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An Overview of Payload Quality Assurance Strategies in HERON Mk. II: Examining the Effects of Microgravity on the GeneEc Pathogenicity and Fluconazole MIC Drug Resistance of Candida albicans using a Microfluidics-based Nanosatellite Background & Hypothesis • Autonomous nanosatellites have created opportuni4es for conduc4ng biological experiments in space without relying on the Interna4onal Space Sta4on 1,5-7 • The microgravity environment in low-Earth orbit has been shown to alter the expression levels of genes involved in pathogenicity, drug resistance, and stress response in the opportunis4c fungal pathogen, Candida albicans (CA) 1 • It is hypothesized that prolonged exposure to microgravity—similar to that of long dura4on space missions—increases suscep4bility to infec4on from residents of the endogenous human microbiota and reduces T-cell ac4vity 2-4,8,16 • Par4cularly, NASA has introduced: • The PharmSat, which is being used to understand how yeast respond to an4fungal drugs in orbit 5,25 • The GeneSat, which is being used to examine how E. coli bacteria behave in space (predecessor of GeneBox) 6,7 • Genes: It is hypothesized that there will be an upregula4on of virulent gene expression in the low- Earth orbit microgravity environment, as quan4fied using the fluorescence detec4on plaTorm 8,9 • MIC: It is hypothesized that the MIC value will increase in low-earth orbit (LEO), depending on exposure 5,10 • There is much NASA literature that supports the LEO immunocompromisa4on of astronauts 2,11,26 • Open-source microbiology plaTorm that is able to autonomously perform scien4fic experiements • We have developed means of placing CA in passive long-dura4on storage such that the cells remain dormant during the pre-launch opera4ons • Our research plaTorm examines gene expression through the use of green fluorescent protein (GFP) as a fusion reporter for 4 candidate virulence factors: MDR1, HSP90, HWP1 and SAP99 12,13,18-23 • On-board fluorescence sensors will quan4fy GFP levels as a measure of real-4me gene expression • Changes in drug resistance will be measured through op4cal density measurements of a minimum inhibitory concentra4on (MIC) assay for fluconazole, a common CA an4fungal medica4on 14 • For the integrity and success of this enterprise, mul4ple other variables will be monitored and controlled, including temperature, humidity, and pressure Materials & Methods Quality Assurance – Biology Acknowledgments & Correspondence Discussion & Challenges References & Further Reading Payload Engineering & Preliminary ScienEfic Results Quality Assurance – Engineering Figure 1 & 2 (é) : The forward and reverse primers were synthesized using open-source bioinforma4cs so^ware, Benchling; the GFP-gene- NAT casse‘e was synthesized using hybrid PCR. These yeast were then plated on NAT media, which only allowed the growth of those strains with the integrated casse‘e. Colony PCR was done a^er the transforma4on to ensure the integra4on of the casse‘e into the yeast genome. Figure 3 (ì) : Fluorescence microscopy of HWP(NAT-GFP)-CA cells. 1. Crabbe A, Nielsen-Preiss SM, Woolley CM, Barrila J, Buchanan K, McCracken J, Inglis DO, Searles SC, Nelman-Gonzalez MA, O‘ CM, et al. Spaceflight Enhances Cell Aggrega4on and Random Budding in Candida albicans. PLoS ONE 2013; 8(12): e80677. 2. Mar4nez EM, Yoshida MC, Candelario TLT, Hughes-Fulford M. Spaceflight and simulated microgravity cause a significant reduc4on of key gene expression in early T-cell ac4va4on. Am J Physiol Regul Integr Comp Physiol 2015; 308(6): 480–488. 3. Ritchie LE, Taddeo SS, Weeks BR, Lima F, Bloomfield SA, Azcarate-Peril MA, Zwart SR, Smith SM, Turner ND. Space Environmental Factor Impacts upon Murine Colon Microbiota and Mucosal Homeostasis. PLoS ONE 2015; 10(6): e0125792. 