Acoustic Stimulation ; Animals ; Auditory Perception ; physiology ; Behavior, Animal ; physiology ; Noise ; adverse effects ; Rabbits ; Sound ; Swine

Objective To express and purify mammalian glycosylation modified trimeric Ebola virus trimeric glycoprotein (EBOV-GP) using novel glycoengineered Pichia pastoris.Methods The EBOV-GP and EBOV-GPΔMLDΔTM genes were cloned into the pPICZ-αA vector,electrochemically converted to glycoengineered Pichia pastoris,and compared with EBOV-GP expressed in HEK-293T cells.Glycosylation was analyzed by PNGaseF and EndoH digestion,while the target protein was purified by affinity chromatography and ion exchange chromatography.N-terminal sequencing was used to determine whether the signal peptide was correctly cleaved during protein translation and gel column analysis was used to find out whether the trimeric structure was formed.Results The results of PNGaseF showed that EBOV-GP expressed by glycoengineered Pichia pastoris and HEK-293T cells had the same relative molecular mass and N-glycosylation degree.EndoH digestion showed that the N-glycosylation modification of EBOV-GPΔMLDΔTM was in a non-high mannose form.N-terminal sequencing showed that the signal peptide of the GP protein itself was correctly excised.Gel column analysis showed that the purified protein was in a trimeric form.Conclusion An EBOV-GP is obtained with complex glycosylation modification based on Glycoengineered Pichia pastoris.

Objective To prepare recombinant human prothrombin-2 expressed in Pichia pastoris, and assay the enzymatic and clotting activities of prothrombin-2 activated by prothrombin activator ecarin.Methods Human prothrombin-2 gene and Echis carinatus ecarin gene were synthesized separately on the basis of the cDNA sequences published in GenBank.The gene of prothrombin-2 was cloned into the expression vector pPICZαA.The expression vector pPICZαA/prothrombin-2 was transformed into glycoengineered P.pastoris, and then prothrombin-2 engineered P.pastoris was screened.The expression products were induced by methanol, purified by two-step chromatography and identified by diges-tion by PNGase F and analysis of pepetide fingerprint.The ecarin gene was cloned into the expression vector pcDNA3.1. The expression vector pcDNA3.1/Ecarin was transformed into HEK 293T cells and the culture supernatant of HEK 293T/Ecarin was collected.The reaction product of HEK 293T/Ecarin cell culture supernatant and purified prothrombin-2 was analyzed by S-2238,which was the chromogenic substrate for thrombin.Fibrinogen was used to measure blood clotting time. Results The purified protein of P.pastoris expressed prothrombin-2 culture supernatant was 37 ×103 .The relative molecular mass(Mr) of the purified protein was reduced to 35 ×103, which was consistent with the theoretical Mr of prothrombin-2 molecular weight.The purified protein was proved to be prothrombin-2 by peptide fingerprint identification. The purified prothrombin-2 processed by HEK 293T/Ecarin culture supernatant could hydrolyze S-2238 to produce yellow pNA, and D405 of pNA increased with the volume of the processed prothrombin-2 that could promote the plasma coagulation.The blood clotting time was close to that of the thrombin kit.Conclusion Prothrombin-2 is prepared by P.pastoris and activated toα-thrombin by ecarin.This technique may replace the method of extraction of prothrombin from plasma and can be used for the treatment of war wounds or for future clinical research.

