Corynebacterium glutamicum SA001 is a mutant with lactate dehydrogenase (ldhA) deletion. In order to increase metabolic flux from isocitrate to succinate, and to improve the production of succinate under anaerobic conditions,we transducted the gene aceA coding isocitrate lyase (ICL) from Escherichia coli K12 into Corynebacterium glutamicum SA001 (SA001/pXMJ19-aceA). After 12 h aerobic induction by adding 0.8 mmol/L of IPTG, the recombinant strain was transferred to anaerobic fermentation for 16 h. Succinate reached 14.84 g/L, with a productivity of 0.83 g/(L x h). Compared to C. glutamicum SA001, the activity of ICL of the recombinant strain was increased 5.8-fold, and the succinate productivity was increased 48%. Overexpression of isocitrate lyase will increase the metabolic flux of glyoxylate bypass flowing to succinate.
Corynebacterium glutamicum ; genetics ; metabolism ; Escherichia coli ; enzymology ; genetics ; Gene Deletion ; Industrial Microbiology ; Isocitrate Lyase ; biosynthesis ; genetics ; L-Lactate Dehydrogenase ; genetics ; Succinic Acid ; metabolism ; Transduction, Genetic

Objective To explore the types and drug resistance of pathogens in patients with type Ⅰ incision surgical site infection in orthopedics department.Methods Patients with type Ⅰ incision surgical site infection in orthopedics department at Peking University Third Hospital from January 2005 to December 2013 were retrospectively collected.Clinical characteristics of patients,distribution and drug resistance of pathogens were analyzed.Results A total of 58.2 thousands patients with type Ⅰ incision surgical site were hospitalized from January 2005 to December 2013 in orthopedics department,and among them 442 patients had infection in the type Ⅰ incision surgical site.The infection rate was 0.8％.Infection was mainly observed in elderly patients.The most common diseases were lumbar canal stenosis (21.7％),cervical spondylosis (20.6％) and lumbar intervertebral disc herniation (14.0％).A total of 453 pathogenic strains were detected,of which 52.9％ were gram-positive bacteria,45.5％ were gramnegative bacteria and 1.6 ％ were fungi.The common pathogens were Staphylococcus aureus (25.2 ％),Staphylococcus epidermidis (14.1 ％),Escherichia coli (11.5 ％),Enterobacter cloacae (7.3 ％),Pseudomonas aeruginosa (6.2 ％) and Acinetobacter baumannii (6.0 ％).The percentage of Meticillinresistant Staphylococcus aureus (MRSA) was 23.7％ and the percentage of Meticillin-resistant Staphylococcus epidermidis (MRSE) was 43.8％.Vancomycin or linezolid-resistant Staphylococcus aureus or Staphylococcus epidermidis were not detected.Proportion of extended-spectrum beta-lactamases (ESBL) producing strains in Escherichia coli was 53.8％,and proportion of ESBL-producing strains in Klebesiella pneumonia was 50.0％.The resistance rates to impenem and meropenem of the three different species in Enterobacteriaceae,including Escherichia coli,Enterobacter cloacae and Klebsiella pneumonia,were 0.Resistance rates of Pseudomonas aeruginosa to cefoperazone-sulbactam,piperacillin-tazobactam were less than 10 ％.Resistance rate of Acinetobacter baumannii to minocyline was 11.1％ and resistance rates of it to other drugs were more than 20％.Conclusions The rate of type Ⅰ incision surgical site infection in orthopedics department is low.Gram-positive and gram-negative bacteria each account for half of the pathogens.The proportion of resistant pathogens is high and empirical treatment is needed to cover these pathogens.

