RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in Human Enterovirus A–D from Clinical Specimens

Objective To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses(HEVs)from clinical samples and to contribute to etiological surveillance of HEV-related diseases.Methods A panel of RT-nPCR assays,consisting of published combined primer pairs for VP1 genes of HEV A–C and in-house designed primers for HEV-D,was established in this study.The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID50 perμL and copies perμL,and the newly established methods were tested in clinical specimens collected in recent years.Results The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID50 perμL and 10 virus copies perμL,and for the complete VP1 gene was 1 CCID50 perμL and 100 virus copies perμL,using serially-diluted virus stocks of five serotypes.As a proof-of-concept,25 serotypes were identified and complete VP1 sequences of 23 serotypes were obtained by this system among 858 clinical specimens positive for HEVs during the past eight surveillance seasons.Conclusion This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A–D,providing rapid,sensitive,and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.

The effects of microRNA-34a regulating Notch-1/NF-κB signaling pathway on lipopolysaccharide-induced human umbilical vein endothelial cells

BACKGROUND: Notch-1/NF-κB signaling plays a key role in the cecal ligation and puncture(CLP)-induced sepsis. This study aims to investigate the intervention effects of microRNA-34a(miR-34a) lentivirus regulating Notch-1/NF-κB signaling pathway on lipopolysaccharide(LPS)-induced human umbilical vein endothelial cells(HUVEC).METHODS: HUVEC were divided into four groups as the following: they were infected with negative control lentivirus(NC group) or miR-34a lentivirus(OE group); LPS(1 g/mL) was added on the third day on the basis of NC group and OE group for 24 hours(NC+LPS group or OE+LPS group). The levels of TNF-α, IL-1β, IL-6, and IL-10 in the cell supernatants, and the mRNA and protein expression of Notch-1 and NF-κB in the HUVEC were evaluated.RESULTS: After 24 hours, the levels of TNF-α, IL-1β, IL-6 in the cell supernatants and the protein expression of NF-κB from NC+LPS group were significantly higher than those of NC group, but IL-10 level and the protein expression of Notch-1 in NC+LPS group were the opposite. After intervention of miR-34a lentivirus, the cell supernatants TNF-α and the protein expression of NF-κB in OE+LPS group after 24 hours markedly decreased compared to NC+LPS group. While the cell supernatants IL-1β and IL-6 and the mRNA expression of NF-κB slightly decreased in OE+LPS group, IL-10 and the mRNA and protein expression of Notch-1 were the opposite.CONCLUSION: miR-34a regulating Notch-1/NF-κB signaling pathway can reduce the HUVEC damage caused by LPS stimulation.

Metallothionein 1 Isoform Gene Expression Induced by Cadmium in Human Peripheral Blood Lymphocytes

Objective To study the gene expression of metallothionein 1 （MT-1） isoforms in human peripheral blood lymphocytes （HPBLs）. Methods The expression of mRNA representing the seven active MT-I genes was determined in HPBLs by quantitative RT-PCR before and after exposure to cadmium. Results Basal expressions of MT-IX, and MT-1A in HPBLs were similar to expression of housekeeping gene. In contrast, the basal gene expressions of MT- 1 H, IF, 1E, and 1G were a little transcripts in human HPBLs. No signal was detected for MT-1B. There was a sex difference （P〈0.05）. in basal gene expression of MT-1E. The levels of gene expression of MT-1A, 1E, IF, 1G, 1H, and 1X increased, but the level of MT-1B did not increase after exposure to cadmium. Conclusions Gene expressions of MT-1 G, MT-1 H, MT-1 F, and MT-1X in HPBLs can be used as a potential biomarker of cadmium exposure.

Analysis of Single Nucleotide Polymorphism in Human Paraoxonase 1 Gene(Q192R) with Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

Introduction Single nucleotide polymorphisms （SNPs） are the most abundant DNA markers in the human genome occurring at a frequency of one in every 500--1000 nucleotides. A variety of methods have been used for the analysis of single nucleotide polymorphisms, including restriction fragment length polymorphism （RFLP）, direct sequencing by using laser-induced fluorescence detectionTM, fluorescence energy transfer, MALDI-TOF MS combined with primer extension or invasive cleavage, and fluorescence polarization. During the past two decades, mass spectrometry has become a very popular tool in the analysis of biomolecules and is perfectly suited to the analysis of single nucleotide polymorphisms （SNPs） due to its speed, low cost, and accuracy. In this work, we used MALDI TOF mass spectrometry to detect the fragments of restriction endonuclease hydrolysis of PCR products flanking a SNP located at paraoxonase 1（Q192R）. Compared with electrophoresis, this method requires less time of analysis and possess a higher accuracy.