Objective To develop an interleukin-4(IL-4) antagonist named M5-IgG1Fc protein constructed by genetic engineering of antibody Fc fragment-cytokine mutein fusion protein which has a long half-life time in plasma.M5-IgG1 Fc protein binds to IL-4 receptor but cannot activate downstream signalling pathway , which provides a basis for drug develop-ment for allergic diseases .Methods The synthesized interleukin-4 mutant gene ( named M5 ) was cloned into the expres-sion vector pBV220 and transformed into E.coli DH5α.Chimeric gene M5-IgG1Fc obtained by overlap extension (SOE) method was transformed into glycoengineered Pichia pastoris GJK01 through expression vector pPICZαA .Then M5-IgGFc fusion protein was obtained by protein purification after being induced by methanol in 72 hours.The anti-IL-4 biologicial ac-tivity assay of M5 and M5-IgG1 Fc was performed with CTLL-2/IL-4R cells and detected with MTT colormetry .Finally,the half-life time of M5 and M5-IgG1 Fc protein in mice was compared by detecting the remaining amount in plasma with ELISA kit.Results The M5 protein expressed in E.coli and M5-IgG1 Fc fusion protein expressed in P.pastoris GJK01 both had IL-4 antagonistic bioactivity .The EC50 of both, which inhibited 5.6 ×10 -2 nmol/ml of IL-4, were 0.31 ±0.05 and 0.77 ± 0.03 nmol/ml,respectively.The maximum of M5 in plasma at 0.5 h was 5.8 ×10 -2 nmol/ml but the remaining amount was 2.8%of the maximum at 2 h.M5 protein could not be detected after administration at 8 h because of the detection line . The maximum of M5-IgG1 Fc fusion protein was 4.7 ×10 -2 nmol/ml,while fusion protein M5-IgG1 Fc decreased to 4.3%of its maximum at 120 h and could not be detected at 168 h.Conclusion M5 protein has IL-4 antagonistic bioactivity .M5-IgG1 Fc fusion protein expressed in glycoengineered P.pastoris GJK01 has IL-4 antagonistic bioactivity and long retention time in mice,which can be potentially used for treatment of allergic diseases .

Aim To evaluate the effects of pantoprazole on various experimental acute ulcer inrats and mice. Methods The model of a gastric ulcer of rats or mice was caused bystree- induced ulcer and ligatel pylurus-induced ulcer. Results & Conclusions At adose of 5, 10, 20 mg? kg-1 of Pantoprazole can markedly decrease the ulcer index ofstree-induced ulcer. Pantoprazole(4, 8, 16 mg? kg -1 ) significantly decrease the areaof ligated pylorus-induced gastric ulcer. It was also found that pantoprazole caninhibit the output of basic gastric acid.

Objective To investigated to implement condition of preventive measure and control effect for endemic fluorosis in Fengshun County from 2005 to 2006.Methods It was investigated according to the National Surveillance Program of Endemic Fluorosis.Hupo,Daizai and Anquan Villages of Tangxi Town in Fengshun County were selected as monitoring spots.The usage of reforming water facilities,fluoride content in drinking water and urine of children aged 8-12 years and the prevalence rate of dental fluorosis of children were investigated.Resul tsin 2005 and 2006.a total of 18 reforming water facilities were surveyed and six of which were damaged or out of service.In 2005,the fluoride content in drinking water in the 3 villages was 2.10,1.22 and 0.15 mg/L The prevalence rate of dental fluorosis of children aged 8-12 years was 54.23%(64/118),38.91%(79/203) and 9.10%(6/66).The urine fluoride content of children was 0.95,0.90 and 1.05 mg/L,respectively.In 2006,the fluoride content in drinking water in Hupo,Daizai and Anquan Village was 2.01,1.57 and 0.21 mg/L.The prevalence rate of dental fluorosis of children aged 8-12 years was 26.47%(27/102),12.50%(23/184)and 6.15%(4/65),respectively.The urine fluoride content of children was 0.97,0.61 and 0.59 mg/L.Conclusions The outcome of surveillance data in Fengshun County has reached the sanle level as that of non-disease area.However,the management of reforming water facilities should be improved.

