Studies on the local structure and spin Hamiltonian parameters for V4+ in Na2 O-PbO-Bi2 O3 -SiO2 glass ceramics.

The local structure, d-d transition band, and spin Hamiltonian parameters (SHPs) are theoretically studied for the V4+ probe in Na2 O-PbO-Bi2 O3 -SiO2 (NPBS) glass ceramics containing V2 O5 dopant with various concentration x (0 ≤ x ≤ 5 mol%) by using the perturbation formulas of the SHPs for tetragonally compressed octahedral 3d1 clusters. The first decreasing (or increasing) and then increasing (or decreasing) d-d transition band (= 10 Dq ) and hyperfine structure constants A// and A⊥ (or g factors g// and g⊥ ) with x can be suitably simulated with the similarly varying Fourier type concentration functions of cubic field parameter Dq , covalence factor N, core polarization constant κ, and reduction factor H (or relative tetragonal compression ratio ρ), with the minima (or maxima) at the middle concentration x = 3 mol%, respectively. The above concentration variations of SHPs and the related quantities may originate from the modifications of local crystal field strength, tetragonal compression, and electron cloud distribution near the impurity V4+ with x, corresponding to the highest [V4+ ]/[V5+ ] ratio at 3 mol%. Present studies would be helpful to explore novel sodium lead bismuth silicate glass ceramics by modifying the concentration of V2 O5 dopant.

[Mitochondrial metabolism's effect on epigenetic change and aging].

Mitochondrion is the metabolic center and powerhouse of cells producing cellular energy which plays an important role in various physiological and pathophysiological processes. Recent research demonstrates that mitochondrial energy metabolism mediates the transmission of mitochondrial-nuclear signals through intermediate products which regulates epigenetic presentation of the chromatin and thereby affects gene expression. Epigenetic modification, a genetic regulatory model, is independent of DNA sequence and plays a major role in establishing and maintaining a specific gene's expression profile. Disorders of mitochondrial metabolism can induce epigenetic reprogramming which in turn initiates aging phenotypes and degenerative diseases. This review introduces recent research progress on the relationship between mitochondrial metabolism and chromatin-related epigenetic modification, discusses the role of mitochondrial stress in chromatin recombination, and suggests future research directions and their application in the study of age-related diseases such as cognitive dysfunction.

An Epigenetic Compound Library Screen Identifies BET Inhibitors That Promote HSV-1 and -2 Replication by Bridging P-TEFb to Viral Gene Promoters through BRD4.

The human HSV-1 and -2 are common pathogens of human diseases. Both host and viral factors are involved in HSV lytic infection, although detailed mechanisms remain elusive. By screening a chemical library of epigenetic regulation, we identified bromodomain-containing protein 4 (BRD4) as a critical player in HSV infection. We show that treatment with pan BD domain inhibitor enhanced both HSV infection. Using JQ1 as a probe, we found that JQ1, a defined BD1 inhibitor, acts through BRD4 protein since knockdown of BRD4 expression ablated JQ1 effect on HSV infection. BRD4 regulates HSV replication through complex formation involving CDK9 and RNAP II; whereas, JQ1 promotes HSV-1 infection by allocating the complex to HSV gene promoters. Therefore, suppression of BRD4 expression or inhibition of CDK9 activity impeded HSV infection. Our data support a model that JQ1 enhances HSV infection by switching BRD4 to transcription regulation of viral gene expression from chromatin targeting since transient expression of BRD4 BD1 or BD1/2 domain had similar effect to that by JQ1 treatment. In addition to the identification that BRD4 is a modulator for JQ1 action on HSV infection, this study demonstrates BRD4 has an essential role in HSV infection.

Effect of histone modifications on hMLH1 alternative splicing in gastric cancer.

