Ultrasonic liquid processor

The OECD-sponsored ZnO-NPs used in this study, Z-COTE (uncoated) and Z-COTE HP1 (coated with triethoxycaprylylsilane), are commercially available from BASF (Mississauga, Canada). Fine ZnO (bulk control particles, micro-sized) and zinc chloride (ZnCl₂, a soluble zinc compound) were purchased from Sigma-Aldrich (Oakville, Canada). ZnO suspensions and dispersions were prepared in deionized water of liquid chromatography and mass spectrometry application grade (LC/MS water, Fisher Scientific, Burlington, Canada) for primary particle analysis. Z-COTE, Z-COTE HP1, or fine-ZnO in powder form were transferred into sterile zinc-free glass tube with caps. Conditions for sonication were optimized for various times and power settings. Sonication was routinely performed with an Ultrasonic Liquid Processor (MISONIX, New York, USA) for 15 min at 30 % amplitude in a water bath while continuously cooling the samples with ice during sonication. The final energy was 76,148 J, and the power was 75–85 W. All stock dispersions were adjusted to a final concentration of 100 µg/mL, and then further diluted in either LS/MS water or appropriate medium to the desired concentration. Working dilutions were used freshly.

For the carotenoid determination, the spectrophotometric analysis was carried out after extraction [22 (link)]. The analyses were carried out in darkness to prevent carotenoid degradation and isomerisation. Before chemical extraction, leaves were homogenised in a blender and an aliquot of 5 g of the sample was weighed into a 50 mL amber coloured flask wrapped with aluminium foil. Then, 100 mL of the solvent mix (hexane/acetone/methanol 2:1:1 v/v/v) was added to the flask and sonicated continuously for 10 min (Misonix Ultrasonic Liquid Processor, Misonix, Inc. 1938, New Highway, Farmingdale, NY, USA).
The extraction was repeated until the sample became colorless. The combined extract was transferred to a separating funnel and 5 mL of distilled water was added to separate polar and nonpolar phases. The nonpolar hexane layer containing carotenoids was collected and concentrated in a rotary evaporator (Heidolph, Schwabach, Germany) until dry. The residue was dissolved in 10 mL of hexane. The total carotenoid content was determined by a spectrophotometric method using a UV-Vis spectrophotometer (Agilent 8453 Technologies, Agilent, Milan, Italy). The absorbance was read at 450 nm. All analyses were performed in triplicate and the results were expressed as mean ± standard deviation (SD).
The results were expressed as g β-carotene/100 g fresh weight of sample.

BL21(DE3) Escherichia coli cells were transformed with pET28 vectors encoding NEDD8, APPBP1, UBA3, Ubc12, their mutants, and fluorescent protein-labeled versions. The transformed bacteria cells were plated on LB agar plates containing 50 μg/mL kanamycin, and single colony cultures were inoculated in 2xYT medium. The expression of poly-histidine-tagged recombinant proteins was induced with 0.2 mM IPTG at 25 °C overnight. Bacterial cells were collected by centrifugation at 8,000 rpm 5 min, resuspended in buffer containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl and 5 mM imidazole, and sonicated with an ultrasonic liquid processor (Misonix, Farmingdale, NY). Cell lysate containing recombinant proteins was cleared by centrifugation at 35,000 g for 30 min. The recombinant proteins were then bound to Ni²⁺-NTA agarose beads (QIAGEN, Valencia, CA) and eluted with buffer containing 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, and 150 mM imidazole. Then the proteins were dialyzed into a buffer of 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 1 mM DTT. SDS-PAGE and Coomassie blue staining confirmed the purity of the proteins. Protein concentrations were determined using Coomassie Plus Protein Assay (Pierce) and concentrations of fluorescent-tagged proteins were determined using fluorescent protein concentration standard curves.