Objective To investigate the protective effects of propofol against acute lung injury(ALI) induced by oleic acid.Methods Forty adult male SD rats weighing 250-290 g were anesthetized with intraperitoneal(i.p.)20% urethrane 6 ml?kg~(-1) and tracheostomized.Left common carotid artery and right internal jugular vein were cannulated for BP monitoring and fluid and drug administration.The animals were randomly divided into 5 groups (n=8 each):Ⅰ control group;Ⅱ ALI group in which ALI was induced by oleic acid 250 mg?kg~(-1) i.v.;Ⅲ,Ⅳ and Ⅴ ALI+propofol group in which propofol was continuously infused i.v.at 4, 8 and 16 mg?kg~(-1)?h~(-1) for 4 h immediately after i.v.oleic acid.The animals were killed at 4 h after oleic acid administration.The lungs were immediately removed for(1)examination of ultrastructure of the lung with transmission electron microseope and(2)determination of SOD and MPO activity,content of MDA,level of IL-10 and IL-18 and expression of NF-kB in lung tissue.Results In group Ⅱ intravenous oleic acid produced damage to mitochondria,rough endoplasmic reticulum and osmiophilic multi-lamellar body in type Ⅱ alveolar epithelial cells. Propofol infusion in group Ⅲ,Ⅳ and Ⅴ attenuated the damage to different degrees.In group Ⅱi.v.oleic acid produced significant decrease in MPO and SOD activity and significant increase in MDA content,IL-10,IL-18 and NF-kB expression in lung tissue.Intravenous propofol infusion attenuated the decrease in MPO and SOD activity, increase in IL-18 expression and MDA content and NF-kB expression in lung tissue produced by i.v.oleic acid, but increased IL-10 expression in lung tissue further.The best protective effect was seen in group Ⅳ.Conclusion Propofol i.v.infusion at 4-16 mg?kg~(-1),h~(-1) can inhibit the oxidative response and inflammatory response and down-regulate NF-kB expression in lung tissue.Propofol infusion at 8 mg?kg~(-1)?h~(-1) provides best protective effects.

Objective To compare the effect of botulinum toxin A(BTX-A)applied according to experi- ence with its effect when the application is guided by electrical stimulation on spasticity in the ankle plantarflexors of children with cerebral palsy(CP).Methods Forty-five children with CP were randomly assigned into 2 groups to receive injections of BTX-A guided by electrical stimulation,or injections of BTX-A guided by experience.All chil- dren received a local injection in the ankle plantar flexors.Physiotherapy and ankle-foot orthoses were applied by a physical therapist 3 days after the BTX-A injections.After the first 10 days,the therapy was administered by the patient's family.Clinical assessments included the patient's passive range of movement(PROM),scoring on the Ash- worth scale(MAS),the composite spasticity scale(CSS),and the D and E dimensions of the gross motor function measure(GMFM),and walking velocity(WV).Assessments were performed before treatment and at 3 days,2 weeks,1,2,and 3 months following the injection with BTX-A.Results All children showed significant decrease in spasticity(PROM,MAS and CSS)after 3 days.The improvement was maintained at 3 months.When compared with the results before the injection,the improvements in standing and walking(GMFM)and in walking velocity were statistically significant after 2 weeks of treatment for both groups,and were maintained at 3 months.The differences in PROM and CSS scores at 3 days,2 weeks,1,2,and 3 months following the injection were statistically significant between the 2 groups.Significant differences were also found between the 2 groups in MAS scores at 3 days,2 and 3 months after treatment,and in GMFM and WV at 2 and 3 months after treatment.Conclusions A BTX-A injec- tion,whether guided by electrical stimulation or experience,in combination with physiotherapy,can reduce spasticity in the ankle plantarflexors of ambulant children with CP and improve their functional performance.BTX-A injection guided by electrical stimulation was more effective than an injection guided by experience.

