African swine fever(ASF)is an acute and highly pathogenic hemorrhagic disease of pigs,causing huge economic losses to pig industry.In order to quantitatively detect clinical samples of ASF and inactivated ASFV antigens,the IgG of ASF positive serum was used as capture anti-body and the HRP-labeled p72 monoclonal antibody was used as detecting antibody.The standard curve was drawn with the cell-cultured ASFV,and a sandwich ELISA detection of antigen was es-tablished.The specificity,sensitivity and stability of the method were evaluated.The effects of dif-ferent inactivation methods and adjuvant addition on antigen detection were further evaluated.The results showed that the minimum detection limits of the recombinant protein and the ASFV were 0.1 mg/L and 103.7 TCID50/mL,respectively.There was no cross-reaction with five common porcine pathogenic viruses,and the coefficient variations between batches was less than 10％.The total co-incidence rate with real-time fluorescence quantitative PCR was 92％(23/25).The sensitivity of antigen detection was significantly reduced when antigen was treated by BEI inactivation,and the detection results were severely interfered by aluminum adjuvant and nano-adjuvant.In summary,the sandwich ELISA antigen detection method established is specific,sensitive,and repeatable,with a good consistency to the qPCR method,which provides an effective clinical diagnostic meth-od for ASFV antigen.

In order to obtain a cell line which high expressed of rabies virus glycoprotein the produc-tion of rabies subunit vaccine,the RABV glycoprotein gene sequence was amplified by RT-PCR and cloned into the lentiviral expression vector.The recombinant plasmid pLV-RVG,envelope plasmid pMD2.0G and packaging plasmid psPAX2 were co-transfected into HEK-293T cells to harvest lentivirus and infect new HEK-293T cells.The 293T-RVG cell line was obtained by puro-mycin pressure screening.Indirect immunofluorescence assay(IFA)and Western blot showed that the 67 kD RABV-G protein was highly expressed in the cells.The neutralizing antibody titer reached 2.19 IU/mL within 14 days after immunization,which was higher than the internationally recognized protection threshold(0.5 IU/mL).This study showed that the cell line 293T-RVG with stable and high expression of rabies virus glycoprotein was successfully constructed.The cell line could protect mice against rabies virus challenge and could be used as a subunit vaccine with poten-tial for large-scale production and application.

ObjectiveTo explore the mechanism of plumbagin as a novel ferroptosis inducer in bladder cancer inhibition. MethodBladder cancer T24 cells were used in this study. The effect of different concentrations of plumbagin （0.1， 1， 2， 3， 6， 12， 24， 48 μmol·L^-1） on the viability of T24 cells was detected by cell counting kit-8 （CCK-8）. The effect of different concentrations of plumbagin （1.5， 3， 6 μmol·L^-1） on the apoptosis of T24 cells was detected by annexin V-fluorescein isothiocyanate （Annexin V FITC）/PI apoptosis kit. Different inhibitors （ferroptosis inhibitor Fer-1， apoptosis inhibitor VAD， and necroptosis inhibitor Nec-1） were used in combination with plumbagin （6 μmol·L^-1）. Reactive oxygen species （ROS） fluorescent probe （DCFH-DA）， malonaldehyde （MDA）， and glutathione （GSH） kits were used to detect the effects of different concentrations of plumbagin （1.5， 3， 6 μmol·L^-1） on the level of ROS and the content of MDA and GSH in T24 cells， respectively. The effect of different concentrations of plumbagin （1.5， 3， 6 μmol·L^-1） on peroxide levels in T24 cells was detected by C11-BODIPY fluorescent probe. Western blot was used to detect the effect of different concentrations of plumbagin （1.5， 3， 6 μmol·L^-1） on the protein expression of solute carrier family 7 member 11 （SLC7A11）， glutathione peroxidase 4 （GPX4）， nuclear factor E₂-related factor-2 （Nrf-2）， and Kelch-like ECH-associated protein 1 （Keap1）. ResultCompared with the blank group， plumbagin could inhibit the activity of T24 cells （P<0.05） with IC₅₀ of 3.52 μmol·L^-1. At the concentrations of 1.5， 3， 6 μmol·L^-1， plumbagin significantly promoted the apoptosis of T24 cells （P<0.05） as compared with the blank group. Compared with the plumbagin group at 6 μmol·L^-1， the ferroptosis inhibitor and apoptosis inhibitor groups could reverse the inhibitory effect of 6 μmol·L^-1 plumbagin on the proliferation of T24 cells （P<0.05）. Compared with the blank group， the plumbagin groups at 1.5， 3， 6 μmol·L^-1 showed increased content of ROS， MDA， and lipid peroxides in T24 cells， decreased GSH level， and reduced SLC7A11， GPX4， and Nrf-2/Keap1 （P<0.05）. Conclusionplumbagin can induce ferroptosis， and its mechanism is related to the Nrf-2/Keap1 signaling pathway.

