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BACKGROUND NOTCH1 plays a key role in the differentiation of ventricles, and mutations are strongly associated with both sporadic and familial bicuspid aortic valves. However, few heart-specific enhancers have been identified to date. RESULTS In this study, we investigated evolutionary conserved regions (ECRs) that might act as potential enhancers within the region approximately 150-kb upstream and downstream of the NOTCH1 gene. Functional validation revealed that one 127-bp ECR located ~85-kb downstream of the NOTCH1 gene drives green fluorescent protein expression in the zebrafish embryo heart. Transcription factor (TF) prediction and core TF distribution analyses were performed to identify the core region. Dissection of ECR3 was performed to identify the 42-bp sequence, which is sufficient for heart-specific expression. In situ hybridization experiments showed that notch1b is expressed in the heart. Overexpression experiments in cells indicated that NKX2-5 is critical for enhancer activity. Mutation of the NKX-5 binding site significantly decreased reporter gene expression. Next, compared with the commonly used myocardium-labeled zebrafish transgenic strain Tg(cmlc2: mCherry), this 42-bp enhancer-labeled stable line mediated a similar expression pattern but with a smaller core region. CONCLUSION This study identified a 42-bp heart-specific enhancer near the NOTCH1 gene and further verified its functional targeting by NKX2-5.

OBJECTIVE: To study the effect of dhfr gene overexpression on ethanol-induced abnormal cardiac and vascular development in zebrafish embryos and underlying mechanisms. METHODS: dhfr mRNA was transcribed in vitro and microinjected into zebrafish fertilized eggs to induce the overexpression of dhfr gene, and the efficiency of overexpression was verified. Wild-type zebrafish were divided into a control group, an ethanol group, and an ethanol+dhfr overexpression group (microinjection of 6 nL dhfr mRNA). The embryonic development was observed for each group. The transgenic zebrafish Tg (cmlc2:mcherry) with heart-specific red fluorescence was used to observe atrial and ventricular development. Fluorescence microscopy was performed to observe the development of cardiac outflow tract and blood vessels. Heart rate and ventricular shortening fraction were used to assess cardiac function. Gene probes were constructed, and embryo in situ hybridization and real-time PCR were used to measure the expression of nkx2.5, tbx1, and flk-1 in the embryo. RESULTS: Compared with the ethanol group, the ethanol+dhfr overexpression group had a significant reduction in the percentage of abnormal embryonic development and a significant increase in the percentage of embryonic survival (P < 0.05), with significant improvements in the abnormalities of the atrium, ventricle, outflow tract, and blood vessels and cardiac function. Compared with the control group, the ethanol group had significant reductions in the expression of nkx2.5, tbx1, and flk-1 (P < 0.05), and compared with the ethanol group, the ethanol+dhfr overexpression group had significant increases in the expression of nkx2.5, tbx1, and flk-1 (P < 0.05), which were still lower than their expression in the control group. CONCLUSIONS: The overexpression of the dhfr gene can partially improve the abnormal development of embryonic heart and blood vessels induced by ethanol, possibly by upregulating the decreased expression of nkx2.5, tbx1, and flk-1 caused by ethanol.

BACKGROUND: Coronary artery aneurysms (CAAs) are a well-known complication of Kawasaki disease (KD), but there are no data on incidence or outcomes of systemic artery aneurysms (SAAs) in the current era. METHODS: From April 1, 2016, to March 31, 2019, we screened for SAAs in 162 patients with KD at risk for SAAs with magnetic resonance angiography or peripheral angiography and analyzed incidence and early outcomes of SAAs. RESULTS: Twenty-three patients had SAAs, demonstrating an incidence of 14.2% (23 of 162) in patients who were screened at 1 month after onset. The proportion of patients with SAAs was estimated to be 2% (23 of 1148) of all patients with KD. The median age at onset of KD with SAAs was 5 months. All patients with SAAs had CAAs, with z scores >8. Of patients with giant CAAs, 38.6% (17 of 44) had SAAs. A total of 129 SAAs occurred in 17 different named arteries. The most common sites for SAAs were the axillary (18.6%), common iliac (12.4%), and brachial (11.6%) arteries. During a median follow-up time of 6 months, 92.9% (79 of 85) of SAAs had some degree of regression, with 80% (68 of 85) of SAAs returning to normal. The overall regression rate was higher for medium to large SAAs than for medium to giant CAAs. CONCLUSIONS: Although the incidence of SAAs may not be as dramatically reduced as we expected compared with previous data, SAAs have a high regression rate during short-term follow-up.

Objective This study investigated the role and mechanism of action of G protein-coupled estrogen receptor (GPER) in melanogenesis. Methods GPER expression was detected in the A375 human melanoma cell line and B16 mouse melanoma cell line. Cell proliferation, melanin content, tyrosinase (TYR) activity, cyclic adenosine monophosphate (cAMP) level, and TYR and microphthalmia-related transcription factor (MITF) expression were measured. GPER activation was altered by agonist and antagonist treatment and its expression was downregulated by gene silencing. Estradiol-induced melanin synthesis and the activation of related signaling pathways were suppressed by inhibiting GPER via antagonist treatment. The relationship between GPER and TYR was evaluated in clinical chloasma samples by immunohistochemistry. Results Upregulation of GPER in A375 cells promoted melanogenesis, favored as indicated by increases in TYR and MITF expression and TYR activity. GPER activated melanin production via the cAMP-protein kinase (PK) A pathway, suggesting that GPER plays an important role in estrogen-induced melanin synthesis. The effect of GPER activation on cAMP-MITF-TYR signaling was also demonstrated in B16 cells. A significant association was observed between GPER and TYR expression in chloasma skin lesions relative to normal skin. Conclusion GPER enhances melanin synthesis via cAMP-PKA-MITF-TYR signaling and modulates the effects of estrogen in melanogenesis. GPER is therefore a potential drug target for chloasma treatment.

