Objective The aim of this study was to clarify the role and mechanism of Pseudomonas aeruginosa vaccine subunit OprI in the fusion protein vaccine rePO(PcrV-OprI).Methods The in vitro stability of rePO,PcrV and OprI at 4 ℃,25 ℃,and 37 ℃ was examined.After immunizing mice with rePO,OprI and PcrV,respectively,the specific antibody potency in serum and the proportion of cells secreting IFN-γ and IL-4 in the spleen were examined;Additionally,detection of the levels of protein uptake by DC2.4 cells in vitro using laser confocal microscopy and flow cytometry,and their ability to promote the maturation of mouse bone marrow-derived dendritic cells(BMDC).Results The heat stability of fusion protein rePO was significantly better than that of PcrV.The induced anti-PcrV IgG and anti-OprI IgG potency of rePO was significantly higher than that of monomeric PcrV and OprI.Additionally,the number of cells secreting IFN-γ and IL-4 induced by immunization with rePO was significantly higher than that of PcrV and OprI.The uptake rate of fusion protein rePO by DC2.4 cells was significantly higher than that of PcrV and OprI.Furthermore,rePO promoted the maturation of mouse BMDC more effectively than PcrV and OprI.Conclusion OprI in the fusion protein rePO can significantly improve its thermal stability and immunogenicity,which lays the foundation for the successful development of Pseudomonas aeruginosa vaccine.

Objective To establish a mouse model of bronchiectasis with acute infection and evaluate the immunogenicity and protective effect of a self-assembling Pseudomonas aeruginosa(PA)nanoparticle vaccine rePO-FN based on fusion of PcrV-OprI(rePO)protein with self-assembling ferritin(Ferritin).Methods ① SPF-grade female C57BL/6 mice(aged 6～8 weeks,weighing 18～20 g)were randomly allocated into normal saline group,and low-,medium-and high-dose elastase groups(n=6).A mouse model of bronchiectasis was established via intratracheal instillation of different doses of elastase(30 μL of normal saline containing 0.65,1.30 and 2.60 IU elastase)for 3 consecutive days.At 14 and 21 d after modeling,ELISA and HE staining were performed respectively to detect the concentration of IL-6 and to observe pathological changes in lung tissue in order to confirm the modeling.② A recombinant plasmid encoding the gene of fusion protein rePO-FN was constructed and expressed in E.coli.The target protein was purified via affinity chromatography and renatured to obtain the desired protein.The physicochemical properties of the rePO-FN protein were characterized using SDS-PAGE protein gel electrophoresis,dynamic light scattering,molecular sieve chromatography,and transmission electron microscopy.③ C57BL/6J mice were randomly divided into PBS group,rePO group,rePO-FN group,and Ferritin group(n=10).The mice in the above groups were immunized intramuscularly with 100 μL PBS buffer alone or containing 10 μg of corresponding proteins on days 0,7,and 14.ELISA was used to measure the specific antibodies in serum.In 7 d after the final immunization,an acute PA infection model was used to compare the survival rates and bacterial colonization among the PBS,rePO,and rePO-FN groups.After establishing a bronchiectasis model by intratracheal instillation of 2.60 IU of elastase in C57BL/6J mice as described above,the mice were randomly divided into bronchiectasis PBS group,bronchiectasis rePO group,and bronchiectasis rePO-FN group(n=10).Immunization was conducted at the same dose and procedure as described above,in 21 d after bronchiectasis modeling.At the 7th d after the final immunization,an acute PA infection model was used to compare the survival rates and bacterial colonization among the groups.Results ①Repeated intratracheal instillation of elastase significantly increased the concentration of IL-6 in the lung tissue when compared to the content of the normal saline group(P＜0.05).Pathological observations revealed varying degrees of bronchial wall destruction,alveolar fusion,edema,neutrophil infiltration,and hemorrhage,with the severity increasing with elastase dose,which confirming successful establishment of the mouse model of bronchiectasis.② Well-dispersed rePO-FN nanoparticles were successfully prepared,with an average particle size of 91.28 nm,a Zeta potential of approximately-6.5 mV,and a polydispersity index(PDI)of 0.306.Molecular sieve chromatography determined the elution volume of rePO-FN protein to be 8.80 mL,corresponding to a molecular weight of approximately 1 400 kDa.③ Under acute PA XN-1 strain infection,the survival rate of the rePO-FN immunization group and the bronchiectasis rePO-FN immunization group were significantly higher than that of the PBS control group(P＜0.05).Additionally,bacterial colonization in the lung tissues was significantly lower in the rePO-FN immune group and the bronchiectasis rePO-FN immune group under acute PA XN-1 strain infection than that in the rePO group and the bronchiectasis rePO group(P＜0.05).Conclusion Our vaccine rePO-FN can effectively trigger a strong humoral immune response and provide significant protection against PA infection in a mouse bronchiectasis model.

