s made by reconstituting in 0.015 mM KOH and 20 mM uracil and uridine stock solution was prepared in water and stored at room temperature. Susceptibility Testing 1) E-test. In vitro antifungal susceptibility to AmB alone or in combination with MPA was determined by 2883-98-9 web E-test according to the manufacturer’s protocol. For C. neoformans and Candida albicans isolates, cells were grown to mid-log phase in YPD broth, washed and diluted to an OD600 
= 0.5. A. fumigatus conidia were dissolved in sterile distilled water, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19650784 filtered through sterile Miracloth and enumerated in hemocytometer slide to yield a final inoculum size of 56105 conidia/mL. Cells were plated with sterile cotton swab to produce confluent growth on AM3 medium supplemented with or without 3.25 mg/mL MPA. Additionally, E-test with wild type C. neoformans was performed on AM3 medium supplemented with 3.25 mg/mL MPA and 20 mg/mL guanine. E-tests with C. neoformans and C. albicans ura2 mutants were also performed on AM3 medium supplemented with 20 mM uracil and 20 mM uridine. Once plates were dry, E-test strips were placed on them and the plates were then incubated at 37uC for 4872 hours. The MIC was determined as the lowest concentration of the drug at which the border of the elliptical zone of growth inhibition intercepted the scale on the strip, including any microcolonies that were present. Drug AmB alone AmB in combination MPA alone MPA in combination FIC index = MICA’/MICA+MICB’/MICB Interaction Replicate 1 0.06 0.0075 30 7.5 0.375 Synergy Replicate 2 0.12 0.0075 30 7.5 0.25 Synergy Replicate 3 0.06 0.015 30 7.5 0.5 Synergy AmB: Amphotericin B, MPA: Mycophenolic acid. MICs of AmB and MPA alone and in combination against C. neoformans in 3 separate experiments and calculated FIC values. doi:10.1371/journal.pone.0087246.t001 4 Nucleotide Biosynthesis and Amphotericin B 2) Checkerboard assay. Susceptibility to AmB and MPA were assessed by a checkerboard assay based on standard protocols proposed by CLSI for broth microdilution antifungal susceptibility test. The media used for analyzing a combination of AmB and MPA against all wild type fungal isolates was AM3-2% Dex, whereas RPMI 1640-2% Dex was used for testing combination of AmB and 5-FC against wild type C. albicans and A. fumigatus. Working solutions of each drug was prepared in the test media at a concentration of four times the targeted final concentration in the assay range. 100 mL of drug mixture with 50 mL of increasing concentrations of one drug and 50 mL of fixed concentration of the other drug used in the combination were mixed and dispensed in the 96 well microtiter plates. One row and column had 50 mL of single drug solution of each concentration. Two wells containing 100 mL each of assay medium and inoculum served as growth control. A single well containing 200mL of only the assay medium served as sterility control. Yeast inocula were prepared to yield a final cell concentration of 16105 CFU/ mL. Final concentration of A. fumigatus conidia used in checkerboard assay was 16105 conidia/mL. 100 mL of the inoculum was dispensed in each well, plates were incubated in air at 37uC and read after 72 hours. Readings were performed spectrophotometrically using an automated plate reader set at 540 nm. MIC endpoint for AmB was defined as the lowest drug concentration, alone and in combination that prevented any discernible growth. For MPA and 5-FC, the MIC was defined as the lowest concentration of drug tested alone and in combin
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