an overview of payload quality assurance strategies in

AnOverviewofPayloadQualityAssuranceStrategiesinHERONMk.II:ExaminingtheEffectsofMicrogravityontheGeneEcPathogenicityandFluconazoleMICDrug

ResistanceofCandidaalbicansusingaMicrofluidics-basedNanosatellite

Background&Hypothesis•  Autonomousnanosatelliteshavecreatedopportuni4es

forconduc4ngbiologicalexperimentsinspacewithoutrelyingontheInterna4onalSpaceSta4on1,5-7

•  Themicrogravityenvironmentinlow-Earthorbithasbeenshowntoaltertheexpressionlevelsofgenesinvolvedinpathogenicity,drugresistance,andstressresponseintheopportunis4cfungalpathogen,Candidaalbicans(CA)1

•  Itishypothesizedthatprolongedexposuretomicrogravity—similartothatoflongdura4onspacemissions—increasessuscep4bilitytoinfec4onfromresidentsoftheendogenoushumanmicrobiotaandreducesT-cellac4vity2-4,8,16

•  Par4cularly,NASAhasintroduced:•  ThePharmSat,whichisbeingusedtounderstand

howyeastrespondtoan4fungaldrugsinorbit5,25•  TheGeneSat,whichisbeingusedtoexaminehow

E.colibacteriabehaveinspace(predecessorofGeneBox)6,7

•  Genes:Itishypothesizedthattherewillbeanupregula4onofvirulentgeneexpressioninthelow-Earthorbitmicrogravityenvironment,asquan4fiedusingthefluorescencedetec4onplaTorm8,9

•  MIC:ItishypothesizedthattheMICvaluewillincreaseinlow-earthorbit(LEO),dependingonexposure5,10

•  ThereismuchNASAliteraturethatsupportstheLEOimmunocompromisa4onofastronauts2,11,26

•  Open-sourcemicrobiologyplaTormthatisabletoautonomouslyperformscien4ficexperiements

•  WehavedevelopedmeansofplacingCAinpassivelong-dura4onstoragesuchthatthecellsremaindormantduringthepre-launchopera4ons

•  OurresearchplaTormexaminesgeneexpressionthroughtheuseofgreenfluorescentprotein(GFP)asafusionreporterfor4candidatevirulencefactors:MDR1,HSP90,HWP1andSAP9912,13,18-23

•  On-boardfluorescencesensorswillquan4fyGFPlevelsasameasureofreal-4megeneexpression

•  Changesindrugresistancewillbemeasuredthroughop4caldensitymeasurementsofaminimuminhibitoryconcentra4on(MIC)assayforfluconazole,acommonCAan4fungalmedica4on14

•  Fortheintegrityandsuccessofthisenterprise,mul4pleothervariableswillbemonitoredandcontrolled,includingtemperature,humidity,andpressure

Materials&Methods

QualityAssurance–Biology

Acknowledgments&Correspondence

Discussion&Challenges

References&FurtherReading

PayloadEngineering&PreliminaryScienEficResults

QualityAssurance–EngineeringFigure1&2(é):Theforwardandreverseprimersweresynthesizedusingopen-sourcebioinforma4csso^ware,Benchling;theGFP-gene-NATcasse`ewassynthesizedusinghybridPCR.TheseyeastwerethenplatedonNATmedia,whichonlyallowedthegrowthofthosestrainswiththeintegratedcasse`e.ColonyPCRwasdonea^erthetransforma4ontoensuretheintegra4onofthecasse`eintotheyeastgenome.

Figure3(ì):FluorescencemicroscopyofHWP(NAT-GFP)-CAcells.

1.  CrabbeA,Nielsen-PreissSM,WoolleyCM,BarrilaJ,BuchananK,McCrackenJ,InglisDO,SearlesSC,Nelman-GonzalezMA,O`CM,etal.SpaceflightEnhancesCellAggrega4onandRandomBuddinginCandidaalbicans.PLoSONE2013;8(12):e80677.

2.  Mar4nezEM,YoshidaMC,CandelarioTLT,Hughes-FulfordM.Spaceflightandsimulatedmicrogravitycauseasignificantreduc4onofkeygeneexpressioninearlyT-cellac4va4on.AmJPhysiolRegulIntegrCompPhysiol2015;308(6):480–488.

3.  RitchieLE,TaddeoSS,WeeksBR,LimaF,BloomfieldSA,Azcarate-PerilMA,ZwartSR,SmithSM,TurnerND.SpaceEnvironmentalFactorImpactsuponMurineColonMicrobiotaandMucosalHomeostasis.PLoSONE2015;10(6):e0125792.

