To screen new antigens for novel tuberculosis(TB)vaccine research,we used bioinformatics to predict the B and T cell epitopes of Rv2318,and evaluated the immunogenicity of Rv2318 and its T/B epitope peptides(Rv2318p).The recombinant plas-mids pET32a-Rv2318 and pET32a-Rv2318p were constructed through gene synthesis methods.The recombinant proteins were ex-pressed in a prokaryotic system and purified with nickel affinity chromatography.Proteins were identified with SDS-PAGE and western blotting.BALB/c mice were immunized subcutaneously with the recombinant proteins to evaluate immunogenicity.Sera were collected,and antigen specific antibody titers were evaluated with ELISA.Splenocytes were isolated,and cytokines and T cell proliferation were analyzed with ELISA and flow cytometry,respectively.Rv2318 included two epitope fragments,aa10-130 and 350-410.SDS-PAGE and western blotting indicated that the target proteins were expressed and purified correctly,and their relative molecular weights were-approximately 68 kD and 42 kD,respectively.Rv2318 and Rv2318p induced stronger humoral immune responses than observed in the control groups(P<0.000 1,n=6).Compared with Rv2318,Rv2318p showed significantly greater enhancement of specific IgG and IgG subclass antibodies(P<0.000 1,n=6).In addition,Rv2318p increased the ratio of IgG2a/IgG1,thus indicating that it primarily induced a cellular immune response biased toward the Th1 type.Cytokine experiments revealed that IFN-γ,IL-2,IL-6,and IL-4 significantly increased after immunization with Rv2318p(P all<0.01,n=6),particularly Th1 type cytokines(IFN-γ and IL-2).Furthermore,Rv2318 increased the expression of only IL-2 and IL-6,particularly IL-6(P all<0.01,n=6).Although Rv2318 in-duced more IFN-γ,we observed no significant difference between Rv2318 and PBS immunized mice.Importantly,Rv2318p stimu-lated mice to express IFN-γ at 842 pg/mL,approximately 3 times the level elicited by Rv2318.Whereas both proteins increased the proportions of CD4+and CD8+T cells,Rv2318p promoted greater proliferation of T lymphocytes.These data indicated that both Rv2318 and its epitope peptides enhanced humoral and cellular immune responses,whereas the epitope peptides notably triggered a stronger Th1 type cellular response.In conclusion,the recombinant protein Rv2318 and its epitope peptides showed favorable immunogenicity,and the immunogenicity of Rv2318p was superior to that of Rv2318.This study provides a theoretical basis for TB vaccine development.

To screen new antigens for novel tuberculosis(TB)vaccine research,we used bioinformatics to predict the B and T cell epitopes of Rv2318,and evaluated the immunogenicity of Rv2318 and its T/B epitope peptides(Rv2318p).The recombinant plas-mids pET32a-Rv2318 and pET32a-Rv2318p were constructed through gene synthesis methods.The recombinant proteins were ex-pressed in a prokaryotic system and purified with nickel affinity chromatography.Proteins were identified with SDS-PAGE and western blotting.BALB/c mice were immunized subcutaneously with the recombinant proteins to evaluate immunogenicity.Sera were collected,and antigen specific antibody titers were evaluated with ELISA.Splenocytes were isolated,and cytokines and T cell proliferation were analyzed with ELISA and flow cytometry,respectively.Rv2318 included two epitope fragments,aa10-130 and 350-410.SDS-PAGE and western blotting indicated that the target proteins were expressed and purified correctly,and their relative molecular weights were-approximately 68 kD and 42 kD,respectively.Rv2318 and Rv2318p induced stronger humoral immune responses than observed in the control groups(P<0.000 1,n=6).Compared with Rv2318,Rv2318p showed significantly greater enhancement of specific IgG and IgG subclass antibodies(P<0.000 1,n=6).In addition,Rv2318p increased the ratio of IgG2a/IgG1,thus indicating that it primarily induced a cellular immune response biased toward the Th1 type.Cytokine experiments revealed that IFN-γ,IL-2,IL-6,and IL-4 significantly increased after immunization with Rv2318p(P all<0.01,n=6),particularly Th1 type cytokines(IFN-γ and IL-2).Furthermore,Rv2318 increased the expression of only IL-2 and IL-6,particularly IL-6(P all<0.01,n=6).Although Rv2318 in-duced more IFN-γ,we observed no significant difference between Rv2318 and PBS immunized mice.Importantly,Rv2318p stimu-lated mice to express IFN-γ at 842 pg/mL,approximately 3 times the level elicited by Rv2318.Whereas both proteins increased the proportions of CD4+and CD8+T cells,Rv2318p promoted greater proliferation of T lymphocytes.These data indicated that both Rv2318 and its epitope peptides enhanced humoral and cellular immune responses,whereas the epitope peptides notably triggered a stronger Th1 type cellular response.In conclusion,the recombinant protein Rv2318 and its epitope peptides showed favorable immunogenicity,and the immunogenicity of Rv2318p was superior to that of Rv2318.This study provides a theoretical basis for TB vaccine development.

