Chloramine t

We measured hydroxyproline content following the manufacturer’s protocol (MilliporeSigma, MAK008). Briefly, liver tissue was homogenized in distilled water and mixed with an equal volume of concentrated hydrochloric acid (~12N HCl), after which homogenates were incubated at 120°C for 3 hours, then oxidized with Chloramine T (MilliporeSigma, MAK008), followed by enzymatic reaction with 4-dimethylaminobenzaldehyde solution. Sample absorbance was measured at 560 nm in duplicate. Hydroxyproline content was expressed as micrograms of hydroxyproline per milligram of liver.

Plasma AOPP concentrations were assessed as chloramine-T equivalents as described previously [18 (link)]. Briefly, 200 μL of each plasma sample (diluted 5 times in PBS) was placed into a 96-well plate, and then 10 μL of 1.16 M potassium iodide (Sigma, St. Louis, MO, USA) and 20 μL of acetic acid (Sigma, St. Louis, MO, USA) were added. Two hundred microlitres of chloramine-T (Sigma) at different concentrations (0, 20, 40, 80, and 100 μM) were used for calibration. The absorbance at 340 nm was read immediately by a Spectra Max M5 spectrophotometer (Molecular Devices, San Francisco, CA, USA). The AOPP concentrations are expressed as μmol/L of chloramine-T equivalents.

To compare the total amount of collagen in lung tissues after nickel nanoparticle exposure, hydroxyproline content in mouse left lungs was measured as previously described [39 (link)]. This method is based on the acid hydrolysis of the lung tissue and subsequent determination of the free hydroxyproline in hydrolyzate. Briefly, left lung tissues were hydrolyzed in 1 mL of 6 N HCl at 100 °C overnight. After cooling to room temperature, the hydrolysate was neutralized with 6 N NaOH to approximately pH 6.0. Then 3% chloramine-T (Sigma-Aldrich, St. Louis, MO, USA) solution containing 50% isopropanol and 0.5 M sodium acetate (pH 6.0) was added to oxidize the free hydroxyproline for the production of a pyrrole, followed by the addition of perchloric acid (Sigma-Aldrich, St. Louis, MO, USA). The addition of Ehrlich’s reagent (5% of 4-dimethylaminobenzaldehyde in methanol) resulted in the formation of a chromophore that can be measured at 560 nm. The amount of hydroxyproline was determined against a standard curve generated by using known concentrations of hydroxyproline (Sigma-Aldrich, St. Louis, MO, USA).

AOPPs were measured in plasma and tissue samples. For plasma determination, 20 μL of plasma and 980 μL of PBS were mixed to 50 μL of KI 1.16 M and 100 μL of acetic acid.
The absorbance of the reaction mixture was read at 340 nm and the AOPPs were quantified in μmol/mg of proteins using Chloramine-T (Sigma-Aldrich, Milan, Italy) as standard for the calibration curve.
AGEs were determined on 100 μL of 1:5 PBS-diluted plasma, reading the fluorescence intensity at 460 nm, after excitation at 355 nm. Results were expressed as arbitrary units (AU).
Tissues were first homogenized using an Ultraturrax homogenizer on PBS and subsequently centrifuged at 13,000 rpm at 4 °C. The supernatant (400 µL) was divided into aliquots used for AOPPs and AGEs determination as described above. Proteins content in plasma and tissue samples was determined by using the Bio-Rad DC protein assay kit (Bio-Rad, Milan, Italy) and albumin as standard for the calibration curve.

Folin-Ciocalteu reagent, potassium acetate, sodium acetate, 2,2-diphenyl-1-Picrylhydrazyl (DPPH), 2,2′-Azino-di-(3-Ethylbenzthiazoline Sulfonic acid) (ABTS), Potassium hexacyanoferrate, thiobarbituric acid, xylenol orange, quinine hemisulfate and chloramine-T were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetic acid (glacial) and formic acid were purchased from Merck KGaA (Darmstadt, Germany). Phytochemical standards, including quercetin, gallic acid, catechin and p-hydroxybenzoic acid, were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Mating interactions between Rm ATCC 52813 and ATCC 52814 were conducted on PDA (Sigma) as described above, and zygospores were analyzed after 7 days of incubation in the dark at 30 °C. A tuft of mycelium containing zygospores was removed from the mating zone and placed in 10% w/v chloramine T (Sigma) for 20 min to kill hyphae. The tuft was subsequently transferred to sterile water and shaken slowly for 5 min at room temperature, followed by two additional water washes lasting for 20 min. The mycelial tuft was then transferred onto a sterile 1.5% water agar and zygospores were collected with sterile forceps, taking care to remove all attached hyphae. A total of 80 zygospores from two separate mating interactions were transferred individually into 0.2-mL PCR tubes, crushed, and subjected to whole-genome amplification (WGA) with the Illustra GenomiPhi Kit v2 (GE) to generate template DNA for multiple PCR reactions per zygospore. A volume of 1 μL of the last water wash was used as a negative control during WGA and subsequent PCR, with a total of 16 negative controls. PCR was performed on the 1/20-diluted WGA products using LR1 and NDL22^{36 (link)} as well as Burkholderia-specific primers^{34 (link)} to detect the presence of fungal and bacterial DNA in zygospores.

The left lungs were removed and the sample was homogenized in 1 mL of PBS and hydrolyzed with 1 mL of HCl for 16 h at 120 °C. The supernatant was centrifuged at 10,000g for 5 min (Model 3740, KUBOTA, Tokyo, Japan), and 5 µL of the supernatant was aliquoted into a 96-well plate. After dispensing 5 µL hydroxyproline standard (Sigma-Aldrich) into each well of the 96-well plate, 5 µL citrate/acetate buffer (deionized distilled water supplemented with 238 mM Citric acid, Sigma-Aldrich, 1.2% glacial acetic acid, Sigma-Aldrich, 532 mM sodium acetate, Sigma-Aldrich, and 850 mM sodium hydroxide, Nacalai Tesque) and 100 µL chloramine T solution (1.0 mL deionized distilled water supplemented with 0.141 g chloramine T, Sigma-Aldrich, 1.0 mL 1-propanol, Sigma-Aldrich, and 8.0 mL citrate/acetate buffer) were added. After 30 min of incubation at 25 °C, 100 µL of Ehrlich's reagent (2.5 g 4-dimethylaminobenzaldehyde, Sigma-Aldrich, 9.3 mL 1-propanol, and 3.9 mL 70% perchloric acid, Sigma-Aldrich) was added and the mixture was incubated at 65 °C for 30 min. After 5 min at 25 °C, the absorbance was measured at 550 nm using a plate reader (iMARK, Bio-Rad, Hercules, CA, USA), as previously described [21 (link)].