Itiswithoutdoubtthattestingplaysapivotalrolethroughoutthewholeofthevinificationprocess.ToproducethebestpossIBLequalitywineandtominimiseprocessproblemssuchas“stuck”fermentationortroublesomeinfections,itisnowrecognisedthatifpossibletestingshouldbeginpriortoharvestingofthegrapesandcontinuethroughtobottling.TrADItionalmethodsofwineanalysisareoftenexpensive,timeconsuming,requireeitherelaborateequipmentorspecialistexpertiseandfrequentlylackaccuracy.However,enzymaticbio-analysisenablestheaccuratemeasurementofthevastmajorityofanalytesofinteresttothewinemaker,usingjustonepieceofapparatus,thespectrophotometer(seepreviousissueNo.116foradetailedtechnicalreview).Grapejuiceandwineareamenabletoenzymatictestingasbeingliquidstheyarehomogenous,easytomanipulate,andcangenerallybeanalysedwithoutanysamplepreparation.

ManyoftheenzymatictestkitsareofficialmethodsofprestigiousorganisationssuchastheAssociationofOfficialAnalyticalChemicals(AOAC)andtheAmericanAssociationofCerealChemists(AACC)inresponsetotheinterestfromoenologists.Megazymedecidedtouseitslonghistoryofenzymaticbio-analysistomakeasignificantcontributiontothewineindustry,bythedevelopmentofarangeofadvancedenzymatictestkits.Thistaskhasnowbeensuccessfullycompletedthroughthestrategicandcomprehensiveprocessofidentifyinglimitationsofexistingenzymaticbio-analysistestkitswheretheyoccurred,andthenusingadvancedtechniques,suchasmolecularBIOLOGy(photo1),torapidlyovercomethem.Noveltestkitshavealsobeendevelopedforanalytesofemerginginteresttotheoenologist,suchasyeastavailablenitrogen(YAN;seepages2-3ofissue117article),orwherepreviouslyenzymesweresimplyeithernotavailable,orweretooexpensivetoemploy,suchasforD-mannitolanalysis.

Increasingtheconductivityoftheelectrolytesusedinmicrobialelectrochemicalsystemsisanessentialprerequisitetothelarge-scalesuccessofthesetechnologies.Microbialbioanodesformedfromasaltmarshinoculumunderconstantacetatefeedinggeneratedupto85A•m^-2inmediacontaining776mMNaCl(45g•L^-1,1.5timesthesalinityofseawater).Thesevalueswerethehighestsalinitiesacceptedbyamicrobialanodesofarandthehighestcurrentdensitiesreportedwithfeltgraphiteelectrodes.

BackgroundandObjectives:Pathogenreductiontechnologies(PRT)forplateletsarenowcompatiblewithbothplasmaandplateletadditivesolutions(PAS).TheaimofthisstudywastoexaminetheeffectofPRTontheplateletstoragelesion,inthepresenceofPASwithlowplasmacarryover.MaterialsandMethods:PRT-treated(Mirasol)anduntreatedbuffycoat-derivedplateletconcentratespreparedin28%plasma/PAS-IIIMwereevaluatedusinginvitrocellqualityparametersondays1,2,5,and7post-collection.Results:Atday5,therewerenosignificantdifferencesbetweencontrolandPRTtreatedplateletsforswirl,viABIlity,pO₂,pCO₂,meanplateletvolumeandadenosinediphosphate-inducedaggregation.PRTtreatmentdidnotaffectthefunctionalintegrityofthemitochondria.However,PRTresultedinadecreaseinpHandenhancementofplateletglycolysisandactivation,evidencedbyincreasedglucoseconsumptionandlactateproductionrates,increasedexpressionofCD62P,CD63,annexinVstainingandincreasedsecretionofcytokines(P<0•05).=""hypotonic=""shock=""response=""and=""aggregation=""in=""response=""to=""collagen=""were=""also=""significantly=""reduced=""in=""prt=""treated=""platelets="">P<0•05).="">Conclusion:DespitetheobserveddifferencesinplateletmetabolismandactivationobservedfollowingPRTtreatmentinPASandlowplasmacarryover,theresultssuggestthattreatmentandstorageofplateletsinPASisnomoredetrimentaltoplateletsthantreatmentandstorageinplasma.

