Silica gel 60 plate

Mosquitoes (200 individuals) were fed ad libitum for six days in the presence or absence of Rv, Compound C, Compound C + Rv, AICAR, polyphenols or an antibiotic mix. Twenty insects were separately homogenized and subjected to lipid extraction using methanol: chloroform: distilled water (2:1:0.8 v/v). The lipid extracts were analyzed by one-dimensional thin-layer chromatography (TLC) on Silica Gel 60 plates (E. Merck, Darmstadt, Germany) for neutral lipids using n-hexane:diethyl ether:acetic acid (60:40:1 v/v) [28 (link),29 (link)]. Cholesterol, cholesteryl-oleate, glycerol-tryoleate, diolein, oleoyl-glycerol and oleic acid (Sigma-Aldrich Co, USA) were used as standards. The lipids were visualized using a charring reagent (Cu₂SO₄) after heating at 200°C for 20 min [28 (link)–30 (link)]. TLCs were scanned, and the densitometry of the bands was assessed using the ImageJ program.

For the analysis of lipids from yeast and C. ellipsoidea, the cells were harvested by centrifugation, and the resulting cell pellets were ground to a fine powder under liquid nitrogen and subsequently treated with isopropanol at 80 °C for 10–15 min to stop the lipolytic activity. Isopropanol was evaporated under nitrogen gas before lipid extraction. The total lipids were extracted according to a modified version of the Bligh and Dyer method [64 (link)], and TAG was separated from the total lipids by thin-layer chromatography (TLC) on Silica Gel 60 plates (Merck, Darmstadt, Germany). The solvents that were used were hexane/diethyl ether/glacial acetic acid (70:30:1, v/v). The lipids were visualized by spraying Primuline (Sigma, 10 mg/100 mL acetone: water (60:40 v/v)) and exposing the plate to UV. Triolein (Sigma) was used as the standard. TAGs were recovered from the TLC plates and then trans-esterified with 5% H₂SO₄ in methanol at 85 °C for 1 h. The fatty acid methyl esters (FAMEs) were extracted with hexane and analysed by GC-MS following the methods described in the following section.

Organ homogenates, hemolymph, and midgut luminal contents were subjected to lipid extraction (Blight and Dyer, 1959 (link)). The organic phases containing the lipids were dried under a stream of nitrogen, and lipids were resuspended in 30 μl of chloroform and analyzed by high-performance thin-layer chromatography (HPTLC) on Silica gel 60 plates (Merck, Darmstadt, Germany) developed in hexane:ethyl ether:acetic acid (60:40:1) as solvent (Kawooya and Law, 1988 (link)). The following commercial lipid standards were used: cholesterol, cholesteryl ester, free fatty acid, monoacylglycerol, diacylglycerol, and TAG (Sigma–Aldrich, St. Louis, MO, United States). Plates were stained with iodine vapor, the spots corresponding to free and esterified cholesterol were scraped, and the lipids were eluted three times from the silica with chloroform:methanol:water (1:2:1). The lipids were extracted from the supernatants in chloroform, and the radioactivity was determined by scintillation counting.

Total parasite lipids were extracted^{56 (link)} and separated by one-dimensional thin layer chromatography (TLC) on Silica Gel 60 plates (Merck, Darmstadt, Germany) in solvent system 1 composed of chloroform:methanol:acetic acid:water (25:15:4:2; v/v/v/v)^{57 (link)}. Lipids were visualized by exposure to iodine vapor. For in vivo labeling experiments, parasites at mid-log phase were cultured in the presence of [³H]-serine for indicated times and lipids were extracted as above. After separation by TLC, [³H]-labeled lipids were analyzed by scanning the air-dried plate with a radioisotope detector (Berthold Technologies, Regensdorf, Switzerland). Quantification of radiolabeled lipids was done using the Rita Control software provided by the manufacturer. On each TLC plate, appropriate lipid standards were carried alongside. For lipid phosphorus quantification, total lipids were separated by two-dimensional TLC using solvent system 2 composed of chloroform:methanol:25% NH₃:water (45:37:6:4; v/v/v/v) for the first dimension and solvent system 3 composed of chloroform:methanol:acetone:acetic acid:water (40:15:15:12:8; v/v/v/v) for the second dimension^{57 (link)}. After exposure to iodine, lipid spots were scraped from the plates and lipid phosphorus was quantified photometrically^{58 (link)}.

NMR spectra were recorded with a Bruker AVANCE-500 spectrometer at a sample temperature of 298 K. NMR spectra were recorded in CDCl3 or CD3OD and calibrate using the TMS signal as internal reference. Mass spectrometric analysis were performed in positive or negative ESI-MS. Mass spectra were recorded on a Thermo Quest Finningan LCQTM DECA ion trap mass spectrometer, equipped with a Finnigan ESI interface; data were processed by Finnigan Xcalibur software system. All reactions were monitored by TLC on silica gel 60 plates (Merck).

FAs were extracted from ~20 mg lung tissue, homogenized in 10 µL phosphate-buffered saline with protease inhibitor (homogenization buffer) per 1 mg tissue, or from ~200 µL RBCs or peripheral blood mononuclear cells (PBMCs) collected as buffy coat. Lipids were extracted from each lipid pool with chloroform: methanol (2:1, v:v; containing 0.01% butylated hydroxytoluene) by a modification of the method of Folch et al. [28 (link)]. The lipid extracts were concentrated and the neutral lipids separated from the phospholipids by thin-layer chromatography (TLC) (silica gel 60 plates, Merck) and eluted with diethyl ether: petroleum ether: acetic acid (30:90:1, v:v:v). The lipid band containing phospholipids was removed from the TLC plate and transmethylated with methanol: sulfuric acid (95:5, v:v) at 70 °C for 2 h to form fatty acid methyl esters (FAME). FAMEs were analyzed with an Agilent Technologies 7890A gas chromatography system equipped with an Agilent Technologies 7000B triple quad mass selective detector (Agilent Technologies, Santa Clara, CA, USA) and quantification performed with Masshunter (B.06.00). Relative percentages of FAs (% w/w) were calculated by taking the concentration of a given FA as a percentage of the total concentration of all FAs identified in the sample.

The MAs and the total extractable lipids were extracted and analyzed as described previously^{16 (link)}, except that the (HP)TLC plates for MAME separation were developed in ether/diethyl ether (9:1, v/v, five runs). Purification of MAMEs and compound Z was performed by preparative TLC using silica gel 60 plates (Merck) developed in the above eluent; the compounds were then scraped off and extracted from silica gel three times with diethyl ether.