Thiamine

GC/MS-grade heptane, boron trifluoride-methanol solution 14% in methanol, and hexane, in addition to LC/MS-grade methanol, water, acetylthiocholine iodide, acetonitrile, folic acid, thiamine, riboflavin, ultrapure water, pyridoxine, niacin, Acetylcholinesterase (from Electrophorus electricus), and 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl, were purchased from Merck (Darmstadt, Germany). Fatty acid methyl esters (unsaturated Kit) and sodium chloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade and were purchased from Chempur (Piekary Slaskie, Poland).
Freeze-dried buffalo worms (Alphitobius diaperinus Panzer), house crickets (Gryllus campestris), mealworms (Tenebrio molitor), and grasshoppers (Chorthippus biguttulus) were purchased from a local commercial source (Delibugs, Lelystad, Netherlands). Before analysis, the insects were shredded.
Insects (10 g) previously ground in a mortar were added to virgin olive oil (1 L). Olive oil with insects was left for 48 h in a cool, dark place.

Healthy cat claws were crushed with a mortar and pestle, sterilized by autoclavation, and subsequently mixed with a minimum medium (MM) containing 15 mM glucose, 10 mM MgSO₄7·H₂O, 29 mM KH₂PO₄, 13 mM glycine, and 3 µM thiamine (all compounds from Merck Millipore, Darmstadt, Germany) to form a suspension of 2% w/v powdered claws. An inoculum of 1 × 10⁶ yeast cells/mL was prepared in MM with or without (control) powdered claws and added to the wells of a 96-well plate in a 200 μL/well volume. Plates were incubated at 35 °C for 7 days, and the growth was analyzed by the absorbance measurement at 530 nm using a microplate reader (SpectraMax M3; Molecular Devices, San Jose, CA, USA). Mitochondrial activity was determined with the CyQUANT XTT assay (Thermo Fisher, Waltham, MA, USA). A mixture of XTT reagent and electron coupling reagent was prepared at a ratio of 6 to 1. Seventy microliters of the mixture were added to each well. Then, the plate was incubated at 37 °C for 4 h in the dark in a 5% CO₂ incubator. The optical densities (ODs) were determined, in two technical replicates, using the SpectraMax^® Plus 384 Microplate Reader (SpectraMax M3; Molecular Devices, San Jose, CA, USA) at 450 nm and 660 nm. Mitochondrial activity was expressed by the decrease in OD (450–660 nm), discounting the average of the ODs of the controls [29 (link)].

The strains and clinical isolates of C. neoformans used in the study are listed in S4 Table. The study was started with H99 strain called H99O that was kindly provided by J. Heitman (Duke University, Durham, NC) in the late 90’s. The reference strain KN99α and strains from the Madhani collection were provided from Kirsten Nielsen's lab and the Fungal Genetic Stock Center [61 (link)], respectively.
C. neoformans strains were grown in liquid Yeast Peptone Dextrose (YPD, 1% yeast extract (BD Difco, Le Pont de Claix, France) 2% peptone (BD Difco), 2% D-glucose (Sigma, Saint Louis, Minnesota, USA)) and in minimal medium (MM, 15mM D-glucose (Sigma), 10 mM MgSO₄ (Sigma), 29.4mM KH₂PO₄ (Sigma), 13mM Glycine (Sigma), 3.0 μM Thiamine (Sigma), [32 (link)]). Minimum inhibitory concentration (MIC) of H99O for fluconazole (FLC) and flucytosine (5FC) (both purchased from lsachim, Shimadzu Group Company, Illkirch-Graffenstaden, France) were determined by the EUCAST method and were 8 and 4 mg/L, respectively.

Artemisinin-sensitive (n = 2) and artemisinin-resistant (n = 5) P. falciparum isolates were obtained from clinical studies conducted on the Thailand-Cambodia border (2 (link), 46 (link)). All parasite isolates were mycoplasma free. Parasites were cultured at 5% parasitemia and 5% hematocrit in culture medium. Culture medium consisted of Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma catalog no. R6504) containing 50 mg/L hypoxanthine (Sigma catalog no. H9377), 3 mg/L thiamine (Sigma catalog no. T1270), 6 mg/L L-ascorbic acid (Sigma catalog no. A5960), 30 mg/L CaCl₂ (Sigma catalog no. C4901), 26 mg/L KH₂PO₄ (Merck catalog no. A681173), 16 mg/L MgSO₄ (Sigma catalog no. M8150), 1 g/L d-glucose (Sigma catalog no. G7021), 5.96 g/L HEPES (Sigma catalog no. H3375), 2 g/L NaHCO₃ (Sigma catalog no. S5761), and 10% human serum. Culture medium was replaced daily and incubated at 37°C in a 5% CO2 incubator.

