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.

Human IgG Fc promotes expression, secretion and immunogenicity of enterovirus 71 VP1 protein

Enterovirus （EV71） can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 （VP1）, plays a critical role in the pathogenicity of EVT1. High level expression and secretion ofVP 1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP 1 DNA vaccines, including wt-VP 1, tPA-VP 1, VP l-d, VP 1-hFc and VP 1 - mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different （P〈0.05）. In this study, we further investigated the protein lev- els of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc （hFc） to VP1. VP 1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VPI-hFc protein for additional studies.

Therapeutic and prevention strategies against human enterovirus 71 infection

Human enterovirus 71(HEV71) is the cause of hand,foot and mouth disease and associated neurological complications in children under five years of age.There has been an increase in HEV71 epidemic activity throughout the Asia-Pacific region in the past decade,and it is predicted to replace poliovirus as the extant neurotropic enterovirus of highest global public health significance. To date there is no effective antiviral treatment and no vaccine is available to prevent HEV71 infection. The increase in prevalence, virulence and geographic spread of HEV71 infection over the past decade provides increasing incentive for the development of new therapeutic and prevention strategies against this emerging viral infection. The current review focuses on the potential, advantages and disadvantages of these strategies. Since the explosion of outbreaks leading to large epidemics in China, research in natural therapeutic products has identified several groups of compounds with anti-HEV71 activities. Concurrently, the search for effective synthetic antivirals has produced promising results. Other therapeutic strategies including immunotherapy and the use of oligonucleotides have also been explored. A sound prevention strategy is crucial in order to control the spread of HEV71. To this end the ultimate goal is the rapid development, regulatory approval and widespread implementation of a safe and effective vaccine. The various forms of HEV71 vaccine designs are highlighted in this review. Given the rapid progress of research in this area, eradication of the virus is likely to be achieved.

Evaluation of Reverse Transcription Loop-Mediated Isothermal Amplification assays for Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 in Clinical Samples

A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for human enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) infection was further evaluated. The one step reaction was performed in a single tube at 65?C for 45 min for EV71 and 35 min for CVA16. The detection limits of RT-LAMP assays for both EV71 and CVA16 were 0.1 of a 50% tissue culture infective dose (TCID50) per reaction, based on 10—Fold dilutions of a titrated EV71 or CVA16 strain. The specific assay showed there were no cross-reactions with Coxsackievirus A (CVA) viruses (CVA 2, 4, 5, 7, 9, 10, 14, and 25), Coxsackievirus B (CVB) viruses (CVB 1, 2, 3, 4, and 5) or ECHO viruses (ECHO 3, 6, 11, and 19). In parallel with commercial quantitative real-time polymerase chain reaction (qRT-PCR) diagnostic kits for EV71 and CVA16, the RT-LAMP assay was evaluated with 515 clinical specimens, the results showed the RT-LAMP assay and the qRT-PCR assay were in complete agreement for 513/515 (99.6%) of the specimens. Two samples with discrepant results from two methods were further verified by nested reverse transcription polymerase chain reaction (nRT-PCR) assay and sequencing to be true positives for CVA16. In conclusion, RT-LAMP assay is demonstrated to be a sensitive and specific assay and have a great potential for the rapid and visual screening of EV71 and CVA16 in China, especially in those resource-limited hospitals and rural clinics of provincial and municipal regions.

Positive Selection Analysis of VP1 Genes of Worldwide Human Enterovirus 71 Viruses

Human enterovirus 71 viruses have been long circulating throughout the world. In this study, we performed a positive selection analysis of the VP1 genes of capsid proteins from Enterovirus 71 viruses. Our results showed that although most sites were under negative or neutral evolution, four positions of the VP1 genes were under positive selection pressure. This might account for the spread and frequent outbreaks of the viruses and the enhanced neurovirulence. In particular, position 98 might be involved in neutralizing antibodies, modulating the virus-receptor interaction and enhancing the virulence of the viruses. Moreover, both positions 145 and 241 might correlate to determine the receptor specificity. However, these positions did not display much difference in amino acid polymorphism. In addition, no position in the VP1 genes of viruses isolated from China was under positive selection.