Objective The aim of this study was to clarify the role and mechanism of Pseudomonas aeruginosa vaccine subunit OprI in the fusion protein vaccine rePO(PcrV-OprI).Methods The in vitro stability of rePO,PcrV and OprI at 4 ℃,25 ℃,and 37 ℃ was examined.After immunizing mice with rePO,OprI and PcrV,respectively,the specific antibody potency in serum and the proportion of cells secreting IFN-γ and IL-4 in the spleen were examined;Additionally,detection of the levels of protein uptake by DC2.4 cells in vitro using laser confocal microscopy and flow cytometry,and their ability to promote the maturation of mouse bone marrow-derived dendritic cells(BMDC).Results The heat stability of fusion protein rePO was significantly better than that of PcrV.The induced anti-PcrV IgG and anti-OprI IgG potency of rePO was significantly higher than that of monomeric PcrV and OprI.Additionally,the number of cells secreting IFN-γ and IL-4 induced by immunization with rePO was significantly higher than that of PcrV and OprI.The uptake rate of fusion protein rePO by DC2.4 cells was significantly higher than that of PcrV and OprI.Furthermore,rePO promoted the maturation of mouse BMDC more effectively than PcrV and OprI.Conclusion OprI in the fusion protein rePO can significantly improve its thermal stability and immunogenicity,which lays the foundation for the successful development of Pseudomonas aeruginosa vaccine.

Objective:To analyze the clinical outcomes of the first frozen-thawed embryo transfer (FET) in patients <35 years old applying natural cycle (NC) and hormone replacement therapy (HRT).Methods:A retrospective cohort study was conducted to analyze 4 814 young infertility patients who underwent the first FET in Reproductive Medicine Center of the First Affiliated Hospital of Zhengzhou University from January 2016 to June 2021. According to different endometrial preparation protocols, they were divided into 2 groups: NC group and HRT group, who were matched the baseline data using 1∶1 propensity score matching (PSM). After the matching, the two groups of their baseline data, pregnancy outcomes and perinatal outcomes were compared, and then we adjusted the confounding factors which affect live birth rate by univariate and multivariate logistic regression analysis. Based on antral follicle count (AFC), number of embryos transferred and number of high-quality embryos transferred, the effect of NC and HRT on the live birth rate were further analyzed.Results:Before PSM, 2 131 patients in NC group and 2 683 patients in HRT group were included. The differences in female age, male age, body mass index (BMI), basal follicle-stimulating hormone (bFSH), anti-Müllerian hormone (AMH), AFC, endometrial thickness on conversion day, and number of embryos transferred were all statistically significant between the two groups (all P<0.05). And the differences in number of high-quality embryos transferred and type of embryos transferred between the two groups were not statistically significant (all P>0.05). After PSM, 1 441 patients in each of NC group and HRT group were included, and there were no significant differences in their baseline characteristics such as female age, male age and BMI between the two groups (all P<0.05). The live birth rate [50.66% (730/1 441)] and the clinical pregnancy rate [60.31% (869/1 441)] in NC group were significantly higher than those in HRT group [44.69% (644/1 441), P=0.001; 54.27% (782/1 441), P=0.001], and the incidence of very low birth weight in NC group was significantly lower than that in HRT group, and there were no statistical significances in other indicators between the two groups (all P>0.05). After adjusting confounders including bFSH, AMH, AFC, endometrial thickness on conversion day, number of embryos transferred and high-quality embryos transferred using multivariate logistic regression analysis, the results showed that NC was an independent protective factor for live birth rate in the first FET cycle ( aOR=1.280, 95% CI: 1.103-1.486, P=0.001). Stratified analysis showed that those with AFC<11, AFC 11-12, 2 embryos transferred and 2 high-quality embryos tranferred in NC group had significantly higher live birth rate [49.03% (151/308), 49.09% (349/711), 56.38% (442/784), 57.85% (350/605)] than those in HRT group [36.36% (120/330), P=0.001; 43.14% (286/663), P=0.027; 48.97% (379/774), P=0.003; 48.68% (294/604), P=0.001]. Conclusion:NC-FET had higher live birth rate and clinical pregnancy rate than HRT-FET in young patients.