4. Rosenzweig JA, Abogunde O, Thomas K, Lawal A, Nguyen YU, Sodipe A, Jejelowo O. Spaceflight and modeled microgravity effects on microbial growth and virulence. Appl Microbiol Biotechnol 2010; 85: 885-891. 5. PharmaSat [Internet]. 2009. Moffet Field (CA): NASA Ames Research Center; [Updated June 12, 2009; cited May 1, 2017]. Available from: h‘ps:// www.nasa.gov/mission_pages/smallsats/pharmasat/main/index.html. 6. Gene-Sat-1 [Internet]. 2006. Moffet Field (CA): NASA Ames Research Center; [Updated March 26, 2007; cited May 1, 2017]. Available from: h‘ps:// www.nasa.gov/centers/ames/missions/2007/genesat1.html. 7. GeneBox [Internet]. 2006. Moffet Field (CA): NASA Ames Research Center; [Updated July 17, 2006; cited May 1, 2017]. Available from: h‘ps://www.nasa.gov/ centers/ames/news/releases/2006/06_52AR.html 8. Horneck G, Klaus DM, Mancinelli RL. Space Microbiology. Microbiology and Molecular Biology Reviews 2010; 74(1): 121-156. 9. Hwang SA, Crucian B, Sams C, Actor JK. Post-Spaceflight (STS-135) Mouse Splenocytes Demonstrate Altered Ac4va4on Proper4es and Surface Molecule Expression. PLoS ONE 2015; 10(5): e0124380. 10. Taylor PW. Impact of space flight on bacterial virulence and an4bio4c suscep4bility. Infec4on and Drug Resistance 2015; 8: 249-262. 11. Verhaar AP, Hoekstra E, Tjan ASW, Utomo WK, Deuring JJ, Bakker ERM, Muncan V, Peppelenbosch MP. Dichotomal effect of spaceflight-associated microgravity on stress-ac4vated protein kinases in innate immunity. Nature Scien4fic Reports 2014; 4: 5468. 12. Bernadis FD, Arancia S, Morelli L, Hube B, Sanglard D, Schafer W, Cassone A. Evidence that Members of the Secretory Aspartyl Proteinase Gene Family, in Par4cular SAP2, Are Virulence Factors for Candida Vagini4s. The Journal of Infec4ous Diseases 1999; 179(1): 201-208. 13. Angiolella L, Stringaro AR, Bernardis FD, Posteraro B, Bonito M, Toccacieli L, Torosantucci A, Colone M, Sanguines M, Cassone A, Palamara AT. Increase of Virulence and Its Phenotypic Traits in Drug-Resistant Strains of Candida albicans. An4microbial Agents and Chemotherapy 2008; 52(3): 927-936. 14. Xie JL, Singh-Babak SD, Cowen LE. Minimum Inhibitory Concentra4on (MIC) Assay for An4fungal Drugs. Bio-Protocol 2012; 2(20): e252. 15. Lee WB, Fu CY, Chang WH, You HL, Wang CH, Lee MS, Lee GB. A microfluidic device for an4microbial suscep4bility tes4ng based on a broth dilu4on method. Biosensors and Bioelectronics 2017; 87: 669-678. 16. Corydon TJ, Kopp S, Wehland M, Braun M, Schu‘e A, Mayer T, Hulsing T, Oltmann H, Schmitz B, Hemmersbach R, Grimm D. Altera4ons of the cytoskeleton in human cells in space proved by life-cell imaging. Nature Scien4fic Reports 2016; 6: 20043. 17. Kim W, Tengra FK, Young Z, Shong J, Marc-Hand N, Chan HK, Pangule RC, Parra M, Dordick JS, Plawsky JL, Collins CH. Spaceflight promotes biofilm forma4on by pseudomonas aeruginosa. PLoS ONE 2013; 8(4): e62437. 18. Ariyachet C, Solis NV, Liu Y, Prasadarao NV, Filler SG, McBride AE. SR-Like RNA-Binding Protein Slr1 Affects Candida albicans Filamenta4on and Virulence. Infec4on and Immunity 2013; 81(4): 1267-1276. 19. Bates S, Hall RA, Cheetham J, Netea MG, MacCallum DM, Brown AJP, Odds FC, Gow NAR. Role of the Candida albicans MNN1 gene family in cell wall structure and virulence. BMS Research Notes 2013; 6(1): 294-302. 20. Desai C, Mavrianos J, Chauhan N. Candida albicans SRR1, a Puta4ve Two-Component Response Regulator Gene, Is Required for Stress Adapta4on, Morphogenesis, and Virulence. Eukaryo4c Cell 2011; 10(10): 1370-1374. 21. Mar4n R, Albrecht-Eckardt D, Brunke S, Hube B, Hunniger K, Kurzai O. A Core Filamenta4on Response Network in Candida albicans Is Restricted to Eight Genes. PLoS ONE 2013; 8(3): e58613. 