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.

Antigens, CD34 ; metabolism ; Cytokines ; metabolism ; Diagnosis, Differential ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Mucin-1 ; metabolism ; Perineum ; pathology ; Phosphopyruvate Hydratase ; metabolism ; Sarcoma ; metabolism ; pathology ; surgery ; Vimentin ; metabolism

Traditional herbal medicines, Panax ginseng, Panax quinquefolium and Panax notoginseng, attract our attention for their extensive and powerful pharmacological activities. Ginsenosides are the main active constituents of these medicinal herbs. The related glycosyltransferases involved in ginsenoside biosynthesis are the key enzymes which catalyze the last important step. Modification of ginsenoside aglycones by glycosyltransferases produces the complexity and diversity of ginsenosides, which have more extensive pharmacological activity. At present, ginsenoside aglycones and compound K have been obtained by synthetic biology. As the last step of ginsenoside biosynthesis, glycosylation of ginsenoside aglycones has been studied intensively in recent years. This review summarizes the basic strategies and research advances in studies on glycosyltransferases involved in ginsenoside biosynthesis, which is expected to lay the theoretical foundation for the in-depth research of biosynthetic pathway of ginsenosides and their production by synthetic biology.
Biosynthetic Pathways ; Ginsenosides ; biosynthesis ; Glycosyltransferases ; metabolism ; Panax ; chemistry ; Plants, Medicinal ; chemistry ; Synthetic Biology

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 investigate the beneficial effect of berberine (BBR) on atherosclerosis in Apo-/-E mice and explore the underlying mechanisms based on attenuating vascular inflammation and modulating calcification in human umbilical vein endothelial cells (HUVECs) and smooth muscle cells(SMCs). METHODS 48 Apo-/-E mice,at 6-8 weeks old,were randomly allocated into 4 groups:normal, model, bbr and atorvastatin (positive control) groups with 12 mice in each group. They were fed with high-fat diet for 4 weeks except those in Normal group and then treated with indicated drugs or solvent for another 4 weeks. The morphology and inflammation infiltration of aortic were examined with HE staining. The expression of BMP- 2 in aortic was examined by immumohistochemical staining. Blood lipid levels were examined by automatic biochemical analyzer. The expression of IL-6, TNF-αand BMP-2 in serum and tissues was detected by ELISA method. The expression of ALP and the content of calcium were detected by commercially-available kits. HUVEC cells were stimulated with TNF-α and incubated with various concentrations of BBR for 24 h. The contents of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM-1), matrix metalloprotein-9 (MMP-9) in the culture supernatant were detected by ELISA method. Calcification was induced with β-glycerophos?phate in SMC cells and the effect of BBR on the content of calcium was examined. RESULTS 4-week berberine treatment markedly lowered serum TC and LDL-c levels and improved the plaquestabilityin Apo-/- E mice fed with a high- fat diet (P<0.05 or P<0.01) which was comparable with the effect of atorvastatin. Berberine also significantly decreased the levels of IL-6 and TNF-α in mice serum and aortic tissues (P<0.05 or P<0.001). Berberine tended to decrease ALP, BMP-2 levels and the content of calcium in mice serum and aortic tissues (P<0.05, P<0.01 or P<0.001) which were not observed in atorvastatin group. Berberine significantly lowered the levels of ICAM-1, VCAM-1, and MMP-9 in TNF-α-stimulated HUVECs. It can also lowered the content of calcium in SMCs. CONCLUSION BBR can profitably regulate the levels of blood lipid in mice fed with a high-fat diet, decrease the injury caused by inflammation, and attenuate vascular calcification. It may improve atherosclerosis and play a role in cardiovascular protection.

OBJECTIVE To investigate the beneficial effect of berberine(BBR) on atherosclerosisin Apo-/-E mice and explore the underlying mechanisms based on attenuating vascular inflammation and modulating calcification in human umbilical vein endothelial cells (HUVECs) and smooth muscle cells (SMCs). METHODS 48 Apo-/-E mice,at 6-8 weeks old,were randomly allocated into 4 groups: normal, model, bbr and atorvastatin (positive control) groups with 12 mice in each group. They were fed with high-fat diet for 4 weeks except those inNormal group and then treated with indicated drugs orsolvent for another 4 weeks. The morphology and inflammation infiltration of aortic were examined with HE staining. The expression of BMP-2 in aortic was examined by immumohistochemical staining. Blood lipid levels were examined by automatic biochemical analyzer. The expression of IL-6, TNF-α and BMP-2 in serum and tissues was detected by ELISA method. The expression of ALP and the content of calcium were detected by commercially-available kits. HUVEC cells were stimulated with TNF-α and incubated with various concentrations of BBR for 24 h. The contents of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM-1), matrix metalloprotein-9(MMP-9) in the culture supernatant were detected by ELISA method. Calcification was induced with β-glycerophosphatein SMC cells and the effect of BBR on the content of calcium was examined. RESULTS 4-week berberine treatment markedly lowered serum TC and LDL-c levels and improved the plaque stability in Apo-/-E mice fed with a high-fat diet(P<0.05 or P<0.01) which was comparable with the effect of atorvastatin. Berberineal so significantly decreased the levels of IL-6 and TNF-α in mice serum and aortic tissues (P<0.05 or P<0.001). Berberine tended to decrease ALP, BMP-2 levels and the content of calcium in mice serum and aortic tissues(P<0.05, P<0.01or P<0.001) which were not observed in atorvastatin group. Berberine significantly lowered the levels of ICAM-1, VCAM-1, and MMP-9 in TNF-α-stimulated HUVECs. It can also lowered the content of calcium in SMCs. CONCLUSION BBR can profitably regulate the levels of blood lipid in mice fed with a high-fat diet, decrease the injury caused by inflammation,and attenuate vascular calcification. It may improve atherosclerosis and play a role in cardiovascular protection.