Objective:To analyze the clinical outcomes of the first frozen-thawed embryo transfer (FET) in patients <35 years old applying natural cycle (NC) and hormone replacement therapy (HRT).Methods:A retrospective cohort study was conducted to analyze 4 814 young infertility patients who underwent the first FET in Reproductive Medicine Center of the First Affiliated Hospital of Zhengzhou University from January 2016 to June 2021. According to different endometrial preparation protocols, they were divided into 2 groups: NC group and HRT group, who were matched the baseline data using 1∶1 propensity score matching (PSM). After the matching, the two groups of their baseline data, pregnancy outcomes and perinatal outcomes were compared, and then we adjusted the confounding factors which affect live birth rate by univariate and multivariate logistic regression analysis. Based on antral follicle count (AFC), number of embryos transferred and number of high-quality embryos transferred, the effect of NC and HRT on the live birth rate were further analyzed.Results:Before PSM, 2 131 patients in NC group and 2 683 patients in HRT group were included. The differences in female age, male age, body mass index (BMI), basal follicle-stimulating hormone (bFSH), anti-Müllerian hormone (AMH), AFC, endometrial thickness on conversion day, and number of embryos transferred were all statistically significant between the two groups (all P<0.05). And the differences in number of high-quality embryos transferred and type of embryos transferred between the two groups were not statistically significant (all P>0.05). After PSM, 1 441 patients in each of NC group and HRT group were included, and there were no significant differences in their baseline characteristics such as female age, male age and BMI between the two groups (all P<0.05). The live birth rate [50.66% (730/1 441)] and the clinical pregnancy rate [60.31% (869/1 441)] in NC group were significantly higher than those in HRT group [44.69% (644/1 441), P=0.001; 54.27% (782/1 441), P=0.001], and the incidence of very low birth weight in NC group was significantly lower than that in HRT group, and there were no statistical significances in other indicators between the two groups (all P>0.05). After adjusting confounders including bFSH, AMH, AFC, endometrial thickness on conversion day, number of embryos transferred and high-quality embryos transferred using multivariate logistic regression analysis, the results showed that NC was an independent protective factor for live birth rate in the first FET cycle ( aOR=1.280, 95% CI: 1.103-1.486, P=0.001). Stratified analysis showed that those with AFC<11, AFC 11-12, 2 embryos transferred and 2 high-quality embryos tranferred in NC group had significantly higher live birth rate [49.03% (151/308), 49.09% (349/711), 56.38% (442/784), 57.85% (350/605)] than those in HRT group [36.36% (120/330), P=0.001; 43.14% (286/663), P=0.027; 48.97% (379/774), P=0.003; 48.68% (294/604), P=0.001]. Conclusion:NC-FET had higher live birth rate and clinical pregnancy rate than HRT-FET in young patients.

