OBJECTIVETo investigate the association between a polymorphism of methylenetetrahydrofolate reductase with Non-syndromic cleft lip with or without cleft palate (NSCL/P) in Chinese population.

METHODSThe polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) technique was used to detect a single nucleotide polymorphism (SNP), rs1801131, at the methylenetetrahydrofolate reductase (MTHFR) gene in both 158 patients with NSCL/P and 192 healthy individuals. The Hardy-Weinberg equilibrium for genotypic distributions was estimated by the goodness-of-fit test. The UNPHASED program was applied to perform the association analysis.

RESULTSThe genotypic distribution of A1298C was not deviated from the Hardy-Weinberg equilibrium in both controls and patients. No association was found between cleft lip with or without palate (CL/P) and controls. There was significant difference of cleft palate only (CPO) and the healthy individuals (χ(2) = 4.256, P = 0.039). The frequency of AC + CC genotype was higher in control group than that in CPO group (OR = 0.8, 95%CI = 0.381 - 1.683), 26 among 100 healthy individuals carried AC + CC genetypes, which were carried by 22% of CPO patients.

CONCLUSIONSThe polymorphism of MTHFR A1298C may be involved in the occurrence of non-syndromic cleft palate only in Chinese population.

Adolescent ; Adult ; Alleles ; Asian Continental Ancestry Group ; genetics ; Child ; Child, Preschool ; Cleft Lip ; genetics ; Cleft Palate ; genetics ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Male ; Methylenetetrahydrofolate Reductase (NADPH2) ; genetics ; Polymorphism, Single Nucleotide ; Young Adult

Objective To investigate the mechanism of endotoxin tolerance (ETT) through observing the expression of OX40 in liver tissues of ETT rats.Methods SD male rats were randomly divided into three groups:normal group (n=6),acute liver failure (ALF) group (n=24) and ETT group (n=24).Lipopolysacharide (LPS) 0.1 mg/kg (ETT groups) or 0.9 ％NaC1 (ALF groups) was administered by five consecutive intraperitoneal injections at 24 h intervals,and at the sixth day,all animals were treated with intraperitoneal injections of D-galactosamine (D-GalN) 800 mg/kg and LPS 8 μg/rat.Blood and liver tissue were collected at 6,12,24 and 48 hours after the injection of D-GalN/LPS.The gene expression of OX40 in the liver was measured by reverse transcription-polymerase chain reaction (RT-PCR).The protein expression of OX40 was estimated by Western blot.The tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels were determined by enzyme-linked immunosorbent assay (ELISA).The data analysis was performed by one-way ANOVA,lease significant difference (LSD) and Dunnett's T3 test.Results The expressions of TNF α and IL-6 were both significantly lower in ETT group compared to ALF group,but still higher than that of control group.The gene expressions of OX40 peaked at 12 hours and decreased gradually in ALF group.The gene expressions of OX40 were significantly lower in ETT group compared to ALF group (6 h:F=5.027,24 h:F=5.539,48 h:F=5.011,all P＜0.05; 12 h:F=36.688; P＜0.01),but still higher than that of normal group.The tendency of OX40 protein expression in ALF group was peaked at 12 hours and decreased at 24 hours.In ETT group,the expression of OX40 was lower,and the difference between ETT group and ALF group had statistical significance (6 h:F=8.658,P＜0.05; 12 h:F=34.611,24 h:F=28.176,48 h:F=16.747; all P＜0.01).Conclusions The level of OX40 is increased in ALF group,while the expressions of OX40,TNF-α and IL-6 are lower in ETT group,which suggested that OX40 may play an important role in the process of ETT.

