A s an im portant part of epigenetic m arker, D N A m ethylation involves in the gene regulation and attracts a w ide spread attention in biological auxology, geratology and oncology fields. In forensic science, because of the relative stable, heritable, abundant, and age-related characteristics, D N A m ethyla-tion is considered to be a useful com plem ent to the classic genetic m arkers for age-prediction, tissue-identification, and m onozygotic tw ins' discrim ination. V arious m ethods for D N A m ethylation detection have been validated based on m ethylation sensitive restriction endonuclease, bisulfite m odification and m ethylation-C pG binding protein. In recent years, it is reported that the third generation sequencing m ethod can be used to detect D N A m ethylation. T his paper aim s to m ake a review on the detection m ethod of D N A m ethylation and its applications in forensic science.

Objective To prepare a capillary electrophoresis sieving medium and apply it in GA118-16A genetic analyzer. Methods The white solid polyacrylamide (LPA) was prepared by polymerization and lyophilized. Through the swelling of the sol buffer, the sieving medium was obtained. The sieving medium was evaluated by 1) characterizing the parameters, including molecular weight, structure and viscosity, 2) applying in the GA118-16A genetic analyzer, including the spatial calibration, the spectral calibration and the STR analysis.. Results The prepared sieving medium Mw 1.8 x 105Da, Mn 1.2 x 105 Da, is of correct structure and high purity. The polydispersity was 1.5The spatial calibration and spectral calibration files can be established successfully in GA118-16A genetic analyzer, and the sieving medium can effectively separate the DNA fragments with 1bp difference. The STR profile is of sharp peaks, no impurity peaks, no tail, and no peak loss. Conclusion The sieving medium prepared by the method can be applied to domestic genetic analyzer such as GA118-16A.

OBJECTIVE: To develop a multiplex allele-specific PCR (AS-PCR) assay with three-color fluorescence labeling for mitochondrial DNA (mtDNA) SNP typing. METHODS: Based on the principle of AS-PCR, the primer sets were designed for 20 SNP located on the coding region of mtDNA and divided into 2 groups labeled with FAM and HEX fluorescence, respectively. A primer set included two forward (reverse) allelic specific primers with different sizes and a generic reverse (forward) primer. Blood samples from 200 unrelated individuals were analyzed by AS-PCR and capillary electrophoresis. Three random samples at least for each SNP site were examined and verified by direct sequencing. The haplotype frequency was investigated. RESULTS: Distinct electropherograms of 200 blood samples were obtained successfully. The typing results of direct sequencing were identical to those obtained from AS-PCR. The minimum detectable DNA concentration was 0.2 pg under the system of 10 microL. The sensitivity of the DNA concentrations ranged from 0.5 to 5 pg. The 200 individuals were assigned into 15 haplotype, and the haplotype diversity was 0.906 0. CONCLUSION AS-PCR is a simple, rapid and efficient method for mtDNA SNP typing, and can be applied to forensic practice.
Alleles ; DNA ; DNA Primers ; DNA, Mitochondrial/analysis* ; Electrophoresis, Capillary ; Haplotypes ; Humans ; Mitochondria ; Polymerase Chain Reaction/methods* ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA

OBJECTIVETo evaluate the incidence of Ketoconazole associated hepatotoxicity and related factor.

METHODSLiterature retrieval was conducted by using multi-databases for meta-analysis on Ketoconazole associated hepatotoxicity. The data were collected with a standardized form. Overall estimation of incidence of hepatotoxicity for specific study type was calculated by using a DerSimonian-Laird random-effects model owing to the substantial differences among the studies.

RESULTSTotally 204 eligible studies were included in the analysis. The incidence of Ketoconazole associated hepatotoxicity was 3.6%-4.2%. The dosage and duration specific subgroup analyses did not show any significant difference among groups, while the age specific subgroup analysis showed the incidence in children and people aged >60 years was 1.4% (95% CI: 0.5%-4.2%) and 3.2% (95% CI: 1.1%-8.7%) respectively. Additionally, the incidence of the hepatotoxicity was higher in people who had oral administration of ketoconazole beyond the provisions of the usage instructions, and the incidence was 5.7% (95% CI: 4.5%-7.2%).

