K2hpo4

To verify that only damaged areas were stained with TB dye, samples were studied using an inverted fluorescent microscope. First, leaves were artificially damaged with a Derma stamp (HudRoller Of Sweden; 36 microneedles; 1 mm) and Derma roller (HudRoller Of Sweden; 1 mm), mimicking dot-like lesions. Damaged leaf samples were immediately cleared. To achieve better visualisation of lesions, samples for microscopy were subjected to dual staining with TB and aniline blue (AB) dyes (Acros Organics). Aniline blue stains callose [59 –62 ], including trauma-induced callose deposited around lesions. Cleared leaves were first soaked in 0.01% TB staining solution in diH₂O (4 h), washed in diH₂O, and stained with 0.01% AB staining solution (2 h) in 150 mM K₂HPO₄ (Merck) [62 ], followed by washing in 150 mM K₂HPO₄. Leaf discs (Ø 10 mm) were then extracted from corresponding sites on TB-stained and dual-stained leaves (N = 5), using a coring tool (Harris Uni-Core). As a negative control for microscopy, discs from cleared leaves without the staining step were imaged (N = 5). Microscopy was carried out using a Zeiss Axio Observer D1 microscope (Carl Zeiss, Jena, Germany) as described previously [62 ].

The electrophoretic monitoring of topological changes in the plasmid DNA (pBR322, New England BioLabs, Ipswich, MA, USA) induced by FeSO₄ × 7H₂O (Lachema, Brno, Czech republic) was used to detect the DNA-damaging and DNA-protective potential of skyrin, as [39 ,40 (link)] described in detail. In brief, the reaction mixture (final volume of 10 μL) consisted of plasmid DNA (200 ng) and either 1 mM FeSO₄ × 7H₂O alone (positive control) or tested concentrations of skyrin (0.0001–100 μM) alone, or combinations of skyrin with 1 mM FeSO₄ × 7H₂O. 0.1 M phosphate buffer (1 M KH₂PO₄, 1 M K₂HPO₄, both purchased from Sigma Aldrich, St. Louis, MO, USA; pH 7.4) was added to all samples and they were incubated 50 min in the dark at room temperature. An analysis of changes in the DNA topology caused by DNA breaks was carried out by gel electrophoresis (in 1% agarose for 90 min/100 V). The DNA was stained with GelRed dye (1 mg/mL, Sigma Aldrich, St. Louis, MO, USA) and visualized by UV illumination (UV Transilluminator MiniBISPro, DNR Bio Imaging Systems Ltd., Neve Yamin, Israel). Increases in DNA strand breakage were assayed by measuring the conversion of supercoiled DNA, form III, to relaxed circular (I) and linear forms (II). Densitometric quantification of plasmid topology forms (%) was carried out in the ImageJ 1.53c program (Wayne Rasband, National Institute of Health, Kensington, MD, USA).

Lactobacillus differential (LBD) agar was used to differentiate P. freudenreichii strains from L. paracasei subsp. paracasei NCC 2511. It contained per litre: 10.0 g of casein enzymatic hydrolysate (Becton Dickinson, Franklin Lakes, NJ, USA), 3.0 g of casein acid hydrolysate (Merck, Darmstadt, Germany), 1.5 g of enzymic digest of soybean meal (Becton Dickinson), 1.0 g of yeast extract (Becton Dickinson), 2.5 g of fructose (Amresco, Solon, OH, USA), 2.5 g of K₂HPO₄ (Sigma-Aldrich), 55 mg of bromocresol green (Sigma-Aldrich), and 15.0 g of agar (Becton Dickinson) [75 (link)]. In addition, TSB agar was used to grow B. amyloliquefaciens NCC 156 for the estimation of colony forming units. It contained per litre: 17.0 g of tryptone (Becton Dickinson), 5.0 g of NaCl, 3.0 g of soytone (Becton Dickinson), 2.5 g K₂HPO₄, and 1.0 mL of 30% silicone antifoam, and 15.0 g of agar (Becton Dickinson) [28 ].

Analytical grade CaCl₂, K₂HPO₄, FeCl₂, FeCl₃, (NH₄)OH aqueous solution (28%), (NH₄)₂CO₃, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), uranyl acetate, poly-(α, β)-dl-aspartic acid sodium salt (pAsp, molecular weight = 2000–11,000 g mol⁻¹) and poly(allylamine hydrochloride) (pAH, mw = 15,000 g mol⁻¹) were purchased from Sigma-Aldrich and used without further purification. Type-I collagen extracted from horse tendon was kindly provided by Prof. Giuseppe Falini (Department of Chemistry, University of Bologna, Italy) and was originally purchased from OPOCRIN Spa^{38 (link)}. Type-I collagen sponge tapes derived from bovine Achilles tendon was purchased from ACE Surgical.

Peptides with the sequences of Pro-Arg, Arg-Pro-Arg and Arg-Pro-Arg-Pro-Pro-Pro-Pro-Cys (≥98%) were synthesized and purified by Synpeptide Co., Ltd. (Shanghai, China). DPP-IV, diprotin A, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), trisodium citrate, 6-mercapto-1-hexanol (MCH), KH₂PO₄ and K₂HPO₄ were purchased from Sigma-Aldrich (Shanghai, China). Other reagents were analytical-grade and used without additional treatment. The peptide stock solution at the concentration of 2 mM was dissolved with deionized water and diluted with a phosphate-buffered saline solution (PBS buffer, 2 mM, pH 7.6) before use. The citrate-stabilized AuNPs with a size of 13 nm were prepared using a trisodium citrate reduction method [45 (link)]. The concentration of synthesized AuNPs was determined by Beer’s law with an extinction coefficient of 2.7 × 10⁸ M⁻¹ cm^–1 [18 (link)]. Unless otherwise noted, the reactions were conducted at room temperature.