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Background: To evaluate the predictive value of serum HBV DNA, HBV RNA, HBcrAg, HBsAg, intrahepatic HBV DNA, and cccDNA for HBeAg clearance and seroconversion during long-term treatment of nucleos(t)ide analogues (NAs) in patients with chronic hepatitis B (CHB).Method: A single center, prospective cohort of CHB patients enrolled between June 2007 and July 2008 was used for this study. Serum HBV RNA levels were retrospectively measured at baseline, 6, 12, 24, 36, 48, 60, 72, and 84 months post-NAs treatment. Serum HBsAg and HBcrAg levels were quantified at baseline, month 6, 60, and 72. Histological sample from liver biopsy at baseline and month 60 were analyzed for intrahepatic HBV DNA and cccDNA.Results: Eighty-three HBeAg patients were enrolled with an median follow-up time of 108 months (range 18-138 months). Of them, 53 (63.86%) patients achieved HBeAg clearance, and 37 (44.58%) achieved HBeAg seroconversion. Only baseline HBV RNA was independently associated with HBeAg clearance (OR=0.50, 95%CI 0.309-0.809, P=0.005) and seroconversion (OR=0.689, 95% CI 0.513-0.925, P=0.013). The independent negative association with HBeAg clearance and seroconversion remained for HBV RNA levels at month 6 (OR=0.42, 95%CI 0.248-0.714, P=0.001; OR=0.44, 95%CI 0.260-0.744, P=0.002) and month 12 (OR=0.39, 95%CI 0.253-0.592, PP=0.001). The AUC of baseline HBV RNA for predicting the HBeAg clearance and seroconversion were 0.81 (95%CI: 0.70-0.89) and 0.68 (95%CI: 0.56-0.78), respectively, higher than that of HBV DNA, HBsAg and HBcrAg.Conclusion: Lower serum HBV RNA at baseline, month 6 and 12 post NAs treatment could predict HBeAg clearance and seroconversion during long-term NAs treatment.

Gene-specific methylation and expression have shown biological and clinical importance for breast cancer diagnosis and prognosis. Integrated analysis of gene methylation and gene expression may identify genes associated with biology mechanism and clinical outcome of breast cancer and aid in clinical management. Using high-throughput microfluidic quantitative PCR, we analyzed the expression profiles of 48 candidate genes in 96 Chinese breast cancer patients and investigated their correlation with gene methylation and associations with breast cancer clinical parameters. Breast cancer-specific gene expression alternation was found in 25 genes with significant expression difference between paired tumor and normal tissues. A total of 9 genes (CCND2, EGFR, GSTP1, PGR, PTGS2, RECK, SOX17, TNFRSF10D, and WIF1) showed significant negative correlation between methylation and gene expression, which were validated in the TCGA database. Total 23 genes (ACADL, APC, BRCA2, CADM1, CAV1, CCND2, CST6, EGFR, ESR2, GSTP1, ICAM5, NPY, PGR, PTGS2, RECK, RUNX3, SFRP1, SOX17, SYK, TGFBR2, TNFRSF10D, WIF1, and WRN) annotated with potential TFBSs in the promoter regions showed negative correlation between methylation and expression. In logistics regression analysis, 31 of the 48 genes showed improved performance in disease prediction with combination of methylation and expression coefficient. Our results demonstrated the complex correlation and the possible regulatory mechanisms between DNA methylation and gene expression. Integration analysis of methylation and expression of candidate genes could improve performance in breast cancer prediction. These findings would contribute to molecular characterization and identification of biomarkers for potential clinical applications.

