Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria.

Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bioproducts, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broad-host-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains.

[Relationship between the levels of maternal serum sTNFR II and the mRNA expression of placental TNFR II in preterm labor with chorioamnionitis and polymorphism in -196 site of tumor necrosis factor-beta receptor II].

OBJECTIVE: To investigate the role of Tumor necrosis factor receptor II (TNFR II) in preterm labor with chorioamnionitis and their gene polymorphisms in genetic susceptibility to preterm labor with chorioamnionitis in Chengdu. METHODS: We collected 46 cases maternal serum and partial placental tissues of preterm labor (21 cases of infectious group with chorioamnionitis, 25 cases of noninfectious group without chorioamnionitis), and 50 cases maternal serum and 20 cases placental tissues of term labor in corresponding period. TNFR II mRNA in placental tissue were tested by RT-PCR, maternal serum levels of sTNFR II were measured by ELISA. According to preliminary studies on TNFR II -196 site of the gene type, we analyze the sites of different genotypes in patients with premature placental TNFR II mRNA and maternal blood levels of sTNFR II difference, and with different genotypes chorioamnionitis relevance. RESULTS: In patients with preterm labor, the results of placental TNFR II mRNA and serum sTNFR II were no statistically significant higher in TG (GG) than in TT (P > 0.05). The levels of maternal serum sTNFR II and the mRNA expression of placental TNFR II in preterm labor with chorioamnionitis were significantly higher than those of preterm labor without chorioamnionitis and term labor (P < 0.05). There were no significant difference between term labor and preterm labor without chorioamnionitis (P > 0.05). Close correlation was observed between the different genotypes and the chorioamnionitis (chi2 = 11.088, P<0.05). The odds ratio (OR)for TG + GG genotype was 12.65, 95 CI 2.359-67.848, with more than 12.65 times probability of chorioamnionitis than that of TT genotype group. CONCLUSION: It suggested that TNFR II -196 polymorphism might not play a role by affecting TNFR II production in preterm labor. The site polymorphism is associated with higher serum sTNFR II and placenta TNFR II mRNA expression in patient with chorioamnionitis. It can be a useful marker for early prediction and diagnosis of preterm labor with chorioamnionitis.

[Gene polymorphism of tumor necrosis factor-alpha receptor II in 196 site and premature births in Chinese Han Population].

OBJECTIVE: To investigate the relationship between tumor necrosis factor receptor II (TNFR II) gene 196T/G polymorphism and preterm labor in Han population in Chengdu. METHODS: Samples were collected from 96 subjects in corresponding period, 46 preterm labor pregnant women (we collected partial placental tissues of preterm labor, 21 cases of infectious group with chorioamnionitis, 25 cases of noninfectious group without chorioamnionitis), and 50 normal labor pregnant women. The DNA was extracted from each sample by using Chelex-100 method, then PCR-RFLP was performed to determine the TNFR II 196 gene polymorphism. RESULTS: 1) TNFR II 196 genotype frequencies of 196M/M (TT), 196M/R (TG) + 196R/R (GG) were 71.7%, 21.7%+ 6.5% and 80.0%, 20.0%+0.0% in preterm labor and normal control group respectively. Allele frequencies of R (G), M (T) were 17.4%, 82.6% and 10.0%, 90.0%, respectively. There were no significant difference in frequencies of genotype and allele in TNFR II 196 gene polymorphism between two groups (P > 0.05, P > 0.05, respectively). 2) Close correlation was observed between the different genotypes and the chorioamnionitis (chi2 = 11.088, P < 0.05). The odds ratio (OR) for TG+GG genotype was 12.65, 95% CI 2.359, 67.848, with more than 12.65 times probability of chorioamnionitis than that of TT genotype group. CONCLUSION: Polymorphism in 196 site of TNFR II gene was not crucial in preterm labor genesis, TG (GG) genotype may contribute to susceptibility to chorioamnionitis in the process of preterm labor in Chinese Han population.

[Relationship among TNF-alpha gene promoter -308 site polymorphism, the levels of maternal serum TNF-alpha, and the mRNA expression placental TNF-alpha in preterm labor].

