Objective To obtain the Tat-GFP fusion proteins with penetrating activity and labeled with green fluorescence protein (GFP), and to explore the cell membrane penetrating activity of Tat-GFP in MCF-7 cells. Methods The plasmid pET-24a-Tat-GFP was transformed into Escherichia coli BL21 cells. Different concentrations (0.5 and 1.0 mmol · L-1 ) of isopropyl-β-D-thiogalactopyranoside (IPTG ) and cell culture temperatures (22℃ and 37℃)were used to optimize the protein expression.The Tat-GFP proteins in supernatant were purified using Ni-IDA resins. Western blotting analysis was used to identify the Tat-GFP protein, and confocal laser scanning microscope (CLSM ) was used to examine the cell penetration of Tat-GFP protein. Results There was no significant difference in the Tat-GFP protein production induced by 0.5 and 1.0 mmol·L-1 IPTG;however,the low temperature (22℃)-induced BL21 cells expressed more Tat-GFP proteins than that at 37℃ induction.The Western blotting analysis results showed that GFP antibody could specifically recognize the proteins in PVDF membranes in dose-dependent manner;the CLSM results indicated the distribution of green fluorescence in cytoplasm and nucleus of MCF-7 cells.Conclusion The Tat-GFP protein highly expresses in the supenatant of Escherichia coli i BL2 1 cells at low temperature;the obtained Tat-GFP protein with green fluorescence preserves the cell penetrating activity.

Objective To study the effect of mechanical ventilation guided by esophageal pressure on hemodynamics and oxygen metabolism of severe acute pancreatitis (SAP) swine model with intra-abdominal hypertension (IAH) Methods By self-controlled study,SAP model was made by infusing sodium taurocholate (5％) into the pancreatic duct in 6 domestic swine.Mechanical ventilator mode was volumeassist control with tidal volume 10 ml/kg ; FiO2 40％ and PEEP 5 cm H2O (routine ventilation) given to SAP model swine.After 3 h ventilation,pneumo-peritoneum was made with N2 gas to increase the intraabdominal pressure (IAP) to 25 mm Hg in SAP swine.Three more hours later,PEEP was adjusted as the measurement of esophageal pressure (Pes guided ventilation) to such a level that trans-pulmonary pressure stayed above 0 cm H2O during end-expiratory occlusion.During the investigation period,heart rate (HR),cardiac output index (CI),central venous pressure (CVP),mean arterial pressure (MAP) and pulmonary arterial wedge pressure (PAWP) were continuously recorded with the aid of Swan-Ganz catheter and ECG monitor,and oxygen partial pressure of artery (PaO2) and carbon dioxide partial pressure of artery (PaCO2) were measured by blood-gas analysis.In addition,systemic oxygen delivery (DO2) and systemic oxygen consumption (VO2) were calculated by using the data of blood-gas analysis of arterial and central venous blood.Results No swine model was subjected to barotrauma.After routine mechanical ventilation,there were significant differences in HR,CI,MAP,CVP,PAWP,Ppeak,Pplat,Pes,pulmonary compliance (Cstat),PaO2,and DO2 between SAP and IAH in swine (all P ＜ 0.05).Compared with routine ventilation,however,PaO2 and Cstat improved significantly with lower CI and increased Pplat after PEEP adjusted according to measurements of esophageal pressure (all P ＜ 0.05).The lactate decreased significantly after esophageal pressure guided ventilation (all P ＜ 0.05).There were no significant changes in PaCO2,HR,MAP,CVP and PAWP in IAH swine after mechanical ventilation with routine parameters (all P ＞ 0.05).Conclusions There were remarkable effects on oxygen metabolism in response to mechanical ventilation guided by esophageal pressure.In case of clinical application of mechanical ventilation,the results of this study are in favor of setting transpulmonary pressure according to measurements of esophageal pressure in SAP patients with IAH in an early stage.

Objective: To investigate the serum anti-mumps IgG antibody levels among children aged 0 to 4 years in Haishu District, Ningbo City, Zhejiang Province, so as to provide insights into improvements for mumps vaccination program. Methods: Children aged 0 to 4 years were sampled from Haishu District using a stratified random sampling method in 2016 (before adjustment of the mumps vaccination program) and 2022 (after adjustment of the mumps vaccination program). Participants' demographics were collected using questionnaires, and the coverage of mumps-containing vaccines was collected from the Ningbo Municipal Immunization Information System. Serum anti-mumps IgG antibody was measured using enzyme-linked immunosorbent assay (ELISA), and the seroprevalence of anti-mumps IgG antibody and geometric mean concentration (GMC) were estimated among children aged 0 to 4 years in 2016 and 2022. Results: A total of 464 children were enrolled in 2016, including 250 boys (53.88%) and 214 girls (46.12%), and there were 301 children receiving mumps-containing vaccines (64.87%). The seroprevalence of anti-mumps IgG antibody were 48.08%, 34.44%, 81.11%, 84.44% and 84.44%, and GMC were 233.86, 351.77, 333.66, 362.29 and 410.72 U/mL. A total of 456 children were recruited in 2022, including 236 boys (51.75%) and 220 girls (48.25%), and there were 427 children receiving mumps-containing vaccines (93.64%). The seroprevalence of anti-mumps IgG antibody were 72.73%, 95.00%, 100.00%, 98.68% and 99.04%, and GMC were 524.05, 1 229.69, 1 623.64, 788.01 and 738.41 U/mL. Higher seroprevalence and GMC of anti-mumps IgG antibody was seen in 2022 than in 2016 among children at all age groups (P<0.05). Conclusion Following adjustment for vaccination programs, the seroprevalence and GMC of anti-mumps IgG antibody significantly increased among children at ages of 0 to 4 years in Haishu District.

