Development and Validation of a Quantitative, High-Throughput, Fluorescent-Based Bioassay to Detect Schistosoma Viability 英文参考文献.docVIP

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Development and Validation of a Quantitative, High-Throughput, Fluorescent-Based Bioassay to Detect Schistosoma Viability 英文参考文献.doc

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Development and Validation of a Quantitative, High-Throughput, Fluorescent-Based Bioassay to Detect Schistosoma Viability 英文参考文献

DevelopmentandValidationofaQuantitative,High- Throughput,Fluorescent-BasedBioassaytoDetect SchistosomaViability EmilyPeak,IainW.Chalmers,KarlF.Hoffmann* InstituteofBiological,EnvironmentalandRuralSciences,AberystwythUniversity,Aberystwyth,UnitedKingdom Abstract Background:Schistosomiasis,causedbyinfectionwiththebloodflukeSchistosoma,isresponsibleforgreaterthan200,000 human deaths per annum. Objective high-throughput screens for detecting novel anti-schistosomal targets will drive ‘genometodrug’leadtranslationalscienceatanunprecedentedrate.Currentmethodsfordetectingschistosomeviability rely on qualitative microscopic criteria, which require an understanding of parasite morphology, and most importantly, mustbesubjectivelyinterpreted.Theselimitations,inthecurrentstateoftheart,havesignificantlyimpededprogressinto wholeschistosomescreeningfornextgenerationchemotherapies. Methodology/Principal Findings: We present here a microtiter plate-based method for reproducibly detecting schistosomulaviabilitythattakesadvantageofthedifferentialuptakeoffluorophores(propidiumiodideandfluorescein diacetate)bylivingorganisms.Wevalidatethishigh-throughputsystemindetectingschistosomulaviabilityusingauranofin (aknowninhibitorofthioredoxinglutathionereductase),praziquantelandarangeofsmallcompoundswithpreviously- described (gambogic acid, sodium salinomycin, ethinyl estradiol, fluoxetidine hydrochloride, miconazole nitrate, chlorpromazine hydrochloride, amphotericin b, niclosamide) or suggested (bepridil, ciclopirox, rescinnamine, flucytosine, vinblastineandcarbidopa)anti-schistosomalactivities.Thisdevelopedmethodissensitive(200schistosomula/wellcanbe assayed),relevanttoindustrial(384-wellmicrotiterplatecompatibility)andacademic(96-wellmicrotiterplatecompatibility) settings,translatabletofunctionalgenomicsscreensanddrugassays,doesnotrequireaprioriknowledgeofschistosome biologyandisquantitative. Conclusions/Significance: The wide-scale application of this fluorescence-based bioassay wil