Integrated analysis of human influenza A(H1N1)virus infectionrelated genes to construct a suitable diagnostic model

The genome characteristics and structural functions of coding proteins correlate with the genetic diversity of the H1N1 virus,which aids in the understanding of its underlying pathogenic mechanism.In this study,analyses of the characteristic of the H1N1 virus infection-related genes,their biological functions,and infection-related reversal drugs were performed.Additionally,we used multi-dimensional bioinformatics analysis to identify the key genes and then used these to construct a diagnostic model for the H1N1 virus infection.There was a total of 169 differently expressed genes in the samples between 21 h before infection and 77 h after infection.They were used during the protein-protein interaction(PPI)analysis,and we obtained a total of 1725 interacting genes.Then,we performed a weighted gene co-expression network analysis(WGCNA)on these genes,and we identified three modules that showed significant potential for the diagnosis of the H1N1 virus infection.These modules contained 60 genes,and they were used to construct this diagnostic model,which showed an effective prediction value.Besides,these 60 genes were involved in the biological functions of this infectious virus,like the cellular response to type I interferon and in the negative regulation of the viral life cycle.However,20 genes showed an upregulated expression as the infection progressed.Other 36 upregulated genes were used to examine the relationship between genes,human influenza A virus,and infection-related reversal drugs.This study revealed numerous important reversal drug molecules on the H1N1 virus.They included rimantadine,interferons,and shikimic acid.Our study provided a novel method to analyze the characteristic of different genes and explore their corresponding biological function during the infection caused by the H1N1 virus.This diagnostic model,which comprises 60 genes,shows that a significant predictive value can be the potential biomarker for the diagnosis of the H1N1 virus infection.

Effects of adenoviral vector-mediated transduction of human p53,B7-1 and GM-CSF genes on liver cancer cells

The potential efficacy and clinical feasibility of gene therapy for liver cancer were tested through therecombinant adenovirus-mediated (Ad-multigenes ) co-transfer of human wild-type p53, B7-l co-stimulation(CD8o) and granulocyte-macrophage colony-stimulating factor (GM-CSF) genes into human hepatocellular carcinoma cell lines. The treated cells underwent apoptosis with specific DNA fragmentation and became more sensitiveto cisplatin, a chemotherapeutic drug. Their growth was partly inhibited. Efficient proliferation and generation ofCTLs and cytokine production were induced in mixed lymphocytes through tumor cell reaction (MLTR) using peripheral blood T lymphocytes from donors as effector cells and Ad-multigenes or Ad-p53-transfected human hepatocellular carcinoma cells (HepG2 or BEL7402) as stimulator cells. Ad-multigenes-transfected rat carcinosarcomaWalker 256 cells were inoculated subcutaneously into normal rats. Fourteen days later, the activity of spleen cellsin rats inoculated with Ad-multigenes-transduced Walker 256 cells was higher than that in Ad-p53-transducedones. These findings suggest that adenovirus-mediated multigenes p53, B7-1 and GM-CSF can induce apoptosis ofliver cancer cells and initiate a potent antitumor immune response against them.

Inhibition of N-Myc down regulated gene 1 in in vitro cultured human glioblastoma cells

