Salmonella enterica serovar Cerro 87, which was isolated from a commercial egg-producing farm, has a phosphorothioated DNA backbone resulting DNA degradation(Dnd) during the pulsed field gel electrophoresis (PFGE) process. In this research, a gene deletion mutant XTG103 was engineered with the entire dnd gene cluster knocked out by double crossover using vector pKOV-kan, and lost Dnd phenotype corre- spondingly. We regulated the DNA phosphorothioation by heterogenous expression of dnd gene cluster with an isopropyl ?-D-1-thiogalactopyranoside (IPTG) inducible promoter PlacZ.

pHZ1080, an E. coli-Streptomyces shuttle expression vector was constructed in order to explore the utilization of lambda phage regulated expression elements in Streptomyces. A 2.7 kb polyketide synthase (PKS) gene from Streptomyces sp. FR-008 was inserted into downstream of lambda phage promoter (PR) to give the shuttle plasmid, pHZ1067. The PKS protein was expressed in Streptomyces lividans carrying pHZ1067 in a heat-dependent manner, as it did in E. coli. The PKS protein expressed in both hosts with same molecular weight was detected by SDS-PAGE and Western-blot. The successful heat-induced expression of PKS suggested that pHZ1080 was useful and convenient for heat-induced expression of heterologous genes in both E. coli and Streptomyces.
Amino Acid Sequence ; Base Sequence ; Blotting, Western ; Cloning, Molecular ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Multienzyme Complexes ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Streptomyces ; enzymology ; genetics ; Temperature

Objective To investigate the influence of iodine excess on expression of TRAIl/TRAIL-sR1 in NOD and Balb/c mice and to study the effect of TRAIl/TRAIL-sR1 on the pathogenesis of experimental autoimmune thyroiditis(EAT). Methods Both Balb/c and NOD mice were divided randomly into control and iodine excess group by feeding with water containing no NaI or 0.05% Nal. The mice were sacrificed after 8 weeks. TRAIL and TRAIL-sR1 mRNA levels were detected by RT-PCR. The function, morphology and apoptosis of thyroids were also observed by ELISA and Tunnel stain. Results Treated by HI, enlarged follicles and flattened epithelium by accumulation of colloid were found in thyroids of both NOD and Balb/c mice. But significant lymphoid cell infiltration and local fibrosis were only found in thyroids of NOD HI group. The relative weight of thyroids of NOD mice in HI group[(104.8±14.5)mg/kg]was heavier than that of control group [(71.8±20.4)mg/kg]. The level of TT4 declined in HI group[(30.77±3.59)mmol/L]compared with control group[(36.43±2.66)mmol/L], meanwhile, the level of TSH was higher in HI group[(6.98±0.66)μg/L]than that in control group [(5.55±0.56)μg/L]. The difference being statistically significant(t=7.773,-9.526,-4.458, all P < 0.05). The relative weight of thyroids of Balb/c mice of HI group[(155.8±20.8)mg/kg]also heavier than that of control group [(105.1±22.0) mg/kg]. The level of TT4 droped in HI group [(19.75±3.32) mmoL/L]was higher than that in control group[(23.46±6.21)mmoL/L], the level of TSH in HI group[(4.14±1.71)μg/L]was higher than that in control group[(3.55±1.41)μg/L], the difference being statistically significant(t=7.554,-7.239,3.140, all P< 0.05). A great deal of apoptotie ceils observed in NOD (3.97±0.91) and Balb/c mice (1.05±0.45) by Tunnel stain were greater than control groups (0.21±0.15, 0.10±0.03), the difference being statistically significant in beth of the two species(t=-7.167,-17.772, both P < 0.05). The apoptosis index of thyroid follicular epithelium in NOD was obviously higher than Balb/c(t=-7.625, P<0.05). The level of TRAIL mRNA did not remarkably change in Balb/c between control group(0.000 59±0.000 39) and HI group(0.001 24±0.000 46, t=-1.940, P>0.05), but it increased apparently in NOD mice HI group(0.018 88±0.005 77) than that of control group(0.009 61± 0.00591, t=-2.71, P<0.05). The level of the expression of TRAIL-sR1 mRNA increased in HI groups of NOD (0.000 53±0.000 15) and Balb/c mice(0.000 42±0.000 09) than that in control groups of NOD(0.000 28± 0.000 05) and Balb/c mice (0.000 17±0.000 06) and the differences were statistically significant between the two species(t=3.050,3.990, all P<0.05). The differences of the expression of TRAIL and TRAIL-sR1 mRNA between the two species were significant(t=-3.37,-4.76, all P<0.05). Conclusions Iodine excess induces colloid goiter in beth species of mice and thyroiditis in NOD mice. The increase of TRAIL and TRAIL-sR1 influenced by iodine excess is one of the molecular bases of follicular epithelium apoptosis and inflammation in thyroids. Genetic factor is a key factor in the pathogenesis of thyroiditis.

