Research on novel pullulanase has major significance on the domestic industrialization of pullulanase and the breakdown of foreign monopoly. A thermophilic bacteria LM 18-11 producing thermostable pullulanase was isolated from Lunma hot springs of Yunnan province. It was identified as Anoxybacillus sp. by 16S rDNA phylogenetic analysis. Full-length pullulanase gene was cloned from Anoxybacillus sp. LM18-11. The optimum temperature of the pullulanase was between 55 and 60 degrees C with a half-life as long as 48 h at 60 degrees C; and its optimum pH was between 5.6 and 6.4. V(max) and K(m) of the pullulanase was measured as 750 U/mg and 1.47 mg/mL, which is the highest specific activity reported so far. The pullulanase crystals structure showed a typical alpha-amylase family structure. The N-terminal has a special substrate binding domain. Activity and substrate binding were decreased when the domain was deleted, the V(max) and K(m) were 324 U/mg and 1.95 mg/mL, respectively. The pullulanase was highly heterologous expressed in Bacillus subtilis by P43 promoter. The extracellular enzyme activity was 42 U/mL, which increased more than 40 times compared to the initial strain. This pullulanase has good application prospects.
Anoxybacillus ; classification ; enzymology ; China ; Glycoside Hydrolases ; metabolism ; Hydrogen-Ion Concentration ; Phylogeny ; RNA, Ribosomal, 16S ; genetics ; Temperature

Objective To study a novel method for the high-dose-rate brachytherapy (HDR) CT image to the intensity modulated radiation therapy (IMRT) CT image deformable image registration and dose accumulation.Methods The applicator in the HDR CT image is first segmented and removed,then a deflation step is performed on the applicator-free HDR CT image by solving the Navier-Stokes equation.Demons algorithm is utilized to register the deflated HDR CT image to the IMRT CT image,along with the HDR dose.The deformed HDR dose is then added on the IMRT dose and yield the final accumulated dose.Results The HDR CT image and IMRT CT image,as well as the corresponding dose distribution,from five cervical cancer patients are used for evaluation of the proposed algorithm,the results show that the proposed method can effectively get rid of the influence of the applicator and produce an accurate accumulated dose.Conclusions Dose accumulation and supervision is an important step in adaptive radiotherapy for accurate dose delivery and treatment plan re-optimization.The proposed method in this study can effectively accumulate the HDR dose to the IMRT dose domain,and the accuracy is proved to be sufficient for clinical needs.

Programmed cell death 5(PDCD5)is a new kind of programmed cell death regulation and control gene. PDCD5′s expression is widespread,and evolution is conservative. PDCD5 has the effect of promo-ting a wide variety of tumor cells to apoptosis and inhibiting proliferation,such as lung cancer,liver cancer et al. Several laboratories′ discoveries show that PDCD5′s expression is significantly decreased when in some dis-ease cases,especially in tumor cases. PDCD5,which is related to the clinical relevance of the tumor,has a great clinical value of discovering,diagnosis and treatment of the tumor.

OBJECTIVETo evaluate biomechanical properties in different methods of internal fixation combined with distal tibiofibular syndesmosis injury, in order to provide a theoretical basis for clinical choice.

METHODSSix lower limbs specimens were collected and divided into 5 groups, including normal group, distal tibiofibular syndesmosis injury (injury group), 3 cortexes group, 4 cortexes group and hook-plate fixation group. Neutral position, plantar flexion position (30°), dorsiflexion (20°) supination external rotation position of foot movement were simulated on universal materials tester. Strength, stiffness and stability of ankle joint in 4 kinds of motion conditions were measured.

RESULTSThere was significant differences in strength and stiffness of ankle joint between injury group and normal group in 4 different kinds of motion conditions (P<0.05). Strength and stiffness of ankle joint in 3 cortexes group, 4 cortexes group and hook-plate fixation group were improved obviously in 4 different kinds of motion conditions, and biomechanical indexes were recovered normally or better than normal group. Stiffness of the three fixation groups were better than normal group,but there was no significant differences among three groups (P > 0.05), while stiffness of hook-plate fixation group was closed to normal group.

