Agrobacterium tumefaciens possesses many advantages as a model bacterium for the study of a wide variety of biological processes. Gene disruption or inactivation is a powerful and direct tool for investigation of in vivo gene functions. The intensive study ofA. tumefaciens has increased the need for simple and highly efficient procedures to manipulate its genome. The sacB gene was used as a counterselectable marker to develop a gene replacement procedure that allows precise insertion, deletion, and allele substitution of any gene sequence in A. tumefaciens without altering the genome in any other way. A kanamycin resistance (KmR) cassette was constructed to the suicide vector as the positive selection marker. The suicide plasmid containing DNA fragments homologous to the flanking sequences of the target gene was integrated into the recipient cell genome at the target gene locus by intermolecular homologous recombination, generating the KmR-single cross-over colonies. The effect of homologous sequence length on the intermolecular homologous recombination was analyzed. The second cross-over colonies generated by intramolecular homologous recombination occurring between two tandem repeats were simply screened out by counter-selection of sacB. Data showed that the intervening sequence length between two repeats significantly affected the intramolecular homologous recombination frequency in A. tumefaciens, indicating that A. tumefaciens adopted the homologous recombination mechanism similar to that in E. coli. All these results demonstrated that investigators could minimize the numbers of colonies to be analyzed and reduce the overall workload by optimizing the relative length of two homologous fragments and using the specific type of single cross-over transformants for screening the second cross-over event. This mutagenesis strategy had successfully been used to generate the double unmarked △vbp2△vbp3 mutant in two A. tumefaciens strains.

Objective To quantitatively assess the left ventricular systolic dyssynchrony in patients with varied degrees of chronic congestive heart failure after old myocardial infarction(OMI) by real-time three-dimensional echocardiography(RT-3DE) and investigate the clinical value of the systolic dyssynchrony index(SDI). Methods Forty patients with congestive heart failure after OMI (infarction group) were divided into the severe dysfunction group (LVEF ≤35 %) and the mild dysfunction group (35 % ＜ LVEF＜50%) ,and 30 normal subjects served as the control. RT-3DE was performed on all subjects to obtain the 17-segmental time-volumetric curves and global systolic function. SDI changes in above groups and the correlation between SDI and LVEF were analyzed. Results The SDI of the infarction group was significantly higher than that of the normal control group ( P ＜0. 01 ). The SDI of the severe dysfunction group was significantly higher than that of the mild group (P＜0.01). SDI and LVEF were negatively correlated ( r = -0.84, P ＜0. 01 ). The dyssynchrony rate in the infarction group was 85 %,in the severe dysfunction group was 100%, in the mild group was 75%. Conclusions Left ventricular systolic dyssynchrony is prevalent in patients with OMI, and it is negatively correlated with the LVEF. SDI is a sensitive indicator in assessing left ventricular systolic dyssynchrony. RT-3DE has a unique advantage in the evaluation of the left ventricular systolic dyssynchrony,especially in the patients with myocardial infarction.

Objective To study the value of MDCT in the preoperative assessment of resectability in patients with carcinoma of pancreas.Methods 53 cases of pancreatic carcinomas with intact MDCT materials proved operation and pathology were collected.Combining the transverse images and multiplanar reformation(MPR),maximum intensity projection(MIP),curved planar reconstruction(CPR) and volume rendering(VR),the resectable possibility of tumors were evaluated preoperatively,inclunding the relationship between tumors,vessels and organs,as well as metastases.Results Of 53 cases,16 had underwent radical resection,2 cases believed unresectable preoperatively,which were overestimating the degrees of vessels involved by tumors,37 cases had palliative operation,in which 5 cases were believed resectable preoperatively.Lymph node or liver metastases were missed in 2 cases and 3 cases were underestimating the degrees of vessels involved by tumor.Conclusion In evaluating the resectability of pancreatic carcinoma preoperatively,MDCT plays an important role.

Objective To investigate the effect of hypoxia-inducible factor-1α expression (HIF-1α) on toll-like receptor 4 (TLR4) signaling pathway-mediated rat lung ischemia-reperfusion injury (LIRI).Method Forty-five S-D rats were randomly divided into Sham group,LIRI group,LIRI+ TLR4-activated group,LIRI+ TLR4-inhibited group,LIRI + ASK1-inhibited group,LIRI + p38-inhibited group,and LIRI + HIF-1α-inhibited group.The interaction between TLR4 signaling pathway [including TLR4,myeloid differentiation factor 88 (MyD88),TIR-domain-containing adapter-inducing interferon-βTIR-domain-containing adapter-inducing interferon-β (TRIF),Apoptosis signal-regulating kinase 1 (ASK1) and p38] and HIF-1α and the role of TLR4-dependent HIF-1α in LIRI in vivo were analyzed.Result In LIRI,HIF-1α accumulation was induced in a TLR4-dependent fashion,and MyD88,but not TRIF,and activation of ASK1 and P38 were found to be critical for TLR4-mediated HIF-1α accumulation.HIF-1α protein played a critical role in TLR4-mediated lung injury of LIRI.HIF-1α up-regulated TLR4 expression in LIRI in a positive feedback manner.Conclusion We identify that HIF-1α has a damaging effect on TLR4 signaling pathway-mediated LIRI and TLR4-HIF-1 may synergistically involved in the development of LIRI.Therefore we suggest that the interaction between them may represent a novel therapeutic target for the development of novel target-based therapies of LIRI.

