Pichia pastoris is a new eukaryotic expression system with great potentials, has unexampled advantages in expressing the heterologous proteins and has gotten more and more wide application. The following aspects were described in detail: the advantages of Pichia pastoris in the expression of heterologous genes, the mechanism of the heterologous gene integrations, the glycosylation of the expressed protein and the modification of the post-translated protein.

Background Establising the culture model of Müller cells for obtaining the highly putified target cells is essential for the study about the physiology and pathology of retinal Müller cells. The exsiting purifing method for culturing Müller cells is dissatisfactory. Objective This study was to establish a method to obtain high purifing Müller cells. Methods The retina from 5 clean newborn SD rats were isolated and digested by 0. 01% trypsin and cultured in DMEM containing 10% fetal bovine serum. The cellular suspension was then prepared,and the target cells were screened using flow cytometry based on the size and the quantity of cells. Cultured and passaged cells were identified by transmission electron microscope and light microscope. Immunocytochemistry was used to detecte the expression of GFAP in cultured cells for the determination of type and purity of the cells. Results The cells showed the similar shape to retinal Müller cells after primarily culture with the large volume, and some small other types of cells could been seen. The growth of cells was quickly 3 weeks later. The fibroblasts were removed using sticking-wall by steps,and neurons were eliminated following passage. Aboundent of cellular organs were seen under the transmission electron microscope. The positive response rate of the cells for CFAP was 100%. Conclution Flow cytometry offer a rapid and feasible approach for purifying Muller cell and it builds the foundation for further study about Müller cells.

NDV strain ZJ1 strain , a highly virulent NDV strain, has been prevalent among the waterfowls in China mainland in the past years. Multi-basic amino acid sequence distribute in the protease cleavage site of F protein of this strain. Recombinant expressing plasmid pCI-FT, was generated by converting multi-basic amino acid sequence of 112, 115, 117 of the protease cleavage site of F_ 0 protein, to the non-basic amino acid sequence characteristic of avirulent NDV strain. The result from co-expression of mutant or parental F protein with homologous HN protein in COS-1 cells revealed that both mutant and parental F protein had fusion activity. The result from co-expression of mutant or parental F protein with homologous HN protein in CEF cells showed that the cleavage activity of mutant F protein was significantly reduced. The study built a foundation for mutagenesis of amino acid sequence of the protease cleavage site of F_ 0 protein at the full-length cDNA clone level, study on factors contributing to virulence and construction of candidate vaccine strain, and so on.

Objective To explore the prevention and treament of the complications for laparoscopic cholecys- teetomy(LC).Methods Clinical datas of 600 eases undergoing LC were collected and analyzed retrospectively.Re- suits Among all the cases,249 cases were diagnosed as cholecystolithiasis,284 cases as gallbladder and 67 cases as chronic cholecystitis.The mean duration of operation was 42min.Conversions to open cholecystectomy were needed in 18 cases(3%).Complications took place in 6 cases(0.5%),involving 2 cases of abdominal bleeding and 1 case of bile leakage.There was no bile duct injury and death.Conclusion Strictly-controlled indications,skilled performance and timely open exploration are very important to prevent and reduce the complications.

