrontofZeebruggeⓑandharbourofOostendeⓒ.Samplepreparationandsolid-phaseextraction
Samplepreparationconsistedof filtering thesamplesuponarrivalat the labusing2.7µmWhatmann filters to remove sediments and debris. To prevent microbial growth during samplestorage(maximum4daysat4°C),thesampleswereacidifiedtopH3withformicacid.Priortosolid-phase extraction (SPE), the samples were neutralized (pH 7) and Na2EDTA.2H2O was added.Thereafter, the samples were extracted in parallel on Oasis® HLB cartridges and on Bakerbondhydrophilicdivinylbenzene(DVB)Speedisks(toallowforacomparison).TheSPE-procedureonOasisHLBandDVBSpeedisksconsistedrespectivelyofloadingavolumeof200mLand1000mLonpre-conditioned cartridges. After washing and drying the cartridges, elution was performed usingmethanol/acetonitrile (50/50, v/v). The eluent was collected in pre-rinsed test tubes, evapo-concentratedandreconstitutedin1mLofmethanol/water(10/90,v/v)with0.1%(v/v)formicacidand0.01%Na2EDTA.2H2O.Finally,theglasstubesweresonicated,vortexedandcentrifuged,andthesupernatantwastransferredintoanHPLCvial.Instrumentation
SamplesoriginatingfrombothOasisHLBandSpeediskextractionwereanalysedusinganin-housedevelopedUHPLC-HRMSmethod.Briefly, thechromatographicanalysiswascarriedoutonareversedphaseHypersilGoldcolumn(50mmx2.1mm,1.9µm).Themobilephaseconsistedofamixtureofwaterandmethanol,bothacidifiedwith0.1%formicacidforanalysisinpositiveionisationmode and 0.01% for negative ionisationmode. The detection was done by a High Resolution Q-ExactiveTMOrbitrapmassspectrometer,equippedwithaheatedelectrosprayionization(HESI)sourceand operating in full scan to allow measurement of known (target) and unknown (untargeted)compounds.Validation
Both analytical methods, i.e. the Oasis HLB and DVB Speedisk-based method, were fullyvalidated using artificial seawater and according to the standard addition approach described byMatuszewskietal.(2003)andVergeynstetal.(2017).Briefly,artificialseawatersamplesspikedbefore(pre-spiked)andafter(post-spiked)extractionwereanalysedintriplicatetogetherwithnon-spikedsamples.Dailyexternalstandardcalibrationwasperformedtoconvertpeakareastoconcentrations.Method precision was determined as relative standard deviation (RSD) of the calibratedconcentrations,measuredintriplicatesamplesonthreedifferentdays.Linearity,methoddetectionlimits,analyterecovery(RE),matrixeffects(ME)andoverallprocessefficiency(PE)weredeterminedasdescribedinVergeynstetal.(2017).
ⓐ ⓑ
ⓒ Oostende Zeebrugge
2 km 1 mi
500 m 1000 ft
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
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Dataprocessing
For targeted analysis, the identification of the compounds was confirmed based on theiraccuratemassandchromatographicretentiontime.ThechromatogramsweremanuallyprocessedusingExactFinder2.0(ThermoFisherScientific,SanFrancisco,USA).Basedonlegislation(EUDirective,2015)andscientificliterature(Willeetal.,2011;Tanwaretal.,2015;Vergeynstetal.,2015),atargetlistwascompiledconsistingof89emergingorganicmicropollutants,including52pharmaceuticals,9personalcareproductsand28pesticides.Untargetedscreeningwasperformedasaproofofconceptmaking use of Compound Discovery 2.1 (Thermo Fisher Scientific, San Francisco, USA) for dataprocessing,followedbymulti-variateanalysisusingSIMCA2.2(Umetrics,Malmo,Sweden).RESULTSANDDISCUSSIONValidationparameters
Methodprecision(RSD,n=3)wasbetterthan20%foratleast80%and97%ofthesamplesextractedwithOasisHLBandSpeediskcartridges,respectively.Methoddetectionlimitswerelowerthan10ng.L-1for67%oftheextractedtargetcompoundswithOasisHLB,whereasthiswasthecasefor80%oftheextractedtargetcompoundswithDVBSpeedisk.LowerMDLsobtainedwithSpeediskextractionclearlyresultfromthelargerextractionvolume(1000mLvs200mL)andpre-concentrationduringsamplepreparation.
The PE values ranged from 0.06% to 103% and from 1.4% to 76% forOasis HLB andDVBSpeediskvalidationsamples,respectively.AdetailedinspectionofthePEanditsallocationintoREandMErevealedthatthedifferencesinPEbetweenthetwoextractionproceduresmainlyoriginatedfromthedifferencesintheextractionrecovery(RE)ratherthanthematrixeffects(ME).Overall,RE-valueswere lower for Speedisk extraction, which could be caused by partial breakthrough for somecompounds. Polar compounds showed higher affinity for Oasis HLB, whereas apolar compoundsshowedhigherrecoverywiththeSpeedisks(Error!Referencesourcenotfound.).
Figure2.SPErecoveries(RE),obtainedwith(●)OasisHLBand(○)DVBSpeediskextraction,asfunctionofpolarity(LogP)forthetargetcompounds.ForallplottedREvalues,thestandarddeviationsare<20%.
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Targetedscreening
The targeted screening was performed on grab samples collected during two samplingcampaignsonthreedifferentlocations.Intotal,60compounds–outofthe89targetcompounds–were quantified in at least one sample. Concentrations ranged between 0.13 - 370 ng.L-1 for thepharmaceuticals,0.65-43ng.L-1forthepersonalcareproducts,and0.10-68ng.L-1forthepesticides.SamplesoriginatingfromtheharbourofOostendeweresignificantlydifferent(p<0.05)fromtheotherlocations.Duetothelowerdetectionlimits,upto62%moretargetcompoundswerefoundingrabsamplesextractedwithSpeediskcomparedtoOasisHLB.Untargetedscreening
Untargetedscreeningrevealedthepresenceofupto1300so-farnon-identifiedcomponentsinasinglesample,i.e.chromatographicpeakswithapeakarealargerthan500000a.u.towhichanaccurate mass could be attributed. The amount of components found in Speedisk extracts wassignificantlylarger(uptoafactorof2.6)thanthosefoundinOasisHLBextracts.Furthermore,multi-variate (discriminative) analysis on all acquired untargeted screening data clearly showed adiscriminationbetweenthetwousedextractiontechniques.CONCLUSION
ThisstudydemonstratestheapplicabilityofanewlydevelopedUHPLC-HR-Q-OrbitrapTM-MSmethodincombinationwithsolid-phaseextractionforbothtargetedanduntargetedscreeningofthemarine environment towards emerging organic micropollutants. The analysis of 18 grab samplescollectedatthreedifferentlocationsintheBelgianPartoftheNorthSea,revealedthedetectionof60pharmaceuticals,personalcareproductsandpesticidesatconcentrationsrangingfrom0.10to370ng.L-1.AlthoughtheextractionrecoveryislowerwithDVBSpeedisksthanwithOasisHLBcartridges,largersamplevolumesandlowerdetectionlimitsoffertheopportunityofdetectingmorecompoundsintargetedanalysisandtracingmorecomponentsinuntargetedanalysis,showingthemeritofthisSPEmethodformonitoringthemarineenvironment.ACKNOWLEDGEMENTS
The authors like to acknowledge the Belgian Science Policy (BELSPO) for funding theNewSTHEPSproject (BR/143/A2/NEWSTHEPS).Thefinancialsupport fromtheHerculesFoundation(Flemish Government; AUGE/11/016) and from the Ghent University Special Research Fund(01B07512) is acknowledged for the UHPLC-Q-ExactiveTM and the automated SPE equipment,respectively.REFERENCESBatt,A.L.,Furlong,E.T.,Mash,H.E.,Glassmeyer,S.T.andKolpin,D.W.(2017)‘Theimportanceofqualitycontrolinvalidating
concentrationsofcontaminantsofemergingconcerninsourceandtreateddrinkingwatersamples’,ScienceoftheTotalEnvironment.ElsevierB.V.,579,pp.1618–1628.doi:10.1016/j.scitotenv.2016.02.127.
Caldas,S.S.,Bolzan,C.M.,Guilherme,J.R.,Silveira,M.A.K.,Escarrone,A.L.V.andPrimel,E.G.(2013)‘Determinationofpharmaceuticals, personal care products, and pesticides in surface and treated waters: Method development andsurvey’,EnvironmentalScienceandPollutionResearch,20(8),pp.5855–5863.doi:10.1007/s11356-013-1650-9.
EUDirective(2015)‘CommissionimplementingDecision(EU)2015/495establishingawatchlistofsubstancesforUnion-widemonitoringinthefieldofwaterpolicypursuanttoDirective2008/105/ECoftheEuropeanParliamentandoftheCouncil’,OfficialJournaloftheEuropeanUnion,L78.
Matuszewski, B. K., Constanzer, M. L. and Chavez-Eng, C. M. (2003) ‘Strategies for the assessment of matrix effect inquantitative bioanalytical methods based on HPLC – MS/MS’, Analytical Chemistry, 75(13), pp. 3019–3030. doi:10.1021/ac020361s.
Stuart, M., Lapworth, D., Crane, E. and Hart, A. (2012) ‘Review of risk from potential emerging contaminants in UK
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groundwater’,ScienceoftheTotalEnvironment.ElsevierB.V.,416,pp.1–21.doi:10.1016/j.scitotenv.2011.11.072.Tanwar,S.,DiCarro,M.,Ianni,C.andMagi,E.(2015)‘OccurrenceofPCPsinNaturalWatersfromEurope’,inM.S.Díaz-Cruz,
D.Barceló(Eds.),TheHandbookofEnvironmentalChemistry,pp.41–53.doi:10.1007/698.Vergeynst, L., Haeck, A., De Wispelaere, P., Van Langenhove, H. and Demeestere, K. (2015) ‘Multi-residue analysis of
pharmaceuticals in wastewater by liquid chromatography-magnetic sector mass spectrometry: Method qualityassessment and application in a Belgian case study’, Chemosphere. Elsevier Ltd, 119, pp. S2–S8. doi:10.1016/j.chemosphere.2014.03.069.
Vergeynst,L.,K’oreje,K.,DeWispelaere,P.,Harinck,L.,VanLangenhove,H.andDemeestere,K.(2017)‘Statisticalproceduresforthedeterminationoflinearity,detectionlimitsandmeasurementuncertainty:AdeeperlookintoSPE-LC-Orbitrapmassspectrometryofpharmaceuticalsinwastewater’,JournalofHazardousMaterials.ElsevierB.V.,323,pp.2–10.doi:10.1016/j.jhazmat.2016.05.077.
Wille, K., Claessens,M., Rappé, K., Monteyne, E., Janssen, C. R., De Brabander, H. F. and Vanhaecke, L. (2011) ‘Rapidquantificationofpharmaceuticalsandpesticidesinpassivesamplersusingultrahighperformanceliquidchromatographycoupledtohighresolutionmassspectrometry’,JournalofChromatographyA.ElsevierB.V.,1218(51),pp.9162–9173.doi:10.1016/j.chroma.2011.10.039.
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BRIDGINGRESOURCERECOVERYTOREUSE:MICROBESASNEXT-GENERATIONFERTILIZER
MyrsiniSakarika1,JanneSpanoghe1,EvaWambacq1,MaartenMuys1,AbbasAlloul1,MarcSpiller1,GustavoGomesdeSousa1,VeerleDerycke2,LutgardStragier3,HarmeenVerstraete3,
KoenFauconnier4,OliverGrunert5,GeertHaesaert2,SiegfriedVlaeminck1
1UniversityofAntwerp,Belgium2GhentUniversity,Belgium
3AvecomNV,Belgium4AgrAqua,Belgium
5GreenyardHorticultureBelgiumNV,Belgium
Organic fertilizers (OF) are conventionallyproduced fromanimal andplantderivedmaterial.
Theirstrikingfeatureisthatnitrogen(N)andpartlyphosphorus(P)arepresentinorganicformandthus are releasedgradually throughdecompositionbymicrobial activity in the soil or the growing
medium.A special typeofOF,microbial biomass (MB), canupgrade inorganic nutrients to ahigh-
qualityOF.Experimentswereconductedusing3differenttypesofMBgrownon(realandsynthetic)
non-feacaly contaminated side-streams: (i) consortium of aerobic bacteria (CAB), (ii) Rhodobactersphaeroides(purplenon-sulfurbacteria)and(iii)Arthrospiraplatensis(Spirulina).Testsusingstruvite
asarecoveredP-sourcerevealedthat theP-releaserates (0.32–4.69gP/L/d)arehigher thanthe
microbial P-uptake rates, indicating that struvite can be directly dosed into the bioreactors as a
sustainable, low-costalternative forcommercialPandNsources.Mineralization testsshowedthatcomparedtothereferencefertilizer (RF) (basedonacombinationofanimalandplantmaterial),N
mineralizationinMBoccursgraduallyduringthefirstweeksofincubation,while25-35%oftheNis
mineralizedafter42daysinallMB(individualandblends)tested.Interestingly,MBblendsprovedto
be useful for steering the NH4-N mineralization. Subsequently, pot trials were performed usingperennial ryegrass (Lolium perenne), parsley (Petroselinum crispum) and tomato (Lycopersicon
esculentum).TheseshowedpromisingresultsastheMB,usedinavarietyofblends(75%CAB:25%
phototrophsto90%CAB:10%phototrophs),performjustaswellastheRFintermsofplantlengthanddryweightincrease,whileduringthediseaseresistancetestsperformedintomatoplantsthepresence
of Spirulina led to a markedly reduced sensitivity to a biotrophic fungus (mildew). In terms of
fertilizationperformance,MBcancompetewithatypicalOF,andCABisfornowthemostcost-efficient
novelsolution.Lifecycleassessment(LCA)resultsindicatethatCABfertilizershaveacomparableoreven lower environmental impact than alternative OF blends (i.e. horn, blood and bone meal).
Efficiency gains in downstream processing and use of renewable energy can even reduce the
environmentalimpact(i.e.greenhousegassemissions)belowthatofmineralfertilizers.Theresearch
wasfundedbyMIPiCleantechfortheMicroNODproject(http://www.micronod.be/).
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
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Lecturesession
KEY-ENABLINGTECHNOLOGIES
48
KEYNOTEIII
LIGHTPOWEREDENZYMATICDEGRADATIONOFLIGNOCELLULOSE:ANEWPHOTO-BIO-CATALYSISFORFUELSANDCHEMICALS
DavidCannella
PhotobiocatalysisUnit,UniversitéLibredeBruxelles,Belgium
E-mail:[email protected]
EverytimeasinglephotonhitsplanetEarth,acomplexsetofreactionsdesignedforcapturing
itsenergystartsrunning,ignitingachainofeventswhoseoutcomesare:theoxygenicphotosynthesis,
thegrowthofbiomassthroughreductionofcarbon,andultimatelytheoriginoflife.Tocloseitscycle,carbonneedstobeoxidizedandreturnedtotheatmosphere.Recentlyhasbeenreportedanefficient
light-drivenenzymaticsystemtooxidizecarbohydratesfromcellulose,whichisthemajorfractionof
the photosynthetically fixed carbon. The system discovered is based on photosynthetic pigmentchlorophyll,andtheubiquitousenzymelyticpolysaccharidemonooxygenase(LPMO)thatdegrades
carbohydrateslikeplantcellwall,starch,andchitinfoundinallecosystems.Sinceitconsumesoxygen
andutilizesthechlorophyllandenergyoflighttodegradetheproductofphotosynthesis(cellulose),it
hasbeencalledinpopularterm“ReversePhotosynthesis”.
This breakthrough provides us with monooxygenases, a major and powerful class of redox
enzyme,asabasictoolto initiateandexpandresearchonthebiggestchallengeswithinchemistry,
physicsandbiology:acompletelyrenewable/greentechnologycapableofconverting light’senergy
into chemical energy (i.e. unique oxidized oligosaccharides, phenols, organic acids, methanol andethanol). Such a technology could indeedmake a real contribution to the 21st century biobased
society.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
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PHOTOREACTIVECROSSLINKINGREVEALSNOVELDIRECTINTERACTIONPARTNERSOFTHEPERSISTERREGULATOROBG.
