Adult ; Drowning ; Humans ; Hydrogen Sulfide ; poisoning ; Male ; Radiography, Thoracic ; X-Ray Film ; Young Adult

Objective To compare and evaluate two type-specific primers PCR genotyping methods of hepatitis B virus ( HBV) which were established by Naito et al ( Naito method) and our lab (new method). Methods The two genotyping methods were applied for detecting the plasmids containing the HBV genomes of genotype A or D or subgenotype B1 or C2 and the plasmids mixed with different proportion of subgenotypes B1 and C2. In addition, the genotypes of 113 serum samples of patients with chronic HBV infection from Shenzhen, Handan and Urumqi cities of China were identified by the two methods, respectively. The results were verified by PCR product based sequencing. Results The sensitivity of the two methods showed no difference when they were applied to detect the plasmids containing the HBV genomes of genotype A or D or subgenotype B1 or C2. While detecting the plasmids mixed with different proportion of subgenotypes B1 and C2, the sensitivity of the new method was superior than that of Naito method. Meanwhile, the specificity of the new method was obviously superior than that of Naito method. Both of the two methods were highly sensitive in identification of HBV genotypes of serum samples with a single genotype. However, the new method showed more sensitive in identification of the B/C mix strains than that of Naito method. The total coincidence rate between the two methods was 83. 2% (94/113), with the discrepancy of 16. 8% (19/113). Fifteen of the 19 inconsistent genotypes by the two methods were selected and their PCR products were sequenced directly. The sequencing results were identical with that of the new methods, but not with that of the Naito method. Conclusion The new method is more sensitive, and its specificity is superior to the Naito method. It could be used for clinical and epidemiological studies on HBV genotype and subgenotype in China.

Antimony ; blood ; Humans ; Spectrophotometry, Atomic ; methods

Objective To assess the effect of peroxisome proliferator-activated receptor γ (PPARγ) agonist on prostate epithelial cells in vitro.Methods The expression of peroxisome proliferator-activated receptor γ(PPARγ) was studied by immunocytochemistry and immunofluorescence study.The RWPE-1 human prostate epithelial cell line was treated with PPARγ agonist rosiglitazone 100 μmol/L for 48 h.Analysis of apoptosis was performed by Caspase 3/7 activity assay.Mitochondria depolarization was measured by using the potential-sensitive color,JC-1.The expression of apoptosis-related proteins-Bax was investigated by immunohistochemistry.Results PPARγ mainly located in nucleus and perinucleus.RWPE-1 cell line treated with PPARγ agonist rosiglitazone showed higher Caspase 3/7 activity (10636±1032 RLU) than in control (5936±620 RLU),P＜0.01 and significantly upregulated Bax level (8250±694 vs.6017±563)than in control group,P＜0.01.In addition,mitochondrial membrane potential was depolarized in rosiglitazone treated cells.Conclusions PPARmay play important roles in the pathophysiology of BPH.The mechanism might be that PPARγ regulates cell apoptosis.It is suggested that the mitochondrial and Bax pathway might be involved in signaling PPARγ induced cell apoptosis.

Objective To establish a hepatitis B virus (HBV) nested PCR (nPCR) for detection of genotypes A-D and subgenotypes B1,B2, C1 and C2. Methods The entire HBV nucleotide sequences of genotypes A-H retrieved from GenBank were compared and analyzed by DNAStar software. The PCR primers were designed by Primer Premier 5.0 software,and the nPCR for genotyping HBV/A-D as well as subgenotyping B1, B2,C1 and C2 were established. There were 3 steps in the process:step 1 for genotypes B, D and subgenotypes C1, C2 with the amplification of Mix A; step 2 for genotype A with the amplification of Mix B; step 3 for subgenotypes B1 and B2 with the amplification of Mix C in the second-ound PCR, based on the first-round amplification procedure. A total of 68 serum samples from patients with chronic HBV infection were detected by nPCR. 15 of 68 sera were selected randomly and their PCR products were directly sequenced to confirm the accuracy of the method. Results Among 68 serum samples of patients with chronic HBV infection detected by the nPCR, 23.53% (16/68) were infected with B2, 11.76% (8/68) with C1,48.53% (33/68) with C2,1.47% (1/68) with D,11.76% (8/68) with B2C2 mix strains,1.47% (1/68) with C2D mix strains and 1.47% (1/68) with B2/C1/D mix strains. The sequencing analysis of the 15 serum samples had the same results as detected by nPCR. Conclusion nPCR is a simple,rapid method and able to detect genotypes A-D and subgenotypes B1 ,B2 ,C1 and C2 subtypes of HBV with both high sensitivity and specificity.

