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.

BACKGROUND:Talus cartilage injury is a common motor system disease.This type of injury will affect the patient's daily life and work ability,and may worsen the condition if left untreated.Surgical treatment is commonly used,but the selection of surgical methods and the evaluation of medium-and long-term follow-up results have always been difficult clinical problems. OBJECTIVE:To explore the influence of T1ρ technique on the range of quantitative evaluation of talus osteochondral injury on the choice of surgical method and the results of medium-and long-term follow-up. METHODS:A total of 154 patients with osteochondral injury of talus admitted to The Second Hospital of Tangshan from January 2019 to August 2022 were retrospectively selected as the study subjects.The lesion site of talus was examined by MRI before operation,and the T1ρ and T2 values of different types were compared.Different surgical methods were selected according to the different T1ρ values.Group A(n=73)was treated with microfracture surgery with T1ρ<45 ms;group B(n=81)was treated with autogenous bone and cartilage transplantation with T1ρ≥45 ms.The general clinical characteristics and curative effects of patients under different surgical methods were compared;the important factors of postoperative recurrence were analyzed by multivariate Logistic regression,and the relationship between T1ρ value and postoperative recurrence was analyzed by restricted cubic spline graph,y=1-1/(1+e-z)regression equation to build a prediction model.The stability of the model was verified by cross-checking method. RESULTS AND CONCLUSION:(1)Classification of talus osteochondral injury in 154 patients(type Ⅰ:36 cases;type Ⅱ:37 cases;type Ⅲ:40 cases;type Ⅳ:41 cases),T1ρ and T2 values of the four groups were statistically significant(P<0.05);pairwise comparison was also statistically significant(all P<0.05).(2)After treatment of 154 patients,7 cases(4.6％)had local swelling,3 cases(2.0％)had pain aggravation,and 5 cases(3.3％)had wound infection.There were 2 cases(1.3％)with poor cartilage healing.(3)After treatment,there were statistically significant differences between groups A and B in terms of American Orthopaedic Foot&Ankle Society score,visual analog scale score,plantar flexor motion range,dorsoextension motion range,subchondral bone marrow edema volume,interleukin-6,interleukin-8,C-reactive protein,procalcitonin,platelet-derived growth factor,transforming growth factor-β1,and efficacy(P<0.05).The total effective rate of group B(90％)was higher than that of group A(85％)(P<0.05).(4)Age(OR=1.589,95％CI:0.305-1.252,P=0.036),interleukin-6(OR=1.737,95％CI:0.974-5.254,P=0.049),interleukin-8(OR=1.385,95％CI:1.066-4.355,P=0.034),C-reactive protein(OR=1.957,95％CI:1.323-2.178,P=0.035),transforming growth factor-β1(OR=1.459,95％CI:0.897-2.455,P=0.038),T1-ρ(OR=1.687,95％CI:0.854-3.321,P=0.026),T2(OR=1.843,95％CI:0.657-2.454,P=0.036),complications(OR=1.719,95％CI:0.654-3.464,P=0.019),and classification of osteochondral injury of talus(OR=3.789,95％CI:1.023-5.897,P=0.028)were independent risk factors for postoperative recurrence.Microfracture surgery(OR=0.751,95％CI:0.321-1.264,P=0.012)and autogenous bone and cartilage grafting(OR=0.649,95％CI:0.246-1.356,P=0.023)were independent protective factors for recurrence after medium-and long-term follow-up.(5)When T1ρ value≤35 ms,the risk of postoperative recurrence decreased rapidly,and when T1ρ value>35 ms,the risk of postoperative recurrence increased rapidly.(6)Further stepwise regression analysis showed that these nine risk factors were most closely associated with postoperative recurrence,and the formula for postoperative recurrence was obtained.The probability of postoperative recurrence was calculated using the regression equation.When P=0.75,the maximum value of Jorden index was 77.728,indicating that the model has a better prediction effect.(7)It is indicated that the quantitative evaluation of T1ρ before operation can effectively guide the selection of surgical methods,improve the success rate of surgery and the quality of life of patients.