hMLH1 is one of the mismatch genes closely related to the occurrence of gastric cancer. Epigenetic regulation may play more important roles than gene mutations in DNA damage repair genes to drive carcinogenesis. In this article, we discuss the role of epigenetic changes, especially histone modifications in the regulation of hMLH1 alternative splicing. Our results showed that hMLH1 delEx10, delEx11, delEx10-11, delEx16 and delEx17 transcripts were ubiquitous in sporadic Chinese gastric cancer patients and gastric cancer cell lines. Lower level of H4K16ac and H3ac was detected in hMLH1 exon 10-11 region in gastric cancer cell lines when compared with human gastric mucosal epithelial cell line GES-1. A significant decrease of hMLH1 delEx11 and delEx10-11 was observed in gastric cancer cell lines after trichostatin A treatment. H3K36me3 and H3K4me2 levels were lower in hMLH1 exon 10-11 and exon 16-17 regions in gastric cancer lines when compared with GES-1. Aberrant transcripts such as hMLH1 delEx11 and delEx10-11 were significantly higher in gastric cancer cell lines after small interfering RNA-mediated knockdown of SETD2 (the specific methyltransferase of H3K36). The hMLH1 delEx10 and delEx10-11 transcripts were increased after interference of SRSF2. Taken together, our study demonstrates that lower level of histone acetylation and specific histone methylation such as H3K36me3 correlate with aberrant transcripts in hMLH1 exon 10-11 region. SRSF2 may be involved in these specific exons skipping as well.

Epigenetic regulation of CDH1 exon 8 alternative splicing in gastric cancer.

BACKGROUND: The tumor suppressor gene CDH1 is critical for intercellular adhesion. In our previous work, we reported a nonfunctional CDH1 transcript that lacks the final 83 base pairs of exon 8 (1054del83). In this work, we probed the role of histone epigenetic modifications as well as DNA methylation in selection of this isoform. METHODS: RT-qPCR was used to detect CDH1 RNA expression. Methylation of CDH1 was analyzed by bisulphite sequencing PCR. ChIP assay was performed to show histones level. Cell lines were treated with DNA methyltransferase inhibitor AZA, HDAC inhibitor TSA, or siRNA oligonucleotides to test regulation of CDH1 splicing. RESULTS: Greater CDH1 1054del83 transcripts were observed in gastric cancer (GC) cell lines than human gastric mucosal epithelial cell line GES-1. All the cell lines showed significant methylation pattern at the CpG sites of CDH1 exon 8. AZA treatment did not influence selection of 1054del83 transcripts. A significant decrease in acetylation for histones H3 and H4K16Ac in an internal region of the CDH1 gene surrounding the alternative exon 8 were detected in GC cell lines. Treatment with TSA preferentially expressed the correctly spliced transcript and not the exon 8 skipped aberrant transcripts, showing that histone acetylation was involved in the splicing regulation. SiRNA-mediated knockdown of SETD2 (The specific methyltransferase of H3K36) decreased exclusion of exon 8, suggesting that the presence of this mark correlates with increased skipping of the final 83 base pairs of CDH1 exon 8. However, CDH1 splicing was not affected by SRSF2 knockdown. CONCLUSIONS: H3K36me3 correlates with increased skipping of the final 83 base pairs of CDH1 exon 8. Histone acetylation was involved in the splicing regulation as well.

The specific methylation characteristics of cancer related genes in Chinese colorectal cancer patients.

Aberrant DNA methylation at CpG islands has been implicated as a critical player in colorectal cancer (CRC). However, its biological role and clinical significance in carcinogenesis have not been clearly clarified in Chinese CRC patients. In order to examine the methylation status of cancer-related genes in CRC progression, 184 tumor tissues were collected from Chinese patients diagnosed with CRC during 2008-2011. Promoter methylation was assessed by combined bisulphite-restriction analysis, methylation-specific PCR, and bisulphite sequencing PCR . The relationship between the gene promoter methylation status and clinicopathological factors/CRC mortality was examined by using the chi-square test/Cox-proportional hazards models. Promoter hypermethylation of MLH1, p16, SFRP2, PHD3, KLOTHO, and IGFBP7 was observed in 1.6, 10.9, 97.3, 44.0, 59.8, and 88.6 % of CRC samples, respectively. KLOTHO promoter methylation reduced with age (P = 0.018) whereas p16 promoter methylation increased with age (P = 0.044) and was more frequent among males (P = 0.017). Tumor tissues (73.9 %) had concurrent methylation of two or more genes, with the most frequent combination as KLOTHO and IGFBP7 (53.8 %). Concurrent methylation of KLOTHO and IGFBP7 occurred more frequently among patients less than 70 years old (P = 0.035) and those with poor differentiation (P = 0.024). CRC-specific mortality was not associated with promoter methylation and clinicopathological features except for age (P = 0.038; risk ratio (RR), 1.96; 95 % confidence interval (CI), 1.04-3.70) and TNM stage (P = 0.034; RR, 3.47; 95 % CI, 1.10-10.92). Methylation frequencies of MLH1, p16, PHD3, KLOTHO, and IGFBP7 in CRC tissues were significantly higher than that in the paired normal tissues, while promoter hypermethylation of SFRP2 was widespread in normal tissues. In conclusion, we suggest that methylation of some genes (MLH1, PHD3, KLOTHO, p16, and IGFBP7) is important in CRC progression whereas SFRP2 methylation is unlikely to contribute to CRC development in Chinese patients. Besides, by identifying the characteristics of concordant methylation, we confirm the multifactorial nature of tumor progression.

