Objective:To prepare a recombinant hemagglutinin trimer (HA-Tri) vaccine against influenza viruses and to study its immunogenicity in a mouse model.Methods:A stable CHO cell line that could express HA-Tri was constructed. Western blot, single radial immunodiffusion, protein particle size detection and N-glycosylation site analysis were performed for qualitative and quantitative analysis of the recombinant protein. According to the different treatment conditions such as dosage and adjuvant, BALB/c mice were divided into 11 groups and subjected to consistent immunization procedures. Serum neutralizing antibody titers were measured on 56 d after the first immunization to evaluate the immunogenicity of HA-Tri.Results:The constructed CHO cells could secret and express HA-Tri proteins. The HA-Tri proteins were biologically active and capable of forming precipitation rings in the single radial immunodiffusion. The particle size of HA-Tri was approximately 18.79 nm and 10 N-glycosylation sites were detected, including high mannose, complex glycoforms and heterozygous glycoforms. After prime-boost immunization, there was no statistically significant difference in the titers of neutralizing antibodies induced in mice by 3.75 μg of HA-Tri in combination with RFH01 adjuvant and 15 μg of monovalent vaccine stock solution ( P=0.431 2, U=36). Serum antibody titers in the HA-Tri+ RFH01 groups were higher than those in the corresponding HA-Tri groups without RFH01 adjuvant, and the highest titer was induced in the 15 μg HA-Tri+ RFH01 group, which was 1 280. Conclusions:The recombinant HA-Tri protein was successfully prepared. HA-Tri in combination with RFH01 adjuvant could induce humoral immune responses against influenza viruses in BALB/c mice, which would provide reference for the development of influenza virus recombinant subunit vaccines.

Objective:To evaluate the immunogenicity of a quadrivalent subunit vaccine combined with RFH01 adjuvant in a mouse model.Methods:Identification tests were performed on four monovalent influenza virus subunit vaccine stock solutions according to the methods described in Part 3 of the Chinese Pharmacopoeia 2020 Edition. In the study of the quadrivalent subunit vaccine combined with RFH01 adjuvant, 460 female BALB/c mice (6-8 weeks old) were randomly divided into 46 groups including experimental groups, vaccine control group, negative control group and blank group with 10 mice in each group. In the study of the quadrivalent subunit vaccine in old and young mice, 80 female 10-month-old and 80 female 10-week-old BALB/c mice were randomly divided into 16 groups ( n=10) including monovalent influenza virus vaccine group, quadrivalent subunit vaccine group, quadrivalent subunit vaccine+ RFH01 adjuvant group, chicken embryo quadrivalent split vaccine control group and PBS group. All mice were immunized by intramuscular injection. At 21 d after the primary immunization, a booster immunization was conducted using the same strategy. Blood samples were collected at 21 d and 42 d after the primary immunization for serum separation. Haemagglutination inhibition (HI) test was performed to detect the antibody levels in mouse serum samples. Results:After the booster immunization, the positive conversion rates in all vaccine+ RFH01 adjuvant groups reached 100%, and the geometric mean titers (GMTs) of serum antibodies were significantly higher than those of the vaccine groups without RFH01 adjuvant. There were significant differences in serum antibody titers between the monovalent/quadrivalent subunit vaccine groups with and without RFH01 adjuvant. After the booster immunization, the titers of serum antibodies against H1N1, H3N2, B/Victoria and B/Yamagata in the 10-week-old mice were significantly higher than those in the 10-month-old mice.Conclusions:The monovalent and quadrivalent influenza virus vaccines in combination with RFH01 adjuvant could elicit higher antibody titers in young (6-10 weeks old) and old (10 months old) mice, showing good immunogenicity.

Objective: To investigate the expression of eukaryotic translation initiation factor 2B1(eIF2B1) in HCC and its correlation with clinical prognosis. Methods: Through the follow⁃up of the clinical data of 743 hepatocellular carcinoma patients in the specimen bank ,91 HBV⁃related liver cancer patients with complete follow⁃up information were screened out , and their postoperative tumor tissues were selected. Meanwhile , the adjacent tumor tissues with a distance of more than 2 cm from the tumor margin were collected to make tissue chips. Western blot and immunohistochemical experiments were performed. Image J was used to analyze the proportion of positive cells stained by tissue chip and the gray value of Western blot results. Clinical data and follow⁃up data of included patients were statistically analyzed by R4. 0. 5 software. Results: Immunohistochemical results suggested that eIF2B1 was more strongly expressed in HCC than in para⁃cancerous tissues. Western blot results showed that eIF2B1 was more strongly expressed in HepG2. 2. 15 cells than in HepG2 cells. The expression of eIF2B1 in HCC tissues was correlated with tumor number (P < 0. 05) . Cox multivariate regression analysis showed that the expression of eIF2B1 was an independent risk factor for the overall clinical prognosis of HBV⁃DNA positive patients (HR = 4. 28 ,P = 0. 018) . Conclusion The expression of eIF2B1 is significantly enhanced in HBV⁃associated HCC tissues and is significantly associated with overall survival in HBV DNA positive patients.

