Transcriptome-wide N6-methyladenosine methylation profile of atherosclerosis in mice.

BACKGROUND: Atherosclerosis (AS) is a critical pathological event during the progression of cardiovascular diseases. It exhibits fibrofatty lesions on the arterial wall and lacks effective treatment. N6-methyladenosine (m6A) is the most common modification of eukaryotic RNA and plays an important role in regulating the development and progression of cardiovascular diseases. However, the role of m6A modification in AS remains largely unknown. Therefore, in this study, we explored the transcriptome distribution of m6A modification in AS and its potential mechanism. METHODS: Methylation Quantification Kit was used to detect the global m6A levels in the aorta of AS mice. Western blot was used to analyze the protein level of methyltransferases. Methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were used to obtain the first transcriptome range analysis of the m6A methylene map in the aorta of AS mice, followed by bioinformatics analysis. qRT-PCR and MeRIP-qRT-PCR were used to measure the mRNA and m6A levels in target genes. RESULTS: The global m6A and protein levels of methyltransferase METTL3 were significantly increased in the aorta of AS mice. However, the protein level of demethylase ALKBH5 was significantly decreased. Through MeRIP-seq, we obtained m6A methylation maps in AS and control mice. In total, 26,918 m6A peaks associated with 13,744 genes were detected in AS group, whereas 26,157 m6A peaks associated with 13,283 genes were detected in the control group. Peaks mainly appeared in the coding sequence (CDS) regions close to the stop codon with the RRACH motif. Moreover, functional enrichment analysis demonstrated that m6A-containing genes were significantly enriched in AS-relevant pathways. Interestingly, a negative correlation between m6A methylation abundance and gene expression level was found through the integrated analysis of MeRIP-seq and RNA-seq data. Among the m6A-modified genes, a hypo-methylated but up-regulated (hypo-up) gene Fabp5 may be a potential biomarker of AS. CONCLUSIONS: Our study provides transcriptome-wide m6A methylation for the first time to determine the association between m6A modification and AS progression. Our study lays a foundation for further exploring the pathogenesis of AS and provides a new direction for the treatment of AS.

Ketogenic diet and ß-Hydroxybutyrate alleviate ischemic brain injury in mice via an IRAKM-dependent pathway.

Ketogenic diet (KD) is a classical nonpharmacological therapy that has recently been shown to benefit cerebral ischemia, but the mechanism remains unclear. This study investigated the neuroprotective effects of KD pretreatment and ß-hydroxybutyrate (BHB, bioactive product of KD) post-treatment in a mouse model of temporary middle cerebral artery occlusion (tMCAO). Neurological function, infarct volume, as well as inflammatory reactions are evaluated 24 h after ischemia. Results showed that both KD pretreatment or BHB post-treatment improved the Bederson score and Grip test score, reduced infarct volume and the extravasation of IgG, suppressed the over-activation of microglia, and modulated the expression of cytokines. Mechanically, we found that both KD pretreatment or BHB post-treatment significantly stimulated the expression of interleukin-1 receptor-associated kinase M (IRAKM) and then inhibited the nuclear translocation of NF-κB. IRAKM deletion (Irakm-/-) exacerbated tMCAO-induced neurovascular injuries, and aggravated neuroinflammatory response. Moreover, KD pretreatment or BHB post-treatment lost their neuroprotection in the tMCAO-treated Irakm-/- mice. Our results support that KD pretreatment and BHB post-treatment alleviate ischemic brain injury in mice, possibly via an IRAKM-dependent way.

Brevinin-GR23 from frog Hylarana guentheri with antimicrobial and antibiofilm activities against Staphylococcus aureus.

Brevinin-GR23 (B-GR23) was a brevinin-2 like antimicrobial peptide, which had antimicrobial activity against Staphylococcus aureus with minimum inhibitory concentration (MIC) of 16 µM. B-GR23 increased the bacterial membrane permeation, leading to the damage of membrane integrity and the leakage of genomic DNA, then causing the cell death. The peptide nearly inhibited all plantonic bacteria to start the initial attachment of biofilm at the concentration of 1 × MIC. Whereas the disruption rates on immature and mature biofilm decreased from 60% to 20%. B-GR23 reduced the production of extracellular polysaccharides (EPS) in the planktonic growth of S. aureus, which is a crucial structure of biofilm formation. B-GR23 with the concentration of ½ × MIC inhibited 50% water-soluble EPS, and 48% water-insoluble EPS, which contributed to the antibiofilm activity. B-GR23 had no significant toxicity to human blood cells under-tested concentration (200 µM), making it a potential template for designing antimicrobial peptides.

Antibacterial and Antibiofilm Activity of Temporin-GHc and Temporin-GHd Against Cariogenic Bacteria, Streptococcus mutans.

Temporin-GHc (GHc) and temporin-GHd (GHd) produced by the frog Hylarana guentheri had shown broad-spectrum antibacterial activities against bacteria and fungi. In this study, we investigated whether they exert antibacterial and antibiofilm activities against cariogenic bacteria, Streptococcus mutans. GHc and GHd adopt the random coil conformation in aqueous solution and convert to α-helix in membrane mimetic environments by using circular dichroism spectroscope. They are positively charged by histidine, with the polar and nonpolar amino acids on opposing faces along the helix. The amphipathicity enabled the peptides to target at bacterial membrane, leading to an increase in membrane permeation and disruption of S. mutans, which allowed the peptides to bind with genomic DNA. GHc and GHd completely impeded the initial attachment of biofilm formation and disrupted preformed S. mutans biofilms. The expression of exopolysaccharide (EPS) biosynthesis genes which synthesize glucosyltransferases in S. mutans was downregulated by exposing to GHc or GHd, contributing to the decrease of soluble and insoluble EPS. GHc and GHd exhibited selectivity toward S. mutans in the presence of human erythrocytes, and no cytotoxicity toward human oral epithelial cells was observed at a concentration of 200 µM. These results laid the foundation for the development of GHc and GHd as potential anti-caries agents.

Molecular cloning and characterization of antimicrobial peptides from skin of Hylarana guentheri.

The cDNAs encoding antimicrobial peptides (AMPs) in the skin of Hylarana guentheri were identified, namely temporin (five peptides, termed temporin-GHa-GHd and temporin-GUa), brevinin-1 (one peptide, brevinin-1GUb), and brevinin-2 (eight peptides, brevinin-2GHd-2GHj, and brevinin-2GHb). Eleven of the 14 peptides have novel primary structures. Synthesized temporin GHa-GHd have broad-spectrum antimicrobial activities against Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli, Vibrio alginolyticus, and Pseudomonas aeruginosa), as well as fungus (Candida albicans). Among these tested strains, S. aureus was the most sensitive to temporin-GHa-GHd with minimum inhibitory concentration (MIC) values between 6.8 and 12.9 µM. They also exhibited antimicrobial activities against Methicillin-resistant S. aureus with the MIC ranging from 12.7 to 51.7 µM. Interestingly, secondary structure prediction shows that there is no α-helix in temporin-GHb, which illustrates that α-helix is not required for the antimicrobial activity of temporin-GHb. NaCl (at final concentrations of 0.15-2 M) decreased the antimicrobial activity of temporin-GHa-GHd slightly, while human serum and S. aureus V8 proteinase had no effect on the antimicrobial activity. Scanning electron microscopy images of E. coli and S. aureus showed that the surface of microbial cells was considerably rough and shrived after 1 h of treatment with temporin-GHa-GHd at 37°C. The stabilities of temporin-GHa-GHd in human serum or in S. aureus V8 proteinase make them to be promising candidates of novel antimicrobial agents or models for the development of novel AMPs.