Early Diagnostic Markers for Esophageal Squamous Cell Carcinoma: Copy Number Alteration Gene Identification and cfDNA Detection.

The high mortality rate of esophageal squamous cell carcinoma (ESCC) is exacerbated by the absence of early diagnostic markers. The pronounced heterogeneity of mutations in ESCC renders copy number alterations (CNAs) more prevalent among patients. The identification of CNA genes within esophageal squamous dysplasia (ESD), a precancerous stage of ESCC, is crucial for advancing early detection efforts. Utilization of liquid biopsies via droplet-based digital PCR (ddPCR) offers a novel strategy for detecting incipient tumor traces. This study undertook a thorough investigation of CNA profiles across ESCC development stages, integrating data from existing databases and prior investigations to pinpoint and confirm CNA markers conducive to early detection of ESCC. Targeted sequencing was employed to select potential early detection genes, followed by the establishment of prediction models for ESCC early detection using ddPCR. Our analysis revealed widespread CNAs during the ESD stage, mirroring the CNA landscape observed in ESCC. A total of 40 CNA genes were identified as highly frequent in both ESCC and ESD lesions, through a comprehensive gene-level CNA analysis encompassing ESD and ESCC tissues, ESCC cell lines, and pan-cancer data sets. Subsequent validation of 5 candidate markers via ddPCR underscored the efficacy of combined predictive models encompassing PIK3CA, SOX2, EGFR, MYC, and CCND1 in early ESCC screening, as evidenced by the area-under-the-curve values exceeding 0.92 (P < .0001) across various detection contexts. The findings highlighted the significant utility of CNA genes in the early screening of ESCC, presenting robust models that could facilitate early detection, broad-scale population screening, and adjunctive diagnosis.

Potential Chemotherapeutic Effect of Selenium for Improved Canceration of Esophageal Cancer.

Esophageal squamous cell carcinoma is the most common type of esophageal cancer and accounts for 5% of malignant tumor deaths. Recent research suggests that chronic inflammation and DNA damage may drive the onset of esophageal squamous cell carcinoma, implying that lowering chronic inflammation and DNA damage compounds may provide chemo-prevention. According to epidemiological and experimental evidence, selenium is linked to a lower risk of several malignancies, including esophageal squamous cell carcinoma. However, its exact mechanism is still unclear. In the present study, we used cell lines and a 4-NQO mice model to explore the anti-cancer mechanism of four types of selenium. Our findings indicated that selenium inhibited the proliferation, colony formation, and ROS level of ESCC cell lines in a time-dependent manner. Intriguingly, selenium treatment impeded 4-NQO-induced high-grade intraepithelial neoplasia and reduced the number of positive inflammatory cells by preserving DNA from oxidative damage. In addition, selenium significantly decreased the expression of Ki-67 and induced apoptosis. This study demonstrates that selenium has a significant chemo-preventive effect on ESCC by reducing high-grade dysplasia to low-grade dysplasia. For the first time, selenium was shown to slow down the progression of esophageal cancer by lowering inflammation and oxidative DNA damage.

Esophageal Squamous Cancer from 4NQO-Induced Mice Model: CNV Alterations.

Squamous esophageal carcinoma is a common pathological type of esophageal carcinoma around the world. The prognosis of esophageal carcinoma is usually poor and diagnosed at late stages. Recently, research suggested that genomic instability occurred in esophageal cells during the development of esophageal squamous cell carcinoma (ESCC). Identifying prognostic and specific genomic characteristics, especially at the early hyperplasia stage, is critical. Mice were given 4-nitroquinoline 1-oxide (4NQO) with drinking water to induce esophageal cancer. The immortalized human esophageal epithelial cell line (NE2) was also treated with 4NQO. We performed histologic analyses, immunofluorescence, and immunohistochemical staining to detect DNA damage at different time points. Whole-exome sequencing was accomplished on the esophagus tissues at different pathological stages to detect single-nucleotide variants and copy number variation (CNV) in the genome. Our findings indicate that all mice were tumor-forming, and a series of changes from simple hyperplasia (ESSH) to intraepithelial neoplasia (IEN) to esophageal squamous cell carcinoma (ESCC) was seen at different times. The expression of γ-H2AX increased from ESSH to ESCC. In addition, mutations of the Muc4 gene were detected throughout the pathological stages. Furthermore, CNV burden appeared in the esophageal tissues from the beginning of ESSH and accumulated more in cancer with the deepening of the lesions. This study demonstrates that mutations caused by the early appearance of DNA damage may appear in the early stage of malignant tissue before the emergence of atypia. The detection of CNV and mutations of the Muc4 gene may be used as an ultra-early screening indicator for esophageal cancer.