Objective To explore the effect of ropivacaine combined with dexmedetomidine in transversus abdominis plane block(TAPB)on postoperative stress hormones and cognitive function in patients undergoing laparoscopic radical gastrectomy.Methods A total of 80 patients undergoing laparoscopic radical gastrectomy at the First Affiliated Hospital of Xinxiang Medical University from April to October 2023 were selected as the research subjects.According to different anesthesia methods,the patients were divided into the observation group and the control group,with 40 patients in each group.Patients in the observation group were injected bilaterally with 2.5 g·L-1 ropivacaine and 0.5 μg·kg-1 dexmedetomidine for TAPB,with 20 mL injection on each side.Patients in the control group were injected bilaterally with 2.5 g·L-1 ropivacaine for TAPB,with 20 mL injection on each side.Mean arterial pressure(MAP)and heart rate(HR)were recorded at the time of admission to the operating room(T1),immediately after endotracheal intubation(T2),40 minutes after pneumoperitoneum(T3),and 15 minutes after extubation(T4).Radioimmunoprecipitation was used to detect serum cortisol(COR)level,and enzyme-linked immunosorbent assay was used to measure serum norepinephrine(NE)and epinephrine(E)levels at 1,6,12,and 24 hours after surgery.Visual analog scale(VAS)was used to assess pain at rest,and Ramsay sedation scale(RSS)was used to evaluate sedation depth.The doses of propofol and sufentanil were compared between the two groups.Serum β-amyloid(Aβ)and S100β protein levels at 1 day before surgery,1 and 3 days after surgery were detected by using the enzyme-linked immunosorbent assay,and cognitive function was assessed at the same time points by using the mini-mental state examination(MMSE).Results At T,and T2,there was no significant difference in MAP and HR between the control group and the observation group(P＞0.05).At T3 and T4,MAP and HR in the observation group were significantly lower than those in the control group(P＜0.05).At 1,6,and 12 hours postoperatively,VAS score in the observation group was significantly lower than that in the control group(P＜0.05).At 24 hours postoperatively,there was no significant difference in VAS score between the control group and observation group(P＞0.05).At 1 and 6 hours postoperatively,RSS score in the observation group was significantly higher than that in the control group(P＜0.05).At 12 and 24 hours postoperatively,there was no significant difference in RSS score between the control group and observation group(P＞0.05).At 1,6,and 12 hours postoperatively,COR,NE,and E levels in the observation group were significantly lower than those in the control group(P＜0.05).At 24 hours postoperatively,there was no significant difference in COR,NE,and E levels between the control group and observation group(P＞0.05).The doses of propofol and sufentanil in the observation group were significantly lower than those in the control group(P＜0.05).One day before surgery,there was no significant difference in MMSE score between the control group and observation group(P＞0.05).At 1 and 3 days postoperatively,MMSE score in the observation group was significantly higher than that in the control group(P＜0.05).One day before surgery,there was no significant difference in serum Aβ and S100β protein levels between the control group and observation group(P＞0.05).At 1 and 3 days postoperatively,serum Aβ and S100β protein levels in the observation group were significantly lower than those in the control group(P＜0.05).Conclusion Ropivacaine combined with dexmedetomidine in TAPB in radical gastrectomy can significantly reduce postoperative pain,increase sedative effect,prolong the duration of TAPB,and benefit patients'postoperative recovery with reduced cognitive impairment.

