Restriction endonucleases are important molecular biology tools for DNA recombination.Because of the cleavage of DNA,their recombinant expression is difficult with low yields and complicated purification processes.In commercial productions,the technology that uses specific methylases to protect host DNA from digestion of the expressed restriction enzymes was cumbersome and practically limited.For solving this problem the expression of restriction enzyme Not Ⅰ was performed by using the DNA methylase M.Sss Ⅰ derived from Spiroplasma sp.MQ1 which specifically kept CpG sequence methylated.The methylated DNA was protected from the cutting of Not Ⅰ whose recognition sequence contained CpG.The gene of methylase M.Sss Ⅰ was introduced into Escherichia coli ER2566 and constitutively expressed,resulting in the CpG methylation pattern of the host DNA.Restriction enzyme Not Ⅰ was successfully expressed in this E.coli strain.Furthermore,by adding a purification tag to one terminus of the enzyme,recombinant Not Ⅰ was prepared as a highly purified and active product through two simple Ni-affinity and anion exchange chromatography steps.This expression system can be applied for the preparation of a series of restriction enzymes with CpG in their recognition sequences.

ObjectiveTo comprehend the change of the characteristics of lifting force produced by a working space marker in process of its cavity-forming. Methods37 patients were successively operated with the surgical mode of minimally invasive video-assisted thyroidectomy (22/37 cases received a lobotomy and others un derwent a partial thyroidectomy) from January to August, 2010. Instead of hand-retraction, a mechanical armworking space marker type I ( WSM-I, MIEO Medinstr Co. Ltd, China) was applied to establish a working space. After pathway making, an interlayer-cavity above the lobe was created by the space maker and endoscopic view was properly built. Following all these steps, a simulated space making procedure was performed in a way of stepwise hook-lifting (5 mm rising per time). The lifting force ( LF)was measured during the process with a modflied force-measure device (FB-50, DESIK company, Germen). Then recorded data were assessed and analyzed statistically. Results①Ascending scope of LF in the process of entire space-forming was 0-27.5 Newton (N).②Along with hook rising, LF ascended correspondingly and 2 specific values emerged: One was 11. 2 ±3.5 N,as the lifting height approached 1.5 cm ( also a approximate position of essential space-forming ( Pe), at which themusculo-cutaneoustissuejustbecame tight) ; the other was 17.5 ± 4.3 N , as the lifting height approached 1.75 cm ( also a approximate position of maximal space-forming (Pmax), at which the musculo-cutaneous tissue appeared real tight, but not in a status of extreme tightness). ③Two types of LF ascending were found when the values transferred to a curve diagram : a palliative linearity increasing while lifting height varied from 0 to 1.5 cm (PO to Pe) and a rapid exponent-like increasing while lifting height varied from 1.5 to 1.75cm ( Pe to Pmax). ④ Dependability analyses yielded a diverse statistical outcome: negative significance of the comparison between incision length and LF value ( P ＞ 0. 05 ), and positive significance of the comparison between skin thickness and LF value ( P ＜ 0. 01 ). Conclusions①LF produced by WSM-I while establishing a working space is proper and relatively small, since the maximal value is merely 27.5N, far less than the stress produced by ordinary cosmetic skin expansion. ②The whole space-forming process can be divided into 2 stages according to the characteristic of LF ascending which correspond also separately to the “essential cavity-forming” and “the maximum cavity-forming” in the real establishing of a working space. ③Attention should be paid to the later stage since in which a rapid LF increasing occurs while the appearance of musculo-cutaneous tissue changes from “just become tight” to “real appear tight”. ④LF control, especially the fine readjustment at or about Pmax should be of necessity in individual space-forming, and then, ideal working space establishment can be archived at a pre cisely balanced LF point: maximum cavity volume acquired and minimal tissue expansion stress produced.

