Protective Effects of Intestinal Gallic Acid in Neonatal Dairy Calves against Extended-Spectrum β-lactamase Producing Enteroaggregative Escherichia coli Infection: Modulating Intestinal Homeostasis and Colitis

Published: 22 February 2022| Version 3 | DOI: 10.17632/6pyjjwmngd.3
Contributor:
Zhiyuan He

Description

Metabolomics preprocessed data, the data set is related to figure 2, Extended Data figure S3.

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Fecal samples (5 mg) from healthy or diarrheal calves were homogenated with zirconium oxide beads for 3 minutes and mixed with 145 μL extraction solution (containing 25 μL of water and 120 μL of methanol) to extract the metabolites. The sample was homogenated for another 3 minutes using a high-throughput tissue disruptor and then centrifuged at 1,800 g for 20 minutes. Acquired supernatant was transferred to a 96-well plate and mixed with 20 μL derivative reagents at 30°C for 60 min, following procedures of Eppendorf epMotion Workstation (Eppendorf Inc., Humburg, Germany). Sample was further diluted with 330 μL of ice-cold 50% methanol, stored at -20°C for 20 minutes, and followed by centrifugation (4,000 g for 30 minutes at 4℃). The supernatants were obtained for LC-MS analysis. The metabolite extracts from calf fecal samples were analysed using an UPLC-MS/MS system (ACQUITY UPLC-Xevo TQ-S, Waters Corp., Milford, MA, USA). Chromatographic separation was performed using an BEH C18 column (2.1 mm×100 mm, 1,7 μm, Waters). The desolvation and source temperatures were set at 500℃ and 150℃, respectively. Mobile phases containing acetonitrile/isopropanol (1:1, 0.1% formic acid) and 0.1% formic acid were used as carried liquid at a constant flow rate of 0.4 mL/min. The raw data were processed using the MassLynx software (version 4.1, Waters, Milford, MA, USA). Each sample was analysed by UPLC-MS/MS in both positive and negative ionization modes to acquire metabolite profiles. Analysis order of all test samples was randomized. The quality-control (QC) samples were pooled samples in which both the metabolite composition of the samples and sample matrix were mixed, and then analyzed using the same methods to evaluate the quality and variance of the acquired data. Self-developed platform iMAP (version 1.0, Metabo-Profile, Shanghai, China) was used for further statistical analyses, including PCA, PLS-DA, univariate analysis and pathway analysis.

Institutions

China Agricultural University

Categories

Bacterial Disease, Emerging Infectious Disease, Alternative Assessment

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