Cooper, Tyler T.

Background Platelet microparticles (PMPs) and their abundance in the blood are a prognostic biomarker in thrombotic disorders and cancer. Nanoscale flow cytometry (nFC) is ideal for high-throughput analysis of PMPs but these clinical assays have not been developed previously. Objective This article demonstrates that nFC is a suitable technology to enumerate PMPs present in plasma samples in a clinical setting. Materials and Methods nFC was performed using the Apogee A50-Micro instrument. Instrument settings and acquisition parameters were developed with the use of fluorescent beads and plasma samples. Sample preparation and handling was also optimized. Results nFC allows for linear detection of particles between approximately 200 and 1,000 nm based on calibration beads and was dependent on dilution factor and flow rate. Linearity in event analysis as samples became more diluted was lost when events approximately 100 nm were gated while linearity was maintained despite dilution of sample in events larger than 200 nm in diameter. Higher flow rates lead to an under-estimation of events analysed per microlitre of analyte and this was more pronounced when plasma samples were not diluted more than 1/20×. Conclusion nFC offers multi-parametric analysis of PMPs when optimal calibration of acquisition and sample processing settings is performed. Analysis of plasmas from metastatic prostate cancer patients and leukaemia patients revealed that PMP levels were larger than 100 nm and were equally abundant in patients that responded to or failed androgen deprivation therapy or between patients representing different stages of leukaemia.

ABSTRACT Kidney stone disease involves the aggregation of stone-forming salts consequent to solute supersaturation in urine. The development of novel therapeutic agents for this predominantly metabolic and biochemical disorder have been hampered by the lack of a practical pre-clinical model amenable to drug screening. Here, Drosophila melanogaster, an emerging model for kidney stone disease research, was adapted as a high-throughput functional drug screening platform independent of the multifactorial nature of mammalian nephrolithiasis. Through functional screening, the therapeutic potential of a novel compound commonly known as arbutin that specifically binds to oxalate, a key component of kidney calculi, was identified. Through isothermal titration calorimetry, high-performance liquid chromatography and atomic force microscopy, arbutin was determined to interact with calcium and oxalate in both free and bound states, disrupting crystal lattice structure, growth and crystallization. When used to treat patient urine samples, arbutin significantly abrogated calculus formation in vivo and outperformed potassium citrate in low pH urine conditions, owing to its oxalate-centric mode of action. The discovery of this novel antilithogenic compound via D. melanogaster, independent of a mammalian model, brings greater recognition to this platform, for which metabolic features are primary outcomes, underscoring the power of D. melanogaster as a high-throughput drug screening platform in similar disorders. This is the first description of the use of D. melanogaster as the model system for a high-throughput chemical library screen. This article has an associated First Person interview with the first authors of the paper.