Objective:To investigate the effects of endotracheal intubation anesthesia versus laryngeal mask anesthesia on the incidence of pulmonary infection in young children undergoing surgery. Methods:A total of 100 young children who underwent surgery under general anesthesia in Hangzhou Children's Hospital from January 2020 to December 2020 were included in this study. They were randomly allocated into the endotracheal intubation anesthesia and laryngeal mask anesthesia groups ( n = 50/group). The symptoms of respiratory tract infection (nasal congestion, cough, sputum ringing, rale, and sputum excretion) and the incidence of pulmonary infection evaluated 24 hours after surgery were compared between the two groups. At the same time, the pathogens of infection and perioperative conditions as well as prognosis were also compared between the two groups. Results:The incidences of nasal obstruction, cough, sputum ringing, rale, sputum excretion, and pulmonary infection were 56.0% (28/50), 42.0% (21/50), 14.0% (7/50), rale 38.0% (19/50), 16.0% (8/50), and 38.00 (19/50) respectively in the endotracheal intubation anesthesia group, which were significantly higher than those in the laryngeal mask anesthesia group [28.0% (14/50), 14.0% (28/50), 4.0% (8/50), 24.0% (12/50) 4.0% (2/50), 6.0%(3/50), χ2 = 12.18, 21.47, all P < 0.001]. A total of 34 strains of pathogens were detected in the two groups, including 4 strains in the laryngeal mask anesthesia group and 30 strains in the endotracheal intubation anesthesia group. The 34 strains of pathogens included 7 strains of gram-positive bacteria (20.6%), 23 strains of gram-negative bacteria (67.6%), and 4 strains of fungi (11.8%). There was no significant difference in pathogen composition between the two groups ( χ2 = 1.04, P = 0.310). The time to recovery and the time to extubation in the laryngeal mask anesthesia group were (10.3 ± 3.1) minutes and (13.5 ± 2.4) minutes, which were significantly shorter than those in the endotracheal intubation anesthesia group [(16.1 ± 5.1) minutes, (19.5 ± 4.1) minutes, t = 3.17, 7.19, both P < 0.05]. There were no significant differences in the proportion of children who needed prolonged hospitalization and the proportion of children scheduled to undergo readmission between the two groups (both P > 0.05). Conclusion:Laryngeal mask anesthesia is simpler to operate, remarkably decreases the incidence of pulmonary infection, and is safer compared with endotracheal intubation anesthesia. Therefore, laryngeal mask anesthesia is preferred for general anesthesia for surgery in young children. The study is of great innovation and science.

Objective:To express the head domain of influenza A virus hemagglutinin (HA) in a prokaryotic expression system and to evaluate its immunogenicity.Methods:The genes encoding the HA head domains of H1N1 and H3N2 influenza viruses were cloned into pET-22b(+ ) prokaryotic expression plasmid. After the induction with IPTG, the fusion proteins rH1N1-HA and rH3N2-HA containing HA head domain and His-tag were expressed and obtained from E. coli BL21. SDS-PAGE and Western blot was used to verify the expression of the recombinant proteins. Rabbits were immunized with multiple doses of the purified recombinant proteins to obtain polyclonal antibodies against the HA head domains of H1N1 and H3N2. The immunogenicity of the recombinant proteins was evaluated in BALB/c mice. Results:rH1N1-HA and rH3N2-HA induced protective antibodies (geometric mean titer ≥40) in mice and could be used as protective antigens. Polyclonal antibodies against rH1N1-HA and rH3N2-HA could be used as important materials for Western blot, ELISA and other immunological assays.Conclusions:The HA head domains prepared in this study could be used as protective antigens to induce protective antibodies in mice. Polyclonal antibodies against the HA head domains could be used for immunological and serological studies of influenza A viruses.