Objective:To evaluate the effects of dexamethasone and etomidate on cortisol secretion in elderly patients undergoing general anesthesia.Methods:One hundred and twenty-five elderly patients of either sex, aged 66-90 yr, of American Society of Anesthesiologists physical statusⅠ-Ⅲ, undergoing minor and medium elective surgeries under general anesthesia, were allocated into 4 groups using a random number table method: propofol and normal saline group (group PN, n=31), propofol and etomidate group (group PD, n=31), etomidate and normal saline group (group EN, n=33) and etomidate and dexamethasone group (group ED, n=30). In PN and EN groups, propofol (2 mg/kg) was used to induce and maintain anesthesia, and normal saline 2 ml and dexamethasone 0.1 mg/kg were intravenously injected, respectively, at 5 min before anesthesia induction.In PD and ED groups, etomidate (0.2 mg/kg) was used to induce and maintain anesthesia, and normal saline 2 ml and dexamethasone 0.1 mg/kg were intravenously injected, respectively, at 5 min before anesthesia induction.The serum cortisol concentrations were measured at 8: 00 after entering the operating room on the morning of operation (T 1), 1 h after the start of anesthesia (T 2), 2 h after the start of anesthesia (T 3), 8: 00 on the next day ofoperation (T 4) and 8: 00 on the 2nd day of operation (T 5). Blood glucose concentrations were measured at T 1-T 3, and the hypotension during the peri-anesthesia period, nausea and vomiting in post-anesthesia care unit, and nausea and vomiting scores were recorded at 24 h after operation. Results:A total of 122 patients completed the trial.Compared with PN group, the concentration of serum cortisol was significantly decreased at T 2-T 5, blood glucose concentrations were increased at T 2 and T 3 ( P<0.05), and no significant change was found in the incidence of hypotension, nausea and vomiting and nausea and vomiting scores in PD group ( P>0.05), and the concentration of serum cortisol was significantly decreased at T 2-T 4, the incidence of hypotension was decreased ( P<0.05), and no significant change was found in the blood glucose concentrations, incidence of nausea and vomiting or nausea and vomiting scores in EN group ( P>0.05). Compared with ED group, the serum cortisol concentration was significantly increased at T 2 and T 3, the incidence of hypotension was increased, the incidence of nausea and vomiting and nausea and vomiting scores were decreased ( P<0.05), and no significant change was found in the blood glucose concentrations in PD group ( P>0.05), and the serum cortisol concentration was significantly decreased at T 2 and T 3 and increased at T 4 and T 5, the serum cortisol concentration was decreased at T 2 and T 3, and no significant change was found in the incidence of hypotension, nausea and vomiting and nausea and vomiting scores in EN group ( P>0.05). Conclusions:Combination of etomidate and dexamethasone significantly enhances the duration and degree of inhibition of cortisol secretion in elderly patients than etomidate or dexamethasone alone.

Objective:To evaluate the effects of different depths of anesthesia on CD4 + T cell function in the patients undergoing radical resection for malignant tumor. Methods:Forty-two American Society of Anesthesiologists physical status Ⅱ patients, aged 36-64 yr, weighing 49-95 kg, undergoing elective radical surgery for malignant tumor, were divided into 2 groups ( n=21 each) using a random number table method: light anesthesia group (L group) and deep anesthesia group (D group). The AAI values were maintained at 30-40 and 20-29 during operation in L group and D group, respectively.The time to eye opening, extubation time and consumption of propofol were recorded.The peripheral venous blood samples were collected immediately before induction of anesthesia (T 0), at 2 h of operation (T 1) and at 4, 24, and 72 h after operation (T 2-4) for determination of serum interferon (IFN)-γ and interleukin-4 (IL-4) concentrations (by enzyme-linked immunosorbent assay), and IFN-γ/IL-4 ratio was calculated. Results:Compared with the baseline value at T 0, the serum IFN-γ concentrations were significantly increased at T 3, 4, the serum IL-4 concentrations were decreased at T 1-4, and IFN-γ /IL-4 ratio was increased in group D ( P<0.05). Compared with group L, the serum IFN-γ concentrations were significantly increased at T 3, 4, the serum IL-4 concentrations were decreased at T 1-4, IFN-γ/IL-4 ratio was increased, the consumption of propofol was increased, and the extubation time was prolonged in group D ( P<0.05), and no significant change was found in the time to eye opening between the two groups ( P>0.05). Conclusion:Deep anesthesia can improve CD4 + T cell function in the patients undergoing radical resection for malignant tumor.