The effects of Wumeiwan (WMW) on TNF-alpha, IL-6, IL-8, IL-10 and NF-kappaBp65 in rats with ulcerative colitis (UC) were investigated, the curative effectiveness of WMW vs salicylazosulfapyridine (SASP) was compared, and the action mechanism was analyzed. Fifty-Six Sprague-Dawley (SD) rats were randomly divided into four groups (n=14 in each group, with equal ratio of male and female): normal control group, model group, SASP group, and WMW group. Except normal control group, the rat UC models in the remaining three groups were established using the method of 2.4-dinitrochlorobenzene (DNCB) immunization and acetic acid local enema. The rats in model group, SASP group, and WMW group were treated with distilled water, SASP, and WMW respectively. The changes in the symptoms and signs were observed, and levels of IL-6, IL-8, TNF-alpha, IL-10 and the expression of NF-kappaBp65 in the colonic tissues were statistically analyzed. The results showed that the levels of IL-6, IL-8, and TNF-alpha were significantly increased (P<0.01), while those of IL-10 significantly reduced (P<0.01) after establishment of rat UC models as compared with normal control group. The levels of IL-6, IL-8, and TNF-alpha were obviously lower, but the level of IL-10 was obviously higher in WMW and SASP groups than those in model group (P<0.05). The levels of IL-6, IL-8, and TNF-alpha were lower, while the level of IL-10 was higher in WMW group than in SASP group. NF-kappaBp65 was expressed negatively or weakly in normal colonic tissues. The positive expression rate of NF-kappaBp65 in WMW group and SASP group was obviously lower than in model group (P<0.01), and there was significant difference between WMW group and SASP group (P<0.05). It was concluded that rat UC model was established successfully. WMW could up-regulate the expression of IL-10, down-regulate the expression of TNF-alpha, IL-6, IL-8, and inhibit the NF-kappaBp65 activity to adjust immune function, indicating WMW had better curative effects on UC in rats.

Objective:To summarize the results and methods of surgical treatment for type A aortic dissection with small true lumen of the descending aorta.Methods:9 patients underwent surgical treatment for type A aortic dissection with small true lumen of the descending aorta between January 2017 and December 2019 were analyzed retrospectively. There were 7 males and 2 females, mean age of (41.6±9.2) years. Acute dissection were 2 cases, and chronic dissection were 7 cases. Preoerative computed tomography was used to diagnose the dissection and evaluate the true lumen of the descending aorta. This procedure was done in all patients via a median sternotomy under hypothermic CPB with SCP. 4-branched prosthetic graft was used to replace the ascending aorta and aortic arch. The procedures involving the descending aorta: Hybrid surgery using TEVAR. Distal intimal flap fenestration. Implanting the intraoperative stent-graft or prosthetic graft at false lumen for second-step operation.Results:There was no in-hospital mortality. Stroke, Spinal cord, visceral ischemia and lower limbs malfunction were not observed. Reintervention was not found in case with acute dissection during follow-up. One patient who reveived fenestration underwent TEVAR, others with chronic dissection underwent thoracoabdominal aortic replacement 3 months after surgery.Conclusion:Hybrid or staged procedures was a suitable alternative to patients with type A aortic dissection with small true lumen of the descending aorta.

In order to explore the differences of chemical constituents of Paeoniae Radix Alba and Paeoniae Radix Rubra, a qualitative analytical method of liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (HPLC-Q-TOF-MS/MS) was developed for identification of multi-constituents and an HPLC-DAD analytical method was developed for simultaneously determining 14 major compounds (gallic acid, protocatechuic acid, paeoniflorin sulfonate, protocatechuic aldehyde, methyl gallate, oxypaeoniflorin, catechin, albiflorin, and paeoniflorin, ethyl gallate, benzoic acid, pentagaloylglucose, benzoyl-paeoniflorin, and paeonol) in Paeoniae Radix Alba and Paeoniae Radix Rubra. Q-TOF/MS qualitative analysis was performed under negative ion mode and inferred 38 components of Paeoniae Radix Alba and 30 components of Paeoniae Radix Rubra. HPLC-DAD quantitative method result showed the contents of 8 ingredients were different between Paeoniae Radix Alba and Paeoniae Radix Rubra. The results indicated that the new approach was applicable in qualitative and quantitative quality control of Paeoniae Radix Alba and Paeoniae Radix Rubra.
Bridged-Ring Compounds ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; Glucosides ; chemistry ; Molecular Structure ; Monoterpenes ; chemistry ; Paeonia ; chemistry ; classification ; Tandem Mass Spectrometry ; methods

Eight fragments were amplified and cloned into pCR2.1 vector with the designed primers.The fragments,amplified with primer Ⅰ to Ⅶ,were subcloned into transcription vector to construct the plasmid pNDVZJI which contained the full-length cDNA of NDV ZJI strain.The eukaryotic expression vector pCI-L was constructed by subcloning the fragments,amplified with the primer Ⅴ,Ⅵ and Ⅷ,into the expression vector pCI-neo.The full-length cDNA clone,pNDVZJI,with three helper plasmids,pCI-NP、pCI-P and pCI-L,were cotranfected into BSR-T7/5 cell expressing T7 RNA polymerase.After inoculation of transfected cell culture into embryonated chicken eggs from specific pathogen free(SPF)flock,The NDV of ZJI strain was rescued successfully,which laid a good foundation for the further related research.