Background Public places are frequently polluted by cigarette smoking, and there is a lack of accurate, real-time, and intelligent monitoring technology to identify smoking behavior. It is necessary to develop a tool to identify cigarette smoking behavior in public places for more efficient control of cigarette smoking and better indoor air quality. Objective To construct a model for recognizing cigarette smoking behavior based on real-time indoor concentrations of PM_2.5 in public places. Methods Real-time indoor PM_2.5 concentrations were measured for at least 7 continuous days in 10 arbitrarily selected places (6 public service providers and and 4 office or other places) from Oct. to Nov. 2022 in Pudong New Area, Shanghai. Indoor nicotine concentrations were monitored with passive samplers simultaneously. Outdoor PM_2.5 concentration data were obtained from three municipal environmental monitoring stations which were nearest to each monitoring point during the same period. Mann-Whitney U test was used to compare indoor and outdoor means of PM_2.5 concentrations, and Spearman rank correlation was used to analyze indoor PM_2.5 and nicotine concentrations. An interactive plot and a random forest model was applied to examine the association between video observation validated indoor smoking behavior and real-time indoor PM_2.5 concentrations in an Internet cafe. Results The average indoor PM_2.5 concentration in the places providing public services [(97.5±149.3) µg·m⁻³] was significantly higher than that in office and other places [(19.8±12.2) µg·m⁻³] (P=0.011). The indoor/outdoor ratio (I/O ratio) of PM_2.5 concentration in the public service providers ranged from 1.1 to 19.0. Furthermore, the indoor PM_2.5 concentrations in the 10 public places were significantly correlated with the nicotine concentrations (r_s=0.969, P<0.001). Among them, the top 3 highly polluted places were Internet cafes, chess and card rooms, and KTV. The results of random forest modeling showed that, for synchronous real-time PM_2.5 concentration, the area under the curve (AUC) was 0.66, while for PM_2.5 concentration at a lag of 4 min after the incidence of smoking behavior, the AUC increased to 0.72. Conclusion The indoor PM_2.5 concentrations in public places are highly correlated with smoking behavior. Based on real-time indoor PM_2.5 monitoring, a preliminary recognition model for smoking behavior is constructed with acceptable accuracy, indicating its potential values applied in smoking control and management in public places.

Objective:To evaluate the efficacy of dexmedetomidine on painless gastroenteroscopy in the patients recovered from COVID-19.Methods:Eighty patients of either sex, aged 18-64 yr, with body mass index <30 kg/m 2, undergoing elective painless gastroenteroscopy, of American Society of Anesthesiologists physical statusⅠor Ⅱ, within 2nd to 7th weeks after diagnosis of COVID-19, who had a negative nucleic acid test or antigen test at present and presented with no respiratory symptoms in our hospital from January to February 2023, were selected and divided into 2 groups ( n=40 each) using a random number table method: dexmedetomidine group (group D) and control group (group C). Sufentanil 0.1 μg/kg was intravenously injected in two groups. Dexmedetomidine 0.2 μg/kg was intravenously injected in group D, and the equal volume of normal saline was given instead in group C. Propofol 1.5-2.0 mg/kg was intravenously injected after 2-3 min observation, and propofol 5-7 mg·kg -1·h -1 was intravenously infused to maintain sedation during operation. The development of bucking and hypoxemia during operation, total consumption of propofol, emergence time, time of hospital discharge, development of bradycardia and hypotension during operation, and scores for patients′ and endoscopic physicians′ satisfaction with anesthesia were recorded. Results:Compared with group C, the incidence of bucking and hypoxemia was significantly decreased, scores for endoscopic physicians′ satisfaction with anesthesia were increased ( P<0.05), and no significant change was found in the other parameters in group D ( P>0.05). Conclusions:Low-dose dexmedetomidine can reduce the risk of bucking and hypoxemia during operation and raise the quality of anesthesia in the patients recovered from COVID-19 undergoing painless gastroenteroscopy.

Objective:To establish a conditional knockout mouse model of polycystic kidney disease 1 ( Pkd1) gene based on CRISPR/Cas9 and Cre-loxP gene editing technology, and to provide an animal model for in-depth research on the role of Pkd1 gene in the development of polycystic kidney disease. Methods:In-Fusion technology was used to construct a targeting vector. Corresponding gRNAs, Cas9 mRNAs, and donor vectors carrying the loxP site were prepared based on the Pkd1 gene, and injected into the fertilized eggs of C57BL/6N mice. The fertilized eggs were transferred to the fallopian tubes of female mice with pseudopregnancy. After the newborn mice were identified by PCR and sequencing analysis, Pkd1 flox/flox F0 generation positive mice were selected. The F0 generation positive mice were bred with wild-type mice, and F1 generation heterozygous mice with Pkd1 flox/+ genotype were selected for offspring. F2 generation homozygous mice with Pkd1 flox/flox genotype were obtained through internal expansion, and then hybridized with Cre positive Ggt1/ Cre mice. F3 generation mice with Pkd1 flox/+Ggt1 Cre genotype were obtained. F4 generation mice with Pkd1 flox/flox Ggt1 Cre genotype were obtained by self crossing or backcrossing with F2 generation Pkd1 flox/flox, namely kidney-specific Pkd1 gene knockout mice ( Ggt1-cKO mice). PCR method was used to identify the genotype of mice, and then the mice were divided into wild-type control (WT) group ( n=6), Pkd1 homozygous control (PKD) group ( n=6), and Ggt1-cKO knockout validation (CKO) group ( n=6) according to the gene identification results. Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression of Pkd1 mRNA in the kidneys and other organs of mice in each group. HE staining was used to detect the pathological changes in renal tissues of mice in each group. The automatic biochemical detector was used to detect the blood urea nitrogen and serum creatinine levels of mice, and the kidney coefficient was calculated. Results:The PCR detection results showed that the genotype of offspring mice in CKO group was consistent with Pkd1floxflox Ggt1 Cre. Pkd1 gene was only specifically expressed in the kidney, but not in other tissues. The RT-qPCR results showed that the relative expression of Pkd1 mRNA in the renal medulla of CKO group was significantly lower than that of WT and PKD groups. The kidney volume of the CKO group had increased by about twice compared to the WT group. Under the microscope, it could be observed that there were multiple vacuoles of varying sizes and shapes in the kidneys of the CKO group, and there was a significant increase in the interstitial space of the medullary tissue. The kidney coefficient, blood urea nitrogen, and serum creatinine in the CKO group were significantly higher than those in the WT and PKD groups (all P<0.05). Conclusion:Based on CRISPR/Cas9 and Cre-loxP gene editing technology, Pkd1 gene kidney conditional knockout mice can be successfully constructed, providing an animal model for further studying the action mechanism of Pkd1 gene in polycystic kidney disease.