Mir-382 Promotes Differentiation of Rat Liver Progenitor Cell WB-F344 by Targeting Ezh2.

BACKGROUND/AIMS: Liver progenitor cells (LPCs) were considered as a promising hepatocyte source of cell therapy for liver disease due to their self-renewal and differentiation capacities, while little is known about the mechanism of LPC differentiate into hepatocytes. This study aims to explore the effect of miR-382, a member of Dlk1-Dio3 microRNA cluster, during hepatic differentiation from LPCs. METHODS: In this study, we used rat liver progenitor cell WB-F344 as LPC cell model and HGF as inducer to simulate the process of LPCs hepatic differentiation, then microRNAs were quantified by qPCR. Next, WB-F344 cell was transfected with miR-382 mimics, then hepatocyte cell trait was characterized by multiple experiments, including that periodic acid schiff staining and cellular uptake and excretion of indocyanine green to evaluate the hepatocellular function, qPCR and Western Blotting analysis to detect the hepatocyte-specific markers (ALB, Ttr, Apo E and AFP) and transmission electron microscopy to observe the hepatocellular morphology. Moreover, Luciferase reporter assay was used to determine whether Ezh2 is the direct target of miR-382. RESULTS: We found that miR-382 increased gradually and was inversely correlated with the potential target, Ezh2, during WB-F344 hepatic differentiation. In addition, functional studies indicated that miR-382 increased the level of hepatocyte-specific genes. CONCLUSIONS: This study demonstrates that miR-382 may be a novel regulator of LPCs differentiation by targeting Ezh2.

Anterior wrist and medial malleolus as the novel sites of tissue selection: a retrospective study on electric shock death through the hand-to-foot circuit pathway.

Our previous work demonstrated that characteristic changes could occur in the anterior wrist and medial malleolus in electric deaths through the hand-to-foot electric circuit pathway in an electric shock rat model. However, whether the same phenomenon occurs in humans is unknown. The aim of the present retrospective study was to ascertain whether the anterior wrist and medial malleolus could also be selected as the promising and significant sites in electric death through the hand-to-foot circuit pathway. Nineteen human cases from the autopsy and one clinical survivor who sustained a severe electric shock through the hand-to-foot circuit pathway were analyzed. Additional ten autopsy patients who died from traffic accidents and sudden cardiac attacks were used as the control group. Histopathological changes in the soft tissues of the anterior wrist and medial malleolus in all autopsy patients, as well as the electric current pathway of the survivor, were observed. The results showed that the nuclear polarizations in the anterior wrist and medial malleolus soft tissues of the electric death were extremely noticeable as compared with the controls. The most severe electrical injury in the survivor occurred in the anterior wrist. These findings suggest that the soft tissues of the anterior wrist and/or the medial malleolus as the narrowest parts of the limbs could be used as the complementary sites for tissue selection and considered as necessary locations for examinations to assess the electric death in medicolegal identification.

Anterior wrist and medial malleolus: the optimal sites for tissue selection in electric death through hand-to-foot circuit pathway.