PaulineHerpels1,ElenLouwagie2,JosepRayoCompany3,AnastassiosEconomou3,
NatalieVerstraeten1,JanMichiels1
1VIB,KatholiekeUniversiteitLeuven,Belgium2KatholiekeUniversiteitLeuven,Belgium
3Rega,KatholiekeUniversiteitLeuven,Belgium
Chronicinfectionsthatarerecalcitranttotreatmentwithantibioticsareposingamajorthreat
tohumanhealth.Thisrecalcitranceispartlycausedbyso-calledpersistercells.Persisterscomprisea
smallfraction(typically<0.1%)oftransientlyantibiotic-tolerantcellsthatareabletosurviveprolongedexposuretoantibiotictreatment.Althoughitsclinicalimportancehasbeenelaboratelydocumented,
theprecisemolecularmechanismsunderlyingpersistencearenotfullyunderstood.Previousresearch
inourgrouphasdemonstratedacentralrolefortheconservedGTPaseObginmediatingpersistence.
Unraveling the precise functioning of Obg-mediated persistence will shed light on this transientphenomenon and identify possible targets for anti-persister therapies. To identify novel direct
interactionpartnersofObg,aninnovativephoto-crosslinkingtechniquewasused.Thismethodallows
covalent binding of direct binding partners in vivo, which is an important feature concerning thetransientnatureofpersistence.Theunnaturalphoto-reactiveaminoacidp-benzoyl-L-phenylalanine
(pBpa)isincorporatedatspecificlocationsoftheObgprotein.UnderinfluenceofUVradiation,the
carbonyloxygenofpBpacrosslinkstoanycarbon-hydrogenbondofmoleculeswithinaradiusof3
angstrom. We used this method to incorporate pBpa in Obg from Escherichia coli (ObgE). Byexperimentaloptimization,wewereabletoefficientlyincorporatepBpaatdifferentresidueslocated
onthesurfaceofObgEandObgED246G(mutantformdeprivedofthepersisterfunction).Crosslinking
ofObgEwithpossibleinteractionpartnerswasdetectedandtheinteractingmoleculeswereidentified
usinghighresolutionliquidchromatography-massspectrometry.Resultsarecurrentlybeingconfirmedand complemented using a bacterial-two hybrid approach. Further testing of the identified direct
interaction partners will result in a selection of genes specifically involved in the Obg-mediated
persistencepathway.The fullunderstandingof thispathwaywill contribute to thedevelopmentof
anti-persister therapies.We hypothesize that targeting Obg-mediated persistencewill significantlyreducethenumberofpersistercellsinbacterialpopulations,therebyfacilitatingclearingofinfections
byconventionalantibiotics.
50
THEEFFECTOFTHELAYERTHICKNESSONTHEPERFORMANCEOFPOROUSLAYEREDRADIALLYELONGATEDPILLARALLAYCOLUMNS.
ShuntaFutagami1,TakeshiHara2,HeidiOttevaere1,HermanTerryn1,GinoV.Baron1,Gert
Desmet1,WimDeMalsche1
1VrijeUniversiteitBrussel,Belgium2KyushuUniversity,Japan
Vast progress in the column efficiency of pillar array columns has been achieved with theadoptionof radiallyelongatedpillars (REPs)asanalternativetoclassicalcylindricalpillars,andthispillar shape can provide a sub-micron minimum plate height. We recently reported that themesoporous silica layerprepared in aREP columnby sol-gel processingdramatically increases theretentioncapacitywhilekeepingitshighcolumnefficiency[1].Inthiscontribution,aREPcolumnwithathickermesoporoussilicalayerisreportedanditsperformanceiscomparedwithamesoporoussilicalayeredREPcolumnpreparedwiththepreviouslyreportedrecipe.
MesoporoussilicalayerwaspreparedinaREPcolumn(1mmwide,18μmdeep,and16.5cmlong)witha sol-gel feed solution,which consistedof amixtureof tetramethoxysilane (TMOS)andmethyltrimethoxysilane(MTMS),polyethyleneglycol(PEG),urea,andaceticacidaqueoussolution.Inordertoprepareathickerlayerthantheonepreviouslyreported[1],thefeedsolutioncontainedmoreTMOS/MTMSmixture, and less PEG to allow for slower gelation. After the C18modification, theretentioncapacityandthecolumnefficiencywereexaminedbychromatographicmeasurementswithalkylphenones.Scanningelectronmicroscopy(SEM)wasappliedtovisuallyinspectthemesoporoussilicalayers.
SEMimagesofamesoporoussilicalayeredREPcolumnshowedstraightanduniformdepositionofthe layer.FromtheSEMimages,thethicknessofthe layerwasdeterminedtobe220nm, . It isnoteworthythatthepreparationconditionforthethickerlayercoveredtheglasslidwithasthickalayerasthatonsilicon. Incontrast, itwasnotthecasewiththepreviouslyreportedcondition. It isassumedthatinthethickerlayercondition,coveringtheglasslidismorefavoredthangrowinganeventhicker layer on silicon. The retention capacity of the PLREP columns was compared by LCmeasurements with alkylphenones with mobile phases of 60, 70, 80 and 90% acetonitrile. ThemeasurementsrevealedthatthePLREPcolumnhavingathickerlayerhas1.3timeslargerretentioncapacity than thePLREP columnhaving a thinner layer. Comparisonof theH-uplotsof thePLREPcolumnsforoctanophenonedemonstratedthattheincreaseintheminimalplateheightof0.94µmto1.43µmwasaccompaniedbytheoptimalvelocityshift from0.10mm/sto0.24mm/swhengoingfromathinnertothickerlayer.Theseverylowplateheightsoutperformclassicalpackedbedcolumnsandhavegreatacademicandindustrialpotential.
References
[1]Futagami,S.etal.J.Chromatogr.A2017,1523,234–241.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
51
MATRXCELL:IMPROVINGCELLCULTUREASSAYSWITHTUNABLEHYDROGELRIGIDITIES
MarieVersaevel1,SylvainGabriele1
1UniversityofMons,Belgium
The development and testing of drug candidates require large amount of upstream in vitro
screening assays. These tests are usually performedwith primary or immortalized cells grown on
culturedishesmadeofrigidpolystyreneorglass,whicharerarelyfunctionalizedwithaspecificproteincoating.Inaddition,plasticandglassculturedishesarecharacterizedbyaveryhighstiffness(~10^9
Pa), far from the softness of physiological tissues (10^2-10^5 Pa). However, many works recently
demonstrated that cells are able to sense themechanical properties of their environment and to
respondbyamodulationoftheirshape,internalstructureandbiologicalfunctions.Asaconsequence,rigid culture substrates can alter the cell sensitivity to drug candidates, that can lead to
misinterpretations in in vitro testings.Cell sensitivity to chemotherapeuticagents forexamplewas
clearlydemonstratedtobedependentonsubstratestiffness.
Indeed, accumulating evidence suggests that the physico-chemical properties of the cellular
microenvironmentmodulateimportantcellularfunctions(e.g.proliferation,migration,differentiation,
…).Weaddressedthischallengebydevelopinginnovativesofthydroxy-PAAmculturesubstratesthat
enableaprecisecontrolofboththestiffnessandthebiologicalcoating(patentUS20150104812andEP2854885).Thisstrategypermitstoobtaintailor-madeculturesubstratesthatmatchthephysico-
chemicalenvironmentofthetissueconcerned.Hereweshowthatbraincells(neuronsandastrocytes)
grownontheseoptimizedculturesubstratespresentamorerelevantphenotypeandoptimalcellular
functionscomparedtoclassiccellcultureconditions.WeaiminlaunchingtheMatrXcellspin-offthatproducesandcommercializesinnovativecelltype-optimizedculturesubstrates.
WeacknowledgetheFIRSTSpin-offprogramoftheWalloonRegionforthefundingofthisproject.
52
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
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POSTERSESSIONS
54
Unevennumbers–PosterSession1Evennumbers–PosterSession2
ChemistryandChemicalEngineering
P1.NathanielBerneman(VUB).Nano-PrecisionConstructionEngineeringatthe1-MicronScale:NewHierarchicalMaterialsandNewApplications.P2. Agata Kryj (VUB). Development of novel column support structures in microchips forelectrochromatographicseparations.P3.MartynaBaca(VUB).Achievingapeakcapacityof1500usingan8mlongchromatographicchip.P4.Benoit Thienpont (VUB).Colorfulmicrospheresmadeof crystallizednanospheres inapolymermatrix.P5. IlseDeLeersnyder(UGent).The influenceofmediacomponentsonthestabilityandtoxicityofsilvernanomaterials.P6.ThomasVirdis(VUB).Non-IdealityinTheAdsorptionOfEthanol/EthylAcetate/WaterMixturesOnZIF-8MetalOrganicFramework.P7. José Luís Dores-Sousa (VUB). Mixed-mode retention mechanisms and selectivity of reversed-phase/weak-anion-exchangecolumnsinliquidchromatography.P8.ChristinaTiflidis(VUB).ElectrochemicalStudyofITOasaWorkingElectrodeforaTransparentChip.P9.JoséLuísDores-Sousa(VUB).Prototypingofmicrofluidicthermoplasticchipsforhigh-performanceliquid-chromatography.P10.AtirujTheppawong(UGent).Synthesis,BiologyandStructure-ActivityRelationshipsofNovelβ-enaminone-,aza-aromatic-andthiazepane-basedCurcuminoids.
AgricultureandFoodTechnology
P11.JoseC.Jimenez-Lopez(EEZ–CSIC,Spain).Flavonoidssyntheticpathwayenzymesasbiomarkersfor the study of biochemical and physiological changes occurring during narrow-leafed lupin seedgerminationandseedlingdevelopment.P12. Oyakhilomen Oyinbo (KUL). Maize Farmers’ Preferences for Site-specific Extension ServiceDeliveryinNigeria:InsightsfromaChoiceExperiment.P13.BrackeJolien(UGent).Nondestructiveopticalcropsensingtomonitornitrogencontentinwoodyornamentals.P14.AliSedaghatDoost(UGent).Formulationofnovelbiopolymernanoparticlesfortargeteddeliveryofbioactivecompounds.P15. Claire Chigwedere (KUL). Influence of storage on the cooking quality of Canadian wondercommonbeans (Phaseolus vulgaris): insight intomechanismof softeningandevolutionof volatilecompounds.P16. Wim Geeraerts (VUB). Lactic acid bacteria species diversity on a selection of cooked meatproductsintheBelgianretail.P17.VincentGreffe (KUL). Improvingtheshelf lifeof rhizobia inoculants:apangenomicsapproachinvestigatingstress-resistanceinSinorhizobiummeliloti.P18.VictorMoreto(KUL).Calibrationofasugarcanecultivarinaquacropmodel.P19.FlorentAudrit(ULB).Effectoftheoxidativeactivityoflaccaseonfoodproducts.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
55
P20.MaremeNiang(CERAAS,Sénégal).Germinationresponsestodroughtstressamongstbaobabs(AdansoniadigitataL)inSenegal.P21.ElsDebonne(UGent).Assessmentoftheantifungalpreservationpotentialofnaturalwateroroilsolublecompoundsinbothin-vitroandbreadbakingtrials.P22.CatherineMermans (UGent).AG326Esubstitution in theglutamate-gatedchloridechannel3(GluCl3) of the two-spotted spider mite Tetranychus urticae abolishes the agonistic activity ofmacrocycliclactones.P23. Niels Langenaeken (KUL). Contribution of small and large barley starch granules to maltoseproductionduringmashing.P24.WillyIrakoze(UCL).Comparativeeffectsofchlorideandsulfatesalinitiesontwocontrastingricecultivars(OryzasativaL.)attheseedlingstage.P25.LauraDeMets(UGent).Crossingborderstomitigatemycotoxins:wheremicrobiology,poly-omicsandcropprotectionmeet.P26.EmielVanReckem(VUB).Theinfluenceofprocessingconditionsonthecommunitydynamicsofcoagulase-negativestaphylococciduringmeatfermentation.P27. Marko Verce (VUB). The composition and functional potential of water kefir fermentationmicrobiotaasrevealedthroughshotgunmetagenomics.P28.WannesVanBeeck(UA).Fermentedcarrots:Anovelfoodmatrixforprobioticinterventions.P29. Louise Vermote (VUB). Cheese brines harbour both halophilic/halotolerant and cheeseingredient-associatedmicroorganisms.P30.BiancaDrepper(KUL).NewweatherrelatedinsurancemodelsfortheFlemishhardfruitsector.P31. Savio Rodrigues (KUL). Hairy Root Disease: Unravelling the molecular interactions betweentomatorootsandrhizogenicagrobacteria.P32.RayangnéwendéAdèleOuédraogo (UCL).Evaluationdedifférentes formulationsdecompostsassociéesounonauTrichodermaetauxchampignonsmycorhiziensarbusculairessurlerendementdelatomate(Solanumlycopersicum)etsurlesolenconditionssemi-contrôlées.P33.PhuongNguyenNhu(VUB).AnovelstressresponsetriggeredbyERD14.P34. Manon Demulder (VUB). Chemical inhibitors of the SOG1 transcription factor to promotealuminiumtoleranceinmaizeandbarley.P35. Anne-SophieMathieu (UCL). Impact of high temperatures on sugarmetabolism and yield inCichoriumintybusL.varsativum.P36.MarieVandermies(ULg).VersatileregulatedpromoterforYarrowialipolytica.P37.AlexisFernandez(ULg).Doespearlingprocessconcentrateβ-Glucansinbarleygrain?P38.AmauryGérard(ULg).WhatisthelandscapeofartisanalcheeseproductioninBelgium?P39.HassinaHafidaBoukhalfa(MohamedKhiderUniversity–Biskra,Algeria).Improvementofsprayretentiononbarleyleaves.P40.QuentinRivière(ULB).Roleofthecircadianclockintheresponseoftheplant-modelArabidopsisthalianatomagnesiumdeficiency.P41.SoundousEl-Hajjaji(ULg).InventoryoftherawmilkbutterproductioninWallonia(Belgium).P42.AmauryGérard(ULg).StudyofthegrowthpotentialofListeriamonocytogenesincheesesmadebyBelgianfarmers.P43. Phuc LeHo (KUL). Changeof electrolyte leakageof dragon fruit (Hylocereus undatus) duringstorage.P44.RedaMelhaoui(UniversitéMohammedPremierOujda,Maroc).TocopherolanalysisinalmondoilsproducedineasternMorocco.
56
P45. Ali Sedaghat Doost (ULg). Fabrication of thymol nano-emulsions as a potential naturalantioxidant.P46.KahramenDeghnouche(MohamedKhiderUniversity–Biskra,Algeria). InventoryandstudyofthenutritionalvalueofspontaneousforageplantsinanaridregionofAlgeria.P47.FinguMabolaJuniorCorneille(ULg). Impactofendophyticentomopathogenicfungusonplantattractivityinaphid-virus-plantinteractions.P48.HassinaHafida Boukhalfa (MohamedKhiderUniversity – Biskra, Algeria). Evualution of sprayretentionvariabilityofhydrophobicplantleaves.P49. Kahramen Deghnouche (Mohamed Khider University – Biskra, Algeria). Evaluation of growthperformanceOuledDjellallambsaccordingtosex,sizeofthelitterandtheparityinasemi-aridregionofAlgeria.P50.JulienLouvieaux(ULB).Below-groundapproachestoimprovenitrogenuseefficiencyinoilseedrape.
Biomathematics
P51.FlorisDalemans(KUL).ApredictionmodelfortheseedandoilyieldofPongamiapinnata.P52.GlebGoussarov(UGent).WholeGenomeStatisticsforComparingBacterialStrains.P53.NicolasDierckxsens(ULB).Targetedassemblyandvariancecallingfromwholegenomedata.P54.JanisBaeten(UGent).Asimplemodelforaerobicgranularsludge.P55.JoëlleDeCaluwé(ULB).Modelingthephotoperiodicentrainmentoftheplantcircadianclock.P56.FrançoisAncien(ULB).Predictionandinterpretationofdeleteriouscodingvariantsintermsofproteinstructuralstability.P57.TamasLazar(VUB).Biophysicalvalidationoffullydisorderedproteinensembles.P58.StijnWittouck(UA).ComparativegenomicsofLactobacillusbacteria.