Human Enterovirus 71 DNA Vaccine Constructs Containing 5’UTR with Complete Internal Ribosome Entry Site Sequence Stimulated Improved Anti-Human Enterovirus 71 Neutralizing Immune Responses

Recent improvement in the technologies for efficient delivery of DNA vaccines has renewed interest in the DNA-based vaccines. Several DNA-based vaccines against human enterovirus 71 (EV71), the causative agent for hand, foot and mouth disease (HFMD) have been developed. Here we examined the potential of improving the vaccines by inserting the EV71 5’ untranslated region (5’ UTR) containing the full length internal ribosome entry site (IRES) sequence to the EV71 VP1-based DNA vaccine constructs. Four vaccine constructs designated as 5’ UTR-VP1/EGFP, VP1/EGFP, 5’ UTR-VP1/pVAX and VP1/pVAX, were designed using the pEGFP-N1 and pVAX-1 expression vectors, respectively. Transfection of Vero cells with the vaccine constructs with the 5’-UTR (5’-UTR-VP1/EGFP and 5’ UTR-VP1/pVAX) resulted in higher percentages of cells expressing the recombinant protein in comparison to cells transfected with vectors without the 5’-UTR (67% and 57%, respectively). Higher IgG responses (29%) were obtained from mice immunized with the DNA vaccine construct with the full length 5’ UTR. The same group of mice when challenged with life EV71 produced significantly higher neutralizing antibody (NAb) titers (>5-fold). These results suggest that insertion of the EV71 5’ UTR sequence consisting of the full length IRES to the EV71 DNA vaccine constructs improved the efficacy of the constructs with enhanced elicitation of the neutralizing antibody responses.

Impact of host genetics on gut microbiome: Take-home lessons from human and mouse studies

The intestinal microbiome has emerged as an important component involved in various diseases.Therefore,the interest in understanding the factors shaping its composition is growing.The gut microbiome,often defined as a complex trait,contains diverse components and its properties are determined by a combination of external and internal effects.Although much effort has been invested so far,it is still difficult to evaluate the extent to which human genetics shape the composition of the gut microbiota.However,in mouse studies,where the environmental factors are better controlled,the effect of the genetic background was significant.The purpose of this paper is to provide a current assessment of the role of human host genetics in shaping the gut microbiome composition.Despite the inconsistency of the reported results,it can be estimated that the genetic factor affects a portion of the microbiome.However,this effect is currently lower than the initial estimates,and it is difficult to separate the genetic influence from the environmental effect.Additionally,despite the differences between the microbial composition of humans and mice,results from mouse models can strengthen our knowledge of host genetics underlying the human gut microbial variation.

Genetic Correction and Hepatic Differentiation of Hemophilia B-specific Human Induced Pluripotent Stem Cells

Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX （F IX） gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay.Results The cell line bore a missense mutation in the 6th coding exon （c.676 C〉T） of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% （10/45） and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.

Detection of CYP2E1,a Genetic Biomarker of Susceptibility to Benzene Metabolism Toxicity in Immortal Human Lymphocytes Derived from the Han Chinese Population

Objective Cytochrome P450 2E1 （CYP2E1） is an important metabolizing enzyme involved in oxidative stress responses to benzene, a chemical associated with bone marrow toxicity and leukemia, We aimed to identify the CYP2E1 genetic biomarkers of susceptibility to benzene toxicity in support of environmental and occupational exposure prevention, and to test whether a model using immortal human lymphocytes might be an efficient tool for detecting genetic biomarkers. Methods Immortalized human lymphocyte cell lines with independent genotypes on four CYP2E1 SNP sites were induced with 0.01% phenol, a metabolite of benzene. CYP2E1 gene function was evaluated by mRNA expression and enzyme activity. DNA damage was measured by Single-Cell Gel Electrophoresis （SCGE）. Results Among the four SNPs, cells with rs2070673TT and rs2030920CC showed higher levels of ~YP2E1 transcription and enzymatic activity than the other genotypes in the same SNP site. Cells with higher gene expression genotypes also showed higher comet rates compared with lower gene expression genotypes. Conclusion These results suggest that CYP2E1 rs2070673 and rs2030920 might be the genetic biomarkers of susceptibility to benzene toxicity and that the immortalized human lymphocytes model might be an efficient tool for the detection of genetic biomarkers of susceptibility to chemicals.

Genetics of inflammatory bowel disease: The role of the HLA complex

The human leucocyte antigen （HLA） complex on chromosome 6p21.3 is the most extensively studied genetic region in Inflammatory bowel disease （IBD）. Consistent evidence of linkage to IBD3 （6p21.1-23）, an area which encompasses the HLA complex, has been demonstrated for both Crohn＇s disease and ulcerative colitis, and a number of replicated associations with disease susceptibility and phenotype have recently emerged. However, despite these efforts the HLA susceptibility gene （s） for IBD remain elusive, a consequence of strong linkage disequilibrium, extensive polymorphism and high gene density across this region. This article reviews current knowledge of the role of HLA complex genes in IBD susceptibility and phenotype, and discusses the factors currently limiting the translation of this knowledge to clinical practice.