CONCLUSIONFor distal tibiofibular syndesmosis injury, 1 screw with 3 cortexes, 4 cortexes and hook-plate had a positive impact on strength, stiffness and stability of distal tibiofibular syndesmosis injury stress,and could restore the normal stabllity of ankle joint after reconstruction. While fixing by screw fixation would limit the rototary motion of ankle joint,ankle mortise could not adapt to changes of talus bone, thus induces screw breakage and traumatic arthritis. Hook-plate fixation is more suitable than 3 cortexes or 4 cortexes fixation for bilmechanical properties,and its' stress is more balance and can reduce postoperative complcations.

Ankle Injuries ; physiopathology ; surgery ; Biomechanical Phenomena ; Fibula ; injuries ; surgery ; Fracture Fixation, Internal ; methods ; Humans ; Joint Instability ; physiopathology ; prevention & control ; Reconstructive Surgical Procedures ; methods ; Tibia ; injuries ; surgery

Objective To study the pathological and immunohistochemical features of alimentary tract mesenchymal tumors and compare with computed tomographic virtue endoscopy (CTVE) imaging technology to evaluate the diagnostic value of CTVE in alimentary tract mesenchymal tumors. Methods Seventy-four pathological specimens of alimentary tract mesenchymal tumors were collected. The pathological features and the expression of CD117, CD34, SMA and S-100 were observed by immunohistochemical method with light microscope. The pathological types and characteristics were determined by pathologists and compared with CTVE imaging technology. Results In the 74 cases of alimentary tract mesenchymal tumors,40 cases were diagnosed as stromal tumor with pathological and immunohistochemical methods (54. 1%).Sixteen of them were malignant, accounting for 40% of the stromal tumor while 33 cases were diagnosed as leiomyoma(44. 6%)and 1 case as schwannoma(1.4%) . In the 74 GIMTs cases ,33 were jejunum GIMTs,21 were ileum GIMTs and 20 were large intestine GIMTs. Immunohistochemistry assay in the 74 GIMTs cases showed that 51.4% GIMTs were positive for CD117, approximately 36. 5% were positive for CD34 , 62.2% were positive for smooth-muscle actin (SMA) and 1. 4% were positive for S-100 protein. In the 74 GIMTs cases,69 cases were diagnosed right in the accuracy for location with CTVE(93. 2%) with 51 cases in small intestinal (94. 4%) and 18 cases in large intestinal (90. 0%). The sensitivity and the specificity of CTVE to distinguish benign from malignant stromal tumors by CTVE characteristics were 84. 2% and 85. 7%respectively. Conclusions GIST is common in GIMTs and is often originated from the small intestinal. The immunohistochemistry has great value in diagnosing alimentary tract mesenchymal tumors. The CTVE imaging technology also has great value in diagnosing alimentary tract mesenchymal tumors which can show the localization, shape size and artery of the tumor clearly. The diagnostic sensitivity and specificity of CTVE are high to distinguish benign from malignant alimentary tract GISTs. CTVE plays an important role in guiding the clinical management of GISTs.

Objective To understand the pattern of and influencing factors on leisure time physical exercises among adults in Zhejiang. Method Totally 17 437 residents aged 18 years and over selected by stratified cluster random sampling in Zhejiang in 2010 were investigated. Result Overall 21.82%(95%CI:17.12%-27.38%) of adults took part in leisure time physical exercises in 2010. The proportion was higher in urban (29.04%,95%CI:23.46%-35.33%) than in rural (18.81%,95%CI:12.89%-26.61%) (χ2=222.06, P<0.05) areas, and also higher in females (22.91%,95%CI:17.66%-29.15%) than in males (20.56%,95%CI:16.39%-25.48%) (χ2=13.94, P<0.05). Adults aged 18-24 and 55-74 years were more likely to take apart in physical exercises (χ2=266.73, P<0.05), and the lowest proportion was found among farmers (5.33%,95%CI:3.99%-7.11%) (χ2=2 078.40, P<0.05). These proportions both increased along with education level and family income increase (χ2=444.87, P<0.05;χ2=332.20, P<0.05). Overall 83.04%and 90.08%of physical exercisers took part in exercises at least 3 times per week and 30 minutes per time, only 30.67% of those reached moderate or vigorous intensity, and overall 5.38% (95%CI: 3.85%-7.48%) of adults took part in regular leisure time physical exercises in 2010. Multiple logistic regression showed that age, education level, occupation and chronic disease history have influence on regular physical exercises. Conclusion The leisure time physical exercises level was generally low among adults in Zhejiang. Young and middle-aged adults with less education and low income especially farmers should be put on emphasis.

