Flavin adenine dinucleotide

The effect of flavins or their analogs on parasites proliferation was evaluated through growth curves. Stationary phase parasites were inoculated in fresh SDM-79 or SDM-20 media (basal 20 nM riboflavin) supplemented to a defined riboflavin concentration. Initial density for T. cruzi Y-GFP, MJ Levin-GFP (referenced as wild-type) and MJ Levin-TcRibJ (referenced as TcRibJ) was 10⁷ parasite/mL, while initial density for T. brucei, L. (L.) mexicana, C. fasciculata and Phytomonas Jma was 10⁶ parasites/mL. Riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) were dissolved in water and the analogs lumiflavin, lumichrome and roseoflavin (all from Sigma-Adrich) dissolved in DMSO. Then, flavins and analogs were added to the culture media at the indicated concentrations. Parasites were daily counted using a hemocytometer chamber. Proliferation was calculated as the percentage of parasite counts relative to control condition values (flavin at 20 nM or without analogs) on the fifth day. Day 5 was chosen because it is when all parasite cultures tested reach stationary phase in control conditions (S1 Fig).

Citrinin, pelargonidin chloride, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), 2′,7′-Dichlorofluorescin diacetate (DCFH₂-DA), dimethyl sulfoxide (DMSO), minimum modified Eagle’s medium (MEM), hoechst 33342, propidium iodide (PI), ethidium bromide, dichlorophenol indophenol, flavin adenine dinucleotide, dicumarol, protease inhibitor cocktail, fluoroshield, RIPA buffer were purchased from Sigma–Aldrich, St. Louis, MO, United States. JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolyl carbocyanine iodide) and fetal bovine serum (Hyclone) was from Invitrogen, United States.

Nicotinamide adenine dinucleotide (NADH) was purchased from Gerbu Biotechnik GmbH (Wieblingen, Germany). Flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), riboflavin, polyethyleneimine (PEI) (MW: 600–1000 kDa), and sulfate-dextran (MW: 100 kDa) were supplied by Sigma-Aldrich Co (St. Louis, IL, USA). Iminodiacetic acid disodium salt monohydrate (IDA) and copper sulphate (II) 5-hydrate were purchased from Fluka (Buchs, Switzerland). Cyanogen bromide 4B Sepharose (Ag-CB) was from GE Healthcare (Uppsala, Sweden). Cross-linked agarose beads (4%) (BCL) were from Agarose Beads Technology (Madrid, Spain). Agarose coated with polyethyleneimine (PEI-Ag) supports were prepared as previously described elsewhere [19 (link)]. Agarose coated with sulfate-dextran agarose beads (DS-Ag) were prepared as previously described [20 ]. Protein concentration was determined using the method of Bradford [21 (link)]. All other used reagents were of analytical grade.