Bovine milk

Synthetic cystic fibrosis sputum medium (SCFM) containing mineral salts, amino acids and 1% (w/v) glucose was used as a base for all liquid media and prepared as previously described [34 (link)]. Additions to SCFM featured 1% (w/v) casein from bovine milk (Sigma-Aldrich, Australia; SCFM+C) or 1% (w/v) mucin from porcine stomach (type III; Sigma-Aldrich, Australia; SCFM+M). Mucin and casein were suspended in Milli-Q H₂O; sodiumhydroxide was added to dissolve casein. These substrates were autoclaved separately and mixed with SCFM just before use. SCFM was sterilized by filtering through a 0.22 μm membrane (Millipore) and final pH of media was adjusted to 5.7 to support fungal growth [35 (link)].

The redox potential (E₀) of the enzyme‐bound FAD was measured by the dye‐equilibration method using the xanthine/xanthine oxidase system as described by Massey 27. The concentrations of enzyme and redox dye were chosen in a way that their absorption maxima were in the same range. The reactions were performed with a Hi‐Tech stopped flow device (SF‐61DX2; TgK Scientific Limited). The measurements took place under anaerobic conditions in a glove box (Belle Technology) at 25 °C in 50 mm HEPES/NaOH, 150 mm NaCl, pH 7.0. The simultaneous reduction of FAD and the redox dye was monitored with a KinetaScanT diode array detector (MG‐6560; TgK Scientific Limited). The reaction was started by mixing a solution containing 300 μm xanthine, 5 μm benzyl viologen, and an appropriate amount of enzyme with a solution containing catalytic amounts of xanthine oxidase (approximately 200 nm, from bovine milk, Grade III purity; Sigma‐Aldrich) and the redox dye indigotrisulfonic acid dipotassium salt (E₀, pH 7.0, 25 °C = −81 mV). The redox potential was calculated using double logarithmic plots, log(ox/red) of the enzyme versus log(ox/red) of the dye, according to Minnaert 39. A linear least‐squares fit was done with Excel 2010 (Microsoft, Redmond, WA, USA).

Fluorescence microscopy was performed on a wide-field microscope (Olympus MT20) equipped with a 150 W mercury-xenon burner, a motorized stage, a × 40 dry objective (Olympus UPLSAPO N/A=0.95) and an EM-CCD camera (Hamamatsu C9100). The GFP signal was acquired using a 474/23 excitation filter and a 525/45 emission filter; the mCherry signal was acquired using 562/40 and 641/75 filters for excitation and emission, respectively. Cell suspensions were transferred to a 96-well glass-bottom plate (Matrical Bioscience) and image acquisition was started after allowing cells to settle down gravitationally for approximately 5 min. For time-lapse experiments using stimulation with synthetic α-factor, wells of the glass-bottom plate were coated with type-IV Concanavalin A (Sigma-Aldrich) prior to the transfer of cell suspensions. synthetic α-factor (Sigma-Aldrich) was prepared as 11 × stocks in 11 μM sodium salt of casein from bovine milk (Sigma-Aldrich) and added to the cell suspensions to reach the desired final α-factor concentration and 1 μM casein concentration. Image acquisition was started immediately after α-factor addition and repeated periodically at defined time intervals over the course of several hours. The average sample size was 227 cells.

The direct inhibitory effect of BLEx and its fractions on XOD activity was evaluated as the conversion of xanthine to Uric acid under XOD (from bovine milk, Sigma). Uric acid, xanthine, and allopurinol (Fujifilm Wako) were dissolved in 0.1 M phosphate buffer (PB; pH 8.0). Next, 50 μL PB, 50 μL samples (appropriate concentrations of BLEx, fractions, or 1 mM allopurinol), and 100 μL of 1 mM xanthine were added to a 96-well plate (UV-star, Greiner Bio-One, Kremsmünster, Austria). Finally, XOD was added to a final concentration at 0.1 mU and reacted for 3 min at 37 °C. The production of Uric acid was measured as the absorbance at 293 nm using SpectraMax (Molecular Devices, San Jose, CA, USA).

LPL activity was determined by measuring the generation of non-esterified fatty acids (NEFA). In a 96-well plate, 0–2 µM of an ApoC2 mimetic peptide, 0.83 nM of LPL from bovine milk (Sigma-Aldrich), ApoC2-deficient ethylenediaminetetraacetic acid (EDTA)-plasma (diluted with PBS to a final TG concentration of 0.67 mg/dl), 2 IU/ml heparin, 0.2% fatty acids free bovine serum albumin (BSA) (ICN Biomedicals), and PBS (Life Technologies) were combined in a final reaction volume of 50 μl. The reaction mixture was pre-incubated on ice for 30 min and incubated at 37°C for 1 h. NEFA were measured with a coupled enzyme reaction (Wako Diagnostics) in a Synergy H1 microplate reader (BioTek, USA).

Galactosyl-cello-oligosaccharides were synthesized using a modified procedure from literature^{43 (link)}. Briefly, 1 mg of cellotriose or cellotetraose was dissolved in 500 µL of 40 mM sodium cacodylate buffer pH 6.8 containing 40 mM MnCl₂, 5 mg of UDP-Gal and 0.5 mg of galactosyl transferase from bovine milk (Sigma-Aldrich). The galactosyl transferase will add one galactose to the non-reducing end of the cello-oligosaccharides, forming a β-(1–4) glycosidic bond⁴⁴ . The reaction was left to proceed for 3 days at 37 °C with shaking at 1,400 rpm. The reaction mixture was left to cool down to room temperature and was directly applied to a CarboGraph column (Grace, Columbia, USA) for desalting of the oligosaccharides, essentially according to a previously published protocol^{45 (link)}.