Fastprep 24 homogenizer

Liver samples (100–120 mg) were homogenized in 1.2 mL methanol in the Fastprep-24 homogenizer (MP Biomedicals, Irvine, CA, USA). For the comparison of extraction methods, a homogenate that corresponded to 50 mg of tissue was extracted, with modifications, according to the method described by Bligh and Dyer [26 (link)], in dichloromethane/methanol/water (2.5:2.5:2.1, v/v/v), and a second homogenate from the same sample that corresponded to 50 mg of tissue was extracted according to the method described by Folch [27 (link)] in dichloromethane/ methanol/NaCl 0.9% in water (2:1:0.2, v/v/v). After centrifugation (1000× g, 15 min, 4 °C), the solutions separated into an upper methanol/water phase (with polar metabolites) and a lower dichloromethane phase (with lipophilic compounds), with an intermediate phase of protein and cellular debris. The aqueous and organic phases were collected and evaporated to dryness. Chloroform was replaced by dichloromethane for security reason [46 (link)]. For the dietary intervention study, liver samples (100–120 mg) were homogenized in 1.2 mL methanol in a Fastprep-24 homogenizer (MP Biomedicals, Irvine, CA, USA). A homogenate that corresponded to 50 mg of tissue was extracted as described above for NMR analysis, and two homogenates from the same sample that corresponded to 1 mg of tissue were extracted for GC and LC-MS analyses.

RNA was extracted from whole blood, plasma, and tissue using RNeasy Protect Animal Blood, QIAamp Viral RNA Mini, and RNeasy Mini kits (Qiagen). Tissue samples were homogenized using a FastPrep^®-24 homogenizer and Lysing Matrix D (MP Biomedicals). qPCR primers and probes ZIKV_1086, ZIKV_1162c, ZIKV_1107_P were synthesized by Integrated DNA Technologies [48 (link)]. Viral loads were quantified by qRT-PCR using the SuperScript^™ III Platinum^™ One-Step qRT-PCR Kit (Thermo Fisher) and a LightCycler 480 (Roche) instrument as previously described [10 (link)]. To determine the concentration of ZIKV RNA in a sample, a synthetic DNA fragment corresponding to the H/PF/2013 genome from positions 1086 to 1107 was amplified and subcloned into pCR4-TOPO (Thermo Fisher). RNA standards were synthesized using a MAXIscript^™ T7/T3 Transcription Kit (Thermo Fisher), quantified by absorbance and Quant-iT Ribogreen RNA Assay Kit (Life Technologies), and serially diluted to generate in-run standard curves for qRT-PCR analysis. To quantify ZIKV NS1-antigenemia, plasma from guinea pigs was subjected to a Zika Virus NS1 ELISA (BioFront Technologies).

From the experiments in 2021, we selected fungal isolates according to morphology from each treatment to confirm their identity using genetic analysis (simple sequence repeats; SSR). Isolates were spread on CM plates covered with filter paper. After 4–5 days of incubation, the mycelia were scraped off the filters, transferred to 2 mL Eppendorf tubes and frozen at -70°C. The frozen mycelia were lyophilized, and cells were disrupted with glass beads (3 mm and 1 mm) in a FastPrep-24 homogenizer (MP Biomedicals, Eschwege, Germany; 25 s at 6 m s^-1). We extracted the DNA (sbeadex plant kit and King Fisher Flex Purification system, Thermo Fisher Scientific, Waltham, Massachusetts) and standardized the samples to 5 ng DNA μL^-1.
We used six SSR markers in two primer pair sets for each species to analyze fungal genotypes (Bb1F4, Bb2A3, Bb2F8, Bb4H9, Bb5F4, Bb8D6 for B. brongniartii; 47 (link); Ma2049, Ma2054, Ma2063, Ma2287, Ma327, Ma195 for M. brunneum; 48 (link), 49 (link)). Reference strains were included for both species (B. brongniartii, Bip2 and Bip4; Metarhizium spp., Ma714, Ma500 and Bip5). Multiplex PCRs and fragment size analyses were performed as described by Mayerhofer et al. (50 (link)) and Fernandez-Bravo et al. (51 (link)).