Systematic calibration of epitranscriptomic maps using a synthetic modification-free RNA library.

Recent years have witnessed rapid progress in the field of epitranscriptomics. Functional interpretation of the epitranscriptome relies on sequencing technologies that determine the location and stoichiometry of various RNA modifications. However, contradictory results have been reported among studies, bringing the biological impacts of certain RNA modifications into doubt. Here, we develop a synthetic RNA library resembling the endogenous transcriptome but without any RNA modification. By incorporating this modification-free RNA library into established mapping techniques as a negative control, we reveal abundant false positives resulting from sequence bias or RNA structure. After calibration, precise and quantitative mapping expands the understanding of two representative modification types, N6-methyladenosine (m6A) and 5-methylcytosine (m5C). We propose that this approach provides a systematic solution for the calibration of various RNA-modification mappings and holds great promise in epitranscriptomic studies.

Targeted RNA N6 -Methyladenosine Demethylation Controls Cell Fate Transition in Human Pluripotent Stem Cells.

Deficiency of the N6 -methyladenosine (m6 A) methyltransferase complex results in global reduction of m6 A abundance and defective cell development in embryonic stem cells (ESCs). However, it's unclear whether regional m6 A methylation affects cell fate decisions due to the inability to modulate individual m6 A modification in ESCs with precise temporal control. Here, a targeted RNA m6 A erasure (TRME) system is developed to achieve site-specific demethylation of RNAs in human ESCs (hESCs). TRME, in which a stably transfected, doxycycline-inducible dCas13a is fused to the catalytic domain of ALKBH5, can precisely and reversibly demethylate the targeted m6 A site of mRNA and increase mRNA stability with limited off-target effects. It is further demonstrated that temporal m6 A erasure on a single site of SOX2 is sufficient to control the differentiation of hESCs. This study provides a versatile toolbox to reveal the function of individual m6 A modification in hESCs, enabling cell fate control studies at the epitranscriptional level.

The RNA m6A reader YTHDC1 silences retrotransposons and guards ES cell identity.

The RNA modification N6-methyladenosine (m6A) has critical roles in many biological processes1,2. However, the function of m6A in the early phase of mammalian development remains poorly understood. Here we show that the m6A reader YT521-B homology-domain-containing protein 1 (YTHDC1) is required for the maintenance of mouse embryonic stem (ES) cells in an m6A-dependent manner, and that its deletion initiates cellular reprogramming to a 2C-like state. Mechanistically, YTHDC1 binds to the transcripts of retrotransposons (such as intracisternal A particles, ERVK and LINE1) in mouse ES cells and its depletion results in the reactivation of these silenced retrotransposons, accompanied by a global decrease in SETDB1-mediated trimethylation at lysine 9 of histone H3 (H3K9me3). We further demonstrate that YTHDC1 and its target m6A RNAs act upstream of SETDB1 to repress retrotransposons and Dux, the master inducer of the two-cell stage (2C)-like program. This study reveals an essential role for m6A RNA and YTHDC1 in chromatin modification and retrotransposon repression.

Clinical Efficacy and Mechanism of Xiao Qinglongtang in Treating Chronic Heart Failure with Cold Phlegm in Lung / 中国实验方剂学杂志

Objective:To observe the clinical efficacy of Xiao Qinglongtang on chronic heart failure with cold phlegm in lung， and explore its mechanism of action. Method:A total of 87 patients with definite chronic heart failure were divided into observation group and control group by random number table method．The two groups received routine western medicine at the same time. Forty-two cases in observation group were treated with Xiao Qinglongtang based on western medicine， and 45 cases in control group received the same dose of placebo. Both groups were treated for 4 weeks， and then their cardiac function， traditional Chinese medicine （TCM） syndrome score and efficacy were compared before and after treatment. Left ventricular ejection fraction （LVEF） and N-terminal pro-brain natriuretic peptide （NT-proBNP） were measured in both groups before and after treatment. The changes of standard deviation of NN intervals （SDNN）， square root of the mean squared differences of successive NN intervals （RMSSD）， high frequency （HF） and low frequency （LF） that reflect autonomic nerve function indexes in heart rate variability （HRV） after treatment were compared between two groups. The changes of inflammatory indicators such as interleukin-6 （IL-6） and highly sensitive C reaction protein （hs-CRP） were detected. Result:After treatment， the total effective rate for cardiac function in observation group was higher than that in control group （P<0.05）. The TCM symptom scores were improved after treatment in both groups （P<0.05）， and the total effective rate in observation group was higher than that in control group （P<0.01）. After treatment， LVEF levels significantly increased （P<0.01） and NT-proBNP levels significantly decreased （P<0.01） in both groups， and the effect in observation group was more obvious （P<0.01）. After treatment， SDNN， RMSD， HF and LF indicators in HRV were all higher than those before treatment in both groups （P<0.01）， and the improvement in observation group was more significant than that in control group （P<0.01）. The levels of IL-6 and hs-CRP decreased after treatment in both groups （P<0.01）， and the level of observation group was significantly lower than that of control group （P<0.01）. Conclusion:Xiao Qinglongtang has certain clinical efficacy in treating chronic heart failure with cold phlegm in lung as it can improve the clinical symptoms of patients， regulate autonomic nervous balance， and inhibit inflammatory factors， providing new clinical ideas to treat chronic heart failure in TCM.

