An animal biosafety level-3 laboratory(ABSL-3)is a high-level biosafety installation that can conduct experiments on animals infected with highly pathogenic microorganisms.In recent years,with the continuous characterization of emerging and re-emerging infectious diseases,high-level biosafety laboratories have played increasingly important roles in pathogenic mechanism and drug and vaccine research and development.The demand for ABSL-3 is increasing year by year.At the same time,there is also a growing demand for personnel who are competent in working in ABSL-3.The systematization,normalization,and standardization of pre-service training have become important to guarantee a reduction in the risks to personnel working in ABSL-3.Training of ABSL-3 staff needs to be carried out in specific simulated laboratories.Therefore,it is necessary to construct simulated ABSL-3 and establish scientific and effective operating standards and mechanisms.This paper comprehensively introduces the design,construction,operation,and functions of a simulated ABSL-3 installation.

Objective:To investigate and analysis of the occurrence and influencing factors of sarcopenia in elderly critically ill patients in the intensive care unit (ICU).Methods:A prospective cohort study was conducted. Elderly patients (aged ≥ 60 years) admitted to the ICU of Tianjin Medical University General Hospital from November 2023 to June 2024 were enrolled. Clinical records were collected, and conduct muscle mass and strength measurements, as well as upper arm circumference and calf circumference were measured. Appendicular skeletal muscle index (ASMI) of less than 7.0 kg/m 2 for males and less than 5.7 kg/m 2 for females was defined as reduced muscle mass, grip strength of less than 28 kg for males and less than 18 kg for females was defined as decreased muscle strength, patients meeting both low muscle mass and low muscle strength criteria were diagnosed with sarcopenia. According to the diagnostic criteria for sarcopenia, patients were divided into sarcopenia group and non-sarcopenia group. Multivariate Logistic regression analysis was applied to identify risk factors for sarcopenia in the elderly and to develop a predictive model for the occurrence of sarcopenia. The predictive value of various risk factors for sarcopenia in elderly critically ill patients were evaluated by receiver operator characteristic curve (ROC curve). The Kaplan-Meier curve for the length of ICU stay of two groups patients were drawn. Results:Finally, 540 elderly critically ill patients were included, including 43 patients with sarcopenia, and the incidence of sarcopenia was 8.0%. Univariate analysis showed that there were significantly differences in body mass index (BMI), number of hospitalizations in the past year, the length of ICU stay, ventilation mode, duration of mechanical ventilation, pre-admission exercise habits, nutritional support methods, upper arm circumference, calf circumference, and albumin infusion between the sarcopenia group and the non-sarcopenia group. Multivariate Logistic regression analysis showed that BMI [odds ratio ( OR) = 0.79, 95% confidence interval (95% CI) was 0.67-0.93, P = 0.004], calf circumference ( OR = 0.64, 95% CI was 0.54-0.76, P < 0.001), and duration of mechanical ventilation ( OR = 1.06, 95% CI was 1.01-1.12, P = 0.034) were associated with an increased risk of sarcopenia in elderly critically ill patients. The ROC curve results showed that the area under the curve (AUC) and 95% CI of BMI, calf circumference, and duration of mechanical ventilation for predicting sarcopenia in elderly critically ill patients were 0.828 (0.767-0.888), 0.889 (0.844-0.933), and 0.397 (0.299-0.496), respectively, with cut-off values of 22.95 kg/m 2, 28.25 cm, and 50.50 days, respectively. The Kaplan-Meier curve showed that the cumulative survival rate of patients with sarcopenia was significantly lower than that of the non-sarcopenia group (Log-Rank test: χ 2 = 5.619, P = 0.018). Conclusion:Lower BMI, smaller calf circumference, and longer duration of mechanical ventilation are associated with an increased risk of sarcopenia in critically ill elderly patients.

