Gen5 software

The comparative in vitro cytotoxicity of D+T NP vs. D+T solution was evaluated using the TZM-bl cell line and CellTiter-Glo^® luminescent assay method, as described previously [45 (link)]. Briefly, the TZM-bl cells (10⁴ cells/well) in complete DMEM medium and PBMCs (10⁵ cells/well) in complete RPMI medium, were treated in triplicate, respectively, with D+T NP or D+T solution, at different concentrations (20, 10, 1, 0.1, 0.01 µg/mL each drug concentration) for 96 h. Similarly, the 5% DMSO-treated cells and 1×PBS (treatment equal volume) treated cells were the positive and negative control, respectively. The cytotoxicity was evaluated by the CellTiter-Glo^® luminescent-cell viability assay kit (Promega; Madison, WI, USA) following manufacturer protocol. The luminescence intensity was read from the Synergy II multi-mode reader with Gen5TM software (BioTek; Winooski, VT, USA). The untreated control cells were considered 100% viable. The percentage cytotoxicity (%Cytotoxicity) was calculated as % normalized viability against the untreated (negative) control group. The experiment was carried out on three independent batches of D+T NPs and D+T solution. The result represents the mean ± SEM of three independent batches of studies. The %Cytotoxicity was evaluated by following Equation (2): $$\%Cytotoxicity=\frac{RL{U}_{Untreated}-RL{U}_{Treated}}{RL{U}_{Untreated}}\times 100$$

Luciferase assay was performed to measure the transcriptional activity of the NF-κB in HCs under pepsin exposure at each pH point compared to control. A NF-κB reporter Vector [pGL4.32(luc2P/NF-κB-RE/Hygro)], a control vector [pGL4.27(luc2P/minP/Hygro)], a Lipofectamine^® 2000 (Invitrogen™, Thermo Fisher Scientific, Waltham, MA, USA) and a firefly Luciferase Assay system (Promega Corporation, Madison, WI, USA) were used, according to manufacturer’s procedure, as previously described [56 (link),57 (link),58 (link)]. The treatment was performed 24 h after transfection. The cells were treated with pepsin (1 mg/mL) at pH 5.0, 6.0 and 7.0 and controls, for 15 min and then media were replaced with serum-free medium. After 4–6 h incubation we measured luminescence using a luminometer and Gen5 software (Synergy1, BIOTEK; Gen5^TM software, BioTek Instruments Inc., Winooski, VT, USA). NF-κB activity was expressed as ratios of mean values of NF-κB reporter (NF-κBLuc2P) against mean values of control (Luc2P) for each condition (pepsin at pH 5.0, 6.0, 7.0 and control). Triplicate assays were performed for each treatment condition.

The resistance of native yeast T. delbrueckii YCPUC10 (non-evolved) and commercial yeast T. delbrueckii Biodiva^TM to ethanol were determined by growing them in YPD medium (20 g/L glucose, 5 g/L peptone, and 5 g/L yeast extract) supplemented with 3, 6, 9, 10, 11, 12, and 14% (v/v) of ethanol. Cell growth was monitored by determining the optical density at 600 nm (OD₆₀₀) using 1 h intervals. The experiments were done in triplicate in a 96-well microplate using 800 TSI plate reader coupled to the Gen5^TM software (BioTek, United States). The specific growth rate (μmax) was estimated from the slope of the growth curve during exponential phase according to the equation lnxt = x0 + mt, where: xt and x0 correspond to the biomass concentration or the optical density (OD) at time t (h) and t = 0, respectively (Barata et al., 2008 (link)). The R² values of the curves were 0.996 or higher in all cases. Lag phase duration was determined mathematically according to Buchanan and Cygnarowicz (1990) (link) as the time when the second derivative of the logarithm of the growth curve reaches a maximum value. Growth efficiency was defined as area under curve (AUC) and expressed as a percentage considering 100% the control condition (Godoy et al., 2016 (link)).

The bioluminescence of bacteria was evaluated using a Synergy 2 microplate reader and Gen5^TM software (BioTek, USA). The luminescent signal was measured kinetically for a period of 6 h with 2.5 min intervals, and relative luminescence units (RLUs) were detected for each sample. During evaluation, the cells were incubated in 1 M sorbitol at room temperature.

The experiments were performed on a Synergy^TM 2 Microplate Reader (BioTek Instruments, Inc., Winooski, VT) with similar filter settings (λ_ex = 485 ± 20 nm, λ_em = 528 ± 20 nm). Sample prepation was similar to the ACE experiments; 15 μL aliquots of mixtures were loaded into a corning 3540 microplate (Corning Incorporated, Corning, NY). Polarization values were calculated from the parallel and perpendicular fluorescence intensities. Calibration was done using the g factor, calculated in the Gen 5TM software (BioTek Instruments, Inc., Winooski, VT). Bound fractions were determined using equation (3):

where: f_a is the bound fraction; P, P_m and P_o are measured polarizations of a sample, complex, and free ssDNA, respectively. The polarization of the complex (P_m) was taken as the plateau with an excess of PLN. The overall fluorescence intensity of the samples with increasing PLN concentrations, was monitored and bound fraction was modified if the overall fluorescence intensity was positively or negatively biased according to the published method49 (link).

To determine whether the cells and tissue slices remained intact during the prolonged incubation, we obtained TPM images of HeLa cells and normal colon tissues co-labelled with ABI-Nu and Pyr-CT and with ABI-Nu and BF-MT after incubation for 0−60 min. The total areas of the nuclei and cytoplasm in the probe-labelled cells and tissues in the 150 × 150 μm panels were measured using the cell analysis program in Gen5^TM software (BioTek, Winooski, VT, USA), which automatically calculates the areas of the fluorescent regions. Since the areas differed depending on the samples, the values measured in each sample at t = 0 were used as a reference and thus were given the value 1.0. The total areas measured at different times from the TPM images of 10 different cell samples and 30 sectional TPM images of the tissue slices obtained at depths of 90−150 μm were averaged. They remained nearly the same within the range of experimental error for 60 min (Fig. S11).

Growth curves were performed to characterize the fitness of the evolved clones of the initial T. delbrueckii YCPUC10 strain [6, 9, 10, 10.5, 11, 11.5, and 12% (v/v)]. Evolved clones were grown in YPD medium (20 g/L glucose, 5 g/L peptone, and 5 g/L yeast extract), and cell growth was monitored by determining the optical density at 600 nm (OD₆₀₀) using 800 TSI plate reader coupled to the Gen5^TM software (BioTek, United States). The experiments were done in triplicate. The specific growth rate, lag phase duration and growth efficiency were determined as described above. From now on, we worked with one of the clones generated.