CONCLUSIONKetoconazole associated hepatotoxicity was common. Off-label use might increase the risk of liver damage. Well-designed large sample studies are needed to identify the risk factors in future.

Antifungal Agents ; administration & dosage ; adverse effects ; Chemical and Drug Induced Liver Injury ; etiology ; Humans ; Ketoconazole ; administration & dosage ; adverse effects ; Off-Label Use

OBJECTIVETo investigate leptin mRNA expressions in subcutaneous (SC) and omental (OM) adipose tissues of patients with nonalcoholic fatty liver disease (NAFLD), and their relationships with insulin resistance (IR), blood leptin, blood triglyceride, total blood cholesterol, blood glucose, body weight index and waist-hip ratio.

METHODSSC and OM adipose tissues were obtained from 10 obese and 11 nonobese NAFLD patients and from 11 obese and 13 nonobese patients without NAFLD, who served as controls. Leptin mRNA expression levels in the subcutaneous and omental adipose tissues were measured using SYBR Green I quantitative real-time PCR. IR was estimated using homeostasis assessment (HOMA). The levels of plasma leptin and insulin were measured using ELISA.

RESULTSThe relative mRNA expression of leptin, HOMA-IR and blood leptin levels in NAFLD differed significantly from those of the controls (P < 0.05). The leptin/GAPDH ratio of the obese and nonobese NAFLD and control cases were 1.32 +/- 0.12, 0.99 +/- 0.05, 1.10 +/- 0.09, 0.87 +/- 0.13 respectively. The expression levels of SC and OM adipose leptin mRNA in NAFLD patients were positively correlated with HOMA-IR (r=0.72, P < 0.05), blood leptin (r=0.69, P < 0.05), blood triglyceride (r=0.32, P < 0.05), body weight index (r=0.57, P < 0.05) and waist-hip ratio (r=0.50, P <0.05).

CONCLUSIONThe primary reason for high levels of blood leptin is high leptin mRNA expression in adipose tissues; in both obese and nonobese patients with NAFLD; high levels of blood leptin and the leptin mRNA expression in adipose tissues and IR exist. These findings suggest that leptin resistance exists in patients with NAFLD and leptin resistance is positively correlated with NAFLD, the same as in insulin resistance.

Adipose Tissue ; metabolism ; Adult ; Aged ; DNA, Complementary ; Fatty Liver ; genetics ; metabolism ; Female ; Gene Expression ; Humans ; Leptin ; genetics ; metabolism ; Male ; Middle Aged ; RNA

OBJECTIVEBy means of phage-display technique, to screen polypeptides that specifically bind to human gastric cancer with high metastatic potential to peritoneum.

METHODSTwo human gastric cancer cell lines were used: GC9811-P with high metastatic potential to peritoneum and its wild type parental GC9811, to carry out subtractive screening with a phage display-12 peptide library.

RESULTSAfter three rounds of screening, 40 phage clones bond to GC9811-P cells were randomly selected. When injected into the peritoneal cavity of nude mice, 6 of the 40 clones did not bind to mouse peritoneum as examined by immunohistochemical staining. They were considered to be capable of binding specifically to GC9811-P cells. Sequence analysis revealed two different exogenous peptides: TLNINRLILPRT and SMSI(X)SPYI(XXX).

CONCLUSIONTwo peptides have been obtained that specifically bind to a gastric cancer cell variant GC9811-P, which easily disseminates to the peritoneum. Whether or not they could block GC9811-P metastasis to peritoneum in vivo remains to be determined.

