L met

Enzyme activity assays were carried out with 5 μM purified FlA or mutants thereof, mixed with 300 μM SAM (New England Biolabs, Ipswich, MA, USA) [or, in some cases, 300 μM of 5'‐ClDA (Cayman Chemical Co., Ann Arbor, MI, USA)] and 1 mM l‐Met (Sigma‐Aldrich Co.) in a buffer containing 30 mM HEPES (pH = 7.8), 150 mM NaCl and 75 mM KF. Reactions were incubated at 37°C for 4 h, with 80‐μl samples taken at specific time points, incubated at 95°C for 5 min and centrifuged for 10 min at 17 000 g. The supernatants were analysed using an Eclipse Plus C18 column (100 × 4.6 mm, 3.5 μm particle size; Agilent Technologies, Santa Clara, CA, USA) connected to a HPLC system (Dionex^TMUltimate 3000; ThermoFisher Scientific) operated at a flow rate of 1 ml min^‐1. A gradient of solvent A [0.05% (v/v) acetic acid in water] and B (acetonitrile) was implemented as follows: 5‐12% (v/v) B in 1.5 min, 12% (v/v) B for 1 min, 12‐30% (v/v) B in 2 min and 30‐70% (v/v) B in 1.5 min. Enzyme kinetics were measured using 2 μM FlA or 1 μM mutants in a buffer containing 30 mM HEPES (pH = 7.8), 150 mM NaCl and 75 mM KF. The SAM concentration was varied between 0 and 800 μM, and samples were taken at specific time points. All samples were denatured by heating, and supernatants were analysed as explained above.

L-Met purchased from Sigma–Aldrich (purity ≥ 98%, St. Louis, MO, USA) was dissolved in phosphate-buffered saline (PBS) and filtered using a 0.22-μm syringe filter.
The fertilized eggs of yellow-feathered broiler chicks, purchased from Qingyuan Chicken Company (Qingyuan, Guangdong, China), were incubated in an automatic hatcher (CFK, Keyu Incubation Equipment Company, Dezhou, Shandong). On E6 unfertilized and dead eggs were removed; then, a total of 80 eggs (45.03 ± 2.25 g) were selected and randomly divided into 4 treatments with 20 eggs per treatment. PBS (control, CON), 5, 10, or 20 mg L-Met was added to the eggs by in ovo injection at E9. The steps were as follows: firstly, a needle was used to make a small hole at 1/3 of the sharp end of the embryo egg after disinfection with a 75% alcohol-soaked cotton ball; secondly, 0.5 mL of the solution was injected into the yolk with a 1 mL disposable sterile syringe. After hatching, the chicks were sacrificed by CO₂ inhalation followed by cervical dislocation to ensure death, then the intestinal samples were collected.

L-Met purchased from Sigma-Aldrich (purity > 98%, St. Louis, MO) and DL-Met purchased from Evonik Nutrition & Care GmbH (purity > 99%, Essen, Germany) were dissolved in PBS and filtered using a 0.22 μm syringe filter.
The target site for injection into the shell was found by illumination (approximately at two-thirds from the blunt end of the embryo egg), and a small hole was drilled by a 5 mL disposable sterile syringe needle (BB000388, Xinjin Shifeng Medical Equipment Co., Ltd., Chengdu, China) at the target site after disinfection with a 75% alcohol-soaked cotton ball. Subsequently, 0.5 mL of solution was injected into the yolk with a 1 mL disposable sterile syringe (BB000242 Xinjin Shifeng Medical Equipment Co., Ltd.), because the yolk is the main source of nutrients for poultry embryos (Uni and Ferket, 2004 ; Oliveira et al., 2008 ). The hole on the shell was immediately sealed with paraffin, and the eggs were returned to the incubator. All the eggs were maintained outside the incubator for less than 30 min during the injection.

Rats were housed and freely habituated for 7 days prior drug treatment. Caffeic acid and L-met were purchased from Sigma Aldrich Chemical Co. (Saint Louis, MO, USA). Rats were randomly chosen into 6 groups (10 rats per group) and 5 rats per cage. Control rats (group 1) were fed with propylene glycol. Group 2: rats received L-met (1.7 g/kg/day) mixed in 0.5% w/v CMC by gastric gavage one time a day on day 1–28. Group 3 and 4 rats received 20 and 40 mg/kg/day of Caffeic acid dissolved in propylene glycol (1 mg/kg/day) by gastric gavage one time a day on day 1–28, respectively. In co-treatment groups (Groups 5 and 6), rats received L-met (1.7 g/kg/day) and Caffeic acid (20 and 40 mg/kg/day) for 28 days. Before starting treatment, all animals were injected with 100 mg/kg BrdU (Sigma Aldrich, Inc., St. Louis, USA) freshly prepared in 0.9% saline solution by i. p. Injection (1 ml/kg/day) for 3 days (Fig. 1) [28 (link)].Fig. 1

Timeline of drug administration and behavioral testing.

Fig. 1