This study aims to analyse the value of CODEHOP RT-PCR in the detection of Flavivirus. According to the amino acid sequences of polyproteins of different flaviviruses published in GenBank, a pair of primers was designed using the CODEHOP method. One-step RT-PCR was used to detect Japanese encephalitis virus strain JEV1201, Dengue virus strain JKD001, and yellow fever virus vaccine YV6161. BLAST analysis and phylogenetic analysis were performed after the RT-PCR products of nucleocapsid genes were sequenced. The results showed that this method could amplify Flavivirus specifically, and the size and sequence of the target fragment accorded with the anticipated result. JEV1201 had the highest homology to Japanese encephalitis virus strain YL2009-4/YC2009-3, belonging to the branch of the phylogenetic tree of Japanese encephalitis virus strains. JKD001 had the highest homology to Dengue virus strain DENV-2/ID/1022DN/1975, belonging to the branch of the phylogenetic tree of Dengue virus strains. YV6161 had the highest homology to Yellow fever virus strain 17D, belonging to the branch of the phylogenetic tree of Yellow fever virus strains. In conclusion, the method of CODEHOP RT-PCR can be effectively used to detect, identify, and phylogenetically analyse Flavivirus.
DNA Primers ; genetics ; Flavivirus ; classification ; genetics ; isolation & purification ; Flavivirus Infections ; virology ; Humans ; Molecular Sequence Data ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Viral Nonstructural Proteins ; genetics

OBJECTIVESTo identify hepatic progenitor cells (HPCs) in patients with severe hepatitis (SH) by detecting their markers and investigate the features of their distribution and location.

METHODSLiver tissues taken from 59 SH patients were tested for the receptor of stem cell factor (c-kit), pi-class glutathione S-transferase (GST-pi), cluster of differentiation 34 (CD34), cytokeratin 19 (CK19), cytokeratin 18 (CK18) and alpha fetoprotein (AFP) by immunohistochemistry (IHC). Meanwhile, 58 patients with acute or chronic hepatitis were also detected to act as controls.

RESULTSHepatic progenitor cells could be seen in SH patients. Most of them existed as ductular cells that had been called "typical ductular proliferation (ADP)" or "typical ductular reaction" in previous research. These ductular cells were mainly located at the portal areas, fibro septa, periportal parenchyma and the border of the pseudolobuli and inflammatory foci. Further, c-kit, GST-pi, CK19 and CK18, but not CD34 and AFP could be detected in these cells. Another kind of HPC was the small hepatocyte-like cell (SHLC), which could express c-kit, GST-pi, and CK18, but not CK19, CD34 and AFP. The semi-quantitative analysis showed that the scope of ADP in SH patients was significantly larger than that in acute and chronic hepatitis patients (chi2= 63.62, P<0.05), and the scope of ADP in subacute severe hepatitis and chronic severe hepatitis patients was also significantly larger than that in acute severe hepatitis patients.

CONCLUSIONIn the course of regeneration of viral hepatitis, different types of pathology have different features. In acute and chronic hepatitis (G1-2), the regeneration is mainly owing to the proliferation of mature hepatocytes, and in chronic hepatitis (G3-4), there is the participation of HPCs, although they are limited. In severe hepatitis, however, since the replicative capacity of normal hepatocytes is impaired or prohibited, liver regenerates and restores mainly by the means of hepatic stem cells activation and proliferation. But the hepatic stem cells don't differentiate into their mature functional compartments directly at all. There are several intermediary or transition populations. In human severe hepatitis, they are mainly ductular cells, and parts of them are small hepatocyte-like cells.

Antigens, CD34 ; analysis ; Cell Division ; Female ; Glutathione S-Transferase pi ; Glutathione Transferase ; analysis ; Hepatitis ; pathology ; Hepatocytes ; pathology ; Humans ; Immunohistochemistry ; Isoenzymes ; analysis ; Keratins ; analysis ; Male ; Proto-Oncogene Proteins c-kit ; analysis ; Stem Cells ; pathology ; alpha-Fetoproteins ; analysis

