DNA damage and repair system in spinal cord ischemia

AbstractBackground and Purpose: Spinal cord ischemia-reperfusion injury may be initiated by a number of mediators, including reactive oxygen species. Recent studies have shown that human MutY homologue (hMYH), human 8-oxo-7,8-dihydrodeoxyguanine (8-oxoG) glycosylase (hOGG1), and human MutS homologue 2 (hMSH2) are important DNA mismatch repair genes. We hypothesized that ischemia-reperfusion injury in spinal cord causes DNA damage manifested by 8-oxoG production and activates the DNA repair system involving hMYH, hOGG1, and hMSH2. Methods: Spinal cords of rabbits were removed at 1, 3, 6, 24, and 48 hours after 30 minutes of infrarenal aortic occlusion. DNA damage was determined with 8-oxoG staining. The expression and localization of DNA repair enzymes, such as hMYH, hOGG1, and hMSH2, were studied with Western blot analysis and immunohistochemical staining. The level of apoptosis was determined with TUNEL study. Activation of caspase-3, an enzyme induced by cellular injury that leads to apoptosis by degrading cellular structural proteins, was also studied. Results: DNA damage monitored with 8-oxoG level was significantly present from 1 hour to 6 hours after reperfusion in gray matter neurons of ischemic spinal cord. The levels of hMYH, hOGG1, and hMSH2 were markedly increased in gray matter neurons at 6 hours after reperfusion. Caspase-3 was also induced at 6 hours to 24 hours after reperfusion in ischemic spinal cord. However, the peak level of TUNEL reactivity was found at 48 hours after reperfusion in spinal cord neurons. Conclusion: This study has shown, for the first time, the rapid expression of DNA damage-repair processes associated with spinal cord ischemia and subsequent reperfusion. (J Vasc Surg 2003;37:847-58.

Automatic Detection of Obstructive Sleep Apnea Events Using a Deep CNN-LSTM Model

Obstructive sleep apnea (OSA) is a common sleep-related respiratory disorder. Around the world, more and more people are suffering from OSA. Because of the limitation of monitor equipment, many people with OSA remain undetected. Therefore, we propose a sleep-monitoring model based on single-channel electrocardiogram using a convolutional neural network (CNN), which can be used in portable OSA monitor devices. To learn different scale features, the first convolution layer comprises three types of filters. The long short-term memory (LSTM) is used to learn the long-term dependencies such as the OSA transition rules. The softmax function is connected to the final fully connected layer to obtain the final decision. To detect a complete OSA event, the raw ECG signals are segmented by a 10 s overlapping sliding window. The proposed model is trained with the segmented raw signals and is subsequently tested to evaluate its event detection performance. According to experiment analysis, the proposed model exhibits Cohen’s kappa coefficient of 0.92, a sensitivity of 96.1%, a specificity of 96.2%, and an accuracy of 96.1% with respect to the Apnea-ECG dataset. The proposed model is significantly higher than the results from the baseline method. The results prove that our approach could be a useful tool for detecting OSA on the basis of a single-lead ECG

cAMP Stimulates SLC26A3 Activity in Human Colon by a CFTR-Dependent Mechanism That Does Not Require CFTR Activity.

Background & aimsSLC26A3 (DRA) is an electroneutral Cl-/HCO3- exchanger that is present in the apical domain of multiple intestinal segments. An area that has continued to be poorly understood is related to DRA regulation in acute adenosine 3',5'-cyclic monophosphate (cAMP)-related diarrheas, in which DRA appears to be both inhibited as part of NaCl absorption and stimulated to contribute to increased HCO3- secretion. Different cell models expressing DRA have shown that cAMP inhibits, stimulates, or does not affect its activity.MethodsThis study re-evaluated cAMP regulation of DRA using new tools, including a successful knockout cell model, a specific DRA inhibitor (DRAinh-A250), specific antibodies, and a transport assay that did not rely on nonspecific inhibitors. The studies compared DRA regulation in colonoids made from normal human colon with regulation in the colon cancer cell line, Caco-2.ResultsDRA is an apical protein in human proximal colon, differentiated colonoid monolayers, and Caco-2 cells. It is glycosylated and appears as 2 bands. cAMP (forskolin) acutely stimulated DRA activity in human colonoids and Caco-2 cells. In these cells, DRA is the predominant apical Cl-/HCO3- exchanger and is inhibited by DRAinh-A250 with a median inhibitory concentration of 0.5 and 0.2 μmol/L, respectively. However, there was no effect of cAMP in HEK293/DRA cells that lacked a cystic fibrosis transmembrane conductance regulator (CFTR). When CFTR was expressed in HEK293/DRA cells, cAMP also stimulated DRA activity. In all cases, cAMP stimulation of DRA was not inhibited by CFTRinh-172.ConclusionsDRA is acutely stimulated by cAMP by a process that is CFTR-dependent, but appears to be one of multiple regulatory effects of CFTR that does not require CFTR activity

