The role of high mobility group box 1 in the injury of Caco-2 epithelial barrier induced by lipopolysaccharide / 中华急诊医学杂志

Objective:To investigate the role and mechanism of high mobility group box 1(HMGB1) in the injury of Caco-2 intestinal epithelial barrier induced by lipopolysaccharide (LPS).Methods:The Caco-2 cellular monolayer barrier was established with Transwell chamber. After the Caco-2 monolayer model was established, the transepithelial electrical resistance (TEER) values were measured. When the TEER value reached 500 Ω·cm 2, the cells were divided into 3 groups: control group, LPS treatment group, and LPS+ ethyl pyruvate (EP) treatment group. The concentration of LPS and EP were 100 μg/mL, 50 μg/mL, separately. Then TEER values were measured at 12, 24, 48 and 72 h, and FITC-dextran permeability was detected at 24 h. The cells were seeded on 6-well plates. After cell density reached 80%, treatments were given as the above. The real-time polymerase chain reaction (RT-PCR) and Western blot were used to measure the changes in the protein and mRNA expressions of Occludin, HMGB1, and nuclear factor-κB (NF-κB). Results:Compared with the control group, the TEER values (Ω·cm 2) reduced at 12, 24, 48 and 72 h in the LPS treatment group [(514.22±12.59) vs (304.96±9.69), (521.65±13.35) vs (276.21±7.82), (523.99±8.18) vs (206.64±15.85), (491.21±6.72) vs (156.33±10.83), all P<0.05]. The FITC-dextran permeability increased significantly at 24 h [(2.58±0.07) vs (1.04±0.06), P<0.05]. The expression levels of Occludin protein and mRNA were decreased (all P<0.05), while the expression levels of HMGB1 and NF-κB protein and mRNA were significantly increased (all P<0.05). Compared with the LPS treatment group, the TEER values (Ω·cm 2) increased significantly at 12, 24, 48 and 72 h in the EP treatment group [(519.00±5.66) vs (304.96±9.69), (504.69±8.57) vs (276.21±7.82), (453.65±10.74) vs (206.64±15.85), (385.28±7.57) vs (156.33±10.83), all P<0.05]. The FITC-dextran permeability decreased at 24 h [(1.23±0.11) vs (2.58±0.07), P<0.05]. The expression level of Occludin protein and mRNA were increased ( P<0.05), while the expression levels of HMGB1 and NF-κB protein and mRNA were significantly decreased (all P<0.05). Conclusions:LPS can injure intestinal barrier directly in vitro and reduces the expression of tight junction proteins between cells. The mechanism may be related to the increased expression of HMGB1 and NF-κB protein.

Metabolic engineering of Escherichia coli for production of hydroxytyrosol / 生物工程学报

Hydroxytyrosol is an important fine chemical and is widely used in food and medicine as a natural antioxidant. Production of hydroxytyrosol through synthetic biology is of important significance. Here we cloned and functionally characterized a hydroxylase encoding gene HpaBC from Escherichia coli BL21, and both subunits of this enzyme can be successfully expressed to convert the tyrosol into hydroxytyrosol. A HpaBC gene integration expression cassette under the tac promoter was constructed, and integrated into the genome of a tyrosol hyper-producing E. coli YMG5A*R using CRISPR-Cas9 technology. Meanwhile, the pathway for production of acetic acid was deleted, resulting in a recombinant strain YMGRD1H1. Shake flask fermentation showed that strain YMGRD1H1 can directly use glucose to produce hydroxytyrosol, reaching a titer of 1.81 g/L, and nearly no by-products were detected. A titer of 2.95 g/L was achieved in a fed-batch fermentation conducted in a 5 L fermenter, which is the highest titer for the de novo synthesis of hydroxytyrosol from glucose reported to date. Production of hydroxytyrosol by engineered E. coli lays a foundation for further construction of hydroxytyrosol cell factories with industrial application potential, adding another example for microbial manufacturing of aromatic compounds.