Objective:To identify the differentially expressed long-chain non-coding RNA(lncRNA) and mRNA using ribonucleic acid sequencing(RNA-seq), and construct a competing endogenous RNA(ceRNA) regulatory network in mice with sepsis-associated encephalopathy.Methods:Ten clean-grade healthy male C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were divided into 2 groups( n=5 each) using a random number table method: sham operation group(group Sham) and sepsis group(group Sepsis). Sepsis was induced by cecal ligation and puncture(CLP) in group Sepsis, while group Sham only underwent laparotomy without CLP. Morris water maze test and contextual fear conditioning test were performed to detect the cognitive function on 1 day before CLP and 3 days after CLP. Three mice were randomly sacrificed in group Sham, and 3 mice with the worst results in the cognitive function test were sacrificed in group Sepsis. The hippocampal tissues were obtained for RNA-seq via the BGISEQ-500 platform, and the differentially expressed mRNA and lncRNA were identified. The differentially expressed mRNAs and lncRNAs were visualized and analyzed by Dr. Tom platform provided by Shenzhen BGI Technology Service Co., Ltd., and the ceRNA regulatory network was constructed using the online visualization tool Cytoscape software. Results:Compared with group Sham, the escape latency was significantly prolonged, and the percentage of time of staying at the target quadrants and percentage of time spent freezing were decreased in group Sepsis( P<0.05). A total of 62 differentially expressed lncRNAs were obtained from RNA-seq, of which the expression of 45 lncRNAs was up-regulated and the expression of 17 lncRNAs was down-regulated.There were 282 differentially expressed mRNAs identified from RNA-seq, of which the expression of 173 mRNAs was up-regulated, and the expression of 109 mRNAs was down-regulated.Gene Ontology enrichment analysis revealed that the differentially expressed mRNAs were involved in biological processes such as memory, learning or memory, inflammatory responses, regulation of aging-related behavioral decline, and regulation of synaptic plasticity. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that differentially expressed mRNAs were enriched in IL-17 signaling pathway, TNF signaling pathway, NF-κB signaling pathway and etc. KDA analysis was performed on the differentially expressed mRNAs to identify the key driver genes, and the results showed that Ch25h, Il6ra, Lcn2, Sgk1, Nr4a3, Osm, Saa3, Ccl7, Sqle, Dhcr24 were the key SAE genes.A competing endogenous RNA regulatory network was successfully constructed based on 9 lncRNAs, 28 mRNAs and 134 miRNAs in the hippocampus of mice with SAE. Conclusions:The results of RNA-seq find that 10 mRNAs including Ch25h, Il6ra, Lcn2, Sgk1, Nr4a3, Osm, Saa3, Ccl7, Sqle, Dhcr24 and lncRNAs such as Rian, Gm35874 and Gm34347 are key genes regulating SAE in mice. Meanwhile, a ceRNA regulatory network based on lncRNA-miRNA-mRNA is successfully constructed in the hippocampus of mice with SAE.

Neuropathic pain is a chronic disease that severely afflicts the life and emotional status of patients, but currently available treatments are often ineffective. Novel therapeutic targets for the alleviation of neuropathic pain are urgently needed. Rhodojaponin VI, a grayanotoxin from Rhododendron molle, showed remarkable antinociceptive efficacy in models of neuropathic pain, but its biotargets and mechanisms are unknown. Given the reversible action of rhodojaponin VI and the narrow range over which its structure can be modified, we perforwmed thermal proteome profiling of the rat dorsal root ganglion to determine the protein target of rhodojaponin VI. N-Ethylmaleimide-sensitive fusion (NSF) was confirmed as the key target of rhodojaponin VI through biological and biophysical experiments. Functional validation showed for the first time that NSF facilitated trafficking of the Cav2.2 channel to induce an increase in Ca²⁺ current intensity, whereas rhodojaponin VI reversed the effects of NSF. In conclusion, rhodojaponin VI represents a unique class of analgesic natural products targeting Cav2.2 channels via NSF.