HumanHealth
P59. Jone Paesmans (VUB). Revealing the function of TBC1D24mutations in epilepsy and relatedneurologicaldiseases.P60.JoseC.Jimenez-Lopez(EEZ–CSIC,Spain).Bluelupinseedβ-conglutinproteinsreversebacktheinducedinsulinresistanceinpancreaticcells.P61.HungNguyen(VUB).Validationofdenovodesignedcalpaininhibitors.P62.MohamedMusrati(VUB).Ly6ClowMonocytesPromoteLiverRegenerationduringDrug-InducedLiverFailure.P63.MateKiss(VUB).Non-canonicalIL-1βreleasebymyeloidcellspromotestumorprogressionandresistancetoimmunotherapy.P64.EvangeliaBolli(VUB).Nanobody-baseddepletionofprotumoraltumor-associatedmacrophagesasnovelcancertherapy.P65.AnnegreteVisnapuu(KUL).Studyingancientmechanismtosynthesizenovelanti-biofilmdrugs.P66.TingWu(UGent).Anti-inflammatoryeffectofAroniapolyphenolsincombinedmicrobialinvitrodigestion/cellculturemodels.P67. KennyAppermans (KUL).Gradients of antimicrobials influence the development of antibioticresistance.
Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018
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P68.ShahbazBashir(KUL).Quantificationofimmuneresponseinskinswellingtestusinganimage-basedplatform.P69.AnaRitaPomboAntunes(UGent).Dissectingtheheterogeneityandfunctionsoftheglioblastomaimmunecompartment.P70.LaureVerstraete(KUL).InvitroandinvivoidentificationofpersisterdeterminantsinnaturalandclinicalPseudomonasaeruginosastrains.P71. Laetitia VanWonterghem (KUL). Identification and characterization of bacterial host factorsinvolvedinconjugativehorizontalgenetransfer.P72. Bram Lories (KUL). Responses to ecological competition link biofilm formation, virulence andantibioticresistanceinSalmonella.P73.MiekeDeBuck(KUL).COOH-terminalSAA1peptidesfailtoinducechemokinesbutsynergizewithCXCL8andCCL3torecruitleukocytesviaFPR2.P74. Helena Van Damme (VUB). The CCL8/CCR8 axis maintains an immunosuppressive tumormicroenvironment.P75. Hanne Vissenaekens (UGent). Cellular quercetin uptake, localization and metabolism understressedconditions.P76. Khaoula Toumi (ULg). Assessment of exposure of workers to pesticide residues in tomatogreenhouses.P77.HassinaHafidaBoukhalfa(MohamedKhiderUniversity–Biskra,Algeria).Operatorexposurerisksinphytosanitarypractices,caseofBiskra–Algeria.P78. Sara Abouelasrar Salama (KUL). Proteolytic cleavage of serum amyloid A (SAA) 1 by matrixmetalloproteinasesfine-tunestheinflammatorycapacityofthisacutephaseprotein.
Environment
P79.HassanMaklad (VUB). Phosphorylationof theTetR transcription factor FadR in thearchaeonSulfolobusacidocaldariusaffectsitsligandbindingability.P80.ZeLiu(UGent).CombinationofO3/H2O2andUVtoassessdegradationofatrazineinaqueoussolution.P81.VereLeybaert(KUL).QuantifyingPrecipitationErrorsviaSoilMoistureDataAssimilation.P82.HuiXu(UGent).Greatercontributionofbelowgroundthanabovegroundmaizebiomasstothestablesoilorganiccarbonpool.P83.MichielMaertens(KUL).TheimpactofvegetationchangesonsoilmoistureovertheDryChacoP84.TessGoessens(UGent).Environmentalscreeningofmulti-classantimicrobialdrugresiduesinsaltandfreshwaterecosystemsusingtargetedanduntargetedhighresolutionmassspectrometry.P85. Nina Ricci Nicomel (UGent). Development of a high-throughput automated platform forstandardizedmetalbiosorptionstudies.P86. AbuakriWumbei (UGent). Pesticides Residue Analysis in Yam from SelectedMarkets AcrossGhanaandBelgium:AnEvaluationoftheQUECHERSMethod.P87.AnuYadav(SCK-CEN).Radiationresistanceinthecyanobacteriumarthrospira.P88. Gilles Olive (Ecole Industrielle et Commerciale de la Ville de Namur). Can Medauroideaextradentata (Brunner von Wattenwyl, 1907) and/or Haaniella echinata (Redtenbacher, 1906)(Phasmatodea:Phasmatidae)helpustofightFallopiajaponica((Houtt.)RonseDecr.,1988)?P89.TriTruong(UGent).DevelopmentawatermonitoringsystembasedonbiologicalindicatorswithBenTreriversystem-thelatestbranchofMekongRiverBasinbeforegoingtothesea.
58
P90. Kamilia Farhi (Mohamed Khider University – Biskra, Algeria). Estimation of the Black belliedsandgrousegroup’ssizeinwateringsiteinSouth-EastofAlgeria.P91.ElenaBorregánOchando(UA).IncorporatingtheUSEPA’s“CompTox”dashboardandthe“For-Ident”platforminworkflowsfortheidentificationandpreliminaryassessmentoforganicemergingmicropollutantsinwater.P92.Marlies Volckaert (UGent). Supply chain of forest products supported by spatial informationsystems.P93. Liesbeth Lemmens (VUB). Transcription factor-mediated gene regulation in response to heatshockstressinthermoacidophilicarchaea.P94.EdelbisLópezDávila(UGent).PesticidesknowledgeandusebyCubanfarmersfromSanctiSpíritusregions.
Key-enablingTechnologies
P95.FréderickMatheuse(VUB).LiquidChromatographyandadvanced3Dprinting:amatchmadeinheaven?P96.LisaTripodi(KUL).Plasticbasedhydrophilic-in-hydrophobicmicrowellarraysfordigitalbioassays.P97.JolienBreukers(KUL).DevelopmentofamicrofluidicplatformforscreeningandsortingofsingleBcells.P98.DavidSybers(VUB).DevelopmentoffluorescentreportersforthermoacidophilicarchaeaP99.KarenVen(KUL).DNA-onlydigitalbioassayP100. Bernd Peeters (KUL). AuNP-labelled FO-SPR sensors to develop innovative DNAzyme-basedbioassayconcepts.P101.MarineLuciano(UMons).Decipheringtheroleofthecurvatureinfoldedepithelialtissues.P102. Laura Alaimo (UMons). Collective cell migration in geometric confinements imposed bymicrochannels.P103.DriesVloemans (KUL). Integrationof passive SAT-basedheating systemonPOCmicrofluidic(i)SIMPLEplatform.P104. Iene Rutten (KUL). DNA origami nano-architectures for well-defined and robust biosensinginterfaces.P105.Michiel Van Tendeloo (UA). Promising nitrogen removal performancewith theMainstreamAnammoxSystem(MAS)forsewagetreatment.P106.SteveSchoonooghe(VUB).Nanobodies:fromresearchtoolstodiagnostics&therapeutics.
POSTERCONTRIBUTIONS
Extendedabstracts
60
FLOWERING AND FRUITING OF YELLOW PASSION FRUIT (PASSIFLORA
EDULIS, VAR F. FLAVICARPA.DEG) AS INFLUENCED BY DIFFERENT FERTILIZER APPLICATIONS IN KIAMBU AND EMBU COUNTIES, KENYA
MWIRIGIPETER1,JOSEPHGWEYI-ONYANGO1ANDMAINAMWANGI1
1DepartmentofAgricultureScienceTechnology,KenyattaUniversity,P.O.Box43844-00100,Nairobi-Kenya
INTRODUCTIONInKenyatheyellowpassionfruit(Passifloraedulis,varf.flavicarpa.deg)varietyhassourandsweetcultivarswhicharemostlysuitedtolowaltitudes.Thevarietyalsothriveswellinmidaltitudesofupto1500mabovesealevelandlatelytherehasbeenincreasedproductionofthisfruitinsemi-aridregionsofEmbu,Meru,TharakaNithiandKirinyagacounties(HCDA,2013).ThestrengthofyellowpassionisthatitisthoughttobemoretoleranttoFusariumwilt,adiseasethathasseverelyreducedpurplepassionfruitproductivity.Worldwide,Brazil isconsideredthecenterofdiversityforthegenusPassiflora,andtheyellowpassionfruit(PassifloraedulisSimsf.flavicarpaDeg.)isthespeciespredominantlygrown(Oliveiraetal.,2012).Brazilisalsothelargestproducerandconsumerofpassionfruitintheworld(JanickandPaul,2008).
ThefruithasemergedasanimportanthighvaluehorticulturalcropinKenyaandhasgainedwideadoptioninMbeere,EmbuCountyduetoitsapparentadaptationtothehotaridconditionsandareadymarketforthefruit.ThefruitismainlyconsumedlocallyandalsoexportedbysomeKenyacompanies(HCDA,2013).Nevertheless,thecurrentproduction levelsofyellowpassion fruit are lowdue to poor agronomic and fertilizermanagement, declining soil fertility levels and erratic rainfallpatterns.
Analysisofsoilandassessmentofplantbehaviorundervaryingamountofnutrientsisrequisiteinoptimizationofnutrientrequirementsforpassionfruitplants.Inaddition,basedonthefactthateachnutrienthasaspecificroleinthephysiologicalfunctionsofplants,thenimbalancesoftenresultsto lowyieldandfruitsofpoorquality. Inadditiontowaterdeficit,theMbeereregionofEmbufacehighsalinity,usuallyduetothehighevapotranspirationdemand,reducedprecipitationandpoormanagementoftillage(DoneenandWestcot,1988).Pooruseoffertilizersbyfarmersfurtherexacerbatesthisproblem.AccordingtoAyersandWestcot(1999),yellowpassionfruit ishighlysensitivetotheactionofsaltshenceirrigationwithsalinewaterandincorrectfertilizerapplicationmayproduceundesirableeffectsinthephysicochemicalqualityofthefruit(Costaetal.,2001;Freireetal.,2010;Diasetal.,2011).Allthesefactorsaffectthefruitformindexleadingtoshriveling,malformationsanddeformedfruits;importantattributesinclassificationandstandardizationhenceaffectingtheacceptanceandjudgmentoftheproductinconsumermarkets.
Themainobjectiveofthisstudywastoassessflowerinductionandfruitformationofyellowpassionundervaryingfertilizertreatmentsintwodifferentecologicalstudysites.
MATERIALSANDMETHODSSixty-fourplantsbelongingtoKPF4varietyweregrownintwositesatKenyattaUniversity(KU)FarmandatUgwerifarmer’sorchardinEmbu.ExperimentaldesignTheexperimentwaslaidoutinaFactorialCompleteRandomizedBlockDesignwithplantingandtopdressingfertilizersasthemainfactors.TheplantingfertilizerswereFarmyardmanureandDiammoniumphosphate(DAP)whilethetopdressingwasCalciumammoniumnitrate(CAN)andNitrabor.Thetreatmentsincluded100gDAP+20kgManure+50gNitrabor;100gDAP+10kgManure+50gNitrabor;100gDAP+50gCAN;100gDAP+50gNitrabor;10kgManure+50gCAN;10kgManure+50gNitrabor; 20kgManure+50gCAN; 20kgManure+50g Nitrabor; 50g DAP+10kgManure+50g CAN, 50g DAP+50g CAN, 50gDAP+50gNitraborandControl(nofertilizer).DatarecordingandanalysisDatarecordingbegunattheonsetofthefirstflowerbudandthenumberofunopenedflowerbuds,openflowersandyoungunripefruits(fruitsformed)wasrecordedonaweeklybasisfortwoconsecutiveseasonsofDecember2016andApril2017.Thedatacollectionwascontinueduntilapproximately80%oftheflowershadformedfruits.AnalysisofvarianceusingSASsoftwareversion9.1wasperformedonthedata.Thedatawascodedasfollows:
Treatment Code Treatment Code100gDAP+20kgManure+50gNitrabor 1 20kgManure+50gCAN 7100gDAP+10kgManure+50gNitrabor 2 20kgManure+50gNitrabor 8
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100gDAP+50gCAN 3 50gDAP+10kgManure+50gCAN 9100gDAP+50gNitrabor 4 Control 10
10kgManure+50gCAN 5 50gDAP+50gCAN 1110kgManure+50gNitrabor 6 50gDAP+50gNitrabor 12RESULTSANDDISCUSSIONSResultsindicatethatthereweresignificantdifferences(≤0.05)amongthetreatments(Table1).Table1:AnalysisofthevariousdependentvariablesforfertilizertreatmentinKenyattaUniversityFarmandUgweriorchard
DependentVariable:Flowerbudinitiation
Source DF SumofSquaresMeanSquare FValue Pr>F
Model 16 68076.8636 4254.804 15.61 <.0001
Error 278 75782.133 272.5976
CorrectedTotal 294 143858.9966
DependentVariable:Openflower
Source DF SumofSquaresMeanSquare FValue Pr>F
Model 16 19701.77955 1231.36122 17.06 <.0001
Error 278 20062.54588 72.16743
CorrectedTotal 294 39764.32542
DependentVariable:Fruitsformed
Source DF SumofSquaresMeanSquare FValue Pr>F
Model 16 116328.2391 7270.5149 12.57 <.0001
Error 278 160753.0965 578.2485
CorrectedTotal 294 277081.3356 Additionally,fortheflowerbudinitiationandopenflowers,thereweredifferencesforthetwositeswithEmbuhavingthehighestaveragenumberofflowerbudsandopenflowers(Figure1).
Fig1:ComparisonofthemeannumberofflowerbudsandopenflowersinthetwostudysitesThecombinedtreatmentsof100gDAP+10kgManure+50gNitraborand20kgManure+50gNitraborshowedthebestresultsintermsofflowerandfruitformationforthetwosites(Figure2).ProbablythecommercialNitraborfertilizer(15.4%N+
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25.9%CaO+0.3%B)hadaneffectonthefloweringpossiblyduetoitsenhancedboroncontentandtheimprovedsolubilityofcalcium.Ontheotherhand,treatments9,10and11whichwerelackingtheNitraborshowedlessnumberofopenedflowersandfruitsformed.ThisstudyfurtheragreeswithfindingsbyFreitasetal.(2006)whichdemonstratedthatnitrogendeficit influences fruitquality, reducing fruitnumberperplant, thepercentageof seed in fruit, °Brixand thevitaminCcontentinthejuice,inadditiontoincreasingskinpercentageandfruitthickness.
Fig2:MeannumberofopenflowersandfruitsformedatthetwositesofstudyThe number of fruits formed increased over the season and had differences in the seasons but there were no majorobservationaldifferencesbetweenthetwosites.However,ahighnumberoffruitdropandespeciallyinEmbuwasobservedwhicharoseasaresultoftheprolongeddryspell.CONCLUSIONThis study has demonstrated that varying fertilizer application can have significant impact on the flowering and fruitformationofyellowpassionwhicharekeydeterminantsofthepotentialyieldofanorchard.REFERENCESAyers,R.S.;Westcot,D.W.(1999).Aqualidadedaáguanaagricultura.CampinaGrande:UFPB,pp.1-158,(FAO:Drainagepaper,29).Costa,J.R.M.;Lima,C.A.A.;Lima,E.D.A.;Cavalcante,L.F.;Oliveira,F.K.D.(2001).Caracterizaçãodosfrutosdemaracujáamareloirrigadoscom
águasalina.RevistaBrasileiradeEngenhariaAgrícolaeAmbiental,5(1):143-146.Doneen,L.D.andD.W.Westcot(1988).Irrigationpracticeandwatermanagement.IrrigationandDrainagePaperNo.1,Rome,FAO.Freitas,M.S.M.,P.H.Monnerat,L.G.R.Pinho,andA.J.C.Carvalho.(2006).Mineraldeficiencyofmacronutrientsandboroninsweet
passionfruit:fruitquality.RevistaBrasileiradeFruticultura28:492–496.[Crossref]).HorticulturalCropDevelopmentAuthority(HCDA).NationalHorticultureValidatedReport.2013.Janick,J.,Paull,R.E.,(2008).TheEncyclopediaofFruitandNuts.CABI,London.Oliveira, E.J., Dias, N.L.P., Dantas, J.L.L., (2012). Selection of morpho-agronomic descriptors for characterization of papaya cultivars.