Characterization of a sucrose phosphorylase from Leuconostoc mesenterides for the synthesis of α-arbutin / 生物工程学报

Sucrose phosphorylase (SPase) gene from Leuconostoc mesenteroides ATCC 12291 was synthesised after codon optimization, and inserted into pET-28a plasmid to generate pET-28a-spase. The recombinant strain Escherichia coli BL21 (DE3)/pET-28a-spase was induced for Spase expression. The recombinant protein Spase was purified and characterized. The specific enzyme activity of SPase was 213.98 U/mg, the purification ratio was 1.47-fold, and the enzyme activity recovery rate was 87.80%. The optimal temperature and the optimal pH of the SPase were identified to be 45 °C and 6.5 respectively, and Km, Vmax and kcat of the SPase for sucrose was 128.8 mmol/L, 2.167 μmol/(mL·min), and 39 237.86 min-1. The recombinant SPase was used for α-arbutin production from hydroquinone and the reaction process was evaluated. The optimal conditions for synthesis of α-arbutin by SPase were 40 g/L hydroquinone, 5:1 molar ratio of sucrose and hydroquinone, and 250 U/mL recombinant SPase at pH 7.0 and 30 °C for 24 h in the dark, and then 500 U/mL glucoamylase was added at 40°C for 2.5 h. Under the optimized process, the yield of α-arbutin reached 98 g/L, and the hydroquinone conversion rate was close to 99%. In summary, the recombinant SPase was cloned and characterized, and its application for α-arbutin production was feasible.

Role of high mobility group box 1 in intestinal mucosal barrier injury in rat with sepsis induced by endotoxin / 中华危重病急救医学

Objective:To investigate the role and mechanism of the high mobility group box 1 (HMGB1) in intestinal mucosal barrier injury in rat with sepsis induced by endotoxin lipopolysaccharide (LPS).Methods:The rats were given intraperitoneal injection of LPS to reproduce a model of sepsis. The effect of HMGB1 inhibitor EP solution (40 mg/kg) on sepsis was observed, and phosphate buffer (PBS) control group was set up. Seventy-two hours after modeling, abdominal aortic blood was obtained, and enzyme-linked immunosorbent assay (ELISA) was used to measure the plasma levels of D-lactic acid and diamine oxidase (DAO) of mucosal barrier permeability. The pathological changes of the intestinal mucosal were observed with light microscope and the Chiu score was recorded. The intestinal mucosal ultrastructural changes were observed with electron microscopy. Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and Western Blot were used to measure the mRNA and protein expressions of Occludin, inflammatory factor HMGB1 and its downstream signal molecule nuclear transcription factor-κB p65 (NF-κB p65) in the rat small intestine.Results:The results of histopathology and ultrastructure of the small intestine showed that in the LPS group, the intestinal mucosa tissue swelled obviously, part of the glands were incomplete, the infiltration of neutrophils increased, themicrovillus cells were absent, arranged indisorder, and the number of tight connections significantly reduced compared with the PBS control group. The levels of D-lactic acid and DAO indicating mucosal barrier permeability, the levels of inflammatory factor HMGB1 and its downstream signaling molecule NF-κB p65 mRNA and protein expressions in the LPS group were significantly higher than those in the PBS control group, and the mRNA and protein expression of Occludin in the small intestine was significantly lower than that in the PBS control group, suggesting that the intestinal mucosal barrier function in septic rats was damaged, permeability increased, and the structure was damaged. After the administration of the HMGB1 inhibitor EP, the intestinal mucosal barrier damage was significantly improved. The performance was as follows: the Chiu score of the small intestine tissue and the plasma D-lactic acid and DAO levels in the EP intervene group were significantly lower than those in the LPS group [Chiu score: 1.60±0.48 vs. 3.40±0.48, D-lactic acid (mmol/L): 3.30±0.22 vs. 5.30±0.16, DAO (U/L): 23.66±0.97 vs. 30.47±1.11, all P < 0.05]. Occludin mRNA and protein expression levels were significantly higher than those in the LPS group [Occludin mRNA (2 -ΔΔCt): 0.82±0.05 vs. 0.37±0.08, Occludin protein (Occludin/β-actin): 1.04±0.09 vs. 0.75±0.11, both P < 0.05], while the mRNA and protein expression levels of HMGB1 and NF-κB p65 were significantly lower than those in the LPS group [HMGB1 mRNA (2 -ΔΔCt): 1.63±0.10 vs. 3.57±0.10, HMGB1 protein (HMGB1/β-actin): 1.40±0.07 vs. 1.87±0.07; NF-κB p65 mRNA (2 -ΔΔCt): 1.47±0.09 vs. 2.62±0.13, NF-κB p65 protein (NF-κB p65/β-actin): 1.24±0.14 vs. 1.60±0.13, all P < 0.05]. Conclusions:Intestinal mucosal barrier function of septic rats was damaged, permeability increased, and structure was damaged. The mechanism may be that the expression of inflammatory factor HMGB1 was up-regulated and promoted the activation of its downstream signaling molecule NF-κB, thereby mediated the inflammatory cascade reaction and caused damage to the intestinal mucosa.