Genetic diversity for grain Zn concentration in finger millet genotypes:Potential for improving human Zn nutrition

Nearly half of the world population suffers from micronutrient malnutrition,particularly Zn deficiency.It is important to understand genetic variation for uptake and translocation behaviors of Zn in relevant crop species to increase Zn concentration in edible parts.In the present study,genetic variation in grain Zn concentration of 319 finger millet genotypes was assessed.Large genetic variation was found among the genotypes,with concentrations ranging from 10 to 86 μg g^(-1)grain.Uptake and translocation studies with Zn/^(65) Zn application in 12 selected low-Zn genotypes showed wide variation in root uptake and shoot translocation,with genotypes GEC331 and GEC164 showing greater uptake and translocation.Genotypes GEC164 and GEC543 showed increased grain Zn concentration.Genotypes GEC331 and GEC164 also showed improved yield under Zn treatment.Appreciable variation in grain Zn concentration among finger millet genotypes found in this study offers opportunities to improve Zn nutrition through breeding.

Optimization of Biodynamic Seated Human Models Using Genetic Algorithms

Many biodynamic models have been derived using trial and error curve-fitting technique, such that the error between the computed and measured biodynamic response functions is minimum. This study developed a biomechanical model of the human body in a sitting posture without backrest for evaluating the vibration transmissibility and dynamic response to vertical vibration direction. In describing the human body motion, a three biomechanical models are discussed (two models are 4-DOF and one model 7-DOF). Optimization software based on stochastic techniques search methods, Genetic Algorithms (GAs), is employed to determine the human model parameters imposing some limit constraints on the model parameters. In addition, an objective function is formulated comprising the sum of errors between the computed and actual values (experimental data). The studied functions are the driving-point mechanical impedance, apparent mass and seat- to-head transmissibility functions. The optimization process increased the average goodness of fit and the results of studied functions became much closer to the target values (Experimental data). From the optimized model, the resonant frequencies of the driver parts computed on the basis of biodynamic response functions are found to be within close bounds to that expected for the human body.

Genetically modified human umbilical cord blood cells as a promising strategy for treatment of spinal cord injury

Spinal cord injury （SCI） continues to be a pressing health and social problem. The injury leads to neuronal and glial cell death accompanied by degeneration of nerve fibers. There are currently no particularly effective treatments. SCI causes profound disabil- ity of people affected and has attracted increased attention in the international field of neuroregeneration. For the past two decades, much hope has been placed in cell therapies for the restoration of both structure and function of the injured spinal cord. Embryonic and neural stem cells, olfactory ensheathing cells, microglia-like cells, Schwann cells, mesenchymal stem cells.

Advances in homology directed genetic engineering of human pluripotent and adult stem cells

The ability to introduce precise genomic modifications in human cells has profound implications for both basic and applied research in stem cells, ranging from identification of genes regulating stem cell self-renewal and multilineage differentiation to therapeutic gene correction and creation of in vitro models of human diseases. However, the overall efficiency of this process is challenged by several factors including inefficient gene delivery into stem cells and low rates of homology directed site-specific targeting. Recent studies report the development of novel techniques to improve gene targeting efficiencies in human stem cells; these methods include molecular engineering of viral vectors to efficiently deliver episomal genetic sequences that can participate in homology directed targeting, as well as the design of synthetic proteins that can introduce double-stranded breaks in DNA to initiate such recombination events. This review focuses on the potential of these new technologies to precisely alter the human stem cell genome and also highlights the possibilities offered by the combination of these complementary strategies.

Genetic diversity of Mansonia altissima A. Chev. under different regimes of human impact in the Akure Forest Reserve,Nigeria

Mansonia altissima is an important West African timber tree species. For the purpose of examining the effect of human impact on its genetic diversity, genetic diversity and spatial genetic structure of the species under different regimes of human impact were investigated in the Akure Forest Reserve, Nigeria, using 504 amplified fragment length polymorphism （AFLP） markers. The results indicate a very low genetic diversity in M. altissima within the forest reserve （He = 0.045; PPL = 16.75%; Br = 1.162）. The highest genetic diversity was observed in the primary forest （H e= 0.062; PPL - 21.00%; Br = 1.204）, with the lowest genetic diversity in the isolated forest patch （He = 0.032; PPL = 9.00%; B r= 1.089）. A significant and pronounced spatial genetic structure was found in the logged forest and in the isolated forest patch. In contrast, the primary forest exhibited very weak spatial genetic structuring. As expected, no spatial genetic structure was found in the planted stands of M. altissima. From a conservation point of view, our results suggest that genetic diversity ofM. altissima is at risk in the forest reserve. The scale of human impact in the study area could pose a serious threat to the maintenance of genetic diversity of the species. These results would offer practical applications in the conservation of other tropical tree species.