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QUANTIFICATION OF IMMUNE RESPONSE WITH A SKIN SWELLING TEST USING AN IMAGE-BASED PLATFORM
SHAHBAZBASHIRA,BERNICELIMA,B,ALIYOUSSEFALIAMERB,TOMASNORTONB,JANPAESHUYSEA
AHostPathogenInteractionLaboratory,DepartmentofBiosystems,FacultyofBioscienceEngineering,KasteelparkArenberg30,3001
Heverlee,KULeuvenUniversity,Belgium.BPrecisionLivestockfarmingLaboratory,DepartmentofBiosystems,FacultyofBioscienceEngineering,KasteelparkArenberg30,3001
Heverlee,KULeuvenUniversity,Belgium.Correspondence:ShahbazBashir([email protected])
INTRODUCTIONEcoimmunologyaddressesthecoreconceptofbiologicalorganizationatthelevelofanindividual´simmunesystemanditsevolution in a given ecological and life history context. Since its emergence, it has widened our knowledge about thedevelopmentofimmunesystemfrommechanismatmolecularleveltoitsphysicaloutcome.Butstilltoday,howtomeasuretheindividualimmunityremainsitscentralconundrum(Brock,Murdock,&Martin,2014).Despiteofanastoundinglargenumber of immunological tests available, complex andmultifaceted nature of the immune systemmakes it difficult tounderstandandencompassitsdiverseanddynamicalaspects(Demas,Zysling,Beechler,Muehlenbein,&French,2011).Inthe field of ecoimmunology and ecotoxicology, Systematized examination of individual's immunocompetence followingexperimentalelicitationofimmuneresponsethroughmitogeninjectionisconsideredasastandardapproach.Skinswellingtest is a cheap and simple approach allowing researcher the opportunity to estimate the cell-mediated immuneresponsivenessofthe individualbyusingdifferentmitogens.Thistest isbasedonthemeasurementoftheskinswellingresponse at specific times following subcutaneous or intradermal administration of experimental pro-inflammatorystimulant(Vinkler,Bainová,&Albrecht,2010).Inthisregards,phytohaematagglutinin(PHA),aplantlectinextractedfromPhaseolusvulgaris,isconsideredasauniversalmitogenofchoice.Forinvivoapplications,PHA-P,acombinationofPHA-L(Leukoagglutinin)andPHA-E(Erythroagglutinin),ispreferred(Kennedy&Nager,2006).SkinswellingresponseofPHAhasmostlybeeninvestigatedinbirds(Adelman&Ardia,2014;Martinetal.,2006;Tollingtonetal.,2015),amphibians(Brown,Shilton,&Shine,2011)andreptiles(Fingeretal.,2013).Incontrast,PHAproinflammatoryresponsehasrarelybeenexploredinmammals,particularlycow.Giventhecomplexityoftheimmuneresponse,theimmunologicalbackgroundmechanismsunderpinningtheskinswellingaftermitogenapplicationisstillnotwellunderstood.Theobjectiveofthestudyathandistousean image-basedplatformto investigateandquantify the inflammatoryresponseandunderlayingthermaldynamicalchanges basedon thewarmth generatedby increasedblood flow at the site of PHA injection in a cow. To best of ourknowledge,thisisthefirststudytounderstandtheinvivothermaldynamicsofanimalskininflammatoryresponseprovokedbyanymitogen.MATERIALANDMETHODSInfraredthermographicimagingandFLIROne™Electromagneticwavelengthspectrumofinfraredradiationrangesfrom700nanometer(nm)to1millimeter(mm).Allbodiesabovezerokelvin(K)emitinfraredradiationsinvisibletonakedhumaneyebetweenelectromagneticwavelengthspectrumrangeof3-15micrometer(μm)andcouldbecapturedbythesensorofthermalcamera.Eachpixelofthegeneratedimagecorresponds to the infrared radiation energy. Total radiation energy emitted from a body follows Stefan-Bolztman Law(Stefan-Bolzmannequation)andisdirectlyproportionaltothebody’ssurfaceareaanditsemissivity(Jiji,2006).
Stefan-Boltzmannequation𝑷=𝑨𝛆𝛔𝑻𝟒P=powerofradiation,A=surfaceareaofanobject,ε=emissivityoftheobject’ssurface,σ=Stefan-BoltzmannconstantandT=absolutetemperatureoftheobject.
To capture the thermal images of the skin swelling response, amobile and inexpensive commercial smartphone basedinfraredthermalcameraFLIROne™(Figure1)wasusedinthisstudyhavingthetemperaturedetectionrangefrom-20to120°Cwithasensitivityof0.1°C.FLIR®systemssoftwarereleasedsoftwaredevelopmentkittoaccesstherawdataofthecameraandcreateacustomsmartphone-basedapplication.
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ExperimentalanimalandtreatmentinoculaThestudywasapprovedbyUniversityEthicalCommitteeforAnimalExperimentationKULeuven(projectnumberP069-2017)andconductedonHolsteincowatZoötechnischCentrum(ZTC)KULeuveninApril,2017.Bothsidesoftheneckwereshavedproperlyandprickedintradermallywith2.5mg/mlPHA-P(Sigma-Aldrich,L8754)inDPBS(Sigma-Aldrich,D5652),5mg/mlHistamine((Sigma-Aldrich,H7125)inDPBSaspositiveandDPBSasnegativecontrol.ExperimentalDesignAspecialcameraframe(Figure2)of15×15×20cmdimensionswasmadefrompolyvinylchloridetosupportthecameraandmobileandtoensuretheconstantdistanceofcameraperpixelfromtheskinatalltimesduringimagecapturing.Intensivethermographic imagingwith FLIROne™ and physicalmeasurementswith digital vernier caliper (POWERFIX®, Lidl)wereperformedat0hourpre-injectionandevery15minutesduringthefirsthourofpostinjections,everyhouruntil15hourspostinjectionsandthenat19,21,24,30and48hourspostinjectiontonoticethethermaldynamicsofinflammation.Thediameteroftheskinswellingareaanddoubleskinfoldthicknessweremeasuredasapartofphysicalmeasurements.ThermalimagescapturedbyFLIROne™werefurtherprocessedinMATLABR2016bbyusingtherawdata.RESULTSSkinswellingresponsewasrapidfollowingallinjections.Maximumswellingdiameterwascausedbyhistamine(80.58mm)followedbyDPBS(35.36mm)andPHA(34.98mm),respectively(Figure3).Themaximumswellingdiameterofhistaminewasmorethan2timesthanthatofcausedbyPHAandDPBS.HistamineelicitedmorerapidandaggressiveswellingascomparedtothegentleswellingofPHA.Theinterestingthingwasthetimingwhenmaximumswellingdiameterwasachieved,whichwasat3hourspostDPBSinjectionandat5hoursposthistamineandPHAinjection.Doubleskinfoldthicknessmeasurementis themost commonmethod to estimate the swelling response.Histamine caused themaximumdouble fold thickness(21.04mm)withalmost350%increment,whilePHAandDPBS(15.65and15.04mm)showedsimilartrendindoubleskinfoldthicknesswithjust150%increment(Figure4).Thermalimagingrevealedthatintradermalinjectionofhistamine(Figure6B)andDPBS(Figure5B)causedaquickreactionandachievedamaximumtotalsurfaceareaoftemperatureincrementwithinfirst1.5hourspost-injectionascomparedtothedelayedreactionshownbyPHA(Figure7B)6hourspostinjection.Thearea of temperature increment caused by PHAwas larger than that of achieved by histamine, despite of the fact thathistaminecauselargerskinswellingandthickness.
Figure 1. FLIR One™ infrared thermal camera for Android
Figure 2. Camera frame
Figure 3. Skin swelling diameter
Figure 4. Double skin fold thickness increment with each hour
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AnotherinterestingfeatureobservedwasthefactthatPHAswellingshowedlocalizedtemperatureincrement(Figure7),whilehistamine(Figure6)andPBS(Figure5)swellingshowedaspreadoutwardtrendoftemperatureincrementfromthesiteofinjectionwithalmostbeingattheborderofthethermalimagesat30hourspostinjection.DISCUSSIONThe physical measurements and imaging was done by the same person to minimize the error, thus enhancing therepeatabilityandprecision.Generally,Skinswellingtestsarebasedontheswellingasaresultofinflammationatthesiteofallergen/mitogeninjection.However,swellingisnottheonlysignofinflammation,heating/warmthcausedbytheincreasedbloodflowtothesiteofinjectionisalsoamongstthecardinalsigns.OurstudyutilizestheproinflammatorypotentialofPHAtounderstandthethermaldynamicsofinflammatoryresponse.Skinswellinganddoublefoldthicknessareininlinewiththestudiesconductedpreviously(Demasetal.,2011;Fingeretal.,2013).LowswellingresponseinourstudytoPHAmightbeduetothefactthatcowwasnotsensitizedtoPHApreviously.ObservationlocalizedtemperatureincrementtrendincaseofPHAanddisseminatedtrendincaseofhistamineandPBSisaninterestingpointtolookfor.
Thisstudyshowsthatobservationoftemperatureincrement,asaresultofallergicreactionatthesiteofmitogeninjection,couldbeapracticaltoolinquantifyingandunderstandingtheimmuneresponseofskinswellingtest.ACKNOWLEDGEMENTSTheauthorwouldliketoacknowledgeDVMWouterMerckxandallthestaffofZoötechnischCentrum(ZTC)KULeuvenfortheirkindcooperationandhelpduringthisstudy.TheauthorwouldalsoliketothankLudoHappaertsforbuildingthecameraframe.REFERENCESAdelman,J.S.,&Ardia,D.R.(2014).Chapter22–Ecoimmunology.InAvianImmunology(pp.391–411).https://doi.org/10.1016/B978-0-
12-396965-1.00022-4
Figure 5. Area of temperature increment processed in MATLAB R2016b provoked by I/D DPBS injection: A) immediate post-injection, B) maximum at 1-hour post injection and C) 30-hours post injection
Figure 6. Area of temperature increment processed in MATLAB R2016b provoked by I/D Histamine injection: A) immediate post-injection, B) maximum at 1.5-hour post injection and C) 30-hours post injection
Figure 7. Area of temperature increment processed in MATLAB R2016b provoked by I/D PHA-P injection: A) immediate post-injection, B) maximum at 6-hour post injection and C) 48-hours post injection
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Brock,P.M.,Murdock,C.C.,&Martin,L.B.(2014).Thehistoryofecoimmunologyanditsintegrationwithdiseaseecology.InIntegrativeandComparativeBiology(Vol.54,pp.353–362).https://doi.org/10.1093/icb/icu046
Brown,G.P., Shilton,C.M.,&Shine,R. (2011).Measuringamphibian immunocompetence:Validationof thephytohemagglutinin skin-swellingassay in thecane toad,Rhinellamarina.Methods inEcologyandEvolution,2(4),341–348.https://doi.org/10.1111/j.2041-210X.2011.00090.x
Demas,G.E.,Zysling,D.A.,Beechler,B.R.,Muehlenbein,M.P.,&French,S.S.(2011).Beyondphytohaemagglutinin:Assessingvertebrateimmunefunctionacrossecologicalcontexts.JournalofAnimalEcology.https://doi.org/10.1111/j.1365-2656.2011.01813.x
EFSAPanelonAnimalHealthandWelfare. (2012).ScientificOpinionontheuseofagamma interferontest for thediagnosisofbovinetuberculosis1.EFSAJournal,10(12),2975.https://doi.org/10.2903/j.efsa.2012.2975.
Finger,J.W.,Adams,A.L.,Thomson,P.C.,Shilton,C.M.,Brown,G.P.,Moran,C.,…Isberg,S.R.(2013).Usingphytohaemagglutinintodetermine immune responsiveness in saltwater crocodiles (Crocodylus porosus). Australian Journal of Zoology, 61(4), 301–311.https://doi.org/10.1071/ZO13041
Jiji,L.M.(2006).Heatconvection.HeatConvection.https://doi.org/10.1007/978-3-540-30694-8Kennedy,M.W.,&Nager,R.G.(2006).Theperilsandprospectsofusingphytohaemagglutinininevolutionaryecology.TrendsinEcology
andEvolution.https://doi.org/10.1016/j.tree.2006.09.017Martin,L.B.,Han,P.,Lewittes,J.,Kuhlman,J.R.,Klasing,K.C.,&Wikelski,M.(2006).Phytohemagglutinin-inducedskinswellinginbirds:
Histological support for a classic immunoecological technique. Functional Ecology, 20(2), 290–299. https://doi.org/10.1111/j.1365-2435.2006.01094.x
Tollington,S.,Greenwood,A.,Jones,C.G.,Hoeck,P.,Chowrimootoo,A.,Smith,D.,…Groombridge,J.J.(2015).Detailedmonitoringofasmallbutrecoveringpopulationrevealssublethaleffectsofdiseaseandunexpectedinteractionswithsupplementalfeeding.JournalofAnimalEcology,84(4),969–977.https://doi.org/10.1111/1365-2656.12348
Vinkler,M.,Bainová,H.,&Albrecht,T.(2010).Functionalanalysisoftheskin-swellingresponsetophytohaemagglutinin.FunctionalEcology,24(5),1081–1086.https://doi.org/10.1111/j.1365-2435.2010.01711.x
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BLUE LUPIN SEED Β-CONGLUTIN PROTEINS REVERSE BACK THE INDUCED INSULIN RESISTANCE IN PANCREATIC CELLS
ELENALIMA-CABELLO1,SONIAMORALES-SANTANA2,VICTORALCHE3,ALFONSOCLEMENTE4,
JUAND.ALCHE1,JOSEC.JIMENEZ-LOPEZ1,5*
1Dept.Biochemistry,Cell&MolecularBiologyofPlants,and4Dept.PhysiologyandBiochemistryofAnimalNutrition;EstacionExperimentaldelZaidin,SpanishNationalResearchCouncil(CSIC),Granada,Spain
2CIBERofFragilityandHealthyAging(CIBERFES),EndocrinologyUnit,EndocrinologyDivision,andProteomicResearchService;BiomedicalResearchInstituteofGranada“IBS.Granada”,UniversityHospitalSanCecilio,Granada,Spain
3AndalusianHealthSystem,HealthCenter"VillanuevadelasTorres",Granada,Spain5TheUWAInstituteofAgricultureandSchoolofAgricultureandEnvironment;TheUniversityofWesternAustralia,Perth,WA,Australia
*Correspondingauthor:[email protected]
INTRODUCTION
Dietscontaininghighlevelsofsaturatedfatsandfructoseplayanimportantroleinobesitydevelopment.Obesityinducesinsulinresistanceintheliver,skeletalmuscles,andadiposetissues,stronglydevelopingintotype2diabetesmellitus(T2DM).Diabetesmellitusisoneofthemostprevalentchronicdisordersworldwide,andtype2diabetesaccountsforover90%ofdiabeticcases.Type2diabetesisfeaturedbyinsulinresistance,whichoccurswhentheinsulin-responsivetissues,mainlyskeletalmuscle,adiposetissue,andtheliver,cannotrespondtoinsulinproperly. Insulinpromotestheglucoseuptakeofinsulin-sensitive tissues and inhibits glucose export from the liver; thus, insulin resistance causes hyperglycaemia andstimulatespancreaticβcellstosecretemoreinsulinformaintaininganormallevelofbloodglucose,whichwillresultinβ-celldamageandthedevelopmentoftype2diabeteswithoutprompttreatment.
Furthermore,insulinresistanceisalsoariskfactorforadiversityofdiseases,includingcardiovascularandkidneydiseases,several cancers and cognitivedisease, increasing themortality risk. Theprevalenceof T2DMhas increaseddramaticallyworldwidenotonlyinadults,butalsoinadolescentsandchildren.