OBJECTIVETo study the indoor environmental factors associated with the prevalence of asthma and related allergies among school children.

METHODSA cluster sampling method was used and the ISAAC questionnaire was conducted. A total of 4612 elementary students under Grade Five of 7 schools were enrolled in the survey for the impact of indoor environmental factors on the prevalence of asthma and related allergies in several urban and suburban schools of Beijing.

RESULTSA total of 4060 sample were finally analyzed including 1992 urban and 2068 suburban. The prevalence of wheeze, allergic rhinoconjunctivitis and atopic eczema in the past 12 months was 3.1% (61/1992), 5.3% (106/1992), 1.1% (22/1992) among urban children while 1.3% (27/2068), 3.1% (65/2068), 1.0% (22/2068) among suburban children respectively. The prevalence of wheeze and allergic rhinoconjunctivitis of the past 12 months in urban were both significantly higher than that in suburban (χ(2) = 14.77, 11.93, P < 0.01). The incidences of having asthma and eczema ever among urban children (5.3% (105/1992), 29.4% (586/1992)) were significantly (χ(2) = 39.03, 147.22, P < 0.01) higher than that among suburban (1.7% (35/2068), 13.8% (285/2068)). Although the distributions of indoor environmental factors were similar in both areas, passive smoking and interior decoration had different influence on the prevalence of asthma and related allergies among school children in the two areas. The significant impact of passive smoking on having asthma ever among suburban children was observed (OR = 2.70, 95%CI = 1.17 - 6.23) while no significant result in urban (OR = 1.06, 95%CI = 0.71 - 1.58); the percentage of interior decoration was 84.0% (1673/1992) among urban children and 80.0% (1655/2068) among suburban children, there was significant impact of interior decoration on the prevalence of having eczema ever among urban children (OR = 1.57, 95%CI = 1.17 - 2.10) but no significant results were found in suburban sample (OR = 1.06, 95%CI = 0.76 - 1.48).

CONCLUSIONThe prevalence of asthma and related allergies among school children is much higher in urban areas than that in suburban areas and the indoor environmental factors such as passive smoking and interior decoration may differently explain the prevalence of asthma and related allergies in the two areas.

Adolescent ; Air Pollution, Indoor ; analysis ; Asthma ; epidemiology ; etiology ; Child ; Child, Preschool ; China ; epidemiology ; Cities ; Environmental Exposure ; Female ; Humans ; Hypersensitivity ; epidemiology ; etiology ; Male ; Prevalence ; Risk Factors ; Students ; Suburban Population ; Surveys and Questionnaires ; Urban Population

In order to investigate the expression of the anti-platelet glycoprotein specific antibodies and anti-HLA antibodies in idiopathic thrombocytopenic purpura (ITP), 45 patients with ITP were selected in this study. An easy PCR-SSP assay was used to detect single-nucleotide polymorphisms or deletion in HPA and HLA systems. The anti-platelet glycoprotein specific antibodies and anti-HLA antibodies in plasma or platelet eluate were tested with a solid phase ELISA. The results indicated that the anti-platelet glycoprotein specific antibodies were detected in plasma or platelet eluate of 45 patients, among which anti-GPIIb/IIIa/and anti-GpIb/IX were most common. Both the anti-platelet glycoprotein specific antibodies and anti-HLA antibodies were found in plasma of 11 patients. Pedigree investigation in 2 patients (case 37 and case 40) was carried out, the results showed that anti-platelet glycoprotein specific antibodies and anti-HLA antibodies detected in 2 patients closely related to incompatibility with platelet antigens and HLA antigens in parents. In conclusion, the results suggested that detection of the anti-platelet glycoprotein specific antibodies and anti-HLA antibodies in plasma or platelet eluate in combination with investigation of clinical manifestation of patients is important for diagnosis of idiopathic thrombocytopenic purpura.
Adolescent ; Adult ; Antibodies, Anti-Idiotypic ; blood ; Antigens, Human Platelet ; immunology ; Child ; Child, Preschool ; Female ; HLA Antigens ; immunology ; Humans ; Infant ; Infant, Newborn ; Male ; Middle Aged ; Platelet Glycoprotein GPIIb-IIIa Complex ; immunology ; Platelet Glycoprotein GPIb-IX Complex ; immunology ; Platelet Membrane Glycoproteins ; immunology ; Purpura, Thrombocytopenic, Idiopathic ; blood ; immunology ; Young Adult

BACKGROUNDAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that primarily affects motor neurons and has no effective treatment. Recently, Iida et al. identified a single-nucleotide polymorphism (SNP) rs2275294 in the ZNF512B gene that is significantly associated with susceptibility to ALS in the Japanese population. Here, we performed a case-control study examining the possible association of rs2275294 with risk of sporadic ALS (SALS) in a large Chinese cohort.

METHODSTo assess this association, we performed a replication study in 953 SALS patients and 1039 age- and gender-matched healthy control subjects, who were recruited from Peking University Third Hospital and the First Affiliated Hospital of Anhui Medical University from January 2004 to December 2013 throughout China. We genotyped the rs2275294 SNP using polymerase chain reaction and direct sequencing.

RESULTSThe allele frequency of rs2275294 in ZNF512B was different between Japanese and Chinese. The association in Chinese between ALS patients and controls did not reach statistical significance (P = 0.54; odds ratio = 0.94; 95% confidence interval = 0.76-1.15).

CONCLUSIONSThe SNP rs2275294 in ZNF512B is not considered to be associated with ALS susceptibility in the Chinese population. Our study highlights genetic heterogeneity in ALS susceptibility in different population. Given our negative results, further replication study involving larger and more homogeneous samples in different ethnicities should be performed in the future.

Adult ; Amyotrophic Lateral Sclerosis ; epidemiology ; genetics ; Asian Continental Ancestry Group ; genetics ; Carrier Proteins ; genetics ; Case-Control Studies ; China ; epidemiology ; Female ; Genetic Predisposition to Disease ; genetics ; Humans ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; genetics

OBJECTIVETo study the levels of pollutions caused by fine particulate matter (PM(2.5)) in the public places and investigate the possible influencing factors.

METHODSA total of 20 public places in four types such as rest room in bath center, restaurant, karaoke bars and cyber cafe in Tongzhou district in Beijing were chosen in this study; indoor and outdoor PM(2.5) was monitored by TSI sidepak AM510. Data under varying conditions were collected and analyzed, such as doors or windows or mechanical ventilation devices being opened, rooms cramped with people and smoking.