Objective:To investigate the predictive value of YKL-40 at admission on stroke-associated pneumonia (SAP) and poor outcome in patients with acute ischemic stroke (AIS).Methods:Patients with AIS admitted to Taixing People’s Hospital from February 2020 to March 2021 were enrolled prospectively. The poor outcome was defined as 3-6 points on the modified Rankin Scale at 90 d after onset. Multivariate logistic regression analysis was used to determine the independent predictors of SAP and poor outcome, and the predictive value of serum YKL-40 on SAP and poor outcome was evaluated by receiver operating characteristic (ROC) curve. Results:A total of 377 patients with AIS were enrolled. The median serum YKL-40 was 127.16 μg/L. One hundred and four patients (27.6%) had SAP, and 126 (33.4%) had poor outcomes at 90 d after onset. Multivariate logistic regression analysis showed that after adjusting for confounding factors, YKL-40 was the independent predictors of SAP (odds ratio [ OR] 1.005, 95% confidence interval [ CI] 1.003-1.008; P=0.001) and poor outcome at 90 d ( OR 1.009, 95% CI 1.006-1.011; P=0.001). The ROC curve analysis showed that the area under the curve of YKL-40 for predicting SAP was 0.769 (95% CI 0.713-0.824; P<0.001), the best cutoff value was 168.70 μg/L, and the sensitivity and specificity were 71.2% and 75.1% respectively; the area under the curve of YKL-40 for predicting poor outcome at 90 d was 0.787 (95% CI 0.735-0.840; P<0.001), the best cutoff value was 195.56 μg/L, and the sensitivity and specificity were 68.3% and 84.1% respectively. Conclusion:Higher serum YKL-40 at admission has a good predictive value for SAP and poor outcome at 90 d in patients with AIS.

Erythrocyte Indices ; Erythrocytes ; Hepacivirus ; Humans ; Liver Diseases ; blood ; virology

Aim To investigate the effect of Rhein on the movement and invasiveness of human ovarian carci-noma cells with directional high lymphatic metastasis SKOV3-PM4 cells and explore the role of Rac1/LIMK1/cofilin signaling pathway. Methods Migration assay and invasion assay were used to observe the effect of Rhein on the metastatic and invasive ability of SK-OV3-PM4 cells in vitro. The effect of Rhein on the morphology and cytoskeleton ultrastructure of ovarian cancer cells was observed by laser scanning confocal microscope and scanning electron microscope. The protein expression level of Rac1,LIMK1,PAK1 and co-filin were detected by Western blot, respectively. Re-sults Rhein inhibited the abilities of cell invasion and migration of SKOV3-PM4 cells,and the inhibitory rate increased along with the increase of the concentration and treatment duration. After treated with 8. 80 μmol· L-1 ,17. 60 μmol · L-1 , 26. 40 μmol · L-1 of Rhein for 24 h,the abilities of migration and invasion of SK-OV3-PM4 cells were inhibited ( P <0. 05 ); the mor-phology and cytoskeleton ultrastructure of SKOV3-PM4 cells were changed, cellular pseudopod reduced, cell microfilament fractured and its distribution disordered, plasma membrane was uneven and cell gap widened . After treatment of Rhein and Rac1 inhibitor , Rac1 protein expression and the expression of P-LIMK1 , P-PAK1 and P-cofilin notably decreased in a dose-de-pendent manner compared with the control group ( P<0. 05 ) . After Rhein and Rhein plus Rac1 inhibitor treatment ,P-LIMK1, P-cofilin, P-PAK1 protein levels of SKOV3-PM4 cells significantly decreased compared with the control group , and the group of Rac1 inhibi-tor plus Rhein treatment, the phosphorylated protein decreased more significantly ( P <0. 05 ) . After Rac1 activator plus Rhein treatment, phosphorylated protein expression of P-LIMK1 ,P-PAK1 and P-cofilin upregu-lated significantly ( P <0. 05 ) . Conclusions Rhein may be a potential inhibitor of Rac1 and can inhibit the migrating and invasive capabilities of directional high lymphatic metastasis SKOV3-PM4 cells through down-regulating the phosphorylation of Rac1/LIMK1 /cofilin pathway associated protein.