Narrow-leafed lupin or NLL (Lupinus angustifolius L.), a legume crop belonging to the Fabaceae family, is a worldwideimportantpulse,whichdisplaysawiderangeofbenefitsforagriculture.Atmolecularlevel,lupinseedproteinsmayalsobeinvolvedinpossibleplantpathogenssuppression(Jimenez-Lopezetal.,2016).Therefore,NLL,withlowcontentinalkaloids,is attracting great attention lately because of their nutritional attributes and human health benefits (nutraceuticalproperties).Thesepropertiesareassociatedtothehighprotein,dietaryfibercontent,absenceofgluten,helpingtoreducebloodpressuretheriskofcardiovasculardisease,andcontributingtoreductionofglucoseandcholesterolbloodlevels,andpreventing obesity anddyslipidemia (Delgado-Andrade et al., 2016).NLL as an important source of proteins for humanconsumptionseemstobeparticularlypromisingasasourceofinnovativefoodingredientsduetoaverageproteincontentsimilartosoybeanandanadequatecompositionofessentialamino-acids.
Recently, it hasbeendemonstrated thatparticular seedproteins suchas conglutinproteinsβ1,β3, andβ6 fight type2diabetesthroughoutthemodulationoftheinsulinmolecularsignallingpathway(Lima-Cabelloetal.,2017).Thesepropertiesmaybeattributedtotheirparticularstructuralfeatures(Lima-Cabelloetal.,2016).Theseproteinsmightbepotentialnewbioactivemolecules with antioxidant, and anti-inflammatory activities (Lima-Cabello et al., 2018), opening the gate forpossibleplant-basedprevention,managementandtherapeuticapproachestotheworldmainhealthconcernandincreasingdiseasediabeticepidemics.
Theaimofthecurrentstudywastoevaluatetheabilityofb-conglutinproteinsfromNLLtoreverttheresponseatmolecularleveloftheinducedinsulinresistancestateofpancreaticPANC-1cells,checkinggenesinvolvedintheinsulinpathwayinT2DMpatients.
MATERIALANDMETHODS
Synthesisofgeneticconstructs
Beta-conglutinsexpressioninbacteriaE.coliwassetupusingthepET28a(+)vectorcontainingeachindividualb-conglutinisoformβ1,β2,β3,β4,andβ6,linkedto6xHis-Tag.
Proteinsexpressionandpurification
ProteinswereexpressedinRosetta™2(DE3)pLysSSingles™CompetentCells(Novagen),andpurifiedfollowingthemethoddevelopedbyJimenez-Lopezetal.(2016).
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Cellcultureandtreatment
PANC-1cellswerepurchasedfromCIC(UniversityofGranada).PANC-1cellsweregrowninpoly-L-lysine-coatedflasks(∼2.0–2.5 × 106 cells/ml) inDulbecco’smodified Eagle’smedium (DMEM) supplementedwith 2 mMglutamine and 10%heat-inactivatedfetalbovineserumat37 °Candin5%CO2/95%air.Cellsweregrownasmonolayersanddetachedfromculturesbytrypsinization,andfrequentlysub-culturedtobeusedintheexponentialgrowthphaseforallexperiments.Cellswerewashedtwicewithphosphate-bufferedsolution(PBS,Sigma)andtreatedwith0.25%tryp-EDTA(Sigma)for10 minin5%CO2/95%airat37 °Chumidifiedatmosphere.Thetrypsinizationeffectwasneutralizedwithculturemedium.Thecellswerecollectedaftercentrifugationat1000×gfor5 minandwashedwithPBSforviabilityassays.CellcountingandviabilitywerecheckedusingaCountessIIFLAutomatedCellCounter(ThermoFisher)atthebeginningandendofeachexperimentusingrepresentativewells.Viabilitywasalways > 95%.
Cellswereculturedto80%confluenceandthentreatedwithβ1,β2,β3β4,orβ6purifiedproteinsfor24halone.Aliquotsofβ1,β2,β3β4,andβ6werestoredat–20°CinPBS.ThepurifiedconglutinswerethawedjustbeforeuseanddilutedinDMEMtodesiredconcentrations.20μgofeachpurifiedconglutinwasaddedtocultures.Afterthetreatments,cellswereharvestedforfurtheranalyses.
InsulinresistancePANC-1cellmodelandglucoseuptake
Panc-1cellswereseededin96-wellplatesinDMEMsupplementedwith10%(V/V)FBS,understandardcellcultureconditions(humidifiedatmosphere,5%CO2and37°C).Thedensityofthecellsineachplatewas2×104cells/mL.Thefollowingprotocolwasusedtodeterminetheoptimaldoseofinsulinandtreatmentdurationrequiredtoestablishinsulin-resistant(IR)cells:WeremovedtheoriginalmediumwhenthecellsbecameadherentandreplaceditwithDMEMcontaining3x10-53x10-63x10-73x10-83x10-9mol/Lof insulin (recombinanthuman insulin, Sigma), respectively, and checking for eachone theglucoseuptakebycells.Themodelofinsulinresistancewasestablishedafter24h.Westablishedtheinsulinconcentration(3x10-7mM)wherecellculturedidusetheloweramountofglucoseuptakeasthepointofinsulinresistanceforthePANC-1cellculture(IRPANC-1).Atthisstate,weconsiderthatcellsareinsulinsensitiveorinsensitivetodeterminewhetherβ-conglutinproteinscanimproveinsulin-dependentglucoseuptakecapacityofthecells.
Glucoseoxidasemethodwasusedtodetectglucoseconsumption(GC)levelsafter24h.Theglucoseoxidasemethodwasusedtodetecttheglucoseconcentrationineachgroup.Glucoseoxidasecatalysesthebreakdownofglucosetoglucoseacidandhydrogenperoxide. Peroxidase then catalyzes the formationof redquinone imide in a reaction involvinghydrogenperoxide,4-aminoantipyrine,andphenol.Theabsorbanceofredquinoneimideatawavelengthof490nmisproportionaltotheconcentrationofglucose.
Quantitativereal-timePCR
Real-timequantitativePCRtechnologywasusedtoassayGLUT-4mRNAexpressionfromeachexperimentalgroup.TotalRNAwasisolatedfromPANC-1andIRPANC-1cellsusingtheRNeasyTissueRNAisolationkit(Qiagen).First-strandcDNAwassynthesizedusingaHigh-CapacitycDNAArchiveKit(AppliedBiosystems).Forgeneexpressionassays,cDNAwasprepared,dilutedandsubjectedtoreal-timePCR,andamplifiedusingTaqMantechnology(LightCycler480quantitativePCRSystem,Roche). Primers and probes were used from the commercially available TaqMan Gene Expression Assays detection(Hs00168966_m1, Applied Biosystems;GenBank accession numberNM_001042.2). Relative changes in gene expressionlevelsweredeterminedusingthe2−ΔΔCtmethod.Thecyclenumberthatdetectedthetranscripts(CT)wasnormalizedtothecyclenumberforβ-Actin(Hs99999903_m1,AppliedBiosystems;GenBankaccessionnumberNM_001101.3)usedasthehousekeepinggene,referredtoasΔCT,wheretherelativemRNAlevelswerepresentedasunitvaluesof2∧[CT(β-Actin)–CT(geneofinterest)],havingCTasthethresholdcyclevaluedefinedasthefractionalcyclenumberatwhichthefluorescenttargetsignalpassesafixedthresholdabovethebaseline.PCRefficiencywasdeterminedbyTaqMananalysisonastandardcurvefortargetsandendogenouscontrolamplifications,whichwerehighlysimilar.
Statisticalanalysis
Valuesshownareexpressedasmeans±S.E.M.ofthethreeindividualexperiments.Statisticalsignificanceofthedatawasassessed using SPSS software by analysis of variance followed by Dunnett analysis. P-values ≤0.05 were consideredstatisticallysignificant.
RESULTS
PancreaticPANC-1insulinresistancecellmodel
PancreaticPANC-1cellsareusedforbiochemicalandnutritionalstudiesasacellculturemodelofhumanbetacellssincetheyretaintheirmorphologyandmostoftheirfunctioninculture.Thus,thiscelllinewouldbeusedtostudytheglucoseuptakeandthemodulationoftheinsulinpathwayinvitro.Insulinresistanceininsulinsensitiveorgansresultsinmetabolicdisorder such as hyperglycaemia, hyperinsulinaemia and hyper triglyceridaemia, which are common features of T2DM.Insulinresistanceinpancreaticcellsmainlycausesimpairedinsulinsynthesis.Inordertoestablishaninvitroinsulinresistantmodelofpancreaticcellsandevaluatetheeffectsofdifferentinsulinconcentrationsonglucosemetabolisminthecellmodel,
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PANC-1cellswereincubatedwith3×10−5,3×10−6,3×10−7,3×10−8,and3×10−9mol/Linsulintobuildaninsulin-resistantcellmodel.Thecellswerestimulatedwithfreshinsulinfor24handtheglucoseuptakebythesecellswascarriedout.Theglucoseuptakebythecellswasdetectedbythemethodofglucoseoxidizes/peroxides(GOD-POD).Following3×10−7mol/LinsulinincubationofPANC-1cells,therewasthemostsignificantdecreaseintheconsumptionofextracellularglucose(P<0.05)comparedwithblankcontrolwithoutinsulinpre-treatment.TheadditionofinsulintoPANC-1cellsgrownin DMEM containing different amounts of glucose induced a statistically significant decrease (P<0.05) in the glucoseconsumptionafterincubationfor24h.
Conglutinproteinsb1,b3andb6increasedGLUT4mRNAexpression
Totestwhetherb-conglutinsreducedbloodglucose levelvia increasingtotalGLUT4proteinexpression,weanalysedm-GLUT4expressionlevelsinIRPANC-1byRT-qPCR.AsshowninFig.2,significantdifferencesinmRNAGLUT4expressionlevelswereobservedbetweencontrolc(-)(non-insulintreatedcells)andIRPANC-1c(+)(p<0.05),whereinsulinsignallingpathwaybiomarkersasINSR-1,Akt,PI3Kremainedlowlevelsunderinsulinresistancestate(Lima-Cabelloetal.,2017).However,b1,b3andb6conglutinproteinsupregulate(reverseback)theinsulinresistancestateinIRPANC-1cellmodelashigherinsulin-inducedupregulatingthemRNA-GLUT4expressioncomparedwiththec(+)control(p<0.05).Furthermore,nosignificantdifferenceswereobservedwhenIRPANC-1cellculturewaschallengedwithb2-ob4-conglutinproteins.
Interestingly,b1-,b3-,andb6-conglutinproteinsincreasetheglucoseuptakeinIRPANC-1cellculture.Thus,theseresultsindicatethatb-conglutinproteinsb1-,b3-andb6-mayalleviatehyperglycaemiaviafacilitatingGLUT4translocationtotheplasmamembraneratherthanviaincreasingthetranslocatorconcentration.
Figure1.EffectofNLLβ1-,β3-,β6-,β2-,andβ4-conglutinproteinsonmRNAexpressionofGLUT-4gene.PANC-1-pancreaticcellswereincubatedfor24 hwitheachpurifiedβ-conglutinproteins(β1,β3β6,β2,β4)alone.mRNAlevelsdeterminedbyreal-timeqRT-PCRofGLUT-4.ThebargraphshowsGLUT-4PANC-1-pancreaticcellsmRNArelative levels.Datarepresentthreeindependentexperiments.C(-):Untreatedcontrolpanc-1culturecells;C(+):insulin-treated(IR)controlPANC-1culturecells;β1,β3,β6,β2,andβ4+ insulin:β-conglutinchallenge IRPANC-1culturecells.p* < .05c(-) versusC(+);p** < .05β-conglutin+insulinversusC(+).
DISCUSSION
In thecurrentstudy,wedemonstrated that treatmentofpancreatic IRPANC-1 induced insulin resistantcell culturewithparticularNLLb-conglutinisoformsincreasedglucoseuptakeandincreaseGLUT-4mRNAexpressionlevels.Thesefindingssuggestthatb-conglutinswouldpotentiallybeabletosignificantlyreducebloodglucoselevelandimproveinsulinresistanceinT2DMinsulinresistantstate.
Finding an effective and natural alternative antidiabetic agent would be of enormous interest and importance for thepreventionandtreatmentofT2DMworldwide.Theseedsofsweetlupinspecies,particularlyNLL,havehighnutritionalvalueandbeneficialnutraceuticaleffects.Despitethesefeatures,NLLislargelyunderexploited,anditsconsumptionisatalongbase,but is increasing.Alternativeapplicationssuchasthe incorporationofNLLseeds innewfunctionalfoodsmayhelpfurther increase the use of NLL grain. In this regard, recent research pointed out the NLL major seed proteins, b-conglutin/vicilinproteinsfamily,asstrongpotentialcandidatefordiabetespreventionandtreatment(Lima-Cabelloetal.,2017;2018).
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CONCLUSION
Thepresentstudyprovidedstrongevidencethatbeta-conglutinproteinsarecapableofalleviationinhyperglycaemiaandinsulinresistanceinpancreaticcells.NormalizationofGLUT-4mRNAexpressionlevelsoftransporterandincreasingglucoseuptakewastheunderlyingmechanismforthebeneficialeffectsofb-conglutinsinthestageofhyperglycaemiaandinsulinresistance,typicalofT2DM.Consequently,dietarysupplementationwithNLLseedproteinsprovidesfeasibilityforitsusageininsulinresistancetherapy,asanovelandsafefunctionalfood,withapotentialroleintheregulationofinflammatory-relatedprocesses.
ACKNOWLEDGEMENTSANDFUNDING
EU Marie Curie grant PIOF-GA-2011-301550; MINECO grants RYC-2014-16536 (Ramon y Cajal research program), andBFU2016-77243-P.
REFERENCES
Delgado-AndradeC.,OliasR.,Jimenez-LopezJ.C.,ClementeA.(2016).Nutritionalandbeneficialeffectsofgrainlegumesonhumanhealth.ARBOR172-779:a313,doi:10.3989/arbor.2016.779n3003.
Jimenez-LopezJC,MelserS,DeboerK,ThatcherLF,KamphuisLG,SinghKB.(2016).Narrow-leafedlupin(LupinusangustifoliusL.)b1-andb6-conglutin proteins antifungal activity, protecting plants against necrotrophic pathogen induced damage from SclerotiniasclerotiorumandPhytophthoranicotianae.FrontiersinPlantScience7:1856,doi:10.3389/fpls.2016.01856.
Lima-CabelloE.,Robles-BolivarP.,AlcheJ.D.,Jimenez-LopezJ.C.(2016).Narrow-leafedlupinb-conglutinproteinsepitopesidentificationandmolecularfeaturesanalysisinvolvedincross-allergenicitytopeanutandotherlegumes.GenomicsandComputationalBiology2(1):e29,doi:10.18547/gcb.2016.vol2.iss1.e29.
Lima-CabelloEetal.(2017).Narrow-leafedlupin(LupinusangustifoliusL.)β-conglutinproteinsmodulatetheinsulinsignallingpathwayaspotentialtype2diabetestreatmentandinflammatory-relateddiseaseamelioration.MolecularNutritionandFoodResearch61(5),doi:10.1002/mnfr.201600819.
Lima-CabelloE,Morales-SantanaS,FoleyRC,MelserS,AlchéV,SiddiqueKHM,SinghKB,AlchéJD,Jimenez-LopezJC.(2018).Exvivoandinvitroassessmentofanti-inflammatoryactivityofseedβ-conglutinproteinsfromLupinusangustifolius.JournalofFunctionalFoods,doi:10.1016/j.jff.2017.11.040.
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FLAVONOIDS SYNTHETIC PATHWAY ENZYMES AS BIOMARKERS FOR THE STUDY OF BIOCHEMICAL AND PHYSIOLOGICAL CHANGES OCCURRING DURING NARROW-LEAFED LUPIN SEED GERMINATION AND SEEDLING
DEVELOPMENT
ELENALIMA-CABELLO1,JUAND.ALCHE1,JOSEC.JIMENEZ-LOPEZ1,2*
1Dept.Biochemistry,Cell&MolecularBiologyofPlants;EstacionExperimentaldelZaidin,SpanishNationalResearchCouncil(CSIC),Granada,Spain
2TheUWAInstituteofAgricultureandSchoolofAgricultureandEnvironment;TheUniversityofWesternAustralia,Perth,WA,Australia*Correspondingauthor:[email protected]
INTRODUCTION
TheLupinusgenuscomprisesover400species.Theirseedscontainapproximately36–52%proteins,30–40%fibers,and5–20%essentialoils,withvaluesdependingontheenvironmentalorgeneticconditions (Ref).OtherchemicalconstituentsdetectedinLupinussp.areoleicandlinoleicacids,flavonoidsincludingalkaloids,carotenoids,andpolysaccharides.Overall,thesweetlupinvarietiessuchasL.albus,L.angustifolius,orL.luteuspresentnutritionalandchemicalcharacteristicsmakingthemmoreedible.