4.  RosenzweigJA,AbogundeO,ThomasK,LawalA,NguyenYU,SodipeA,JejelowoO.Spaceflightandmodeledmicrogravityeffectsonmicrobialgrowthandvirulence.ApplMicrobiolBiotechnol2010;85:885-891.

5.  PharmaSat[Internet].2009.MoffetField(CA):NASAAmesResearchCenter;[UpdatedJune12,2009;citedMay1,2017].Availablefrom:h`ps://www.nasa.gov/mission_pages/smallsats/pharmasat/main/index.html.

6.  Gene-Sat-1[Internet].2006.MoffetField(CA):NASAAmesResearchCenter;[UpdatedMarch26,2007;citedMay1,2017].Availablefrom:h`ps://www.nasa.gov/centers/ames/missions/2007/genesat1.html.

7.  GeneBox[Internet].2006.MoffetField(CA):NASAAmesResearchCenter;[UpdatedJuly17,2006;citedMay1,2017].Availablefrom:h`ps://www.nasa.gov/centers/ames/news/releases/2006/06_52AR.html

8.  HorneckG,KlausDM,MancinelliRL.SpaceMicrobiology.MicrobiologyandMolecularBiologyReviews2010;74(1):121-156.9.  HwangSA,CrucianB,SamsC,ActorJK.Post-Spaceflight(STS-135)MouseSplenocytesDemonstrateAlteredAc4va4onProper4esandSurfaceMolecule

Expression.PLoSONE2015;10(5):e0124380.10.  TaylorPW.Impactofspaceflightonbacterialvirulenceandan4bio4csuscep4bility.Infec4onandDrugResistance2015;8:249-262.11. VerhaarAP,HoekstraE,TjanASW,UtomoWK,DeuringJJ,BakkerERM,MuncanV,PeppelenboschMP.Dichotomaleffectofspaceflight-associated

microgravityonstress-ac4vatedproteinkinasesininnateimmunity.NatureScien4ficReports2014;4:5468.12.  BernadisFD,AranciaS,MorelliL,HubeB,SanglardD,SchaferW,CassoneA.EvidencethatMembersoftheSecretoryAspartylProteinaseGeneFamily,in

Par4cularSAP2,AreVirulenceFactorsforCandidaVagini4s.TheJournalofInfec4ousDiseases1999;179(1):201-208.13. AngiolellaL,StringaroAR,BernardisFD,PosteraroB,BonitoM,ToccacieliL,TorosantucciA,ColoneM,SanguinesM,CassoneA,PalamaraAT.Increaseof

VirulenceandItsPhenotypicTraitsinDrug-ResistantStrainsofCandidaalbicans.An4microbialAgentsandChemotherapy2008;52(3):927-936.14.  XieJL,Singh-BabakSD,CowenLE.MinimumInhibitoryConcentra4on(MIC)AssayforAn4fungalDrugs.Bio-Protocol2012;2(20):e252.15.  LeeWB,FuCY,ChangWH,YouHL,WangCH,LeeMS,LeeGB.Amicrofluidicdeviceforan4microbialsuscep4bilitytes4ngbasedonabrothdilu4onmethod.

BiosensorsandBioelectronics2017;87:669-678.16.  CorydonTJ,KoppS,WehlandM,BraunM,Schu`eA,MayerT,HulsingT,OltmannH,SchmitzB,HemmersbachR,GrimmD.Altera4onsofthecytoskeletonin

humancellsinspaceprovedbylife-cellimaging.NatureScien4ficReports2016;6:20043.17.  KimW,TengraFK,YoungZ,ShongJ,Marc-HandN,ChanHK,PanguleRC,ParraM,DordickJS,PlawskyJL,CollinsCH.Spaceflightpromotesbiofilmforma4onby

pseudomonasaeruginosa.PLoSONE2013;8(4):e62437.18. AriyachetC,SolisNV,LiuY,PrasadaraoNV,FillerSG,McBrideAE.SR-LikeRNA-BindingProteinSlr1AffectsCandidaalbicansFilamenta4onandVirulence.

Infec4onandImmunity2013;81(4):1267-1276.19.  BatesS,HallRA,CheethamJ,NeteaMG,MacCallumDM,BrownAJP,OddsFC,GowNAR.RoleoftheCandidaalbicansMNN1genefamilyincellwallstructure

andvirulence.BMSResearchNotes2013;6(1):294-302.20. DesaiC,MavrianosJ,ChauhanN.CandidaalbicansSRR1,aPuta4veTwo-ComponentResponseRegulatorGene,IsRequiredforStressAdapta4on,

Morphogenesis,andVirulence.Eukaryo4cCell2011;10(10):1370-1374.21. Mar4nR,Albrecht-EckardtD,BrunkeS,HubeB,HunnigerK,KurzaiO.ACoreFilamenta4onResponseNetworkinCandidaalbicansIsRestrictedtoEightGenes.