22. Tan X, Fuchs BB, Wang Y, Chen W, Yuen JG, Chen RB, Jayamani E, Anastassopoulou C, Pukkila-Worley G, Coleman JJ, Mylonakis E. The Role of Candida albicans SPT20 in Filamenta4on, Biofilm Forma4on and Pathogenesis. PLoS ONE 2014; 9(4): e94468. 23. Xu N, Qian K, Dong Y, Chen Y, Yu Q, Zhang B, Xing L, Li M. Novel role of the Candida albicans ferric reductase gene CFL1 in iron acquisi4on, oxida4ve stress tolerance, morphogenesis and virulence. Research in Microbiology 2014; 165(3): 252-261. 24. Ricco T. Integrated Microfluidic Systems in Challenging Environments: Biological Studies in Earth Orbit [Internet PDF]. NASA Ames Research Center, Moffet Field, CA. 25. O/OREOS [Internet]. 2010. Moffet Field (CA): NASA Ames Research Center; [Updated November 19, 2010; cited May 1, 2017]. Available from: h‘ps:// www.nasa.gov/mission_pages/smallsats/ooreos/main/index.html. 26. Ma4n AC, Wang J-H, Keyhan M, Singh R, Benoit M, Parra MP, Padgen MR, Ricco AJ, et al. Payload hardware and experimental protocol development to enable future tes4ng of the effect of space microgravity on the resistance to gentamicin of uropathogenic Escherichia coli and its σs-deficient mutant. Life Sciences in Space Research 2017; 15(1): 1–10. • The authors thank: (1) The Cowen Laboratory, Department of Molecular Gene4cs, University of Toronto; (2) The Faculty of Engineering and Applied Sciences, University of Toronto; (3) The University of Toronto Aerospace Team (UTAT); (4) The Wheeler Laboratory, Ins4tute of Biomaterials and Biomedical Engineering, University of Toronto; (5) The Guenther Laboratory, Department of Mechanical and Industrial Engineering, University of Toronto; (6) Department of Physics, University of Toronto; (7) The Navarre Laboratory, Department of Molecular Gene4cs, University of Toronto; (8) The NASA Ames Research Centre (ARC), Mountain View, CA, USA • The following advisors are acknowledged: Dr. Lucy Xie (1); Sang Hu Kim (1); Erin Wong (7); Professor Christopher J. Damaren (University of Toronto Ins4tute for Aerospace Studies–UTIAS); Professor Jason Harlow (6); Antonio J. Ricco (NASA ARC and Stanford University) • The following past members are acknowledged: Keenan Burne‘ (UTIAS), Ben Makarchuk, Jeffrey Osborne (Kepler Communica4ons–KC), Stephen Lau (KC), Alejandro Duque (Dept. of Molecular Gene4cs, UofT), Samantha Murray (Dept. of Electrical and Computer Engineering, UofT) • The authors state no conflicts of interest, neither internally nor externally • Corresponding author: Avinash N. Mukkala | [email protected], www.utat.ca • Recent literature has been published around the issues of long-term spaceflight and how it directly affects the innate and adap4ve immune system (T-cells, macrophages) 2,8,9,11 • Mul4ple reports have been published that depict the gene4c and pharmacological changes that microbial popula4ons experience in orbit 1,8,17 • The inherent mul4disciplinary nature of our project mandates enormous amounts of 4mely collabora4on between student engineers and biologists Avinash N. Mukkala , 1,2, * Ali Haydaroglu, 2,3 Dylan Vogel, 2,3 Luca Talamo, 1,2 Marieke de Korte, 1,2,+ Amna Zulfiqar, 1,2,+ Elisabeth Grande, 1,2,+ Priyank Purohit, 2,4 Shimon Smith, 1,2 Bruno Almeida, 2,3 Russell Brown; 2,3 | Kathleen Gwozdecky, 2,3 Adityapal Bha4a, 2,3 Shrey Parikh, 2,3 Victor Nechita, 2,3 Arash Nourimand, 2,3 Haley Blinn, 1,2 Siddharth Mahendraker, 1,2 Joanna Hon, 1,2 Mitchell Au 2,3 Quality Assurance – Team Management 1 Faculty of Arts and Science, University of Toronto, Toronto, Canada; 2 University of Toronto Aerospace Team, Toronto, Canada; 3 Faculty of Applied Science and Engineering, University of Toronto, Toronto, Canada; 4 Department of Cell and Systems Biology, University of Toronto, Toronto, Canada; + Equal contribu4on; *Corresponding author: ANM – [email protected] • Tri-annual internal design reviews: industry leaders, professional