In this paper, metabolic networks of the Corynebacterium glutamicum GWY020 and the two de-rivatives carrying additional mutations HUI821and GUI089 were established and modified. The concentra-tions of extra-cellular metabolites were determined under sub-steady-state (50 h~52 h) of the batch culture. The metabolic flux distribution maps of the three strains were obtained, compared and analyzed. These re-sults indicate that the introduction of analog supersensitive marker or analog resistant marker skew the metabolic flux towards the formation of L-Arginine. This study revealed the usefulness of the metabolic flux analysis as a tool for verification of existing production strains. The analysis may play an important role in helping us to rationally re-design metabolism for further improvement of fermentation process.

This study is to establish the methods for determination of iridoid glycosides and triterpenic acids in Corni Fructus and provide technical support for the quality control of Corni Fructus. Morroniside, loganin and sweroside were determined by HPLC-UV method with acetonitrile and 0.1% formic acid as the mobile phase, and the detective wavelength was set at 240 nm. Oleanolic acid and ursolic acid were determined by HPLC-ELSD method with methanol-0.5% ammonium acetate (87:13) as the mobile phase. The results showed that the linear ranges of morroniside, loganin and sweroside were 5.335-213.4 mg · L(-1) (r = 0.9999), 5.515-220.6 mg · L(-1) (r = 1.0000), 1.992-79.68 mg · L(-1) (r = 1.0000), respectively. The average recoveries of the above three iridoid glycosides were 98.49%-99.28% with RSDs of recoveries being less than 2%. The linear ranges of oleanolic acid and ursolic acid were 7.74-154.8 mg · L(-1) (r = 0.9964), 10.82-216.4 mg · L(-1) (r = 0.9996), respectively. The average recoveries of the above two triterpenic acids were 98.11%-99.27% with RSDs of recoveries being less than 3%. The method established in this research is simple, rapid and reliable, and can be used for quality control of Corni Fructus. Furthermore, the research provided experimental data for the improvement of present quality standard of Corni Fructus, which has important significance to guarantee its quality and clinical curative effect.
Cornus ; chemistry ; Drug Stability ; Oleanolic Acid ; analysis ; Quality Control ; Triterpenes ; analysis

Objective To investigate the accuracy and the consistency with pathological diagnosis of confocal laser endomicroscopy (CLE) in real-time diagnosis of gastric malignancy and precancerous diseases.MethodsFrom January 2010 to March 2011, the out-patients and hospitalized patients of suspected gastric malignancy or precancerous diseases in Shanghai Ruijin Hospital were screened and undergone confocal laser endomicroscopy.Fluorescein sodium was used as fluorescent agent in the examination.A four-tiered diagnostic system was applied in the real time diagnosis with CLE images, and targeted biopsy was performed accordingly. Surgery was performed in CLE diagnosed or highly malignancy suspected patients.The diagnosis of common endoscopy, CLE and pathology was reviewed.ResultsA total of 160 patients were enrolled in this study, of those one patient withdrew because of fluorescin allergy and the rest 159 patients completed the CLE examination.A total of 194 lesions were inspected, among them, 130 cases each with one lesion, 23 cases each with two lesions and 6 cases each with three lesions.Overall accuracy rate of CLE was 93.3％ (181/194).And the sensitivity and specificity of CLE in gastric malignancy diagnosis was 93.6 ％ and 99.3 ％ respectively.Ideal correlation was identified between confocal laser endomicroscopy and final pathology results (Kappa=0.876).The incidence of marked fluorescin-related adverse events was only 0.6％ (1/160). ConclusionsCLE is consistent with histopathology and is helpful to make accurate diagnosis of gastric malignancy and precancerous diseases.It is important in early diagnosis of gastric malignancy and helps to avoid misdiagnosis and missed diagnosis.