Objective To study the effect of endotoxin tolerance (ETT) on chemokine receptor 7 (CXCR7) in the liver tissue of rats with acute liver failure (ALF).Methods SD male rats were randomly divided into three groups:normal group,ALF group and ETT group.The rats in the ETT group and ALF group were injected with lipopolysacharide (LPS) 0.1 mg/kg or saline respectively,one time / day for 5 days.At 24 hours after the 5th-day injection,all rats were injected with D-GalN 800 mg/kg and LPS 8μg/rat.Blood sample and liver tissue were collected on 2,6,12,24 and 48 hours after injection.The gene expressions of CXCR7 in the liver were measured by RT-PCR,and the protein expressions of CXCR7 were determined by Western Blot.The data analysis was performed by LSD,Dunnett's t test.Results The histological damage in the liver tissue was significantly mider in ETT group compared to ALF group.The gene expressions of CXCR7 were significantly milder in ETT group compared to ALF group (2 h:F =29.222,6 h:F=166.892,12 h:F=38.975,24h:F=34.603,48 h:F=18.929,allP＜0.01),but still severer than that of normal group.The CXCR7 protein expression in ALF group and ETT group peaked at 24 hours,but the expression of CXCR7 in ETT group was lower compared with that in in ALF group (2h:F=11.155,6 h:F=42.553,12h:F=17.082,all P＜0.01; 24 h:F=7.242,P＜0.05).Conclusions During the process of endotoxin tolerance,LPS pretreatment and D-GalN can decrease the liver injury,down-regulate the expressions of CXCR7mRNA and CXCR7.This suggests that CXCR7 may play an important role in the ETT.

Objective To obtain the information of the 2009 influenza outbreak and the variations of influenza virus strains in quanzhou, and explore the relationship between the genetic variation of influenza virus and influenza epidemic. Methods During the influenza outbreak in quanzhou,one hundred and ninetyeight throat swabs specimens from the patients with influenza were collected. Viruses were isolated with MDCK cells and identified with serological test, followed by real-time RT-PCR. RNA of four influenza virus strains were extracted, then HA1 gene was amplified by RT-PCR. The purified PCR products were sequenced. The data were analyzed with the software DNAstar megalign. Results Total 98 pieces of H3N2 subtype influenza virus nucleic acid were detected in 198 throat swabs specimens,among which 62 influenza virus strains were identified as subtype influenza A( H3N2 ). The sequencing results of HA1 gene in these positive strains showed that their genetic characterization were more closed to strains A/Ningbo/333/2008 with a nucleotide homology of 98.7%, which was 96.8% as compared with A/Xiamen/70/2004. The amino acids sequences deduced from the nucleotide sequences in HA1 region of the isolated strain had 7 mutant sites compared with A/Brisbane/10/2007 vaccine strain. One variant amino acids were found located in the antigenic determinant sites A( 144 ), two were in the sites B( 158,189 ). Phylogenetic analysis also confirmed the difference in HAl domain. Conclusion The influenza virus strains causing the flu outbreak among some communities of quanzhou in 2009 are subtype influenza A ( H3N2 ), whose genetic characterization and antigenicity were different from the vaccine strain.

Objective To investigate the influenza H1N1 virus surveillance of 2009 in Quanzhou,and analyze the HA and NA gene of influenza H1N1 virus, explore its genetic variation and molecular characteristics. Methods During the influenza H1N1 virus surveillance in Quanzhou,specimens of throat swabs from the patients with influenza were collected, and detected by real-time RT-PCR. Viruses were isolated with MDCK cells and identified with serological test. Two influenza virus isolates were extracted, and their HA and NA genes were amplified by RT-PCR. The purified PCR products were sequenced. The data obtained were analyzed with the software DNAMAN. Results Of 1020, influenza H1N1 virus RNA was detected in 200 specimens, seasonal influenza virus RNA was detected in 70 specimens. A total of 29 influenza A H1N1 virus strains were isolated. The nucleotide homology in the HA gene was highly homologous with that of pandemic influenza virus in North America. The amino acids sequences deduced from the nucleotide sequences in HA region of the isolated strain had 22 variations compared with A/Brisbane/59/2007 vaccine strain recommend by WHO,the characteristics of α2,6 sialic acid receptor binding remained. The analysis of amino acids sequences of NA indicated that this virus possessed Oseltamivir sensitivity. Conclusion The causative influenza H1N1 strains in Quanzhou is highly homologous with that of pandemic influenza in North America, and it is antigenically and genetically different from the vaccine strain.