Specific morphological changes may be absent in some cases of electrocution shocked by the voltage of 220 V or lower. In this study, we attempted to demonstrate that the anterior wrist and medial malleolus were the optimal sites with promising and significant changes in electric death through the hand-to-foot circuit pathway. We established an electric shock rat model and observed histopathologic changes in the anterior wrist and medial malleolus. The results showed that the current intensities in the left anterior wrist and right medial malleolus were remarkably higher than those in the other sites, and the nuclei long/short (L/S) axis ratios of the arterial endotheliocyte and the skeletal muscle cell in these two areas were significantly higher than those in other parts of the body. These findings suggested that the anterior wrist and/or medial malleolus soft tissues as the narrowest parts of the limbs could be used as promising and useful sites for the assessment of electrical shock death, especially in forensic pathologic evaluation.

Metabolic Characterization of Myocardial Infarction Using GC-MS-Based Tissue Metabolomics.

Understanding the metabolic features of myocardial infarction (MI) is critical to its prevention and treatment. Here, we aimed to characterize the metabolic features of early MI using a tissue metabolomics method based on gas chromatography-mass spectrometry (GC-MS). Thirty-four pairs of infarcted myocardia and their matched non-infarcted myocardia were collected from 34 rats that underwent coronary artery ligation (CAL); their metabolic profiles were compared by GC-MS-based tissue metabolomics to characterize the metabolic features of MI. On the basis of differential metabolites, their diagnostic potential for MI was analyzed, and MI-related metabolic pathways were investigated. Serum samples before and post MI were used to validate the results obtained in myocardia. The metabolic profile of the infarcted myocardia was obviously different from that of the non-infarcted myocardia, as indicated by partial least squares discriminate analysis (PLS-DA) plots. Twenty-two metabolites were identified to be different between the infarcted myocardia and non-infarcted myocardia. These metabolic alterations reflect energy deficit, acidosis, oxidative stress, ionic imbalance, and cardiac injury post MI. Glutamine, glutamate, and lactate were confirmed to jointly confer a favorable potential for diagnosing MI, which can be well validated in serum.

TLR3 Expression is a Potential Prognosis Biomarker and Shapes the Immune-Active Tumor Microenvironment in Esophageal Squamous Cell Carcinoma.

BACKGROUND: Toll-like receptor 3 (TLR3) not only plays a crucial role in innate immune and inflammation but also in anti-cancer immunity. Nevertheless, the clinicopathological outcome of TLR3 in ESCC is still ambiguous. METHODS: Immunohistochemistry was performed to investigate TLR3 expression and its impact on survival in 137 ESCC patients (including paired esophageal tissues with different stages of early lesions from 37 patients). Furthermore, we downloaded ESCC RNA-seq datasets (including phenotype and survival data) from The Cancer Genome Atlas (TCGA). The relationship between TLR3 and prognosis, biological landscape, and immune infiltration was assessed to verify the immunohistochemical results of our tissue samples, explore the possible mechanism of prognostic outcomes, and predict the sensitivity of immunotherapy. RESULTS: TLR3 protein expression displayed an increasing trend in the progression through different grades of cellular atypia, from normal, esophageal simple hyperplasia (ESSH), intraepithelial neoplasia (IEN) to ESCC (P < 0.0083). TLR3 protein had a positive association with inflammation level (Rho = 0.341, P < 0.001). TLR3 mRNA expression was significantly higher in comparison to adjacent normal tissues (P < 0.001). Cox regression analysis indicated high TLR3 protein and mRNA expression conferred good prognosis in our samples and TCGA, especially for advanced ESCC patients (TNM stage III and IV). Overexpression of TLR3 resulted in an immune-active microenvironment via the recruitment of immune-active cells including cytotoxic lymphocytes (CTLs), CD8+ T cells, NK cells, dendritic cells, and M1-type macrophages. TLR3 expression was correlated with the pro-inflammatory cytokines and chemokines relating to anti-tumor immunity. Moreover, GSEA analysis indicated upregulated expression of TLR3 could activate the apoptotic pathway. CONCLUSION: High TLR3 expression in ESCC patients was associated with a more favorable prognosis, immune-active cell infiltration, and an activated apoptotic pathway. TLR3 has potential applications for immunotherapy and immune response prediction in patients with ESCC.