Objective:To get norovirus (NoV) GII.6 P protein through prokaryotic expression and prepare the polyclonal antibody.Methods:NoV GII.6 P region gene was amplified and cloned into prokaryotic expression vector pET28a, and the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cell. The recombinant protein GII.6 P was expressed by induced Isopropyl β-D-Thiogalactoside (IPTG) and then purified with Ni-NTA Affinity Column. The binding ability of recombinant GII.6 P was determined by oligosaccharide binding assay and the polyclonal antibody serum was prepared by immunizing BALB/c mice. The titer of GII.6 P polyclonal antibody was determined by ELISA, and the specificity of the antibody was detected by western blot (WB). The effectiveness of GII.6 P polyclonal antibody was assessed. Results:The recombinant GII.6P-pET28a plasmid was constructed successfully and the recombinant GII.6 P protein was expressed with relative molecular mass of 40 ×10 3. The purity of GII.6P protein was more than 90% after purification. The oligosaccharide binding showed that the GII.6 P protein binds to B, le b and H2, but does not bind to A, H1, and le a type; the titer of GII.6 P polyclonal antibody was 1∶160 000. WB indicated that the antibody had high specificity and the cross experiments did not show affinity to GII.4. Conclusions:The GII.6P protein has been expressed successfully and the GII.6 P polyclonal antibody with high titer was prepared, which provides an effective tool for detection and vaccine development for NoV GII.6.

Objective: To explore the correlation between sleep and the severity of coronary artery stenosis, and to further guide the prevention and control of coronary heart disease. Methods: A total of 302 patients, including 183 males and 119 females, were enrolled in the department of cardiology of Changhai hospital from February to June 2019. The patients were divided into three groups according to the degree of stenosis (atherosclerotic group, atherosclerotic group with degree of stenosis <50%, atherosclerotic group with degree of stenosis 50%-70%, and atherosclerotic group with degree of stenosis >70%). General information, comorbidities and Pittsburgh sleep quality index (PSQI) of patients in each group were analyzed to compare the differences and analyze the risk factors of aggravated coronary artery stenosis. Results: There were statistically significant differences in age, gender and diabetes among all groups (P<0.05). There was no statistically significant difference in the incidence of hypertension and hyperlipidemia in each group (P>0.05). Spearman correlation analysis showed that PSQI score was positively correlated with the degree of atherosclerosis (P<0.01). In the multivariate regression model, poorer sleep quality (OR=1.75, 95% CI: 1.14-2.69, P<0.05), shorter sleep time (OR=1.64, 95% CI: 1.05-2.55, P<0.05) and more diurnal dysfunction (OR=1.45, 95% CI: 1.11-1.88, P<0.01) were correlated with increased coronary artery stenosis. Higher PSQI total score (OR=1.13, 95% CI: 1.06-1.20, P<0.01) and lower PSQI evaluation grade (very bad vs very good: 13.85, 95% CI: 1.56-122.82, P<0.05) were also related to the increase of coronary artery stenosis. Conclusions Poorer sleep quality, shorter sleep time and more diurnal dysfunction were independent risk factors for the aggravation of coronary artery stenosis. Higher Pittsburgh sleep quality index was significantly associated with the severity of coronary artery stenosis.

Objective To evaluate the efficacy of oxycodone for improvement of general anesthesia for laparoscopic cholecystectomy in elderly patients.Methods A total of 160 patients of both sexes,aged 65-75 yr,with body mass index ＜30 kg/m2,of American Society of Anesthesiologists physical status Ⅰ-Ⅲ,scheduled for elective laparoscopic cholecystectomy,were divided into 2 groups (n =80 each) using a random number table method:general anesthesia group (group GA) and oxycodone + general anesthesia group (group OX+GA).Anesthesia induction:propofol was given by closed-loop infusion at the initial target plasma concentration of 2 μg/ml,the target bispectral index (BIS) value was set at 50,and 2 min later remifentanil was given by target-controlled infusion at the target plasma concentration of 4 ng/ml,and cisatracurium 0.2 mg/kg was intravenously injected when BIS value was decreased to 70.Laryngeal mask airways were inserted and the patients were mechanically ventilated when BIS value was decreased to 50 and TOF ratio was decreased to 25％,and end-tidal pressure of carbon dioxide was maintained at 35-45 mmHg.Anesthesia maintenance:propofol was given by closed-loop infusion,the target BIS value was set at 50,cisatracurium 0.1 mg/kg was intravenously injected when TOF ratio was increased to 10％;remifentanil was given by target-controlled infusion at the target plasma concentration of 4-6 ng/ml.Oxycodone 0.07 mg/kg was intravenously injected at 5 min before stretching internal organs.Before anesthesia,at 5 min after laryngeal mask airway placement,at skin incision and while stretching internal organs,analgesia nociception index value and perfusion index value were recorded,the development of intraoperative cardiovascular events,emergence time,time for removal of laryngeal mask airway,time of post-anesthesia care unit stay and development of nausea and vomiting and back and shoulder pain within 48 h after surgery were also recorded.Results Compared with group GA,the analgesia nociception index value and perfusion index value were significantly increased while stretching internal organs,and the incidence of intraoperative hypertension,tachycardia,and nausea and vomiting and back and shoulder pain within 48 h after surgery were decreased in group OX-GA (P＜0.05).Conclusion Oxycodone can inhibit nociceptive stimuli,is helpful in maintaining stable hemodynamics and reduces postoperative complications in elderly patients undergoing laparoscopic cholecystectomy under combined general anesthesia.