Phenolicscompoundssuchasflavonesandisoflavonesarepresentinseveralpartsoflupin,includingtheseeds.However,germinationhasbeenwidelyusedforitsabilitytodecreaselevelsofanti-nutritionalfactorspresentinlegumeseeds,atthesame time improving the concentration and bioavailability of their nutrients. In this regard, germination of lupin seedsmodifies the compositionof theprotein fraction,andminimises thepresenceofphenolic (alkaloids) compounds,whichprotectionagainstdiseasemaybeassociatedwiththepowerfulantioxidantandfreeradicalscavengingpropertiesofthesecompounds,alsoactingasfungicides,bacteriostats,etc.Oneofthemostabundantalkaloidinlupinseeds,quinolizidine,isusedasanitrogensourceforseedlings,andtheyalsoplayadefensiveroleagainstpredatorsintheplant.
Alkaloid synthesis takes place in the stromaof leaf chloroplasts, following a light-regulatedbiosynthesis. Then they aretransportedviathephloemandstoredinvacuolesinalltheorgansoftheplant.Theseedsareespeciallyrichinalkaloids,containingupto5%(dryweight)whichrepresentsabout8–10%ofthetotalnitrogenstoredinlupinseeds.
Germination involves a great number of physiological changes, including synthesis, degradation and transformation ofdifferentcompounds(Zienkiewiczetal.,2011;Jimenez-Lopezetal.,2016a).Duringgermination,someoftheseedreservematerials,particularlyseedstorageproteins,e.g.lupinsvicilins,aredegradedandusedforrespirationandsynthesisofnewcellconstituentsofthedevelopingembryo,leadingtosignificantchangesinseedatthebiochemicalandphysiologicallevel.Furthermore,germinationprocessesfavourasignificantincreaseintheantioxidantcapacityoflupinseed(Jimenez-Lopezetal.,2016b),whichislinkedtochangesofphenoliccompoundsthatseemtobeassociatedwithanincreaseinthefreeradicalscavengingcapacity.Thisalsoincreasespotentialnutritivevalueandhealthqualitiesoftheseedsbyraisingthelevelsofbioactivecompoundsfornutraceuticalandfunctionalityoftheseeds(Lima-Cabelloetal.,2017;Lima-Cabelloetal.,2018).
Seedgerminationmayreducethetotalalkaloidcontent.However,thisdegradationcouldbeattributedtomobilizationofalkaloidalnitrogen,whichalsomaybeinfluencedbynitrogenmobilizationfromseedstorageproteinsalonggermination.ThepresentstudyaimstoidentifykeyplayersdrivingthefunctionalinterplaybetweenspecificmembersoftheNLLseedconglutinfamiliesandthealkaloidmetabolicpathwayduringseedgermination.
MATERIALANDMETHODS
Samplesandgerminationprocess
SeedsofL.angustifoliusL.wereusedinthisstudy.Severalassayswereperformedinordertoselectthebestconditionsforthe germination of the seeds such as different media (water, MS), different temperature conditions, and the optimalgerminationconditionswereperformedonwetfilterpaper,intoagerminationchamberunderenvironmentalcontrolledconditions:16hnight/25Cfor8hlightperdayexposure,andwateringoftheseedsduringgerminationtokeepthepaperalwayswet. L.angustifolius seedswerewashedwith sodiumhypochlorite solution (0.2gCl/l), thenwith steriledistilledwater,andscarifiedpriortogermination,inordertoavoidfungusproliferationandimprovethegerminationoftheseeds.Samplesweretakenat0(1-dayimbibition),2,3,4,5,7,9and11germinationdays.
GeneexpressionanalysesbyqPCR
Real-timequantitativePCRtechnologywasusedtoassaythedifferentgenes(mRNAexpression)inNLLofthefourconglutinfamilies(alpha,beta,gammaanddelta),andthekeyenzymesresponsibleofthealkaloidsmetabolic(synthetic)pathwaysuchaslysinedecarboxylase(L/ODC),lysineoxidase(LO),Twoacyltransferases(ATs):(–)-13α-hydroxymultiflorine/(+)-13α-
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hydroxylupanine O-tigloyltransferase (HMT/HLT; EC 2.3.1.93), and p-coumaroyl-CoA/feruloyl-CoA: (+)-epilupinine/(–)-lupinine O-coumaroyl/feruloyltransferase (ECT/EFT-LCT/LFT). Total RNA was isolated from seed tissue at differentgerminationstages,using theRNeasyTissueRNA isolationkit (Qiagen).First-strandcDNAwassynthesizedusingaHigh-CapacitycDNAArchiveKit(AppliedBiosystems).Forgeneexpressionassays,cDNAwasprepared,dilutedandsubjectedtoreal-timePCR,andamplifiedusingTaqMantechnology(LightCycler480quantitativePCRSystem,Roche).SpecificFwdandRv primers were designed for each gene and Real-time PCR using QuantiTec SYBER green PCR master mix (AppliedBiosystems).Relativechangesingeneexpressionlevelsweredeterminedusingthe2−ΔΔCtmethod.Thecyclenumberthatdetectedthetranscripts(CT)wasnormalizedtothecyclenumberforubiquitin(usedasthehousekeepinggene)detection,referredtoasΔCT,wheretherelativemRNAlevelswerepresentedasunitvaluesof2∧[CT(β-Actin)–CT(geneofinterest)],havingCTasthethresholdcyclevaluedefinedasthefractionalcyclenumberatwhichthefluorescenttargetsignalpassesafixedthresholdabovethebaseline.Eachassayincludedatemplate-omittedcontrolandaRTnegativecontrol.
RESULTS
L.angustifoliusseedgermination
Wehavestudied the initial timecourseofNLLseedgerminationuntil the first leavesaredifferentiated,andcotyledonscontinue thedegradation.ThematureNLL seedconsistsofabrownseedcoat,absentendospermand largecotyledons(Figure1).Inthematureembryo,twocotyledonsandaradicleareclearlydistinguishable.Invitrogerminationwasfolloweduntiltheembryoturnedintoayoungsmallgreenplant.Thisgerminationperiodtakeplaceabout11days.Afterthisperiodofculture,seedlingsconsistedoftwocotyledons,stemandaroot,completelyturnedintoagreencolour.
Figure4.SeedgerminationstagesofL.angustifolius.Picturesincludefromimbibitionto11daysafterimbibition(DAI).
Wecanconsiderthisstageastheendoftheseedlingstageandstartoftheplantgrowth.Finally,after11days,theplantwasreadyforcontinuousgrowinginapot.
GeneexpressionanalysisofConglutinfamiliesandalkaloidenzymaticpathway
Geneexpressionanalysiswasperformedforallmembersofthedifferentconglutinfamilies.MostofthegenesexhibitedacharacteristicexpressionpatternwheremRNA%decreasedas thegerminationprocessprogresseduntil11days.Al thisstage,the%ofmRNAfortheseconglutingeneswasalmostabsent.However,Figure2showsacharacteristicpatternofexpressionforb3tob6,gamma1and2geneswhereinitialandmiddlestagesofgermination(3to5days)arecharacterizedbyabundanttranscriptsquantity,andwhereseedexhibitedanactivemetabolism.Interestingly,theexpressionanalysisoftheabovementionedgenes fromseedgerminated inwaterandMSmediumexhibiteda comparable levelof transcriptexpression,withfewexceptionsinsomegerminationsteps.
Interestingly,geneexpressionanalysiswasalsoperformedforkeyenzymesinvolvedinalkaloidsyntheticpathway,L/ODC,LO,HMT/HLTandECT/EFT-LCT/LFT,asdepictedinFigure3.
LOandECT/EFT-LCT/LFTexhibitedanincreasingexpressionpatternfrominhibitionstateto11daysofgermination.LODandHMT/HLTexhibitedamRNAhighexpressionpatternbetween3to5daysofgermination.
AlltheseenzymesexhibitedabundanttranscriptsinthesamestagesofgerminationcomparedtoNLLseedstorageproteins(betaandgammaconglutins).
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Figure 2. Expression analysis of conglutin genes during seed germination. The housekeeping gene used as control wasubiquitin(UBQ).Relativegeneexpressionlevelsweredeterminedbyusingthe2−ΔΔCTmethod.ThecyclenumberatwhichthetranscriptsweredetectablewasnormalizedtothecyclenumberofUBQ,referredtoasΔCT.X-axisrepresentsDAI.Blackbars:IMBandgreybars(seedgerminatedwithoutMSmedia).Dotsbars:seedgerminatedinMSmedium.
Figure3.Expressionanalysisofalkaloidsyntheticpathwaygenesduringseedgermination.Thehousekeepinggeneusedascontrolwasubiquitin(UBQ).Relativegeneexpressionlevelsweredeterminedbyusingthe2−ΔΔCTmethod.ThecyclenumberatwhichthetranscriptsweredetectablewasnormalizedtothecyclenumberofUBQ,referredtoasΔCT.X-axisrepresentsDAI.Blackbars:IMBandgreybars(seedgerminatedwithoutMSmedia).Dotsbars:seedgerminatedinMSmedium.
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DISCUSSION
Themainlimitingfactorofusinglupinasproteinsourcearealkaloidswhichintoxicdosescauseneuromuscularblockage,respiratorydepression,cyanosis,crampsandcardiacarrest.Forthatreason,manystudiesondecreaseofalkaloidcontentin lupin were carried out. Among many technological methods used for decrease of alkaloid content in lupin seedsgerminationhasbeenrecentlyconsideredasparticularlypromising.
The current study showedan increased transcripts expressionof alkaloids synthetic enzymes, and a general decreasingpatternofdifferentgenesrelatedtoconglutinfamilies.However,atthemiddleofthegerminationprocess(3to5days),bothtypeofgenesexhibitedabundanceoftranscripts.
PreviousstudiesshowedthatgerminationcausesanincreaseoftotalphenolsinsproutsofLupinusangustifolius.Anincreaseisalsoobservedatfourdaysandamorepronouncedoneatninedays.Ariseintotalphenolsofchickpeawasalsoreportedafterfivedaysofgermination.Alltheseresultsagreewithourstudyaboutthealkaloidenzymaticexpressionanalysis.
Thegerminationcausedacleardecreaseoftotalalkaloidscontentsinboththecases.However,thecontentsofthesamealkaloids during germination increased. For example, in the case of L. angustifolius, the increase of tigloyloxylupanine,3OHlupanine,angustifolineisnoted.ForL.luteustheincreaseofgraminecontentisobserved.Generally,thealkaloidcontentinsproutsisdependentonthelupinspecies,temperatureandtimeofgermination.
CONCLUSION
Severalvariationswereobservedingeneexpressionofconglutinandalkaloidssyntheticenzymesduringthedifferentdaysofgermination.However,comparablepatternofexpressionbetweenbothtypesofgeneswereobservedbetween3to6daysofgermination.ThisdatacouldinferthefunctionalinterplayinmetabolicprocessesduringseedsgerminationofNLLseedstorageproteins(conglutins)andthealkaloidmetabolicpathwaybeyondtheroleasnitrogensource.Thus,weproposethese gene families as molecular markers of the germination progressing, and particularly to dissect this physiologicalcomplicateprocess(germination)infunctionalsteps.
ACKNOWLEDGEMENTSANDFUNDING
EU Marie Curie grant PIOF-GA-2011-301550; MINECO grants RYC-2014-16536 (Ramon y Cajal research program), andBFU2016-77243-P.
REFERENCES
Jimenez-Lopez JC, Zienkiewicz A, Zienkiewicz K, Alche JD, Rodríguez-García MI. (2016a). Biogenesis of protein bodies during leguminaccumulationindevelopingolive(OleaeuropaeaL.)seed.Protoplasma253(2):517-530.doi:10.1007/s00709-015-0830-5.
Jimenez-LopezJC,MelserS,DeboerK,ThatcherLF,KamphuisLG,SinghKB.(2016b).Narrow-leafedlupin(LupinusangustifoliusL.)b1-andb6-conglutin proteins antifungal activity, protecting plants against necrotrophic pathogen induced damage from SclerotiniasclerotiorumandPhytophthoranicotianae.FrontiersinPlantScience7:1856,doi:10.3389/fpls.2016.01856.
Lima-CabelloEetal.(2017).Narrow-leafedlupin(LupinusangustifoliusL.)β-conglutinproteinsmodulatetheinsulinsignallingpathwayaspotentialtype2diabetestreatmentandinflammatory-relateddiseaseamelioration.MolecularNutritionandFoodResearch61(5),doi:10.1002/mnfr.201600819.
Lima-CabelloE,Morales-SantanaS,FoleyRC,MelserS,AlchéV,SiddiqueKHM,SinghKB,AlchéJD,Jimenez-LopezJC.(2018).Exvivoandinvitroassessmentofanti-inflammatoryactivityofseedβ-conglutinproteinsfromLupinusangustifolius.JournalofFunctionalFoods,doi:10.1016/j.jff.2017.11.040.
ZienkiewiczA,Jimenez-LopezJC,ZienkiewiczK,AlcheJD,Rodriguez-GarciaMI.(2011).Developmentofthecotyledoncellsduringolive(OleaeuropaeaL.)invitroseedgerminationandseedlinggrowth.Protoplasma248(4):751-765.doi:10.1007/s00709-010-0242-5.
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TOCOPHEROL CONTENT OF ALMOND OILS PRODUCED IN EASTERN MOROCCO
ABMELHAOUIR,BFAUCONNIERM-L,CSINDICM,AADDIM;AABIDM,
AMIHAMOUA.,ASERGHINI-CAIDH,AELAMRANIA
ALaboratoiredeBiologiedesplantesetdesmicro-organismes,FacultédesSciences,UniversitéMohamedIer,Oujda;Maroc.
BLaboratoiredeQualitéetSécuritédesProduitsAlimentaires,GemblouxAgro-BioTech,UniversitédeLiège;Belgique.CLaboratoiredeChimieGénéraleetOrganique,GemblouxAgroBio-Tech,UniversitédeLiège;Belgique
INTRODUCTION
AlmondisthemostimportanttreenutcropinMoroccointermsofacreageandproductionvalue.Almondplantationscoveratotalareaof151,000hawithanestimatedaverageannualproductionof99.000tonsofshelledproducts.9%ofthisareawhichprovidesupto14%ofMoroccanproductionofalmondsislocatedineasternMorocco(MAPM,2014).RecentlyeasternMoroccoregionwassupportedbytheBelgiandevelopmentagency"BTC"throughthe"PROFAO*"projectforplanting6000haofalmondtreesandfortheimprovementofalmondvaluechainatthepost-harvestlevel,particularlytheevaluationofalmondsanditsderivedproducts(oilandotherco-products).Almondsareagooddietarysourceoftocopherols(vitaminE),sterols,andflavonoids,suggestingplayingaroleinhealthpromotion.Furthermoreconsumptionoftocopherolshavebeenassociated with the natural antioxidant benefits and health outcomes (Maguire et al., 2004). The concentrations oftocopherols in fully ripenedalmondkernelshavebeenstudied indifferent regionsofMorocco (Kodadetal.,2014),buttocopherolcontentsineasternMoroccohasnotyetbeenstudied.Thegoalofthisstudyconcernstocopherolanalysis inalmondoilsproduced ineasternMorocco.Thevarietiesused inthisstudyare:Ferragnes,Feraduel (F/F)andFournatdeBrezenaudoriginatedfromFrance;MarconafromSpainandBeldilocalecotypefromeasternMorocco.PROFAO*:ProjetFilièreAmandesdeL’Oriental,“ThealmondvaluechainineasternMorocco,PillarIIoftheMoroccanGreenPlan,supportedbyBelgiandevelopmentagency(BTC2011-2017)
MATERIALANDMETHODSPlantmaterialTodeterminetocopherolcontentsasaqualityparameterofalmondoils(AO)producedineasternMorocco,AOoffivemainvarietiesgrowninthisregion(Fournat,Marcona,Ferragnes/FerraduelcoupleandlocalecotypeBeldi)weretested.Almondsweretrituratedusinganoilscrewpress(KOMETModèleDD85G).Thealmondsvarieties(Figure1)arecollectedinapilotareaintheeasternMorocco(SIDIBOUHRIA:34°44’13.6’’N,002°20’15.0’’W),
FournatdeBrezenaud Marcona Ferragnes/Feraduel BeldiecotypeFigure1:ThemostcommonalmondvarietiesgrownineasternmoroccoTocopherolcontentsanalysisThedifferenttocopherolisoforms(α-,β-,γ-andδ-tocopherols)wereevaluatedfollowingtheAOCSmethodCe8-89(AOCS1989).AnoilhexanesolutionwasanalyzedbyHPLCwithfluorescencedetectorHPLC-FLD(AgilentTechnologiesseries1200system,AgilentTechnologies),equippedwithanautomaticinjector,onanUptisphere120A°NH2column(150mm*3mm,3µm) Interchim (Montluçon, France) andmaintained at 30 °C. The injection volumewas 10µL. Themobile phasewashexane/2-propanol(99:1,v/v)elutedataflowrateof1mLmin-1.Thetocopherolswereidentifiedandquantifiedbyexternalstandardization(Mixtureoftocopherols:α-tocopherol,β-tocopherols,γ-tocopherols,δ-tocopherols)obtainedfromSigma-Aldrich(Steinheim,Germany).
