0.45 μm filter

For production of infectious HIV-1, HEK293T cells were transfected with a plasmid encoding full-length HIV-1_NL4.3 by calcium-phosphate DNA precipitation (Takara Bio Europe). Virus-containing culture supernatants were harvested 48–72 h post transfection, filtered using 0.45 μm filters (VWR International), and concentrated via ultracentrifugation through a 20% sucrose cushion (Sigma-Aldrich). The infectious titer of the virus stock was defined by a β-galactosidase-based blue cell assay using TZM-bl cells46 (link).

Soluble phenolic compounds were extracted according to Lopez-Martinez and co-workers [24 (link)] with some modifications: 0.5 g of ground whole grain were extracted with 10 mL of an ethanol:water 80:20 (v/v) mixture and maintained under stirring for 2 h in the dark at room temperature. Samples were centrifuged at 2000× g for 15 min at 4 °C (Avanti J-25, Beckman Coulter, CA, USA). The supernatant was collected, evaporated using a Rotavapor, freeze-dried, and finally resuspended in 10 mL of water. The extracts were passed on 0.45 μm filters (VWR International, Fontenay-sous-Boys, France) and stored until use at −20 °C. Each extraction was performed in triplicate.

The plasmid SV-Psi⁻-Env⁻-MLV and L-LUC-SN were co-transfected with or without an envelope glycoprotein plasmid (pHCMV-VSV-G/pSARS-CoV-1/pSARS-CoV-2/pHIV-1 LAI gp160) into HEK293FT cells using Lipofectamine™ 3000 (ThermoFisher, US). Cell supernatants containing viruses were collected after 2 days of transfection. Viruses were filtered through a 0.45 μm filter (VWR, US) and centrifuged at 4 °C, 6500 rpm for 18 h over a 20% sucrose cushion. Viruses were resuspended in 500 μL cell culture medium and stored at − 80 °C. We measured a 25% loss in infectivity due to one cycle of freeze/thaw for the SARS-CoV-2 pseudotype, and no loss for the VSV-G pseudotype.

The Total Phenolic Content (TPC) of extracts was determined according to the Folin–Ciocalteu assay [24 ]. Samples, both before and after digestion, were solubilized in ethanol:water 60:40 (v/v) at the final concentration of 2 mg extracts/mL and filtered using a 0.45 μm filter (VWR International, Fontenay-sous-Boys, France). A calibration curve was prepared using gallic acid standard solutions in the range of 5–50 μg/mL. The amount of 300 μL of diluted samples or standards was added with 1.5 mL of 0.2 N Folin–Ciocalteau reagent and 1.2 mL of 7.5% sodium carbonate. In parallel, the digestive blank or water were analyzed to remove interference. Solutions were maintained for 30 min in the dark and the absorbance was detected at 765 nm in a UV–visible spectrophotometer (Varian Cary 50 SCAN, Palo Alto, CA, USA). Results were expressed as mg/g equivalent of gallic acid (GAE).

The pooled A. tumefaciens was infiltrated into the underside of four- to six-week-old Nicotiana benthamiana new leaves using a needleless plastic syringe. The tobacco plants were grown in 16-h light and 8-h dark periods at 21 °C with a relative humidity of 60–80% and photon intensity of 120–150 μmol/m². Three leaves were infiltrated for each experimental set, and each set was completed on a single plant. As a negative control, A. tumefaciens transformed with pEAQ_GFP was infiltrated into a separate plant. Plants were maintained in dark for 12 h to increase the agrobacterial infection and then shifted to light. The plants were maintained in normal conditions for four to six days. Once the fluorescence from GFP was intense when exposed to UV light, all infiltrated leaves were detached from the petiole, snap-frozen in liquid nitrogen, and ground into a fine powder. Metabolites were extracted with 1 ml 100% methanol (Fisher Scientific, Waltham, MA, USA) and heating at 65 °C for 10 min. They were centrifuged at 17,000 g for 10 min, filtered through a 0.45 μm filter (VWR, Randor, PA, USA), and stored at −20 °C before LC/MS analysis.