Congenital heart disease (CHD) is one of most prevalent birth defects in the world. However, the underlying molecular mechanism(s) have not been fully understood. Here we report that increased CHD susceptibility is associated with genetic polymorphisms for de novo nucleotide biosynthesis in northern Chinese population, which has been reported with lower plasma folate levels. Nine tagSNPs of four genes (GART, ATIC, MTHFD1 and SHMT1) in de novo nucleotide biosynthesis were sequenced in 802 sporadic CHDs patients and 1,093 controls from two Han Chinese populations, located in north China (Shandong) and South China (Shanghai). Six SNPs were found to be significantly associated with CHDs or septation defects only in the Shandong population dataset, but none displayed significant association with any CHDs in the Shanghai population dataset as well as in the combined dataset. We also showed that the minor A allele of rs7279549 in GART reduced transcriptional activity and displayed lower affinity for unknown transcription factor(s), demonstrating the allele is a functional risk factor for CHD in Shandong population. Our study indicates that dysregulation of de novo nucleotide biosynthesis pathway may conditionally contribute to CHD pathogenesis in northern Chinese.

Congenital heart defects (CHDs) are one of the most common human birth defects worldwide. TBX20 is a crucial transcription factor for the development of embryonic cardiovascular system. Previous studies have demonstrated that mutations in the TBX20 coding region contribute to familial and sporadic CHD occurrence. However, it remains largely unknown whether variants in the TBX20 regulatory region are also related to CHDs. In this study, we sequenced the 2 kb region upstream of the TBX20 transcription start site in 228 CHD patients and 292 controls in a Han Chinese population. Among the 8 single nucleotide polymorphisms (SNPs) identified, six SNPs are in strong linkage disequilibrium and the minor alleles are associated with lower CHD risk (for rs10235849 chosen as tag SNP, p = 0.0069, OR (95% CI) = 0.68 (0.51–0.90)). Functional analysis showed that the minor alleles have lower transcriptional activity than major alleles in both human heart tissues and three cell lines. The electrophoretic mobility shift assay suggested that TBX20 minor alleles may exhibit higher binding affinity with certain transcription repressors. Our results indicate that a moderately lower TBX20 activity potentially reduces CHD risk in the Han Chinese population, providing new insight in the study of CHD etiology.

Recent studies have reported the role of Wnt/β-catenin signaling in hair cell (HC) development, regeneration, and differentiation in the mouse cochlea; however, the role of Wnt/β-catenin signaling in HC protection remains unknown. In this study, we took advantage of transgenic mice to specifically knockout or overactivate the canonical Wnt signaling mediator β-catenin in HCs, which allowed us to investigate the role of Wnt/β-catenin signaling in protecting HCs against neomycin-induced damage. We first showed that loss of β-catenin in HCs made them more vulnerable to neomycin-induced injury, while constitutive activation of β-catenin in HCs reduced HC loss both in vivo and in vitro. We then showed that loss of β-catenin in HCs increased caspase-mediated apoptosis induced by neomycin injury, while β-catenin overexpression inhibited caspase-mediated apoptosis. Finally, we demonstrated that loss of β-catenin in HCs led to increased expression of forkhead box O3 transcription factor (Foxo3) and Bim along with decreased expression of antioxidant enzymes; thus, there were increased levels of reactive oxygen species (ROS) after neomycin treatment that might be responsible for the increased aminoglycoside sensitivity of HCs. In contrast, β-catenin overexpression reduced Foxo3 and Bim expression and ROS levels, suggesting that β-catenin is protective against neomycin-induced HC loss. Our findings demonstrate that Wnt/β-catenin signaling has an important role in protecting HCs against neomycin-induced HC loss and thus might be a new therapeutic target for the prevention of HC death.

Many cancer drugs are toxic to cells by activating apoptotic pathways. Previous studies have shown that mitochondria have key roles in apoptosis in mammalian cells, but the role of mitochondrial DNA (mtDNA) copy number variation in the pathogenesis of tumor cell apoptosis remains largely unknown. We used the HEp-2, HNE2, and A549 tumor cell lines to explore the relationship between mtDNA copy number variation and cell apoptosis. We first induced apoptosis in three tumor cell lines and one normal adult human skin fibroblast cell line (HSF) with cisplatin (DDP) or doxorubicin (DOX) treatment and found that the mtDNA copy number significantly increased in apoptotic tumor cells, but not in HSF cells. We then downregulated the mtDNA copy number by transfection with shRNA-TFAM plasmids or treatment with ethidium bromide and found that the sensitivity of tumor cells to DDP or DOX was significantly increased. Furthermore, we observed that levels of reactive oxygen species (ROS) increased significantly in tumor cells with lower mtDNA copy numbers, and this might be related to a low level of antioxidant gene expression. Finally, we rescued the increase of ROS in tumor cells with lipoic acid or N-acetyl-L-cysteine and found that the apoptosis rate decreased. Our studies suggest that the increase of mtDNA copy number is a self-protective mechanism of tumor cells to prevent apoptosis and that reduced mtDNA copy number increases ROS levels in tumor cells, increases the tumor cells’ sensitivity to chemotherapeutic drugs, and increases the rate of apoptosis. This research provides evidence that mtDNA copy number variation might be a promising new therapeutic target for the clinical treatment of tumors.