In our previous work, we found that trivalent dimethylarsinous acid (DMA(III)) have high affinity binding to cysteine residue 13 of rat hemoglobin. However, it is still unknown why arsenic intermediate metabolite DMA(III) has high binding affinity for Cysl3 but not for other cysteine residues 93, 140, 111 and 125. In order to better understand the molecular mechanism of DMA(III) with rat hemoglobin, we have done current study. So, SD rats were divided into control and arsenic-treated groups randomly. Arsenic species in lysate of red blood cells were analyzed by HPLC-ICP-MS, and then determined by a hybrid quadrupole TOF MS. In addition, trivalent DMA(III) binds to different cysteine residues in rat hemoglobin alpha and beta chains were also simulated by Molecular Docking. Only Cys13 in alpha chain is able to bind to DMA(III) from the experiment results. Cys13 of alpha chain in rat hemoglobin is a specific binding site for DMA(III), and we found that amino acids compose pockets structure and surround Cys13 (but not other cysteine residues), make DMA(III) much easy to bind cysteine 13. Taken together, the DMA(III) specific binding to Cys13 is related to spatial structure of Cys13.
Animals ; Arsenic ; metabolism ; Binding Sites ; Cacodylic Acid ; analogs & derivatives ; chemistry ; Chromatography, High Pressure Liquid ; Cysteine ; metabolism ; Hemoglobins ; metabolism ; Mass Spectrometry ; Peptide Fragments ; metabolism ; Rats

Objective To investigate the expression of hypoxia inducible factor-1α(HIF-1α)in the transplanted liver in rats and the role of HIF-1α in the JNK signaling pathway.Methods Ninety-six SD rats were randomly divided into normal control(NC)group,autotransplantation(AT)group,hypoxia preconditioning (HP)+ AT group according to simple random sampling method.No treatment was applied to the rats in the NC group except for blood vessel separation.Stable rat models of 70％ AT was established in the AT group.Rats in the HP + AT group were given 8％ oxygen mixed gas for 90 minutes before the operation.The levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were detected at 1,2,12,24 hours after operation,and the expression of HIF-1α in the hepatic tissue,mRNA expression of c-Jun,and protein expression of Cleaved Caspase-3 were detected by immunohistochemical staining,reverse transcription-polymerase chain reaction and Western blot,respectively.All data were analyzed using the analysis of variance or t test.Results The levels of ALT and AST in the HP + AT group were significantly lower than those in the AT group,while higher than those in the NC group at 1,2,12 and 24 hours after the operation(F=2631.371,1177.642,810.383,682.848;743.618,1095.522,375.995,580.613,P ＜0.05).The protein expression levels of HIF-1α in the AT group were significantly lower than those in the HP + AT group,but higher than those in the NC group at 1,2,12 and 24 hours after operation(F = 191.737,284.482,459.419,213.782,P ＜ 0.05).The mRNA expression levels of c-Jun in the HP + AT group were significantly lower than those in the AT group,while higher than those in the NC group at 1,2 and 12 hours after operation(F = 66.211,53.169,9.645,P ＜ 0.05).There was no significant difference in the mRNA expression of c-Jun among the 3 groups at 24 hours after operation(F = 1.100,P ＞ 0.05).The protein expressions of Cleaved Caspase-3 in the HP + AT group were significantly lower than those in the AT group,while higher than those in the NC group at 1,2,12 and 24 hours after the operation(F =23.133,31.158,14.347,29.043,P ＜ 0.05).Conclusion High expression of HIF-1α after HP inhibits apoptosis of hepatic cells and alleviates ischemia reperfusion injury of hepatic tissues by suppressing JNK activation,down-regulating protein expression of Cleaved Caspase-3 after orthotopic liver transplantation in rats.