Purpose To use two-dimensional speckle tracking imaging(2D-STI)to evaluate left ventricular myocardial strain in term and preterm neonates.Materials and Methods A total of 81 neonates who accepted echocardiography in Soochow University Affiliated Children's Hospital from February to August 2018 were retrospectively enrolled,including 42 term neonates and 39 preterm neonates.Longitudinal and circumferential peak strain of each segment of left ventricle were measured by 2D-STI,and the average global longitudinal peak strain(GLPS-avg)and the average global circumferential peak strain(GCPS-avg)of left ventricular were measured too.Left ventricular ejection fraction(LVEF-M,LVEF-Simpson),short-axis shortening rate(LVFS-M),left ventricular end-diastolic diameter(LVEDD),and tissue Doppler imaging parameters(E',A' and E'/A')of the septal mitral annulus were also measured.The differences in each segment of left ventricle and echocardiography between term neonates and preterm neonate were further compared.Results There were statistically significant differences in GLPS-avg,the global longitudinal peak strain from the apical two-chamber view(GLPS-a2c),GCPS-avg and the global circumferential peak strain in the middle and apical segments(GCPS-M ang GCPS-A)(t=-3.60--2.18,all P<0.05)between term neonates and preterm neonates,while there were no significant differences between the global longitudinal peak strain from the apical four-chamber and three-chamber view(GLPS-a4c and GLPS-a3c),the global circumferential peak strain in the basal segments(GCPS-M)(all P>0.05).There were statistically significant differences in GLPS-a4c,GLPS-a2c,GLPS-a3c in preterm neonates group(F=3.20,P=0.044),and the strain value GLPS-a4c was the highest.There were statistically significant differences in GCPS-B,GCPS-M,GCPS-A in preterm neonates group(F=79.15,P<0.001).There were statistically significant differences in GCPS-B,GCPS-M,GCPS-A in term neonates group(F=97.95,P<0.001),and the strain value increased from the basal segment to the apical segments.There was statistically significant difference in LVEDD between term neonates and preterm neonates(all P<0.05).There were no statistically significant differences in LVEF-M,LVEF-Simpson,LVFS-M,E',A' and E'/A' between term neonates and preterm neonates(all P>0.05).Conclusion 2D-STI may be a useful method of measuring left ventricular myocardial systolic function and can provide early detection of differences in left ventricular myocardial contraction between term and preterm neonates.

Objective To investigate the diagnostic value of CT thin-section target reconstruction technique in patients with pulmonary small size groud glass nodules (sGGN).Methods A total of 109 patients with pulmonary sGGN certified post-operation and/or follow up were analyzed retrospectively,and the clinical and CT images of all patients were completely collected.There were 23 cases of benign group,21 cases of adenocarcinoma in situ group,29 cases of minimally invasive adenocarcinoma group and 36 cases of invasive adenocarcinoma group,respectively.The differential diagnostic value using CT thin-section target reconstruction technique was analyzed between the benign and malignant pulmonary sGGN groups,and the CT feature detection rates were compared between CT thin section target and common reconstruction techniques.Results The CT thin-section target reconstruction technique had improved the diagnostic efficiency of pulmonary sGGN.The diagnostic accuracy,area under curve(AUC),sensitivity and specificity were 85.32 ％,0.679,90.80 ％,63.64 ％ in malignant group and 77.06％,0.764,83.72％,52.17％ in benign pulmonary sGGN group,respectively.The CT features as solid component in whole nodule,"halo sign",speculation sign,pleural retraction sign,lobulation sign were more detected by using the thin-section target reconstruction technique than that by using the common reconstruction among adenocarcinoma in situ group,minimally invasive adenocarcinoma group and invasive adenocarcinoma group (P＜0.05) but not speculation sign in the invasive adenocarcinoma group(P=0.126).Conclusion The CT thin section target reconstruction technique can improve the diagnostic value of pulmonary sGGN.

OBJECTIVETo explore the effects of acupuncture on the expression of protein kinase B (PKB/AKT) in lung tissues of asthma rats.

METHODSForty SPF male SD rats were randomly divided into a blank group, a model group, an acupuncture group and a blocker group, 10 rats in each one. The rat model of asthma was established by egg albumin stimulation in the model group, acupuncture group and blocker group. Since the establishment of rat model, the rats in the acupuncture group were treated with acupuncture at "Dazhui" (GV 14), "Feishu" (BL 13) and "Fengmen" (BL 12) before atomization; the rats in the blocker group were treated with intervention of blocker LY294002, once every two days, for 7 times. There was no treatment in the blank group and model group. HE staining was applied to observe the morphologic changes of lung tissues; the immunohistochemical method was applied to test the protein expression of AKT in lung tissue.

