The direct nucleic acid repair dioxygenase FTO is an enzyme that demethylates N6-methyladenosine (m6A) residues in mRNA in vitro and inside cells. FTO is the first RNA demethylase discovered that also serves a major regulatory function in mammals. Together with structure-based virtual screening and biochemical analyses, we report the first identification of several small-molecule inhibitors of human FTO demethylase. The most potent compound, the natural product rhein, which is neither a structural mimic of 2-oxoglutarate nor a chelator of metal ion, competitively binds to the FTO active site in vitro. Rhein also exhibits good inhibitory activity on m6A demethylation inside cells. These studies shed light on the development of powerful probes and new therapies for use in RNA biology and drug discovery.
Abstract Notoginsenoside R 1 (NGR 1 ), a diagnostic protopanaxatriol‐type (ppt‐type) saponin in Panax notoginseng , possesses potent biological activities including antithrombotic, anti‐inflammatory, neuron protection and improvement of microcirculation, yet its pharmacokinetics and metabolic characterization as an individual compound remain unclear. The aim of this study was to investigate the exposure profile of NGR 1 in rats after oral and intravenous administration and to explore the metabolic characterization of NGR 1 . A simple and sensitive ultra‐fast liquid chromatographic–tandem mass spectrometric method was developed and validated for the quantitative determination of NGR 1 and its major metabolites, and for characterization of its metabolic profile in rat plasma. The blood samples were precipitated with methanol, quantified in a negative multiple reaction monitoring mode and analyzed within 6.0 min. Validation parameters (linearity, precision and accuracy, recovery and matrix effect, stability) were within acceptable ranges. After oral administration, NGR 1 exhibited dose‐independent exposure behaviors with t 1/2 over 8.0 h and oral bioavailability of 0.25–0.29%. A total of seven metabolites were characterized, including two pairs of epimers, 20( R )‐notoginsenoside R 2 /20( S )‐notoginsenoside R 2 and 20( R )‐ginsenoside Rh 1 /20( S )‐ginsenoside Rh 1 , with the 20( R ) form of saponins identified for the first time in rat plasma. Five deglycometabolites were quantitatively determined, among which 20( S )‐notoginsenoside R 2 , ginsenoside Rg 1 , ginsenoside F 1 and protopanaxatriol displayed relatively high exploration, which may partly explain the pharmacodynamic diversity of ginsenosides after oral dose.
N 6 -methyl-adenosine (m6A) is a prevalent RNA modification in many species. Abnormal m6A methylation levels can lead to RNA dysfunction and can cause diseases. Tobacco mosaic virus (TMV) is one of the most devastating viruses for agricultural plants. It has many hosts, particularly including tobacco and other members the family Solanaceae. However, it remains unclear whether the abnormal growth induced by TMV is associated with the m6A level.A rapid and accurate analytical method using ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HR - MS/MS) was developed to analyse the adenosine (A), cytidine (C), guanosine (G), uridine (U), and m6A contents in the tobacco leaf, and the m6A/G ratio was used to evaluate the m6A level. Subsequent protein sequence alignments were used to find the potential methylases and demethylases in Nicotiana tabacum (N. tabacum). Finally, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was used to analyse the gene expression levels of the potential methylases and demethylases in the N. tabacum leaf.The results showed that TMV reduced the m6A level. Moreover, protein sequence alignments revealed partial homology among human ALKBH5, Arabidopsis (NP_001031793), and Nicotiana sylvestris (XP_009800010). The gene expression level of the potential demethylase XM_009801708 increased at 14 and 21 days in N. tabacum infected with TMV, whereas all of the potential methylases decreased.The reversible m6A modification in N. tabacum mRNA might represent a novel epigenetic mechanism involved in TMV.
It has been established that Cutaneous T-Cell lymphomas (CTCL) are caused by the monoclonal proliferation of T lymphocytes in the skin. This heterogeneous group of diseases represents a significant source of distress to patients since the diagnosis and treatment are often challenging. As one of the most abundant internal modifications in mRNA in higher eukaryotes, N6-methyladenosine (m6A) is widely recognized to affect the development and progression of cancers. However, knowledge on the involvement of m6A in CTCL is still limited. In this work, we revealed the role of METTL3-mediated m6A modification in CTCL progression. ELISA, western blot, and qRT-PCR assays demonstrated that METTL3 was significantly downregulated in CTCL cells both in vivo and in vitro. CCK-8, EdU, flow cytometry, and transwell assays showed that the decline in METTL3 levels was responsible for CTCL cell proliferation and migration. Furthermore, using small interfering RNAs against METTL3 and the RIP assay, we showed that CDKN2A was a key regulator during this process in vitro and in vivo, and insufficient methylation modification blocked the interaction between CDKN2A and m6A reader IGF2BP2, resulting in mRNA degradation. To the best of our knowledge, this is the first study to depict the role of m6A in CTCL development and provide potential bio-targets for therapy.
Abstract N6-methyladenosine (m 6 A) is considered as the most common and important internal transcript modification in several diseases like type 2 diabetes, schizophrenia and especially cancer. As a main target of m 6 A methylation, long non-coding RNAs (lncRNAs) have been proved to regulate cellular processes at various levels, including epigenetic modification, transcriptional, post-transcriptional, translational and post-translational regulation. Recently, accumulating evidence suggests that m 6 A-modified lncRNAs greatly participate in the tumorigenesis of cancers. In this review, we systematically summarized the biogenesis of m 6 A-modified lncRNAs and the identified m 6 A-lncRNAs in a variety of cancers, as well as their potential diagnostic and therapeutic applications as biomarkers and therapeutic targets, hoping to shed light on the novel strategies for cancer treatment.
Although the onset time of chemical reactions can be manipulated by mechanical, electrical, and optical methods, its chemical control remains highly challenging. Herein, we report a chemical timer approach for manipulating the emission onset time of chemiluminescence (CL) reactions. A mixture of Mn 2+ , NaHCO 3 , and a luminol analog with H 2 O 2 produced reactive oxygen species (ROS) radicals and other superoxo species (superoxide containing complex) with high efficiency, accompanied by strong and immediate CL emission. Surprisingly, the addition of thiourea postponed CL emission in a concentration-dependent manner. The delay was attributed to a slow-generation-scavenging mechanism, which was found to be generally applicable not only to various types of CL reagents and ROS radical scavengers but also to popular chromogenic reactions. The precise regulation of CL kinetics was further utilized in dynamic chemical coding with improved coding density and security. This approach provides a powerful platform for engineering chemical reaction kinetics using chemical timers, which is of application potential in bioassays, biosensors, CL microscopic imaging, microchips, array chips, and informatics.
We present a maximum likelihood (ML) deconvolution algorithm with bandwidth and total variation (TV) constraints for degraded image due to atmospheric turbulence. The bandwidth limit function is estimated in view of optical system parameters and Fourier optical theory. With the aid of bandwidth and TV minimization as compelling constraints, the algorithm can not only suppress noise effectively but also restrict the bandwidth of point-spread function (PSF) that may lead to trivial solution. Compared with the conventional ML method, the proposed algorithm is able to restore a noise-free image, and the detailed texture is better than that of ML.
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