Expression and Significance of MyD88 in Patients With Gastric Cardia Cancer in a High-Incidence Area of China.

Background: Gastric cardia cancer (GCC) arises in the area of the stomach adjoining the esophageal-gastric junction and has unique risk factors. It was suggested that the involvement of Helicobacter pylori is associated with GCC from high-risk population. Myeloid differentiation factor 88 (MyD88) is a crucial adaptor molecule in Toll-like signaling pathway recognizing H. pylori. Its role in GCC has not been elucidated yet. In this study, our purpose is to investigate the expression and significance of MyD88 in GCC tissue. Methods: Expression of MyD88 and nuclear factor κB (NF-κB) p105/p50 and infection of H. pylori were detected by immunohistochemistry in gastric cardia tissue. The correlation of MyD88 expression to NF-κB p105/p50 expression, H. pylori infection, and clinicopathologic characteristics in gastric cardia tissue was analyzed. The involvement of MyD88 in patient prognosis was also analyzed. Results: Our data showed that the expression of MyD88 elevated from normal mucosa to inflammation (p = 0.071). The expression of MyD88 was enhanced in GCC tissues by contrast to non-malignant cardia mucosa (p = 0.025). What's more, overexpression of MyD88 was detected in intestinal-type adenocarcinoma with inflammation. Patients with high MyD88 staining revealed a better differentiation (p = 0.02). MyD88 also positively correlated with NF-κB p105/p50 expression (p = 0.012) in cancer tissue. Expression of MyD88 was increased but not significantly in biopsies with H. pylori infection compared with non-infected biopsies. Multivariate analyses revealed lymph node metastasis but not MyD88 expression was an independent predictor for patient survival. Conclusion: These findings provide pathological evidence that upregulating MyD88 and inducing inflammation might be involved in gastric cardia carcinogenesis in high-risk population. MyD88 plays a role in gastric cardia carcinogenesis with NF-κB pathway activation. Higher MyD88 expression is not a major prognostic determinant in GCC, but it may relate to the tumor cell differentiation.

[Effects of Huoxue Tongmai Lishui method on fundus fluorescein angiography of non-ischemic retinal vein occlusion: a randomized controlled trial].

BACKGROUND: Huoxue Tongmai Lishui method, a traditional Chinese medicine treatment for eliminating water, activating and promoting blood circulation, could inhibit fundus hemorrhage on experimental retinal vein occlusion (RVO) with high obvious effective rate, and improve symptoms in traditional Chinese medicine. The action mechanism may be related to reducing plasma viscosity and non-perfusion area, and the formation of collateral circulation. OBJECTIVE: To explore the therapeutic effects of Huoxue Tongmai Lishui method (Sanxue Mingmu Tablet) on fundus fluorescent angiograph of non-ischemic retinal vein occlusion (RVO). DESIGN, SETTING, PARTICIPANTS AND INTERVENTIONS: Thirty-four patients with non-ischemic RVO in Department of Ophthalmology, the First Affiliated Hospital, Hunan University of Traditional Chinese Medicine from April 2005 to April 2009 were included. All the patients were diagnosed as qi stagnation and blood stasis syndrome or hyperactivity of liver yang syndrome, and they were randomly divided into two groups, with 17 eyes of 17 patients in treatment group treated by Sanxue Mingmu Tablet combined with conventional treatment, and 18 eyes of 17 patients in control group treated by Xueshuantong Tablet combined with conventional treatment. The patients were treated for two months. MAIN OUTCOME MEASURES: Fundus colour photography, and fundus fluorescent angiograph were detected in two groups before and after the treatment. RESULTS: The curative effect of Sanxue Mingmu Tablet was better than that of Xueshuantong Tablet. Huoxue Tongmai Lishui method could significantly shorten the retinal circulation time, reduce the non-perfusion area, decrease the formation of angiogenesis and promote the formation of collateral circulation. CONCLUSION: Huoxue Tongmai Lishui method is an effective traditional Chinese medicine treatment with high obvious effective rate in reducing non-perfusion area and avoiding venous occlusion and formation of collateral circulation.