RESULTSThe average concentration of indoor PM(2.5) in 20 public places was (334.6 +/- 386.3) microg/m(3), ranging from 6 microg/m(3) to 1956 microg/m(3); while in bath center, restaurant, karaoke bars and cyber cafe were (116.9 +/- 100.1)microg/m(3), (317.9 +/- 235.3) microg/m(3), (750.6 +/- 521.6)microg/m(3) and (157.5 +/- 98.5) microg/m(3) respectively. The concentrations of PM(2.5) in restaurant (compared with bath center: Z = -10.785, P < 0.01; compared with karaoke bars: Z = -10.488, P < 0.01; compared with cyber cafe: Z = -7.547, P < 0.01) and karaoke bars (compared with bath center: Z = -16.670, P < 0.01; compared with cyber cafe: Z = -15.682, P < 0.01) were much higher than those in other two places. Single-factor analysis revealed that the average concentration of indoor PM(2.5) in 20 public places was associated with the number of smokers per cube meters(9.13 x 10(-3); r = 0.772, F = 26.579, P < 0.01) and ventilation score [(2.5 +/- 1.5) points; r = 0.667, F = 14.442, P < 0.01], and there were significant correlation between the average indoor and outdoor levels in restaurant [(317.9 +/- 235.3) microg/m(3), (67.8 +/- 78.9) microg/m(3); r = 0.918, F = 16.013, P = 0.028] and cyber cafe [(157.5 +/- 98.5) microg/m(3), (67.7 +/- 43.7) microg/m(3); r = 0.955, F = 30.785, P = 0.012]. Furthermore, significant correlation was observed between the average concentration of indoor PM(2.5) [(157.5 +/- 98.5) microg/m(3)]and the number of people per cube meters (288.7 x 10(-3)) in cyber cafe (r = 0.891, F = 11.615, P = 0.042). Multiple regression analysis showed that smoking (b' = 0.581, t = 3.542, P = 0.003) and ventilation (b' = -0.348, t = -2.122, P = 0.049) were the major factors that may influence the concentration of indoor PM(2.5) in four public places. With cluster analysis, the results showed that the major factors that influence the concentration of indoor PM(2.5) was the outdoor PM(2.5) levels [(49.6 +/- 39.5) microg/m(3); b = 1.556, t = 3.760, P = 0.007] when ventilation (score > 2) was relatively good. The number of smokers per cube meters (14.7 x 10(-3)) became the major influence factor when the ventilation score

CONCLUSIONThis study indicated that smoking was the main source of indoor PM(2.5) in public places. Outdoor PM(2.5) should be correlated with indoor PM(2.5) concentration under drafty situation.

Air Pollution, Indoor ; analysis ; Environmental Monitoring ; methods ; Particulate Matter ; analysis ; Public Facilities ; Tobacco Smoke Pollution ; analysis

OBJECTIVETo assess the effectiveness and the possible adverse effects of catgut implantation at acupoints for allergic rhinitis (AR).

METHODSThis systematic review was carried out in accordance with the Cochrane Handbook version 5.1.0 and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement. Extensive literature searches were conducted in PubMed, Excerpta Medical Databases, the Cochrane Library, the China National Infrastructure, Wanfang Chinese Digital Periodical and Conference Database, and the Weipu Chinese Science and Technique Journals Database. The Chinese Clinical Trial Registry Center was also searched for ongoing trials up to September 2012. Randomized controlled trials (RCTs) or quasi-RCTs were included. Risk of bias assessment was performed using the Cochrane tool for assessing risk of bias.

RESULTSFive RCTs with 285 participants were found from 49 relevant studies, but there was just one RCT which met the inclusion criteria for this review. The study showed that treatment of catgut implantation at acupoints could lead to a better alleviation of the signs and symptoms of AR than the crude herb moxibustion. No adverse events were reported in this study.

CONCLUSIONSBecause of the methodological shortcoming and the risk of bias of the included trial, catgut implantation was proved with only limited evidence for the treatment of AR. Robust RCTs with high quality and larger sample size in this field are hoped to be carried out in the future.

Acupuncture Points ; Catgut ; adverse effects ; Clinical Trials as Topic ; Humans ; Publication Bias ; Rhinitis, Allergic ; Rhinitis, Allergic, Perennial ; therapy ; Risk Factors

OBJECTIVETo explore the impact of Foxp3 expression and CD(4)(+)CD(25)(+) regulatory T cells on pathogenesis of childhood asthma.

METHODSTotally 15 patients with acute asthma exacerbation, 15 children with asthma remission and 10 children who were hospitalized for skeleton deformity without atopic disorders or history of allergic diseases or respiratory infections within a month as controls were recruited in this study from Sep. 2004 to Mar. 2005. The percentage of CD(4)(+)CD(25)(+) T cells were detected by 2-color flow cytometry. The levels of interleukin (IL)-4, IL-10, interferon (IFN)-gamma, transforming growth factor (TGF)-beta in plasma and supernatant were assayed by ELISA. Both the asthmatic children and the control children were selected to induce sputum by hypertonic saline. Sputum was processed for detecting the expression of Foxp3-mRNA. The expression of Foxp3-mRNA in both sputum and PBMC was detected by semi-quantitative RT-PCR with beta-actin as internal control.