Objective: To investigate the awareness of air pollution protection knowledge and its influencing factors among primary school students in Shennongjia Forest District, Hubei Province, so as to provide insights into targeting implementation of health education on air pollution protection knowledge. Methods: Students in Grade 3 to 5 in Shennongjia Shiyan primary school were enrolled by stratified cluster sampling method, and students' demographic features and awareness of air pollution protection knowledge were investigated using the Investigation on the Effects of Air Pollution Health Protection of Pupils (Volume A). Factors affecting the awareness of air pollution protection knowledge among primary school students were identified using a multivariable logistic regression model. Results: A total of 897 questionnaires were allocated, and 877 valid questionnaires were recovered, with an effective rate of 97.77%. The respondents included 446 men (50.86%) and 431 women (49.14%), 301 third grade students (34.32%), 284 fourth grade students (32.38%), and 292 fifth grade students (33.30%), and had a mean age of (10.32±0.93) years. The overall awareness of air pollution protection was 55.76%, and the awareness rates of basic concepts, basic knowledge, and basic behaviors and skills were 42.99%, 53.48% and 57.24%, respectively. Multivariable logistic regression analysis identified age (OR=1.453, 95%CI: 1.053-2.005), living with parents (OR=2.638, 95%CI: 1.571-4.429), mother's educational level (below primary school, OR=0.270, 95%CI: 0.084-0.862; primary school, OR=0.169, 95%CI: 0.069-0.416; junior high school, OR=0.309, 95%CI: 0.138-0.691; high school, OR=0.352, 95%CI: 0.160-0.773) and average annual family income (50 000 to 100 000 Yuan, OR=1.629, 95%CI: 1.162-2.282; 100 000 to 150 000 Yuan, OR=1.802, 95%CI: 1.101-2.948; ≥150 000 Yuan, OR=1.939, 95%CI: 1.065-3.529) as factors affecting the awareness of air pollution protection knowledge among primary school students. Conclusion The awareness of air pollution protection knowledge is 55.76% among primary school students in Shennongjia Forest District, and is affected by age, mother's educational level, average annual family income and living with parents.

Aim To investigate the protective effect of remimazolam(Re)combined with thoracic sympathetic nerve block(TSNB)on myocardial ischemia/reperfusion(MI/R)rats.Methods Rats were randomly separated into control group,MI/R group,Re group,TSNB group,and Re+TSNB group,with 12 rats in each group.Except for the control group,the remaining rats were subjected to left anterior descending coronary artery(LAD)ligation to construct an MI/R model.In the Re group,20 mg/kg Re was intraperitoneally injected 30 min before ischemia.In TSNB group,0.2％ropivacaine 50 μL was injected into the thoracic epidural catheter 30 min before ischemia.In the Re+TSNB group,20 mg/kg Re was intraperitoneally injected and 0.2％ropivacaine 50 μL was injected into the thoracic epidural catheter 30 min before ischemia.The control group and MI/R group were injected with normal saline only.Rats in each group were evaluated for cardiac function and infarct size.HE staining and TUNEL staining were applied to observe pathological changes in myocardial tissue and myocardial cell apoptosis.Serum myocardial injury markers creatine kinase(CK)and aspartate transaminase(AST),cardiac troponin(cTnⅠ),myocardial inflammatory factors interleukin-8(IL-8),tumor nec-rosis factor-α(TNF-α),and oxidative stress factors malondialdehyde(MDA),superoxide dismutase(SOD)were detec-ted.Western blot was applied to detect the expression of IL-8,TNF-α,B lymphoblastoma-2-associated X protein(Bax),and B-lymphoblastoma-2(Bcl-2)in myocardial tissue.Results Compared with the control group,the myocardial cells of rats showed edema and myocardial fiber disorder in the MI/R group,the left ventricular developmental pressure(LVDP),maximal left ventricular pressure rising