DACT1 Involvement in the Cytoskeletal Arrangement of Cardiomyocytes in Atrial Fibrillation by Regulating Cx43.

OBJECTIVE: To determine the role of the dishevelled binding antagonist of beta catenin 1 (DACT1) in the cytoskeletal arrangement of cardiomyocytes in atrial fibrillation (AF). METHODS: The DACT1 expression and its associations with the degree of fibrosis and ß-catenin in valvular disease patients were analyzed by immunohistochemistry and Masson's staining. DACT1 was overexpressed in the atrial myocyte cell line (HL-1) and the cardiac cell line (H9C2) by adenoviral vectors. Alterations in the fibrous actin (F-actin) content and organization and the expression of ß-catenin were detected by flow cytometry, immunofluorescence, and Western blotting. Additionally, the association of DACT1 with gap junctions connexin 43 (Cx43) was detected by immunohistochemistry, immunofluorescence, and Western blotting. RESULTS: Decreased cytoplasmic DACT1 expression in the myocardium was associated with AF (P=0.037) and a high degree of fibrosis (weak vs. strong, P=0.028; weak vs. very strong, P=0.029). A positive association was observed between DACT1 and ß-catenin expression in clinical samples (P=0.028, Spearman's rho=0.408). Furthermore, overexpression of DACT1 in HL-1 and H9C2 cells induced an increase in ß-catenin and subsequent partial colocalization of DACT1 and ß-catenin. In addition, F-actin content and organization were enhanced. Interestingly, DACT1 was positively correlated with the Cx43 expression in clinical samples (P=0.048, Spearman's rho=0.370) and changed the Cx43 distribution in cardiac cell lines. CONCLUSION: DACT1 proved to be a novel AF-related gene by regulating Cx43 via cytoskeletal organization induced by ß-catenin accumulation in cardiomyocytes. DACT1 could thus serve as a potential therapeutic marker for AF.

Artesunate prevents rats from the clozapine-induced hepatic steatosis and elevation in plasma triglycerides.

Clozapine is an atypical antipsychotic with therapeutic efficacy in treatment-resistant schizophrenia patients and low incidence of extrapyramidal side effects. However, the use of clozapine has been limited by its adverse effects on metabolism. Artesunate is a semisynthetic derivative of artemisinin and was shown to decrease the plasma cholesterol and triglyceride in rabbits and rats in recent studies. The aim of this study was to examine possible effects of artesunate on the clozapine-induced metabolic alterations in rats given saline, clozapine, artesunate, or clozapine plus artesunate for 6 weeks. The clozapine group showed significantly high plasma levels of triglyceride, hepatic steatosis, and fibrosis along with high levels of C-reactive protein, alanine aminotransferase, and aspartate aminotransferase compared to the saline group. But the treatment had no effect on weight gain and caused no hyperglycemia, hyperinsulinemia, and behavioral changes in the rats. More significantly, these clozapine-induced changes were not seen in rats coadministered with clozapine plus artesunate. These results added evidence supporting psychiatrists to try add-on treatment of artesunate in schizophrenia patients to ameliorate clozapine-induced adverse metabolic effects.

Intramedullary schwannoma of the upper cervical spinal cord: a case study of identification in pathologic autopsy.

Intramedullary schwannoma of the upper cervical spinal cord is rarely reported in forensic medicine. We herein report a case involving a patient who died of compression from an intramedullary schwannoma in the upper cervical spinal cord. A 30-year-old man initially presented with a five-day history of pain in the left chest that progressed to weakening in the left arm. Although the patient was treated with analgesic poultices, he developed inspiratory dyspnoea and died while working the next day without having undergone any medical imaging examination or surgical treatment. Anatomical and histopathological examinations revealed an intramedullary schwannoma in the left cervical spinal cord (C3-C5) underneath the spinal nerve root. The cause of death might have been asphyxia secondary to the tumour, which interfered with the nerve function in the respiratory muscles. This finding suggests that an autopsy is essential for pathologists and medicolegists to comprehensively undertake their due obligation to obtain "the first evidence", especially when there is a lack of directly related evidence. As part of the central nervous system, the spinal cord could be systematically included in a routine pathological autopsy in some cases.