[The epigenetic effect on pre-mRNA alternative splicing].

Alternative splicing is a crucial step of the gene expression process in eukaryotes. It is a major cause for protein diversity and plays critical roles in differentiation, development, and disease. The studies on the mechanism of alternative splicing have traditionally focused on RNA sequence elements and their related splicing factors, but recent groundbreaking studies have shown that epigenetic factors play a key role in alternative splicing regulation. DNA methylation, chromatin structure and histone modifications interact with each other and regulate the process of alternative pre-mRNA splicing, forming a large and complex regulatory network. These findings suggest that epigenetic regulation not only determines the initiation of gene expression but also influences the outcome of pre-mRNA splicing. This review mainly focuses on the recent research progress in epigenetic regulation of pre-mRNA alternative splicing, including the functions of DNA methylation, chromatin structure and histone modifications in pre-mRNA alternative splicing, and speculates on its far-reaching effects on the study of human disease.

[Establishment of a hMSH2/hMSH6 protein interaction system and functional evaluation of hMSH2 gene missense mutations].

OBJECTIVE: To construct a hMSH2/hMSH6 protein interaction system, and to use it for evaluating missense mutations detected in hMSH2 gene. METHODS: Recombinant plasmids pGADT7-hMSH2, pGBKT7-hMSH6 and 7 recombinant pGBKT7 plasmids with different hMSH6 domains were constructed through genetic engineering. Subsequently, site-directed mutagenesis was used to construct 10 mutant pGADT7-hMSH2 plasmids, which were transformed into yeast AH109. The growth of transformants was observed on a histidine-deficient culture. RESULTS: Both hMSH6 MutSII-V and MutSIII-V could interact with hMSH2 in yeast AH109. Yeast two-hybrid transformants pGADT7-hMSH2/pGBKT7-hMSH6 MutSII-V were used to construct a hMSH2/hMSH6 protein interaction system. Compared with wild-type hMSH2, yeast two-hybrid transformants c.505A>G, c.1168C>T, c.1255C>A, c.1261C>A could grow normally, c.1223A>G, c.1886A>G, c.2108C>A and c.2516A>G grew slowly, c.518T>G and c.1664 delA could not grow in a histidine-deficient medium in yeast AH109. CONCLUSION: A hMSH2/hMSH6 protein interaction system has been constructed with yeast two-hybrid system, which has been used for functional evaluation of hMSH2 gene missense mutations. c.518T>G is a pathological mutation. c.1223A>G, c.1886A>G, c.2108C>A, c.2516A>G may in part affect the hMSH2 function. And c.505A>G, c.1168C>T, c.1255C>A, c.1261C>A were innocuous variants.

Influence of eight unclassified missense variants of the MLH1 gene on Lynch syndrome susceptibility.

Missense mutations in MLH1 have frequently been detected in patients with Lynch syndrome, but their genetic significance has not been extensively assessed. In this study, we attempt to evaluate the etiological role of eight MLH1 missense variants. The variants were analyzed for their ability to affect MLH1 protein interaction with its partner PMS2 in vivo employing a yeast two-hybrid system. In addition, a SIFT (sorting intolerant from tolerant) algorithm was adopted to predict the effects of amino acid substitutions. Finally, scanning of mutations in a normal Chinese population and assay of the clinical characteristics have all been taken into account. Our results demonstrated that the MLH1 variants D485E and L653R cause functional alterations of the human MutLα complex significantly. The R265C, D304V, A586P, and R755S variants affect partial interaction. The remaining two variants, N38D and L559R, could be nonfunctional polymorphisms or might affect the mismatch repair system through other mechanisms.

Mutyh deficiency downregulates mitochondrial fusion proteins and causes cardiac dysfunction via α-ketoglutaric acid reduction with oxidative stress.