Objective To investigate the effects of Panax notoginseng saponins(PNS)on both the excitatory and inhibitory synaptic transmission in the pyramidal neurons in hippocampal CA1 region of rats.Methods Wistar male rats(3—4 weeks)were killed by cervical dislocation and hippocampal slices(400 ?m)were prepared,blind whole-cell voltage-clamp recordings were performed on the CA1 pyramidal cells in hippocampal slices to examine and analyze the effects of PNS(0.05—0.4 g/L)on CA1 afferent fiber-evoked excitatory postsynaptic currents(EPSCs)and inhibitory postsynaptic currents(IPSCs),respectively.Moreover,the Schaffer collateral/commissural pathway was stimulated with paired pulses(interpulse interval was 50 ms)and the paired-pulse facilitation(PPF)was analyzed by EPSC2/EPSC1(P2/P1)ratio.Results PNS(0.1—0.4 g/L)significantly depressed amplitude of EPSCs in neurons in the hippocampal CA1 region(P0.05).Conclusion The inhibitory effect of PNS on EPSCs in hippocampal CA1 pyramidal neurons is not due to the reinforcement of the inhibiting interneurons.It may be a result of direct inhibition on excitatory synaptic transmission.The increasing of P2/P1 ratio after PNS application suggests that PNS depresses the excitatory synaptic transmission by presynaptic mechanism.

Objective To investigate the accelerating role of recombinant human parathyroid hormone (PTH) in bone fracture repair.Methods 2-month old male Sprague-Dawley rats underwent closed unilateral femoral fracture and intramedullary nail fixation.The rats were divided into 2 equal groups randomly: the treatment group receiving subcutaneous injection of rhPTH(1-34) 10 μg/(kg·d) immediately after operation and for 2,7,14,21 and 42 d,respectively, and the control group receiving subcutaneous injection of normal saline in the same volume.X-ray and micro-CT were conducted at 2, 7, 14, 21 and 42 days after surgery.Results The continuity of porosis between fracture sides was better and fracture line has been blurred in the PTH-treated group at 21 days after fracture compared with the control group, the bone volume (BV),BV/TV, bone mineral density(BMD)and trabecular pattern factor (Tb.Pf) were significantly higher, and trabecular separation (Tb.Sp) and degree of anisotropy (DA) were significantly lower in the PTH-treated group at 42 days after fracture.Conclusions Our findings suggest that a low dose recombinant human parathyroid hormone can accelerate the bone fracture healing, probably through improving the BV, BV/TV, Tb.P and BMD, and decreasing the Tb.Sp and DA.

Objective To establish embryonic stem cells (ESC) that can express green fluorescent protein (CFP) and differentiate them into neurons. It would provide tagging neurons for clinical transplantation to cure neural system diseases. Methods ESC (R1) was transfeeted with a plasmid containing the GFP by electroporation. A transgeuic cell line was obtained after selection with G418. The ESCs were characterized by AKP staining. Monolayer differentiation method was used to induce neural differentiation derived from GFP-ESC and immunofluorescence method was used to identify Tuj1 positive cells. Results There was no significant difference(X2=3.14,P0.05) in transfect rates between liposome and electroporation (65% vs 79%). The AKP staining of GFP-ESC was positive. GFP-ESC could be differentiated into neural cells. Conclusions These results show that ESC expressing GFP has been estabhshed, which can be differemiated into neurons.

Objective To study the effects of Liensinine on apoptosis of 5637 cells, and its mechanism thereof. Meth?ods CCK-8 method and the colony formation test were used to detect cell viabilities, and then inhibition rates were calcu?lated. Flow cytometry was used to detect the effects of Liensinine on apoptosis of 5637 cells. Western blot assay was used to detect Caspase-7 protein expression. Results CCK-8 assay and colony formation test indicated that Liensinine inhibited the cell proliferation significantly. Results of flow cytometry indicated that Liensinine induced early apoptosis of 5637 cells. Western blot assay showed that Liensinine improved the expression of Caspase-7 and enhanced the activation of Caspase-7 in 5637 cells. Conclusion Liensinine could inhibit the proliferation of 5637 cells, induce early apoptosis, which may be re?lated with the enhanced expression of Caspase-7 and its activation.