Metabolic risk factors associated with sudden cardiac death (SCD) during acute myocardial ischemia.

Sudden cardiac death (SCD) is the leading cause of death worldwide. Myocardial ischemia (MI) is the most common underlying causal disorder for SCD. Metabolic risks leading to SCD during acute MI are still not fully understood. Here, using tissue metabolomics, we aimed to investigate myocardial metabolic alterations relevant to SCD events in an acute MI rat model induced by coronary artery ligation (CAL). Thirty-four rats were successfully performed CAL, of which 13 developed lethal ventricular tachyarrhythmia (LVTA)-SCD and 7 developed severe atrioventricular block (AB)-SCD. Fourteen rats that survived within 70 min after the ligation were served as peer controls. The partial least squares-discriminant analysis plots demonstrated clear separations between the SCD rats and controls, indicating obvious differences in myocardial metabolome between these rats. The levels of isoleucine, lactate, glutamate choline, phosphorylcholine, taurine and asparagine in ischemic myocardia were positively associated with LVTA-SCD events; in contrast, the levels of alanine, urea, phenylalanine, linoleic acid, elaidic acid and stearic acid were inversely correlated with LVTA-SCD events. The levels of glutamate and urea were positively and negatively relevant to AB-SCD events, respectively. The dangerous metabolites indicated that lower levels of energy substrates, severe hypoxia, the inhibition of transamination and hyper sympathetic excitement and reactive oxygen species in myocardia were vulnerable to SCD during acute MI. The results suggest fatal metabolic alterations correlated with SCD events during acute MI, which could offer novel clues for the prevention or treatment of acute MI-related SCD.

Non-targeted metabolomics identified a common metabolic signature of lethal ventricular tachyarrhythmia (LVTA) in two rat models.

Lethal ventricular tachyarrhythmia (LVTA) is the predominant underlying mechanism of sudden cardiac death (SCD). The aim of this study is to characterize the metabolic features of myocardia following LVTA, and identify potential biomarkers to diagnose LVTA. We developed two LVTA rat models induced by aconitine injection or coronary artery ligation, which represent cardiac ion channel disease-related and cardiac ischemia-related SCD, respectively. The myocardial metabolic profile was investigated by gas chromatography-mass spectrometry and proton nuclear magnetic resonance-based metabolomics. Twenty-three aconitine-injected and 14 coronary artery ligation-treated rats developed LVTA SCD. A total of 38 differential metabolites of myocardia were identified in aconitine-induced LVTA rats, of which 31 metabolites showed a similar change in coronary artery ligation-related LVTA rats. Fatty acids (stearic, palmitic, linoleic, elaidic, and myristic) and branched-chain amino acids (valine, leucine, and isoleucine) were the most down-regulated metabolites. Furthermore, elevated ADP, phosphate, lactate, glutamate, aspartate, threonine, choline and arginine were also observed. Major pathways regarding these dysregulated metabolites post LVTA are energy excessive consumption and deficit, ionic imbalance, oxidative stress, cardiac cytotoxicity and membrane injury. Valine, stearic acid and leucine collectively enable a precision of 92.9% to distinguish LVTA from its control, and are correlated with several arrhythmia indices. Our results uncovered a common metabolic feature of LVTA in myocardia in two rat models, which represent cardiac ion channel disease and cardiac ischemia, respectively. l-Valine, l-leucine and stearic acid jointly confer good potential for distinguishing LVTA, which might be potential biomarkers of LVTA-related SCD.

Trace Element Concentrations in Human Tissues of Death Cases Associated With Secondary Infection and MOF After Severe Trauma.