OBJECTIVE: To investigate the diagnosis and management of non-pituitary lesions in sphenoidal sinus. METHOD: All cases with non-pituitary lesions in sphenoidal sinus were confirmed by CT scan. Eight cases with localized lesions underwent operation by trans-sphenoidal approach. Two cases with juvenile nasopharyngeal angiofibroma with invasion to the sphenoid sinus were treated by trans-septal approach. The rest received operation by trans-superior meatal or trans-ethmoidal approach. RESULT: Forty-five of these cases underwent complete or major resection of the lesion by endoscopic sphenoid sinus surgery, including 23 cyst and pus cyst of sphenoidal sinus, 8 fungal sphenoid sinusitis, 2 bleeding polyp of sphenoidal sinus, 1 post- hypophysectomy granulation hyperplasia of sphenoidal sinus , 5 papilloma of sphenoid sinus, 1 cerebrospinal rhinorrhea of sphenoid sinus, 2 ossified fibroma of sphenoid sinus,2 juvenile nasopharyngeal angiofibroma with invasion to the sphenoid sinus, 1 meningioma of ethmoid and sphenoid sinus. Three cases with hematoma in sphenoidal sinus and pseudoaneurysm in internal carotid artery underwent nasal endoscopic examination, and the diagnosis was established by DSA, and they received interventional therapy. Three cases with malignancy of sphenoidal sinus received major mass resection of sphenoidal sinus by trans-ethmoidal approach, and followed with radio therapy and chemotherapy. Two cases with NPC involving sphenoidal sinus were treated by radio therapy and chemotherapy after pathological examination. CONCLUSION Headache and visual loss were two common symptoms for the lesions in sphenoidal sinus. Imaging study including CT, MRI and DSA is very important for the diagnosis of the lesions in sphenoidal sinus. There are various surgical pathways to deal with sphenoidal sinus diseases under nasal endoscope. The operation will be direct, safe and minimal invasive if we choose the pathway properly.
Adolescent ; Adult ; Aged ; Child ; Endoscopy ; Female ; Humans ; Male ; Middle Aged ; Paranasal Sinus Diseases ; surgery ; Sphenoid Sinus ; Sphenoid Sinusitis ; surgery

Objective TostudytheinfluenceofmeteorologicalconditionschangingontheactivityintensityofinfluenzaA (H1 N1 )and to establish the prediction model of H1 N1 positive rate associated with meteorological factors.Methods TheinfluenzaA(H1N1)datafromAprilof2009toJanuaryof2011insentinelhospitalanddailymeteorologicaldatain the same period were collected,which were analyzed by Spearman correlation analysis.The prediction model was establishedusingChi-squaredautomaticinteractiondetector(CHAID).Results Weeklyaverageatmospheric pressure(r =0.50),highest atmospheric pressure (r =0.51 ),lowest atmospheric pressure (r =0.50),average temperature (r=-0.40),highest atmospheric temperature(r=-0.41),lowest atmospheric temperature(r=-0.39), precipitation(r=-0.23 )and average wind speed (r=-0.22 )were positively correlated with the activity intensity of H1N1(all P<0.05).Factors that affected H1N1 positive rate were lowest atmospheric pressure,average wind speed and precipitation(P<0.05 ).The prediction model of H1 N1 positive rate showed that the correct rate of prediction was 66.67%.Conclusion Lowestatmosphericpressure,averagewindspeedandprecipitationarecloselyassociatedwith the activity intensity of influenza A (H1 N1 ).CHAID method can be used to predict the H1 N1 epidemics.