MutY homolog (MUTYH), an important protein in base excision repair (BER) system, excises adenine in the nascent strand opposite 8-oxoguanine in template DNA and restores G:C base-pair to maintain the fidelity of DNA replication. The loss of MUTYH causes oxidative stress and influences cardiac function, but the mechanism remains to be addressed. Here we demonstrate that Mutyh deficiency alters mitochondrial structure and impairs mitochondrial function through downregulation of mitochondrial fusion protein Mfn2 and alteration of the ratio of L-Opa1/S-Opa1 accompanied by reduction of α-ketoglutaric acid (α-KG) under oxidative stress condition. Further analysis reveals that the Mutyh deficiency may cause downregulation of histone demethylases and DNA demethylases and inhibition of the Mfn2 transcription. Oxidative stress associated with tert-butyl hydroperoxide (t-BHP) exposure results in the degradation of L-Opa1 and impairs the balance of L-Opa1/S-Opa1. Interestingly, α-KG supplementation alleviates the damage associated with Mutyh deficiency, restores the expression of Mfn2 and prevents degradation of L-Opa1. The current study demonstrates the relationship among Mutyh deficiency-coupled oxidative stress, the altered expressions of Mfn2 and Opa1, and the mitochondrial dysfunction, in which an intermediate in the tricarboxylic acid (TCA) cycle, α-KG has a key regulatory role.

Two functional variations in 5'-UTR of hoGG1 gene associated with the risk of breast cancer in Chinese.

8-Hydroxy-2'-deoxyguanine (8-OHdG) is produced by the oxidative stress-induced damage in DNA, which could pair with adenine (A) during DNA replication, leading to G-T transversion mutations. Glycosylase hOGG1 can recognize and excise oxidized guanines from duplex DNA. This work aims to investigate the relationship between the functional variations in 5-untranslated region (5'-UTR) of hOGG1 gene and the risk of breast cancer. Genotypes were analyzed in 518 sporadic breast cancer patients and 777 health controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. Risk-stratified subgroup analysis was performed to reveal the associations between the detected variations and the risk of characteristic breast cancer. In addition, immunohistochemistry was carried out to assess the functional effect of these variations on hOGG1gene expression. Five variations in 5'-UTR of hOGG1 gene are found in this study. Three of them, c.-18G>T, c.-23A>G, and c.-53G>C, are known single nucleotide polymorphisms, the other two, c.-45G>A and c.-63G>C, are rare variations. The frequency of c.-18G/T and c.-53G/C was significantly higher in breast cancer patients than those in healthy controls (P = 0.03, OR 2.01, 95% CI 1.04-3.90; and P = 0.01, OR 2.43, 95% CI 1.17-5.04, respectively). Both variations were especially prevalent in premenopausal status, and in the triple (estrogen receptor, progesterone receptor, and human epidermal growth factor Receptor 2) negative subgroups, respectively. Moreover, the variation of c.-18G>T could cause a reduced expression of hOGG1 gene.

The MLH1 2101C>A (Q701K) variant increases the risk of gastric cancer in Chinese males.

BACKGROUND: Gastric cancer is one of the most common cancers affecting East Asians, and MLH1 could play a critical role during tumorigenesis in this condition. METHODS: Samples from 236 Chinese patients suffering from gastric cancer were screened for MLH1 germline mutations. Carrier frequencies of the mutations were compared between gastric cancer patients and 240 cancer-free controls. Bioinformatic analysis was used to predict the effect of these mutations on protein function and mRNA splicing. RESULTS: Six MLH1 sequence alterations were identified in gastric cancer patients including two promoter region substitutions, -93G>A and -28A>G, and four missense mutations 649C>T (R217C), 655A>G (I219V), 1151T>A (V384D) and 2101C>A (Q701K). Compared with the MLH1 2101CC genotype, the 2101CA genotype was associated with a risk of gastric cancer (OR = 8.42, 95% CI = 1.04-68.06) in males. Furthermore, the MLH1 2101C>A mutant was predicted by in silico analysis to affect exon splicing ability. Immunohistochemistry of one index patient carrying the MLH1 2101C>A mutation demonstrated a loss of MLH1 protein and normal expression of MSH2 and E-cadherin. No significant differences were demonstrated between cases and controls for the other five MLH1 variants but the data indicated an ethnic difference in the frequency of these variations between Eastern Asians and Western populations. CONCLUSIONS: An ethnic-specific MLH1 mutation spectrum occurred in Chinese gastric cancer patients. The MLH1 2101C>A mutation could be a marker for susceptibility to gastric cancer, particularly in males.