PLoSONE2013;8(3):e58613.22.  TanX,FuchsBB,WangY,ChenW,YuenJG,ChenRB,JayamaniE,AnastassopoulouC,Pukkila-WorleyG,ColemanJJ,MylonakisE.TheRoleofCandidaalbicans

SPT20inFilamenta4on,BiofilmForma4onandPathogenesis.PLoSONE2014;9(4):e94468.23.  XuN,QianK,DongY,ChenY,YuQ,ZhangB,XingL,LiM.NovelroleoftheCandidaalbicansferricreductasegeneCFL1inironacquisi4on,oxida4vestress

tolerance,morphogenesisandvirulence.ResearchinMicrobiology2014;165(3):252-261.24.  RiccoT.IntegratedMicrofluidicSystemsinChallengingEnvironments:BiologicalStudiesinEarthOrbit[InternetPDF].NASAAmesResearchCenter,Moffet

Field,CA.25. O/OREOS[Internet].2010.MoffetField(CA):NASAAmesResearchCenter;[UpdatedNovember19,2010;citedMay1,2017].Availablefrom:h`ps://

www.nasa.gov/mission_pages/smallsats/ooreos/main/index.html.26. Ma4nAC,WangJ-H,KeyhanM,SinghR,BenoitM,ParraMP,PadgenMR,RiccoAJ,etal.Payloadhardwareandexperimentalprotocoldevelopmenttoenable

futuretes4ngoftheeffectofspacemicrogravityontheresistancetogentamicinofuropathogenicEscherichiacolianditsσs-deficientmutant.LifeSciencesinSpaceResearch2017;15(1):1–10.

•  Theauthorsthank:(1)TheCowenLaboratory,DepartmentofMolecularGene4cs,UniversityofToronto;(2)TheFacultyofEngineeringandAppliedSciences,UniversityofToronto;(3)TheUniversityofTorontoAerospaceTeam(UTAT);(4)TheWheelerLaboratory,Ins4tuteofBiomaterialsandBiomedicalEngineering,UniversityofToronto;(5)TheGuentherLaboratory,DepartmentofMechanicalandIndustrialEngineering,UniversityofToronto;(6)DepartmentofPhysics,UniversityofToronto;(7)TheNavarreLaboratory,DepartmentofMolecularGene4cs,UniversityofToronto;(8)TheNASAAmesResearchCentre(ARC),MountainView,CA,USA

•  Thefollowingadvisorsareacknowledged:Dr.LucyXie(1);SangHuKim(1);ErinWong(7);ProfessorChristopherJ.Damaren(UniversityofTorontoIns4tuteforAerospaceStudies–UTIAS);ProfessorJasonHarlow(6);AntonioJ.Ricco(NASAARCandStanfordUniversity)

•  Thefollowingpastmembersareacknowledged:KeenanBurne`(UTIAS),BenMakarchuk,JeffreyOsborne(KeplerCommunica4ons–KC),StephenLau(KC),AlejandroDuque(Dept.ofMolecularGene4cs,UofT),SamanthaMurray(Dept.ofElectricalandComputerEngineering,UofT)

•  Theauthorsstatenoconflictsofinterest,neitherinternallynorexternally•  Correspondingauthor:AvinashN.Mukkala|avinash.mukkala@mail.utoronto.ca,www.utat.ca

•  Recentliteraturehasbeenpublishedaroundtheissuesoflong-termspaceflightandhowitdirectlyaffectstheinnateandadap4veimmunesystem(T-cells,macrophages)2,8,9,11

•  Mul4plereportshavebeenpublishedthatdepictthegene4candpharmacologicalchangesthatmicrobialpopula4onsexperienceinorbit1,8,17

•  Theinherentmul4disciplinarynatureofourprojectmandatesenormousamountsof4melycollabora4onbetweenstudentengineersandbiologists

AvinashN.Mukkala,1,2,*AliHaydaroglu,2,3DylanVogel,2,3LucaTalamo,1,2MariekedeKorte,1,2,+AmnaZulfiqar,1,2,+ElisabethGrande,1,2,+PriyankPurohit,2,4ShimonSmith,1,2BrunoAlmeida,2,3RussellBrown;2,3|KathleenGwozdecky,2,3AdityapalBha4a,2,3ShreyParikh,2,3VictorNechita,2,3ArashNourimand,2,3HaleyBlinn,1,2SiddharthMahendraker,1,2JoannaHon,1,2MitchellAu2,3

