Relationship between HBV genotype,PreS/S gene mutation and immunoprophylaxis failure to prevent HBV mother-to-child transmission

AIM：To investigate the relationship between hepatitis B virus (HBV) genotype, PreS/S gene mutation and immunoprophylaxis failure to prevent HBV mother-to-child transmission.
METHODS：Pregnant women with positive HBV surface antigen (HBsAg) and HBV DNA≥1×1010 IU/L were divided into case group (15 cases) and control group (45 cases) according to their neonates with immunoprophylaxis failure or not. The genotypes of HBV and the mutation rate and mutational hot spots in PreS/S gene were detected by PCR amplification technique in the two groups.
RESULTS：(1) Genotypes B and C of HBV were detected in both case and control groups, and the majority of HBV genotype was B in the two groups. Genotype distribution difference between case and control groups was not statistically significant (P>0.05). (2) There was no significant difference in the mutation rate of PreS/S gene between case and control groups (P>0.05). The mutation rates of PreS/S gene between genotypes B and C were significantly different (P<0.05), but when the HBV genotype was the same, the mutation rate of PreS/S gene had no significant difference between case and control groups. Homology tree model based on PreS2/S gene formed genotype B and genotype C clusters, and in each cluster, the sequences of case and control groups did not formed smaller different clusters further. (3) 529G-A, 530A-G, 826A-G1 and 166het-dupC were hot spots of mutation in PreS2/S gene and were found in 4 cases in case group, respectively. A530T (1 case), A530G (2 cases), T531C (3 cases) were found in control group.
CONCLUSION：(1) The mutation rates of PreS/S gene are different in various genotypes. (2) The mutation in PreS/S gene of HBV is prevalent, but not all of the mutations are related to immunoprophylaxis failure to prevent HBV mother-to-child transmission. To find mutational hot spots which are related to immunoprophylaxis failure is more important.     

Effects of ethanol on HBV gene mutations in patients with chronic hepatitis B by gene chip

AIM: To study ethanol influence on gene mutations of HBV DNA and to offer testimony for clinical diagnosis and treatment of chronic hepatitis B. METHODS: 85 patients with chronic hepatitis B were divided into alcoholic group and non-alcoholic group. Gene chip technique was used to detect gene mutations located in Pre-C nt G1896A and nt A1814C, basal core promoter (BCP) nt A1762T and nt G1764A, P nt C528A and nt T552C. RESULTS: The mutation frequency on BCP nt A1762T and nt G1764A in alcoholic group was significantly higher than that in non-alcoholic group (P<0.05). No difference of mutation frequency on pre-c nt G1896A nt A1814C and P nt C528A nt T552C between alcoholic and non-alcoholic group was observed (P>0.05). CONCLUSION: Ethanol stimulates HBV gene mutations on BCP nt A1762T and nt G1764A, enhances HBV DNA replication and gene expression, deteriorates the state of the illness.     

Cloning of human β thalassemic mutation β IVS II654(C→T) and establishment of its mammalian expression system

AIM: To clone human β-globin gene carrying a thalassemic mutation IVS II654(C→T) and establish a eukaryotic expression system for high-level expression of human β IVS II654 gene in mouse erythroleukaemia(MEL) cells. METHODS: The fragments of human β 654 gene isolated from the β thalassemia patients homozygous for the β 654 mutation were amplified by PCR, and cloned to plasmid pBGT51. Then, the human β LCR and β 654 gene were subcloned from plasmid pBGT51 to the stable mammalian expression vector pcDNA3. 1+ together, and the MEL cells were transfected with this vector using commercially available cationic lipid FuGENE6. The MEL cells were induced for further maturation by DMSO and the expression of human β 654 gene in the MEL cells was identified by RT-PCR. RESULTS: A mammalian expression system of human β thalassemic mutation βIVS II654(C→T) was established. CONCLUSION: The level and the reliability of expression of human β 654 gene in the MEL cells in vitro are similar to that in vivo in human body. This may be a valuable gene therapy model for human β thalassemic mutation βIVS II654(C→T).     