Objectives To investigate the effects of different degrees of carotid artery stenosis on cognitive function and neuronal apoptosis of hippocampal CA1 region in rats and to analyze the possible mechanisms of cognitive impairment. Methods According to the random number table,50 male Wistar rats were allocated into a sham operation group,a unilateral mild stenosis group,a unilateral moderate stenosis group,a unilateral severe stenosis group,a bilateral mild stenosis group,a bilateral moderate stenosis group,
a bilateral severe stenosis group,and a sham operation group. Aneedle-controlled suture method was used to induce a carotid stenosis model with different degrees of stenosis in rats. Water maze was to localize navigation and spatial search test was used to evaluate the cognitive function with different degrees of carotid artery stenosis in rats. Immunohistochemical method was used to observe the numbers of positive cells of P75 neurotrophin receptor (p75NTR),Bax,Bcl-2,neurotrophic factor-3 (NT-3 ),nerve growth factor (NGF)in hippocampal CA1 region under the light microscope. The conditions of neuronal apoptosis were observed. Results In 50 rats,6 died,1 rat in poor health failed to complete the Morris water maze test. The degree of bilateral carotid artery stenosis in 1 rat failed to meet 30%-60% at the same time,they were all removed. The remaining 42 rats were 6 in each group. (1)Compared with the sham operation group,the mean escape latency was prolonged (39 ± 6 s,32 ± 5 s,69 ± 7 s,respectively vs. 23 ± 4 s;all P < 0. 01),and the percentage of swimming distance in the platform quadrant was decreased (35 ± 4%,44 ± 4%,22 ± 5%,respectively vs. 53 ± 7%;all P < 0. 01),and the cognitive function was decreased with the degree of stenosis in the unilateral severe stenosis group,bilateral moderate stenosis group,and bilateral severe stenosis group. Compared with the unilateral severe stenosis group,the mean escape latency was prolonged and the percentage of swimming distance in the platform quadrant was decreased in the bilateral severe stenosis group (P < 0. 01). (2)Compared with the numbers of positive cells of Bax,p75NTR and Bcl-2 (8. 8 ± 3. 1,4. 2 ± 2. 3,and 5. 8 ± 1. 8,respectively)in the sham operation group,the numbers of positive cells of Bax,p75NTR,and Bcl-2 were increased (25. 5 ± 3. 5,11. 0 ± 2. 2,12. 3 ± 2. 7;15. 8 ± 3. 7,8. 9 ± 2. 2, 10. 5 ± 2. 9;and 47. 9 ± 6. 3,24. 7 ± 3. 0,12. 8 ± 2. 5,respectively)in the unilateral severe stenosis group, bilateral moderate stenosis group,and bilateral severe stenosis group (all P < 0. 01). The numbers of Bax and p75NTR positive cells were increased with the degree of stenosis. When the stenosis was severe,the numbers of Bax and p75NTR positive cells were increased in the bilateral severe stenosis group compared with those of the unilateral severe stenosis group (P < 0. 01). The numbers of NGF and NT-3 positive cells in each stenosis group had an increased trend compared with sham operation group,but there were no significant differences (F =1. 034,and 1. 358;P = 0. 420 and 0. 259 respectively). Conclusions Carotid stenosis can cause cognitive disorder in rats,and it is correlated with the degree of carotid stenosis. Ischemia caused neuronal apoptosis in hippocampal CA1 region may be one of the mechanisms of cognitive impairment after carotid artery stenosis in rats.

Objective To investigate the effects of intensive antihypertensive treatment and guidelinerecommended standard blood pressure control on early reperfusion and outcomes after intravenous recombinant tissue plasminogen activator (rtPA) thrombolysis in patients with acute ischemic stroke.Methods A total of 50 patients with acute ischemic stroke (systolic blood pressure,150-185 mmHg;1 mmHg=0.133 kPa) and received intravenous rtPA therapy were enrolled prospectively.They were randomly divided into either a intensive antihypertensive treatment group or a guideline antihypertensive treatment group.In the the intensive antihypertensive treatment group,systolic blood pressure was decreased to 140-150 mmHg in 60 min for at least 72 h.In the guideline antihypertensive treatment group,systolic blood pressure was decreased to the target value ＜ 180 mmHg according to the guideline recommendation.Multi-mode MRI was completed at 24 h before and after thrombolysis.The primary endpoints were the modified Rankin Scale (mRS) score at day 90 and the mortality at day 90;the secondary endpoints were the early reperfusion rate in ischemic brain tissue,recanalization rate,and incidence of symptomatic intracranial hemorrhage.Results There was no significant difference in demographics and baseline data between the 2 groups.Within 24,48,and 72 h after thrombolysis the mean systolic blood pressure in the intensive antihypertensive treatment group was significantly lower than those in the guideline antihypertensive treatment group,while there was no significant difference in diastolic blood pressure.There were no significant differences in favorable outcome rate at day 90 (mRS score 0-2:68％ vs.64％;x2 =0-089,P=0.765),mortality (4％ vs.12％;x2 =1.087,P=0.297),incidence of symptomatic intracranial hemorrhage (4％ vs.8％;x2 =0.355,P =0.552),reperfusion rate after thrombolysis (76％ vs.68％;x2 =0.397,P =0.529),and recanalization rate (56％ vs.52％;x2 =0.081,P =0.777) between the intensive antihypertensive treatment group and the guideline antihypertensive treatment group.Conclusions Early intensive antihypertensive treatment in patients with acute ischemic stroke received intravenous rtPA thrombolysis does not have adverse effect on reperfusion rate,and does not increase the risk of death or disability either.