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Fat mass and obesity-associated protein (FTO) is a crucial eraser of RNA N6- methyladenosine (m6A) modification, and abnormal FTO expression level is implicated in pathogenesis of numerous cancers. Herein, we demonstrate the construction of a label-free fluorescent biosensor for homogeneous detection of m6A eraser FTO in breast cancer tissues. When FTO is present, it specifically erases the methyl group in m6A, inducing the cleavage of demethylated DNA by endonuclease Dpn II and the generation of a single-stranded DNA (ssDNA) with a 3′-hydroxyl group. Subsequently, terminal deoxynucleotidyl transferase (TdT) promotes the incorporation of dTTPs into the ssDNA to obtain a long polythymidine (T) DNA sequence. The resultant long poly (T) DNA sequence can act as a template to trigger hyperbranched strand displacement amplification (HSDA), yielding numerous DNA fragments that may be stained by SYBR Gold to produce an enhanced fluorescence signal. This biosensor processes ultrahigh sensitivity with a detection limit of 1.65 × 10−10 mg/mL (2.6 fM), and it can detect the FTO activity in a single MCF-7 cell. Moreover, this biosensor can screen the FTO inhibitors, evaluate enzyme kinetic parameters, and discriminate the FTO expression levels in the tissues of breast cancer patients and healthy persons. [Display omitted] • We construct a new biosensor for label-free detection of FTO activity. • This biosensor exhibits high sensitivity and good specificity. • This biosensor can detect the FTO activity in a single MCF-7 cell. • It can screen inhibitors and measure enzyme the kinetic parameters. • It can discriminate breast cancer tissues from healthy person tissues. [ABSTRACT FROM AUTHOR]

[Display omitted] • We demonstrate simultaneous single-molecule detection of FTO and ALKBH3. • Only a single DNA ligase can achieve multi-cyclic cascade amplification. • This method exhibits high sensitivity and good specificity. • This method can screen inhibitors and detect cellular RNA demethylases. • This method can discriminate breast cancer patients from healthy persons. RNA demethylases play a crucial role in gene expression regulation by controlling RNA methylation levels, and their abnormal expressions are associated with many human diseases. Herein, we develop a single-molecule detection method for simultaneous quantification of N6-methyladenosine (m6A) demethylase FTO and N1-methyladenosine (m1A) demethylase ALKBH3 based on RNA demethylation-directed multi-cyclic ligase chain reaction (LCR) cascades. We design a ssDNA containing m6A modification as the substrate of FTO and a ssDNA containing m1A modification as the substrate of ALKBH3. FTO and ALKBH3 catalyze the demethylation of m6A and m1A to generate normal adenine (A), respectively. Afterwards, Taq ligase initiates the LCR reaction with the demethylated ssDNAs as the templates to produce abundant biotinylated Cy3/Cy5-labeled signal probes. Subsequently, the resultant biotinylated probes are captured by streptavidin-coated magnetic beads (MBs) to form the MB-ssDNA-Cy3/Cy5 complexes. After magnetic separation, the ssDNAs in the MB-ssDNA-Cy3/Cy5 complexes are digested by DNase I, liberating abundant Cy3 and Cy5 fluorophores that can be counted by single-molecule detection. Remarkably, only a single DNA ligase is employed to achieve target signal conversion and multi-cyclic cascade amplification. This method displays extremely high sensitivity with a limit of detection of 6.33 × 10−18 mol/L for FTO and 1.44 × 10−17 mol/L for ALKBH3. Moreover, it can measure the enzyme kinetic parameters, screen inhibitors, and quantify cellular FTO and ALKBH3 activities with single-cell sensitivity. Furthermore, it can differentiate the FTO and ALKBH3 levels in the tissues of healthy human and breast cancer patients, offering a robust platform for biomedical research and clinical diagnosis. [ABSTRACT FROM AUTHOR]

Abstract Background Androgen has been implicated in aging-related cardiac remodeling, but its precise role in aging heart remains controversial. We aimed to investigate the role of testosterone in the development of aging-related cardiac remodeling and the mechanisms involved. Methods Wild type and Axl knockout mice (Axl−/−) were randomized into three groups: the young group (n = 30, 3 months old), the aging group (n = 30, 18 months old), the testosterone undecanoate treatment group (TU, n = 30, 18 months old). Mice in the TU group were given testosterone undecanoate (39 mg/kg) by subcutaneous injection on the back at fifteen-months-old, once a month, a total of three times. The old group received solvent reagent (corn oil) by the same method. Results The aging mice exhibited a decrease in serum testosterone, and Gas6 levels and an increase in apoptosis, and manifested cardiac fibrosis. Testosterone injection to wild type mice increased the levels of testosterone and Gas6 in serum and decreased cardiac apoptosis and fibrosis. Axl−/−mice receiving testosterone injection exhibited no obvious improvement in cardiac remodeling although the levels of testosterone and Gas6 in serum elevated. Conclusions These data indicated that testosterone replacement therapy (TRT) alleviates cardiac fibrosis and apoptosis, at least in part by enhancing Gas6 expression. Moreover, deletion of Axl disables testosterone, which indicated that Axl is an important downstream regulator of testosterone. TRT would improve aging-related cardiac remolding via Gas6/Axl signaling pathway, implicating its therapeutic potential to treat aging-related heart disease. Highlights • Testosterone decreases cardiomyocyte apoptosis in aging mice. • Testosterone ameliorates cardiac fibrosis in aging mice. • Testosterone increases the level of Gas6. • Testosterone protects from cardiac aging in a Gas6/Axl dependent manner. [ABSTRACT FROM AUTHOR]