Effects of bone marrow mesenchymal stem cells on the expressions of inflammatory factors in rats with multiple organ dysfunction syndrome / 中华急诊医学杂志

Objective To investigate the effects of bone marrow mesenchymal stem cells (BMSC) on the expression of inflammatory factors in rats with multiple organ dysfunction syndrome (MODS). Methods BMSC extracted from the 4-week-old Sprague-Dawley (SD) rats was cultivated and identified in vitro, then the 4th passage of which was used in the experimental study. Sixty SD rats were randomly(random number) divided into three groups (n=20 in each group): Sham group (SG), MODS group (MG) and BMSC group (BG). Rats in the MG was injected by 1 mg/kg lipopolysaccaride (LPS) via great saphenous vein, rats in the SG injected with the same volume sterile phosphate buffer saline and rats in the BG infused by 1×106/cells BMSCs through the tail vein at 2 h after LPS injection. The survival rate, tissue pathological changes of the lung, liver and heart by hematoxylin and eosin (HE) staining, organ dysfunction measurement by blood gas analysis and biochemical indicators as well as the related inflammatory factors by protein microarray and enzyme linked immunosorbent assay (ELISA), were detected 72 h post operation. Multi-group quantitative data was analyzed by one way ANOVA, paired-comparisons by LSD-t test and the comparisons of survival curves in the three groups by Log-rank test. The value of P<0.05 was considered statistically significant. Results The survival rate in SG, MG and BG was 100％, 60％ and 80％, respectively. The survival curves showed that the survival rate of SG was higher than the MG and BG (SG vs. MG, χ2=9.798, P=0.0017; SG vs. BG, χ2=4.333, P=0.0374), but there was no significant difference comparing the BG to the MG (χ2=2.408, P=0.1207). The tissue congestion and edema, and inflammatory cells infiltration in the lung, liver, and heart of the MG were observed by HE staining, while these changes reduced in the BG. Compared with the SG, the levels of pH and PaCO2 and lactic acid (Lac) increased significantly (all P<0.01), the level of total bilirubin (TB) significantly increased [(0.801±0.501)U/L vs. (2.533±0.382)U/L, P=0.003], while the albumin(ALB) level decreased significantly[(35.471±4.015)U/L vs. (23.202±4.872)U/L, P<0.01], and creatine kinase (CK) level increased significantly in MG [(315.670±41.402) vs. (708.250±219.201), P=0.042]. After BMSC treatment, the organ function improved significantly (all P<0.05). Gamma interferon (IFN-γ) and monocyte chemotactic protein 1 (MCP-1) were the differential expression factors in protein chips. The results of ELISA were similar to the protein chips: compared with the SG, IFN-γ and MCP-1 expressions in the MG increased significantly (P<0.01). Compared with MG, the expressions of IFN-γ and MCP-1 decreased significantly in the BG (P<0.01). Conclusion BMSC administration could modulate the inflammatory response of MODS rats by inhibiting the levels of IFN-γ and MCP-1, and improve the organ function and the survival rate.

Role of HMGB1-RAGE / TLRs-NF-κB signaling pathway on bone mesenchymal stem cells transplantation therapy for lipopolysaccaride-induced coagulation disorder rats / 中华危重病急救医学