SNPs analysis of the METTL4 gene in high myopia groups

AIM: To investigate the single nucleotide polymorphisms (SNPs) in the METTL4 gene which was mapped to 18p11.31, and the relationship between the SNPs and high myopia. METHODS: Genomic DNA was collected from 71 control subjects and 177 individuals with high myopia. Among them, there were 59 autosomal dominant high myopia probands (AD group), 46 autosomal recessive probands (AR group) and 72 patients non-transmitted (SF group). The exons of METTL4 gene were analyzed by polymerase chain reaction, heteroduplex-single strand conformation polymorphism (HA-SSCP) and sequencing. RESULTS: There were 2 SNPs of METTL4 gene in high myopia individuals and control subjects: SNP7438A→C, Glu230Asp, which hadn't been reported in GenBank;and SNP131C→A, Gln310Lys. SNP7438A→C genotypes between controls and high myopia groups were not different. SNP131C→A genotypes between controls and AR or SF groups were not different, while SNP131C→A genotypes showed a significant difference between AD group and control subjects. CONCLUSION: In METTL4 gene, SNP7438A→C is not responsible for high myopia. Further studies are needed to confirm whether SNP131C→A is responsible for autosomal dominant high myopia.     

Epidermal growth factor contents in human milk,cow's milk and cow's-milk-based infant formulas

AIM: To determine EGF contents in human milk, frech cow's milk and cow's milk-based infant formulas and the relationship between EGF content of human milk and neonatal maturity. METHODS: EGF contents in 57 human colostrum from mothers delivering prematurely and at term, 4 different fresh cow's milk and 8 different cow's-milk-based infant formulas with hydrolyzed and non-hydrolyzed proteins were determined by radioimmunoassay (RIA). RESULTS: Human milk from mothers of premature infants had higher EGF content compared to that from mothers of term infants . There was a negative correlation between EGF content of human milk and gestational age, birth weight of neonates. The values in fresh cow's milk were similar to that in human term milk. The contents in non-hydrolyzed protein formulas were much lower than that in human milk and fresh cow's milk. No immunoreactive EGF was detected in all hydrolyzed protein formulas. CONCLUSION: The occurrence of high EGF concentration in premature milk may represent a maternal compensatory mechanism to accelerate the growth and maturation in immature infants. Lack of EGF in formulas suggests that they may not suitable for those newborns with immature or damaged gastrointestinal tract.     

Study on mutation of mitochondrial gene from rat breast cancer

AIM: To know the variations of the cytochrome b gene in cancer tissue, paracarinoma tissue and normal tissue and to inquire into the relationship between mutations of mitochondrial genome and carcinogenesis. METHODS: Cellular total DNA was extracted.The cytochrome b genes of three tissues were amplifyed with polymerase chain reaction(PCR). PCR products were analysed by DNA auto-sequencing method. RESULTS: The cytochrome b gene of cancer tissue had the C to G mutation at nt 14931, the C to G mutation at nt 15004 and the T to C mutation at nt15435,respectively. The cytochrome b gene of paracarinoma tissue had the A to C mutation at nt 15436. The cytochrome b gene of normal tissue had not mutation. CONCLUSION:Mitochondrial DNA mutations could be the endogenous factors that induce nuclear genome mutation. It could promoto carcinogenesis. The paracarinoma tissue was abnormal in DNA molecular level.     

Relationship between matrix metalloproteinase 2-735C→T genetic polymorphism in promoter region and coronary atherosclerosis