EZH2 promotes malignant behaviors via cell cycle dysregulation and its mRNA level associates with prognosis of patient with non-small cell lung cancer.

Epigenetic silencing is a common mechanism to inactivate tumor suppressor genes during carcinogenesis. Enhancer of Zeste 2 (EZH2) is the histone methyltransferase subunit in polycomb repressive complex 2 which mediates transcriptional repression through histone methylation. EZH2 overexpression has been linked to aggressive phenotypes of certain cancers. However, the mechanism that EZH2 played in promoting malignancy in non-small cell lung cancer (NSCLC) remains unclear. In addition, the correlation of EZH2 overexpression and the prognosis of NSCLC patients in non-Asian cohort need to be determined.Up-regulation of EZH2 was found in NSCLC cells compared with normal human bronchial epithelial cells by western blot assay. Upon EZH2 knockdown using small interfering RNA (siRNA), the proliferation, anchorage-independent growth and invasion of NSCLC cells were remarkably suppressed with profound induction of G1 arrest. Furthermore, the expression of cyclin D1 was notably reduced whereas p15(INK4B), p21(Waf1/Cip1) and p27(Kip1) were increased in NSCLC cells after EZH2-siRNA delivery. To determine whether EZH2 expression contributes to disease progression in patients with NSCLC, Taqman quantitative real-time RT-PCR was used to measure the expression of EZH2 in paired tumor and normal samples. Univariate analysis revealed that patients with NSCLC whose tumors had a higher EZH2 expression had significantly inferior overall, disease-specific, and disease-free survivals compared to those whose tumors expressed lower EZH2 (P = 0.005, P = 0.001 and P = 0.003, respectively). In multivariate analysis, EZH2 expression was an independent predictor of disease-free survival (hazard ratio = 0.450, 95% CI: 0.270 to 0.750, P = 0.002).Our results demonstrate that EZH2 overexpression is critical for NSCLC progression. EZH2 mRNA levels may serve as a prognostic predictor for patients with NSCLC

∆DNMT3B4-del Contributes to Aberrant DNA Methylation Patterns in Lung Tumorigenesis

Aberrant DNA methylation is a hallmark of cancer but mechanisms contributing to the abnormality remain elusive. We have previously shown that ∆DNMT3B is the predominantly expressed form of DNMT3B. In this study, we found that most of the lung cancer cell lines tested predominantly expressed DNMT3B isoforms without exons 21, 22 or both 21 and 22 (a region corresponding to the enzymatic domain of DNMT3B) termed DNMT3B/∆DNMT3B-del. In normal bronchial epithelial cells, DNMT3B/ΔDNMT3B and DNMT3B/∆DNMT3B-del displayed equal levels of expression. In contrast, in patients with non-small cell lung cancer NSCLC), 111 (93%) of the 119 tumors predominantly expressed DNMT3B/ΔDNMT3B-del, including 47 (39%) tumors with no detectable DNMT3B/∆DNMT3B. Using a transgenic mouse model, we further demonstrated the biological impact of ∆DNMT3B4-del, the ∆DNMT3B-del isoform most abundantly expressed in NSCLC, in global DNA methylation patterns and lung tumorigenesis. Expression of ∆DNMT3B4-del in the mouse lungs resulted in an increased global DNA hypomethylation, focal DNA hypermethylation, epithelial hyperplastia and tumor formation when challenged with a tobacco carcinogen. Our results demonstrate ∆DNMT3B4-del as a critical factor in developing aberrant DNA methylation patterns during lung tumorigenesis and suggest that ∆DNMT3B4-del may be a target for lung cancer prevention

cAMP Stimulates SLC26A3 Activity in Human Colon by a CFTR-Dependent Mechanism That Does Not Require CFTR ActivitySummary