Objective To determine the effect of bone mesenchymal stem cells (BMSCs) in transplantation therapy for lipopolysaccharide (LPS)-induced coagulation disorder and the underlying mechanism of high mobility group protein B1-receptors for advanced glycation end products / Toll-like receptors-nuclear factor-κB (HMGB1-RAGE / TLRs-NF-κB) signaling pathway.Methods BMSCs of female Sprague-Dawley (SD) rats ageing 4-5 weeks old were extracted and cultivatedin vitro, and the fourth-passaged BMSCs phenotype was identified by flow cytometry for transplantation in the following experimental study. The rats were randomly divided into normal saline (NS) control group, LPS group, and BMSC group according to the random number table with 15 rats in each group. Coagulation disorders model was reproduced by injection of 1 mg/kg LPS via saphenous vein, and the rats in the NS control group was injected with equal volume NS. Those in the BMSC group were infused BMSC 0.5 mL containing 1×106 cells via tail vein at 2 hours after LPS injection, and the rats in other groups were injected with equal volume NS. Abdominal aorta blood was collected at 1, 3 and 7 days post operation. Coagulation indexes such as platelet count (PLT), platelet volume distribution width (PDW), mean platelet volume (MPV), plateletcrit (PCT), platelet large cell ratio (P-LCR), activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), international normalized ratio (INR), and fibrinogen (FIB) were determined. The mRNA levels and contents of HMGB1, RAGE, TLR2/4 and NF-κB were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively.Results ① The cells culturedin vitro were spindle shaped or flat. The fourth-passaged BMSCs phenotype was successfully identified by flow cytometry technology. ②Coagulation indexes: compared with NS control group, PLT, PCT and FIB in LPS group were significantly decreased, PDW, MPV, P-LCP, and INR were significantly increased, and APTT, PT, and TT were significantly prolonged from the first day. Furthermore, those in LPS group were gradually ameliorated with prolongation of LPS induction time. The coagulation function abnormality induced by LPS was reversed by BMSCs with significant difference at 1 day as compared with LPS group [PLT (×109/L):398.8±17.9 vs. 239.1±15.8, PCT (％): 0.35±0.04 vs. 0.23±0.06, FIB (g/L): 1.7±0.6 vs. 0.8±0.1, PDW (％):12.4±1.6 vs. 16.2±1.5, MPV (fl): 11.0±1.6 vs. 13.7±1.1, P-LCP (％): 13.0±2.1 vs. 15.3±2.7, INR: 1.52±0.17 vs. 1.82±0.19, APTT (s): 66.3±4.1 vs. 89.5±4.5, PT (s): 18.3±0.7 vs. 25.1±1.9, TT (s): 87.5±7.8 vs. 115.0±9.7, allP < 0.05], till 7 days. ③ HMGB1-RAGE / TLRs-NF-κB signaling pathway related molecules: compared with NS control group, the mRNA expressions and contents of HMGB1, RAGE, TLR2/4 and NF-κB were significantly increased in LPS group from the first day. However, the mRNA expressions and contents of the molecules in LPS group were gradually decreased with prolongation of LPS induction time. After BMSC intervention, the mRNA expressions and contents of molecules at 1 day were significantly lower than those of LPS group [HMGB1 mRNA (2-ΔΔCt): 10.77±0.04 vs. 24.51±3.69, HMGB1 content (μg/L): 0.48±0.01 vs. 0.95±0.06; RAGE mRNA (2-ΔΔCt): 11.57±1.11 vs. 18.08±0.29, RAGE content (μg/L): 0.73±0.04 vs. 1.37±0.06; TLR2 mRNA (2-ΔΔCt): 2.60±0.22 vs. 12.61±0.27, TLR2 content (μg/L): 0.81±0.03 vs. 1.59±0.09; TLR4 mRNA (2-ΔΔCt): 2.95±0.52 vs. 4.06±0.11, TLR4 content (μg/L):0.80±0.09 vs. 1.18±0.11; NF-κB mRNA (2-ΔΔCt): 1.29±0.06 vs. 7.79±0.25, NF-κB content (μg/L): 1.22±0.24 vs. 2.42±0.26, allP < 0.05], till 7 days.Conclusion BMSCs administration could ameliorate the coagulation function in LPS-induced coagulation disorder rats and these might be associated with HMGB1-RAGE / TLRs-NF-κB signaling pathway inhibition.

Surface display of phytase on Saccharomyces cerevisiae for efficient bioethanol production from corn starch / 生物工程学报

Production of bioethanol using starch as raw material has become a very prominent technology. However, phytate in the raw material not only decreases ethanol production efficiency, but also increases phosphorus discharge. In this study, to decrease phytate content in an ethanol fermentationprocess, Saccharomyces cerevisiae was engineered forheterologous expression of phytase on the cell surface. The phy gene encoding phytase gene was fused with the C-terminal-half region of α-agglutinin and then inserted downstream of the secretion signal gene, to produce a yeast surface-display expression vector pMGK-AG-phy, which was then transformed into S. cerevisiae. The recombinant yeast strain, PHY, successfully displayed phytase on the surface of cells producing 6.4 U/g wet cells and its properties were further characterized. The growthrate and ethanol production of the PHY strain were faster than the parent S. cerevisiae strain in the fermentation medium by simultaneous saccharification and fermentation. Moreover, the phytate concentration decreased by 91% in dry vinasse compared to the control. In summary, we constructed recombinant S. cerevisiae strain displaying phytase on the cell surface, which could effectively reduce the content of phytate, improve the utilization value of vinasse and reduce the discharge of phosphorus. The strain reported here represents a useful novel engineering platform for developing an environment-friendly system for bioethanol production from a corn substrate.