Background:TheINTERCEPTBloodSystemusesamotosalen-HClandUVAlighttocross-linkDNAandRNA,therebyinhibitingpathogenreplication.Althoughpreviousstudieshaveshownthatthistreatmentaltersinvitroplateletquality,moststudieshaveassessedapheresisplateletsorplateletspooledfrom5or6donors.InAustralia,plateletsarepreparedusingbuffy-coatsfrom4donors,withSSP+andhavelowerplasmacarryoverthanrecommendedbythemanufacturer(32–47%).Assuch,itiscurrentlyunknownwhethertheseplateletconcentratesaresuitableforINTERCEPTtreatment.Materialsandmethods:Plateletconcentrateswerepreparedbypoolingfourbuffy-coatswithSSP+,resultingin30%plasmacarryover.Twoplateletunitswerepooledandsplittogeneratepairedunits,withoneunittreatedwiththeINTERCEPTSystem(n = 6),whilsttheotherremaineduntreated(n  = 6).Allunitswerestoredforsevendaysat22°Cwithagitation.Results:INTERCEPTtreatmentresultedin10•4 ± 4•3%lossofplatelets,butdidnotsignificantlyaffectthefunctionalintegrityofmitochondria.INTERCEPT-treatedplateletsdemonstratedadecreasedpH,acceleratedlactateproductionandglucoseconsumption,aswellashighersurfaceexpressionandincreasedsecretionofP-selectinandreducedcollagen-inducedaggregation.Thesechangeswereparticularlyevidentfromday5ofstorage.Conclusion:TheobservedincreaseinplateletglycolysisfollowingINTERCEPTtreatmentisconsistentwithpreviousliteraturereports.Importantly,theinvitrochangeswerelessmarkedthanpreviouslyreportedindicatingthattheplateletssuspendedinSSP+withreducedplasmacarryoverareofsuitableinvitroqualityfollowingINTERCEPTtreatmentandstorage.

Theopportunityforamutationtoinvadeapopulationcandramaticallyvarydependingonthecontextinwhichthismutationoccurs.Suchcontextdependenceisdifficulttodocumentasitrequirestheabilitytomeasurehowamutationaffectsphenotypesandfitnessandtomanipulatethecontextinwhichthemutationoccurs.Weidentifiedamutationinageneencodingaglobalregulatorinoneoftwoecotypesthatdivergedfromacommonancestorduring1200generationsofexperimentalevolution.Wereplacedtheancestralallelebythemutantallele,andviceversa,inseveralclonesisolatedduringthetimecourseoftheevolutionexperiment,andcomparedthephenotypeandfitnessofclonesisogenicexceptforthefocalmutation.Weshowthatthefitnessandphenotypeofthemutationarestronglyaffectedbyepistaticinteractionsbetweengenesinthesamegenome,aswellasbyfrequencydependentselectionresultingfrombioticinteractionsbetweenindividualsinthesamepopulation.Weconcludethatamongstthereplicatepopulationinwhichitspread,themutationweidentifiedisonlyadaptivewhenoccurringinspecificgenomesandcompetingwithspecificindividuals.Thisstudythusdemonstratesthattheopportunityforanadaptivemutationtospreadinanevolutionarylineagecanonlybeunderstoodinthelightofitsgenomicandcompetitiveenvironments.

Manybacteriaarenaturallycapableofaccumulatingbiopolyesterscomposedof3-hydroxyfattyacidsasintracellularinclusions,whichserveasstoragegranules.Recently,theseinclusionshavebeenconsideredasnano-/microbeadswithsurface-attachedproteins,whichcanbeengineeredtodisplayvariousprotein-basedfunctionsthataresuitableforbiotechnologicalandbiomedicalapplications.Inthisstudy,thefood-grade,generally-regarded-as-safegram-positiveorganismLactococcuslactiswasengineeredtorecombinantlyproducethebiopolyesterpoly(3-hydroxybutyrate)andtherespectiveintracellularinclusions.Thecodon-optimizedpolyhydroxybutyratebiosynthesisoperonphaCABfromCupriavidusnecatorwasexpressedusingthenisin-controlledgeneexpressionsystem.RecombinantL.lactisaccumulatedupto6%(wt/wt)poly(3-hydroxybutyrate)ofcellulardryweight.Poly(3-hydroxybutyrate)granuleswereisolatedandanalyzedwithrespecttoboundproteinsusingbiochemicalmethodsandwithrespecttoshape/sizeusingtransmissionelectronmicroscopy.TheimmunoglobulinG(IgG)bindingZZdomainofStaphylococcusaureusproteinAwaschosenasanexemplaryfunctionalitytobedisplayedatthegranulesurfacebyfusingittotheNterminusofthegranule-associatedpoly(3-hydroxybutyrate)synthase.Thepresenceofthefusionproteinatthesurfaceofisolatedgranuleswasconfirmedbypeptidefingerprintingusingmatrix-assistedlaserdesorptionionization-timeofflight(massspectrometry).ThefunctionalityoftheZZdomain-displayinggranuleswasdemonstratedbyenzyme-linkedimmunosorbentassayandIgGaffinitypurification.Inbothassays,theZZbeadsfromrecombinantL.lactisperformedatleastequallytoZZbeadsfromEscherichiacoli.Overall,inthisstudyitwasshownthatrecombinantL.lactiscanbeusedtomanufactureendotoxin-freepoly(3-hydroxybutyrate)beadswithsurfacefunctionalitiesthataresuitableforbiomedicalapplications.