Objective To investigate the dynamic expressions of epidermal growth factor receptor (EGFR) in mice with liver fibrosis and the effect of bone morphogenetic protein-7 (BMP-7) intervention on the expression of EGFR,and to explore a new therapy target for fibrosis.Methods A total of 30 healthy male ICR mice were randomly divided into three groups:6 mice in control group,18 mice in hepatic fibrosis group and 6 mice in BMP-7 intervention group.The model of mice with liver fibrosis was established by subcutaneous injection of carbon tetrachloride (CCl4) for 12 weeks.After administration of CCl4 for 8 weeks,human recombinant BMP-7 was given into mice in intervention group by intraperitoneal injection for 4 weeks.Hematoxylin-Eosin and Masson staining of liver tissues were employed to observe the pathological changes,and the semi-quantitative analysis of liver fibrosis was performed.Blood withdrawn from inferior vena cava was detected for levels of alanine aminotransferase (ALT),aspartate aminotransferase (AST) and albumin (Alb).The expressions of transforming growth factor-β1 (TGF-β1)mRNA and TGF-β1,EGFR,phosphorylation EGFR (pEGFR) protein in each group were detected using quantitative real time polymerase chain reaction and Western blot.Measurement date was compared using analysis of variance and Pearson correlation analysis.Results The model of mice with liver fibrosis was successfully established.In model group,the serum levels of ALT and AST increased,while the level of Alb decreased gradually.All these biochemical index improved after intervention of BMP-7 (ALT:[153.9±18.1] U/L vs [191.3±24.5] U/L;AST:[177.8±19.2] U/L vs [206.6±25.0] U/L;Alb:[25.4±0.9] g/L vs [22.2±1.2] g/L; all P＜0.05).With the progress of fibrosis,TGF-β1,EGFR and pEGFR protein expressions increased gradually in model group and reached a peak at week 12,which was significantly different compared to the control group (all P＜0.05).In BMP-7 intervention group,the expressions of the three proteins decreased significantly compared to model group (TGF-β1:0.256 ± 0.006 vs 0.287±0.014,EGFR:1.061±0.017 vs 1.094±0.014,pEGFR:0.855±0.053 vs 1.007±0.063;all P＜0.05).Additionally,linear correlation analysis showed that expressions of both EGFR and pEGFR proteins were positively correlated with TGF-β1 protein (rs =0.895 and 0.859,respectively; both P＜0.05).Conclusions BMP-7 can suppress the pathogenesis of mouse liver fibrosis.The mechanism may rely on the regulation of EGFR and TGF-β1 expressions.

Objective To study the expression of the three autophagy-associated proteins, BECN1, LC3 and p62, after the injury of the skeletal muscle of rats and to explore its application in differentiation between antemortem and postmortem injury. Methods The 72 healthy Sprague-Dawley rats were randomly divided into the undamaged control group, the antemortem injury group （0.5 h, 1 h, 2 h, 4 h, 8 h, 16 h and 24 h） and postmortem injury group （0.5 h, 1 h, 2 h and 4 h）. A model of the injured right hind limb of rats was constructed. The expressions of the autophagy-associated proteins, BECN1, LC3-2/LC3-1 and p62, in the control group, the antemortem injury group and postmortem injury group were detected by Western blotting method. The data were respectively centralized and standardized and the orthogonal partial least square-discrimination analysis （OPLS-DA） identification model of antemortem and postmortem injury groups was constructed. Results The expression of BECN1, p62 protein and LC3-2/LC3-1 after the injury of the skeletal muscle of the rats showed different degrees of changes, but the differences among the 3 groups had no statistical significance. Antemortem and postmortem injury groups can be distinguished by centralizing and standardizing the expression levels of autophagy protein BECN1 and the ratio of LC3-2/LC3-1. The principal components extracted from OPLS-DA model of antemortem injury and postmortem injury had a relatively good interpretation of the model （R_x²=0.563, R_y²=0.439）, but it were less predictive （Q²=0.366）. Conclusion The expression of BECN1 and the ratio of LC3-2/LC3-1 in injured local tissue of the rat skeletal muscle can be used for the differentiation of antemortem injury group and postmortem injury group.
Animals ; Autophagy ; Muscle, Skeletal ; Postmortem Changes ; Proteins/metabolism* ; Rats ; Rats, Sprague-Dawley