Proper trace element level is crucial for the organs in maintaining normal physiological functions. Multiple organ failure (MOF) might be added to critically ill patients due to a lack of trace elements. Alterations of trace element levels in brain, heart, liver, and kidney after severe trauma, however, have been little studied so far. In this study, tissue samples of the frontal cortex of the brain, interventricular septum of the heart, right lobe of the liver, and upper pole of the kidney were obtained from forensic autopsies, of which 120 cases died during the 5th to 15th day of hospitalization, whereas the trauma death group and 43 cases immediately died due to severe craniocerebral trauma as the control group. Copper (Cu), iron (Fe), zinc (Zn), and selenium (Se) were quantified by inductively coupled plasma atomic emission spectrophotometry (ICP-AES). Cu, Fe, Zn, and Se concentrations in the brain, heart, liver, and kidney in the trauma group decreased dramatically (p<0.05) compared to the control group. The incidence of secondary infection and multiple organ failure (MOF) in the trauma death group were 78.33 and 29.17%, respectively. The concentrations of all elements exhibited a significant correlation with secondary infection and MOF (p<0.01). Our data suggest that low concentrations of Cu, Fe, Zn, and Se in pivotal organs may contribute to the incidence of secondary infection and MOF after severe trauma, which to some extent results in death.

Dact1 involvement in the cytoskeletal arrangement of cardiomyocytes in atrial fibrillation by regulating cx43

Abstract Objective: To determine the role of the dishevelled binding antagonist of beta catenin 1 (DACT1) in the cytoskeletal arrangement of cardiomyocytes in atrial fibrillation (AF). Methods: The DACT1 expression and its associations with the degree of fibrosis and β-catenin in valvular disease patients were analyzed by immunohistochemistry and Masson's staining. DACT1 was overexpressed in the atrial myocyte cell line (HL-1) and the cardiac cell line (H9C2) by adenoviral vectors. Alterations in the fibrous actin (F-actin) content and organization and the expression of β-catenin were detected by flow cytometry, immunofluorescence, and Western blotting. Additionally, the association of DACT1 with gap junctions connexin 43 (Cx43) was detected by immunohistochemistry, immunofluorescence, and Western blotting. Results: Decreased cytoplasmic DACT1 expression in the myocardium was associated with AF (P=0.037) and a high degree of fibrosis (weak vs. strong, P=0.028; weak vs. very strong, P=0.029). A positive association was observed between DACT1 and β-catenin expression in clinical samples (P=0.028, Spearman's rho=0.408). Furthermore, overexpression of DACT1 in HL-1 and H9C2 cells induced an increase in β-catenin and subsequent partial colocalization of DACT1 and β-catenin. In addition, F-actin content and organization were enhanced. Interestingly, DACT1 was positively correlated with the Cx43 expression in clinical samples (P=0.048, Spearman's rho=0.370) and changed the Cx43 distribution in cardiac cell lines. Conclusion: DACT1 proved to be a novel AF-related gene by regulating Cx43 via cytoskeletal organization induced by β-catenin accumulation in cardiomyocytes. DACT1 could thus serve as a potential therapeutic marker for AF.

Design, synthesis, and biological evaluation of novel 4-alkylamino-1-hydroxymethylimidazo[1,2-a]quinoxalines as adenosine A(1) receptor antagonists.

A series of 4-alkylamino-1-hydroxymethylimidazo[1,2-a]quinoxalines have been synthesized and evaluated for their adenosine A(1) receptor inhibitory activity in the radioligand binding assays. The compounds were tested for the inhibition percent (IP) and the affinity toward A(1)AR (K(i)) that IP were more than 90% in the nanomolar range. 4-Cyclopentylamino-7,8-dichloro-1-hydroxymethylimidazo[1,2-a]quinoxaline 18 is the most potent compound in this series, having K(i)=7nM, which is remarkably higher than that of IRFI-165 (K(i)=48). 1-Hydroxymethyl groups of the tricyclic heteroarmatic compounds displayed the potent affinities toward A(1)AR.