rate(+dp/dtmax),maximal left ventricular pressure decreasing rate(-dp/dtmax),SOD activity,and level of Bcl-2 were significantly reduced,the myocardial infarction area,cell apoptosis rate,levels of cTnⅠ,CK,AST,IL-8,TNF-α,MDA,and Bax were increased(P＜0.05).Compared with the MI/R group,the morphology of myocardial fibers and myocardial cells was significantly improved in the Re group,TSNB group and Re+TSNB group,the LVDP,±dp/dtmax,SOD activity,and level of Bcl-2 were significantly increased,the myocardial infarction area,cell apoptosis rate,levels of cTnI,CK,AST,IL-8,TNF-α,MDA,and Bax were significantly decreased(P＜0.05).Compared with the Re group and TSNB group,the LVDP,±dp/dtmax,SOD activity,and level of Bcl-2 were significantly increased in the Re+TSNB group,the myocardial infarction area,cell apoptosis rate,levels of cTnⅠ,CK,AST,IL-8,TNF-α,MDA,and Bax were significantly decreased(all P＜0.05).Conclusion The combination of Re and TSNB may protect against MI/R injury by reducing myocardial infarction and myocardial cell apoptosis,and inhibiting inflammatory response and oxidative stress.

Objective:To evaluate the effect of superior cervical ganglion block (SCGB) on cardiac function and nucleotide like receptor protein 3 (NLRP3) signaling pathway in a rat model of myocardial ischemia-reperfusion (I/R).Methods:Sixty healthy SPF male Sprague-Dawley rats, weighing 250-300 g, aged 2-3 months, were divided into 4 groups ( n=15 each) using a random number table method: sham operation group (sham group), myocardial I/R group (IR group), myocardial I/R + normal saline group (IR+ NS group), and myocardial I/R + SCGB group (IR+ SCGB group). Myocardial I/R model was developed by ligation of the left anterior descending branch of the coronary artery for 45 min followed by restoration of blood flow in anesthetized aninals. IR+ SCGB group received SCGB (0.25% ropivacaine 0.1 ml) at 10 min before reperfusion once a day for 2 consecutive weeks, while 0.9% sodium chloride was given instead of ropivacaine in IR+ NS group. Blood samples were collected at 24 h and 14 days of reperfusion for determination of serum concentrations of norepinephrine (NE), troponin T (TnT), tumor necrosis factor-alpha (TNF-α), interleukin-18 (IL-18) and IL-1β by enzyme-linked immunosorbent assay. Echocardiography was performed before ischemia and at 14 days of reperfusion, and left ventricular short axis shortening rate (FS), ejection fraction (EF), and cardiac output (CO) were measured. The rats were sacrificed at 14 days of reperfusion and the hearts were taken for determination of the contents of norepinephrine (NE) in myocardial tissues in the infarction area (by enzyme-linked immunosorbent assay), percentage of myocardial fibrosis area (by Masson staining), M1 macrophage marker CD68 + cell count in the infarction area (by immunohistochemical method), and expression of NLRP3 and gasdermin D (GSDMD) in myocardial tissues (by Western blot). Results:Compared with Sham group, the serum concentrations of TnT, TNF-α, IL-18 and IL-1β, percentage of myocardial fibrosis area, and NE levels in serum and myocardial tissues were significantly increased, the expression of NLRP3 and GSDMD in myocardial tissues was up-regulated, CD68 + cell count was increased, and EF, CO and FS were decreased in IR group ( P<0.05). Compared with IR group, the serum concentrations of TnT, TNF-α, IL-18 and IL-1β, percentage of myocardial fibrosis area, and NE levels in serum and myocardial tissues were significantly decreased, the expression of NLRP3 and GSDMD in myocardial tissues was down-regulated, CD68 + cell count was decreased, and EF, CO and FS were increased in IR+ SCGB group ( P<0.05), and no statistically significant changes were found in the parameters mentioned above in IR+ NS group ( P>0.05). Conclusions:SCGB can improve the cardiac function in a rat model of myocardial I/R, and the mechanism may be related to the inhibition of NLRP3 signaling pathway.