OBJECTIVE: To investigate the role of tumor necrosis factor-alpha (TNF-alpha) in preterm labor and gene polymorphisms of TNF-alpha in genetic susceptibility to preterm labor. METHODS: We collected maternal serum and partial placental tissues from 46 cases of preterm labor(21 cases of infectious proup with chorioamnionitis, 25 cases of noninfectious group without chorioamnionitis), while obtained maternal serum of 50 cases and placental tissues of 20 cases of term labor in contemporary period. TNF-alpha mRNA in placental tissue was tested by RT-PCR, Maternal serum level of TNF-alpha was measured by ELISA. Based on the genotype results of preliminary studies on TNF-alpha promoter -308 site, we analyzed the difference of placental TNF-alpha mRNA, maternal serum levels of TNF-alpha in different genotypes, and the relationship of different genotypes to chorioamnionitis. RESULTS: The levels of maternal serum TNF-alpha and the mRNA expression of placental TNF-alpha in preterm labor with chorioamnionitis were significantly higher than those of preterm labor without chorioamnionitis and term labor (P < 0.05). There was no significant difference between those of term labor and preterm labor without chorioamnionitis (P > 0.05). In patients with preterm labor, the levels of placental TNF-alpha mRNA and serum TNF-alpha were statistically significant higher in GA(AA) than in GG(P < 0.05), and GA(AA) has significant relationship with chorioamnionitis Codds ratio (OR) 4.22, 95% CI 1.197, 14.8963. CONCLUSION: It suggested that TNF-alpha -308 polymorphism was associated with the genetic susceptibility to preterm labor. TNF-alpha and its gene polymorphism can be a useful marker for early prediction and diagnosis of preterm labor and intrauterine infection, and may be a genomic index of poor prognosis.

[Influence of dexamethasone on expression of 11beta-HSD2 in primary cultured cytotrophoblasts from human preterm placenta].

OBJECTIVE: To study the influence of dexamethasone (DEX) on the expression of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) in primary cultured cytotrophoblasts from human preterm placenta. METHODS: Placenta villous cytotrophoblasts from preterm birth were cultured with the protocol of enzymatic dissociation and tissue incubation method. After isolation and identification, cytotrophoblasts were treated with the repeated administration of DEX (100 nmol/L), or DEX-RU486 (1 micromol/L) for 7 days, in which DEX was not added into the culture at 4th day. Cytotrophoblasts were collected everyday, and the expression levels of 11beta-HSD2 mRNA and protein were determined by real-time fluorescence quantitative PCR and Western blot method. RESULTS: After the treatment of DEX (100 nmol/L), the expression of 11beta-HSD2 mRNA and protein in cytotrophoblast increased in the first three days (P < 0.05). At 4th day, 11beta-HSD2 mRNA and protein declined in the absence of DEX. In 5th-7th day, the increase of 11beta-HSD2 expression were resumed when cytotrophoblasts received DEX again (P < 0.05). With the treatment of DEX and RU486 (l micromol/L), both mRNA and protein level of 11beta-HSD2 in cytotrophoblasts were lower than those with DEX alone, but there was not significantly different (P > 0.05). CONCLUSION: Repeated administration of DEX can upregulate the expression level of 11beta-HSD2 in primary cultured cytotrophoblasts from preterm placenta. Cytotrophoblasts from preterm birth may have the ability to protect the infants by the mechanism of 11beta-HSD2 regulation.

[Effect of N-acetylcysteine on lipopolysaccharide stimulating IL-8 expression of human uterine smooth cell].

OBJECTIVE: To investigate the effects of N-acetylcysteine (NAC)on the expression of IL-8 and the activity of NF-kappaB, which are induced by lipopolysaccharide (LPS) in human uterine smooth cell. METHODS: Human uterine smooth cells were primarily cultured and stimulated by LPS after pretreated by 5,10,15 mmol/L NAC and normal saline (NS). ELISA was performed to measure the expression of IL-8 protein in supernatants and RT-PCR was used to detect the IL-8 mRNA expression. The expression of NF-kappaB P65 protein was detected by Western Blotting. Furthermore, the expression of IL-8 mRNA and protein were detected at 0, 3, 6, 12, 24 and 48 h after LPS challenge in human smooth cell pretreated by 10 mmol/L NAC. RESULTS: (1) NAC significantly inhibited IL-8 mRNA expression and production in human uterine smooth cells with a concentration-dependent way (5-15 mmol/L). (2) The decreasing expression of NF-kappaB P65 protein was accompanied with increasing concentration of NAC in accordance with IL-8 mRNA (P < 0.01). CONCLUSION: NAC may inhibit the induction IL-8 gene and protein by LPS inhibiting NF-kappaB activation in human uterine smooth cell.