AIM: To study short ds RNA oligonucleotides(si RNA)as a potent tool for artificially modulating gene expression of N-Myc down regulated gene 1(NDRG1) gene induced under different physiological conditions(Normoxia and hypoxia) modulating NDRG1 transcription, m RNA stability and translation. METHODS: A cell line established from a patient with glioblastoma multiforme. Plasmid DNA for transfections was prepared with the Endofree Plasmid Maxi kit. From plates containing 5 × 107 cells, nuclear extracts were prepared according to previous protocols. The p SUPERNDRG1 vectors were designed, two sequences were selected from the human NDRG1 c DNA(5'-GCATTATTGGCATGGGAAC-3' and 5'-ATGCAGAGTAACGTGGAAG-3'. reverse transcription polymerase chain reaction was performed using primers designed using published information on β-actin and hypoxia-inducible factor(HIF)-1α m RNA sequences in Gen Bank. NDRG1 m RNA and protein level expression results under different conditions of hypoxia or reoxygenation were compared to aerobic control conditions using the Mann-Whitney U test. Reoxygenation values were also compared to the NDRG1 levels after 24 h of hypoxia(P < 0.05 was considered significant).RESULTS: si RNA- and iodoacetate(IAA)-mediated downregulation of NDRG1 m RNA and protein expression in vitro in human glioblastoma cell lines showed a nearly complete inhibition of NDRG1 expression when compared to the results obtained due to the inhibitory role of glycolysis inhibitor IAA. Hypoxia responsive elements bound by nuclear HIF-1 in human glioblastoma cells in vitro under different oxygenation conditions and the clearly enhanced binding of nuclear extracts from glioblastoma cell samples exposed to extreme hypoxic conditions confirmed the HIF-1 Western blotting results. CONCLUSION: NDRG1 represents an additional diagnostic marker for brain tumor detection, due to the role of hypoxia in regulating this gene, and it canrepresent a potential target for tumor treatment in human glioblastoma. The si RNA method can represent an elegant alternative to modulate the expression of the hypoxia induced NDRG1 gene and can help to monitor the development of the cancer disease treatment outcome through monitoring the expression of this gene in the patients undergoing the different therapeutic treatment alternatives available nowadays.

Down-regulation of Notch-1 gene expression inhibits growth of TJ-905 glioblastoma

Objective To study the inhibitory effect of siRNA on glioblastoma (GBM) Notch-1 gene expression in addition to the growth of TJ-905 glioblastoma. Methods Three small interference RNAs (siRNAs) targeting Notch1 gene,named siRNA1,siRNA2,siRNA3,synthesized chemically in vitro with gene bank BLAST. TJ-905 cells were transfected twice with the siRNA by using Oligofectamine

Anti-tumor effects induced by gene vaccines co-expressing truncated human prostate specific membrane antigen gene and mouse 4-1BBL

Objective To investigate the influence of m4-1BBL on anti-tumor effects induced by truncated human prostate specific membrane antigen ( tPSMA ) gene in mice. Methods A eukaryotic expression plasmid encoding tPSMA and m4-1BBL ( pDC316-tPSMA-IRES m4-1BBL) ,pDC316-tPSMA and pDC316 were constructed.

Variation and evolution of NP genes of human avian H_5N_1 virus strains

In order to reveal variation and revolution of NP genes of human avian H5 N1 influenza virus strains, the NP gene of a human avian H5 N1 influenza virus strain in Guangdong was sequenced and the global NP genes of strains were retrieved. The sequences were analyzed by DNAStar 5.0, and the evolutionary speed was studied with reference to the epidemiological data. It was found that NP genes of 45 strains during 1997-2006 were homologically classified into three groups： strains in 1997-1998, strains in 2004-2005 and strains from 2003 to 2006. There were 35 substitutions in NPs in all strains accounting for a ratio of 7.03% （35/498）. An additional glycoprotein domain （NGT430-432） was found in NP genes in the strains of 2003-2006, the mutation of N370S in GD-01-06 resulted in occurrence of one more glycoprotein domain （NES368-370）. In the synonymous variation, Ks values in NP were 2.03 × 10^-5-2.55 × 10^-5 Nt/d and K. values in NP were 1.58 × 10^-6-3.10 × 10^-6 Nt/d. There didn＇t exist obviously selective pressure. An additional glycoprotein domain in every strain of 2003-2006 and one more in strain GD-01-06 might change the antigenicity of human avian H5 N1 influenza virus. The variation on human avian H5 N1 influenza strains occurred frequently in the natural world, which would result in high probability of human-human transmission along with the natural evolution of the virus.

Progress of CACNA1S Gene and Hypokalemic Periodic Paralysis

CACNA1 S gene is the gene encoding L-type calcium channel αa-subunit. CACNA1 S gene mutations can cause hypokalemic periodic pa- ralysis （HOKPP）. The related research speculated that CACNA1 S gene was the candidate genes which affect meat quality traits. In the present ar- ticle, the biological characteristics of CACNA1 S gene, structure, genetic diseases and the research development were respectively reviewed so as to provide a reference for further research.