BACKGROUND:At present, a heparin-coated extracorporeal circulation pipe is used widely, but the price is expensive and limits its wide application.
OBJECTIVE:To screen the optimal coating condition of oxidated sodium alginate as a coating material to coat the medical polyvinyl chloride pipe and to evaluate the anticoagulant properties and the stability of the coated pipe.
METHODS:Oxidized sodium alginate was prepared with sodium periodate, and the mole ratio of sodium periodate and sodium alginate was 1:8, 1:10 and 1:12, respectively. Oxidized sodium alginate with different
degree of oxidation was coated on the surface of medical polyvinyl chloride pipes by chemical methods, then to select the best degree of oxidation. The optimal coating condition was screened through an orthogonal
experiment based on concentrations of sulfuric acid, polyethyleneimine and oxidized sodium alginate, and pH
value and temperature of oxidized sodium alginate. Additional y, the anticoagulant properties and expulsion rates of the prepared pipes were evaluated in comparison with the blank control group and heparin-coated group.
RESULTS AND CONCLUSION:The optimal oxidation degree for oxidized sodium alginate was 1:10 for the mole ratio of sodium periodate and sodium alginate. The best coating conditions were 50%concentrated sulfuric acid, 0.05%polyethyleneimine, 40 ℃ of reaction temperature, 2 g/L oxidized sodium alginate, and pH value=3.5. The oxidized sodium alginate coating group had a similar trend compared with heparin-coated group in the expulsion rate. The anticoagulant properties of oxidized sodium alginate coating group were little weaker than those of
heparin-coated group (P<0.05), but significantly better than those of the blank control group. The oxidized sodium alginate-coated extracorporeal circulation pipe has a good anticoagulant property and stability.

Objective To prepare and evaluate the biocompatibility of polycarbonate coated with low molecular weight heparin (LMWH) and partial oxidation sodium alginate(OSA).Methods Coating material was prepared by means of chemical graft-modification and the feature of the material was determined with infrared spectrum and the stablity of the coating in fluid was examined.Biocompatibility was evaluated by contact angle and in-vitro tests including protein adhesion,platelet adhesion and caugulation.Results LMWH or OSA was tightly combined with polycarbonate.After being coated,the contact angle,albumin and fibrinogen adhering to materials were decrease (P＜0.05).The anticoagulant activity was notably promoted by coating.Compared with LMWH coated material,the contact angle,albumin and fibrinogen adhering were decreased significantly,but the improvement of anticaugulation was limited(P＜0.05).Conclusion Chemical graft-modification LMWH or OSA can be applied to polycarbonate.The biocompatibility of the coated materials was significantly promoted.

Objective: To investigate the influencing factors of sulfated modification of Bupleurum chinense polysaccharides (BCP),and to elucidate the possible mechanism of improving the antioxidant ability of sulfated BCP (S-BCP).Methods:BCP was sulfated by chlorosulfonic acid-pyridine method.The degree of substitution (DS)of S-BCP was observed by adjusting the volume ratios of chlorosulfonic acid to pyridine (1:2,1:4,and 1:8).The structures of BCP and S-BCP were analyzed by infrared (IR)spectroscopy,the morphology of BCP and S-BCP were observed under scanning electron microscopy (SEM).The antioxidant model was established by using 1, 1-diphenyl-2-picrylhydrazyl (DPPH)free radical scavenging.The experiment was divided into positive control group,BCP group and S-BCP group,and the scavenging rates of DPPH free radical in various groups were compared. Results:When the volume ratio of chlorosulfonic acid to pyridine was 1 : 4,the reaction time was 2 h and the reaction temperature was 60 ℃,the maximum sulfur content percentage of S-BCP was 18.62% and the DS was the highest (DS = 2.32 ).Compared with BCP group, the scavenging rate of DPPH free radical of S-BCP was significantly increased (P <0.05).Conclusion:The volume ratio of chlorosulfonic acid to pyridine can affect the DS of S-BCP.The sulfated modification can increase the anti-oxidant capacity of BCP by changing its physic-chemical characters and spatial conformation.