AIM: To investigate the relationship between matrix metalloproteinase 2 ( MMP-2 )-735C→T polymorphism in the promoter region and coronary atherosclerosis (CAS) in Han population of China. METHODS: This study was conducted with a CAS group including 309 patients confirmed by angiography and 311 control healthy subjects. Genotype of -735C→T functional promoter polymorphism of the MMP-2 gene was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The relationship between the polymorphism in MMP-2 gene and CAS was analyzed. RESULTS: The frequency of CC genotype (86.1%) in CAS group was significantly higher than that in control group (79.7%), but the frequency of CT+TT genotype (13.9%) in CAS group was significantly lower than that in control group (20.3%). The statistical difference between CAS group and controls was significant(χ²=4.398,P<0.05). The frequency of -735C in CAS group (92.6%) was higher than that in control group (89.1%) and the frequency of -735T in CAS group (7.4%) was lower than that in control group (10.9%), with the statistical significant difference (χ²=4.521, P<0.05). The degree of stenosis in coronary artery did not significantly relate to the MMP-2 gene -735C→T polymorphism in the promoter region. CONCLUSION: The genetic polymorphism in MMP-2 promoter region (-735C→T) is associated with the susceptibility to CAS in Han population of China. CC genotype and C allele may be a genetic marker. The -735C→T polymorphism may be useful as a predictor of CAS.     

Changes of DNA methylation at CpG sites in promoter region of TNF-α gene in DENV2-infected peripheral blood mononuclear cells

AIM：To examine DNA methylation at CpG sites in the promoter region of tumor necrosis factor-alpha (TNF-α) gene in dengue virus type 2 (DENV2)-infected peripheral blood mononuclear cells (PBMC).
METHODS：DNA methylation in the promoter region of TNF-α gene was measured by bisulfite sequencing PCR.
RESULTS：The promoter region of TNF-α gene was from -294 bp to +58 bp, including 11 CpG sites. The PCR products identified by aga-rose gel electrophoresis were consistent with the theoretical size. Two sites were methylated at 0 h and 6 h and 6 sites were methylated at 12 h in TNF-α gene promoter region in DENV2-infected PBMC. The average methylation rates were 103%, 121% and 255% at 0 h, 6 h and 12 h, respectively. Significant differences between 0 h and 12 h and between 6 h and 12 h were observed.
CONCLUSION：The DNA methylation in the promoter region of TNF-α gene is increased in DENV2-infected PBMCs.     

Role of renal preS1/S2-Ag and other HBV antigen determination in diagnosis of HBV-associated glomerulonephritis

AIM：To detect the expression of preS1/S2 antigen (preS1/S2-Ag) and other antigens of hepatitis B virus (HBV) in renal tissues of patients with HBV-associated glomerulonephritis (HBV-GN), and to analyze their roles in the diagnosis of HBV-GN.
METHODS：Patients hospitalized in our department from January in 2003 to January in 2013 were retrospectively studied. A total of 49 patients with positive HBV surface antigen (HBsAg) serology, clinical manifestations of hematuria and/or proteinuria, and pathological diagnosis of glomerulonephritis, without systemic lupus erythematosus, anaphylactic purpura, diabetes or hepatitis C, were selected. PreS1/S2-Ag, HBV e antigen (HBeAg), HBsAg and HBV core antigen (HBcAg) in the renal tissues were examined. Five cases of glomerular minimal-change disease (MCD) with negative HBsAg and 5 cases of non-glomerulonephritis with positive HBsAg served as controls. RESULTS：The positive rates of preS1/S2-Ag, HBeAg, HBsAg and HBcAg in the renal tissues from the 49 patients of glomerulonephritis with HBV infection were 32.7% (16 cases), 38.8% (19 cases), 14.3% (7 cases) and 46.9% (23 cases), respectively. Total antigen positive rate was 70.2% (36 cases). The expression of preS1/S2-Ag was located in the cytoplasm of renal tubular epithelial cells, glomerular epithelial cells, endothelial cells and mesangial cells, and positively correlated with the expression of HBcAg (r=0.459, P<001). The 4 antigens were not detected in the 5 cases of HBsAg-negative patients with glomerular MCD. In the 5 cases of HBsAg-positive patients with non-glomerulonephritis, there were 2 cases expressing HBeAg and 1 case expressing HBcAg, but no cases expressing preS1/S2-Ag or HBsAg. CONCLUSION：The expression of preS1/S2-Ag in renal tissues suggests that HBV may invade the cells of renal tissue. Combined detection of the 4 antigens could elevate the rate of diagnosis of HBV-GN.     