Background:Tissue microarray has been increasingly used in research of malignancies. It has been revealed that TGF-β signaling pathway contributes to the tumorigenesis and progress of malignancies. Aims:To determine the expressions of Runx3,Smad4,Cdk2 and p21,the key molecules in TGF-β signaling pathway by tissue microarray,and investigate their correlations with clinicopathological features and prognosis of gastric cancer. Methods:A total of 378 paraffin embedded tissue blocks,including 130 gastric cancer tissue and 248 para-cancer tissue from 130 patients undergoing radical resection of gastric cancer were obtained. Tissue microarray and immunohistochemistry were used to determine the expressions of Runx3,Smad4,Cdk2 and p21. Results:The aberrant expression rates of Runx3,Smad4, Cdk21 and p21 in gastric cancer tissue were significantly higher than those in para-cancer tissue(67. 7% ,35. 4% , 63. 8% and 70. 0% vs. 14. 1% ,12. 5% ,18. 1% and 37. 1% ,P < 0. 05,respectively). Aberrant expression of Runx3 was closely correlated with histological grade and lymph node metastasis of gastric cancer( P < 0. 05),while aberrant expressions of Smad4 and p21 were correlated with histological grade only(P < 0. 05);aberrant expression of Cdk2 was correlated with histological grade,lymph node metastasis and TNM staging(P < 0. 05). Pairwise correlations were seen among aberrant expressions of Runx3,Smad4 and p21 in gastric cancer,while Cdk2 was correlated with Runx3 only. Kaplan-Meier survival curve showed that 5-year survival rates in Runx3,Smad4 and p21 aberrant expression groups were significantly lower than those in normal expression groups(P <0. 05). Furthermore,Cox proportional hazard model indicated that Runx3 and Smad4 were independent prognostic factors for gastric cancer. Conclusions:Runx3,Smad4,Cdk2 and p21 might play pivotal roles in tumorigenesis and progression of gastric cancer. As interactions occurred among these four proteins,whether Runx3 and Smad4 could be used for predicting the prognosis of gastric cancer needs to be further studied.

Objective To investigate the distribution of air microorganisms in soldies′living quarters of one under-ground tunnel during airtight survival training .Methods Anderson sampling was carried out in Area A and Area B ( living quarters) and Area C (a toilet) at designated time and place.After sampling, the culture and identification of microorgan-isms were finished .Results ①In the living quarters of the whole underground tunnel , the total number of bacteria ranged from 125 to 37 800/m3,but it was 2692, 1844 and 2199/m3, respectively in Area A, B and C.The mean number of bac-teria was 2245/m3 .The number of fungi ranged from 0 to 10 017/m3 .The total number of fungi of Area A , B and C was 1064 , 883 and 1011/m3 .The mean number was 986/m3 .②The number of bacteria in the living areas presented three fea-tures:the total bacteria showed overall three peaksin Area A and B, buttwin peaksin Area C.In Area A and B, the number of bacteria exhibited low colony at first but fluctuated heavily later .In Area C, it decreased gradually to the mini-mum,and the fungi showed a wavy and irregular trend .③The air microbial species included cocci ( Micrococcus, Coriolis of Staphylococcus aureus, S.epidermidis and S.equorum) , bacilli ( Acinetobacter baumannii, A.lwoffii and Alcaligenes faeca-lis),and fungi (Mucor and Saccharomyces).Conclusion Although the content of microorganisms was up to the military hygiene standards , it was higher than in the same kind of tunnels .Themulti peakphenomenon of microbial distribution suggests that the change of air microorganisms in tunnels has its own characteristics .Most of the air microorganisms are con-ditioned pathogens that may cause illness if they are not under control .

Background and purpose:Quinonoids can change the cell cycle distribution of tumor cells, and effect the radiosensitizing. This study aimed to investigate the radiosensitization effects, cell cycle and apoptosis of cryptotanshinone on H22 hepatoma-bearing mice. Methods:The mouse hepatoma H22 model was established, then divided into blank control group, irradiation alone group, high dose of cryptotanshinone group, cryptotanshinone (low, medium and high)+IR groups. After irradiated, observed the growth of tumor’s conditions, record epigenetic tumor irradiation time, calculated the delay time of tumor growth and enhancement factor (EF). After 22 days, mice were killed, stripped tumor, and calculated the inhibition rate. The cell cycle distribution and apoptosis were measured by lfow cytometry. Results:Cryptotanshinone (low, medium and high) groups inhibited the tumor growth better than the blank group, and had the signiifcant radiosensitizing effect. The enhancement factor was 1.22, 1.43,2.19, respectively. Cells were treated with cryptotanshinone which had significant effects on cell cycle, and induced apoptosis, which indicated signiifcant G2/M phase arrest and a decrease in S phase. Conclusion:Cryptotanshinone inhibited the tumor growth and had the radiosensitizing effects on H22 hepatoma-bearing mice. One of the mechanism may be that it might make significant G2/M phase arrest and S phase decreased, and induced apoptosis. So cells were more sensitive to radiation.

