Objective The study aims to investigate the immunological functions of the nucleocapsid (N) protein of the novel coronavirus Omicron (BA.1, BA.2) and evaluate the differences among different N proteins of mutant strains in immunogenicity. Methods By aligning sequences, the mutation sites of the Omicron (BA.1, BA.2) N protein relative to prototype strain of the novel coronavirus (Wuhan-Hu-1) were determined. The pET-28a-N-Wuhan-Hu-1 plasmid was used as template to construct pET-28a-BA.1/BA.2-N through single point mutation or homologous recombination. The three kinds of N protein were expressed in prokaryotic system, purified through Ni-NTA affinity chromatography, and then immunized into mice. The titer and reactivity of the polyclonal antibody, as well as the expression level of IL-1β and IFN-γ in mouse spleen cells, were detected using indirect ELISA and Western blot assay. Results The constructed prokaryotic expression plasmids were successfully used to express the Wuhan-Hu-1 N, BA.1 N, and BA.2 N proteins in E.coli BL21(DE3) at 37 DegreesCelsius for 4 hours. The indirect ELISA test showed that the titers of polyclonal antibody prepared by three N proteins were all 1:51 200. All three N proteins can increase the expression of IFN-γ and IL-1β cytokines, but the effect of Omicron N protein in activing two cytokines was more obvious than that of Wuhan-Hu-1 N protein. Conclusion The study obtained three new coronavirus N proteins and polyclonal antibodies, and confirmed that mutations in the amino acid sites of the N protein can affect its immunogenicity. This provides a basis for developing rapid diagnostic methods targeting N protein of different novel coronavirus variants.
Animals ; Mice ; SARS-CoV-2/genetics* ; Coronavirus Nucleocapsid Proteins/immunology* ; Nucleocapsid Proteins/isolation & purification* ; COVID-19/immunology* ; Antibodies, Viral/immunology* ; Mice, Inbred BALB C ; Interferon-gamma/metabolism* ; Interleukin-1beta/metabolism* ; Female ; Escherichia coli/metabolism* ; Mutation ; Humans

Objective To explore the correlation between the expression level of serum Receptor for Advanced Glycation End-Product(RAGE)and High-Mobility Group Protein B1(HMGB1)expression with the occurrence of acute respiratory distress syndrome(ARDS)and interferon-γ/interleukin-4(IFN-γ/IL-4)ratio in patients with severe pneumonia(SP).Methods A prospective investigation was carried out on one hundred children with SP admitted to our hospital from March 2020 to February 2022,and the participants were classified into ARDS group(n = 56)and control group(n = 44)based on the occurrence of secondary ARDS.General informations werec-ollected.The expression of RAGE,HMGB1,IFN-γ and IL-4 in peripheral blood was measured using Enzyme-Linked Immunosorbent Assay(ELISA).Then multivariate Logistic regression analysis was conducted to screen the influencing factors of secondary ARDS in SP children,and the correlation with IFN-γ/IL-4 ratio was verified by pearson correla-tion analysis,moreover,receiver operating characteristic(ROC)curve was plotted to evaluate the value of RAGE and HMGB1 expression in predicting the occurrence of ARDS in SP children.Results There were no statistical difference in gender,age,body temperature and onset season between the two SP groups.The ARDS group had more types of pathogenic bacteria,larger ratio of the partial pressure of oxygen in arterial blood to the inspired oxygen fraction(PaO2/FiO2),higher Acute Physiological Score(APS),and up-regulated expression of RAGE,HMGB1,IFN-γ and IL-4,as well as larger IFN-γ/IL-4 ratio than those of control group,with statistical difference(all P<0.05).Multivariate Logistic regression analysis revealed that pathogen type,PaO2/FiO2 ratio,RAGE,HMGB1,IFN-γ,IL-4 and IFN-γ/IL-4 were the influencing factors for the occurrence of ARDS in children with SP.Pearson correlation test denoted that the serum RAGE and HMGB1 expression levels of SP children were positively correlated with IFN-γ,IL-4 and IFN-γ/IL-4 ratio(P<0.05).ROC curve found that the AUC of serum RAGE and HMGB1 in predicting the occurrence of ARDS in SP children was 0.707 and 0.750,with a sensitivity of 73.2%and 64.3%,and a specificity of 68.2%and 77.3%.The combined test of RAGE and HMGB1 in predicting the occurrence of ARDS in SP children reached an AUC of 0.848,providing a sensitivity and specificity of 80.4%and 81.8%respectively.Conclusions Serum RAGE and HMGB1 expression levels are elevated in SP children with ARDS,and the two are positively correlated with IFN-γ/IL-4 ratio.Therefore,monitoring serum RAGE and HMGB1 expression in children with ARDS secondary to SP has predictive value for the risk of ARDS in SP children.