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 investigate the effect of pioglitazone on white matter injury after cerebral ischemia-reperfusion in mice and its mechanism.Methods:Forty-two young male C57BL/6J mice were randomly divided into sham operation group, model group, and pioglitazone group ( n=14 in each group). The model of cerebral ischemia-reperfusion was induced by transient middle cerebral artery occlusion with suture-occluded method. On the 3 rd and 7 th day after the establishment of the model, the neural function was assessed by the adhesive removal test. The mice were killed on the 7 th day after the establishment of the model. HE staining was used to detect the extent of cerebral infarction. Immunofluorescence staining and Western blot analysis were used to detect the degree of white matter damage and the changes of microglia phenotype. Results:On the 7 th day after cerebral ischemia-reperfusion, the adhesive removal time in the PGZ group was significantly shortened compared with the model group ( P<0.05), the percentage of cerebral infarction volume was significantly reduced ( P<0.05), the ratio of MBP/NF200 fluorescence intensity in the cortical and striatal areas was significantly increased (all P<0.05), and the number of CD16 +/Iba1 + microglia was significantly decreased ( P<0.01), while the number of CD206 +/Iba1 + microglia tended to increase, but there was no statistical difference. Conclusion:Pioglitazone may reduce the degree of white matter injury and nerve function damage in mice with cerebral ischemia-reperfusion, and its mechanism may be associated with regulating the transformation of microglia from M1 type to M2 type.

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 effect of down-regulation of RACK1 expression on growth and radiosensitivity of oral squamous cell carcinoma cells.Methods:The shRNA vector for RACK1 gene was constructed and transfected into HSC-3 cells by lipofectamine. The stably-transfected cell line was obtained by constructing G418. The expression levels of RACK1 mRNA and protein were detected by RT-PCR and Western blot. The cell proliferation was detected by CCK8 assay. Cell apoptosis was examined by flow cytometry. The invasive and metastatic capabilities of cancer cells were assessed by cell invasion assay in vitro.The effect of X-ray irradiation combined with down-regulation of RACK1 expression upon cell proliferation was assessed by clone formation assay. The xenograft tumor nude mouse model was established to observe the inhibitory effect of down-regulating RACK1 gene expression combined with X-ray irradiation on oral squamous cell carcinoma. Results:RT-PCR revealed that the expression level of RACK1 mRNA of transfected HSC-3 cells was significantly down-regulated ( P<0.05). Western blot showed that the expression level of RACK1 protein was significantly down-regulated ( P<0.05). CCK8 assay demonstrated that down-regulation of RACK1 expression could remarkably inhibit the growth of HSC-3 cells ( P<0.05). RACK1 gene shRNA interference combined with X-ray irradiation significantly enhanced the apoptosis rate of HSC-3 cells ( P<0.05). The number of invasion cells in vitro in the RACK1 silencing group was evidently decreased ( P<0.05). Clone formation assay showed that the survival fraction in the shRACK1 group was significantly lower than that in the control group. The sensitization enhancement ratio was 1.37(ratio of D 0 value). Xenograft tumor experiment in nude mice showed that tumor growth was significantly inhibited in the shRACK1 group, the tumor volume was significantly decreased and the tumor mass was significantly lower than those in the control group (all P<0.05). Conclusion:Down-regulating RACK1 expression can enhance the radiosensitivity of oral squamous cell carcinoma cells, providing novel thinking to improve the radiosensitivity of oral squamous cell carcinoma.