Benefits of Genetic Engineering to the Environment and Human Health

The purpose of this essay is to argue that the genetic engineering may bring about benefits to human health and the environment.By means of research of secondary source collection,relevant evidence is selected,evaluated and organized into three main parts:improving agricultural environment,providing effective medical therapy and supplying safe and nutrition food to human body.In order to explain the benefits that created by genetic engineering technologies,examples based on opinions of experts and results of experts' experiments are used.The research results strongly suggest that the genetic engineering has positive effects on environment and mankind.Base on those finds,the argument is justified that genetic engineering is certainly beneficial to the environment and human health.In the future,more attention and researches should be focus on the genetic engineering with the purpose of benefiting human beings and the natural worlds.

Human a Type Genetic Engineering Interference Essence Injection

The highest-level interference essence against virus and turnour genetic engineering medicine is a new type created in the 1980s. Compared with chemical medicines, the interference essence has a special effect in the treatment of viruses and tumours. The human a, type genetic engineering interference essense is prepared by the Institute of Viruses of the Chinese Academy of Preventive Medical Sciences, the Shanghai Vaccine

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.

Survival of transplanted neurotrophin-3 expressing human neural stem cells and motor function in a rat model of spinal cord injury

BACKGROUND： Many methods have been attempted to repair nerves following spinal cord injury, including peripheral nerve transplantation, Schwann cell transplantation, olfactory ensheathing cell transplantation, and embryonic neural tissue transplantation. However, there is a need for improved outcomes. OBJECTIVE： To investigate the repair feasibility for rat spinal cord injury using human neural stem cells （hNSCs） genetically modified by lentivirus to express neurotrophin-3. DESIGN, TIME AND SETTING： In vitro cell biological experiment and in vivo randomized, controlled genetic engineering experiment were performed at the Third Military Medical University of Chinese PLA and First People＇s Hospital of Yibin, China from March 2006 to December 2007. MATERIALS： A total of 64 adult, female, Wistar rats were used for the in vivo study. Of them, 48 rats were used to establish models of spinal cord hemisection, and were subsequently equally and randomly assigned to model, genetically modified hNSC, and normal hNSC groups. The remaining 16 rats served as normal controls. METHODS： hNSCs were in vitro genetically modified by lentivirus to secrete both green fluorescence protein and neurotrophin-3. Neurotrophin-3 expression was measured by Western blot. Genetically modified hNSC or normal hNSC suspension （5 × 10^5） was injected into the rat spinal cord following T10 spinal cord hemisection. A total of 5μL Dulbecco＇s-modified Eagle＇s medium was infused into the rat spinal cord in the model grop. Transgene expression and survival of transplanted hNSCs were determined by immunohistochemistry. Motor function was evaluated using the Basso, Beattie, and Bresnahan （BBB） scale. MAIN OUTCOME MEASURES： The following parameters were measured： expression of neurotrophin-3 produced by genetically modified hNSCs, transgene expression and survival of hNSCs in rats, motor function in rats. RESULTS： hNSCs were successfully genetically modified by lentivirus to stably express neurotrophin-3. The transplanted hNSCs primarily gathered at, or around, the injection site two weeks following transplantation, and gradually migrated towards the surrounding tissue. Transplanted hNSCs were observed 7.0-8.0 mm away from the injection site. In addition, hNSCs were observed 10 weeks after transplantation. At week 4, BBB locomotor scores were significantly greater in the genetically modified hNSC and normal hNSC groups, compared with the model group （P 〈 0.05）, and scores were significantly greater in the genetically modified hNSC group compared with the normal hNSC group （P 〈 0.05）. CONCLUSION： hNSCs were genetically modified with lentivirus to stably secrete neurotrophin-3. hNSCs improved motor function recovery in rats following spinal cord injury.

Immunogenetic characteristics of patients with autoimmune gastritis

AIM:To explore whether predisposition to autoimmune gastritis (AIG) is found in human leukocyte antigen (HLA), cytokine or killer cell immunoglobulin-like receptor (KIR) gene variations.METHODS: Twelve Finnish patients with autoimmunetype severe atrophy of the gastric corpus were included. The patients' serum was analyzed for pepsinogen-interleukin (IL)-1 gene cluster, IL-2, IL-4, IL-6, IL-10, IL-12, interferon γ, transforming growth factor β, and tumor necrosis factor α. Variation in KIR genes was also explored. The results were compared with prevalence of the polymorphisms in Finnish or European populations.RESULTS: All patients had pepsinogen-CONCLUSION: As explored with modern DNA-based methods, HLA-DRB1*04 and DQB1*03 alleles, but not HLA-B8-DRB1*03, may predispose to AIG.