Characterization of Highly Pathogenic Avian Influenza H5N1 Viruses Isolated from Domestic Poultry in China / 生物医学与环境科学（英文）

The highly pathogenic avian influenza (HPAI) H5N1 virus has caused several outbreaks in domestic poultry. Despite great efforts to control the spread of this virus, it continues to evolve and poses a substantial threat to public health because of a high mortality rate. In this study, we sequenced whole genomes of eight H5N1 avian influenza viruses isolated from domestic poultry in eastern China and compared them with those of typical influenza virus strains. Phylogenetic analyses showed that all eight genomes belonged to clade 2.3.2.1 and clade 7.2, the two main circulating clades in China. Viruses that clustered in clade 2.3.2.1 shared a high degree of homology with H5N1 isolates located in eastern Asian. Isolates that clustered in clade 7.2 were found to circulate throughout China, with an east-to-west density gradient. Pathogenicity studies in mice showed that these isolates replicate in the lungs, and clade 2.3.2.1 viruses exhibit a notably higher degree of virulence compared to clade 7.2 viruses. Our results contribute to the elucidation of the biological characterization and pathogenicity of HPAI H5N1 viruses.

Construction and identification of mutation of pgm gene attenuate the virulent of Brucella melitensis strain 16 / 中国地方病学杂志

Objective The construction of suicide plasmid vector could be used to make mutation of pgm gene which attenuates the virulent of Brucella melitensis strain 16, the research may lay a foundation for the development of novel live attenuated vaccines. Methods Sucrose sensitive gene as forward screening sign and fusion sequences of kanamycin resistance gene were constructed based on plasmid pucl9; pucS1.6K suicide plasmid vector was established by modifying pgm gene with fusion sequences of kanamycin resistance gene (insertion mutation); pgm gene mutation of Brucella melitensis strain 16 was obtained by electro transformation and mutation was confirmed by PCR amplification. Results The results showed that the identified Brucella melitensis strain 16 pgm gene was inactivated after insertion of kanamycin resistance gene, and the mutant pgm gene DNA fragment length was approximately 3525 bp, in line with expectations, Brucella pgm gene mutant melitensis strain 16 was successfully constructed. Conclusions The construction of suicide plasmid vector and precise mutation of Brucella melitensis strain 16 is successful, the study is not only provided an effective technology platform for constructing mutants of Brucella but also lays a foundation for the development of novel live attenuated vaccines.

Generation and characterization of a cold-adapted attenuated live H3N2 subtype influenza virus vaccine candidate / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>H3N2 subtype influenza A viruses have been identified in humans worldwide, raising concerns about their pandemic potential and prompting the development of candidate vaccines to protect humans against this subtype of influenza A virus. The aim of this study was to establish a system for rescuing of a cold-adapted high-yielding H3N2 subtype human influenza virus by reverse genetics.</p><p><b>METHODS</b>In order to generate better and safer vaccine candidate viruses, a cold-adapted high yielding reassortant H3N2 influenza A virus was genetically constructed by reverse genetics and was designated as rgAA-H3N2. The rgAA-H3N2 virus contained HA and NA genes from an epidemic strain A/Wisconsin/67/2005 (H3N2) in a background of internal genes derived from the master donor viruses (MDV), cold-adapted (ca), temperature sensitive (ts), live attenuated influenza virus strain A/Ann Arbor/6/60 (MDV-A).</p><p><b>RESULTS</b>In this presentation, the virus HA titer of rgAA-H3N2 in the allantoic fluid from infected embryonated eggs was as high as 1:1024. A fluorescent focus assay (FFU) was performed 24-36 hours post-infection using a specific antibody and bright staining was used for determining the virus titer. The allantoic fluid containing the recovered influenza virus was analyzed in a hemagglutination inhibition (HI) test and the specific inhibition was found.</p><p><b>CONCLUSION</b>The results mentioned above demonstrated that cold-adapted, attenuated reassortant H3N2 subtype influenza A virus was successfully generated, which laid a good foundation for the further related research.</p>