Basic medium (B medium) for Staphylococci consisted of Soy Peptone (10 g; Plato, Koblenz, Germany), Yeast Extract (5 g; Deutsche Hefewerke, Nuernberg, Germany), NaCl (5 g; Carl-Roth, Karlsruhe, Germany), Glucose (1 g; Carl Roth) and K₂HPO₄ (1 g; Applichem, Darmstadt, Germany). Deionized water was added to a final volume of 1 liter and pH was adjusted to 7.2.
LB medium for E. coli consisted of Peptone (10 g; Plato), Yeast Extract (5 g; Deutsche Hefewerke) and NaCl (5 g; Carl Roth). Deionized water was added to a final volume of 1 liter and pH was adjusted to 7.2. MacConkey agar was prepared according to the manufactures’ instructions (Carl Roth). Minimal media M9 for staphylococci consisted of Na₂HPO₄ (6 g; Carl Roth), KH₂PO₄ (3 g; Fisher Chemicals), NH₄Cl (1 g; Merck Darmstadt, Germany) and NaCl (0.5 g; Carl Roth). Deionized water was added to a final volume of 1 L, the media was autoclaved and supplemented with thiamine (2 µg/mL; Sigma Aldrich, Munich, Germany), MgSO₄ (1 mM; Carl Roth) and casamino acids (0.2%; BD Bioscience, Heidelberg, Germany). Cells were routinely grown at 37 °C either in liquid culture in baffled Erlenmeyer flasks (1:5 ratio) with shaking or on 1.5% agar plates.

Monoclonal anti-actin antibody, Prussian Blue, pantothenate, pantethine, vitamin E, L-carnitine, thiamine, Luperox^® and trypsin were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-mitochondrial acyl carrier protein (mtACP), anti-NF-Y, anti-FOXN4 and anti-hnRNPA/B were purchased from Invitrogen/Molecular Probes (Eugene, OR). Anti-phospho-PGC1α was purchased from RD systems. NFS1 antibody and Omega 3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PANK2, anti-PGC1α, complex 1 activity kit and PDH activity kit were purchased from Abcam (Cambridge, UK). Anti-TFAM was purchased from Cell Signaling. BODIPY^® 581/591 C11 was purchased from Thermo-Fisher (Waltham, MA). A cocktail of protease inhibitors (complete cocktail) was purchased from Boehringer Mannheim (Indianapolis, IN). The Immun Star HRP substrate kit was from Bio-Rad Laboratories Inc. (Hercules, CA).

Thiamine, AuNPs, nGr, PIX, PIXCoCl, monosodium phosphate, disodium phosphate, vitamin B12, ascorbic acid, maltodextrin, fructose, glucose, ammonium chloride, iron (II) sulphate hydrate, and sodium acetate were obtained from Sigma Aldrich (Milwaukee, WI, USA). Fluka supplied the paraffin oil (d420, 0.86 g cm⁻¹) (Buchs, Sweden). Phosphate buffer solution (PBS, 0.1 mol L⁻¹) was made by combining monosodium phosphate and disodium phosphate in a solution-making process. To achieve the desired range of pH values (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0), the pH of the buffer solution was modified by adding varying amounts of solutions with a concentration of 0.1 mol L⁻¹ of either NaOH or HCl.
For the preparation of Thiamine solutions, PBS (pH 2.0) was utilized. The stock solution (10⁻² mol L⁻¹) was prepared only with PBS (pH 2.0) and had a concentration of 10⁻² mol L⁻¹. The remaining solutions (10⁻³ mol L⁻¹ −10⁻¹² mol L⁻¹) were prepared using PBS and 0.1 mol L⁻¹ NaNO₃ as supporting electrolytes. When not in use, all solutions were stored in a dark, dry place, at room temperature.