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Sepsis continues to overwhelm hospital systems with its high mortality rate and prevalence. A strategy to reduce the strain of sepsis on hospital systems is to develop a diagnostic/prognostic measure that identifies patients who are more susceptible to septic death. Current biomarkers fail to achieve this outcome, as they only have moderate diagnostic power and limited prognostic capabilities. Sepsis disrupts a multitude of pathways in many different organ systems, making the identification of a single powerful biomarker difficult to achieve. However, a common feature of many of these perturbed pathways is the increased generation of reactive oxygen species (ROS), which can alter gene expression, changes in which may precede the clinical manifestation of severe sepsis. Therefore, the aim of this study was to evaluate whether ROS-related circulating molecular signature can be used as a tool to predict sepsis survival. Here we created a ROS-related gene signature and used two Gene Expression Omnibus datasets from whole blood samples of septic patients to generate a 37-gene molecular signature that can predict survival of sepsis patients. Our results indicate that peripheral blood gene expression data can be used to predict the survival of sepsis patients by assessing the gene expression pattern of free radical–associated -related genes in patients, warranting further exploration. [ABSTRACT FROM AUTHOR]

Background Chromodomain helicase DNA binding protein 5 (CHD5) has recently been identified as a potent tumour suppressor by acting as a master regulator of a tumour-suppressive network. Its inactivation resulted from aberrant methylation in the promoter occurs in several types of human malignancy and is associated with malignant tumour behaviour. In human hepatocellular carcinoma (HCC), CHD5 gene expression, methylation status and tumour-suppressive function have not been elucidated. Aims In this study, we focused on the epigenetic modification and tumour-suppressive mechanism of CHD5 gene in HCC. Methods CHD5 expression in nine HCC cell lines and 30 pairs of HCC specimens and adjacent non-cancerous tissues were analysed by quantitative reverse transcription PCR and Western blotting. Methylation-specific sequencing and methylation-specific PCR were performed to examine DNA methylation status of the CHD5 promoter in HCC cell lines and samples. The effect of CHD5 restoration on proliferation, colony formation, senescence, apoptosis and tumourigenicity were examined. Results CHD5 expression was sinificantly down-regulated in HCC cell lines and tissues examined, and the −841 to −470 region of CHD5 promoter was hypermethylated in these samples. Treatment with DNA methyltransferase inhibitor 5-aza-2-deoxycytidine resulted in a striking regional demethylation of the −841 to −470 region of CHD5 promoter and an increase in CHD5 expression. The restoration of CHD5 expression inhibited tumour cell proliferation, colony formation and tumourigenicity and caused cellular senescence. Conclusions Our findings demonstrate that CHD5 is a potential tumour suppressor gene epigenetically silenced in HCC. [ABSTRACT FROM AUTHOR]

Chromodomain helicase DNA binding protein 5 (CHD5) was previously proposed to function as a potent tumor suppressor by acting as a master regulator of a tumor-suppressive network. CHD5 is down-regulated in several cancers, including leukemia and is responsible for tumor generation and progression. However, the mechanism of CHD5 down-regulation in leukemia is largely unknown. In this study, quantitative reverse-transcriptase polymerase chain reaction and western blotting analyses revealed that CHD5 was down-regulated in human leukemia cell lines and samples. Luciferase reporter assays showed that most of the baseline regulatory activity was localized from 500 to 200 bp upstream of the transcription start site. Bisulfite DNA sequencing of the identified regulatory element revealed that the CHD5 promoter was hypermethylated in human leukemia cells and samples. Thus, CHD5 expression was inversely correlated with promoter DNA methylation in these samples. Treatment with DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) activates CHD5 expression in human leukemia cell lines. In vitro luciferase reporter assays demonstrated that methylation of the CHD5 promoter repressed its promoter activity. Furthermore, a chromatin immunoprecipitation assay combined with qualitative PCR identified activating protein 2 (AP2) as a potential transcription factor involved in CHD5 expression and indicated that treatment with DAC increases the recruitment of AP2 to the CHD5 promoter. In vitro transcription-factor activity studies showed that AP2 over-expression was able to activate CHD5 promoter activity. Our findings indicate that repression of CHD5 gene expression in human leukemia is mediated in part by DNA methylation of its promoter. [ABSTRACT FROM AUTHOR]