Objective To discuss the optimal operation mode and operation path in minimally invasive technique for living donor nephrectomy.Methods From September 2013 to August 2015, 68 living donor nephrectomy was retrospectively reviewed. Thirty-one patients were performed with robotic-assisted laparoscopic living donor nephrectomy(robotic group), twenty-nine patients underwent totally retroperitoneal laparoscopic living donor nephrectomy(non hand assisted group),and eight patients were performed with hand assisted retroperitoneal laparoscopic living donor nephrectomy(hand assisted group). Operation time, warm ischemia time, intraoperative hemorrhage volume, hospitalization time, complications and preoperative and postoperative serum creatinine value of the recipients between the two groups were compared.Results The operations of three groups were all performed successfully. Intraoperative hemorrhage volume in the three groups were(39±15)ml,(62±37)ml and(53±19)ml, and there were significant differences between these groups(P<0.05). But hospitalization time ,operation time, warm ischemia time and complications occurred rate in the three groups had no significant difference(P>0.05). In robotic group,2 donors occurred with splenic injury during operation and 1 donor was detected with hemorrhage after operation. In non-hand assisted group, 1 donor occurred with urinary tract infection, 1 donor occurred with external iliac vein thrombosis. In hand assisted group 1 donor was detected with wound fat liquefaction after operation. All the donors were followed up for more than 9 months, no hypertension, proteinuria and renal dysfunction complications were detected. The blood creatinine in three groups of recipients after operation of 5th day and 28th day were(118±26)μmol/L, (130±33)μmol/L,(128±41)μmol/L and(114±17)μmol/L,(116±34)μmol/L,(115±29)μmol/L, respectively, and there was no statistical difference(P>0.05).Conclusions Minimally invasive technique for living donor nephrectomy is beneficial to patients' recovery. Surgery doctors should combine personal experience and the hospital's hardware conditions and other factors. The principle is to ensure the donor's safety and to balance the interests of the donor and the recipient, to choose their own most skilled way of surgery.

Objective: To analyze the risk factors for metastasis of lymph nodes between sternocleidomastoid and sternohyoid muscle (LNSS) in papillary thyroid cancer (PTC). Methods: Papillary thyroid cancer patients with clinically positive lateral lymph node metastasis (cN1) who underwent surgery including LNSS dissection between May 1, 2013 and May 31, 2016 at the Department of Head and Neck Surgery, Fudan University Shanghai Cancer Center were retrospectively studied. Logistic regression analysis was performed to evaluate possible clinicopathological factors related to LNSS metastasis. Results: In 85 patients, 54 patients (63.5%) showed LNSS in their surgical specimen, and 20 patients (23.5%) had pathologically positive LNSS metastasis. Patients with LNSS showed preoperatively higher levels of serum thyroid stimulating hormone (TSH) and thyroid peroxidase antibody (TPO-Ab) compared to patients only with fibrofatty tissues between sternocleidomastoid and sternohyoid muscle (P<0.05), and they also displayed a higher proportion of multifocality in ipsilateral thyroid lobe (P<0.05). Multi-factor analysis indicated that LNSS metastasis was correlated with original tumor size (OR=1.819, 95%CI 1.050-3.850, P=0.002) and Level Ⅳ lymph node metastasis (OR=2.190, 95%CI 1.132-2.334, P=0.005). Furthermore, the number of positive LNSS was tightly correlated to that of level Ⅳ lymph node metastasis(P<0.05). Conclusion LNSS metastasis is occult but not quite rare in PTC. Patients with extensive lymph node metastasis in Level Ⅳhave a higher risk for metastasis of LNSS.