Menadione

A semiquantitative measure of biofilm formation was calculated by using an XTT [2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetra-zolium-5-carboxanilide] reduction assay, adapted from previous reports. XTT was prepared in a saturated solution at 0.5 g/liter in Ringer’s lactate. The solution was filter sterilized through a 0.22-μm-pore-size filter, aliquoted, and stored at -70°C. Prior to each assay, an aliquot of stock XTT was thawed, and menadione (Sigma; 10 mM prepared in acetone) was added to a final concentration of 1 μM. A 100 μl aliquot of the XTT-menadione solution was then added to each prewashed biofilm and to control wells (for the measurement of background XTT-reduction levels). The plates were then incubated in the dark for up to 2 h at 37°C. The activity of the fungal mitochondrial dehydrogenase reduces the tetrazolium salt XTT to formazan salts, resulting in a colorimetric change that correlates with cell viability. The colorimetric change was measured using an ELISA reader (Microplate Reader iMarkTM; BIO-RAD) at 490 nm. In all experiments, RPMI 1640 medium free of biofilm formation was included as a negative control (Mowat et al., 2007 (link); Pitangui et al., 2012 (link)).

C. parapsilosis biofims were obtained in vitro on the surface of 96-well polystyrene microtiter plates as described previously [25 (link)]. Briefly, 100 μL of the standardized mutants of C. parapsilosis suspension (1 × 10⁶ blastoconidia/mL) in RPMI 1640 was allowed to adhere and form biofilms at 37 °C for 24 h. After biofilm formation, the medium was aspirated, and non-adherent cells were removed by washing three times with sterile PBS. Quantification of biofilms was performed by (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) (MTT, Sigma Chemicals, Saint Louis, MO, USA) reduction assay. MTT was prepared as a saturated solution at 0.5 g L
⁻¹ in PBS, filter-sterilized though 0.22 μm pore-size filters, aliquoted, and stored at −70 °C. An aliquot of the MTT stock solution was thawed prior to each assay, and 10 mM menadione (Sigma Chemicals, Saint Louis, MO, USA) in acetone was added to give a final concentration of 25 μM. An aliquot of 100 μL of the MTT–menadione solution was added to each well, and the plates were incubated for 2 h at 37 °C. The metabolic activity of sessile C. parapsilosis cells was assessed quantitatively by measuring absorbance in a microtiter plate reader (Asys Jupiter) at 540 nm. The tetrazolium salt that accumulated after MTT reduction by cellular dehydrogenases was proportional to the number of viable cells present in the biofilm.

We used a UVB irradiation model and a menadione-induced oxidative stress model. Stress induction was performed after exposing NHEK to RJ for three days. In the UVB irradiation model, NHEK cells were washed with PBS and irradiated by using a UV transilluminator (95-0343-01, Analytik Jena AG, Jena, Germany). The intensity of UVB was monitored using a UV meter (UV-340A, Kenis, Tokyo, Japan). After irradiation, the medium was replaced with fresh KGM2 containing RealTime-Glo™ MT Cell Viability Assay (Promega, Madison, WI, USA) components. Luminescence was measured after 24 h using an Envision plate reader (PerkinElmer, Waltham, MA, USA).
NHEK cells were treated with menadione (Sigma-Aldrich) for 1 h and then washed with PBS for the menadione-induced oxidative stress model. The NHEK cells were further cultured in KGM2 containing RealTime-Glo™ MT Cell Viability Assay (Promega) components. Luminescence was measured after 24 h using an Envision plate reader (PerkinElmer). For evaluating the NQO1 activity in menadione-stimulated oxidative stress, the NQO1 inhibitor, ES936, was added 30 min before menadione treatment. Viability was measured using a WST-8 based assay with Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan).