[Gene polymorphism of tumor necrosis factor-alpha promoter region in -308 site and premature births in Chinese Han populations].

OBJECTIVE: To investigate the relationship between the polymorphism of tumor necrosis factor-alpha gene--308G/A and premature births. METHODS: DNA was extracted from the blood samples taken from 96 women using chelex-100 method, among whom 46 gave premature births and 50 gave normal births. Then PCR-RFLP was performed to determine the TNF-alpha-308 gene polymorphism. RESULTS: (1) The frequencies of TNF-alpha -308 genotype TNF1/1 (GG) and TNF1/2 + TNF2/2 (GA + AA) were 60.9% and 32.6% +/- 6.5% for the women given premature births and 82.0% and 12.0% + 6.0% for the women given normal births, respectively. The allele frequencies of TNF1(G) and TNF2(A) were 77.2% and 22.8% for the women given premature births and 88.0% and 12.0% for the women given normal births, respectively. The differences were statistically significant (P < 0.05). (2) The women with genotype TNF1/2(GA) and TNF2/2(AA) were 2.929 (95% CI 1.152-7.447) times of those with TNF1/1(GG) in risk of giving premature births. The women with allele (TNF2) were also more likely to give premature births, with an odds ratio (OR) of 2.169 (95% CI 0.999, 4.709) compared to those with TNF1. CONCLUSION: Gene polymorphism of Tumor Necrosis Factor-alpha promoter region in--308 site is associated with increased risk of premature births. TNF2(A) allele may be a risk factor of premature births in Chinese populations.

[Effect of N-acetylcysteine on lipopolysaccharide induced preterm labor in mice].

OBJECTIVE: To test the effect of N-acetylcysteine (NAC) on the mice with infection associated preterm labor. METHODS: The pregnant C57BL/6 mice were randomly distributed into five groups, each with 20 mice and were given lipopolysaccharide (LPS), saline solution (NS), NAC, LPS+NAC (therapy), and NAC+ LPS (prevention) respectively. The LPS (20 microg) was injected intraperitoneally to the mice every 12 hours since day 16 of gestation to induce preterm labor. The NAC was orally administered (0.5 g/kg) every 12 hours since day 15 or day 16 for the prevention and therapy groups respectively. The latency interval (from LPS injection to delivery of the first pup) and fetal survival rates were recorded in eight mice from each group. The remaining mice were killed at 4, 8, 12, and 24 hours after the LPS injection (three for a group each time). The NF-kappaB activity and IL-8 mRNA in uterine and maternal and fetal hepatic GSH-PX and serum IL-8, MDA, and SOD were examined. RESULTS: The average latency interval of LPS-treated mice was (15.1 +/- 1.9) hours, with a fetal survival rate of 4.3%. The NAC therapy extended the latency interval of LPS-treated mice to (35.4 +/- 2.1) hours, with a fetal survival rate of 69.0%. The NAC prevention extended the latency interval of LPS-treated mice to (44.8 +/- 2.6) hours, with a fetal survival rate of 84.3%. The expression of NF-KB P65 was activated and reached the peak 4 hours after the LPS injection. The uterine IL-8 mRNA and serum IL-8 and MDA reached the peak whereas the maternal and fetal hepatic GSH and SOD declined to the lowest 8 hours after the LPS injection. The NAC significantly inhibited the effect induced by the LPS (P < 0.01). CONCLUSION: The NAC has a therapeutic effect on the LPS-induced preterm labor in mice. It is even better to be used in preventing preterm labor. The mechanism of the protective effect of NAC may include the deactivation of the NF-kappaB/IL-8 and the reduce of the production of ROS.