Ultrasonic processor

Cellular uptake of rutin in TBFEs was measured as described by Zhang et al. and Go et al. [36 (link),37 ], with slight modification. Cells (1 × 10⁶ cells/2 mL) were incubated overnight at 37 °C and treated with TBFEs (24-fold diluted) or standard rutin (equivalent to the rutin amount in the autoclaved sample) for 24 h. After centrifugation (2500× g, 3 min) at 4 °C, the cell pellets were resuspended in RIPA buffer and lysed using an ultrasonic processor (Sonics & Materials Inc., Newtown, USA). After centrifugation (2500× g, 3 min) at 4 °C, two volumes of acetonitrile were added to the supernatants. After vortexing, the solutions were centrifuged (10,000× g, 3 min) at 4 °C, and the supernatants were concentrated using a nitrogen evaporator (MG-3100, Eyela, Tokyo, Japan). Finally, rutin concentrations were determined by HPLC.

At indicated time points, worms were washed off the plate with ddH₂O and washed three times to remove bacteria. Worms were resuspended in lysis buffer (20 mM HEPES pH 7.5, 25 mM KCl, 0.5% NP-40) and disrupted by two 5 s pulses using an Ultrasonic Processor (Sonics) at 50% amplitude. Protein concentrations were determined using Coomassie Plus Protein Assay Reagent (Thermo Scientific). Protein samples were incubated for 10 min at 95°C in NuPage LDS Sample Buffer (Invitrogen) containing 10 mM DTT then subjected to electrophoresis using NuPage 4–12% Bis-Tris gels (Invitrogen) and transferred to Amersham Hybond ECL nitrocellulose (GE Healthcare). Blots were incubated with mouse anti-FLAG antibody (Sigma cat# F3165) along with monoclonal β-tubulin antibody (MP Biomedicals LLC cat# 08691261) in 1x Tris-buffered saline (0.01% Tween-20) containing 5% non-fat dry milk. Horseradish peroxidase-conjugated secondary antibodies (Jackson Laboratories) were visualized using Western Lighting Plus-ECL Chemiluminescence Substrate (Perkin Elmer). Three biological replicates were performed and NHR-14::GFP::FLAG was quantified by densitometry analysis using Image J (NIH).

SLNs containing 125, 375, or 500 µL L⁻¹ of 2-nonanone or 2-tridecanone were synthesized by high shear homogenization, followed by an ultrasonication method [30 ,55 (link)]. The lipid phase composed of glyceryl tristearate (Sigma-Aldrich^®) (5% w/v) was dissolved in 4 mL of hexane (MerckMilipore^®, Bedford, MA, USA) at 70 °C. Subsequently, 125, 375, or 500 µL L⁻¹ of 2-nonanone or 2-tridecanone (Sigma-Aldrich, St. Louis, MO, USA) was added to the lipid phase. The aqueous phase of Tween 80 at 1.5% w/v was dissolved in 40 mL of distilled water at 70 °C. The aqueous phase was gradually added to the lipid phase and homogenized at 10,000 rpm for 5 min using Ultraturrax OV5 (Velp^®, Scientifica, Usmate Velate, Italy). Then, the resulting solution was sonicated with an ultrasonic processor (Sonics and Materials, Newtown, CT, USA) for 5 min (5 s on/off, 35% amplitude) and cooled at 4 °C. Then, SLNs containing 2-ketones were kept at 4 °C until their use (vertical refrigerated cabinet, 2–8 °C, 290 L, HYC-290, Haier^®, Qingdao, China).

The bacterial adherence to the surface was determined using a 12-well polystyrene plate (Bar-Naor, Petah Tikva, Israel). Ten μL of bacterial suspension were mixed with 3 mL of TSB containing 2.5% lactose and seeded in each well. The plate was incubated at 37 °C under static conditions for different times (0 h–8 h, 16 h, 17 h, 24 h, 32 h, 48 h, and 72 h). Viable bacteria were quantified after washing the wells twice with phosphate-buffered saline (PBS) solution, dislodging the adhered bacteria by scraping and re-suspending in PBS. The biofilms generated at different time points (16 h, 17 h, 24 h, 32 h, 48 h, and 72 h) were subjected to mild sonication with an Ultrasonic processor (Sonics, Newtown, CT, USA) for 20 s (amplitude, 20%; pulse, 10 s; pause, 10 s). After vortexing, the culture was serially diluted, plated on LB agar, and colonies were counted. The biofilm samples were also stained with 0.1% crystal violet (CV; Merck, Darmstadt, Germany) following a previously described procedure [22 (link)]. Briefly, the wells were washed twice with DDW, stained with crystal violet, incubated for 20 min, washed twice with DDW, and dried overnight. The crystal violet was then eluted with 33% acetic acid for 30 min, and the absorbance was measured at 595 nm using a spectrophotometer.

Initially, a starter bacterial suspension was generated for each of the tested bacteria. Thus, the cells of B. licheniformis and P. aeruginosa were grown in LB at 37°C in a shaker incubator at 150 rpm for 5 h. Meanwhile, the peptide-coated and uncoated stainless steel surfaces were placed into a sterile 12-well culture plate. Then, 10 μl of the bacterial suspensions were dripped onto the surfaces and incubated for 30 min at room temperature to allow the initial adhesion of bacteria to the surfaces. Afterward, 3 ml of suitable growth medium was added to each plate and the samples were incubated for 18, 42, or 66 h at 37°C. Next, the surfaces were washed using distilled water in order to remove non-adherent bacteria. The surfaces were then transferred into 15-ml test tubes with 1 ml of sterile distilled water and mildly sonicated for 20 s – amplitude, 20%; pulse, 10 s; pause, 10 s – with an ultrasonic processor (Sonics, Newtown, USA). The bacterial counts were detected using the colony-forming unit (CFU) method on LB agar plates that were incubated at 37°C overnight.