engineers, scien4sts and professors • Annual external design reviews: CSDC • Technical consulta4ons with NASA for GeneSat and PharmaSat at the NASA Ames Research Centre • Standard CubeSat qualifica4on tes4ng: vibra4on, thermal and radia4on • Task: high-fidelity func4onality – pre-build reviews • Ensuring transforma4on efficiency via NAT pla4ng • Ensuring proper casse‘e integra4on via repeated colony PCR assays and rou4ne microscopy • Ensuring sta4s4cal relevancy from repeated measurements: e.g. figure 10, 20 data points per well averaged with extremely low standard error • Replica4ve assurance by conduc4ng en4re assays in tandem with one another • On-ground reproducibility in exact same laboratory condi4ons for easier and more reliable comparison • Biggest challenge for a student team is the ability to retain membership and knowledge across years • No full-4me employees; all volunteer-based; turnover • Self-create tests to measure QA for processes/ applica4ons that are understudied in literature • Systems integra4on knowledge is personnel-limited • Approaching the knowledge reten4on issue by crea4ng updated documenta4on for all subsystems Figure 4 & 5 (çè) : (4) Side-view cross sec4on of the fluidics card, PCB, payload structure design. 1 microfluidics chip sandwiched by 2 PCBs, and encompassed by heaters and conductors. The current design has a total 33 wells for both gene4c and pharmacological experimenta4on. (5) Full-view cross sec4on of the fluidics card structure within the primary pressure vessel structure. The empty spaces are to be filled by fluid actua4on micro-machinery for on-board dilu4ons, pumping and waste management. Figure 6, 7 & 8 (é) : Func4onal diagrams of the three sensor modes: (6) sta4c mode, (7) fluorescence mode and, (8) op4cal density mode. (7) The 470nm excita4on LED hits GFP within CA samples. GFP will emit green light at ~508nm, picked up by our photodiode sensors. The 495nm longpass filter will exclude excita4on wavelength light. (8) 600nm light will be emi‘ed from the LED PCB, and hits, sca‘ers then passes through sample à OD600. Figure 9 (é) : ~5-month, full humidity, stasis test results. Figure 10 (é) : Ini4al proof-of-concept GFP-CA-HSP90 light detec4on in 3 dilu4ons. Figure 11 (è) : Sta4c diagram cross sec4on of microfluidics chip architecture. Solenoid valves prevent premature growth media (YPD) and stasis media (H2O) mixing before experiment start commands are sent via telemetry. Membranes allow cell exclusion and gas exchange with pressure vessel air reservoir. YPD and FLC are stored together ini4ally before actua4on. Piston system creates necessary pressure to force stasis media out, while keeping cells in. A^er which, growth media (YPD) with various FLC dilu4ons are pumped in. Figure 12 (î) : Payload instrumenta4on hardware func4onal architecture. CAN commands are sent from OBC to payload SSM PCB. The payload SSM PCB controls: (1) MF actua4on, (2) MOSFET heater drivers, (3) thermistor circuitry for MF chip temperature modula4on. It also houses various environmental sensors: PCB temperature, humidity, pressure and accelerometer. The payload SSM communicates with sensor PCBs, LED PCBs via SPI command protocols. The sensor PCBs measure photocurrents on the order of 1pA; designed amplifier chain to achieve be‘er satura4on of signal. Measurements taken every 30min over 48hours, all readings taken in triplicates, from a total of 15 gene wells and 18 drug resistance wells. NASA AAQ [2017-10-13]: Huntsville, AL, USA

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Page 1: 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|[email protected],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–[email protected]

•  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

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