BACKGROUND:Chitosanase is an enzyme for efficient and special hydrolysis of chitoan, and hence its effective and stable expression with enzymatic activity wil contribute to improving gene therapeutic effect.
OBJECTIVE: To construct a chitosanase gene for the efficient and specifical hydrolysis of chitosan, and to investigate its expression inEscherichia coli and the main influencing factors of enzymatic activity.
METHODS:According to the sequences of endo-chitosanase ofAspergilus sp. CJ22-326 provided in Genbank (EU302818), primers were designed and synthesized. The Asperguilus endo-chitosanase gene was amplified by successive extension PCR. And then the recombinant pET28a-His6-CSN was constructed and expressed in
Escherichia coli BL21. Finaly the recombinant His6-CSN fusion protein was analyzed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE), the western blot and dinitrosalicylic acid assay for detecting the enzyme activity of eluted His6-CSN fusion protein. The influence of different pH value and temperature on the enzyme activity of the recombinant chitosanase was investigated.
RESULTS AND CONCLUSION: SDS-PAGE showed that 29 kDa proteins were expressed and the western blot assay showed that His6-CSN expressed successfuly in the host. Dinitrosalicylic acid assay determined the
enzymatic activity of His6-CSN was significantly higher than that of lysozyme, but lower than that of chitosanase from Streptomyces griseus (P < 0.05). The recombinant chitosanase displayed the maximal activity at temperature of 50℃ and pH value of 6.0. There were a higher enzymatic activity remaining at pH value of 4.0-7.0 and
temperature of 30-50℃.

Objective To explore the applicative value of contrast enhanced ultrasound (CEUS) through rectumin the T-staging of rectal cancer.Methods 24 cases with rectal cancer were examined preoperatively by endorectal ultrasound (ERUS) and CEUS during May 2012 to November 2013 in our hospital.The characteristics of contrast enhanced ultrasound imaging in early and late phase of the rectal cancers were studied,and the imaging distribution and enhancement mode were observed.The preoperative staging diagnosis was conducted by consulting T staging criteria.The diagnostic results were compared with the postoperative pathologic diagnosis.Results The contrast enhanced time in rectal cancer began at 14-35s.The early tumor showed uneven nodular,ribbon or ring in high enhancement.At the advanced late,angiographic tumor showed homogeneous enhancement and continued to be observed during the whole time.Normal rectal wall ultrasonography showed spotty low enhancement or no enhancement.The overall accuracy of ERUS in T-staging was 66.7％ (16/24) and the overall accuracy of CEUS was 83.3％ (20/24) respectively.There was a significant difference in the overall accuracy between ERUS and CEUS (P＝0.018).The sensitivity,accuracy and positive predictive value of CEUS were 100.0％ (2/2),100.0％ (2/2),100.0％ (2/2) inT1 stage,83.3％ (10/12),83.3％ (10/12),100.0％ (10/10) in T2 stage,100.0％ (8/8),88.9％ (8/9),88.9％(8/9) in T3 stage,and 100.0％ (2/2),100.0％(2/2),100.0％ (2/2) in T4 stage,respectively.Conclusions As compared toroutine transrectal ultrasound,transrectal contrast enhanced ultrasound imaging on rectal cancer shows the typical sonographic characteristic imaging on microvascular focus size and borders,on characteristic infiltration zone.Therefore,contrast enhanced ultrasound improves the diagnostic accuracy of preoperative T-staging in rectal cancer surgery.

To develop a handling platform for loading, unloading, fixation, deployment, withdrawal and etc of medical materials in field conditions. Classified handling was performed for different function units with the de-vice developed, which was composed of a trolley, a railroad, a hydraulic handling board, a fixation belt and a vehicle-mounted tractor. The platform could enhance the efficiency of materials handling, which behaved well in the hos-pital without any handling device. The modular handling platform is easy to unload, carry, transport, fix and maintain, and thus is worthy popularizing practically.

The data collected from 34 type 2 diabetic patients receiving intensive insulin therapy for six years showed that the yearly mean HbA1C was less than 7.0％,and none of the patients showed severe hypoglycemia,occurrence or evident progression of retinopathy or nephropathy,and the islet β cell function gained improvement.The DQOL score,used to evaluate the quality of patients' life had no significant change during the observation ( P ＞0.05 ).It is satisfactory and safe to maintain long-term glycemic control with prolonged intensive insulin therapy in patients with type 2 diabetes,and that such therapy does not induce untoward influence on the quality of diabetic patients life.