RESULTSHE staining indicated the infiltration and aggregation of a variety of inflammatory cells around airways, as well as bronchial smooth muscle spasm and confined lumen in the model group; in the acupuncture group and blocker group the inflammatory cells were less and confined lumen was relieved. Compared with the blank group, the protein expression of AKT was higher in the model group (<0.05); compared with the model group, the protein expression of AKT in the acupuncture group and blocker group was reduced (both<0.05); the differences between the acupuncture group and blank group, blocker group were not significant (both>0.05).

CONCLUSIONSAcupuncture could reduce the protein expression of AKT in lung tissue in asthma rats, leading to relieved inflammation reaction and airway remodeling.

OBJECTIVETo explore the regulation of acupuncture for transforming growth factor β1 (TGF-β1) expression in the lung tissues of bronchial asthmatic rats.

METHODSForty SPF male SD rats were randomly divided into a blank group, a model group, an acupuncture group and a shallow fast acupuncture group, 10 rats in each group. Asthma model was established in the model, acupuncture, and shallow fast acupuncture groups, and no intervention was applied in the blank and model groups. Before atomization excitation, 20 min acupuncture was applied at "Dazhui" (GV 14) "Feishu" (BL 13) "Fengmen" (BL 12) in the acupuncture group. Acupuncture only into skin without needle retained at the above acupoints was used in the shallow fast acupuncture group before atomization excitation from the day of model established. All the treatment was given 7 times, once the other day. The pathological lunge tissues of rats were observed by HE staining; the protein expression of TGF-β1 in the lung tissues was examined by immunohistochemical staining and immunofluorescence staining. The TGF-β1 expressions in bronchoalveolar lavage fluid (BALF) and serum were showed by ELISA.

RESULTSThere were changes in the lung tissues in the model group, including mucus plug in the bronchial lumen, irregular bronchial epithelium, thick airway smooth muscle, local break, narrow lumen, a large number of inflammatory cell infiltration. The phenomena in the acupuncture group were improved and those in the shallow fast acupuncture group were not apparently changed compared with those in the model group. The IOD value of TGF-β1 expression in the model group increased compared with that in the blank group (<0.05); the positive expression in the acupuncture group was lower than those in the model and shallow fast acupuncture groups (both<0.05). The contents of TGF-β1 in BALF and serum of the acupuncture group were lower than those in the model and shallow fast acupuncture groups (all<0.05).

CONCLUSIONSAcupuncture may improve airway inflammatory response and remolding of asthma by inhibiting TGF-β1 expression.

Objective To observe the antidepressant effect of the volatile oil of Myristica in mice and investigate its mechanism.Methods Healthy male Kunming mice were divided into control group,fluoxetine hydrochloride group,high,medium and low dose group,and all of the mice were gavaged for 7 days.The role of antidepressant on the mice were observed with tail suspension tests and forced swimming tests.Then serotonin(5-HT),dopamine(DA) and norepinephrine (NE) in the brain of the mice were determined by enzyme-linked immunosorbent assay.Results Compared with the control group((82.60 ± 24.70)s),the medium dose group could shorten the immobility time of the tail suspension tests ((54.40± 15.87) s),and showed statistical significance (P＜ 0.05).The content of 5-HT,DA,NE in the brain of the medium group were (19.35±2.79) ng/ml,(12.16±0.71)pg/ml,(0.27±0.12) ng/ml,and control group were (14.95±4.83) ng/ml,(11.32±0.95) pg/ml,(0.20±0.11) ng/ml.Compared with the control group,the content of 5-HT of the medium dose group was increased and showed statistical significance(P＜0.05).Conclusion The volatile oil of Myristica fragrans Houtt has antidepressant effect,and may be related to raising the content of 5-HT,DA,NE in the brain of the mice,especially 5-HT.