Methylation of IRAK3 is a novel prognostic marker in hepatocellular carcinoma

AIM: To examine the methylation levels of interleukin-1 receptor-associated kinase 3(IRAK3) and GLOXD1 and their potential clinical applications in hepatocellular carcinoma(HCC).METHODS: m RNA expression and promoter methylation of IRAK3 and GLOXD1 in HCC cells were analyzed by reverse transcription-polymerase chain reaction(RTPCR) and methylation-specific PCR(MSP), respectively. Using pyrosequencing results, we further established a quantitative MSP(Q-MSP) system for the evaluation of IRAK3 and GLOXD1 methylation in 29 normal controls and 160 paired HCC tissues and their adjacent nontumor tissues. We also calculated Kaplan-Meier survival curves to determine the applications of gene methylation in the prognosis of HCC.RESULTS: IRAK3 and GLOXD1 expression was partially restored in several HCC cell lines after treatment with 5-aza-2′-deoxycytidine(DNA methyltransferase inhibitor; 5DAC). A partial decrease in the methylated band was also observed in the HCC cell lines after 5DAC treatment. Using GLOXD1 as an example, we found a significant correlation between the data obtained from the methylation array and from pyrosequencing. The methylation frequency of IRAK3 and GLOXD1 in HCC tissues was 46.9% and 63.8%, respectively. Methylation of IRAK3 was statistically associated with tumor stage. Moreover, HCC patients with IRAK3 methylation had a trend toward poor 3-year disease-free survival(P < 0.05). CONCLUSION: IRAK3 and GLOXD1 were frequently methylated in HCC tissues compared to normal controls and nontumor tissues. IRAK3 methylation was associated with tumor stage and poor prognosis of patients. These data suggest that IRAK3 methylation is a novel prognostic marker in HCC.

Weekly pattern of emergency room admissions for peptic ulcers:A population-based study

AIM:To investigate variations in the incidence of peptic ulcers(PUs) in Taiwan by day of the week within age subgroups.METHODS:Ambulatory care data were retrieved from the Longitudinal Health Insurance Database 2000.There were 7204 subjects≥18 years-old with an emergency room admission claim for the treatment of PUs,resulting in a total of 9234 emergency room visits for PUs between 2009 and 2011.Data was divided into the seven days of the week and an additional variable for holidays.One-way analysis of variance was used to examine associations among the daily mean number of PU emergency room admissions and holidays/weekends/weekdays.RESULTS:One-way analysis of variance showed that there was a significant difference in emergency room admissions for PUs by the day of the week(P<0.001),with admission more likely to occur on Sundays or holidays than weekdays within the total and working populations.The weekday patterns of admission were similar for the patients aged 18-64 years and≥65years of age.Holidays,followed by Sundays,had higher PU admissions than the mean daily PU emergency room admissions.Furthermore,inclusion of only those treated for PUs with hemorrhage or perforation,Sundays and holidays had higher mean emergency room admissions than other days.Inclusion of patients who diagnosed with Helicobacter pylori infection,only holidays had higher mean emergency room admissions than other days.Inclusion of patients who had been prescribed non-steroidal anti-inflammatory drugs(NSAIDs)for over30 d,Sundays and holidays had higher mean PU ER admissions than other non-holiday weekdays.CONCLUSION:There is a higher incidence of emergency room admission for PUs on weekends than on weekdays for the total and working populations.

Functional and molecular mechanism of intracellular pH regulation in human inducible pluripotent stem cells

AIM To establish a functional and molecular model of the intracellular pH(pH_i) regulatory mechanism in human induced pluripotent stem cells(hiPSCs).METHODS hiP SCs(HPS0077) were kindly provided by Dr. Dai from the Tri-Service General Hospital(IRB No. B-106-09). Changes in the pH_i were detected either by microspectrofluorimetry or by a multimode reader with a pH-sensitive fluorescent probe, BCECF, and the fluorescent ratio was calibrated by the high K^+/nigericin method. NH_4Cl and Na-acetate prepulse techniques were used to induce rapid intracellular acidosis and alkalization, respectively. The buffering power(β) was calculated from the ΔpH_i induced by perfusing different concentrations of(NH_4)_2SO_4. Western blot techniques and immunocytochemistry staining were used to detect the protein expression of pH_i regulators and pluripotency markers.RESULTS In this study, our results indicated that(1) the steadystate pH_i value was found to be 7.5 ± 0.01(n = 20) and 7.68 ± 0.01(n =20) in HEPES and 5% CO_2/HCO_3^- buffered systems, respectively, which were much greater than that in normal adult cells(7.2);(2) in a CO_2/HCO_3^--buffered system, the values of total intracellular buffering power(β) can be described by the following equation: β_(tot) = 107.79(pH_i)~2-1522.2(pH_i) + 5396.9(correlation coefficient R^2 = 0.85), in the estimated pH_i range of 7.1- 8.0;(3) the Na^+/H^+ exchanger(NHE) and the Na^+/HCO_3^- cotransporter(NBC) were found to be functionally activated for acid extrusion for pHi values less than 7.5 and 7.68, respectively;(4) V-ATPase and some other unknown Na^+-independent acid extruder(s) could only be functionally detected for pHi values less than 7.1;(5) the Cl^-/OH^- exchanger(CHE) and the Cl^- /HCO_3 anion exchanger(AE) were found to be responsible for the weakening of intracellular proton loading;(6) besides the CHE and the AE, a Cl^--independent acid loading mechanism was functionally identified; and(7) in hiPSCs, a strong positive correlation was observed between the loss of pluripotency and the weakening of the intracellular acid extrusion mechanism, which included a decrease in the steady-state pH i value and diminished the functional activity and protein expression of the NHE and the NBC.CONCLUSION For the first time, we established a functional and molecular model of a pHi regulatory mechanism and demonstrated its strong positive correlation with hiPSC pluripotency.

