Diethylenetriaminepentaacetic acid

1,4-Benzoquinone, dimethyl sulfoxide, oligomycin A, adenosine 5’-triphosphate (ATP) disodium salt hydrate, 1-octane-sulfonic acid, sodium phosphate, acetonitrile, glutathione, glutathione disulfide, and diethylenetriaminepentaacetic acid were obtained from Sigma Aldrich (St. Louis, MO, USA).

Three P. aeruginosa strains (ATCC 27853, ATCC 2110, and ATCC 2112) were selected for this assay. The strains ATCC 2110 (resistant to ampicillin, cefazolin, cefotaxime, cefoxitin, nitrofurantoin, trimethoprim-sulfamethoxazole, and tetracycline) and ATCC 2112 (resistant to amoxicillin-clavulanic acid, ampicillin, cefazolin, cefpodoxime, ceftriaxone, cefotaxime, cefuroxime, cefoxitin, nitrofurantoin, trimethoprim-sulfamethoxazole tetracycline, and tigecycline) were chosen due to their multidrug-resistant characteristics. The strain ATCC 27853 was selected due to the absence of antibiotic resistance. Each strain was evaluated solely in triplicate.
To simulate in vivo conditions, SCFM2 artificial sputum medium (4 g DNA salmon sperm, GoldBio, St Louis, MO, USA; 5 g swine stomach mucin, Sigma-Aldrich; 5 g casamino acids, Difco; 5.9 mg diethylenetriamine pentaacetic acid, Sigma-Aldrich; 5 g NaCl, Dynamic; 2.2 g KCl, Dynamic; 5 mL egg yolk emulsion; and 1000 mL distilled water, pH = 6.9) that mimics a cystic fibrosis model was employed [42 (link)]. After connecting to the flow system, phage cocktail-coated and non-coated tubes were supplied with a continuous flow of SCFM2 culture medium inoculated with 1 × 10⁵ CFU/mL of P. aeruginosa strains for 24 h. After 24 up to 168 h, the system was supplied with sterile SCFM2 culture medium without recirculation [33 (link)].

The peptides PSELT (H-Phe-Gln-Ile-Cys-Val-Ser-Sec-Gly-Tyr-Arg-OH), [Ser46,49] PSELT [called inert PSELT (I-PSELT) as a control], and PSELT-dansyl were synthesized by Fmoc solid phase methodology on an automated peptide synthesizer (CEM, Saclay, France) as previously described [19 (link)]. KCl, NaCl, NaHCO₃, CaCl₂, MgSO₄, KH₂PO₄, NaH₂PO₄, Na₂HPO₄, mannitol, glucose, Na-pyruvate, β-nicotinamide adenine dinucleotide (NADH), reduced disodium salt hydrate, 2,4 dinitrophenylhydrazine (DNPH), 2-thiobarbituric acid (TBA), bovine serum albumin (BSA), butylated hydroxyanisole, diethyl ether, ethylenediaminetetraacetic acid (disodium salt), diethylenetriamine pentaacetic acid, pyrogallol, streptomycin sulfate, tween-20, hydrogen peroxide (H₂O₂), and urea were purchased from Sigma Aldrich (St. Louis, MO, USA). Tunicamycin (TM) was from Cayman Chemical (Ann Arbor, MI, USA), Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12), penicillin/streptomycin and fetal bovine serum (FBS) were provided by Thermo Fisher Scientific (Waltham, MA, USA). All the solutions were freshly prepared before starting the experiments. Absolute ethanol, ethyl acetate, hydrochloric acid, methanol, and trichloroacetic acid (TCA) were from Carlo Erba Reagents (Cornaredo, Milano, Italy).

BMDMs were isolated and cultured for 7 days as described above. On day 7, cells were incubated with 50, 200 or 500 µM sodium ascorbate (Sigma Aldrich, St. Louis, MO, USA) and harvested at 30 min or 24 h to measure ascorbate content, or with 500 μM sodium ascorbate for 0, 2, 4, 6, 8 and 24 h. Wells were washed twice with PBS (Life Technologies, Carlsbad, CA, USA) and cells detached with two rounds of incubation in TrypLE (Life Technologies, Carlsbad, CA, USA) (200 µL each) and vigorous trituration. Cells were pelleted in microfuge tubes using a swing out rotor (500× g for 3 min at RT) and extracted with 0.54 M perchloric acid containing Diethylenetriamine penta-acetic acid (Sigma Aldrich, St. Louis, MO, USA) for ascorbate analysis [33 (link)]. Precipitated proteins were pelleted (10,000× g for 10 min at 4 °C) and ascorbate was measured in the supernatant using HPLC with electrochemical detection as previously described [34 (link)]. The concentration was assessed relative to standards ranging from 1.25 to 40 μM ascorbate made fresh for each analysis.

A pool of dialyzed HDLs isolated from healthy subjects (4 mg protein/mL) was incubated with 1 mmol/L and 10 mmol/L potassium cyanate (KCN) in PBS containing 100 μmol/L diethylenetriaminepentaacetic acid (Sigma-Aldrich) for 4 h at 37 °C, as previously described [27 (link)]. Control HDLs were incubated under the same conditions with PBS instead of potassium cyanate. HDLs were then immediately and extensively dialyzed three times against sterilized 0.01% (wt/vol.) EDTA endotoxin-free PBS for 18 h at 4 °C in the dark, and then immediately used for analysis.