Objective:Before the radiotherapy was performed, patients with pelvic tumors were analyzed for the consistency of bladder filling in the three steps of " Immobilization" , " CT Simulation" and " X-ray Simulation" .Methods:In 2014, 105 patients (68 cases of cervical cancer, 32 cases of rectal cancer, 3 cases of vaginal cancer and 2 cases of prostate cancer) with pelvic tumor radiotherapy were randomly assigned to monitor bladder urine volume to a target urine volume of 400 ml. First, patient were exhorted to empty the bladder, and the bladder volume meter BVI 9400 was used to measure the urine volume of the patient after emptying of the bladder. The patient immediately drank about 540 ml of water and suppressed urine, measurements were taken every 0.5 h. At the same time, when the patient complained of " urgency of urine" , bladder urine volume would be measured again and the time would also be recorded. Every other half an hour (emptying, 0.5 h after emptying, 1.0 h after emptying), when complaining of " urgency of urine" , when actually performing urine volume and time were described as: U 0 and t 0, U 0.5 and t 0.5, U 1.0 and t 1.0, U t and t, U T and T. Results:There was a statistically significant difference in gender and age, and women had stronger ability to urinate than men U 1.0( P=0.003), young people had stronger ability to urinate than middle-aged U 1.0( P=0.002). In the three-step comparison, there was no statistically difference between 1 hour after emptying urine volume U 1.0( P=0.177) and the actually performing urine volume U T ( P=0.052). And the final urine volume was concentrated at 298-526 ml. After the patient emptied the urine volume and complained of " urgency of urine" , the time slot was t=(75.2±49.9) min, with the urine volume of U t=(331.2±140.3) ml. And there was no statistically difference between U t and U T ( P=0.198) at X-ray Simulation. Conclusions:The patient emptied the bladder and immediately drank 540 ml of water. After 1 hour of suppressing urine, he complained of " urgency of urine" and achieved the target urine volume (400 ml). At this time, the bladder urine volume U 1.0 was consistency in the immobilization, CT Simulation, and X-ray Simulation.

AIMTo study a new way to prepare high-dosage paclitaxel entrapped magnetic targeted nanoparticles and evaluate its quality.

METHODSFe3O4 nanoparticles are prepared by co-depositing, at the same time ultrasonic is used to decrease soft agglomerate of nanoparticles and increase disperse level of it. The property of nanoparticles surface is improved to make the integrating of liposome and nanoparticle to be tighter. At last, paclitaxel entrapped magnetic solid liposome nanoparticles have been prepared by microemulsion-curing under low-temperature. The loading efficiency and encapsulating rate were determined by reverse-phase high-perfomance chromatography.

RESULTSThe nanoparticles have spherical shape. Diameter of nanoparticle ranged from 150 nm to 170 nm. 98.29% of the drug is entrapped in the particle.

CONCLUSIONMagnetic susceptibility of nanoparticles is high, and the nanoparticles meet with the demand of targeted delivery system.

Antineoplastic Agents, Phytogenic ; administration & dosage ; chemistry ; Drug Compounding ; methods ; Drug Delivery Systems ; Emulsions ; Ferrosoferric Oxide ; chemistry ; Hydrogen-Ion Concentration ; Liposomes ; Nanoparticles ; administration & dosage ; chemistry ; Paclitaxel ; administration & dosage ; chemistry ; Particle Size ; Temperature ; Ultrasonics

Bone Transplantation ; Dental Research ; Humans ; Tooth

Objective To express the hemagglutinin of H7N9 in Pichia pastoris and analyze its immunogenicity. Methods The HA [1 -525 amino acids(aa)] of H7N9 [A/Hangzhou/1/2013(H7N9)] lacking the C-terminal transmembrane anchor-coding sequence was amplified by PCR and cloned into the expression vector pPICZαA.The plasmid pPICZ-HA7-S-was transformed into P.pastoris and the HA1-525 was detected by ELISA.Then the HA1-525 was precipitated by PEG20000.After immunization with the HA1-525 , the anti-HA7 antibody in mouse serum was detected by ELISA and hemagglutinin inhibition ( HI) test.Results The HA1-525 was expressed in P.pastoris after being induced with methanol. Western blotting confirmed that there were specific dispersion bands and the molecular weight of HA1-525 decreased to 58 × 103 after being digested by endo H.Anti-HA7 antibody was found in serum of HA1-525 immunized mice and the hemaggluti-nation-inhibition titers reached 1∶700 after the third doses.Conclusion This study shows the HA1-525 expressed in P.pastoris can induce the neutralizing antibody in mice.