Objective:To study the influence of 5′-untranslated region modification of pPIC9 on expression of LL-37 in Pichia pastoris.Methods:The sequence GGATCCAA was deleted from 5′-UTR of pPIC9 and the modified product was trans- formed into E.coli DH5?to construct a modified eukaryotic vector pPIC9-EDIT.After PCR and sequencing,pPIC9-EDIT was ligated with LL-37 sequence coded by the biased codon of yeast,the product was then transformed into E.coli DH5?to con- struct the recombinant expression vector pPIC9-EDIT-LL-37,the latter was transformed into P.pastoris GS115 by spheroplas- ting and the insert was confirmed by PCR.The bacteriolytic activity to E.coli.DH5?was analyzed to screen the highest ex- pressing strain and to determine the best inducing time and concentration of methanol.The fermentation product was analyzed by Tricine-SDS-PAGE and Western blotting.The antibacterial activities of expression products of pPIC9-LL-37 and pPIC9-ED- IT-LL-37 were compared,and the changes of LL-37 protein expression were determined before and after modification.Results: pPIC9-EDIT and pPIC9-EDIT-LL-37 were successfully constructed.Expression of LL-37 gene was confirmed by PCR in P.pastoris after pPIC9-EDIT-LL-37 transformation.The highest expressing strain was identified;the best inducing time was 72 h and the best concentration of methanol was 0.5%.Tricine-SDS-PAGE and Western blotting analysis showed that the ex- pression product was LL-37.The expression level of LL-37 protein increased by 35 times after modification.Conclusion:Modifi- cation of pPIC9 5′-UTR can obviously improve expression of LL-37 protein in P.pastoris;it is worth to be used in the research of other heterogenous protein.

Objective: : T o study the microanatomy of the facial nerve in the cerebellopontine angle (CPA) a nd the internal auditory meatus (IAM) for the preservation of the facial nerve i n the acoustic neurinomas surgery. Methods: Forty sides of CPA f acial nerve of 20 adult cadaver heads were examined. Results: In the pontomedullary sulcus the facial nerve (1.98? 0.10) mm anterior to the vestibulocochlear nerve,(8.76?1.42) mm lateral to the abducent nerve at the points where the nerves join the brain stem at the lateral end of the sulcu s, (8.15?2.18) mm above the junction of the Ⅸ nerve with the medulla. There were 62 nutritious artery supplying the facial nerve in the CPA, mainly from the anterior inferior cerebellaris artery (AICA) and its branches; and 17 in the IA M were mostly from the internal auditory arteria (IAA). Conclusion: To be familiar with the microanatomy of the facial nerve in CPA and IAM is h elpful for localizing the facial nerve and improve its preservation in acoustic neuroma surgery.

Objective To assess the feasibility of intestitial MR lymphangiography (MRL) with subcutaneous injection of a commercially available,non-ionic,extracellular paramagnetic contrast agent,to visualize lymphatic vessels in patients with primary lymphedema.Methods Forty lower extremities in 31 patients with clinically advanced stages of primary lymphedema were examined with magnetic resonance lymphangiography.A 1 ml mixed liquor of gadobenate dimeglumine and mepivacainhydrochloride were injected subcutaneously into the dorsal aspect of both feet For MRL,a 3D fast spoiled gradient-recalled echo T1-weighted images with a fat saturation technique (T1 high resolution isotropic volume excitation,THRIVE) were performed after subcutaneous application of the contrast material To outline lymphatic vessels,source images were used to reconstruct images of MIP.The SNR and CNR of enhanced lymphatic vessels and veins were measured and calculated respectively.The significance of the differences of the data comparisons was assessed using an unpaired student t teat.Results Of the 40 lower extremities,the beaded appearance of dilated lymphatic vessels was detected in 36 lower legs (90.0%) and 17 upper legs (42.5%).The numbers of the dilated lymphatic vessels displayed in all segments of lower extremities added up to 365 and its mean diameter was (3.4 ± 0.1) mm on MRL MIP image.The average SNR and CNR were 257 ±130,207 ± 113 in the dilated lymphatic vessels and 218 ± 129,152 ± 113 in the vein respectively,which was statistically significant (SNR t = -2.649,CNR t = -3.404,P <0.01).Contrast enhancement was observed in 30/40 inguinal lymph node groups (75.0%).In 26 lower extremities (65.0%) collateral vessels with dermal back-flow areas between lymphatic vessels were seen.Conclusions As a novel tool to image the pathologically modified lymphatic vessels in patients with clinically advanced stage of primary lymphedema,magnetic resonance lymphangiography is a safe,useful technique.

Objective To evaluate invitro antibacterial activity of Gymnema sylvestre extractant.Methods The antibacterial activity of Gymnema sylvestre extractant against staphyloccus aureus,bacillus coli,bacillus proteus,bacillus aeruginosus was detected by agar dilution method.Results The minimum inhibitory concentration were 0.65,1.25,1.25,2.5 mg/mL respectively.Conclusion Gymnema sylvestre extractant had strong antibacterial activity against various bacteria.