Regulatory effect of Egr-1 promoter sequences induced by doxorubicin in transcriptional targeting on expression of GM-CSF gene / 中国实验血液学杂志

In order to explore the regulatory effects of Egr-1 promoter sequences induced by doxorubicin (ADM) in transcriptional targeting on the expression of hematopoietic growth factor genes. The human GM-CSF cDNA and enhanced green fluorescent protein (eGFP) cDNA were linked together with internal ribozyme entry site (IRES) and then inserted into the expression vector pCIneo under control of the Egr-1 promoter (Egr-EG). The vector was transferred into human bone marrow stromal cell line HFCL by lipofectin. The transfected cell clones (HFCL/EG) were selected by the addition of G418. The cells were exposed to the clinically important anticancer agent doxorubicin. The activity of eGFP in HFCL/EG cells was detected by flow cytometry. The amounts of GM-CSF in HFCL/EG postchemotherapy were confirmed with ELISA. The effect of GM-CSF in HFCL/EG cultural supernatants on expansion of CFU-GM derived from cord blood was also studied. The effect of N-acetylcysteine (a free radical scavenger) on GM-CSF production following exposure to ADM was examined. The results indicated that the activity of eGFP and the amounts of secreted GM-CSF in HFCL/EG cells exposed to ADM increased as compared to non-ADM group. The effect of GM-CSF in HFCL/EG cultural supernatants on expansion of CFU-GM was significantly higher than that of non-ADM group. N-acetylcysteine significantly decreased the concentration of GM-CSF produced by HFCL/EG treated with ADM. It is concluded that these in vitro data provide an experimental basis for the use of gene therapy of hematopoietic growth factor gene regulated by Egr-1 promoter to protect hematopoiesis from ADM-injury.

Investigation of semen quality of 18-35 year old Chinese army men / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the semen quality of the Chinese army men.</p><p><b>METHODS</b>Ten-item sperm quality analyses were made by manual methods and the computer assisted sperm analysis system in 1054 young Chinese army men. The subjects were divided into 4 age groups (18-20 yrs., 21-25 yrs., 26-30 yrs and 31-35 yrs.), and the results of the analyses were compared.</p><p><b>RESULTS</b>Among the 1 054 young males investigated, the semen volume was (2.6 +/- 1.4) ml, sperm density (55.9 +/- 46.5) x 10(6)/ml, sperm grade a + b motility (47.1 +/- 19.0)%, sperm viability (70.6 +/- 22.1)%, morphologically normal sperm (84.7 +/- 10.2)%, and acrosomal integrity (86.1 +/- 7.2)%. As for the percentages of the quality indexes that met WHO standards, the sperm volume was 73.5%, liquefaction time 91.1%, pH 93.0%, grade a + b motility 45.5%, viability 86.7%, sperm density 80.4%, morphologically normal sperm 98.2%, and the sperm total number 78.0%. Those who accorded with all the WHO standards accounted for 40.2%.</p><p><b>CONCLUSION</b>The semen quality of the 18-35 year old army men was better than previously reported in the similar literature. And that of the 26-30 yrs. group was the best among all the age groups.</p>

Long-term exposure to low intensity microwave radiation affects male reproductivity / 中华男科学杂志

<p><b>OBJECTIVE</b>To evaluate the effect of Long-term exposure to low intensity microwave radiation on male reproductivity.</p><p><b>METHODS</b>A total of 289 married male radar operators were included in the radar group and 148 married men unexposed to microwave radiation were enrolled as controls. Questionnaires were used and the intensity of microwave radiation in different working areas was detected.</p><p><b>RESULTS</b>The rate of sexual dysfunction was 43.6% in the radar group and 24.4% in the control group (P < 0.01). The natural pregnancy rate was 53.6% within 1 year of marriage and 46.4% after 1 year of marriage in the radar group, as compared with 81.1% and 18.9% in the control group (P < 0.01).</p><p><b>CONCLUSION</b>Long-term exposure to low intensity microwave radiation evidently increased the sexual dysfunction rate and decreased natural pregnancy rate in men.</p>