Metallothioneins (MTs) are ubiquitous metal-binding, cysteine-rich, small proteins and play a major role in metal homeostasis and/or detoxification in all organisms. In a previous study, a novel full length MT gene was isolated from the freshwater crab ( Sinopotamon henanense), a species widely distributed in Shanxi and Henan Provinces, China. In this report, the gene for the crab MT was inserted into a PET-28a-6His-SUMO vector and recombinant soluble MT was over-expressed as fusions with SUMO in Escherichia coli. The recombinant fusion protein was purified by affinity chromatography and its biochemical properties were analyzed. In addition, on the basis of constructing SUMO-MT, two mutants, namely SUMO-MTt1 and SUMO-MTt2, were constructed to change the primary structure of SUMO-MT using site-directed mutagenesis techniques with the amino acid substitutions D3C and S37C in order to increase metal-binding capacity of MT. E. coli cells expressing SUMO-MT and these single-mutant proteins exhibited enhanced metal tolerance and higher accumulation of metal ions than control cells. The results showed that the bioaccumulation and tolerance of Zn, Cu and Cd in these strains followed the decreasing order of SUMO-MTt1 > SUMO-MTt2 > SUMO-MT. E. coli cells have low tolerance and high accumulation towards cadmium compared to zinc and copper. These results show that the MT of S. henanense could enhance tolerance and accumulation of metal ions. Moreover, we were able to create a novel protein based on the crab MT to bind metal ions at high density and with high affinity. Therefore, SUMO-MT and its mutants can provide potential candidates for heavy metal bioremediation. This study could help further elucidate the mechanism of how the crab detoxifies heavy metals and provide a scientific basis for environment bioremediation of heavy metal pollution using the over-expression of the crab MT and mutant proteins. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) can function as tumor suppressors or oncogene promoters during tumor development. In this study, low levels of expression of miR-196b were detected in patients with chronic myeloid leukemia. Bisulfite genomic sequencing PCR and methylation-specific PCR were used to examine the methylation status of the CpG islands in the miR-196b promoter in K562 cells, patients with leukemia and healthy individuals. The CpG islands showed more methylation in patients with chronic myeloid leukemia compared with healthy individuals (P<0.05), which indicated that low expression of miR-196b may be associated with an increase in the methylation of CpG islands. The dual-luciferase reporter assay system demonstrated that BCR-ABL1 and HOXA9 are the target genes of miR-196b, which was consistent with predictions from bioinformatics software analyses. Further examination of cell function indicated that miR-196b acts to reduce BCR-ABL1 and HOXA9 protein levels, decrease cell proliferation rate and retard the cell cycle. A low level of expression of miR-196b can cause up-regulation of BCR-ABL1 and HOXA9 expression, which leads to the development of chronic myeloid leukemia. MiR-196b may represent an effective target for chronic myeloid leukemia therapy. [ABSTRACT FROM AUTHOR]

Abstract: Chronic myeloid leukemia (CML) is associated with overexpression of BCR-ABL1, a nonreceptor tyrosine kinase critical for malignant transformation. We investigated whether non-coding microRNAs (miRNAs) targeting BCR-ABL1 mRNA contribute to the pathogenesis of CML. Indeed, miR-30a targeted BCR-ABL1 and was underexpressed in bone marrow from CML patients. In K562 leukemia cells, overexpression of miR-30a reduced ABL1 and BCR-ABL1 protein expression, decreased proliferation, and arrested cell cycle progression between G1 and S. These findings strongly suggest that miR-30a acts as a tumor suppressor by downregulating ABL1 and BCR-ABL1 expression. Upregulation of miR-30a in hematopoietic cells may have therapeutic efficacy against CML. [Copyright &y& Elsevier]

Abstract: Outbreaks of hand, foot, and mouth disease caused by enterovirus 71 (EV71) have become considerable threats to the health of infants and young children. To identify the cellular long noncoding RNAs (lncRNAs) involved in the host response to EV71 infection, we performed comprehensive lncRNA and mRNA profiling in EV71-infected rhabdomyosarcoma cells through microarray. We observed the differential expression of more than 4800 lncRNAs during infection. Further analysis showed 160 regulated enhancer-like lncRNA and nearby mRNA pairs, as well as 313 regulated Rinn’s lncRNA [M. Guttman I. Amit, M. Garber, C. French, M.F. Lin, D. Feldser, M. Huarte, O. Zuk, B.W. Carey, J.P. Cassady, M.N. Cabili, R. Jaenisch, T.S. Mikkelsen, T. Jacks, N. Hacohen, B.E. Bernstein, M. Kellis, A. Regev, J.L. Rinn, E.S. Lander. Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 458 (2009) 223–227, A.M. Khalil, M. Guttman, M. Huarte, M. Garber, A. Raj, D. Rivea Morales, K. Thomas, A. Presser, B.E. Bernstein, A. van Oudenaarden, A. Regev, E.S. Lander, J.L. Rinn. Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc. Natl. Acad. Sci. USA 106 (2009) 11667–11672] and nearby mRNA pairs. The results provided information for further research on the prevention and treatment of EV71 infection, as well as on distinguishing severe and mild EV71 cases. [Copyright &y& Elsevier]

Abstract: Chromodomain helicase DNA binding protein 5 (CHD5) is a potent tumor suppressor that serves as a master regulator of a tumor-suppressive network. Examination of the role played by CHD5 in a wide range of human cancers is warranted. In this study, we focused on the epigenetic modification and tumor-suppressive role of CHD5 in lung cancer. We measured CHD5 mRNA and protein expression in lung cancer cells, lung cancer tissues, and their corresponding noncancerous lung tissues using real-time PCR and Western blot analysis. We then determined the methylation status of the CHD5 promoter in these samples using methylation-specific sequencing and analyzed CHD5 re-expression in lung cancer cells treated with or without 5-aza-2-deoxycytidine, an inhibitor of DNA methylation. Next, the lung cancer cell clones stably expressing EGFP-CHD5 protein or EGFP protein, respectively, were obtained and the effects of restored CHD5 expression on cell proliferation, colony formation, and tumorigenicity were assessed. CHD5 expression ranged from low to absent in the lung cancer cell lines and tissues examined; the CHD5 promoter was hyperethylated in these samples. Treatment with 5-aza-dC resulted in a localized decrease in methylation density and an increase in CHD5 expression. Clonogenicity and tumor growth were abrogated in A549 and H1299 cells upon restoration of CHD5 expression. A significant reduction in clonogenicity was observed; an average of 47.83±4.6% reduction for A549-EGFP-CHD5 was observed compared to A549-EGFP, and an average of 56.39±5.3% reduction for H1299-EGFP-CHD5 was observed compared to H1299-EGFP. A549-EGFP exhibited an average tumor size of 452.3±36.5mm3, whereas A549-EGFP-CHD5 exhibited an average tumor size of only 57.7±18.5mm3. Thus, our findings indicate that CHD5 is a potential tumor suppressor gene that is inactivated via an epigenetic mechanism in lung cancer. [Copyright &y& Elsevier]