RESULTSThe percentage of CD(4)(+)CD(25)(+) regulatory T cells in exacerbation and remission asthmatic children was significantly lower than that of the control children both prestimulation [(10.1 +/- 2.1)% vs. (15.5 +/- 2.7)%, (11.7 +/- 2.5)% vs. (15.5 +/- 2.7)%, P < 0.05] and poststimulation with PHA [(12.4 +/- 2.3)% vs. (26.9 +/- 3.8)%, (17.3 +/- 3.2)% vs. (26.9 +/- 3.8)%, P < 0.05]. The percentage of CD(4)(+)CD(25)(+) regulatory T cells was significantly higher after PHA stimulation in normal children [(15.5 +/- 2.7)% vs. (26.9 +/- 3.8)%, P < 0.01]. The expression of Foxp3-mRNA (Foxp3/beta-actin) in asthmatic children was significantly lower than that in the control children in both PBMC and induced sputum. The expression of Foxp3-mRNA in PBMC was significantly higher after PHA stimulation in the control children (0.77 +/- 0.22 vs. 1.07 +/- 0.21, P < 0.05). However, there was no significant difference in Foxp3-mRNA expression in asthmatic children pre and post PHA stimulation. A significant positive correlation between the Foxp3-mRNA expression and the percentage of CD(4)(+)CD(25)(+) regulatory T cells was detected. The levels of IFN-gamma and TGF-beta were significantly lower in asthmatic children than those in the control children, and the levels of IFN-gamma and TGF-beta correlated positively with Foxp3-mRNA expression and the percentage of CD(4)(+)CD(25)(+) regulatory T cells. The level of IL-4 both in plasma and supernatant was higher in asthmatic children. The levels of IL-10 was higher only in exacerbation than in control children, the levels of IL-4 and IL-10 had no correlation with Foxp3-mRNA expression and the percentage of CD(4)(+)CD(25)(+) regulatory T cells.

CONCLUSIONInsufficient secretion of TGF-beta, decreased Foxp3 expression, insufficient number of CD(4)(+)CD(25)(+) regulatory T cells and the defective ability of converting CD(4)(+)CD(25)(-) T cells to CD(4)(+)CD(25)(+) regulatory T cells might play an important role in pathogenesis of asthma.

Asthma ; physiopathology ; Case-Control Studies ; Child ; Cytokines ; analysis ; blood ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Forkhead Transcription Factors ; genetics ; metabolism ; Humans ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sputum ; chemistry ; T-Lymphocytes, Regulatory ; physiology

This study was aimed to investigate the characteristics of human bone marrow mesenchymal stem cells during ex-vivo expansion, MSCs were isolated from human bone marrow. At each passage, the characteristics of proliferation kinetics, osteogenic and adipogenic differentiation potential were analyzed, and cell morphology, surface markers were investigated as well. The karyotype analysis was done in different passage cells. The infection HIV, HCV, HBV and TP were detected by ELISA. Mycoplasma contamination in vitro was detected by PCR method. HLA-SBT was used to reanalyze the results of HLA antigens and alleles. STR genetic loci were detected by PCR in the MSC1, MSC2, MSC3 and MSC4. The results indicated that the proliferative ability and osteogenic potential decreased with the increase of passage number during culture expansion. The multiple differentiation potential of MSCs was maintained during their life span. Karyotype analysis showed that MSCs from 4 groups before passage 8 were normal. The expression of CD29, CD44, CD105, CD166 and CD73 were positive. The expression of CD14, CD34, CD45, CD80, CD86 were all negative. SBT was used to identify HLA-A, B, Cw, DRB1, DRPB1, DQ alleles in the MSC1, MSC2, MSC3, MSC4. The genetype of STR in the MSC1, MSC2, MSC3, MSC4 was different. MSC 3 was examined by TP-ELISA to confirm the infectious disease of TP. MSC2 was contaminated by mycoplasma at passage 5. It is concluded that culture expansion causes MSCs to gradually lose their stem cell properties. During ex-vivo expansion of MSCs, the osteogenic differentiation potential is decreased. MSCs before passage 8 can be a valuable subject for basic research and clinical application.
Adipogenesis ; Adult ; Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Cells, Cultured ; Female ; Humans ; Karyotyping ; Male ; Mesenchymal Stromal Cells ; cytology ; Osteogenesis