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StatisticalanalysisTocopherol analyseswere carried out on triplicate for each sample of almondoil varieties. All statistical analyseswereperformedwiththeSPSSsoftwareforWindows(SPSS.21,USA).Valuesofdifferentparameterswereexpressedasthemean(X¥±standarddeviation).ThenormaldistributionwasverifiedaccordingtoShapiroWilktest.Duncan'smethodwasusedformeancomparison.Onlyvariableswithaconfidencelevelsuperiorto95%(P<0.05)wereconsideredassignificant.
RESULTS
AlmondOilsextractedfromlocalBeldiecotypeandthemainintroducedvarietiesFerragnes/Ferraduel(F/F),MarconaandFournatwereanalyzedfortheirquantitativeandqualitativetocopherolcontents.Theaverageoftotaltocopherolquantitiesobservedis528.98mg/kgAO,withtheα-tocopherolasadominantcompoundinalmondsoil.Totaltocopherolscontentandproportionofdifferenttocopherolshomologuescontent(α-,β-,γandδtocopherols)ofanalyzedalmondoilsbyHPLC-FLDaresummarizedinTable1.
Table1:Totalcontentandproportionofmaintocopherolhomologuesinfivescrew-pressedalmondoils(mg/kgAO)AlmondoilTocopherolsmg/kgAO
Fournat Marcona Ferragnes/Feraduel Beldi
α-tocopherol 483.98ab±7.83 456.44bc±2.87 425.03c±7.59 517.02a±8.56β-tocopherol 3.17a±0.78 1.77c±0,01 2.28bb±0.25 2.95a±0.33Ɣ-tocopherol 14.27a±0.82 2.75b±0.01 5,80c±0.14 9.01d±1.01δ-tocopherols ND ND ND NDTotaltocopherols 501.43ab±8.05 460.96bc±2.88 433.11c±7.88 528.98a±9.71Significantdifferencesareshownbydifferentletters(a-d).ND:notdetected
DISCUSSION
Tocopherolanalysisshowedthepresenceof3tocopherolisoforms(α-,β-and,γ–tocopherol).SimilarresultswerefoundbyKornsteineretal.,(2006),Zhuetal.,(2015a,b)andZhuetal.,(2017).Thehigherα-tocopherolcontent(517mg/kgoil)wasfound inAOBeldi ecotype.While the lower contentswere registered in the introduced varietieswhich range between484mg/kgoilforFournatand425mg/kgoilforthecoupleF/F.Regardingtoβandγtocopherols,ourresultsshowthatβ-tocopherolcontentsrangefrom1.77to3.17mg/kg,andγ-tocopherolfrom2,75to14,27mg/kgoilrespectivelyforMarconaand Fournat, while intermediate values are recorded for F/F. The total tocopherol content of the introduced varietiescultivatedineasternMoroccoismuchhigher(Table1)comparedtothesamevarietiescultivatedinSpain(Marcona:374.1,Fournat:402.4,F/F:385.2mg/kgoil)(Kodadetal.,2011)Thisisanimportantparameterforthevalorizationoftheseproductsfortheirusesasfoodstarsandincosmeticsaswell.AlthoughtheBeldiecotypeischaracterizedbyhighdiversityrelatedtonaturalhybridizationbetween thenativesalmond trees (Melhaouietal., 2017)andbya strongpresenceofdoubles itstocopherolrichnesscanbeconsideredasanaddvalueforthelocalproductionandcouldbeexclusivelyorientatedtowardvirginalmondoilextraction.Sincealmondoilextractionbyscrewpresscanpreservenaturalantioxidantsandfat-solublebioactivecompoundswhichmakethisoilwithinterestingnutritionalandcosmeticproperties.
CONCLUSION
Thisstudyshowsa largevariabilityofcontents fordifferent tocopherolhomologues in theanalyzedalmondoilswithα-tocopherolasadominantcompound.Thisvariationisrelatedtothegenotypeaswellastothegeographicallocalization.Moreover,tocopherolrichnessinBeldiecotypecouldbeusedasanaddvalueforvirginalmondoilextractionwithinterestingnutritionalandcosmeticproperties.
REFERENCES
Jambazian,P.R.,Haddad,E.,Rajaram,S.,Tanzman,J.,Sabaté,J.,2005.Almondsinthedietsimultaneouslyimproveplasmaalpha-tocopherolconcentrationsandreduceplasmalipids.J.Am.Diet.Assoc.105,449–454.https://doi.org/10.1016/j.jada.2004.12.002Kodad,O.,Estopanan,G.,Juan,T.,Company,R.S.i,2014.TocopherolconcentrationinalmondoilfromMoroccanseedlings:Geographicaloriginandpost-harvestimplications.J.FoodCompos.Anal.33,161–165.https://doi.org/10.1016/j.jfca.2013.12.010Kodad, O., Estopanan, G., Teresa Juan, AliMamouni, Rafel Socias i Company, 2011. Tocopherol Concentration in AlmondOil: GeneticVariationandEnvironmentalEffectsunderWarmConditions.JAgricFoodChem6137–6141.Kornsteiner,M.,Wagner, K.H., Elmadfa, I., 2006. Tocopherols and total phenolics in 10 different nut types. Food Chem. 98, 381–387.https://doi.org/10.1016/j.foodchem.2005.07.033
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Maguire, L.S.,O’Sullivan, S.M.,Galvin, K.,O’Connor, T.P.,O’Brien,N.M., 2004. Fatty acid profile, tocopherol, squalene andphytosterolcontent of walnuts, almonds, peanuts, hazelnuts and the macadamia nut. Int. J. Food Sci. Nutr. 55, 171–178.https://doi.org/10.1080/09637480410001725175MAPM,2014.Ministèredel’agricultureetdelapechemaritimeVeille-économique-secteur-amandier-Notestrategique,N°99.Availableat:http://www.agriculture.gov.ma/pages/veille/veille-economique-secteur-amandier-juillet-2014,.Melhaoui,R.,Abid,M.,Mihamou,A.,Sindic,M.,Caid,H.S.,Elamrani,A.,2017.Flowering,aCriticalPhenologicalStageasaLimitingFactorforAlmondNativeEcotypesCultivationinEasternMorocco.Appl.Microsc.47,157–159.Zhu,Y.,Taylor,C.,Sommer,K.,Wilkinson,K.,Wirthensohn,M.,2015a.Influenceofdeficitirrigationstrategiesonfattyacidandtocopherolconcentrationofalmond(Prunusdulcis).FoodChem.173,821–826.https://doi.org/10.1016/j.foodchem.2014.10.108Zhu, Y.,Wilkinson, K.L.,Wirthensohn,M.,Al, E., 2017. Changes in fatty acid and tocopherol content during almond (Prunusdulcis, cv.Nonpareil)kerneldevelopment.Sci.Hortic.225,150–155.https://doi.org/10.1016/j.scienta.2017.07.008
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IMPROVEMENT OF SPRAY RETENTION ON BARLEY LEAVES
H.H.BOUKHALFA,M.BELHAMRA
DPAZAlaboratory,UniversityMohamedKhiderBiskra,FacultyofSciencesandsciencesofnatureandlifeDept.Agronomy,BP145RP07000Biskra,Algeria
CorrespondingauthorE-mail:[email protected]
SUMMARYThe droplet size distribution of agricultural sprays is a key parameter during the plant protection product applications.Phenomena governing spray retention on plants are investigated for a very long time in order to optimise pesticideapplication.Thesecanbedividedinphysicalandchemicalpropertiesofeitherthetargetorthedrop.Leafmorphologymayplay an important role in spray deposition. Different leaf structures such aswax, hairs, edges and veins are importantimpingement and retention variables. Surfactants are nowadays very useful additives to improve the effectiveness ofphytosanitarytreatments.Theycontributetochangetheimpacttypesandthustheamountofsprayretainedbytheleavesofthetreatedplant.
WeperformedtestsofretentiononwholebarleyplantsonBBCH-scale12andsmallpiecesofbarleyleavesatthesamestageofgrowth.Sprayingwasdoneinthreeways:waterwithoutsurfactant,waterwithBreak-Thru®S240andwaterwithLi700®.Thethreeslurriesoffluoresceincontainedinanamountof0.2g/l.Fluoresceinretainedbytheleavesinbothcasesisthenmeasuredbyaspectrofluorometer.Theretentiontestsonwholeplantsshowthatit istripledbythefirstsurfactantanddoubledbythesecond.Byconsonsmallpiecesofbarleyleaves,theamountwasincreasedbytheuseofsurfactantsbutnottothesamescale.Thisstudyconcludedthattheuseofsurfactantsinspraypesticidesmayincreasetheamountofretentionasafunctionofleafareaandthesurfactantused.
INTRODUCTIONRetentionismainlyassociatedwithdropletprimaryadhesion,whilebouncingandsplashingareseenasdetrimental.Impactoutcomesdependonleafsurfaceandspraymixtureproperties.Onsuperhydrophobicspecies,surfactantsareoftenusedtoenhancesprayformulationperformancesbyaffectingthephysicochemicalpropertiesofdroplets.
Theaimofthestudyistotesttwosurfactantsusingsimultaneousobservationofdropletimpactsbyhighspeedimagingandfluorescenttraceranalysisofdeposits.WorksofHollowayetal.(2000)confirmedalsothat,tank-mixadjuvantscanhaveaconsiderable influenceon theefficiencyof deliveryofwater spays. Theyaffect thephysicochemical propertiesof spraydropletsintermsoftheirultimateimpactionandspreadingbehaviour.Surfactantsarethemostwidelyusedadjuvanttypeinagrochemicalapplicationsandthemostefficientinenhancingtheirefficacy(Wangetal.,2007).
MATERIALSANDMETHODSTheexperimentalmethodologyexplained inBoukhalfaetal. (2014)wasassociatedtoanexperimentonwholeplantsofbarley.Foreachformulationtensprayswererealizedonfivebarleyplantsatthetwoleavesgrowthstage(BBCHstage12)grown indoor incontrolledconditions.Theamountof sprayactually retainedbybarleyplantswasassessedbydosingafluorescenttraceraddedtothemixtures.Barleyplantswereplacedlinearlybelowthecenterofamovingnozzleatthespeedof2m/susingapressureof2bars.Thelaststepwasmeasuringleafareaforeachsprayandcalculatingtheamountofspraysretainedbybarleyplants.RESULTSANDDISCUSSION
Dropletsimpactbehaviour:Results obtained by image analysis for the three spraymixtures are represented by volumetric distribution of differentimpactbehaviourofdropletsrelatingtotheirdiametersandvelocities.Table1:Volumetricdistributionofdropletsimpactbehaviour
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Resultsofwatershowthehigheramountofbouncing.SplashingoccursinbothCassie-BaxterandWenzelwettingregimesaswellasadhesion.
For the mixture containing Break Thru S240, bouncing is not observed at the same scale of water. This behaviour ischaracteristicofthe lowdynamicsurfacetensionofthissuper-spreaderorganosiliconeadjuvant.CassieBaxterregime isobservedveryfewtimes.Break-thruincreasegreatlyadhesionanddecreasehighlyreboundcomparingtowater.
TheresultsforthewettingagentLi700,showthereforethatsplashingoccursonlyinWenzelwettingregime.Decreaseofadhesion is compensated by the increase of shattering inWenzel regime related to the higher VMD (volume mediandiameter).
Retentionrelatedtothemixture:Sprayretentionforeachmixturewerecomparedonfigure1.
Figure1.Comparisionretentionforthreemixtures
Figure 1 represents volumes of spray retained by hole plants of barleymeasured by spectrofluorometer. The diagramconfirmstheeffectofadjuvantsonthespraycharacteristics.Inhisstudies,Stock(1997)confirmedthatthesuperspreadingeffectoforganisiliconematerialscanhaveasignificantimpactuponretentionefficiency.Buthethoughtthatthesubsequentsuperspreadingmay,dependingonsprayvolume, leadtoexcessiverunoffandareducednetretentionwhilsttheinitialeffectonretentionmaybebeneficial.Ourresultsconfirmthattheuseoftankmixsurfactantscanimprovesprayretentiononbarleyleaves.CONCLUSIONTank-mixadjuvantsaffectpositivelyretentionandimproveapplicationefficiency.Sprayretentionisdifferentfromaplanttoanotherone,sothenumberoftrialsmustbeincreasedinordertoobtainasignificantresult.Dependingonthespraymixture,dropletsfragmentedinWenzelregimeaccountedfor28-46%ofretentionatfirstimpact,withaclearrankingasafunctionofDST.LITERATUREBoukhalfa, H. H., Massinon, M., Belhamra, M., & Lebeau, F., (2014). Contribution of Spray Droplet Pinning Fragmentationto CanopyRetention.CropProtection,56,91-97.
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Boukhalfa,H.H.,Massinon,M.,&Lebeau,F.,(2013).High-speedimagingusetopredictsprayretentiononbarleyleaves.CommunicationsinAgriculturalandAppliedBiologicalSciences,Vol78(2)(1-386(2013)),31-36.Massinon,M.,BoukhalfaH.H.,Lebeau,F.,(2014).Theeffectofsurfaceorientationonsprayretention.PrecisionAgriculture15:241–254.Massinon,M.,Boukhalfa,H.H.,&Lebeau,F., (2014).Sprayretentionassmentcombininghigh-speedshadowimageryandfluorescencetechniques.InternationalAdvancesinPesticideApplication:AspectsofAppliedBiology122,2014(pp.219-225).Warwick,UK:AssociationofAppliedBiologists.Massinon,M.,Boukhalfa,H.H.,Marechal,P.,&Lebeau,F.(2012,Juillet).Theeffectofleaforientationonsprayretentiononblackgrass.Paperpresentedat11thinternationalconferenceonprecisionAgriculture,Indianapolis,USA.SmithD.B.,AskewS.D.,MorrisW.H.,ShawD.R.&BoyetteM.(2000).Dropletsizeandleafmorphologyeffectsonpesticidespraydeposition.TransactionsoftheASAE.43(2):255-259.StockD. (1997).Doweneedadjuvants?Mechanisticstudiesand implicationsfor futuredevelopments.Proc.50thN.Z.PlantprotectionConf.:185-190.WangC.J.&LiuZ.Q.(2007).Foliaruptakeofpesticides.Presentstatusandfuturechallenge.PesticideBiochemistryandPhysiology.87:1-8.