Objective:To construct the expression vector of the fusion protein of human serum albumin (HSA) and thymopentin (TP5) and to express it in Pichia pastoris,and to elucidate the biological activity of fusion protein.Methods:The HSA-TP5 fusion gene was constructed by gene recombination and transfected into Pichia pastoris to construct the eukaryotic expression system of HSA-TP5.The recombinant eukaryotic expression plasmid of PPICZα-HSA-TP5 was obtained by agarose gel electrophoresis and purification reagent.The two step fermentation method was used to ferment gene engineering bacteria of HSA-TP5 in high density,and the fermentation supernatant protein was precipitated and concentrated;the purified fusion protein was obtained by cation exchange chromatography and hydrophobic chromatography and analyzed by SDS-polyacrylamide gel electrophoresis.The effect of the fusion protein on the proliferation of lymphocytes was detected by MTT assay.Results:The HSA target gene fragment with length of 1 845 bp was achieved by PCR method.The HSA-TP5-pPICZαC fusion plasmid was identified by restriction endonuclease digestion,and the fragment length was 707 bp.The sequence analysis showed that the HSA and TP5 sequences of the target genes were identical with the gene sequences reported in GenBank and were fused by forward fusion.PCR method confirmed that the eukaryotic recombinant plasmid PPICZ αC-HSA-TP5 was integrated into the yeast genome,and compared with control group,the target gene PCR product length was found to be 1 860 bp.SDS-PAGE analysis showed that the expression level of HSA-TP5 fusion protein was gradually increased with the induction time within 72 h.HSA-TP5 fusion protein was purified by cation exchange chromatography and AKTA multifunctional protein purification system.The MTT assay results showed that HSA-TP5 fusion protein was consistent with TP5 protein in promoting lymphocyte proliferation activity.Conclusion:HSA-TP5 fusion protein can be obtained by constructing the eukaryotic expression system of Pichia pastoris and owns the biological activity.

OBJECTIVE: To evaluate the therapeutic results of endoscopic orbital decompression for thyroid-associated ophthalmopathy. METHOD: The records of nine patients (twelve orbits) received endoscopic orbital decompression for thyroid-associated ophthalmopathy were analyzed for changes in visual acuity, intraocular pressure, proptosis, corneal ulceration and movement. The follow-ups ranged from two months to thirty-six months. RESULT: Twelve orbits (100%) had improvement in visual acuity (range 0.1-0.7). Ten orbits (83.3%) decreased in intraocular pressure (range 0.2-21.4 mm Hg). Eight orbits (66.70%) decreased in proptosis (one-five mm). The orbit with corneal ulcer was healed after decompression. Diplopia was cured in one of four patients. CONCLUSION Endoscopic orbital decompression is a safe and effective procedure for the treatment of thyroid-associated ophthalmopathy.
Adult ; Decompression, Surgical ; methods ; Endoscopy ; Female ; Graves Ophthalmopathy ; surgery ; Humans ; Male ; Middle Aged ; Orbit ; surgery ; Retrospective Studies ; Treatment Outcome

ObjectiveTo investigate the clinical features of infection in patients with acute-on-chronic liver failure (ACLF) and bacterial infection and the influencing factors for 90-day survival rate. MethodsThe patients with ACLF who were admitted to The Fifth Medical Center of Chinese PLA General Hospital from January 2014 to December 2015 were enrolled, and related clinical data were collected and analyzed, including infection time and site, microbial culture, biochemical parameters and inflammatory markers, and 28- and 90-day prognosis after infection. The t-test or the Mann-Whitney U test was used for comparison of continuous data between two groups, and the chi-square test was used for comparison of categorical data between two groups. The life-table method was used to plot survival curves. The indices affecting prognosis in the univariate analysis were further included in the multivariate logistic regression analysis. ResultsA total of 1074 patients with ACLF were admitted, among whom 609 had bacterial infection, and the incidence rate of bacterial infection was 567%. Among these 609 patients, 16 underwent liver transplantation within 90 days and related data statistics were obtained for the remaining 593 patients. As for infection site, among the patients with infection, 70.15% had abdominal infection, 41.15% had pulmonary infection, 11.97% had sepsis, 5.40% had urinary system infection, 4.89% had thoracic infection, and 8.6% had infection at other sites. Among the patients with infection, 64.76% had infection at a single site. The positive rates of microbial culture of ascites, phlegm, urine, and pleural effusion were 22.70%, 52.82%, 40.63%, and 35.71%, respectively. Escherichia coli was the most common bacterium in ascites and accounted for 43.82%; Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii accounted for 22.67%, 2400%, and 22.67%, respectively, in phlegm; Escherichia coli, Klebsiella pneumoniae, and Staphylococcaceae accounted for 33.80%, 29.58%, and 15.49%, respectively, in blood culture. The 90-day survival rate after infection was 38.11% (226/593) in the patients with ACLF, and the multivariate logistic regression analysis showed that age (P=0.006), stage of hepatic encephalopathy (HE) (P＜0001), stage of liver failure (P＜0.001), and acute kidney injury (AKI) (P＜0.001) were independent risk factors for 90-day survival in patients with ACLF and infection. ConclusionThere is a high incidence rate of infection in patients with ACLF. The 90-day survival rate is relatively low, and the presence of AKI, HE stage III or above, and advanced liver failure may indicate poor prognosis.