Du800

TRIzol reagent (Invitrogen) was used to extract the total RNA of intestinal mucosal scrapings following its manufacturer's instructions. The purity and content of RNA were determined spectrophotometrically (Beckman Coulter DU800; Beckman Coulter Inc.) (Liu, Zhang, Li, Yan, & Zhang, 2019b). Then, the RNA samples met the requirements were reverse‐transcribed into complementary DNA with the PrimeScript RT reagent kit (Takara). RT‐PCR for quantification analysis of target genes was performed on the Opticon DNA Engine (Bio‐Rad) with SYBR Green PCR reagents (Takara). Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (GenBank NM001206359) and β‐actin (GenBank DQ452569) were selected as the housekeeping gene. The primers of β‐actin, GAPDH, NF‐κB, MyD88, claudin‐1, ZO‐1, Occludin, Bax, and Bcl‐2 were synthesized commercially by Life Technologies Limited (Table S2). The cycle profile consisted of denaturation at 95°C for 2 min, followed by 40 cycles of 95°C for 20 s, 63°C for 30 s, and 72°C for 60 s. The PCR products were then analyzed for homogeneity by melting curve analysis. The housekeeping genes did not vary between diets (p = .81) in duplicates. Each sample and standard were moved simultaneously in duplicate on the same PCR plate, and the average of each duplicate copy was used for statistical analysis.

TGFα/c-myc mice with confirmed liver tumours according to Gd-EOB-DTPA (Dotarem)-enhanced MRI or nude mice with HepG2 tumours received iRGD/CEND-1 or RGD control peptide (4 μmol/kg each), or PBS by tail vein injection. Evans blue (33.3 mg/kg, MP Biomedicals, Eschwege, Germany) was injected intravenously 15 min later. Another 30 min later, the mice were terminally perfused with Ringer solution by cannulation of the left heart ventricle. Organs were inspected macroscopically following laparotomy, and the liver, tumour and other organs were excised. Evans blue was extracted from tissues in N,N-dimethylformamide for 24 h at 37 °C and quantified spectrophotometrically (Beckman Coulter DU 800, Beckman Coulter, Brea, CA, USA) at 600 nm.

TRIzol Reagent (TaKaTa, Dalian, China) was used to extract the total RNA from the liver and thymus on the basis of the manufacturer's instructions. Spectrophotometer (Beckman Coulter DU800; Beckman Coulter Inc.) was applied to measure the concentration and purity of RNA at 260 and 280 nm to ensure the ratios of absorption (OD260/OD280 nm) various between 1.8 and 2.0 for all samples. The integrity of RNA was verified by formaldehyde gel electrophoresis. Reverse transcription of each sample was conducted by PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Dalian, China) following the manufacturer's instructions. The primers were synthesized commercially by Life Technologies Limited and exhibited in Table 2.
The analysis of the expression abundance of TNF-α, IL-6, IL-1β, TLR4, NF-κB, Nrf-2, and HO-1 was calculated by quantitative real-time PCR in the liver and thymus using SYBR Premix Ex Taq II (Tli RnaseH Plus) reagents (TaKaRa, Dalian, China) and the CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Richmond, CA). The β-actin gene was chosen as the reference gene to normalize the mRNA expression of target genes. The relative expression ratio of target genes relative to the reference gene was calculated using the 2^−ΔΔCT method [16 (link)]. Each sample was simultaneously performed on the same PCR plate.

Frozen jejunum samples were weighed and homogenized in 9 times the volume of 50 mM of Tris-HCl buffer (pH 7.0) on ice with a homogenate machine (Homogenizer Power Gen 125™, Fisher Scientific, Pittsburgh, PA, USA). Homogenate was centrifuged at 3000 g and 4 °C for 10 min, and the supernatant was collected and stored at −20 °C for an enzyme assay. Total protein was extracted, and the concentration was determined according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering, Nanjing, China). Disaccharidase (including maltase, sucrase, and lactase) activities were measured with commercial kits according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering, Nanjing, China). The absorbance was determined with a spectrophotometer (Beckman Coulter DU-800; Beckman Coulter, Inc., Brea, CA, USA). The activities of disaccharidases were presented as U/mg protein. One unit (U) was defined as 1 nmol of maltose, sucrose, and lactose for the enzymatic reaction, which was instituted according to the manufacturer’s instructions [8 (link)].

According to the previous study, the thawing samples of jejunum and ileum were weighed (approximately 2 g), then 9 times volume of 50 mM Tris–HCl buffer (pH 7 · 0) than the sample weight were added and homogenized for 40 s by homogenate machine (Homogenizer Power Gen 125™, ThermoFisher Scientific, MA, USA) and centrifuged at 3000 g for 10 min, the supernatant was collected and stored at −20 °C [11 (link)]. Total protein was extracted from the supernatant and protein concentration was determined by bicinchoninic acid protein assay with bovine serum albumin as the standard (Solarbio, Inc., Beijing, China). Activities of disaccharidase including maltase, sucrase and lactase were measured using commercial kits (Nanjing Jiancheng Bioengineering, Nanjing, China). The absorbance at 450 nm was determined with spectrophotometer (Beckman Coulter DU-800; Beckman Coulter, Inc., CA, USA). Activities of disaccharidase were presented as U/mg protein. One unit (U) was defined by 1 nmol of maltose, sucrose and lactose as a substrate for the enzymatic reaction, respectively.

Michaelis-Menten kinetic parameters for KPC-2 and the variant enzyme-substrate pairs were determined at 25°C in 50 mM sodium phosphate buffer, pH 7.0, containing 0.1 mg/mL BSA using variable amounts of enzyme depending on the enzyme-substrate pair. The initial velocities of β-lactam hydrolysis were measured on a Beckman-Coulter spectrophotometer model DU-800 (Fullerton, CA) using the following extinction coefficients: imipenem, Δε₂₉₅ = -9000 M^-1cm^-1; meropenem, Δε₂₉₅ = -10,940 M^-1cm^-1; ceftazidime Δε₂₉₅ = -7600 M^-1cm^-1; ampicillin, Δε₂₃₅ = -900 M^-1cm^-1. GraphPad Prism 5 was used to obtain the steady-state parameters by non-linear least squares fit of the data to the Michaelis-Menten equation v = k_cat[S]/(K_m + [S]). The velocity of ceftazidime hydrolysis could not be saturated by measurable concentrations due to a high K_m. Thus, the second order rate constant at steady-state, k_cat/K_m, was determined by fitting the progress curves to the equation v = k_cat/K_m[E][S], where [S] << K_m (eq. 1).

Purified Arabidopsis mitochondrial MDH (EC 1.1.1.37)68 and purified recombinant wild-type and mutant YUC6 proteins at the indicated molar ratios were incubated in 40 mM HEPES (pH 7.5) buffer at 45 °C. The absorbance at 340 nm, indicating aggregation of MDH, was monitored for 30 min using a Beckman DU-800 spectrophotometer (Beckman Coulter) attached to a thermostatic cell holder assembly58 (link)60 (link)68 .