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EVAUATION OF SPRAY RETENTION VARIABILITY ON HYDROPHOBIC PLANT LEAVES
H.H.BOUKHALFA,M.BELHAMRA
LaboratoryDEDSPAZA,DepartmentofAgronmicalSciences,MohamedKhiderUniversity
BiskraAlgeria,BP145RPBiskra,Algeria
INTRODUCTIONSpraying iswidespreadinagriculturalpracticetoprotectcropsfromavarietyofpests. Inpracticeanamountofproductalwaysgreaterthanrequiredisappliedtoprovidethelevelofcropprotectionneeded.SprayretentionismainlyassociatedwithdropletadhesionandsplashinginWenzelwettingstate.Impactoutcomesdependonleafsurfaceandspraymixtureproperties.Barleyfoliageisorientedmainlyverticallyandtheleavesaredifficulttowetbecauseoftheirdensecoveringofmicrocrystalline epicuticularwax. And that affect spray retention. Drop behaviour during impact affects also retention.ObservationofdropimpactsusinghighspeedimagingshowsthatfragmentationinWenzelwettingregimecancontributetoretention.
MATERIALANDMETHODSMeasurements of retention were done for two surfactants and compared to water on barley leaves (BBCH12) usingspectrofluorometry.Break-Thru®S240(Organosiliconesurfactant)attheconcentrationof0,1%andLi700®(Phospholipidsurfactant)attheconcentrationof0,25%wereappliedtofoliageinaqueoussprays.Thethreetankmixturesweremarkedbyfluoresceintracerataconcentrationof1g/l.
Thesprayswereproducedbyaflat-fannozzleTeejet11003andapressureof2bars,mounted50cmheightabovethetargetonarampmovingataspeedof2m/s.Sprayingswereperformedinlaboratoryatatemperatureof18°Candrelativehumidityof40%.
Foreachformulationtensprayswererealizedonfivebarleyplantsatthetwoleavesgrowthstage(BBCHstage12)grownindoorincontrolledconditionsandtenspraysrealizedonindividualbarleyplantsatthesamegrowthstage.Theamountofsprayactuallyretainedbybarleyplantswasassessedbydosingfluorescenttracer.Barleyplantswereplacedlinearlybelowthecenterofthemovingnozzle.Thelaststepwasmeasuringleafareaforeachsprayandcalculatingtheamountofspraysretainedbybarleyplantstodeterminethevariabilityofsprayretention.
RESULTSSprayingon5barleyplantsSprayretentionmeasuredon5linearbarleyplantsarerepresentedin(Figure1).Thereisavisiblevariabilitybetweenthetentrials forall tankmixturestested.Theuseofadjuvantmay increasesprayretentionbutthere isno influenceonthevariability.
Figure1.Retentionvariabilitymeasuredon5barleyplantsfromthethreemixtures.
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OnewaysprayingonindividualbarleyplantsSprayretentionmeasuredonindividualbarleyplantsfromonewayspryingarerepresentedin(Figure2).Thediagrammeshowsthesameresultsobtainedwith5linearbarleyplants.Thereisgreatsprayretentionvariabilitybetweeneachplant.
Figure2.Retentionvariabilitymeasuredforonewaysprayingonindividualbarleyplantsfromthethreemixtures.DoublesprayingonindividualbarleyplantsSprayretentionmeasuredon5linearbarleyplantsarerepresentedin(Figure3).Thesamevariabilityobtainedbyone-waysprayingisobtainedwithedabblesprays.Weperformedthistrialinordertotesttheinfluenceofintersectionofsprayjetsapplied in field applications. Spray retention variability between each plant is the same in all cases tested and for allformulationsusedinthisstudy.
Figure3.Retentionvariabilitymeasuredfordabblesprayingonindividualbarleyplantsfromthethreemixtures.DISCUSSIONA 3D virtual spraying model predicting spray droplet interception and retention by single plant architecture has beendevelopedandusedforfocusingonthevariabilitybyMassinonetal.(2015).It investigatedasingle3Dplantmodelasafunctionofthesprayquality,thevolumeperhectareappliedandtheplantsizefortwocontrastedformulationscenariosrepresentativeoflowandhighsprayliquidwettingproperties.Resultsobtainedbythismodelshowedthatretentionrangedfrom6.8%to96.6%ofa fulladhesiondependingonsprayqualityandformulationscenaro.Theseresultscooborateourresults.Massinonetal.(2015)explanedthatthevariabilityofdepositsincreaseswithdecreasingsprayfineness,appliedvolumeperhectareandplantsizebecauseofthereducednumberofdropletcontributingtoretention. InourexperimentthesameresultisobservedwitnformulationcontainingBreaktruewitchiscaracterizedbyfinenessofdropletsandthoseobtanedwithformulationcontainingLi700caracterizedbylownumberofdroplets.
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CONCLUSIONTheaimofthisworkistoobservetheretentionvariabilitythatcanoccurduringatreatmentdependingontheequipment,tankmixformulationandcropproperties.Tank-mixadjuvantsaffectpositivelyretentionandimproveapplicationefficiency,buttheyhaven’tanyeffectonsprayretentionvariability.Sprayretentionisdifferentfromaplanttoanotherone,sothenumberoftrialsmustbeincreasedinordertoobtainasignificantresult.REFERENCESBoukhalfa, H. H., Massinon, M., Belhamra, M., & Lebeau, F., (2014). Contribution of Spray Droplet Pinning Fragmentationto Canopy
Retention.CropProtection,56,91-97.Boukhalfa,H.H.,Massinon,M.,&Lebeau,F.,(2013).High-speedimagingusetopredictsprayretentiononbarleyleaves.Communications
inAgriculturalandAppliedBiologicalSciences,Vol78(2)(1-386(2013)),31-36.Massinon,M.,Dumont,B.,DeCock,N.,OuledTalebSalah,S.,&Lebeau,F.,(2015).Studyofretentionvariabilityonanearlygrowthstage
herbaceousplantusinga3Dvirtualsprayingmodel.CropProtection,78,63-71.Massinon,M.,BoukhalfaH.H.,Lebeau,F.,(2014).Theeffectofsurfaceorientationonsprayretention.PrecisionAgriculture15:241–254.Massinon,M.,Boukhalfa,H.H.,&Lebeau,F., (2014).Sprayretentionassmentcombininghigh-speedshadowimageryandfluorescence
techniques. International Advances in Pesticide Application: Aspects of Applied Biology 122, 2014(pp.219-225).Warwick, UK:AssociationofAppliedBiologists.
Massinon,M., Boukhalfa, H. H,Marechal, P, Lebeau, F. (2012). The effect of leaf orientation on spray retention on blackgrass. 11 thinternationalconferenceonprecisionAgriculture,Indianapolis,USA.
SmithD.B.,AskewS.D.,MorrisW.H.,ShawD.R.&BoyetteM.(2000).Dropletsizeandleafmorphologyeffectsonpesticidespraydeposition.TransactionsoftheASAE.43(2):255-259.
StockD.(1997).Doweneedadjuvants?Mechanisticstudiesandimplicationsforfuturedevelopments.Proc.50thN.Z.PlantprotectionConf.:185-190.
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EVALUATION OF GROWTH PERFORMANCE OF OULED DJELLAL LAMBS BY SEX AND LITTER SIZE IN A SEMI-ARID REGION OF ALGERIA
KDEGHNOUCHE,BNACERCHERIF,HHBOUKHALFA
LABORATORYDEDSPAZA.MOHAMEDKHIDERUNIVERSITY.BISKRA.07000.ALGERIA
INTRODUCTION RuminantlivestockisoneofthekeysectorsofAlgerianagricultureinwhichthecomponent(smallruminant)predominates.AccordingtothestatisticsofthespecializedservicesoftheMinistryofAgricultureandRuralDevelopmentin2015,sheepfarming accounts for nearly 80%of the total numberof livestock in the country andAlgeria is ranked5th in theworldproductionofsheepmeat.Despitetheexceptionalgrowthofthissector,sheepmeatremainsinaccessibleforalargepartofAlgerianhouseholdswithmediumandlowincomes.ThelowproductivityofdomesticherdsisattributedaccordingtoBencherif(2011)toamisbehaviorofbreedingandfeedingofherdswhichisoftenofextensivetype.Theobjectiveofthisworkistostudyandanalyzetheimpactsexofthelamb,andthesizeofthelitteronmeasurablecriteria(birthweight,weightattypicalages,averagedailygainsatagestypes)intheOuledDjellallambsfrombirthtoweaninginordertoprovidethebreederwithassistanceinmanagingtheherdonthebasisofreproductionandgrowthcriteriaontheonehandandtoallowgeneticevaluationewesbasedonprolificacyanddairyvalue.MATERIALSANDMETHODSAnimalsThestudyconcernedanumberof200lambs(maleandfemale)ofOuledDjellalbreedfrommultiparousandprimiparousewesthatdidnotundergoheatsynchronization.TheywerebornbetweenOctober10thandDecember9th,2016.Theywereraisedinsemi-intensive,inadditiontobreastmilk,theyhadbarleyhayattheirdisposalandtheywerepreviouslyidentifiedwithnumberedearrings,theywerehousedinsheepfoldwithnaturallight.Weighingthe lambswasdoneusingascalewithamaximumcapacityof50kg±100g,andall informationfrombirthtoweaningisrecordedonindividualgrowthrecordsforeachlamb.Thestudiedvariables:Controloftheevolutionoflambgrowthfrombirthtoweaningrepresentedbyliveweight(WV)andmeandailygain(ADG)wasperformedasfollows:liveweightin(kg)atd0(atbirth),d10,d20,d30andd90(weaning).-theADGin(g)between:d0-d10;d10-d20;d20-J30andd30-d90.Thefactorsofvariation:-Thesexoftheproduct(male,female)-Thesizeofthelitter(single,double)Statisticalanalysis:Descriptivestatisticsandone-wayanalysisofvariance(ANOVA)wereperformedwiththeSPSSsoftware(2013version20)forPVandGMQanalysis.-ThecorrelationresultswerecalculatedbythePearsontestfortheevaluationoftherelationshipbetweenliveweightatdifferentgrowthphasesoflambs.Alltheaveragesoftheresultswerecalculatedwiththeirmeanstandarderrors(mean±E.S.M).Thestatisticaldifferencewasreportedat(P<0.05).-Thegeneralizedlinearmodel(GLM)wasusedtotesttheeffectsofthefactorsonthevariables,bytheapplicationoftheTtestforindependentsamplestoestimatethemeaningorthehomogeneitybetweenthedifferentsetsofdata(comparisontestbetweentheaverages).-ThefrequenciesofeachquantitativevariableweregraphicallyrepresentedusingExcel(2007).RESULTS:Growthperformancefrombirthtoweaning:I-Dependingonthesizeofthelitter:1-Weightatbirth:Themeanliveweightofthelambsatbirthis4.19±0.94kg.Singlelambstendtohavehigherbirthweightscomparedtodoubles(4.70±0.54kgvs.3.88±0.30kg)respectively(Figure1).2-Weightattypicalages(10,20,30and90):Singlelambstendtohavegreaterweightsthantwinsthroughouttheexperiment(Figure2)
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Meanweightsobservedat10,20,30and90jcorrelateverysignificantlywithbirthweight(r=0.56P=0.009;r=0.57P=0.008;r=0.54P=0.013andr=0.63P=0.003respectively).Birthweightandmeanweightsat4typicalagesdiffersignificantlywithlittersize(P<0.05)
3-AverageDailygainsItappearsthatsinglestendtohavehigherADGscomparedtodoubles(Figure3).Thecorrelationsaresignificantbetweentheaveragedailygainsatthedifferenttypicalages,(the30dayADGiscorrelatedwiththe20-dayADGwithacorrelationcoefficientr=0.68andP=0.001,itisalsocorrelatedwiththeADGat90dayswithacorrelationcoefficientr=0.64andP=0.002,alsothe90dayADGiscorrelatedwiththe20dayADGwithacorrelationcoefficientr=0.57andP=0.008).Thereisnosignificantcorrelationbetweenbirthweightandaveragedailygainat30daysafterbirth(P>0.05/P=0.30),whereasitisamilkproductionindex.4-Evolutionoftheweight:"Growthcurve"Theliveweightinsingleanddoublelambsevolvesaccordingtoafirstorderpolynomialregressioncurve,theequationsarerespectivelyy=4.108x-1.163andy=2.478x-0.362ofwhich(xisthetimeandyistheweight).Singlebirthlambsalwaysgrowlargerthandoubles.Theytendtohavehigherliveweightsthandoublesfrombirthtoweaning.
II- Accordingtosex:
1-Birthweight:Theaverageliveweightof lambsatbirth is4.19±0.94kg.Male lambsshowedaslightdifferenceinbirthweightcomparedtolambs(4.37±0.5vs4±0.42kgrespectively)Birthweightdidnotdiffersignificantlywithlambsex(P>0.05)/P=0.39)2-Weightattypicalages(10,20,30and90days):Malelambstendtohavehigherweightsthanewelambsforthedurationoftheexperiment(Figure5).Theweightsatthedifferenttypicalagesdonotdiffersignificantlywiththesexofthelamb(P>0.05/P=0.23,P=0.16,P=0.29,P=0.86respectivelyatthetypicalages10,20,30and90days).
Figure 1: Difference in birth weightbetweensingleanddoublelambs
Figure 2: Variations in weights at differenttypical ages (kg) by litter size
Figure3:Variationsinaveragedailygains(g)bylittersizeinOuledDjellallambs.
Figure4:EvolutionofweightrelativetolittersizeinOuledDjellallambs
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Figure5:Variationsinweight(kg)bysexinOuledDjellallambs.3-Theaveragedailygains:ItappearsthatmaleshavehigherADGsthanewesfrombirthupto20days,andthentheopposite(Figure6).Alsoatthetypicalages,ADGsdonotdiffersignificantlywiththelambsex(p>0.05/P=0.40,P=0.34,P=0.62andP=0.59respectivelyattheADGsat10,20,30and90days).(Figure6)4-Evolutionoftheweight«Growthcurve»Liveweight inmaleand female lambsevolvesaccording toa firstorderpolynomial regression curve, theequationsarerespectivelyy=3.092x+0.027andy=3.031x-0.699ofwhich(xisthetimeandyistheweight).Malelambsarestillgrowingfasterthanewelambs(Figure7).Theytendtohavehigherliveweightsthanfemalesfrombirthtoweaning(Figure7).
DISCUSSIONThemeanliveweightoflambsatbirth(4.19kg)issimilartothemeanbirthweightreportedinlambsbornfromcrossesbetweenOuledDjellalfemalesandD'menmales(3.99kg)(Lebiedetal.2015)TheweightresultsforthedifferenttypicalagesfoundinthisstudyareverysimilartothosereportedbyBendiabandDekhili(2012)forlambsofthesamebreed.ThesuperiorityinweightgaininfavorofsimplebirthtypehasalsobeenreportedbyKerfaletal,(2005).CONCLUSIONTheresultsobtainedattheendofthisstudyallowedustoshowthat:-LiveweightandaveragedailygainsofOuledDjellallambsfrombirthtoweaningarecloselyrelatedtolittersize.-Similarly,averysignificantcorrelationwasobservedbetweenmeanweightsattypicalagesandbirthweight.-Thegrowthperformanceofpre-weanedOuledDjellallambsisnotrelatedtothesexofthelamb.-TheseweightandgrowthperformancestestifytothesatisfactorygrowthpotentialoflambsoftheOuledDjellalbreed.Attheendoftheseresults,itappearsthatthe"sizeoflitter"factorhasasignificantinfluenceongrowthperformance,whichmustbetakenintoaccount.Finally,thisworkshouldbesupplementedbytheidentificationandstudyoftheeffectsofotherfactorswhichcaninducethegrowthoflambs,futurebreeders,inotherwordsthefutureoftheOuledDjellalbreed.LITERATUREBENDIABNetDEKHILIM.,(2012).FacteursinfluençantlacroissancedesdansleNordestAlgérien.RevueAgriculture.3-4p.BENCHERIF S., (2011). L'élevage pastoral et la céréaliculture dans la steppe algérienne Evolution et possibilités de développement.Agriculture,economyandpolitics.AgroParisTech.French.269p.
Figure6:Variations inaveragedailygains(g)bysexforOuledDjellallambs.
Figure 7: Evolution of weight by sex inlambsOuledDjellal.
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