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. 2016 Apr 5;7(2):e02210-15. doi: 10.1128/mBio.02210-15

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Copyright © 2016 Chen et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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FIG 7 — RSV reduced TMAO levels by decreasing TMA generation via remodeling microbiota in ApoE^−/− mice. Eight-week-old female ApoE^−/− mice (n = 5 per group) were fed chow, chow with RSV (0.4%), chow with choline (1%), or chow with choline (1%) plus RSV (0.4%) in the absence or presence of Abs for 4 months. (A) Serum TMA and TMAO levels were measured by LC/MS. (B) Western blotting detection of FMO3 expression in the liver. (C) Expression levels of FMO3 gene mRNAs were quantified using qPCR assays. (D) Liver FMO activity was assessed as described in Materials and Methods. (E) 16S rRNA gene sequencing analysis of cecal content at the phylum level. (F) Heat map of 16S rRNA gene sequencing analysis of cecal content at the genus level. The scale reflects the data as follows: red indicates high values whereas blue indicates low values for the spercentage of reads that were classified at that rank. (G) Linear discriminant analysis (LDA) coupled with effect size measurements identifies the most differentially abundant taxons at the genus level between the choline diet and the RSV-plus-choline diet. RSV-plus-choline-diet-enriched taxa are indicated with a positive LDA score (green), and taxa enriched in the choline diet have a negative score (red). Only taxa meeting an LDA significance threshold of >2 are shown. (H) Correlation heat map demonstrating the association between the indicated microbiota taxonomic genera and TMA and TMAO concentrations (all reported as means ± SD in micromoles) from mice grouped by dietary status (choline and RSV plus choline). Red denotes a positive association, blue a negative association, and white no association. A single asterisk indicates a significant FDR-adjusted association of P values of ≤0.05, and a double asterisk indicates a significant FDR-adjusted association of P values of ≤0.01. (I) Production of TMA from choline by cecal content in vitro. Values are expressed as means ± SD (n = 5). a, P < 0.05; b, P < 0.01 (versus vehicle-treated control group); c, P < 0.05; d, P < 0.01 (versus choline-treated group); #, P < 0.05 (versus group cotreated with RSV and choline).