A novel label-free fluorescence strategy for methyltransferase activity assay based on dsDNA-templated copper nanoparticles coupled with an endonuclease-assisted signal transduction system.

Evaluating DNA methyltransferase (MTase) activity has received considerable attention due to its significance in the fields of early cancer clinical diagnostics and drug discovery. Herein, we proposed a novel label-free fluorescence method for MTase activity assay by coupling double-stranded DNA (dsDNA)-templated copper nanoparticles (CuNPs) with an endonuclease-assisted signal transduction system. In this strategy, dsDNA molecules were first methylated by DNA adenine methylation (Dam) MTase and then cleaved by the methylation-sensitive restriction endonuclease DpnI. The cleaved DNA fragments could not act as efficient templates for the formation of fluorescent CuNPs and thus no fluorescence signal was produced. Under optimized experimental conditions, the developed strategy exhibited a sensitive fluorescence response to Dam MTase activity. This strategy was also demonstrated to provide an excellent platform to the inhibitor screening for Dam MTase. These results demonstrated the great potential for the practical applications of the proposed strategy for Dam MTase activity assay.

Wnt1-overexpressing skeletal myoblasts as an improved cell therapy for cardiac repair following myocardial infarction.

AIM: Recent findings highlight the critical role of the Wnt signaling pathway in cardiac repair and stem cell regulation. Our previous study shows that lithium chloride (LiCl) optimizes skeletal myoblast (SkM) for transplantation by mimicking the Wnt/ß-catenin signaling activities. In this study, we evaluate the therapeutic potential of SkMs genetically modified with Wnt1gene (Wnt1 SkMs) in a rat model with myocardial infarction (MI). METHODS: We harvested neonatal SkMs using Wistar rats (1-3-day old) transfected with p-EGFP-C3-Wnt1 plasmid. RT-PCR and immunofluorescence showed a higher expression of Wnt1 in the Wnt1 SkMs. We observed that Wnt1 SkMs increased connexin 43 (Cx43) expression, reduced apoptosis induced by hydrogen peroxide (H2O2) and decreased caspase-3 expression via the canonical Wnt signaling pathways compared to the empty vector transfected SkMs (control SkMs). For in vivo studies, the myocardial infarction model was developed in the Wistar rats. The rats were grouped to receive 100 µL basal DMEM without cells or containing 1.5×106SkMs and Wnt1 SkMs. Histological studies revealed improved survival of SkMs, reduced cardiomyocytes apoptosis, and upregulated expression of Cx43 in Wnt1 SkMs therapy group. Echocardiography monitored four weeks after therapy showed improvement of the left ventricular function in rats treated with Wnt1SkMs compared to other groups. CONCLUSION: Transplantation of Wnt1 SkMs improves rat myocardial function and enhances anti apoptotic properties of both SkMs and cardiomyocytes and upregulation of tissue Cx43 after infarction via the canonical Wnt/ß-catenin signaling activities.

Estrogen regulates the expression and activity of epithelial sodium channel in mouse osteoblasts.

Estrogen plays an important role in bone metabolism and only high dose can stimulate osteoblast bone formation. However, the underlying mechanisms have not been elucidated. The epithelial sodium channel (ENaC) is a key pathway for sodium transport in epithelia, vascular endothelium, and other tissues; although the expressions of α and γ ENaC mRNA were found in osteoblasts, the regulation of ENaC by estrogen in osteoblasts has not been studied. Our recent data confirmed the ENaC expression in mouse primary osteoblasts by immunocytofluorescence, RT-PCR, western blot, and patch clamp. Furthermore, we found estrogen (10(-5)M) increased the expression of α and γ ENaC subunits at both the mRNA and protein levels in osteoblasts. On the other hand, 17ß estradiol (20 nM) increased inward Na+ currents which were inhibited by amiloride. The estrogen dose used in patch clamp is much lower than those of mRNA and protein analysis, which means single cell ENaC electrophoretic mobility is much more sensitive to estrogen than the mRNA and protein production by estrogen stimulation. Our results suggest that estrogen regulates expression and function of ENaC in osteoblasts may provide a new clue that the mechanism of high dose of estrogen influence osteoblast bone formation via ENaC activity.