蔷薇属 3 个野生种中45S rDNA 和5S rDNA 的物理定位

 采用双色荧光原位杂交（FISH）技术对3 个二倍体蔷薇野生种：多苞蔷薇（Rosa multibracteata Helm. et Wils.）、川滇蔷薇（R. soulieana Crép.）和金樱子（R. laevigata Michx.）的体细胞中期染色体进行 了45S rDNA 和5S rDNA 物理定位。结果表明：45S rDNA 在这3 种蔷薇的染色体上的数量和分布模式较 一致,都有1 对位点,均位于一对亚中部着丝点异形同源染色体的短臂上。5S rDNA 在多苞蔷薇上有1 对位点,在川滇蔷薇和金樱子上各有2 对位点,都分布于染色体长臂的近着丝点处。这3 种蔷薇各自的 rDNA 位点数量、所在染色体和信号强弱有明显差异。研究结果为识别3 种蔷薇各自的染色体提供了明确 有效的分子细胞遗传学标记。     

S-gene polymorphism of hepatitis B virus in Hakka area of Guangdong

AIM: To examine the surface (S) gene variability of hepatitis B virus (HBV) in Hakka area of Guangdong province in China. METHODS: The S genes from 40 HBsAg and HBV DNA positive patients in Hakka area of Guangdong province were amplified and sequenced directly for its molecular characterization, then these sequences were compared with the consensus sequences. RESULTS: In all 40 patients, 36 patients belongs to subtype adw and 4 patients to subtype adr. In 36 patients of subtype adw, it was found that at the position 131 all amino acid residues were Thr not Asn, at the position 127, 133, 134 and 143 all were also substituted by other amino acids. In 4 patients of subtype adr, amino acid residues were found to be highly conservative. CONCLUSION: The different HBV subtypes have different patterns of geographic distribution. Subtype adw is the major type in Hakka area of Guangdong. S gene sequence of HBV has its variability, especially in subtype adw.     

Changes of Kupffer cells during tree shrew chronically infected with hepatitis B virus

AIM：To explore the changes and significance of Kupffer cells in the process of tree shrew chronically infected with hepatitis B virus (HBV). METHODS：The animals were divided into 3 groups. Group A consists of 6 tree shrews that were identified as persistently infected with HBV; group B consists of 3 tree shrews that were suspected as persistently infected with HBV; group C consists of 4 tree shrews that were not inoculated with HBV and were applied as normal controls. Liver biopsies were collected regularly from all animals, and the Kupffer cells were isolated, purified and primarily cultured. The techniques of flow cytometry, immunohistochemistry, lysosomal fluorescent probe staining and real-time RT-PCR were applied to determine the number and function of these Kupffer cells. RESULTS：The result showed that the count and proportion of CD163+ cells in group A were significantly higher than those in group B and group C (P<0.05). Meanwhile, the fluorescence intensity levels of lysosomal, the number of lysozyme-positive cells and the mRNA expression level of TNF-α in the Kupffer cells in group A were significantly lower than those in group B and group C (P<0.05). CONCLUSION：Kupffer cells may play a regulatory role during host’s chronic HBV infection.     

番茄抗黄化曲叶病毒基因Ty-1的双重SNP标记的开发

本试验旨在开发一种新的设计共显性SNP标记的方法,以提高SNP标记检验的效率,并将其成功应用于番茄抗黄化曲叶病毒基因Ty-1的SNP标记开发中,提高了种质资源鉴定和育种分离世代材料筛选的效率。通过对抗病基因Ty-1和等位感病基因ty-1的cDNA序列进行比对,挑选了两个稳定扩增的多元非同义突变位点,分别用来设计Ty-1和ty-1的SNP标记NL3和NL2,将两个标记进行混合得到双重SNP标记NL2-3。通过用NL2-3检验已知的抗病和感病番茄材料,验证了其多态性和稳定性。并用NL2-3对抗感病材料的F₃代杂交分离群体和普通自交系进行了Ty-1的筛选,并经过田间观察进一步验证了该标记的可靠性。此方法开发得到的双重SNP标记只需要进行一次PCR反应和一次凝胶电泳就可以区分纯合抗、杂合抗、纯合感3种基因型,提高了育种选择的效率。     