Objective:To evaluate the effectiveness and safety of 3.5% lidocaine hydrochloride ophthalmic gel for eye surface anesthesia.Methods:A multicenter, randomized, double-blind, placebo controlled clinical trial was conducted in 10 hospitals in China from August 2018 to April 2019 under the approval of an Ethics Committee of Beijing Hospital (No.2018BJYYEC-014-02). A total of 220 fellow eyes of 220 subjects who received ocular surgery in one eye were actually enrolled according to a same included criteria in different institutes.The 220 eyes were randomly divided into control group and trail group.Two drops of lidocaine hydrochloride gel were dropped at about 5 mm away from corneal limbus at 6 o'clock direction of experimental eye of the trail group, and the blank gel was used in the eyes of the control group in the same way.The pain sensation was assessed with a 0.3 mm toothless micro forceps on conjunctiva within a specified time, and ''pain'' or ''no pain'' was answered by the subjects.The primary effective indexes, namely the number of eyes and percentage of ''no pain'' within 5 minutes following dropping, as well as the secondary indexes including the onset time point of the drug and the duration of anesthesia were recorded and evaluated.Safety evaluation took ocular and system adverse events into account.Results:Within 5 minutes after dropping, ''no pain'' occurred in 104 eyes (94.55%) and 29 eyes (26.36%) in the trail group and control group, respectively, showing a significant difference between the two groups ( χ2=106.948, P<0.001). And there was a significant difference in anesthesia onset time between the trail group and control group (40.0 seconds vs. 300.0 seconds) ( Z=-15.17, P<0.001). The duration of anesthesia was 860.5 (577.5, 1 180.0) seconds in the trail group and 676.0 (280.0, 1 401.0) seconds in the control group, with no statistically significant difference between the two groups ( Z=0.898, P>0.05). The incidence of adverse events in the trail group and control group were 5.45% (6/110) and 4.55% (5/110), respectively, without statistical significance between them ( P=1.000). Conclusions:The 3.5% lidocaine hydrochloride is a safe, effective, easy to use and high-quality surface anesthesia drug for eye surgery.

Objective To investigate the effect of vacuum-assisted closure(VAC)on the circulating number of endothelia progenitor cell(EPCs)in diabetic patients with mild to moderate degrees of ischemic foot ulcer and their related factors. Methods A total of 84 diabetic patients with foot ulcer duration for at least 4 weeks and ankle brachial index(ABI)0.5~0.9 were selected and divided into and assigned to two groups according to 2: 1 randomization:vacuum-assisted closure(VAC)treatment group(n=56)and Non-VAC treatment group(n=28). The control group (NC) was composed of 18 patients who had normal glucose tolerance and lower extremity ulcer without arteriovenous disease. VAC was performed on the ulcer wound after debridement for 1 week in both VAC group and NC group,and the patients in Non-VAC group received conventional treatment process. The circulating number of EPCs was measured before and after various treatments and the influencing factors of their changes were analyzed. Results After VAC treatment,the circulating number of EPCs significantly increased in both VAC group and NC group[(85.3 ± 18.1)vs(34.1 ± 12.5)/106cells,(119.9 ± 14.4)vs(66.1 ± 10.6)/106cells,both P<0.05]. By contrast,the circulating number of EPCs had no significant change in Non-VAC group[(45.2 ± 19.4)vs(34.7 ± 16.8)/106cells, P>0.05]. In addition,the circulating levels of vascular endothelial growth factor(VEGF)and the protein expressions of VEGF and stromal cell-derived factor-1α(SDF-1α)in the granulation tissue also significantly increased after VAC treatment in both VAC group and NC group,but no significant change in Non-VAC group. Compared with Non-VAC group,the changes of VEGF and SDF-1α levels in the sera and granulation tissue were all significantly higher in both VAC group and NC group(P<0.05 or P<0.01). There were no significant differences in changes of the circulating number of EPCs, and VEGF and SDF-1α in the sera and granulation tissue between VAC group and NC group. Correlation analysis showed that the change of the circulating number of EPCs was correlated with the changes of VEGF and SDF-1α levels in the sera and granulation tissue of VAC group and NC group(P<0.05). Conclusion VAC treatment may increase the circulating number of EPCs in diabetic patients with mild to moderate ischemic foot ulcer as in non-diabetic controls,which may be attributed to the upregulation of systemic and local VEGF and SDF-1α levels.