Effect of chemoinducible GM-CSF gene expression regulated by Egr-1 promoter on chemotherapy damage of tumor bearing mice / 中国癌症杂志

Background and purpose:Ionizing radiation(IR) activates the early growth response- I(Egr1) promoter through specific cis-acting sequences termed CArG elements by production of radical oxygen intermediates(ROls).Egr-EG,an expression vector pCIneo containing CArG elements cloned upstream of the cDNA for human recombinant GM-CSF,was used to treat hematopoietic damage due to chemotherapy.Commonly used chemotherapeutic agents can cause tumor cell death by producing DNA damage and generating ROIs.We therefore hypothesized that clinically employed chemotherapeutic agents that increase ROIs could also be employed to activate Egr-EG in a chemoinducible gene therapy strategy.This study was done to explore the chemo-protective effect of the expression of hematopoietic growth factors regulated by Egr-1 promoter on chemotherapy induced damage. Methods:The human GM-CSF cDNA and EGFP cDNA were linked together with internal ribosome entry site(IRES) and then inserted into the eukaryotic expression vector pCI-neo with the Egr-1 promoter(Egr-EG),and was further transduced into human bone marrow stromai cell lines HFCL(HFCL/EG).The HFCL/EG cells were transplanted i.v.into BI6 melanoma in C.B-17 combined immunodeficient(SCID) mice.5-FU was given i.p.on day 3 and 4.The white blood cell amount in peripheral blood,the expression of EGFP and GM-CSF in human stromal cell engrafted in recipient mice were detected by flow cytometry,RT-PCR,Western blot and colony-forming units for granulocytes and macrophages(CFU-GM),respectively.Results:In contrast to the two control groups,HFCL/EG(the Egr-regulatory element-derived expression of GM-CSF gene therapy) resulted in a proportional increase in the number of the white blood cell after chemotherapy,no significant diifferences were found for CFU-GM in bone marrow cells and the inhibition ratio on tumor in recipient mice.Chemotherapy could markedly increase the expression of EGFP and GM- CSF mRNA/protein as compared with that of non-chemotherapy control groups and HFCL group.Conclusion: Chemoinducible GM-CSF gene therapy regulated by Egr-1 promoter can ameliorate the toxic effect on 5-FU chemotherapy-inducible hematopoietic damage.

Conditions for SARS-CoV cultivation and inactivation / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the method for cultivation and inactivation of SARS-CoV.</p><p><b>METHODS</b>In order to choose the sensitive cell strain and the best infection dose of the virus, Vero, Vero-E6 and 2BS cell lines were infected with SARS-CoV. The cultivation temperature was selected among 25 degrees C, 33 degrees C and 37 degrees C. The best inactivating time and effect were observed with beta-propiolactone whose concentration ranged from 1:2000 to 1:20,000 at room temperature.</p><p><b>RESULTS</b>Vero and Vero-E6 cell lines were sensitive to SARS-CoV. The cytopathic changes of the cells were 75% at 37 degrees C in 5 percent CO2 incubator after infection. SARS-CoV was inactivated completely in beta-propiolactone (1:4000). The toxicity of beta-propiolactone was hydrolyzed completely when the inactivated virus was cultured for 16 hours at 2 degrees C, 8 degrees C and in water bath for 2 hours at 37 degrees C.</p><p><b>CONCLUSION</b>The titer of SARS-CoV was the highest when it was cultured in Vero or Vero-E6 cells for 72 hours at 37 degrees C in 5 percent CO2 incubator. SARS-CoV was inactivated completely in beta-propiolactone when its concentration was 1:4000 and the interaction time was 1 hour.</p>