Oxidative stress induces different tissue dependent effects on Mutyh-deficient mice.

8-oxoguanine (8-oxoG) is one of the most prevalent genotoxic lesions, and it is generated in DNA attacked by reactive oxygen species (ROS). Adenine misincorporated opposite to 8-oxoG during replication is excised by MutY homolog (MUTYH), an important protein of the base excision repair (BER) system. Mutyh plays an important role in the maintenance of genomic integrity, but the functional consequences of Mutyh deficiency are not fully understood. In the current study, we investigated the histological and functional changes of five tissues (hippocampus, heart, liver, kidney and lung) and their molecular basis in Mutyh-/- and wild-type mice exposed to D-galactose (D-gal). Our data indicated that Mutyh deficiency hindered the weight gain of experimental mice and induced substantial alterations of 8-oxoG content and superoxide dismutase (SOD) activity, but no significant histological and functional impairment appeared in the investigated tissues of Mutyh- deficient mice without D-gal exposure. Under low-dose D-gal exposure, Mutyh deficiency altered expression of genes involved in mitochondrial unfolded protein response (UPRmt) in the heart, liver and lung, and caused an enhanced expression of mitochondrial dynamics proteins (MDPs) in hippocampus and liver. The stress responses could maintain mitochondrial proteostasis and function. However, such responses were not noted when experiencing excessive damage burden induced by high-dose D-gal exposure, in which Mutyh deficiency increased accumulation of 8-oxoG and aggravated mitonuclear protein imbalance, as well as histological lesions in heart, liver and kidney. A higher sensitivity to ROS-induced cardiotoxicity with high-dose D-gal exposure was noticed in Mutyh-/- mice. However, no differences in learning and memory impairments were observed between Mutyh-/- and wild-type mice with high-dose D-gal exposure. In conclusion, our data demonstrated that Mutyh deficiency has different impacts on various tissues based on the degree of oxidative stress.

Analysis of hMLH1 missense mutations in East Asian patients with suspected hereditary nonpolyposis colorectal cancer.

PURPOSE: Germ line mutations in the DNA mismatch repair gene hMLH1 are a frequent cause of hereditary nonpolyposis colorectal cancer and about one-third of these are missense mutations. Several missense mutations in hMLH1 have frequently been detected in East Asian patients with suspected hereditary nonpolyposis colorectal cancer, but their pathogenic role has not been extensively assessed. The aim of this study was to perform functional analyses of these variants and their association with gastrointestinal cancer in East Asians. EXPERIMENTAL DESIGN: Altogether, 10 hMLH1 variants were analyzed by yeast two-hybrid and coimmunoprecipitation assays. RESULTS: The carboxyl-terminal replacements Q542L, L549P, L574P, and P581L in hMLH1 resulted in complete loss of activity in both yeast two-hybrid and coimmunoprecipitation tests and thus might be considered as pathogenic. The amino-terminal variants S46I, G65D, G67R, and R217C did not affect complex formation with hPMS2 in coimmunoprecipitation, but partly or fully lost their activity in yeast two-hybrid assay, and we suggested that these variants might reduce the efficiency of the heterodimer to go into the nucleus and thus the mismatch repair function might be blocked or reduced. The V384D and the Q701K variant resulted in the interaction of hMLH1 with hPMS2 at reduced efficiency and might raise the gastrointestinal cancer risk of the mutation carriers. CONCLUSIONS: This work availably evaluated the functional consequences of some missense mutations not previously determined in the hMLH1 gene and might be useful for the clinical diagnosis of hereditary gastrointestinal cancer, especially in East Asians.

Serum miR-20a and miR-486 are potential biomarkers for discriminating colorectal neoplasia: A pilot study.