Inhibition of mouse acrosome reaction and sperm-zona pellucida binding by anti-human sperm membrane protein 1 antibody

Aim： To investigate the possible functions of human sperm membrane protein （hSMP-1） in the process of fertilization. Methods： A 576-bp cDNA fragment of HSD-1 gene coding for the extracellular domain of hSMP-1 was cloned and expressed. The localization of this protein on human and mouse sperm was determined by indirect immunofluorescent staining by using anti-recombinant hSMP-1 （anti-rhSMP-1） antibodies. Sperm acrosome reaction and spermzona pellucida （ZP） binding assay were carried out in 10-week-old BALB/c mice. Results： Recombinant hSMP-1 was successfully cloned and expressed. The expression of the native protein was limited on the acrosome of human and mouse sperm. Treatment of anti-rhSMP-1 antibodies significantly decreased the average number of sperms bound to each egg. Meanwhile, the percentage of acrosome reaction was decreased in comparison to pre-immune control after treatment with anti-rhSMP-1 （P 〈 0.05）. Conclusion： The results suggest that anti-rhSMP-1 antibody inhibited mouse acrosome reaction and sperm-ZP binding.

Cloning and characterization of a novel gene (C17orf25) from the deletion region on chromosome 17p13.3 in hepatocelular carcinoma

Using a combination of hybridization of PAC to a cDNA library and RACE technique, we isolated a novel cDNA, designated as C17orf25 (Chromosome 17 open rea(ling frame 25, previously named it HC71A), from the deletion region on chromosome 17p13.3. The cDNA encodes a protein of 313 amino acids with a calculated molecular mass of 34.8 kDa. C17orf25 is divided into 10 exons and 9 introns, spanning 23 kb of genomic DNA. Northern blot analysis showed that the mRNA expression of C17orf25 was decreased in hepatocellular carcinoma samples as compared to adjacent noncancerous liver tissues from the same patients. The transfection of C17or25 into the hepatocellular carcinoma cell SMMC7721 and overexpression could inhibit the cell growth. The above results indicate that C17orf25 is a novel human gene, and the cloning and preliminary characterization of C17orf25 is a prerequisite for further functional analysis of this novel gene in human hepatocellular carcinoma.

Cytochrome P450 2E1 genetic polymorphism and gastric cancer in Changle,Fujian Province

AIM: Genetic polymorphism in enzymes of carcinogen metabolism has been found to have the influence on the susceptibility to cancer. Cytochrome P450 2E1 (CYP2E1) is considered to play an important role in the metabolic activation of procarcinogens such as N-nitrosoamines and low molecular weight organic compounds. The purpose of this study is to determine whether CYP450 2E1 polymorphisms are associated with risks of gastric cancer. METHODS: We conducted a population based case-control study in Changle county, Fujian Province, a high-risk region of gastric cancer in China. Ninety-one incident gastric cancer patients and ninety-four healthy controls were included in our study. Datas including demographic characteristics, diet intake, and alcohol and tobacco consumption of individuals in our study were completed by a standardized questionnaire.PCR-RFLP revealed three genotypes:heterozygote (C1/C2) and two homozygotes (C1/C1 and C2/C2) in CYP2E1. RESULTS: The frequency of variant genotypes (C1/C2 and C2/C2) in gastric cancer cases and controls was 36.3% and 24.5%, respectively. The rare homozygous C2/C2 genotype was found in 6 individuals in gastric cancer group(6.6%), whereas there was only one in the control group (1.1%). However, there was no statistically significant difference between the two groups (two-tailed Fisher's exact test P=0.066). Individuals in gastric cancer group were more likely to carry genotype C1/C2 (odds ratio, OR=1.50) and C2/C2 (OR=7.34) than individuals in control group (chi(2) =4.597, for trend P=0.032). The frequencies of genotypes with the C2 allele (C1/C2 and C2/C2 genotypes) were compared with those of genotypes without C2 allele (C1/C1 genotype) among individuals in gastric cancer group and control group according to the pattern of gastric cancer risk factors. The results show that individuals who exposed to these gastric cancer risk factors and carry the C2 allele seemed to have a higher risk of developing gastric cancer. CONCLUSION: Polymorphism of CYP2E1 gene may have some effect in the development of gastric cancer in Changle county, Fujian Province.