Pectinisamajorcomponentoftheprimarycellwallofhigherplants.SomegalacturonylresiduesinthebackboneofpectinaceouspolysaccharidesareoftenO-acetylatedattheC-2orC-3position,andtheresultingacetylesterschangedynamicallyduringthegrowthanddevelopmentofplants.Theprocessesinvolvebothenzymaticacetylationanddeacetylation.Throughgenomicsequenceanalysis,weidentifiedapectinacetylesterase(PAE1)fromblackcottonwood(Populustrichocarpa).RecombinantPtPAE1exhibitedpreferentialactivityinreleasingtheacetatemoietyfromsugarbeet(Betavulgaris)andpotato(Solanumtuberosum)pectininvitro.OverexpressingPtPAE1intobacco(Nicotianatabacum)decreasedthelevelofacetylestersofpectinbutnotofxylan.Deacetylationengendereddifferentialchangesinthecompositionand/orstructureofcellwallpolysaccharidesthatsubsequentlyimpairedthecellularelongationoffloralstylesandfilaments,thegerminationofpollengrains,andthegrowthofpollentubes.Consequently,plantsoverexpressingPAE1exhibitedseveremalesterility.FurThermore,incontrasttotheconventionalview,PAE1-mediateddeacetylationsubstantiallyloweredthedigestibilityofpectin.Ourdatasuggeststhatpectinacetylesterasefunctionsasanimportantstructuralregulatorinplantsbymodulatingtheprecisestatusofpectinacetylationtoaffecttheremodelingandphysiochemicalpropertiesofthecellwall"spolysaccharides,therebyaffectingcellextensibility.

Gardencompostleachatewasusedtoformmicrobialbioanodesunderpolarizationat−0.4,−0.2and+0.1V/SCE.Currentdensitieswere6.3and8.9Am^-2onaverageat−0.4and+0.1V/SCErespectively,withacetate10mM.Thecatalyticcyclicvoltammetry(CV)showedsimilarelectrochemicalcharacteristicsforallbioanodesandindicatedthatthelowercurrentsrecordedat−0.4V/SCEwereduetotheslowerinterfacialelectrontransferrateatthispotential,consistentlywithconventionalelectrochemicalkinetics.RNA-andDNA-basedDGGEevidencedthatthethreedominantbacterialgroupsGeobacter,AnaerophagaandPelobacterwereidenticalforallbioanodesanddidnotdependonthepolarizationpotential.Onlynon-turnoverCVsshoweddifferencesintheredoxequipmentofthebiofilms,thehighestpotentialpromotingmultipleelectrontransferpathways.Thisfirstdescriptionofapotential-independentelectroactivemicrobialcommunityopensuppromisingProspectsforthedesignofstablebioanodesformicrobialfuelcells.

Microalgaearereceivingincreasingattentionasalternativeproductionsystemsforrenewableenergysuchasbiofuel.ThephotosyntheticalgaChlamydomonasreinhardtiiiswidelyrecognizedasthemodelsystemtostudyallaspectsofalgalphysiology,includingthemolecularmechanismsunderlyingtheaccumulationofstarchandtriacylglycerol(TAG),whicharetheprecursorsofbiofuel.Allofthesepathwaysnotonlyrequireacarbon(C)supplybutalsoarestronglydependentonasourceofnitrogen(N)tosustainoptimalgrowthrateandbiomassproduction.InordertogainabetterunderstandingoftheregulationofCandNmetabolismsandtheaccumulationofstoragecarbohydrates,theeffectofdifferentNsources(NH₄NO₃andNH₄⁺)onprimarymetabolismusingvariousmutantsimpairedineitherNIA1,NIT2orbothlociwasperformedbymetabolicanalyses.Thedatademonstratedthat,usingNH₄NO₃,nia1straindisplayedthemoststrikingphenotype,includinganinhibitionofgrowth,accumulationofintracellularnitrate,andstrongstarchandTAGaccumulation.ThemeasurementsofthedifferentCandNintermediatelevels(amino,organic,andfattyacids),togetherwiththedeterminationofacetateandNH₄⁺remaininginthemedium,clearlyexcludedthehypothesisofaslowerNH₄⁺andacetateassimilationinthismutantinthepresenceofNH₄NO₃.TheresultsprovideevidenceoftheimplicationofintracellularnitrateandNIT2inthecontrolofCpartitioningintodifferentstoragecarbohydratesundermixotrophicconditionsinChlamydomonas.Theunderlyingmechanismsandimplicationsforstrategiestoincreasebiomassyieldandstorageproductcompositioninoleaginousalgaearediscussed.