辣椒恢复基因连锁标记的适用性评价与应用

为了更准确、高效地利用分子标记辅助选择技术进行辣椒细胞质雄性不育育性基因的选择,本试验利用36份已知基因型(Rf Rf或rfrf)的辣椒自交系对11对已报道的辣椒恢复基因连锁标记进行适用性检验。结果表明,显性标记CRF3S1S和CRF-SCAR的适用性最广,其准确率分别为91.67%、88.89%;在6对共显性标记中Ca Rf-FL-M2的适用性最广,其准确率为80.56%。育种实际应用中可以利用标记CRF3S1S或CRF-SCAR对辣椒材料进行初次鉴定,对其中含有Rf基因的辣椒材料利用标记Ca Rf-FL-M2进行再次鉴定。     

西瓜二倍体及同源多倍体遗传差异的AFLP分析

试验以蜜枚和JM西瓜品种的纯合二倍体及其人工诱导的同源四倍体、三倍体为材料,对不同倍性西瓜AFLP标记的分子遗传变异进行了比较。结果表明:共筛选48对引物组合,获得4800～5000条位于50～600bp的片段,大部分引物组合扩增出的不同倍性之间的AFLP条带没有明显差异;蜜枚品种西瓜具有多态性的条带有3条,其中1条为四倍体特有带,1条为三倍体特有带,1条为二倍体特有带;JM西瓜具有多态性的条带有7条,3条为JM4x特有带,2条为JM3x特有带,2条为JM2x特有带,不同倍性西瓜之间多态性较低。同源四倍体和相应的二倍体相比,有特异性片段的消失或增加,也有扩增出双亲皆有或皆无而三倍体F1消失或增加的DNA片段。     

Role of calcineurin in cTnI Asp128Tyr mutation-induced myocardial hypertrophy

AIM：To investigate the role of calcineurin (CaN) signaling pathway in myocardial hypertrophy induced by cardiac troponin I (cTnI) Asp128Tyr mutation. METHODS：The adenovirus containing cTnI Asp128Tyr mutation was transfected into the cultured neonatal rat cardiomyocytes. Cardiac hypertrophy was evaluated by determining the mRNA expression of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) in the transfected cells. The effects of CaN inhibitors cyclosporine A (CsA) and FK506 on cardiac hypertrophy were also observed. The mRNA expression of ANF, BNP and CaN was measured by real-time quantitative PCR. The activity of CaN was also detected. RESULTS：Compared with blank control group, negative control virus group or virus containing wildtype cTnI gene group, the mRNA expression of ANF and BNP in mutation group (transfected with the adenovirus containing cTnI Asp128Tyr mutation) significantly increased, and decreased after treatment with CsA or FK506. The mRNA expression and activity of CaN increased in the mutation group compared with blank control group, negative control virus group or virus containing wild-type cTnI gene group. At the same time, the mRNA expression and the activity of CaN decreased after treatment with CsA or FK506. CONCLUSION：Calcineurin signaling pathway is involved in the cardiac hypertrophy induced by cTnI Asp128Tyr mutation.     

Single nucleotide polymorphism and mutation of miRNA binding sites at BCL11B-3'UTR in healthy individuals and patients with T-ALL

AIM:To analyze the microRNA (miRNA) binding sites at B-cell lymphoma/leukemia 11B gene 3'-untranslated region (BCL11B-3'UTR), and to establish the method of identifying the single nucleotide polymorphism (SNP) and mutation of these miRNA binding sites in healthy individuals and patients with T-cell acute lymphoid leukemia (T-ALL). METHODS:TargetScan was used for screening and predicting the miRNA binding sites at BCL11B-3'UTR. PCR and sequencing were used to identify the miRNA binding sites at BCL11B-3'UTR. The polymorphisms in DNA sample of peripheral blood mononuclear cells from 20 healthy individuals and 21 patients with T-ALL at this region were analyzed. RESULTS:Twenty-four highly conserved miRNA binding sites were screened according to the criteria of context ++ score and seed match categories. The nucleotide exchange (T>C) located at site 2 402 of BCL11B-3'UTR was detected in one case out of 21 cases of T-ALL samples, which had been registered as SNP (rs184678181) in dbSNP. No polymorphism or mutation in BCL11B-3'UTR miRNA binding sites was identified in the samples from the healthy individuals. CONCLUSION:Polymorphism or mutation in BCL11B-3'UTR is rare in healthy individuals and T-ALL patients. To our best knowledge, it is the first identification of BCL11B-3'UTR SNP (T>C at site 2 402), which is involved in hsa-miR-6814-5p binding site in a patient with T-ALL. Further investigation will focus on its effect for BCL11B expression regulation.     

