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Next Generation Sequencing Facilitates Quantitative Analysis of ApoE-/-/SAHHWT, ApoE-/-/SAHH+/- mice and ApoE-/-/SAHH+/- +Betaine mice [WGBS]

ABSTRACT: Objective: the next generation sequencing (NGS) has revolutionized the system-based analysis of cellular pathways. The purpose of this study was to explore the mechanism of methylation changes caused by SAHH knockout and dietary betaine addition in atherosclerosis.

ORGANISM(S): Mus musculus

PROVIDER: GSE186253 | GEO | 2021/10/22

REPOSITORIES: GEO

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Project description:In a transcriptome based trail, we figured out that the deletion of luxS has a massive influence to cell growth and metabolism of Streptococcus sanguinis SK36. The biofilm defective luxS deletion mutant was comlemented by transgenic sahH from Pseudomonas aeruginosa. Thus 209 of 216 influenced genes of the luxS mutant compared to the isogenic wildype were restored in their expression (fold change of n M-bM-^IM-% 3; p M-bM-^IM-$ 0.05), including genes involved in cell division processes, stress response and catabolite control. Phenotypically, the reduced biofilm depth of S. sanguinis SR DELTAluxS was elevated to wild type level by the complementation with sahH. Neither the addition of physiological concentrated artificial autoinducer 2 (AI-2) to the culture of S. sanguinis SR DELTAluxS nor the presence of the wild type strain in a trans-well assay had a cumulative effect on biofilm thickness of the luxS mutant. Furthermore, we identified 9 genes in the sahH complemented luxS mutant, which were regulated directly by AI-2 (fold change M-bM-^IM-% 3; p M-bM-^IM-$ 0.05), amongst them genes involved in competence development. So we concluded that AI-2 has no influence on biofilm growth, but regulative functions in competence development in S. sanguinis. Further, we figured out the suitability of transgenic sahH for the complementation of the interrupted Pfs/LuxS pathway without restoration of AI-2 release acquiring an essential tool for further investigations on AI-2. Aim: Is the presence of AI-2 necessary for the biofilm formation of S. sanguinis SK36? What is the impact of a deletion of luxS, is a disrupted activated methionine cycle the reason for the hampered biofilm depth causes by its inactivation? Materials and Methods: S. sanguinis SK36, S. sanguinis SR M-NM-^TluxS, and S. sanguinis SR M-NM-^TluxS/sahH were grown in CDM/sucrose for 24 h anaerobically; biofilm depth was measured by safranine and crystal violet stain. Biofilm morphology was investigated by scanning electron microscopy. The AI-2 release was monitored hourly to identify the time period of its release. For transcriptome analysis total RNA was isolated after 8 hours of growth and used for microarray analysis. The chip study used total RNA recovered from S. sanguinis SK36, its luxS mutant, and the sahH complementated luxS mutant. Gene expression analysis was done with three independent replicates, each. Chip content: 10186 genes (1883 genes of P. gingivalis W83, 1964 genes of F. nucleatum DSMZ 25586, 2244 genes of S. sanguinis SK36, 2168 genes of A. actinomycetemcomitans HK1651, 1927 genes of S. mutans UA159) with up to thirteen 60-mer probes per gene, with three-fold technical redundancy and additionally 3510 random sequence probe. In this study only the S. sanguinis and RANDOM probes were used for analysis.

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Next Generation Sequencing Facilitates Quantitative Analysis of ApoE-/-/SAHHWT, ApoE-/-/SAHH+/- mice and ApoE-/-/SAHH+/- +Betaine mice [WGBS]

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