Objective:To investigate specificity of neurovascular compression in patients with primary trigeminal neuralgia (PTN) by three-dimension reconstruction and computational fluid dynamics.Methods:Clinical characteristics and preoperative magnetic resonance imaging (MRI) data of 20 patients with both PTN and single artery compression (PTN group) and 10 patients without PTN but having neurovascular contact in MRI images (control group) in the Second Affiliated Hospital of Zhengzhou University from January 2018 to December 2019 were collected and analyzed. After three-dimension reconstruction of the MRI images, curvature of the arterial loop, angle between the plane of arterial loop and the trigeminal nerve and location of the compression were observed. Then bidirectional structure-fluid coupling based on the optimized stereolithography models of arterial loop and nerve were processed by ANSYS 19.2 software. In the location of the compression of contact, equivalent stress (ES) of arterial loop on the nerve, shearing stress (SS) of the blood flow and local deformation of the nerve were iteratively computed. All parameters were analyzed and compared between the PTN group and the control group, and the correlation analysis was proceeded between the anatomical parameters and hemodynamical parameters.Results:The curvature of arterial loop [0.21(0.12) mm -1vs 0.13(0.07) mm -1, U=34.00, P<0.05], the angle between vascular loop and nerve [69.70(30.67)° vs 43.40(37.21)°, U=38.00, P<0.05] in the PTN group were significantly greater than those in the control group, and the location of compression was significantly closer to the root of nerve in the PTN group [PTN group: (4.23±1.29) mm vs control group: (5.54±1.85) mm, t=-2.26, P<0.05]. The average SS [15 952.48(5 365.56) Pa vs 12 501.97(6 355.26) Pa, U=53.00, P<0.05], ES [24 965.65(7 693.22) Pa vs 14 992.99(9 824.08) Pa, U=32.00, P<0.05] in the PTN group were significantly greater than those in the control group. The curvature of arterial loop was positively correlated with the SS ( r=0.931, P<0.05) and ES ( r=0.962, P<0.05), and the latter two ( r=0.787, P<0.05; r=0.853, P<0.05) were positively correlated with the local neural deformation. Conclusions:In patients with PTN, offending artery compresses the root of nerve by greater arterial curvature and angle between the arterial loop and nerve. These anatomical differences will cause significantly greater SS, ES and local neural deformation.

Objective:To evaluate the role of heme oxygenase-1 (HO-1) in sevoflurane-induced improvement in sepsis-associated encephalopathy (SAE) in mice.Methods:A total of 136 adult male mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups ( n=34 each) using a random number table method: sham operation group (group Sham), SAE group, SAE+ sevoflurane group (group SAE+ Sevo) and SAE+ sevoflurane+ HO-1 inhibitor Zn Protoporphyrin Ⅸ (ZnPPⅨ) group (group SAE+ Sevo+ ZnPPⅨ). The model of SAE was established by cecal ligation and puncture (SAE) in anesthetized mice.In SAE+ Sevo and SAE+ Sevo+ ZnPPⅨ groups, 2% sevoflurane-33% oxygen was inhaled for 2 h starting from the time point immediately after establishment of the model, while 33% oxygen was inhaled for 2 h in Sham and SAE groups.ZnPPⅨ 25 mg/kg was intraperitoneally injected at 30 min before the model was established in group SAE+ Sevo+ ZnPPⅨ.Six mice were sacrificed at 6, 12 and 24 h after establishment of the model for determination of levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), high mobility group protein B1 (HMGB1) and HO-1 in cortical tissues (by enzyme-linked immunosorbent assay) and the expression of HO-1 (by Western blot). Another 6 mice were sacrificed for determination of apoptosis in cortical tissue (by TUNEL staining), and apoptotic index (AI) was calcultated.Ten mice in each group were selected, Y maze test was performed at 3, 5, 7 and 14 days after establishment of the model, and the percentage of spontaneous alternation was calculated. Results:Compared with Sham group, the levels of TNF-α, IL-1β and HMGB1, AI, HO-1 activity and its expression level in cortex were significantly increased, and the percentage of spontaneous alternation was decreased in SAE, SAE+ Sevo and SAE+ Sevo+ ZnPPⅨ groups ( P<0.05). Compared with group SAE, the levels of TNF-α, IL-1β and HMGB1 and AI were significantly decreased, and HO-1 activity and its expression level and the percentage of spontaneous alternation were increased in group SAE+ Sevo, the levels of TNF-α, IL-1β and HMGB1 in cortex were decreased ( P<0.05), and no significant change was found in the other parameters in group SAE+ Sevo+ ZnPPⅨ ( P>0.05). Compared with group SAE+ Sevo, the levels of TNF-α, IL-1β and HMGB1 and AI were significantly increased, and HO-1 activity and its expression level and the percentage of spontaneous alternation were decreased in group SAE+ Sevo+ ZnPPⅨ ( P<0.05). Conclusion:The mechanism by which sevoflurane improves SAE is related to increasing HO-1 activity and reducing inflammatory response in cortical tissues of mice.