Animals ; Binding Sites ; Cell Line, Tumor ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Peptide Library ; Peptides ; metabolism ; Peritoneal Neoplasms ; secondary ; Protein Array Analysis ; methods ; Protein Binding ; Sensitivity and Specificity ; Stomach Neoplasms ; metabolism ; pathology

Clopidogrel was believed to be superior to aspirin by the well-known CAPRIE trial. However, no other large clinical trials demonstrated the same results, but all focused on the combination use of clopidogrel with aspirin, and combination therapy in CREDO was called the "Emperor's New Clothes". However, no one overturned the results of these clinical trials by quantitatively analyzing them. We reviewed ten large-scale clinical trials about clopidogrel. On the basis of results of CAPRIE, CREDO and CHARISMA trials, we re-estimated their minimal sample sizes and their powers by three well-established statistical methodologies. From the results of CAPRIE, we inferred that the minimal sample size should be 85 086 or 84 968 but its power was only 30.70%. A huge gap existed. The same was also true of CREDO and CHARISMA trials. Moreover, in CAPRIE trial, 0 was included in the 95% confidence interval and 1 was included in the 95% confidence interval for the relative risk. There were some paradoxical data in CAPRIE trial. We are led to conclude that the results in CAPRIE, CREDO, and from the subgroup analysis in CHARISMA trials were questionable. These results failed to demonstrate that clopidogrel was superior to aspirin or that clopidogrel used in combination with aspirin was better than aspirin alone. The cost-effectiveness analyses by some previous studies were not reliable.

Adult ; Aged ; Carcinoma, Hepatocellular ; genetics ; pathology ; Female ; Gene Expression ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; Liver Neoplasms ; genetics ; pathology ; Male ; Middle Aged ; Neoplasm Metastasis ; RNA, Messenger ; genetics ; Young Adult

Inherited retinal diseases (IRDs), including retinitis pigmentosa, Usher syndrome, Cone-Rod degenerations, inherited macular dystrophy, Leber's congenital amaurosis, Leber's hereditary optic neuropathy are the most common and severe types of hereditary ocular diseases. So far more than 200 pathogenic genes have been identified. With the growing knowledge of the genetics and mechanisms of IRDs, a number of gene therapeutic strategies have been developed in the laboratory or even entered clinical trials. Here the progress of IRD research on the pathogenic genes and therapeutic strategies, particularly gene therapy, are reviewed.
Biomedical Research ; methods ; trends ; Clinical Trials as Topic ; Genetic Predisposition to Disease ; genetics ; Genetic Therapy ; methods ; trends ; Humans ; Mutation ; Retinal Diseases ; genetics ; therapy ; Treatment Outcome

Objective:To preliminarily disclose the biological properties of recombinant human tau441 (P301S) protein, such as aggregation, antigenicity and immunogenicity.Methods:The recombinant plasmid tau441 (P301S) was expressed by prokaryotic expression system and purified by nickel column affinity chromatography. The protein concentrations were determined via BCA kit. The purity of protein was determined by SDS-PAGE gel coomassie brilliant blue staining. Western blot (WB) and negative staining transmission electron microscopy (TEM) were used to identify the recombinant proteins. The antigenicity was detected through indirect enzyme linked immunosorbent assay (ELISA), and the immunogenicity was detected by specific antibody titers of mouse immune serum.Results:The purity of recombinant human tau441 (P301S) was 70%. WB showed specific bands at relative molecular mass (Mr.×10 3) 64 and higher relative molecular mass. Negative staining TEM showed that tau441 (P301S) was aggregated, and the area was significantly larger than tau wild-type control protein (t=6.439, P=0.003). After 9 days of incubation at 4 ℃, tau441 (P301S) formed obvious fibrotic structure. Indirect ELISA result showed that tau441 (P301S) could be recognized by anti-tau monoclonal antibody HT7 (1∶80 000). The specific antibody titer of the immunized serum was 1∶128 000 and WB showed that the immunized serum recognized the brain lysate extract of Alzheimer’s disease (AD) transgenic mice. Conclusions:The recombinant human tau441 (P301S) protein had the characteristics of enhanced aggregation in vitro, but its antigenicity and immunogenicity were not changed.