Objective To study the effect of liensinine on the proliferation of human bladder cancer T24 cells.Methods T24 cells were treated with different concentrations of liensinine.Its influence on the cell proliferation was detected by the CCK-8 exper-iment and the clonogenic experiment.After staining of T24 cells,the influence of liensinine on the cell cycle was examined by the flow cytometry.The mRNA change of p2 1 gene was determined by real-time quantitative PCR.Results Compared with the con-trol,liensinine significantly inhibited the proliferation of T24 cells in different doses groups(1.562 5,3.125 0,6.250 0,12.500 0, 25.000 0μg/mL),the differences had statistical significance and showed the dose-dependence;the cell cycle detection results re-vealed that liensinine arrested the T24 cells at the S phase;the real-time quantitative PCR detection results showed that liensinine increased mRNA of p21 gene in T24 cells.Conclusion Liensinine inhibits the proliferation of T24 bladder cancer cells and arrests the T24 cells at S phase,its mechanism may be related with the upregulation of p21 expression.

Objective To investigate the effect of high mobility group box-1 protein (HMGB1) on cytokine expression in splenic dendritic cell (DCs). Method DCs isolated from the spleens of male Wistar rats were seed-ed on 96-well (1×10s cells/well) cell culture plates, and the cells were stimulated with HMGB1 for various length of time or in different concentrations. (1) The time-dependent response between HMGB1 and tumor necrosis factor-α(TNF-α) as well as interleukin-12 (IL-12) gene/protein expressions: 24 wells of DCs were dividedly into six groups including 24 h-normal controls (n=4), 48 h-normal controls (n=4), 72 h-normal controls (n=4), and 24 h-HMGB1 treated group (n=4), 48 h-HMGB1 treated group (n=4) as well as 72 h-HMGB1 treated group (n=4), respectively. Among three HMGB1-treated groups, DCs were stiraulated by 1 μg/mL HMGB1. DCs were denatured in cell culture plates to determine gene expression of IL-12 as well as TNF-α, and supernatants were harvested to determine Il-12 as well as TNF-α protein levels. (2) The dose-dependent response between HMGB1 and TNF-α as well as IL-12 gene/protein expressions: 16 wells of DCs were dividedly into four groups in-cludingnormal controls (n=4), 0.1 μg/mL HMGB1 treated group (n=4), 1 μg/mL HMGB1 treated group (n =4), and 10 μg/mL HMGB1 treated group (n=4), respectively. After stimulated for 48 h, DCs were dena-tured in cell culture plates to determine gene expression of IL-12 as well as TNF-α, and supernatants were harvest-ed to determine IL-12 as well as TNF-α protein levels. Total RNA was extracted from cells using the single-step technique of acid guanidinium thiocyanate-chloroform extraction according to the manufacturer' s instruction (Promega, Madison, WI). mRNA for TNF-α and IL-12 were quantified by SYBR Green two-step, real-time re-verse transeription-polymerase chain reaction taking glyceraldehyde-3-phesphate dehydrogenase (GAPDH) as an internal standard. Levels of IL-12 and TNF-α in cell culture supernatants were determined with ELISA, strictly fol-lowing the protocols provided by the manufacturer. Data were analyzed with a one-way ANOVA. A P-values <0.05 were considered statistically significant. Results After stimulation with 1 μg/mL HMGB1, IL-12 and TNF-α protein and gene expressions in rat splenic DCs were markedly up-regulated at 24 h to 72 h (P<0.05 or P<0.01), and the expression levels of IL-12 and TNF-α peaked at 48 h (P<0.01). When DCs were cultured in the presence of 0.1 μg/mL, 1 μg/mL, and 10 μg/ml HMGBI for 48 h, expressions of IL-12 and TNF-α were also significantly up-regulated (P<0.01), and values of these cytokines were highest in 1 μg/mL HMGB1-treated group (P<0.01). Conclusions These data suggest that HMGB1 appears to be a potential immunostimulatory signal that induced DC maturation, and HMGB1 stimulation can result in marked up-regulation of IL-12 as well as TNF-α synthesis and release in splenic DCs.