ObjectiveTo access the value of combined application of high-resolution interstitial MR lymphangiography (MRL) and heavily T2WI for the visualization of lymphatic vessels in patients with primary lymphedema.Methods Forty lower extremities in 31 patients with primary lymphedema were examined by heavily T2 WI and interstitial MRL with a 3.0 T MR unit (Philips Medical Systems,Best,the Netherlands).Two experienced radiologists analyzed the images and tried to determine the differences in number of lymphatic vessels,and their maximum diameters,SNR and CNR,and accumulated lymph fluid in the tissue.Statistical analyses were conducted by using Wilcoxon test and t test.ResultsDilated lymphatic vessels in 73 leg segments of 40 lower extremities were visualized on heavily T2WI (median 5,1 to 24),which were more than those on MRL ( median 3,1 to 16) (Z =-2.92,P ＜ 0.01 ).The maximum diameter of lymphatic vessels was (4.3 ± 1.5 ) mm on heavily T2 WI,whereas it was ( 3.4 ± 1.0) mm on MRL ( t =6.90,P ＜ 0.0 1 ).The average SNR and CNR in the dilated lymphatic vessels were 257 ± 130,207 ± 113 on MRL and 169 ± 91,135 ± 82 on heavily T2 WI,which was statistically significant ( SNR t =- 5.95,CNR t =-5.10; P ＜ 0.01 ).The visualization of regions of accumulated lymph fluid on heavily T2WI (median 1 ) had a higher score than that on MRL ( median 0 ) ( Z =- 5.64,P ＜ 0.01 ).Conclusions The heavily T2WI has greater sensitivity and the MRL image has better SNR and CNR.Combining these two MR techniques can provide adequate information for clinicians in the therapeutic planning of patients with advanced stages of lymphedema.

ObjectiveTo evaluate the ability of high resolution magnetic resonance (MR) imaging at 3.0 tesla to depict the characterization of human carotid arterial vessel wall and detect atherosclerotic lesions ex vivo in comparison to histopathologic results. MethodsEighteen carotid arteries obtained from 9 elderly donors underwent fat-suppressed T1 - and T2-weighted MR imaging at 3.0 tesla MR system with a mouse coil. Corresponding histological sections were obtained for the comparison. Correlation between MR images and histopathologic slices was obtained by Pearson or Spearman correlation coefficient.Cohen K was computed to quantify the agreement between MRI and histopathologic findings.Results Lumen area,intima and media area measured on fat-suppressed T2-weighted images showed stronger correlation with the corresponding histopathologic slices [ MRI vs.histopathology:( 27. 53 ± 6. 77 ) mm2 vs.( 25.83 ±6. 69 ) mm2, r2 ＝ 0. 91,P2 ＝ 0. 024, ( 12.31 ± 3. 31 ) mm2 vs.( 12. 28 ± 3.71 ) mm2, r2 ＝ 0. 70, P2 ＝ 0. 020,Median 12. 29 mm2(Min 1.12 mm2, Max 33.18 mm2) vs.Median 11.62 mm2(Min 0.89 mm2, Max 32. 84 mm2 ), r2 ＝ 0. 74, P2 ＝ 0. 016, respectively]. The Cohen K score between the MR imaging and American Heart Association classifications was 0. 74, which corresponds to a good agreement. Conclusions 3.0 T high-resolution multi-sequence MRI can clearly show the structure of ex vivo carotid artery wall and allow quantitative assessment.Fat-suppressed T2Wimaging has a greater advantagein presenting atherosclerotic lesions.

Objective To evaluate the application of cervical lateral mass plate fixation in the treatment of fracture and dislocation of lower cervical spine. Methods From February 2001 to June 2003, 21 cases of lower cervical spine injury were treated by cervical lateral mass plating fixation, received spinal decompression and reduction according to the types of fracture and dislocation. A cervical lateral mass plate was applied in each lateral mass. The screw prick point was defined at 1-2 mm inner and lower to the mass center. The sagital angle, horizontal angle of internal fixation screw were 45 degrees and 25-30 degree respectively. Results The follow up ranged from nine months to two years and nine months (mean 13 months). All cases were encouraged to sit up, wearing soft collar 4-7 days after the surgery. The mean off-bed time of those cases without spinal cord injuries less than Frankel C grade were seven days (4-14 days) after operation. All cases obtained solid bony fusion 4-6 months postoperatively. Sixteen cases with spinal cord injury improved for one grade according to American Spinal Injury Association. Three cases with nerve root injury obtained complete recovery after operation. There was no severe complication such as vertebral artery nerve root or spinal cord injuries or aggravation of spinal cord injury. One case had uneven reduction and two suffered screw loosening. Conclusion Cervical lateral mass plate fixation is an efficient and reliable technique for segmental posterior fixation, for it has the advantages of wide indication, relatively simple and safe operating as well as strong stability.