AIM: Recent advances in circulating microRNAs (miRNAs) as noninvasive biomarkers have provided promising prospect in detecting colorectal cancer (CRC). However, the capability of miRNAs for detecting colorectal neoplasia (CRN, including precancerous lesions and curable stage CRCs) remains unclear. This study aimed to identify the potential of serum miRNAs (miR-20a, miR-486, miR-92a, and miR-135b) selected from the literature for discriminating CRN patients. MATERIALS AND METHODS: The serum samples from 46 CRN patients and 33 healthy controls were analyzed with quantitative reverse transcription-polymerase chain reaction. RESULTS: Serum miR-20a and miR-486 were significantly downregulated in CRN patients compared to that of in healthy controls (fold change = 0.697 and 0.696, P = 0.01 and 0.05, respectively). The serum level of miR-92a was not significantly different between two groups, while miR-135b level in serum was too low to be accurately quantified. In addition, serum miR-486 level was much more downregulated in tubulovillous adenoma and high-grade intraepithelial neoplasia patients than that of in healthy controls. For miR-20a and miR-486, the area under the receiver operating characteristic curve for discriminating CRN patients were 0.676 and 0.629, respectively, while their combined value was 0.698. No significant correlation was observed between miR-20a and miR-486 serum levels with age, gender, location, or lesion size. CONCLUSION: The results suggested that serum miR-20a and miR-486 could be potential noninvasive biomarkers for identifying CRN patients.

Variations in exon 7 of the MSH2 gene and susceptibility to gastrointestinal cancer in a Chinese population.

Epidemiologic, structural, and bioinformatic analyses were used to evaluate variants in the MSH2 and MLH1 genes in 187 subjects with suspected hereditary gastrointestinal cancer in China. An increased frequency of variants was observed in exon 7 of the MSH2 gene; there was a statistical difference (P < 0.05) between the colorectal cancer (CRC) group (6/82, or 7.32%) or the gastric cancer (GC) group (8/105, or 7.62%) and the controls (1/112, or 0.89%). The odds ratio (OR) was 8.76 for CRC and 9.15 for GC, suggesting an association between the presence of variants in exon 7 of the MSH2 gene and risk of gastrointestinal cancer in the studied population. In addition, MSH2 1168T showed trends toward association with CRC and GC in young (<50 yr) sporadic disease patients (OR = 10.97 and 17.15, respectively). The c.1168C>T (p.Leu390Phe), c.1255C>A (p.Gln419Lys), and c.1261C>A (p.Leu421Met) in exon 7 and c.518T>G (p.Leu173Arg) in exon 3 of MSH2 were suspected as predisposing to gastrointestinal cancer. Variants c.505A>G (p.Ile169Val), c.1221C>G (p.Leu407Leu) and c.1223A>G (p.Tyr408Cys) in MSH2 and c.655 A>G (p.Ile219 Val) and c.927C>T (p.Pro309Pro) in MLH1 might be merely polymorphisms. Consequences of the variant c.2101C>A (p.Gln701Lys) in MLH1 remain to be elucidated.

Effective protection of Terminalia catappa L. leaves from damage induced by carbon tetrachloride in liver mitochondria.

The protective effects of chloroform extracts of Terminalia catappa L. leaves (TCCE) on carbon tetrachloride (CCl4)-induced liver damage and the possible mechanisms involved in the protection were investigated in mice. We found that increases in the activity of serum aspartate aminotransferase and alanine aminotransferase and the level of liver lipid peroxidation (2.0-fold, 5.7-fold and 2.8-fold) induced by CCl4 were significantly inhibited by oral pretreatment with 20, 50 or 100 mg/kg of TCCE. Morphological observation further confirmed the hepatoprotective effects of TCCE. In addition, the disruption of mitochondrial membrane potential (14.8%), intramitochondrial Ca2+ overload (2.1-fold) and suppression of mitochondrial Ca2+-ATPase activity (42.0%) in the liver of CCl4-insulted mice were effectively prevented by pretreatment with TCCE. It can be concluded that TCCE have protective activities against liver mitochondrial damage induced by CCl4, which suggests a new mechanism of the hepatoprotective effects of TCCE.

[Application of a novel method to collect large amount of fecal mucosa in screening colorectal cancer].