Relationship between Microsatellite Alterations of RASSF1A Gene and Development of Cervical Carcinoma

Objective： To explore the relationship between microsatellite alterations of RASSFIA gene and the development of cervical carcinoma, and its relationship with HPV16 infection. Methods： Two sites of microsatellite polymorphism of RASSFIA gene were selected. Polymerase chain reaction （PCR） technique was used to detect LOH and MSI in 50 cases of cervical carcinoma and 40 cases of cervical intraepithelial neoplasia （CIN）, and to detect the infection state of HPV16. Results： At D3S1478 and D3S4604, the LOH rates of cervical carcinomas were 32.6% （14/43） and 48.9% （23/47）, the MSI rates were 14% （6/43） and 19.1% （9/47）, respectively. The LOH rates of CINs were 31.4% （11/35） and 39.5% （15/38）, the MSI rates were 11.4% （4/35） and 15.8% （6/38）, respectively. There were no significant differences between cervical carcinomas and CINs in respect to their positive rates of LOH and MSI at D3S1478 and D3S4604 （P〉0.05）. There were significant differences in LOH rates at D3S1478 and D3S4604 between the stage Ⅰ-Ⅱ and Ⅲ-Ⅳ cervical carcinomas and between the well/moderately differentiated cervical carcinomas and the poorly differentiated cervical carcinomas （P〈0.05）. The positive rates of LOH and MSI for CIN Ⅲ and noninvasive cervical carcinomas were higher than those in CIN Ⅰ-Ⅱ. The rates of infection of HPV16 in cervical cancer was obviously higher than that in CIN and in normal cervical tissues （P〈0.05）, and the incidence of LOH of RASSFIA gene was higher in HPV16（＋） than that in HPV16（-） （P〈0.05）. Conclusion： The RASSFIA gene change is a relatively late event in cervical carcinomas. The detection of LOH and MSI of RASSFIA gene might be helpful to the early diagnosis and the screening of cervical carcinoma. It might also be useful for predicting the prognosis of cervical carcinoma.

EFFECT OF MATRINE ON EXPRESSION OF HCCR1 AND HCCR2 PROTEINS IN CULTURAL HUMAN HEPATOCELLULAR CARCINOMAS CELLS

Objective： To explore the effects of matrine on HCCR1 and HCCR2 expression in cultural human hepatocellular carcinomas （HCC） cells at the level of gene and protein. Methods： Three methods, representational difference analysis （RDA） of cDNA, microarrays and fluorescence in situ hybridization （FISH） were used to detect levels of mRNA and protein expression of HCCR1 and HCCR-2 before and after treatment of matrine. Results： Matrine had inhibitory effect on the mRNA and protein expression of HCCR1 and HCCR2 in cultural HCC cells. Conclusion： Matrine has inhibitory effect on gene transcription, protein expression of HCCR 1 and HCCR2 in cultural HCC cells.

Construction and expression of an optimized, novel human immunodeficiency virus type-1 lentiviral vector containing green fluorescent protein

The human immunodeficiency virus （HIV） lentiviral vector is an ideal vector for gene therapy. In the present study, the wild-type HIV-1 genome was segregated into four plasmids, and an optimized novel HIV-1 lentiviral vector containing green fluorescent protein and vesicular stomatitis virus G pseudo-capsule was constructed. The plasmids were pHR-CMV-EGFP, pCMVΔ8.9, pRSV-Rev, pCMV-VSV-G. The four plasmid system was co-transfected into 293T cells, and green fluorescent protein expression was observed. The present study obtained lentiviral particles by high-speed centrifugation, and the lentiviral particle titer was 4 × 108 TU/mL after centrifugation. Thus, an optimized novel HIV-1 lentiviral vector was successfully constructed.

Oxidative stress regulated heme-oxygenase-1 and glutathione S-transferase-m1 gene expression changes in cell lines exposed to melanins

To investigate the effects of oxidative stress on substantia nigra neuronal degeneration and death in patients with Parkinson＇s disease, we treated neuroblastoma cells （SK-N-SH） and glioma cells with Fenton＇s reagent, iron chelating agent, neuromelanin and dopamine melanin. We investigated the changes in expression of nine oxidative stress-related genes and proteins. The levels of mRNAs for heme-oxygenase-1 and glutathione S-transferase-ml were significantly reduced in SK-N-SH cells exposed to oxidative stress, and increased in glial cells treated with deferoxamine. These results revealed that SK-N-SH neurons react sensitively to oxidative stress, which implies different outcomes between these two types of cells in the substantia nigra. Moreover, the influences of neuromelanin and dopamine melanin on cell function are varied, and dopamine melanin is not a good model for neuromelanin.