Effect of antisense oligonucleotides of ryanodine receptor on proliferation and Ca2+i of airway smooth muscle cells / 中国应用生理学杂志

<p><b>AIM</b>To investigate the effect of antisense oligonucleotides (ASON) of ryanodine receptor on proliferation and [Ca2+]i concentration of airway smooth muscle cells (ASMCs).</p><p><b>METHODS</b>ASMCs were cultivated with collagen enzyme digestion method. Different concentrations of ASON were added to the cultures with Lipofectamine 2000 to observe the ASMCs proliferation using MTS/PES method. The changes of ASMCs [Ca2+]i were also observed by flow cytometry. The expression of mRNA of subtypes of RyR was assayed by RT-PCR.</p><p><b>RESULTS</b>RyR ASON restrained the proliferation of ASMCs, decreased the expression of RyR and reduced the concentration of [Ca2+]i.</p><p><b>CONCLUSION</b>RyR ASON could inhibit the proliferation of ASMCs by influencing the concentration of [Ca2+]i after excited.</p>

A cross-sectional study on nonionizing radiation to male fertility / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between microwave radiation and male reproductivity.</p><p><b>METHODS</b>After filling out questionnaire and body check, we carried out molecular epidemiological studies, using single cell gel electrophoresis (SCGE) and sperm automatic analysis among people working on radar.</p><p><b>RESULTS</b>Quality of semen and semi-clinical injury of sperm among the people working on radar had changed when radar electromagnetic wave frequency distance, intensity, lasting time and protection shield were changing. Dose-response relationship was noticed and the increase of sperm dysmorphia played a principal role. The results between exposed group and control group showed significant difference (P < 0.01).</p><p><b>CONCLUSION</b>People working on radar who suffered from non-ionization for long time and had bad radar shield protection would show semi-clinical injury on sperm and bad semen quality. However, it did not affect the male reproductive function. It was necessary to reinforce the protection of non-ionization and to improve male reproductive health care of people working on radar.</p>

Expression of ryanodine receptor isoforms in airway smooth muscle cells of rats / 中国应用生理学杂志

<p><b>AIM</b>To detect the expression of ryanodine receptor (RyR) subtypes in normol rat airway smooth muscle cells(ASMCs) and changes during chronic asthma formation.</p><p><b>METHODS</b>ASMCs were cultured with collagen enzyme digestion method. The expression of subtypes of RyR were detected by RT-PCR. Purified PCR product linked with pGEM-T vector to make DNA sequence assay. Chronic asthma model was made with OVA, the changes of RyRs detected by RT-PCR.</p><p><b>RESULTS</b>All subtypes of RyR were expressed in airway smooth muscle cells of normol rat. The expression of RyR1 increased obviously compared with control group (P < 0.05) on chronic asthma.</p><p><b>CONCLUSION</b>Co-expression of three subtypes of RyR in ASMCs of normal rat, indicate that there are complicated intercellular Ca2+ regulation mechanism in ASM, moreover RyR1 might play a role during asthma development.</p>

Study on sperm quality of males living in cold area / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the effects on male reproductive function working under cold area.</p><p><b>METHODS</b>After on site investigation, advanced molecular lab analysis-single-cell gel electrophoresis (SCGE) and sperm chromatin structure assay (SCSA) which are combined with semen routine analysis were used to evaluate semen quality and sperm sub-clinical injury.</p><p><b>RESULTS</b>Semen routine analysis showed that the semen parameters of the males working in cold area were within normal range, but level I comet cell percentage in SCGE increased significantly, which was 4.4%, compared to the contrast group (1.9%) with significant difference. During sperm chromatin structure assay parameters, comp alpha t increased, with an average value of 22.26%. The two kinds of results both showed single and double strand breakages in sperm.</p><p><b>CONCLUSION</b>Long-term exposure to cold could induce sperm DNA injury, but not affect sperm quality. The results suggested that it was important to reinforce the reproductive care in males working in cold areas.</p>