QualityAssurance–TeamManagement

1FacultyofArtsandScience,UniversityofToronto,Toronto,Canada;2UniversityofTorontoAerospaceTeam,Toronto,Canada;3FacultyofAppliedScienceandEngineering,UniversityofToronto,Toronto,Canada;4DepartmentofCellandSystemsBiology,UniversityofToronto,Toronto,Canada;+Equalcontribu4on;*Correspondingauthor:ANM–avinash.mukkala@mail.utoronto.ca

•  Tri-annualinternaldesignreviews:industryleaders,professionalengineers,scien4stsandprofessors

•  Annualexternaldesignreviews:CSDC•  Technicalconsulta4onswithNASAforGeneSatand

PharmaSatattheNASAAmesResearchCentre•  StandardCubeSatqualifica4ontes4ng:vibra4on,

thermalandradia4on•  Task:high-fidelityfunc4onality–pre-buildreviews

•  Ensuringtransforma4onefficiencyviaNATpla4ng•  Ensuringpropercasse`eintegra4onviarepeated

colonyPCRassaysandrou4nemicroscopy•  Ensuringsta4s4calrelevancyfromrepeated

measurements:e.g.figure10,20datapointsperwellaveragedwithextremelylowstandarderror

•  Replica4veassurancebyconduc4ngen4reassaysintandemwithoneanother

•  On-groundreproducibilityinexactsamelaboratorycondi4onsforeasierandmorereliablecomparison

•  Biggestchallengeforastudentteamistheabilitytoretainmembershipandknowledgeacrossyears

•  Nofull-4meemployees;allvolunteer-based;turnover•  Self-createteststomeasureQAforprocesses/

applica4onsthatareunderstudiedinliterature•  Systemsintegra4onknowledgeispersonnel-limited•  Approachingtheknowledgereten4onissuebycrea4ng

updateddocumenta4onforallsubsystems

Figure4&5(çè):(4)Side-viewcrosssec4onofthefluidicscard,PCB,payloadstructuredesign.1microfluidicschipsandwichedby2PCBs,andencompassedbyheatersandconductors.Thecurrentdesignhasatotal33wellsforbothgene4candpharmacologicalexperimenta4on.(5)Full-viewcrosssec4onofthefluidicscardstructurewithintheprimarypressurevesselstructure.Theemptyspacesaretobefilledbyfluidactua4onmicro-machineryforon-boarddilu4ons,pumpingandwastemanagement.

Figure6,7&8(é):Func4onaldiagramsofthethreesensormodes:(6)sta4cmode,(7)fluorescencemodeand,(8)op4caldensitymode.(7)The470nmexcita4onLEDhitsGFPwithinCAsamples.GFPwillemitgreenlightat~508nm,pickedupbyourphotodiodesensors.The495nmlongpassfilterwillexcludeexcita4onwavelengthlight.(8)600nmlightwillbeemi`edfromtheLEDPCB,andhits,sca`ersthenpassesthroughsampleàOD600.

Figure9(é):~5-month,fullhumidity,stasistestresults.

Figure10(é):Ini4alproof-of-conceptGFP-CA-HSP90lightdetec4onin3dilu4ons.

Figure11(è):Sta4cdiagramcrosssec4onofmicrofluidicschiparchitecture.Solenoidvalvespreventprematuregrowthmedia(YPD)andstasismedia(H2O)mixingbeforeexperimentstartcommandsaresentviatelemetry.Membranesallowcellexclusionandgasexchangewithpressurevesselairreservoir.YPDandFLCarestoredtogetherini4allybeforeactua4on.Pistonsystemcreatesnecessarypressuretoforcestasismediaout,whilekeepingcellsin.A^erwhich,growthmedia(YPD)withvariousFLCdilu4onsarepumpedin.

Figure12(î):Payloadinstrumenta4onhardwarefunc4onalarchitecture.CANcommandsaresentfromOBCtopayloadSSMPCB.ThepayloadSSMPCBcontrols:(1)MFactua4on,(2)MOSFETheaterdrivers,(3)thermistorcircuitryforMFchiptemperaturemodula4on.Italsohousesvariousenvironmentalsensors:PCBtemperature,humidity,pressureandaccelerometer.ThepayloadSSMcommunicateswithsensorPCBs,LEDPCBsviaSPIcommandprotocols.ThesensorPCBsmeasurephotocurrentsontheorderof1pA;designedamplifierchaintoachievebe`ersatura4onofsignal.Measurementstakenevery30minover48hours,allreadingstakenintriplicates,fromatotalof15genewellsand18drugresistancewells.

NASAAAQ[2017-10-13]:Huntsville,AL,USA