Cacl2 2h2o

Presence of Ag, Ca, and P ions due to material leaching following 1-day incubation in an aqueous environment was detected using an Agilent ICP-MS apparatus (Agilent Technologies, Santa Clara, CA, USA). Coated PMMA sheets (n = 3 in each group) were incubated in 5 mL of distilled water for 24 h, in a 37 °C incubator. Samples that were used for longer-term assessments were incubated in ATF, pH 7.4. The ATF was prepared as previously described by mixing 0.67% (w/v) of NaCl (Sigma-Aldrich), 0.22% (w/v) of NaHCO₃ (Sigma-Aldrich), 0.008% (w/v) of CaCl₂·2H₂O (Sigma-Aldrich), and 0.14% (w/v) of KCl (Sigma-Aldrich) in distilled water [48 (link)]. The ATF was refreshed daily. The ATF was substituted with distilled water, 24 h before the pre-determined collection timepoints (days 14 and 28). The substitution was to eliminate the detection of Ca and P elements present in the ATF, through the ICP-MS, in the final analysis. The following parameters of ICP-MS were applied to assess the trace elements in water—1500W radio frequency (RF) power; 1.8V RF matching; 10 mm sampling depth; 0.2 mm torch-H; −0.6 mm torch-V; 1.07 L/min gas flow rate; 0.1 rps nebulizer pump; and 2 °C spray chamber (S/C) temperature.

The bacterial strains and plasmids used in this study are listed in supplementary Table S1. All the cultures were stored in 10% (v/v) skim milk at − 80 °C. PAO1, the non-mucoid P. aeruginosa strain was grown at 37 °C in biofilm minimal medium (BMM)^{16 (link)}, which contained (per liter): 9.0 mM sodium glutamate, 50 mM glycerol, 0.02 mM MgSO₄, 0.15 mM NaH₂PO₄, 0.34 mM K₂HPO₄, and 145 mM NaCl, 200 µl trace metals, 1 ml vitamin solution. Trace metal solution (per liter of 0.83 M HCl): 5.0 g CuSO₄.5H₂O, 5.0 g ZnSO₄.7H₂O, 5.0 g FeSO₄.7H₂O, 2.0 g MnCl₂.4H₂O. Vitamins solution (per liter): 0.5 g thiamine, 1 mg biotin (Gold bio). The pH of the medium was adjusted to 7.0. When needed, 5 mM CaCl₂^.2H₂O (Sigma) was added. PAO1 was used to obtain genomic DNA for cloning efhP. For DNA manipulations, E. coli and P. aeruginosa cultures were grown in Luria–Bertani (LB) broth (per liter: 10 g tryptone, 5 g yeast extract, 5 g NaCl) at 37 °C with shaking at 200 rpm. Antibiotics used for E. coli were (per ml) 50 μg kanamycin (Kan) and 100 μg ampicillin (Amp) and for P. aeruginosa, (per ml) 60 μg tetracycline (Tet), and 100 µg carbenicillin (Carb).
No live vertebrate animals were used in this study.

For determination of glucose-stimulated insulin secretion, 20 human islets per well in duplicate were pre-incubated in a 24-well plate in 400 μL modified Krebs-Ringer buffer (115 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L KHP₂O₄, 1.2 mmol/L MgSO₄, 5 mmol/L NaHCO₃ (all from Merck, Darmstadt, Germany), 2.6 mmol/L CaCl₂ × 2H₂O, 0.2% BSA (w/v), 2 mmol/L glutamine (all from Sigma-Aldrich, Soeborg, Denmark), 20 mmol/L Hepes (Life Technologies, Naerum, Denmark), pH adjusted to 7.4; KRBH) supplemented with 2 mmol/L glucose (Sigma-Aldrich, Soeborg, Denmark) for 1.5 h at 37 °C in a humidified atmosphere with 5% CO₂. Islets were subsequently moved to fresh KRBH supplemented with 2 mmol/L glucose for 30 min at 37 °C, followed by collection of 200 μL supernatant. Two-hundred μL KRBH supplemented with 38 mmol/L glucose were added to wells for a final concentration of 20 mmol/L glucose for 30 min at 37 °C. Supernatants were collected and islets were lysed in complete lysis buffer. Twenty-four h insulin release was measured in supernatants from GLT or vehicle treated INS-1E or EZH2 KD and HET cells. Insulin concentrations of supernatants and lysates were determined using an in-house rat insulin ELISA [63 (link)] or Human Insulin ELISA kit (Sigma-Aldrich, Soeborg, Denmark).