Objective To investigate whether gemcitabine (GEM) could enhance radiosensitivity of human non-small cell lung cancer cells and its related mechanism.Methods Clonogenic assay was used to analyze radiosensitivity enhancement by GEM on p53 mutant human lung adenocarcinoma cell line 973.Alterations of cell cycle distribution and apoptosis were measured by flow cytometry.Results Mild radiosesitizing effect was observed when 10 nmol/L GEM was administrated before or after irradiation.Marked radiosesitizing effect was demonstrated when 100 nmol/L GEM was administrated before or after irradiation, with much stronger effect of pre-irradiation GEM treatment.Mutation of p53 gene affected cell cycle redistribution and cell apoptosis, but had no relationship with radiosensitivity enhancement of GEM.Conclusions 100 nmol/L GEM could significantly enhance radiosensitivity of human lung cancer cells.However, this effect may not be associated with p53 gene mutation, cell cycle redistribution or cell apoptosis.

Objective To evaluate lower extremity deep venous hemodynamic changes and lower extremity deep venous disease in abdominal obesity.Methods To compare venous flow parameters of the lower limbs as assessed by Duplex ultrasound scanning in obese and nonobese individuals according to body mass index(BMI).Venous hemodynamics were studied in a prospective cohort study in nonobese( BMI ＜ 25 kg/m2) and obese individuals( BMI ＞ 30 kg/m2 ).Diameter,peak,mean,and minimum velocities were assessed.Results The study examined 45 limbs in 24 nonobese individuals and 44 limbs in 22 obese individuals.The diameter of the femoral vein was significantly greater in obese vs nonobese limbs ( 10.4 ± 1.3) mm vs (7.1 ± 1.0) mm,P ＜ 0.01 ).Calculation of venous amplitude and shear stress showed significantly higher values in nonobese vs obese limbs [ ( 11 ± 5 ) vs ( 8 ± 3 ) cm/s ],( P ＜ 0.01 ) and [ (0.50 ± 0.17 ) vs ( 0.30 ± 0.09 ) ] dyn/cm2,( P ＜ 0.01 ).Spearman rank correlation revealed a significant inverse correlation between waist-to-hip ratios and waist circumference and velocities amplitude (peak ve locity-minim velocity),and shear stress.Conclusions Lower limb venous flow parameters differ significantly between obese and nonobese individuals.These findings suggest the mechanical role of abdominal adipose tissue potentially leading to elevated risk for both chronic venous insufficiency and venous thromboembolism.

The Expert Consensus on the Diagnosis and Therapy of myelodysplastic syndrome (MDS) (2014) will be interpreted in this paper focusing on whether it is scientific and reasonable.Some advises and views will be put forward,hoping that it will be useful to improve the diagnostic and therapeutic ability on MDS for clinicians in our country.

Traditional Chinese medicines (TCM) is one of the most important sources of new drugs.The rapid development of modern science and technology has brought new opportunities for TCM.Admittedly,new academic theory is getting into a golden period of innovation.Key technologies that enbody TCM features and adapt to modern drug-screening requirements are urgently needed.After five years' endeavor,the authors' group has made great progress in the new theories and methodologies for the discovery of bioactive compounds from TCM.In this review,a total of five key technologies:library-bioactivity-structure integration,biological and chemical fishing technology,ligand-and receptor-based virtual screening,profile-bioactivity relationship and the technology for discovering bioactive equivalent combinatorial components (BECCs),were introduced.In the text,several valuable demonstrations over the TCM-based drug discovery were provided,for uncovering the scientific basis of TCM and accelerating the process of TCM modernization.

OBJECTIVE:To prepare Resveratrol-poloxamer 188-solid dispersion(RES-P188-SD)and study its dissolving char-acteristic in vitro and antibacterial activity. METHODS:With P188 as the carrier, solvent method was used to prepare RES-P188-SD. With the mass ratio of drug to carrier,melting temperature and mesh number as the observed factors,and solubility and yield of RES as the observed indexes,L9(34)orthogonal test was designed to optimize the preparation technology,and compre-hensive score of observed indexes were analyzed. The verification test was carried out. After RES-P188-SD was prepared by the op-timal technology,basket method was used to determine its dissolution rate and then its accumulative dissolution rate was calculat-ed,scanning electron microscopy to analyze phase characterization and cylinder-plate method to determine antibacterial activity. RE-SULTS:The optimal technology was as follows as the mass ratio of drug to carrier of 1∶10,melting temperature of 70 ℃ and mesh number of 80. For the sample prepared by the optimal technology,average solubility was 0.51 mg/ml (RSD=1.96%,n=3),average yield was 91%(RSD=0.64%,n=3),average 15 min accumulative dissolution rate was up to 83%(RSD=0.69%, n=3);the drug in amorphous form was homogenously distributed in the carrier;RES-P188-SD could inhibit Staphylococcus aure-us and Escherichia coli. CONCLUSIONS:The optimal technology is stable and feasible,by which RES-P188-SD has been pre-pared successfully,providing reference for increasing the solubility,dissolution rate and antibacterial activity of RES.