AIM:To determine whether acellular porcine cornea stroma (APCS) could support the growth of the rabbit corneal cells in vitro.METHODS: APCS was prepared. The rabbit's corneal epithelium and stromal cells were cultured and seeded on, APCS in vitro.The observation of phase contrast photograph and histological examination were performed.RESULTS: Histological examination showed the epithe- lium grew on the scaffold of APCS in 2-3 layers at 10th day. The stromal cells adhered to the surface of the scaffold after 24 hours and invaded into the interlaminar of the material at 5th day.CONCLUSION: These results indicate that APCS can support the growth and proliferation of the corneal epithelium and stromal cells in vitro.

Background The pathogenesis of primary open angle glaucoma(POAG) and high myopia are very complex.To construct the regulatory network of virulence genes and relevant genes that involved in pathogenicity are helpful for reveal of the pathogenesis.Objective The aim of this study was to investigate myocilin(Myoc),a gene that contributes to POAG and high myopia in eyes of BXD Recombinant Inbred(BXD RI)mice and construct the regulatory network of Myoc.Methods The affymetrix microarray system was used to detect the differential expression of Myoc in the eyes of C57BL/6J(B6),DBA/2J(D2) and BXD RI mice.Expression quantitative trait loci (eQTL) mapping was performed to construct the regulatory network of Myoc gene.Results The average expression level of the Myoc gene in the BXD strains was 10.83,and the gene exhibited expression levels ranging from 8.39 in BXD55 mice tol 1.43 in B6 mice.The eQTL mapping for the Myoc gene showed a significant likelihood ratio statistic (LRS) of 21.78.The QTL was mapped in chromosome 2,and Myoc was located on chromosome 1,indicating that the Myoc gene was a trans-acting QTL.Olfml2a was identified to be a candidate upstream gene of Myoc by analysis of bioinformatics.Genetic regulatory network analysis demonstrated that a series of genes associated with Myoc probably played roles in the pathogenesis and development of POAG and high myopia.Conclusions The genetical genomics approach provides a powerful tool for constructing pathways that contribute to complex traits,such as POAG and high myopia.

OBJECTIVE: To test the technical parameters of GlobalFiler® PCR Amplification Kit for its application to forensic application value and to investigate the genetic polymorphisms. METHODS: The validation was conducted in sensitivity, mixed samples, species specificity, adaptability, survivability, consistency, peak height balance and stability. The amplification and detection of the genomic DNA from 373 unrelated individuals from Beijing Han nationality were extracted by automation workstation. RESULTS: Global-Filer® PCR Amplification Kit was adaptive to some mixed, degraded and inhibited samples. The power of sensitivity and adaptability and peak height balance showed well. The distributions of genotype frequencies for 21 STR loci in the population were all in accordance with Hardy-Weinberg equilibrium (P > 0.05). The PIC value of the 21 STR loci was among 0.536 to 0.940; the H value was among 0.558 to 0.933; the DP value was among 0.783 to 0.992; the PE value was among 0.243 to 0.874. CONCLUSION GlobalFiler® PCR Amplification Kit is suitable for criminal cases and DNA database in forensic practice. And 21 STR loci in Beijing Han nationality have high polymorphism, which have application value in forensic practice and population genetics.
Asian People/genetics* ; Beijing ; Databases, Nucleic Acid ; Ethnicity ; Gene Frequency ; Genetic Loci/genetics* ; Genetics, Population ; Genotype ; Humans ; Polymerase Chain Reaction/standards* ; Polymorphism, Genetic ; Reproducibility of Results ; Species Specificity