Objective:To preliminarily evaluate the immunogenicity and efficacy of two novel tuberculosis vaccine candidates (a fusion multicomponent protein EPDPA015f and a mixed multicomponent protein EPDPA015m) and to provide a new antigen combination for the development of tuberculosis vaccines.Methods:Recombinant plasmids for the expression of EPDPA015f and EPDPA015m proteins were constructed. Six-week-old BALB/c mice were immunized with EPDPA015f or EPDPA015m in combination with aluminium adjuvant (50 μg/mouse) for three times with an interval of 10 d. The mice were sacrificed 10 d after the last immunization to collect blood and spleen samples. Serum antibody titers and cytokine levels were measured by ELISA, Luminex technique and enzyme-linked immunospot assay (ELISPOT). Mycobacterial growth inhibition assay (MGIA) was used to detect the ability of mouse splenocytes to inhibit the growth of Mtb in vitro. One-way analysis of variance and t-test were used for statistical analysis. Results:Both EPDPA015f and EPDPA015m could induce the production of various cytokines and IgG antibodies at a high level. The levels of cytokines related to Th1 (IL-2, TNF-α, IFN-γ), Th2 (IL-4, IL-6, IL-10) and Th17 (IL-17) as well as other proinflammatory cytokines (GM-CSF, IL-12) were higher in the EPDPA015f group than in the adjuvant group ( P<0.05). The titer of IgG antibody induced by EPDPA015f was as high as 1∶4×10 6. The results of MGIA showed that the numbers of Mtb (lgCFU) in the PBS, adjuvant, EPDPA015f and EPDPA015m groups were 3.46±0.11, 3.51±0.06, 2.98±0.09 and 3.19±0.08, respectively. The number of colonies in the EPDPA015f group was the least as compared with that in the other three groups ( P<0.001, P<0.001, P<0.01). Conclusions:The vaccine candidate EPDPA015f could elicit more comprehensive and high-level cellular and humoral immune responses, and exhibited superior in vitro inhibitory activity against the growth of Mtb. EPDPA015f had the potential to be used as a preventive vaccine or a booster vaccine

Objective:To evaluate the immunogenicity and protective effect of a multicomponent recombinant protein vaccine EPRHP014 constructed independently and provide a scientific basis for developing new tuberculosis (TB) vaccine and effective prevention and control of TB.Methods:Three full-length Mycobacterium ( M.) tuberculosis protein antigens (EsxH, Rv2628, and HspX) and two epitope-predicted and optimized epitope-dominant protein antigens (nPPE18 and nPstS1) were selected, from which five protein antigens were used to construct a protein antigen composition EPRHP014, including a fusion expression multi-component protein antigen (EPRHP014f) and a multi-component mixed protein antigen (EPRHP014m) formed with the five single protein using clone, purification, and purification respectively. Multicomponent protein vaccines EPRHP014f and EPRHP014m were prepared with aluminum adjuvant, and the BCG vaccine was used as a control. ELISA detected the titer of serum-specific antibodies, the secretion of various cytokines was detected by ELISpot and Luminex, and immune protection was observed by the M.tuberculosis growth inhibition test in vitro. The results were statistically analyzed by t-test or rank sum test, and P<0.05 was considered a statistically significant difference. Results:Mice Immunized with EPRHP014m and EPRHP014f could produce highly effective IgG antibodies and their subtypes IgG1 and IgG2a, and the antibody titers were similar to those of mice immunized with BCG, with no statistical significance ( P>0.05). The number of spot-forming cells (SFC) secreting IFN-γ and IL-4 induced by EPRHP014f group was significantly higher than those by EPRHP014m group and BCG group ( P<0.05), but there was no significant difference in the number of SFC for IFN-γ and IL-4 induced between EPRHP014m group and BCG group ( P>0.05). The secretion levels of GM-CSF and IL-12p70 induced by the EPRHP014m group were higher than those of the BCG group ( P<0.05), but there was no significant difference in the levels of IL-6 and IL-10 induced between EPRHP014m group and BCG group ( P>0.05). There was no significant difference in the secretions of IL-6, IL-10, IL-12, and GM-CSF between the EPRHP014f and BCG groups ( P>0.05). EPRHP014m group, EPRHP014f group, and BCG group had obvious antibacterial effects in vitro, and the difference was insignificant ( P>0.05). Conclusion:Both EPRHP014f and EPRHP014m can induce strong humoral and cellular immune responses in mice after immunization, and have a strong ability to inhibit the growth of M. tuberculosis in vitro, indicating that the antigen composition EPRHP014 has good potential in the development and application of TB vaccine.