Objective To establish a method for determination of escitalopram in biological samples by ultrasound-assisted ionic liquid-dispersive liquid-liquid microextraction combined with gas chromatography-tandem mass spectrometry （GC-MS/MS） and provide evidences for forensic determination of cases related to escitalopram. Methods The 1-hexyl-3-methylimidazolium hexafluorophosphate （[C₆MIM][PF₆]） was selected as an extract solvent to process biological samples. Ultrasound-assisted extraction was used on the samples. Then the samples were detected by GC-MS/MS. Results The linear range of escitalopram in blood and liver were 5.56-1 111.10 ng/mL and 0.025-5.00 mg/g, respectively. The correlation coefficient （r） were greater than 0.999, limit of detection （LOD） were 4.00 ng/mL and 2.00 μg/g, limit of quantitation （LOQ） were 14.00 ng/mL and 6.00 μg/g, respectively. The extraction recovery rates were all greater than 50%, the interday and intraday precision were less than 20%. Escitalopram was detected in blood and liver samples from the actual poisoning case by this method with a content of 1.26 μg/mL and 0.44 mg/g, respectively. Conclusion The ultrasound-assisted ionic liquid-dispersive liquid-liquid microextraction combined with GC-MS/MS is environment friendly, rapid, has good enriching effect and consumes less organic solvent and can be used for forensic determination of escitalopram related cases.
Citalopram ; Gas Chromatography-Mass Spectrometry ; Limit of Detection ; Liquid Phase Microextraction ; Tandem Mass Spectrometry

OBJECTIVES: To explore the mRNA differential expressions and the sequential change pattern in acute myocardial infarction (AMI) mice. METHODS: The AMI mice relevant dataset GSE4648 was downloaded from Gene Expression Omnibus (GEO). In the dataset, 6 left ventricular myocardial tissue samples were selected at 0.25, 1, 4, 12, 24 and 48 h after operation in AMI group and sham control group, and 6 left ventricular myocardial tissue samples were selected in blank control group, a total of 78 samples were analyzed. Differentially expressed genes (DEGs) were analyzed by R/Bioconductor package limma, functional pathway enrichment analysis was performed by clusterProfiler, protein-protein interaction (PPI) network was constructed by STRING database and Cytoscape software, the key genes were identified by Degree topological algorithm, cluster sequential changes on DEGs were analyzed by Mfuzz. RESULTS: A total of 1 320 DEGs were associated with the development of AMI. Functional enrichment results included cellular catabolic process, regulation of inflammatory response, development of muscle system and vasculature system, cell adhesion and signaling pathways mainly enriched in mitogen-activated protein kinase (MAPK) signaling pathway. The key genes of AMI included MYL7, TSC22D2, HSPA1A, BTG2, NR4A1, RYR2 were up-regulated or down-regulated at 0.25-48 h after the occurrence of AMI. CONCLUSIONS The functional signaling pathway of DEGs and the sequential expression of key genes in AMI may provide a reference for the forensic identification of AMI.
Animals ; Computational Biology/methods* ; Gene Expression Profiling/methods* ; Mice ; Mitogen-Activated Protein Kinases/metabolism* ; Myocardial Infarction/metabolism* ; RNA, Messenger ; Ryanodine Receptor Calcium Release Channel/metabolism* ; Transcriptome

Objective To study the DNA methylation of nucleated cells in peripheral blood of patients died from anaphylactic shock caused by cephalosporin drugs and to provide a new research direction and basis for the forensic diagnosis of shock caused by drug hypersensitiveness. Methods Methylation microarray was used to detect DNA methylation of nucleated cells in peripheral blood of patients died from anaphylactic shock caused by cephalosporin drugs and normal subjects. Sequencing data and chip data were analyzed for differences in DNA methylation using R language methylkit, ChAMP package. Random forest algorithm was used to evaluate the importance of the DNA methylation differential sites. Results Differential sites of DNA methylation highly associated with anaphylaxis caused by cephalosporin drugs were obtained at loci such as ETS1, PRR23B and GNAS. Conclusion Cephalosporin allergy is associated with DNA methylation, and DNA methylation may be a new strategy for forensic identification of anaphylactic shock and death.
Anaphylaxis/genetics* ; DNA Methylation ; Forensic Medicine ; Humans