Zebrafish as a disease model for studying human hepatocellular carcinoma

Liver cancer is one of the world's most common cancers and the second leading cause of cancer deaths. Hepatocellular carcinoma(HCC), a primary hepatic cancer, accounts for 90%-95% of liver cancer cases. The pathogenesis of HCC consists of a stepwise process of liver damage that extends over decades, due to hepatitis, fatty liver, fibrosis, and cirrhosis before developing fully into HCC. Multiple risk factors are highly correlated with HCC, including infection with the hepatitis B or C viruses, alcohol abuse, aflatoxin exposure, and metabolic diseases. Over the last decade, genetic alterations, which include the regulation of multiple oncogenes or tumor suppressor genes and the activation of tumorigenesis-related pathways, have also been identified as important factors in HCC. Recently, zebrafish have become an important living vertebrate model organism, especially for translational medical research. In studies focusing on the biology of cancer, carcinogen induced tumors in zebrafish were found to have many similarities to human tumors. Several zebrafish models have therefore been developed to provide insight into the pathogenesis of liver cancer and the related drug discovery and toxicology, and to enable the evaluation of novel smallmolecule inhibitors. This review will focus on illustrativeexamples involving the application of zebrafish models to the study of human liver disease and HCC, through transgenesis, genome editing technology, xenografts,drug discovery, and drug-induced toxic liver injury.

Innate immune responses regulate morphogenesis and degeneration:roles of Toll-like receptors and Sarm1 in neurons

The central nervous system is recognized as an immunoprivileged site because peripheral immune cells do not typically enter it. Microglial cells are thought to be the main immune cells in brain. However, recent reports have indicated that neurons express the key players of innate immunity, including Toll-like receptors （TLRs） and their adaptor proteins （Sarml, Myd88, and Trif）, and may produce cytokines in response to pathogen infection. In the absence of an immune challenge, neuronal TLRs can detect intrinsic danger signals and modulate neuronal morphology and function. In this article, we review the recent findings on the involvement of TLRs and Sarml in controlling neuronal morphogenesis and neurodegeneration. Abnormal behaviors in TLR- and Sarml-deficient mice are also discussed.

Induction of Apoptosis in Human Hep3B Hepatoma Cells by Norcantharidin through a p53 Independent Pathway via TRAIL/DR5 Signal Transduction

Objective： To investigate the inhibitory activities of norcantharidin （NCTD）, a demethylated analogue of cantharidin, on Hep3B cells （a human hepatoma cell line） with deficiency of p53. Methods： The survival rate of the Hep3B cells after treating with NCTD was measured by MTT assay. Cell cycle of treated cells was analyzed by flow cytometry, and DNA fragmentation was observed by electrophoresis. The influence of inhibitors for various caspases and anti-death receptors antibodies on the NCTD-induced apoptosis in the cells was determined. Results： NCTD treatment resulted in growth inhibition of Hep3B cells in a dose- and time-dependent manner. Cell cycle analysis of the cells after treatment with NCTD for 48 h shows that NCTD induced G2M phase arrest occursat low concentration （≤25 μ mol/L） but G0G1 phase arrest at high concentration （50 μ mol/L）. The addition of both caspase-3 and caspase-10 inhibitors completely inhibited DNA fragmentation. Addition of anti-TRAIL/DR5 antibody significantly inhibited DNA fragmentation. Conclusion： NCTD may inhibit the proliferation of Hep3B cells by arresting cell cycle at G2M or G0G1 phase, and induce cells apoptosis via TRAIL/DR5 signal transduction through activation of caspase-3 and caspase-10 by a p53-independent pathway,

No increased risk of dementia in patients receiving androgen deprivation therapy for prostate cancer： a 5-year follow-up study