Objective:To evaluate the role of nucleotide-binding oligomerization domain-2 (NOD2) in dorsal root ganglion in the development of neuropathic pain (NP) in rats.Methods:Thirty-two adult male SPF Sprague-Dawley rats, weighing 240-260 g, aged 2-3 months, were divided into 4 groups ( n=8 each) using a random number table method: control group (group C), NP group (group S), negative control siRAN group (group N), and NOD2-siRNA group (group R). In N and R groups, 1×10 8 IFU/ml negative control siRNA and NOD2-siRNA 10 μl were intrathecally injected, respectively, once a day for 3 consecutive days.Normal saline 10 μl was intrathecally injected once a day for 3 consecutive days in C and S groups.The model of NP was established using spared nerve injury (SNI) at 2 weeks after intrathecal injection.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before surgery and 1, 3, 7, 10, 14 and 28 days after SNI.Animals were sacrificed after measuring pain threshold on day 28, and the dorsal root ganglions (DRGs) of the lumbar segment (L 4-6) were removed for determination of the expression of NOD2 (by Western blot) and expression of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), IL-6 and NOD2 mRNA (using quantitative real-time polymerase chain reaction). Results:Compared with group C, MWT was significantly decreased at each time point after SNI, and the expression of NOD2 protein and mRNA and TNF-α, IL-1β and IL-6 mRNA in DRGs was up-regulated in group NP ( P<0.01). Compared with group NP, MWT was significantly increased at each time point after SNI, and the expression of NOD2 protein and mRNA and TNF-α, IL-1β and IL-6 mRNA in DRGs was down-regulated in group R ( P<0.01), and no significant change was found in the parameters mentioned above in group N ( P>0.05). Conclusion:The mechanism underlying the development of NP may be related to the up-regulation of NOD2 expression in DRGs, thus further promoting the expression of pro-inflammatory factors in rats.