Mostoceanographicinstrumentsontheseafloorhavenoconnectionswiththesurfaceandthereforehavetorunonbatteriesandstoredatauntilrecovery.Todemonstrateadevelopingtechnology,sensorsandacousticmodemswerepoweredwithenergyharvestedfromtheseafloor,anddatawererelayedacousticallyinnear–realtimetotheMontereyAcceleratedResearchSystem(MARS)observatoryinMontereyBay,California,andtosurfaceresearchvessels.MARSisacabledobservatoryindeepwater(~890m)attheedgeofMontereyCanyon.AnacousticmodemwasattachedtotheMARSnodeandconfiguredtosendoutcommandsto,andrelaydatareceivedfrom,remotemodems.Twobenthicmicrobialfuelcells(BMFCs)positionedapproximately0.5kmawayfromMARSsuppliedpowertotheremotemodemsandsensors.Attheirpeakperformance,theseBMFCsproducedcontinuouspowerdensitiesof~35mWm^-2(footprintarea).Themodemsutilizedinthisstudycontainedanintegratedpowermanagementplatform(PMP)designedtomanageandstoretheelectricalenergygeneratedbyeachBMFCandtorecordBMFCperformanceparametersandsensordataonanhourlybasis.Temperatureandeitheroxygenorconductivitysensorswerechosenbecauseoftheircommonuseandenvironmentalrelevance.AcousticallytransmitteddatarecordsshowthattheBMFCsrenewedenergystoresandthattheoceanographicsensorsmeasureddissolvedoxygen,temperature,andconductivityreliablythroughouttheoperationallifeofeachBMFCsystem(~6months).Thesesystemsremainedinplaceformorethan12months.

Thedevelopmentofeconomically-efficientmicrobialelectrochemicaltechnologiesremainshinderedbythelowionicconductivityoftheculturemediausedastheelectrolyte.Toovercomethisdrawback,halotolerantbioanodesweredesignedwithsaltmarshsedimentusedastheinoculuminelectrolytescontainingNaClat30or45 g/L(ionicconductivity7.0or10.4 S·m^-1).Thebioanodeswereformedatfourdifferentpotentials− 0.4,− 0.2,0.0and0.2 V/SCEtoidentifytheeffectontheelectrochemicalkineticparameters,thebiofilmstructuresandthecompositionofthemicrobialcommunities.Thebioanodesformedat− 0.4 V/SCEwerelargelydominatedbyMarinobacterspp.Voltammetryshowedthattheyprovidedhighercurrentsthantheotherbioanodesintherangeoflowpotentials,butthemaximumcurrentswerelimitedbythepoorsurfacecolonization.Thebioanodesformedat− 0.2,0.0and0.2 V/SCEshowedsimilarratiosofMarinobacterandDesulfuromonasspp.andhighervaluesofthemaximumcurrentdensity.Thecombinedanalysisofkineticparameters,biofilmstructureandbiofilmcompositionshowedthatMarinobacterspp.,whichensuredahigherelectrontransferrate,werepromisingspeciesforthedesignofhalotolerantbioanodes.ThechallengeisnowtoovercomeitslimitedsurfacecolonizationintheabsenceofDesulfuromonasspp.