20个苹果品种的S基因型鉴定

以‘富士’、‘华瑞’、‘华硕’和‘华星’等20个苹果品种为材料,利用S等位基因高度保守氨基酸序列FTQQYQ和anti-1/MIWPNV设计的S基因通用引物,以及S等位基因多态性序列设计的19对特异引物,PCR扩增、测序以鉴定20个品种的S基因型;并用‘华瑞’和‘华硕’分别与‘美八’、‘锦秀红’、‘华冠’和‘富士’进行授粉试验验证S基因型的准确性。PCR结果表明:通用引物扩增S等位基因时,仅‘富士’、‘华瑞’、‘华硕’、‘华星’、‘美八’和‘红脆宝’6个品种有效地扩增出2条特异的S等位基因条带,其S基因型有S1S_9、S_9S_(24)、S_5S_9和S_5S_(24)等4种;19对特异引物扩增S等位基因时,‘华帅’等14个品种扩增得到2条特异性条带,S基因型有S_(10)S_(19)、s_2s_3、s_2S_5、s_3S_(10)、s_2S_9、S_5S_(24)、S_9S_(10)、s_3S_(10)和S_5S_9等9种。因此,20个苹果品种的S基因型分别为:‘富士’S1S_9,‘华瑞’和‘华硕’S_9S_(24),‘华星’、‘美八’和‘红珍珠’S_5S_9,‘红脆宝’、‘华玉’和‘99-1-29’S_5S_(24),‘华帅’S_(10)S_(19),‘金玉’s_2s_3,‘早红’、‘华美’和‘嘎拉’s_2S_5,‘Seokwang’s_3S_(10),‘锦秀红’、‘蜜玉’和‘华冠’s_2S_9,‘绿佳’S_9S_(10),‘信浓红’s_3S_(10)。2015和2016年‘华瑞’与‘华硕’的正反交组合坐果率较低(低于15.52%);而‘华瑞’和‘华硕’分别与‘美八’、‘锦秀红’和‘华冠’、‘富士’品种的正反交组合坐果率较高(高于46.30%)。因此,本试验中相同S基因型的授粉组合其坐果率较低,不同S基因型的授粉组合其坐果率较高,授粉试验支持S基因型鉴定结果。     

不同产地中国李资源遗传多样性SSR分析

利用均匀分布在8个染色体连锁群上的16对SSR引物对来自3类产区的24份中国李品种的遗传多样性进行分析,结果表明：各引物的多态信息含量（PIC）在0.547 ~ 0.783间变化,其中引物CPSCT005最低,引物CPSCT022最高。不同引物间有效等位基因数（Ne）、Nei’s基因多样性（H）和Shannon’s指数（I）分析均存在显著差异,且均为引物CPSCT039最高,引物CPSCT031最低。3类产区所有引物分析表明,Nei’s基因多样性（H）、Shannon’s指数（I）和有效等位基因数（Ne）的关系是：南方品种和北方品种相差不大,均大于国外品种,且南方品种与国外品种先聚到一类。16对SSR引物总共扩出条带86条,其中多态性条带81条,多态性比率为94.19%,平均每对引物扩增位点5.38个。品种聚类分析表明,在遗传距离0.35处,24份中国李材料可分为2大类,大部分北方品种聚为一类,南方品种和国外品种聚为一类,从分子水平支持了以前提出的国外品种起源于中国的说法。