Objective:To evaluate the effects of vitamin K 2 on sevoflurane-induced cognitive decline in aged mice. Methods:A total of 72 SPF healthy female C57BL/6J mice, aged 12 months, weighing 20-25 g, were divided into 4 groups ( n=18 each) using a random number table method: control+ corn oil group (group Con+ Oil), sevoflurane+ corn oil group (group Sevo+ Oil), control+ vitamin K 2 group (group Con+ K 2) and sevoflurane+ vitamin K 2 group (group Sevo+ K 2). The mice in Sevo+ Oil and Sevo+ K 2 groups were anesthetized with 2.5% sevoflurane+ 33% oxygen for 2 h. The mice in Con+ Oil and Con+ K 2 groups were treated with 33% oxygen only.The animals in Con+ Oil and Sevo+ Oil groups were intraperitoneally injected with corn oil 100 μl at 30 min before oxygen or sevoflurane inhalation.Vitamin K 2 (dissolved in corn oil, concentration 1 mg/ml) 100 mg/kg was injected intraperitoneally in Con+ K 2 and Sevo+ K 2 groups.At 24 h after sevoflurane inhalation, 8 mice from each group were randomly selected and sacrificed, and the hippocampal tissues were removed for determination of activity of ATPase, contents of interleukin-1beta (IL-1β), IL-6 and tumor necrosis factor-alpha (TNF-α) (by enzyme-linked immunosorbent assay) and the expression of AT8 and PHF1 (by Western blot). The remaining 10 mice in each group received standardized feeding, and the cognitive function was assessed using Y-maze at 1, 3, 5, 7 and 14 days after sevoflurane inhalation. Results:Compared with group Con+ Oil, the contents of IL-1β, IL-6 and TNF-α were significantly increased, expression of AT8 and PHF1 were up-regulated, activity of ATPase was decreased, and spontaneous alternation percentage was decreased at 1, 3, 5, 7 and 14 days after sevoflurane inhalation in group Sevo+ Oil ( P<0.05). Compared with group Sevo+ Oil, the contents of IL-1β, IL-6 and TNF-α were significantly decreased, expression of AT8 and PHF1 were down-regulated, activity of ATPase was increased, and spontaneous alternation percentage was increased at 1, 3, 5, 7 and 14 days in group Sevo+ K 2 ( P<0.05). There was no significant difference in the above indicators between group Con+ K 2 and group Sevo+ K 2 ( P>0.05). Conclusion:Vitamin K 2 can improve sevoflurane-induced cognitive decline in aged mice, the mechanism is related to increasing activity of ATPase and inhibiting the up-regulation of AT8 and PHF1 expression in hippocampus.

Objective:To explore the efficacy of precision hepatectomy in the treatment of single hepatocellular carcinoma with microvascular invasion (MVI) of and the risk factors of positive incisal margin after operation.Methods:The clinical data of 212 patients with single hepatocellular carcinoma with MVI treated in Affiliated Hospital of Panzhihua University from July 2016 to July 2019 were analyzed retrospectively. 152 patients were treated with precision hepatectomy and 60 patients with traditional hepatectomy. According to the pathological results of postoperative liver resection, the patients treated with precision hepatectomy were divided into two groups: negative group ( n=129) and positive group ( n=23). The operation-related indexes, postoperative complications and disease-free survival rate of precision hepatectomy and traditional hepatectomy were compared, and the general data of patients with negative and positive liver cutting edge were compared. multivariate analysis of the factors affecting the positive liver cutting edge after operation; to construct a line chart prediction model to predict the positive liver cutting edge after operation, and to evaluate its predictive efficiency. Normally distributed measurement data are represented by mean±standard deviation ( Mean± SD), independent t-test is used for comparison between groups; count data are represented by the number of cases and percentages, and χ2 test is used for comparison between groups. Results:The operative time, intraoperative blood loss, postoperative hospital stay, positive rate of surgical margin, total incidence of postoperative complications, AFP negative conversion rate 6 months after operation, and 1-year disease-free survival rate of precision hepatectomy were (328.62±38.74) min, (496.83±59.76) mL, (15.28±3.61) d, 15.13% (23/152), 3.95% (6/152), 81.58% (124/152), 67.11% (102/152), respectively. The mean values of traditional hepatectomy were (315.29±40.95) min, (681.46±58.27) mL, (23.87±4.65) d, 28.33% (17/60), 21.67% (13/60), 