Rational design and construction of an overproducing shikimic acid Escherichia coli by metabolic engineering / 生物工程学报

Shikimic acid (SA), as a hydroaromatic intermediate in the common pathway of aromatic amino acid biosynthesis, is the starting material for the synthesis of neuraminidase inhibitors and other useful compounds. The fermentative production of SA by metabolically engineered microorganisms is an excellent alternative to the extraction from fruits of the Illicium plant. In this study, Escherichia coli was metabolically engineered by rational design and genetic manipulation for fermentative production of SA. Firstly, blocking the aromatic amino acid pathway after the production of SA was carried out by deletion of aroL and aroK genes encoding SA kinase. Secondly, the ptsG gene encoding protein EIICBglc were removed in the aroL/aroK mutant strain to make the phosphotransferase system (PTS) system default. In the resulting strain, the phosphoenolpyruvate-dependent PTS pathway, a main pathway for glucose transport, were replaced by ATP-dependent GalP (galactose permease). Thus, more PEP flux was used to produce SA as a critical precursor of SA. Furthermore, ydiB gene (encoding quinic acid/SA dehydrogenase) was deleted to prevent SA precursors of 3-dehyroquinic acid into the byproduct of quinic acid. Thus, the engineered strain with four genes deletion was constructed and 576 mg/L SA was produced in the shake flask fermentation. Results show that SA produciton was increased 90 times compared to the parent strain E. coli CICIM B0013.

Temperature-switched high-efficiency D-lactate production from glycerol / 生物工程学报

Glycerol from oil hydrolysis industry is being considered as one of the abundent raw materials for fermentation industry. In present study, the aerobic and anaerobic metabolism and growth properties on glycerol by Esherichia coli CICIM B0013-070, a D-lactate over-producing strain constructed previously, at different temperatures were investigated, followed by a novel fermentation process, named temperature-switched process, was established for D-lactate production from glycerol. Under the optimal condition, lactate yield was increased from 64.0% to 82.6%. Subsequently, the yield of D-lactate from glycerol was reached up to 88.9% while a thermo-inducible promoter was used to regulate D-lactate dehydrogenase transcription.

Elimination of succinate and acetate synthesis in recombinant Escherichia coli for D-lactate production / 生物工程学报

When Escherichia coli CICIM B0013-030 (B0013, ack-pta, pps, pflB) was used for D-lactate production, succinate and acetate were the main byproducts (as much as 11.9 and 7.1% the amount of lactate respectively). In order to decrease the byproduct levels, we inactivated succinate and acetate synthesis in B0013-030. Two recombinant plasmids containing mutation cassettes of frdA::difGm and tdcDE::difGm respectively were constructed first. The mutation cassettes were used to delete the target genes on the chromosomal by Red recombination. Subsequently, the antibiotic resistance gene was excised from the chromosomal by Xer recombination. Thereby, mutants B0013-040B (B0013-030, frdA) and B0013-050B (B0013-040B, tdcDE) were produced. D-lactate producing abilities of the engineered strains were tested both in shake flasks and in bioreactors using two-phase fermentation (aerobic growth and anaerobic fermentation) with glucose as the sole carbon source. When fermentation was carried out in shake flasks, inactivation of frdA in B0013-030 to produce B0013-040B reduced succinate accumulation by 80.8%. When tested in a 7-liter bioreactor, B0013-040B accumulated 114.5 g/L D-lactate of over 99.9% optical purity. However, 1.0 g/L succinate and 5.4 g/L acetate still remained in the broth. Further inactivation of tdcD and tdcE genes in B0013-040B to produce B0013-050B decreased acetate and succinate accumulation to 0.4 g/L and 0.4 g/L respectively, and lactate titer was as much as 111.9 g/L (tested in the 7-liter bioreactor). In lightof the lower byproduct levels and high lactate production, strain B00 13-050B may prove useful for D-lactate production.

Hemodynamics of deep vein in lower limb during perioperative period of total hip arthroplasty / 中华创伤杂志

Objective To investigate hemodynamic changes of deep vein in lower limb during the perioperative period of total hip arthroplasty (THA) and early diagnosis of deep vein thrombosis (DVT). Methods Doppler ultrasound, hemorheology detection and plasma D-dimer testing were done on 62 patients treated with THA. Statistical analysis was carried out on the data of patients with or without DVT to study the early diagnosis of DVT. Results The results of Doppler ultrasound showed DVT in 8 patients. Compared with postoperative concentration of plasma D-dimer, the preoperative con-centration of plasma D-dimer was significandy higher in patients with or without DVT (P < 0.05). The levels of hemorheological indices were significantly increased at postoperative day 7 (P < 0.05). Con-clusions Doppler ultrasound combined with plasma D-dimer testing and hemorheology detection are helpful in early diagnosis of DVT.