Electroacupuncture Prevents Osteoarthritis of High-Fat Diet-Induced Obese Rats.

The effects of acupuncture on osteoarthritis (OA) pathogenesis have been demonstrated in vitro and in animal models. However, the potential for acupuncture to mediate protective effects on obese-induced OA has not been examined. Here, we investigated the effects of different acupuncture patterns on OA pathogenesis in high-fat diet- (HFD-) induced obese rats. After 12-week diet-induced obesity, obese rats were treated with three acupuncture protocols for 2 weeks, including ST36, GB34, and ST36+GB34. The results showed that the three acupuncture protocols both prevented obesity-induced cartilage matrix degradation and MMP expression and mitigated obesity-induced systemic and local inflammation but had different regulatory effects on lipid metabolism and gut microbiota disorder of obese-induced OA rats. Furthermore, the three acupuncture protocols increased the microbial diversity and altered the structure of community of feces in obese rats. We found that ST36 and GB34 could inhibit proinflammatory shift in the gut microbiome with an increase in the ratio of Bacteroidetes/Firmicutes and promote the recovery of relative abundance of Clostridium, Akkermansia, Butyricimonas, and Lactococcus. Although both ST36 and GB34 had an anti-inflammatory effect on serum inflammatory mediators, only the acupuncture protocol with both ST36 and GB34 could effectively inhibit LPS-mediated joint inflammation in obesity rats. Therefore, relieving obesity-related chronic inflammation, lipid metabolism disorder, and gut microbiota disorder may be an important mechanism for acupuncture with ST36 and GB34 to promote OA recovery.

Single-base mapping of m6A by an antibody-independent method.

N 6-methyladenosine (m6A) is one of the most abundant messenger RNA modifications in eukaryotes involved in various pivotal processes of RNA metabolism. The most popular high-throughput m6A identification method depends on the anti-m6A antibody but suffers from poor reproducibility and limited resolution. Exact location information is of great value for understanding the dynamics, machinery, and functions of m6A. Here, we developed a precise and high-throughput antibody-independent m6A identification method based on the m6A-sensitive RNA endoribonuclease recognizing ACA motif (m6A-sensitive RNA-Endoribonuclease-Facilitated sequencing or m6A-REF-seq). Whole-transcriptomic, single-base m6A maps generated by m6A-REF-seq quantitatively displayed an explicit distribution pattern with enrichment near stop codons. We used independent methods to validate methylation status and abundance of individual m6A sites, confirming the high reliability and accuracy of m6A-REF-seq. We applied this method on five tissues from human, mouse, and rat, showing that m6A sites are conserved with single-nucleotide specificity and tend to cluster among species.

Study on the Bioemulsifier Produced by a Hydrocarbon- degrading Strain T7-2 and Its Physic-chemical Properties / 微生物学通报

A low-temperature hydrocarbon-degrading strain T7-2, isolated from sea-mud of Bohai polluted area and identified as Rhodococcus erythropolis, was found to produce an extracellular, nondialyzable emul- sifying agent (referred to as bioemulsifier) when grew with hexadecane as carbon source. The results showed that, this bioemulsifier which could remarkably emulsify hydrocarbons such as diesel oil, is consisted of three parts-carbohydrates, lipids and proteins, the proportion of which was 55.43:31.24:12.65. The mono- saccharide compositions were identified as mannose and rhamnose; the lipid compositions included de- canoic acid, lauric acid, hexadecanoic acid and stearic acid, and the protein constituents were composed of sixteen amino acids. Besides, according to the study of the physic-chemical properties of the bioemulsifier, it possesses the obvious advantages of character stability, high function efficiency and wide adaptation range, therefore this bioemulsifier is believed to have extensive application values for bioremediation of marine oil pollution, petroleum exploitation and etc.