ONE-seq: epitranscriptome and gene-specific profiling of NAD-capped RNA.

The hub metabolite, nicotinamide adenine dinucleotide (NAD), can be used as an initiating nucleotide in RNA synthesis to result in NAD-capped RNAs (NAD-RNA). Since NAD has been heightened as one of the most essential modulators in aging and various age-related diseases, its attachment to RNA might indicate a yet-to-be discovered mechanism that impacts adult life-course. However, the unknown identity of NAD-linked RNAs in adult and aging tissues has hindered functional studies. Here, we introduce ONE-seq method to identify the RNA transcripts that contain NAD cap. ONE-seq has been optimized to use only one-step chemo-enzymatic biotinylation, followed by streptavidin capture and the nudix phosphohydrolase NudC-catalyzed elution, to specifically recover NAD-capped RNAs for epitranscriptome and gene-specific analyses. Using ONE-seq, we discover more than a thousand of previously unknown NAD-RNAs in the mouse liver and reveal epitranscriptome-wide dynamics of NAD-RNAs with age. ONE-seq empowers the identification of NAD-capped RNAs that are responsive to distinct physiological states, facilitating functional investigation into this modification.

Identification and functional analysis of circulating small extracellular vesicle lncRNA signatures in children with fulminant myocarditis.

Fulminant myocarditis (FM) is the most serious type of myocarditis. However, the molecular mechanism underlying the pathogenesis of FM has not been fully elucidated. Small extracellular vesicles (sEVs) play important roles in many diseases, but any potential role in paediatric FM has not been reported. Here, the differential signatures of lncRNAs in plasma sEVs were studied in FM children and healthy children using transcriptome sequencing followed by functional analysis. Then immune-related lncRNAs were screened to study their role in immune mechanisms, the levels and clinical relevance of core immune-related lncRNAs were verified by qRT-PCR in a large sample size. Sixty-eight lncRNAs had increased levels of plasma sEVs in children with FM and 11 had decreased levels. Functional analysis showed that the sEVs-lncRNAs with different levels were mainly related to immunity, apoptosis and protein efflux. Seventeen core immune-related sEVs-lncRNAs were screened, functional enrichment analysis showed that these lncRNAs were closely related to immune activation, immune cell migration and cytokine pathway signal transduction. The results of the study show that sEVs-lncRNAs may play an important role in the pathogenesis of fulminant myocarditis in children, especially in the mechanism of immune regulation.

Quantitative Acetylomics Reveals Dynamics of Protein Lysine Acetylation in Mouse Livers During Aging and Upon the Treatment of Nicotinamide Mononucleotide.

Lysine acetylation is a reversible and dynamic post-translational modification that plays vital roles in regulating multiple cellular processes including aging. However, acetylome-wide analysis in the aging process remains poorly studied in mammalian tissues. Nicotinamide adenine dinucleotide (NAD+), a hub metabolite, benefits health span at least in part due to the activation of Sirtuins, a family of NAD+-consuming deacetylases, indicating changes in acetylome. Here, we combine two antibodies for the enrichment of acetylated peptides and perform label-free quantitative acetylomic analysis of mouse livers during natural aging and upon the treatment of beta-nicotinamide mononucleotide (NMN), a NAD+ booster. Our study describes previously unknown acetylation sites and reveals the acetylome-wide dynamics with age as well as upon the treatment of NMN. We discover protein acetylation events as potential aging biomarkers. We demonstrate that the life-beneficial effect of NMN could be partially reflected by the changes in age-related protein acetylation. Our quantitative assessment indicates that NMN has mild effects on acetylation sites previously reported as substrates of Sirtuins. Collectively, our data analyze protein acetylation with age, laying critical foundation for the functional study of protein post-translational modification essential for healthy aging and perhaps disease conditions.

Altered plasma exosome miRNAs and novel potential biomarkers in pediatric fulminant myocarditis.

Previous studies have indicated that exosome-mediated intercellular microRNAs (miRNA) can influence fulminant myocarditis (FM) pathogenesis between immune and cardiac cells. This study explored plasma exosome miRNA profile in pediatric FM using a small RNA microarray. As per our analysis, we observed the differential expression of 266 miRNAs, including 197 upregulated and 69 downregulated candidate genes. Differentially expressed mRNAs in pediatric FM patients' peripheral blood mononuclear cells (PBMCs) were intersected with miRNA target genes predicting tools to screen for FM-specific target genes. The hub genes and their biological and mechanistic pathways related to inflammation and/or the immune system were identified. CeRNA networks of lncRNAs, circRNAs, miRNAs, and mRNAs between cardiomyocytes and PBMCs were finally established. Furthermore, we verified that hsa-miR-146a-5p, hsa-miR-23a-3p, and hsa-miR-27a-3p had higher expression levels in exosomes of pediatric FM patients by qRT-PCR, and hsa-miR-146a-5p shown high sensitivities and specificities for FM diagnosis. Overall, the results demonstrate that the exosome miRNAs play a regulatory role between immune and cardiac cells and provide research targets.

Subacute Toxicity Study of Nicotinamide Mononucleotide via Oral Administration.

Nicotinamide mononucleotide (NMN), a key precursory metabolite of NAD+, has been shown to elevate the cellular level of NAD+ and ameliorate various age-related diseases. Despite these progresses, systemic evaluation pertaining to the subacute toxicity of NMN remains to be determined. Here, we examine the subacute toxicity of NMN in mice and beagle dogs. Mice were gavaged with a saturated concentration of NMN solution at the maximum intragastric dose once or twice per day for 7 days. Dogs were gavaged twice per day for 14 days. In mice, NMN administrated once per day for 7 days is well tolerated with minimal deleterious effects. Upon higher dosage, we observe slightly increased level of alamine aminotransferase, while other biomarkers remain unchanged. Consistently, administration of NMN in beagle dogs only results in mild increases in creatinine and uric acid. Together, our study highlights the safety of NMN, providing a possible safe dose range for oral administration of NMN.