OBJECTIVE: To explore the application of a novel device of collecting large amount of fecal mucosa for detecting the DNA methylation and screening colorectal cancer. METHODS: Preoperative complete fecal sample and surgical specimen of 10 patients with colorectal cancer, and complete fecal sample and normal bowel mucosal samples confirmed by colonoscopy of 6 hospitalization cases at The Third Affiliated Hospital, Nanjing University of TCM from March to April 2014 were collected. A self-made bowel mucosa collector (consisting of upper, middle, lower three containers of 1 000 ml volume, with filter screen in each bottom whose pore diameter is 100, 200 and 300 mesh.) was used to collect mucosal exfoliation cells. Fecal DNA kit was applied to extract DNA of exfoliation cells and the concentration and purity of DNA were measured by UV spectrophotometer (A260/A280), meanwhile DNA methylation of fecal fluid and mucosal tissues was detected by bisulfite sequencing pCR(BSP). RESULTS: DNA methylation sequencing showed that FBN1, SPG20, and SNCA genes presented methylation in CpG island in fecal fluid and cancer tissues from 10 colorectal cancer patients, but did not presented methylation in fecal fluid and mucosa from 6 control cases. When fecal amount was below 100 g, collection rate of fecal fluid was 60% to 80%; when fecal amount was over 100 g, collection rate of fecal fluid was unstable. When fecal amount was 50 to 100 g, DNA A260/A280 value was 1.6 to 1.8, and DNA concentration was 5.0 to 56.1 ng/L. CONCLUSION: Collection rate of fecal fluid with this self-made fecal mucosa collector is quite stable when managing fecal amount of 50 to 100 g once, and can obtain higher purity and concentration of DNA, meeting the demand of methylation detection for screening colorectal cancer.

[Screening and diagnostic value of the molecular markers of DNA methylation in colorectal neoplasma].

OBJECTIVE: To screen the molecular markers of DNA methylation with potential diagnostic value, and to explore their methylation features in Chinese colorectal neoplasma in order to find out ones with higher diagnostic value. METHODS: Tissue samples of colorectal cancer and normal adjacent mucosa(>10 cm distance to tumors) from 10 colorectal cancer patients undergoing operation, and tissue samples of colorectal adenoma from 10 patients undergoing endoscopic resection in our center from June to August 2013 were collected respectively. Methylation status of 8 genes, such as SNCA, MAL, INA, SPG20, FBN1, CNRIP1, TFPI2, OSMR, was detected by BSP and qMSP to screen genes with potential diagnostic valua. ROC curve was drawn to analyze its diagnostic value. RESULTS: BSP measurement showed that the rate of DNA methylation of SNCA, SPG20 and FBN1 was 100% in colorectal cancer and adenoma, while no methylation was found in normal adjacent mucosa. The other 5 genes expressed in different extent in cancer, adenoma and normal adjacent mucosa. Among 10 cancer tissues and normal adjacent mucosa detected by qMSP method, positive SNCA methylation was found in 5 cases and 1 case respectively; positive SPG20 in 8 cases and 1 case respectively; positive FBN1 in 7 cases and 0 cases respectively, whose differences were significant (P=0.070, P=0.003 and P=0.007). The area under curve(AUC) of SNCA, SPG20, and FBN1 methylation for diagnosing colorectal cancer was 0.890, 0.730 and 0.880 respectively. CONCLUSION: SNCA, SPG20 and FBN1 are potential genes with screening value for colorectal neoplasma.

Hepatoprotective activity of Terminalia catappa L. leaves and its two triterpenoids.

The aim of this study was to evaluate the effect of the chloroform extract of Terminalia catappa L. leaves (TCCE) on carbon tetrachloride (CCl(4))-induced acute liver damage and D-galactosamine (D-GalN)-induced hepatocyte injury. Moreover, the effects of ursolic acid and asiatic acid, two isolated components of TCCE, on mitochondria and free radicals were investigated to determine the mechanism underlying the action of TCCE on hepatotoxicity. In the acute hepatic damage test, remarkable rises in the activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (5.7- and 2.0-fold) induced by CCl(4) were reversed and significant morphological changes were lessened with pre-treatment with 50 and 100 mg kg(-1) TCCE. In the hepatocyte injury experiment, the increases in ALT and AST levels (1.9- and 2.1-fold) in the medium of primary cultured hepatocytes induced by D-GalN were blocked by pre-treatment with 0.05, 0.1, 0.5 g L(-1) TCCE. In addition, Ca(2+)-induced mitochondrial swelling was dose-dependently inhibited by 50-500 microM ursolic acid and asiatic acid. Both ursolic acid and asiatic acid, at concentrations ranging from 50 to 500 microM, showed dose-dependent superoxide anion and hydroxyl radical scavenging activity. It can be concluded that TCCE has hepatoprotective activity and the mechanism is related to protection of liver mitochondria and the scavenging action on free radicals.