DSP was purchased from Carbosynth Ltd. (Compton, UK). Low methoxy amidated pectin (CF 005, degree of esterification 35%; degree of amidation 15%; further denoted as pectin) was kindly donated by Herbstreith & Fox (Neuenbürg, Germany). Hypromellose (Metolose^® SH 4000) was obtained by courtesy of Shin-Etsu Chemical Co., Ltd., Tokyo, Japan. Lactose monohydrate (GranuLac^® 200; further denoted as lactose) was obtained from Meggle (Wasserburg am Inn, Germany). Mannitol was purchased from BDH Prolabo (Lutterworth, UK). Simulated Nasal Fluid (SNF) was prepared by dissolving NaCl (150.0 mM; Kemig, Zagreb, Croatia), KCl (40.0 mM; Kemig) and CaCl₂ × H₂O (5.3 mM; Sigma-Aldrich, Munich, Germany) in distilled water.
For cell biocompatibility and permeability studies in vitro, Hank’s balanced salt solution with 5.3 mM Ca²⁺ (HBSS-Ca²⁺; pH 7.4) was prepared by dissolving KCl (5.4 mM), NaHCO₃ (4.2 mM), NaCl (136.9 mM), D-glucose monohydrate (5.6 mM) (all purchased from Kemig), KH₂PO₄ (0.4 mM; Kemika, Zagreb, Croatia), Na₂HPO₄ × 2H₂O (0.3 mM; Fluka Chemie AG, Buchs, Switzerland) and CaCl₂ × 2H₂O (5.3 mM) (Sigma-Aldrich) in distilled water.

High density polyethylene (HDPE, Q3802, Q-Chem, Doha, Qatar) and expanded graphite (EG, GFG200, SGL Carbon, Wiesbaden, Germany) having an average size of 200 μm were used for the composite preparation. CaCl₂ 2 H₂O (Sigma Aldrich^®, St. Louis, MO, USA, purity ≥ 99 %) and Na₂SO₄ (Sigma Aldrich^®, purity 99.5%), formamide (Sigma Aldrich^®, purity ≥ 99.8%), and ethylene glycol (Sigma Aldrich^®, purity ≥ 99%) were used. Ultrapure water was obtained using an Ultrapure Water System NW Series (Heal Force Bio-Meditech Holdings, Ltd., Shanghai China).

Various chemicals including GO (40%), MGO (40%), methanol, hydrochloric acid, acetonitrile, sodium hydroxide, 4-nitro-1,2-phenylenediamine, sodium acetate (CH₃COONa), pancreatin (8 × USP specifications from pig pancreas), lipase (Type II from pig pancreas, 100–500 U/mg protein), alpha-amylase (from Aspergillus oryzae powder, 1.5 U/mg), pepsin (from pig gastric mucosa solid, lyophilized powder, 250 U/mg), mucin, NaCl, KCl, CaCl₂·2H₂O, NaHCO₃, urea, bovine serum albumin, uric acid, bile salts, and other chemicals were obtained from Sigma–Aldrich (St. Louis, MO, USA).

For peptaibol production, approximately 10⁷Trichoderma spores were added to a 500 mL Erlenmeyer flask containing 200 mL of TLE medium [CaCl₂.2H₂O 0.3 g.L^-1 (Sigma-Aldrich), KH₂PO₄ 2.0 g.L^-1 (Vetec), (NH₄)₂SO₄ 1.4 g.L^-1 (Vetec), MgSO₄.7H₂O 0.3 g.L^-1 (Vetec), urea 0.3 g.L^-1 (Vetec), peptone 1.0 g.L^-1 (MicroMed), trace elements solution (Fe²⁺, Zn²⁺, Mn²⁺, Cu²⁺) 0.1% (v/v) and glucose 0.3% (w/v, Sigma-Aldrich). The flasks were incubated on a rotary shaker at 28°C, with 120 rpm in the dark for 5 days.