Empower pro software

For ¹H-NMR, a solution was prepared (6% (w/w)) of MeHA polymer in D₂O and analyzed on a 300 mHz Gemini Spectrometer (Varian, Palo Alto, CA). All samples were measured with a relaxation delay of 0.6 seconds for 512 scans. The degree of methacrylate substitution was calculated by comparing the integrals of the peaks of the methacrylate groups at 1.9, 5.7 and 6.1 ppm relative to the integrals originating from the protons of HA.
For the HPLC measurements, 15 mg of MeHA was dissolved in 10 ml of 0.02 M NaOH solution, and incubated at 37°C for 30 minutes to ensure complete hydrolysis of the ester bonds. After that, 2 ml of 2 M acetic acid (Merck) was added to acidify the solution. Samples were filtered over 0.2 μm filters and injected onto a Sunfire RP-18 column (Lichrospher, Darmstadt, Germany). Samples were analyzed using a HPLC Waters 2695 system equipped with an UV detector model 2487 (λ = 210 nm, Waters Inc., Dublin, Ireland). The mobile phase consisted of a mixture of acetonitrile (Actu-All Chemicals, Oss, the Netherlands) and water (ratio: 15/85) adjusted to pH 2 with perchloric acid (HClO₄, 70%, Sigma-Aldrich) and a flow rate of 1 ml/min was used. A calibration curve was obtained by injection of methacrylic acid solutions in eluent with a concentration range of 0 to 160 μg/ml. Empower Pro software (Waters) was used to calculate the concentration of methacrylic acid.

The HPLC analysis was performed on a liquid chromatograph with a DAD detector (M2998), operated by the Empower-Pro software (Waters). The separation was carried out on an RP-18 column (Luna 3 µm, 4.6 mm x 150mm) in the following gradient system: A—100% ACN, B—redistilled water, C—100% MeOH: 0–15 min, 18–22% AC, 15–27 min, 22–25% AC, 27–35 min, 25–30% AC, 35–45 min, and 30–50% AC. The flow rate was 1 mL/min, and the sample volume dispensed was 20 µl. Detection of the obtained chromatographic separations was carried out at two wavelengths—λ = 280 nm and λ = 330 nm.

Chromatographic analysis was carried out in a Waters 2695 separation module system with a Waters 2695 photodiode matrix detector and Empower Pro software (Waters Corporation, Milford, MA, USA). Chemical separation was performed using a Supelcosil LC-F column (4.6 mm × 250 mm i.d., particle size 5 μm) (Sigma-Aldrich, Bellefonte, PA, USA). The mobile acid phase was performed using 0.5% triflouroacetic, aqueous solution (solvent A), and acetonitrile (solvent B) gradient: 0–1 min, 0% of B; 2–3 min, 5% of B; 4–20 min, 30% of B; 21–23 min, 50% of B; 24–25 min, 80% of B; 26–27 min, 100% of B; 28–30 min, 0% of B. The flow rate was 0.9 mL/min with a volume of 10 μL sample. Absorbance was measured at 270 nm [46 (link)]. A preliminary identification of the peaks resolved was performed by comparison with t_R and UV-Vis characteristic bands of known standards and literature data.

The HPLC system consisted of a Waters Alliance 2695 chromatograph equipped with a column heater and an autosampler with a sample cooling device, coupled to a Waters 2996 U/V–Vis photodiode array detector. Data acquisition and integration were performed with Empower Pro Software from Waters. Separation conditions were as described previously [21] (link). Briefly, the column was an Aquapore RP-300, C8, 7 µm, 220×2.1 mm i.d. (Brownlee) and was maintained at 50°C. Mobile Phase A (MP A) consisted of 0.05% trifluoroacetic acid (TFA) in 10% acetonitrile/90% water; Mobile Phase B (MP B) was 0.05% TFA in 90% acetonitrile/10% water; Mobile Phase C (MP C) was 0.05% TFA in acetonitrile. The column was equilibrated with a mixture of MP A and MP B (70∶30) until a stable baseline was obtained. Elution was carried out using a multi-step gradient consisting of MP A/MP B (70∶30) for 1 min (at 0.7 ml/min), linear gradient to MP A/MP B (65∶35) over 5 min (at 0.7 ml/min), linear gradient to MP A/MP B (61∶39) over 19 min (at 0.7 mL/min), linear gradient to MP A/MP B (50∶50) over 10 min (at 0.7 mL/min), linear gradient to MP C over 5 min (at 1.0 mL/min), MP C for 12 min, linear gradient to MP A/MP B (70∶30) over 3 min. The effluent was monitored at 220 nm.

Thousand seed weight, a measure of yield, was determined as five times the mass of three groups of 200 randomly selected seeds as described [56 ]. To determine DON contamination, three or five biological replicates of 1.0 g of pooled ‘Fielder’ grains or individual spikes, respectively, were ground to a fine powder in liquid nitrogen. DON was extracted in five volumes of 84 % (v/v) acetonitrile by shaking at 220 rpm, 25 °C for 2 h. DON was quantified, relative to a commercial DON standard (Sigma-Aldrich; St. Louis, MO), by LC–MS through modification of [57 (link)]. Briefly, DON was separated with a Waters 2695 LC coupled with a Waters Symmetry C18 column (100 × 2.1 mm ID, 3.5 µm) with mobile phases of 0.3 % (v/v) acetic acid (A) and 95 % (v/v) methanol: 0.3 % (v/v) acetic acid (B) using a gradient elusion from 0 to 7 min: 99 % A, 7 to 25 min: 67 % A 33 % B, and 25 to 30 min 99 % A. The flow rate and column temperature were maintained at 0.2 ml/min and 25° C, respectively. Mass spectrum analysis was performed using a Waters 3100 Mass Detector fitted with ESI in negative ion mode and an optimized 40 V cone voltage. DON was detected at m/z of 355.3 Da and analyzed using Empower Pro Software (Waters, Milford, MA).

Recombinant human iNOS and nNOS, were purchased from Enzo Life Sciences, Inc. (New York, USA), while bovine recombinant eNOS was purchased from Cayman Chemicals (Ann Arbor, Michigan, USA). HPLC analyses were performed using a Waters system composed of a P600 model pump, a 2475 multi‐fluorescence detector, and a 7725i model sample injector (Rheodyne, Cotati, CA, USA) equipped with a 5 μL loop. The column was a XTerra MS C8 (250×4.6 mm i.d., 5 m particle size; Waters). A column thermostat oven module Igloo‐Cil (Cil Cluzeau Info Labo, France) was used. Chromatograms were recorded on a Fujitsu Siemens Esprimo computer and data were processed by the Empower Pro software (Waters). The appropriate mobile phase was prepared daily, filtered through a 0.45 μm WTP membrane (Whatman, Maidstone, UK), sonicated and degassed before use. Column temperature was kept constant at 20 °C. All assays were performed in triplicate.

Mice were fasted overnight and then sacrificed. Blood samples were obtained from the retro-orbital plexus. Triglyceride (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) were determined by commercially enzymatic methods (test kits, Nanjing Jiancheng Biotech Inc. Shanghai, China), according to the manufacturer’s instructions. The sterol analyses were performed using high-performance liquid chromatography (HPLC) analysis (Model 2790, controlled with Empower Pro software; Waters Corp., Milford, MA)[20 (link)].