Prior studies suggested that the use of androgen deprivation therapy （ADT） in patients with prostate cancer （PC） might cause the impairment of cognitive function which is one of the common symptoms of dementia; however, the association between ADT and cognitive impairment still remains controversial. This retrospective cohort study aimed to investigate the relationship between ADT and subsequent risk of dementia using a population-based dataset, Data for this study were taken from the Taiwan （China） Longitudinal Health Insurance Database 2005. We included 755 PC patients who received ADT in the study cohort and 559 PC patients who did not receive ADT in the comparison cohort. Each patient was individually tracked for a 5-year period to define those who subsequently received a diagnosis of dementia. Results show that the incidence rates of dementia per 100 person-years were 2.35 （95% confidence interval [95% CI]： 1.82-2.98） and 1.85 （95% Ch 1.35-2.48） for PC patients who received ADT and those who did not receive ADT, respectively. The adjusted hazard ratio （HR） for dementia for PC patients who received ADT was 1.21 （95% Ch 0.82-1.78, P = 0,333） compared to those who did not receive ADT. In addition, the adjusted HRs for dementia for PC patients receiving ADT with gonadotropin-releasing hormone （GnRH） agonists and without GnRH agonists were 1.39 （95% Ch 0.80-2.40, P = 0.240） and 1.13 （95% CI： 0.75-1.71, P = 0.564）, respectively, compared to PC patients not receiving ADT. We concluded that there was no difference in the risk of subsequent dementia between PC patients who did and those who did not receive ADT.

Healthcare utilization and costs in patients with benign prostatic hyperplasia： a population-based study

This study aimed to investigate differences in healthcare service utilization between patients with and those without benign prostatic hyperplasia （BPH） using Taiwan＇s National Health Insurance population-based database. A total of 7413 patients with BPH and 7413 age-matched patients without BPH were included. The outcome variable was 1-year utilization of healthcare services Jncluding the number of outpatient visits, inpatient days, and the costs of outpatient and inpatient treatments. In addition, we separated healthcare services into urology services and nonurology services for analysis. We found that as to the utilization of outpatient urological services, patients with BPH had more outpatient services （7.84 vs 0.52, P 〈 0.001）, higher outpatient costs （US$372 vs US$34, P 〈 0.001）, a longer length of inpatient stay （0.55 vs 0.11, P 〈 0.001）, higher in-patients costs （US$149 vs US$32, P 〈 0.001）, and higher total costs （US$521 vs US$67, P 〈 0.001） than the comparison group. As for nonurological services, patients with BPH also had more outpatient services （49.11 vs 24.79, P〈 0.001）, higher outpatient costs （US$1794 vs US$1014, P〈 0.001）, a longer length of in-patient stay （3.72 vs 2.04, P〈 0.001）, higher inpatient costs （US$874 vs US$486, P〈 0.001）, and higher total costs （US$2668 vs US$1500, P 〈 0.001） compared to comparison patients. We also found that the average total cost was about 2-fold greater for patients with BPH than comparison patients. We concluded that patients with BPH had higher healthcare utilization than comparison patients without BPH.

Antiarrhythmic drug usage and prostate cancer： a population-based cohort study

Even though the relationship between antiarrhythmic drug usage and subsequent prostate cancer （PCa） risk has recently been highlighted, relevant findings in the previous literature are still inconsistent. In addition, very few studies have attempted to investigate the association between sodium channel blockers or potassium channel blockers for arrhythmia and the subsequent PCa risk. Therefore, this cohort study aimed to find the relationship between antiarrhythmic drug usage and the subsequent PCa risk using a population-based dataset. The data used in this study were derived from the Longitudinal Health Insurance Database 2005, Taiwan, China. We respectively identified 9988 sodium channel blocker users, 3663 potassium channel blocker users, 65 966 beta-blocker users, 23 366 calcium channel blockers users, and 7031 digoxin users as the study cohorts. The matched comparison cohorts （one comparison subject for each antiarrhythmic drug user） were selected from the same dataset. Each patient was tracked for a 5-year period to define those who were subsequently diagnosed with PCa. After adjusting for sociodemographic characteristics, comorbidities, and age, Cox proportional hazard regressions found that the hazard ratio （HR） of subsequent PCa for sodium channel blocker users was 1.12 （95% confidence interval [CI]： 0.84-1.50）, for potassium channel blocker users was 0.89 （95% CI. 0.59-1.34）, for beta-blocker users was 1.08 （95% Ch 0.96-1.22）, for calcium channel blocker users was 1.14 （95% Ch 0.95-1.36）, and for digoxin users was 0.89 （95% Ch 0.67-1.18）, compared to their matched nonusers. We concluded that there were no statistical associations between different types of antiarrhvthmic drug usage and subsequent PCa risk.