Objective To investigate the correlation between the expression of circulating exosome miR-485-3p and STYX with the risk of premature coronary heart disease.Methods A total of 50 inpatients with early onset coronary heart disease diagnosed by coronary angiography or CT angiography (CTA) in Af-filiated Puyang Municipal People's Hospital of Xinxiang Medical College from August to December 2023 were selected as the study group and 50 patients with excluded coronary artery disease by examination during the same period were included in the control group.The general clinical data of the two groups were collected,the plasma exosome miR-485-3p and STYX levels were detected.The degree of coronary arterial lesions in the pa-tients of the study group was evaluated by the Gensini score.The Spearman correlation analysis was used to analyze the relationship between plasma exosome miR-485-3p and STYX with LDL and Gensini score.The re-ceiver operating characteristic (ROC) curve was used to analyze the diagnostic value of plasma exosome miR-485-3p and STYX in the diagnosis of premature coronary heart disease.The multivariate logistic regression was used to determine the independent risk factors for premature coronary heart disease.Results Compared with the control group,the family history of coronary heart disease,smoking history,LDL and plasma exo-some miR-485-3p level in the study group were increased,the plasma STYX level was decreased and the differences were statistically significant (P<0.05);the Spearman correlation analysis showed that miR-485-3p was positively correlated with LDL (r=0.546) and Gensini score (r=0.485),and negatively correlated with STYX (r=-0.576).STYX was negatively correlated with LDL (r=-0.389) and Gensini score (r=-0.531).The ROC curve showed that the area under the curve of miR-485-3p,STYX and their combination in the diagnosis of premature coronary heart disease was 0.821 (95％CI:0.736-0.906),0.850 (95％CI:0.772-0.927) and 0.899 (95％CI:0.837-0.960) respectively.Conclusion The expression of circulating exosome miR-485-3p in premature coronary heart disease is up-regulated and the expression of STYX is down-regulated,the both are closely related to the degree of coronary artery lesion,which could be used as the po-tential biomarkers for the diagnosis of premature coronary heart disease.

Objective:To investigate the expression level and regulatory mechanism of 15-hydroxyprostaglandin dehydrogenase（HPGD） in chronic rhinosinusitis with nasal polyps（CRSwNP）. Methods:The expression pattern and level of HPGD in CRSwNP and control was observed using immunofluorescence, and western blot was used for analysis of HPGD expression in nasal polyp tissues. The effect of recombinant human high mobility group box-1（HMGB1） on HPGD expression in primary human nasal epithelial cells was observed, and the potential blocking effect of RAGE neutralizing antibody on HMGB1-induced HPGD expression was investigated. Results:The expression of HPGD was elevated in CRSwNP patients compared to the control, while the protein mainly localized at CD68-positive cells and epithelial cells. Recombinant human HMGB1 stimulated an increase in HPGD expression in primary human nasal mucosal epithelial cells at a time-dependent manner. Additionally, increased phosphorylation levels of MEK and elevated RAGE expression were also observed at 12 hours, but decreased at 24 hours after the incubation of HMGB1. The increase in the expression of HPGD induced by HMGB1 in primary human nasal epithelial cells was partly inhibited with RAGE neutralizing antibody. Conclusion:Elevated HPGD expression is observed in CRSwNP, predominantly in macrophages and epithelial cells. HMGB1 regulates HPGD expression through the RAGE-MEK signaling pathway, potentially providing a new target for future regulation of PGE2levels in CRSwNP.
Humans ; Antibodies, Neutralizing/metabolism* ; Chronic Disease ; HMGB1 Protein/metabolism* ; Mitogen-Activated Protein Kinase Kinases/metabolism* ; Nasal Mucosa/metabolism* ; Nasal Polyps/metabolism* ; Rhinitis