Objective To study the regularity changes in serum levels of C-reactive protein(CRP)and mannose-binding lectin(MBL)in patients of community acquired pneumonia(CAP)with different syndrome patterns of traditional Chinese medicine(TCM),and to explore the new objective markers to differentiate the syndrome patterns of TCM. Methods According to The Guideline on TCM diagnosis and treatment of CAP(2011 edition),104 patients with CAP were selected and their syndromes were cassified into 3 classes and 8 patterns of syndrome:excessive class〔including following patterns:wind-heat invading lung(fengrexifei),exopathic cold and interior heat(waihanneire), accumulation of heat in lung(tanreyongfei),accumulation of phlegm-dampness in lung(tanshiyongfei)〕,deficient vital QI leading to lingering of pathogen class〔qi deficiency of lung and spleen(feipiqixu),both qi and yin deficiency (qiyinliangxu)〕,TCM critical class〔heat falling into pericardium(rexianxinbao),pathogen invasion and vital qi deterioration(xiexianzhengtuo)〕. In the same period,after physical examinations,100 healthy volunteers were chosen as healthy control group. The serum levels of CRP and MBL were detected before treatment and after treatment for 4 days and 7 days. Results Among the 104 CAP patients,the most popular class of syndrome was the excessive one(63.5%),followed by deficient vital QI leading to lingering of pathogen(19.2%)and TCM critical class(17.3%). The serum CRP level in CAP patients at each time point was higher than that in healthy control group,which had a different tendency to change over time in different syndrome patterns of TCM. With the prolongation of treatment time,the serum CRP levels in fengrexifei and waihanneire patterns returned to a normal level on the 7th day(mg/L:13.51±11.48,7.07±1.84 vs. 6.96±2.19,both P>0.05),in feipiqixu and qiyinliangxu patterns the CPR levels were higher,but its descent rate was relatively fast,and on the 7th day it was approximately normal in spite of being higher than the level in healthy control group(25.25±25.90,18.17±23.19 vs. 6.96±2.19,both P<0.05);in tanreyongfei and tanshiyongfei patterns,although the CPR levels were decreased,they still maintained at relatively high levels on the 7th day after treatment(51.70±27.33,49.28±30.57),and no downtrend of CPR was seen in rexianxinbao and xiexianzhengtuo patterns. Before treatment,the serum MBL levels in CAP patients with fengrexifei,waihanneire, tanreyongfei,tanshiyongfei,feipiqixu and qiyinliangxu patterns were higher than the level in healthy control group, and in rexianxinbao and xiexianzhengtuo patterns,the levels were lower than those in other patterns and kept being at relatively lower levels along with the prolongation of the therapy. Conclusion Serum CRP can be used as a reference marker for different syndrome patterns of TCM in patients with CAP,and low serum MBL level was a risk factor of severe syndrome patterns of TCM and a poor prognosis in CAP.

Objective To construct eukaryotic expression vector of human antimicrobial peptide LL-37 pPIC9-LL-37, and transform the plasmid pPIC9-LL-37 into P.pastoris GS115 to obtain the recombinant P.pastoris strains.Methods The full-length of antimicsobial peptide LL-37 gene was artificially synthesized by overlap extension method and was fused to pPIC9 and then the fused plasmid was transformed into E.coli DH5?.After analysis by PCR and sequencing,the plasmid pPIC9-LL-37 was transformed into P.pastoris.The colonies exhibiting the phenotype of His+Mut+ or His+Mut-were screened by means of MM and MD plates and the insertion was confirmed by PCR.Results The results of PCR and sequencing confirmed that the LL-37 gene was correctly inserted into pPIC9. The colonies of 10 His+Mut+ and 9 His+Mut-were obtained by means of MM and MD plates screening and were confirmed by PCR.Conclsion The recombinant P.pastoris strains containing LL-37 were successfully obtained.

Objective:To explore and compare the clinical efficacy of lyophilized recombinant human brain natriuretic peptide (Lrh-BNP) and dobutamine (Dob) in the treatment of patients with acute heart failure (AHF) and impacts on the plasma galectin (Gal)-3,Cystatin C (CysC) and endothelin (ET-)-1 levels.Methods:114 cases of patients with AHF in our hospital from February 2015 to February 2017 were selected as the research objectives and randomly divided into two groups.Dob group was treated by Dob,while Lrh-BNP group was treated by Lrh-BNP.The cardiac function parameters,plasma Gal-3,CysC,ET-1 levels before and after treatment,clinical comprehensive efficacy and incidence of adverse reactions were compared between two groups.Results:The FS,LVEF levels of both groups at 72 hours after treatment were significantly higher than those before treatment (P＜0.01),but the LVEDD,plasma Gal-3,CysC,ET-1 levels were obviously decreased (P＜0.01),the index mentioned above of Lrh-BNP group improved more significantly than those of the Dob group(P＜0.01).The overall effective rate of Lrh-BNP group was 89.5 ％,which was significantly higher than that of the Dob group (73.7％,P＜0.05).No significant difference was found in the incidence of adverse reaction between two groups(P＞0.05).Conclusion:Lyophilized recombinant human brain natriuretic peptide was more effective in the treatment of AHF than Dobutamine with equal safety,which might be related to the decrease of plasma Gal-3,CysC,ET-1 levels.