Objective:To investigate the relationship between serum lipocalin-2 level and the risk of cardiovascular disease(CVD) in patients with type 2 diabetes.Methods:A total of 279 type 2 diabetic patients were enrolled in this study. Basic information and clinical data were collected. These patients were divided into CVD group and non-CVD group according to their cardiovascular disease status. Serum lipocalin-2 level was assessed by enzyme linked immunosorbent assay.Results:Compared to non-CVD group, serum lipocalin-2 level was significantly higher in CVD group( P<0.01). The Spearman correlation analysis showed that serum lipocalin-2 level was positively correlated with waist circumstance, diastolic blood pressure, uric acid, triglyceride, and HbA 1C( P<0.05), while negatively correlated with high density lipoprotein-cholesterol level( P<0.01). In addition, the univariate and multivariate logistic regression analysis revealed that serum lipocalin-2 was an independent risk factor for CVD( P<0.01)after adjustment for potential confounders. Moreover, receiver operating characteristic curve analysis demonstrated that the area under curve value of lipocalin-2 was 0.74, with the optimal cutoff value of lipocalin-2 66.84 ng/mL. Conclusion:Serum lipocalin-2 is closely associated with CVD in patients with type 2 diabetes, which might be considered as one of the predictors for CVD in type 2 diabetes mellitus.

OBJECTIVETo investigate the effect of HS3ST3B1 on hepatitis B virus (HBV) replication.

METHODSHepG2 cells were classified into 7 groups according to the plasmids transfected: (1) Blank group, no plasmid transfected; 2. Positive control, transfected with pCH9-HBV which permits HBV replication; (3) Negative control, transfected with pCH9-HBV + pcDNA3.1 + pTZU6+1; (4) Treatment A, transfected with pCH9-HBV + pCDNA3.1-HS3ST3B1 + pTZU6+1; (5) Interference A, transfected with pCH9-HBV + pCDNA3.1-HS3ST3B1 + psh1126 (a plasmid to interfere HS3ST3B1 expression); (6) Treatment B, transfected with pCH9-HBV + pTZU6+1; (7) Interference B, transfected with pCH9-HBV + psh1126. The levels of HBV DNA were detected in the above groups by Southern blotting. HBV total RNA of Negative control, Treatment A and Interference A were quantified by Real-time PCR to determine the influence of HS3ST3B1 over-expression on the HBV RNA transcription. The activity of the four HBV promoters [core promoter (cp), x promoter(xp), surface antigen promoter1(sp1), surface antigen promoter2 (sp2)] were assayed by Dual-Luciferase Reporter Assay System. The data was analyzed using one way ANOVA, with P < 0.05 indicating statistically meaningful difference.

RESULTSouthern blot data revealed the level of HBV DNA in Treatment A and Interference A accounted for 10% +/- 2% and 31% +/- 4% of that in control. Compared with control, a statistical difference existed between Treatment A and Control, with F value equalling to 20.8 and P value equalling to 0.034 respectively. A statistical difference also existed between Interfere A and Treatment A, with F value equalling to 24.9 and P value equalling to 0.021 respectively. The level of HBV DNA in Experiment B was raised by 130% +/- 11% as compared to that in Interference B, and the levels of HBV DNA showed a dose-dependent decrease when H7 cells were transfected with 0.5, 1.0, 1.5 microg pCDNA3.1-HS3ST3B1 respectively. Statistical differences existed between control and H7 transfected with different dose of pCDNA3.1-HS3ST3B1, with F values equalling to 22.7, 20.3, 26.5 and P values equalling to 0.029, 0.041 and 0.015 respectively. Real-time PCR revealed that the HBV total RNA in Treatment A accounted for 17.0% +/- 2.7% of that in control and there was a statistical difference between Treatment A and control, with F value equalling to 25.6 and P value equalling to 0.018. In addition, HBV DNA in Interference A was restored to 74.0% +/- 3.9% of that in control, and there was also a statistical difference between Treatment A and Interference A, with F value equalling to 21.3 and P value equalling to 0.032. However, the down regulation of HBV total RNA had nothing to do with HBV promoters activity.

CONCLUSIONHS3ST3B1 can inhibit HBV replication and reduce the level of HBV total RNA, but the downregulation of HBV total RNA may not be the result of direct interaction of HS3ST3B1 and HBV promoters.

DNA Replication ; DNA, Viral ; biosynthesis ; Hep G2 Cells ; Hepatitis B virus ; genetics ; physiology ; Humans ; Plasmids ; Sulfotransferases ; genetics ; Transfection ; Virus Replication