development and validation of a quantitative, high-throughput, fluorescent-based bioassay to detect schistosoma viability开发和验证的定量、高通量fluorescent-based生物测定检测血吸虫生存能力.pdfVIP

Development and Validation of a Quantitative, High- Throughput, Fluorescent-Based Bioassay to Detect Schistosoma Viability Emily Peak, Iain W. Chalmers, Karl F. Hoffmann* Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, United Kingdom Abstract Background: Schistosomiasis, caused by infection with the blood fluke Schistosoma, is responsible for greater than 200,000 human deaths per annum. Objective high-throughput screens for detecting novel anti-schistosomal targets will drive ‘genome to drug’ lead translational science at an unprecedented rate. Current methods for detecting schistosome viability rely on qualitative microscopic criteria, which require an understanding of parasite morphology, and most importantly, must be subjectively interpreted. These limitations, in the current state of the art, have significantly impeded progress into whole schistosome screening for next generation chemotherapies. Methodology/Principal Findings: We present here a microtiter plate-based method for reproducibly detecting schistosomula viability that takes advantage of the differential uptake of fluorophores (propidium iodide and fluorescein diacetate) by living organisms. We validate this high-throughput system in detecting schistosomula viability using auranofin (a known inhibitor of thioredoxin glutathione reductase), praziquantel and a range of small compounds with previously- described (gambogic acid, sodium salinomycin, ethinyl estradiol, fluoxetidine hydrochloride, miconazole nitrate, chlorpromazine hydrochloride, amphotericin b, niclosamide) or suggested (bepridil, ciclopirox, rescinnamine, flucytosine, vinblastine and carbidopa) anti-schistosomal activities. This developed method is sensitive (200 schistosomula/well can be