Current hepatitis C virus (HCV) genotyping techniques are often highly technical, costly, or need improvements in sensitivity and specificity. These limitations indicate the need of novel methods for HCV genotyping. The present study aimed to develop a novel genotyping method combining high-resolution melting (HRM) analysis with Bayes discriminant analysis (BDA). Target gene fragment including 5'-untranslated and core region was selected. Four or five inner amplicons for every serum were amplified using nested PCR, HRM was used to determine the melting temperature of the amplicons, and HCV genotypes were then analyzed utilizing BDA. In initial genotyping (HCV genotypes were classified into 1b, 2a, 3a, 3b, and 6a), both the overall accuracy rate and the cross-validation accuracy rate were 92.6 %, external validation accuracy rate was 95.0 %. To enhance the accuracy rate of genotyping, HCV genotypes were firstly classified into 1b, 3a, 3b, and 2a-6a, followed by a supplementary genotyping for 2a-6a. Both the overall accuracy rate and the cross-validation accuracy rate reached 97.5 %, and external validation accuracy rate was 100 %. Comparing adjusted HRM genotyping with type-specific probe technique, the difference in accuracy rates was not significant. However, the limit of detection and cost were lower for HRM. Comparing with sequencing, the limit detection of HRM was the same as the former, but the cost of HRM was lower. Hence, HRM combined with BDA was a novel method that equipped with superior accuracy, high sensitivity, and lower cost and therefore could be a better technique for HCV genotyping.

Gene-specific methylation alterations in breast cancer have been suggested to occur early in tumorigenesis and have the potential to be used for early detection and prevention. The continuous increase in worldwide breast cancer incidences emphasizes the urgent need for identification of methylation biomarkers for early cancer detection and patient stratification. Using microfluidic PCR-based target enrichment and next-generation bisulfite sequencing technology, we analyzed methylation status of 48 candidate genes in paired tumor and normal tissues from 180 Chinese breast cancer patients. Analysis of the sequencing results showed 37 genes differentially methylated between tumor and matched normal tissues. Breast cancer samples with different clinicopathologic characteristics demonstrated distinct profiles of gene methylation. The methylation levels were significantly different between breast cancer subtypes, with basal-like and luminal B tumors having the lowest and the highest methylation levels, respectively. Six genes (ACADL, ADAMTSL1, CAV1, NPY, PTGS2, and RUNX3) showed significant differential methylation among the 4 breast cancer subtypes and also between the ER +/ER- tumors. Using unsupervised hierarchical clustering analysis, we identified a panel of 13 hypermethylated genes as candidate biomarkers that performed a high level of efficiency for cancer prediction. These 13 genes included CST6, DBC1, EGFR, GREM1, GSTP1, IGFBP3, PDGFRB, PPM1E, SFRP1, SFRP2, SOX17, TNFRSF10D, and WRN. Our results provide evidence that well-defined DNA methylation profiles enable breast cancer prediction and patient stratification. The novel gene panel might be a valuable biomarker for early detection of breast cancer.

Circulating cell-free DNA (cfDNA) has been considered as a potential biomarker for non-invasive cancer detection. To evaluate the methylation levels of six candidate genes (EGFR, GREM1, PDGFRB, PPM1E, SOX17, and WRN) in plasma cfDNA as biomarkers for breast cancer early detection, quantitative analysis of the promoter methylation of these genes from 86 breast cancer patients and 67 healthy controls was performed by using microfluidic-PCR-based target enrichment and next-generation bisulfite sequencing technology. The predictive performance of different logistic models based on methylation status of candidate genes was investigated by means of the area under the ROC curve (AUC) and odds ratio (OR) analysis. Results revealed that EGFR, PPM1E, and 8 gene-specific CpG sites showed significantly hypermethylation in cancer patients' plasma and significantly associated with breast cancer (OR ranging from 2.51 to 9.88). The AUC values for these biomarkers were ranging from 0.66 to 0.75. Combinations of multiple hypermethylated genes or CpG sites substantially improved the predictive performance for breast cancer detection. Our study demonstrated the feasibility of quantitative measurement of candidate gene methylation in cfDNA by using microfluidic-PCR-based target enrichment and bisulfite next-generation sequencing, which is worthy of further validation and potentially benefits a broad range of applications in clinical oncology practice. Quantitative analysis of methylation pattern of plasma cfDNA by next-generation sequencing might be a valuable non-invasive tool for early detection of breast cancer.