Carbohydrateresponsiveelementbindingprotein(ChREBP)iscentralfordenovofattyacidsynthesisunderphysiologicalconditionsandinthecontextofnonalcoholicfattyliverdisease.Weexploreditscontributiontoalcohol-inducedsteatosisinamousemodelofbingedrinkingasacuteethanol(EtOH)intoxicationhasbecomeanalarminghealthproblem.Within6hours,ChREBPacetylationanditsrecruitmentontotargetgenepromoterswereincreasedinliverofEtOH-fedmice.AcetylationofChREBPwasdependentonalcoholmetabolismbecauseinhibitionofalcoholdehydrogenase(ADH)activitybluntedChREBPEtOH-inducedacetylationinmousehepatocytes.Transfectionofanacetylation-defectivemutantofChREBP(ChREBP^K672A)inHepG2cellsimpairedthestimulatoryeffectofEtOHonChREBPactivity.Importantly,ChREBPsilencingintheliverofEtOH-fedmicepreventedalcohol-inducedtriglycerideaccumulationthroughaninhibitionofthelipogenicpathwaybutalsoled,unexpectedly,tohypothermia,increasedbloodacetaldehydeconcentrations,andenhancedlethality.ThisphenotypewasassociatedwithimpairedhepaticEtOHmetabolismasaconsequenceofreducedADHactivity.WhiletheexpressionandactivityoftheNAD⁺dependentdeacetylasesirtuin1,aChREBP-negativetarget,weredown-regulatedintheliverofalcohol-fedmice,theywererestoredtocontrollevelsuponChREBPsilencing.Inturn,ADHacetylationwasreduced,suggestingthatChREBPregulatesEtOHmetabolismandADHactivitythroughitsdirectcontrolofsirtuin1expression.Indeed,whensirtuin1activitywasrescuedbyresveratrolpretreatmentinEtOH-treatedhepatocytes,asignificantdecreaseinADHproteincontentand/oracetylationwasobserved.Conclusion:ourstudydescribesanovelroleforChREBPinEtOHmetabolismandunravelsitsprotectiveeffectagainstsevereintoxicationinresponsetobingedrinking.

Theobjectivewastoreplacesyntheticmediumbywastewaterasastrategytodesignlow-costscalablebioanodes.Theadditionofactivatedsludgewasnecessarytoformprimarybioanodesthatwerethenusedastheinoculumtoformthesecondarybioanodes.Bioanodesformedinsyntheticmediumwithacetate10 mMprovidedcurrentdensitiesof21.9 ± 2.1 A/m²,whilebioanodesformedinwastewatergave10.3 ± 0.1 A/m².Thedifferencewasexplainedintermsofbiofilmstructure,electrochemicalkineticsandredoxchargecontentofthebiofilms.Inbothmedia,currentdensitieswerestraightforwardlycorrelatedwiththebiofilmenrichmentinGeobacteraceaebut,insidethisfamily,GeobactersulfurreducensandanunculturedGeobactersp.weredominantinthesyntheticmedium,whilegrowthofanotherGeobactersp.wasfavouredinwastewater.Finally,theprimary/secondaryproceduresucceededindesigningbioanodestotreatfoodwastesbyusingwastewaterasdilutionmedium,withcurrentdensitiesof7 ± 1.1 A/m².

Theperformanceofmicrobialfuelcellsandotherrelatedmicrobialelectrochemicalprocessesisseentodeteriorateseverelywhentheyarescaledup.Thiscrucialproblemisaddressedherebycomparingthekineticsofmicrobialanodeswithprojectedsurfaceareasof9and50cm²underwell-controlledelectrochemicalconditions.Themicrobialanodekineticswerecharacterizedbylowscanratevoltammetry.The9-cm²anodesshowedNernstianbehaviour,whilethe50-cm²anodesshowedsignificantlylowerperformance.Thedistributionoftheelectrostaticpotentialintheexperimentalset-upwasmodellednumerically.Themodelpredictedthegeneraltrendofthevoltammetrycurvesrecordedwiththe50-cm²anodeswell,showingthatpartoftheperformancedeteriorationwasduetoohmicdropandtonon-uniformityofthelocalpotentialovertheanodesurface.Furthermore,thebiofilmpresentedslightlydifferentelectrochemicalcharacteristicswhengrownonthe9-cm²or50-cm²anodes,andthedifferenceinlocalpotentialoverthe50-cm²anodesinducedspatialheterogeneityinbiofilmdevelopment.Theeffectoflocalpotentialonbiofilmcharacteristicswasanadditionalcauseofthelowerperformanceobtainedwiththe50-cm²anodes.Inthecurrentstateoftheart,thesoundestwaytodesignlarge-sizedmicrobialanodesistoadoptthedualmainaimofminimizingtheohmicdropwhilekeepingthemostuniformpossiblepotentialovertheelectrodesurface.Modellingpotentialdistributioninsidethereactorshouldmakeanessentialcontributiontothis.