Objective To evaluate the relationship of clinical symptom to plasmic levels of D-dimer, activated factorⅦ (FⅦa) and tissue factor pathway inhibitor (TFPI)/X a in patients with urticaria. Methods A total of 27 patients with chronic urticaria (CU), 27 patients with acute urticaria (AU) and 26 normal human controls were included in this study. Symptom score was determined and disease course was surveyed in these patients. ELISA was used to detect the plasma levels of D-dimer, FⅦa and (TFPI)/Xa in patients and controls. The relation of clinical symptom and disease course to plasma levels of these parameters was assessed. Results In patients with AU and normal controls, the plasma level of D-dimer was 450.57± 242.13 ng/mL and 266.81±40.68 ng/mL, respectively, the level of FⅦa, 2.23± 0.74 ng/mL and 5.23±1.35 ng/mL, respectively, and the level of TFPI/Xa 0.87±0.13 nmol/L and 0.88 ~ 0.12 nmol/L, respectively. There was a significant difference in the level of both D-dimer and FⅦa (both P < 0.01 ), whereas no differ-ence was observed in that of TFPI/X a (P > 0.05) between patients with AU and normal controls. In addi-tion, increased level of D-dimer and decreased level of FⅦa were noticed in patients with CU compared with those in normal controls (593.80±294.04 ng/mL vs 266.81±40.68 ng/mL, 3.98±0.35 ng/mL vs 5.23± 1.35 ng/mL, both P < 0.01 ), but there was no significant difference in the plasma level of TFPI/Xa (0.87± 0.16 nmol/L vs 0.88±0.12 nmol/L, P > 0.05). Significant difference was observed in the plasma level of D-dimer and FⅦa between patients with AU and CU (450.57±242.13 ng/mL vs 593.80 ±294.04 ng/mL, P < 0.05; 2.23± 0.74 ng/mL vs 3.98± 0.35 ng/mL, P<0.01 ). The plasma level of D-dimer positively corre-lated to the symptom score of patients with CU and those with AU (r= 0.68, P< 0.01; r= 0.82, P< 0.01),but was independent of discase course (P> 0.05). Neither the level of FⅦa nor that of TFPI/Xa correlated to symptom score or disease course of patients (all P > 0.05). Conclusions There is an overactivation of coagulation cascade, consumption of blood coagulation factors and secondary fibrinolysis in patients with urticaria, suggesting that plasma D-dimer and FⅦa may be associated with the clinical symptoms of urticaria.

Objective To study the relationship of T, B and NK lymphocytes with the pathogenesis of chronic urticaria. Methods Flow cytometry was applied to assess the proportion of T, B and NK lymphocyte subgroups in the peripheral blood of 51 patients with chronic urticaria and 30 sex and age-matched human controls. The CD4:CD8 ratio was calculated. Moreover, the symptoms, disease course and response to antihistamines of these patients were evaluated by one physician. Results The percentage of CD8+ T and NK cells, CD4:CD8 ratio were (27.20±8.22)%, (21.20±10.84)% and 1.48±0.62, respectively, in these patients,(29.9±3.74)%, (17.5±3.56)%, 1.24±0.27, respectively, in the controls; the differences were significant between the two groups (all P＜0.05). Decreased levels of CD3+ T cells, CD8+ T cells and B cells were noted in patients resistant to antihistamines compared with those responsive to antihistamines[(61.81±11.70)% vs (75.74±2.36)%, (24.00±7.79)% vs (34.22±9.30)%, (10.78±2.07)% vs (15.25±4.10)%, P＜0.05, 0.01, 0.05, respectively)], while the CD4:CD8 ratio and percentage of NK cells were increased in antihistamine-resistant patients compared to those in antihistamine-sensitive patients [1.67±0.76 vs 1.17±0.41, (28.61±12.62)% vs (12.78±6.02)%, both P＜0.01 ]. In these patients with chronic urticaria, the percentages of CD3+ T and CD8+ T cells were negatively correlated with the symptom scores (R = -0.31, -0.28, respectively, both P＜0.05 ), while the percentage of B cells was positively correlated with the symptom scores and disease course (R = 0.53, 0.55, respectively, both P＜0.01 ). Conclusions There is an abnormality in the proportion of T, B and NK lymphocyte subgroups in patients with chronic urticaria,which indicates that humoral immunity may be involved in the pathogenesis of chronic urticaria and the mechanism for responsiveness to antihistamine.

Objective To investigate the genotypes of β-lactamases produced by Klebsiella pneumoniae.Methods Plasmid conjugation,PCR amplification,gene cloning and DNA sequencing,isoelectric focusing electrophoresis and extended-spectrum β-lactamase(ESBLs)confirmatory test were carried out for analyzing the encoding gene of β-lactamases in clinical strains of Klebsiella pneumoniae collected from hospital wards.Results Totally 75 clinical strains of Klebsiella pneumoniae were collected,in which 48 strains were confirmed to produce genotype of β-laetamases(64.0%),including 39 ESBLs-producing Btraim(52.0%).Among 48 strains,17 isolates(35.4%)carried 2 types of ESBLs genes,7(14.6%)carried 3 types of ESBL8 genes,and 5(10.4%)carried 4 types of ESBLs genes.CTX-M was the most comon type(30/48,62.5%),followed by TEM(26/48,54.2%)and SHV(25/48,52.1%).Among 9 isolates with DHA-1 AmpC β-laetamase,8 produced AmpC β-lactamases and ESBLs.Class A carbapenemase KPC-2 was produced in 3 isolates.False negative rate of ESBLs confirmatory test was 23.1%(9/39).Condusion Genotypes of β-lactamases produced by Klebsiella pneumoniae are complicated,which results in multi-drug resistance in clinic.