The dissociation of iron from heme is a major factor in iron metabolism and the cellular concentrations of the metal correlate with heme degradation. We tested the hypotheses that (1) exposure to a product of heme catabolism, carbon monoxide (CO), alters iron homeostasis in the lung and in cultured respiratory epithelial cells; (2) this response includes both decreased uptake and increased release of cell metal; and (3) the effects of CO on cell function track changes in metal homeostasis. In rats exposed to 50 ppm CO for 24 hours, non-heme iron concentrations decreased in the lung and increased in the liver. In respiratory epithelial cells cultured at air-liquid interface, CO exposure decreased cell non-heme iron and ferritin concentrations within 2 hours and the effect was fully reversible. CO significantly depressed iron uptake by epithelial cells, despite increased expression of divalent metal transporter-1, while iron release was elevated. The loss of non-heme iron after CO reduced cellular oxidative stress, blocked the release of the proinflammatory mediator (interleukin-8), and interfered with cell cycle protein expression. We conclude that CO reduces the iron content of the lung through both the metal uptake and release mechanisms. This loss of cellular iron after CO is in line with certain biological effects of the gas that have been implicated in the protection of cell viability.

Sodium glucose co-transporter 2 (SGLT2) is a renal type III integral membrane protein that co-transports sodium and glucose from filtrate to epithelium in the proximal tubule. Human subjects with homozygous or compound heterozygous mutations in SLC5A2 exhibit glucosuria without hypoglycemia or other obvious morbidity, suggesting that blockade of SGLT2 has the potential to promote normalization of blood glucose without hypoglycemia in the setting of type 2 diabetes. This report presents the in vitro and in vivo pharmacological activities of EGT1442, a recently discovered SGLT2 inhibitor in the C-aryl glucoside class. The inhibitory effects of EGT1442 for human SGLT1 and SGLT2 were evaluated in an AMG uptake assay and the in vivo efficacy of treatment with EGT1442 was investigated in rats and dogs after a single dose and in db/db mice after chronic administration. The effect of EGT1442 on median survival of SHRSP rats was also evaluated. The IC(50) values for EGT1442 against human SGLT1 and SGLT2 are 5.6μM and 2nM, respectively. In normal rats and dogs a saturable urinary glucose excretion was produced with an ED(50) of 0.38 and 0.09mg/kg, respectively. Following chronic administration to db/db mice, EGT1442 dose-dependently reduced HbA(1c) and blood glucose concentration without affecting body mass or insulin level. Additionally, EGT1442 significantly prolonged the median survival of SHRSP rats. EGT1442 showed favorable properties both in vitro and in vivo and could be beneficial to the management of type 2 diabetic patients.

Human exposure to gadolinium-based contrast agents can be complicated by nephrogenic systemic fibrosis (NSF). Demonstration of significant quantities of insoluble gadolinium in the skin of NSF patients suggested transmetallation as a mechanism of toxicity of this injury. An alternative pathway for the biological effect of gadolinium is a disruption of iron homeostasis. We tested the postulate that cell exposure to gadolinium increases iron uptake to disrupt intracellular metal homeostasis and impact inflammatory events. Alveolar macrophages, THP1 cells, NHBE cells, and BEAS-2B cells all demonstrated a capacity to import gadolinium from both GdCl(3) and Omniscan. All four cell types similarly imported iron following exposure to ferric ammonium citrate (FAC). Exposure of all cell types to gadolinium and iron resulted in increased iron import relative to cell concentrations following incubation with FAC alone. To analyze for further evidence of changes in iron homeostasis, cell ferritin concentration was determined. Relative to incubation with FAC alone, co-incubation of BEAS-2B cells with gadolinium and FAC resulted in significant increases in ferritin level. Finally, potential effects of gadolinium uptake and associated changes in iron homeostasis on the inflammatory response were evaluated by measuring IL-8. Co-incubation of BEAS-2B cells with both gadolinium and iron resulted in diminished release of IL-8 relative to levels of the cytokine following incubation with gadolinium alone. We conclude that gadolinium impacts cell iron homeostasis to change import and storage of the metal and biological effects of exposure.