m6A demethylase ALKBH5 attenuates doxorubicin-induced cardiotoxicity via posttranscriptional stabilization of Rasal3.

The clinical application of anthracyclines such as doxorubicin (DOX) is limited due to their cardiotoxicity. N6-methyladenosine (m6A) plays an essential role in numerous biological processes. However, the roles of m6A and m6A demethylase ALKBH5 in DOX-induced cardiotoxicity (DIC) remain unclear. In this research, DIC models were constructed using Alkbh5-knockout (KO), Alkbh5-knockin (KI), and Alkbh5-myocardial-specific knockout (ALKBH5flox/flox, αMyHC-Cre) mice. Cardiac function and DOX-mediated signal transduction were investigated. As a result, both Alkbh5 whole-body KO and myocardial-specific KO mice had increased mortality, decreased cardiac function, and aggravated DIC injury with severe myocardial mitochondrial damage. Conversely, ALKBH5 overexpression alleviated DOX-mediated mitochondrial injury, increased survival, and improved myocardial function. Mechanistically, ALKBH5 regulated the expression of Rasal3 in an m6A-dependent manner through posttranscriptional mRNA regulation and reduced Rasal3 mRNA stability, thus activating RAS3, inhibiting apoptosis through the RAS/RAF/ERK signaling pathway, and alleviating DIC injury. These findings indicate the potential therapeutic effect of ALKBH5 on DIC.

Quantitative Kinetics of the Reaction between CH2OO and H2O2 in the Atmosphere.

Criegee intermediates (CIs) are generated from the ozonolysis of unsaturated hydrocarbons in the atmosphere. They have an important role in determining the implications of atmospheric bimolecular reactions with other atmospheric species. The reaction between CH2OO and H2O2 plays a crucial role in understanding how CIs impact the HOx budget in the atmosphere. The reaction mechanism and kinetics are critical to atmospheric modeling, which is a prominent challenge in present-day climate change modeling. This is particularly true for bimolecular reactions that involve complex reaction sequences. Here, we report the mechanism and quantitative kinetics of the CH2OO + H2O2 reaction by using a novel dual-level strategy that contains W3X-L//CCSD(T)-F12a/cc-pVTZ-F12 for the transition state theory and M11-L/MG3S functional method for direct dynamics calculations using canonical variational transition state theory with small-curvature tunneling to obtain both recrossing effects and tunneling. The present work shows that the CH2OO + H2O2 reaction has a negative temperature dependency with the decrease in the rate constant of CH2OO + H2O2 from 1.31 × 10-13 cm3 molecule-1 s-1 to 3.80 × 10-14 cm3 molecule-1 s-1 between 200 and 350 K. The calculated results also show that the CH2OO + H2O2 reaction can have an impact on the H2O2 profile under certain atmospheric conditions. The present findings should have implications for the quantitative kinetics of Criegee intermediates with other hydroperoxides.

[Analysis of the phase error non-linearity of FTS and discussion about Mertz method].

Fourier transform spectrometer is an important instrument in the remote sensing applications. There are phase error problems in the Fourier transform spectrometer signal processing procedure. In the present paper, the cause of phase error of Fourier transform spectrometers is shown firstly. It is mainly because of inaccuracy of sampling. Then the nonlinearity of phase error is analyzed. It is suggested that it is because that the interferogram is of finite length and the interferogram is discrete that this nonlinearity exists. The authors studied this problem with a new method. The nonlinearity is shown by rigorous derivation and the authors draw the conclusion by reasoning. Then through the nonlinearity of phase error, the authors have a discussion on the possible error in the Mertz phase correcting method. The possible error lies in the phase interpolation procedure, a part of Mertz method. A method consisting of zero adding and transforming is given to reduce this error. The methods are compared and illustrated by an experiment which uses simulated interferogram from standard spectrum library. The experiment demonstrates that the method of zero adding and transforming can reduce the phase error of phase interpolation and improve the problem of rapid phase change under some circumstances, which can help get better spectrum.

Broad profiling of DNA-binding transcription factor activities improves regulatory network construction in adult mouse tissues.

Molecular systematics involves the description of the regulatory networks formed by the interconnections between active transcription factors and their target expressed genes. Here, we have determined the activities of 200 different transcription factors in six mouse tissues using an advanced mouse oligonucleotide array-based transcription factor assay (MOUSE OATFA). The transcription factor signatures from MOUSE OATFA were combined with public mRNA expression profiles to construct experimental transcriptional regulatory networks in each tissue. SRF-centered regulatory networks constructed for lung and skeletal muscle with OATFA data were confirmed by ChIP assays, and revealed examples of novel networks of expressed genes coregulated by sets of transcription factors. The combination of MOUSE OATFA with bioinformatics analysis of expressed genes provides a new paradigm for the comprehensive prediction of the transcriptional systems and their regulatory pathways in mouse.

Novel high-throughput profiling of human transcription factors and its use for systematic pathway mapping.

Transcription factors (TFs) are crucial components of regulatory networks that control gene transcription. Current TF assays are limited to the analysis of a single TF or require TF-specific antibodies. Here we report the Single Primer Amplification assisted Oligonucleotide Array-based Transcription Factor Assay (SPA-OATFA) which can directly analyze the binding activities of 240 human TFs simultaneously. Examining early events during serum-stimulation of HeLa cells as a model, we demonstrated the utility of SPA-OATFA combined with whole genome gene expression to systematically map the temporal activation of signaling pathways. Both TFs known to function in this stimulation response such as EGR1 and AP1 and new TFs such as HSF1 were identified. This information, combined with mRNA profiling, provided novel insights into the activities of regulatory pathways, and illustrates the potential of SPA-OATFA in detailed systems biology analysis of cell responses.

Rapid chip-based capillary electrophoretic mobility shift assay with negative pressure injection for the binding study of transcription factor Abf1 in Saccharomyces cerevisiae.

The study on the interactions of nucleic acids with transcription factor (TF) is critical to understand the gene expression at the molecular level. In the present study, a rapid chip-based capillary electrophoretic mobility shift assay with LIF detection has been developed on a PDMS-quartz chip. We used a simple negative pressure injection procedure to avoid the bias of electrokinetic injection and to allow loading of the high salt buffered sample. We observed signals for Cy3-labelled oligonucleotides with a 2.6% RSD in peak height. The specific binding of TF autonomously replicating sequence-binding factor 1 (Abf1), either recombinant Abf1 or endogenous Abf1 in yeast cell extracts, with Cy3-labelled specific capture dsDNA could be analyzed on the uncoated chip filled with 2% hydroxypropylcellulose sieving matrix within 100 s. The specificity of the TF-DNA complex was confirmed by both competition experiment and supershift assay. The interactions between Abf1 in the range from 0.156 to 80 nM and dsDNA capture molecules were examined and a detection limit of 0.156 nM Abf1 was found. The uncoated chip capillary electrophoretic mobility shift assay method described here demonstrated great potential for fast, qualitative and quantitative analysis of protein-DNA interaction with low consumption of samples.

Parallel profiling of active transcription factors using an oligonucleotide array-based transcription factor assay (OATFA).

Cell/tissue-specific gene expression are tightly regulated by various combinations of multiple transcription factors (TFs). Here, we present an oligonucleotide array-based transcription factor assay (OATFA), which allows the simultaneous assay of multiple active TFs. In this proof-of-principle work, both purified TFs and cell extracts were analyzed using OATFA and further antibody-based validation confirmed the chip data. This method could simplify the assay of multiple TFs and may facilitate high-throughput profiling of large numbers of TFs.