In order to evaluate the long-term antibacterial effect of quercetin-doped adhesive, additional specimens were prepared and placed in a thermocycling machine from 5 °C to 55 °C for 10,000 cycles, with the dwell time set at 15 s, to simulate an entire year’s clinical physiological ageing. After that, biofilm-coated specimens from each group was prepared as mentioned above. The XTT/Menadione reagent was prepared by mixing XTT (Sigma, St. Louis, MO, USA) solutions (1 mg/mL) with Menadione (Sigma, St. Louis, MO, USA) (1 mM) in the volume ratio of 12.5:1. Three biofilm-coated specimens from each group were transferred into separate centrifuge tubes containing 4 mL of sterile PBS (0.01 mM, pH 7.3), then 54 μL of XTT/Menadione reagent was added and the solutions were mixed gently. After an incubation period of 4 h at 37 °C with light protection, the microtubes were centrifuged at 3000 rpm for 3 min to form bacterial suspension solutions. Three readings of each tube from each group (n = 9 each group) was measured under 492 nm using a spectrophotometer (Powerwave 340, Bio-tek Instruments, Winooski, VT, USA). Since quercetin has intrinsic fluorescence^{42 (link)}, the background absorbance of each group was subtracted from OD492 value.

The synthetic community was assembled by 14 oral bacterial strains, eight commensal (Streptococcus sanguinis LMG14657, Streptococcus salivarius TOVE-R, Streptococcus gordonii ATCC 49818, Streptococcus mitis DSM 12643, Streptococcus oralis (clinical isolate), Actinomyces naeslundii ATCC 51655, Actinomyces viscosus DSM 43327 and Veillonella parvula DSM 2007) and six pathobionts (Porphyromonas gingivalis ATCC 33277, Fusobacterium nucleatum ATCC10953, Aggregatibacter actinomycetemcomitans ATCC 43718, Prevotella intermedia ATCC 25611, Streptococcus mutans ATCC 25175 and Streptococcus sobrinus ATCC 33478). The strains were maintained on blood agar No2 (Oxoid, Hampshire,UK) supplemented with hemin (5 mg/mL) (Sigma Aldrich, Belgium), menadione (1 mg/mL) (Sigma Aldrich, Belgium) and 5% sterile horse blood or cultured in liquid medium in Brain Hearth Infusion (BHI) (Roche, Belgium) broth under anaerobic (80% N2, 10% H2, and 10% CO2) conditions.
BHI medium was used for the assembled synthetic community. This medium is enriched with 2.5 g/L Mucin from porcine stomach type III (Sigma, Diegem, Belgium), 1.0 g/L Yeast extract (Oxoid, Hampshire, UK), 0.1 g/L cysteine (Merck—Calbiochem), 2.0 g/L sodium bicarbonate (Sigma Aldrich, Belgium), 0.25% glutamic acid (Merck—Calbiochem), 5.0 mg/L hemin (Sigma Aldrich, Belgium), 1.0 mg/L menadione (Sigma Aldrich, Belgium).

Experiments were started 24 h after isolation of hepatocytes (20 h after attachment). At this time point, it was assumed that no effects of insulin or dexamethasone are present. As a model of oxidative stress-induced apoptosis, we used menadione (2-methyl-1,4-naphthoquinone). The quinone menadione undergoes one- electron reduction catalyzed by NADPH-cytochrome P-450 reductase or NADH ubiquinone oxidoreductase, yielding a semiquinone reactive intermediate that undergoes redox cycling to generate superoxide anions (O₂^•−) (Thor et al. 1982 (link)). This model has been described before and induces predominantly apoptotic cell death in primary cultures of rat hepatocytes (Conde de la Rosa et al. 2006 (link)). Cells were exposed to menadione (50 μmol/L; Sigma) for the indicated time and/or metformin (0.1–0.5 mmol/L), added 10 min before menadione treatment. The concentrations of metformin used were based on our previous study in which the protective effect of metformin on bile acid-induced apoptosis was described and represent the two lowest effective concentrations of metformin against menadione-induced apoptosis (Woudenberg-Vrenken et al. 2013 (link)). In some experiments, the insulin-sensitizing drug rosiglitazone (0.5–10 μmol/L) and insulin (50 nmol/L) was used.