Objective To investigate the role of TRIM65 on DSS induced colitis and the underlying molecular mechanisms. Methods Trim65+/+ and Trim65-/- mice were administered with 3% (w/v) DSS in their drinking water for 5 consecutive days and then were switched to sterile water for 2 days. DSS treated mice were monitored daily for the clinical symptoms (bodyweight, stool consistency and rectal bleeding score). Mice were sacrificed on day 7 to measure colon length. Colon homogenates were collected to measure MPO activity and detect cleaved caspase-1 and mature IL-1β by Enzyme linked immunosorbent assay (ELISA) and Western blot. Trim65-/- mice were intraperitoneally injected with NLRP3 inflammasome inhibitor MCC950, and were given the above treatment to determine the effect of MCC950 on colitis in Trim65-/- mice. Results The results showed that deletion of Trim65 significantly enhanced weight loss and colon shortening in DSS mice, increased disease activity index and histopathological score, induced the activity of MPO, and promoted the F4/80+ immune cell infiltration, the activation of caspase-1 and the secretion of mature IL-1 in the colon of DSS mice. The NLRP3 inflammasome inhibitor MCC950 alleviated DSS induced colitis symptoms and inflammation levels in trim65 deficient mice. Conclusion TRIM65 plays an anti-inflammatory role in DSS induced colitis mice by inhibiting the activation of NLRP3 inflammasome.

Objective:To evaluate the effects of stellate ganglion block (SGB) on the activation of M1 microglia during cerebral ischemia-reperfusion (I/R) in rats.Methods:Fifty-four SPF healthy male Sprague-Dawley rats, aged 8-10 weeks, weighing 240-270 g, were divided into 3 groups ( n=18 each) using a random number table method: sham operation group (group Sham), cerebral I/R group (group IR) and SGB group.Blood vessels were only exposed, without occlusion in group Sham.Cerebral I/R was induced by occlusion of the middle cerebral artery for 90 min followed by reperfusion in group IR.Cervical sympathetic trunk transaction was performed to induce left SGB immediately after onset of reperfusion in group SGB.Blood samples were collected from the apex of the heart at 6, 12 and 24 h of reperfusion for determination of the concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β in the serum (using enzyme-linked immunosorbent assay). The animals were sacrificed after the neurological function was evaluated at 24 of reperfusion, and brain tissues were removed for microscopic examination of the pathological changes in cortex, for determination of percentage of cerebral infarct size (by TTC staining), for assessment of cell apoptosis and apoptosis rate in cortex (by TUNEL), and for determination of the expression of microglial biomarker Iba-1 and activated M1 microglia biomarker CD68 (by Western blot). Results:Compared with group Sham, the neurological function score, percentage cerebral infarct size, apoptosis rate in cortex, concentrations of TNF-α, IL-6 and IL-1β in the serum, and the expression of Iba-1 and CD68 were significantly increased in IR and SGB groups ( P<0.05). Compared with group IR, the neurological function score, percentage cerebral infarct size, apoptosis rate in cortex, concentrations of TNF-α, IL-6 and IL-1β in the serum, and the expression of Iba-1 and CD68 were significantly decreased in group SGB ( P<0.05), and the pathological changes of brain tissues were significantly attenuated in group SGB. Conclusion:The mechanism by which SGB reduces cerebral I/R injury is related to inhibiting activation of M1 microglia in rats.