66.67% (40/60) and 46.67% (28/60), respectively, the difference was statistically significant ( P<0.05). Univariate analysis showed that the positive liver resection margin after precision liver resection was related to the maximum diameter of the tumor, vascular tumor thrombus, TNM staging, BCLC staging, liver cirrhosis, AFP 2 months after surgery, and the distance between the tumor and the resection margin ( OR=3.645, 5.248, 4.285, 4.462, 3.883, 3.964, 3.872; 95% CI: 2.875-4.415, 4.426-6.070, 3.271-5.299, 3.354-5.570, 3.062-4.704, 3.248-4.680, 2.987-4.757; P<0.05). Maximum tumor diameter >5 cm, vascular tumor thrombus, TNM stage Ⅲ, BCLC stage C, liver cirrhosis, postoperative AFP ≥20 μg Uniql, the distance between the tumor and the resection margin was <1 mm were the risk factors of positive incisal margin after precision hepatectomy in patients with single liver cancer with MVI( OR=6.685, 8.425, 7.758, 7.854, 7.124, 7.246, 6.926; 95% CI: 5.828-7.542, 7.6385-9.212, 6.926-8.590, 7.062-8.646, 6.583-7.665, 6.618-7.874, 6.028-7.824; P<0.05). The constructed line chart prediction model had better differentiation and higher accuracy. Conclusions:Precision hepatectomy in the treatment of single hepatocellular carcinoma with MVI has the advantages of less intraoperative bleeding, faster postoperative recovery, less postoperative complications, low positive rate of liver incisal margin and high disease-free survival rate. The construction of a risk prediction model with positive surgical margin provides a reference for improving the survival rate of patients in clinic.

Objective: To analyze the epidemiological history, clinical manifestations, treatment and the short-term prognosis of 31 cases of 2019 novel coronavirus(2019-nCoV) infection in children from six provinces (autonomous region) in northern China. Methods: A retrospective analysis of the epidemiological history, clinical symptoms, signs, laboratory examinations, chest imaging, treatment and the short-term prognosis of 31 cases of 2019-nCoV was conducted. The patients were diagnosed between January 25th, 2020 and February 21st, 2020 in 21 hospitals in 17 cities of six provinces(autonomous region) of Shaanxi, Gansu, Ningxia, Hebei, Henan and Shandong. Results: The age of the 31 children with 2019-nCoV infection was 7 years and 1 month (6 months -17 years). Nine cases (29%) were imported cases. Other 21 cases (68%) had contact with confirmed infected adults. One case (3%) had contact with asymptomatic returnees from Wuhan. Among the 31 children, 28 patients (90%) were family cluster cases. The clinical types were asymptomatic type in 4 cases (13%), mild type in 13 cases (42%), and common type in 14 cases (45%). No severe or critical type existed. The most common symptom was fever (n=20, 65%), including 1 case of high fever, 9 cases of moderate fever, 10 cases of low fever. Fever lasted from 1 day to 9 days. The fever of fifteen cases lasted for ≤3 d, while in other 5 cases lasted > 3 d. Other symptoms included cough (n=14, 45%), fatigue (n=3, 10%) and diarrhea (n=3, 9%). Pharyngalgia, runny nose, dizziness, headache and vomiting were rare. In the early stage, the total leukocytes count in peripheral blood decreased in 2 cases (6%), the lymphocytes count decreased in 2 cases (6%), and the platelet count increased in 2 cases (6%).Elevation of C-reactive protein (10%, 3/30), erythrocyte sedimentation rate(19%,4/21), procalcitonin(4%,1/28), liver enzyme(22%, 6/27) and muscle enzyme (15%, 4/27) occurred in different proportions. Renal function and blood glucose were normal. There were abnormal chest CT changes in 14 cases, including 9 cases with patchy ground glass opacities and nodules, mostly located in the lower lobe of both lungs near the pleural area. After receiving supportive treatment, the viral nucleic acid turned negative in 25 cases within 7-23 days. Among them, 24 children (77%) recovered and were discharged from hospital. No death occurred. Conclusions In this case series, 2019-nCoV infections in children from six provinces (autonomous region) in northern China are mainly caused by close family contact. Clinical types are asymptomatic, mild and common types. Clinical manifestations and laboratory examination results are nonspecific. Close contact history of epidemiology, nucleic acid detection and chest imaging are important bases for diagnosis. After general treatment, the short-term prognosis is good.