Background & Aims: SLC26A3 (DRA) is an electroneutral Cl-/HCO3- exchanger that is present in the apical domain of multiple intestinal segments. An area that has continued to be poorly understood is related to DRA regulation in acute adenosine 3′,5′-cyclic monophosphate (cAMP)-related diarrheas, in which DRA appears to be both inhibited as part of NaCl absorption and stimulated to contribute to increased HCO3- secretion. Different cell models expressing DRA have shown that cAMP inhibits, stimulates, or does not affect its activity. Methods: This study re-evaluated cAMP regulation of DRA using new tools, including a successful knockout cell model, a specific DRA inhibitor (DRAinh-A250), specific antibodies, and a transport assay that did not rely on nonspecific inhibitors. The studies compared DRA regulation in colonoids made from normal human colon with regulation in the colon cancer cell line, Caco-2. Results: DRA is an apical protein in human proximal colon, differentiated colonoid monolayers, and Caco-2 cells. It is glycosylated and appears as 2 bands. cAMP (forskolin) acutely stimulated DRA activity in human colonoids and Caco-2 cells. In these cells, DRA is the predominant apical Cl-/HCO3- exchanger and is inhibited by DRAinh-A250 with a median inhibitory concentration of 0.5 and 0.2 μmol/L, respectively. However, there was no effect of cAMP in HEK293/DRA cells that lacked a cystic fibrosis transmembrane conductance regulator (CFTR). When CFTR was expressed in HEK293/DRA cells, cAMP also stimulated DRA activity. In all cases, cAMP stimulation of DRA was not inhibited by CFTRinh-172. Conclusions: DRA is acutely stimulated by cAMP by a process that is CFTR-dependent, but appears to be one of multiple regulatory effects of CFTR that does not require CFTR activity. Keywords: Cl-/HCO3- Exchange, CFTR, Colon, Secretory Diarrhea, Enteroid

Impact of 24 h continuous hypothermic perfusion on heart preservation by assessment of oxidative stress

Despite investigating numerous solutions, additives, and techniques over the last two decades, extending donor heart preservation beyond 4-6 h has not been achieved. Hypothermic heart preservation (HP) induces oxidative stress (OS) with reactive oxygen species (ROS) production, causing DNA cleavage and impairing repair. Quantification of cardiomyocyte concentrations of DNA damage by-products (8-oxoG) and mismatch repair enzymes (MYH, OGG-1, MSH2) reflects the severity of OS. If increased repair enzyme production is insufficient to repair injury, cell death occurs and functional outcomes are impacted. We investigated continuous hypothermic perfusion (CHP), a new form of HP, and the mechanism of injury associated with hypothermic storage, by assessing functional outcome and OS after allotransplantation of canine hearts. Fourteen canine hearts were harvested using standard techniques after baseline echocardiograms and haemodynamic parameters were obtained. The hearts were implanted after 24 h CHP (n = 10) or 4 h static preservation (SP; n = 4). After weaning from cardiopulmonary bypass (CPB), recipients were kept alive for 6 h. Repeat echocardiograms and haemodynamic parameters were obtained. Quantification of MYH, OGG-1, and MSH2 concentrations were performed on biopsies using immunohistochemistry and Western blot analysis. Twelve out of 14 hearts (8/10 CHP; 4/4 SP) were successfully weaned on moderate inotropic support. Post-implant echocardiogram, completed in 6/10 CHP and 2/4 SP hearts, demonstrated hyperdynamic function and normal wall motion. The expression and activity of DNA repair enzymes was identical between normal baseline and CHP hearts. CHP reduces OS associated with prolonged hypothermic preservation and may allow longer preservation periods without compromising function. CHP offers several potential advantages: (1) resuscitation of non-beating heart donor organs, (2) time for HLA tissue typing, (3) facilitate interventions improving graft function, and (4) increased organ sharing