Objective To investigate the efficacy of real-time visual feedback on improving the quality of manual chest compression in ambulance.Methods Ten pre-hospital doctors with cardiopulmonary resuscitation experience,aged under 40 years,were recruited to this randomized,crossover,manikin research and randomly assigned into control group (n=5) and feedback group (n=5) by the sealed envelope method.The setting place was a moving ambulance with the velocity of 25～50 km/ h.The whole process consisted of two sessions.In control group,which received feedback in the second session,chest compressions were performed without interruption during each of the three 2 min phases per session,resting for 2 min between phases and for 5 min between sessions.In feedback group,which received feedback in the first session,chest compressions were performed without interruption during each of the three 2 min phases per session,resting for 2 min between phases and for 5 min between sessions.Data of compression rate,compression depth,compression detention and compression accuracy rate were collected.Results In control group,the compressions rate was lower and compression detention was shorter during the second session compared with those during the first session [(109.8±±4.7) r/min vs.(121.2± 10.1) r/min,(6.5±2.1) r/min vs.(10.4±2.8) r/min,all P＜0.05],while the compression accuracy rate during the second session was higher than that during the first session [(28.2±±14.3) ％ vs.(16.8±9.9) ％,P＜0.05].There was no significant difference in compression rate between the two sessions in control group;Compression frequency,compression depth,compression detention and compression accuracy rate did not significantly change between the two sessions in feedback group (all P＞0.05).In the whole process,the compression rate was lower and compression detention was shorter in the feedback group compared with the control group [(111.1±5.1) r/min vs.(115.5±9.7) r/min,(6.5±1.8) vs.(8.4±4.6) r/min,all P＜0.05],and the compression accuracy rate in the feedback group was higher than that in the control group[(22.5±13.4) ％ vs.(26.7±16) ％,P＜0.05].There was no significant difference in compression rate between the two groups during whole process (P＞0.05).Conclusions Although real-time visual feed back improved the quality of manual chest compression in ambulances,which demonstrated more reasonable compression rate,less compression detention and higher compression accuracy,the overall quality of reuscitation was still not enough to achieve effective treatment.This implies that more optimal methods are required to transfer the patients suffering cardiac arrest.

Objective:To investigate the effects of Liuweidihuang pill on the insulin levels in sera and pancreatic islets from spontaneous mouse models of human type 2 diabetes administrated with different doses .Methods:The 6-8 week-old KK-Ay mice were randomly divided into three groups including no drug control group ,low-dose group and high-dose group,in addition C57BL/6J mice were used as a genetic control group .All the animals were given with different dose Liuweidihuang pill solutions or sterile distilled water by intragastrical administration for fifteen weeks .The fasting blood glucose ,body mass and food consumption were measured weekly .The serum insulin levels were surveyed by ELISA .And the insulin levels in the pancreas islets were detected by immunofluorescence and immunohistochemistry .Results:Decreased fasting blood glucose ,controlled body mass and food consumption ,and lower levels of insulin in the sera and pancreas islets were confirmed from the KK-Ay mice administered with Liuweidihuang pill .Furthermore,the low dose program exhibits a stronger effect .Conclusion:Liuweidihuang pill has exhibited relatively therapeutic effects in the spontaneous type 2 diabetes mice including controls of hyperglycemia and body mass and relieving insulin resistance .In addition , the low-dose regimen showed even better treatment in controlling insulin levels in the sera and pancreas islets .

Using forward and reverse genetics and global gene expression analyses, we explored the crosstalk between the IκB kinase β (IKKβ) and the transforming growth factor β (TGFβ) signaling pathways. We show that in vitro ablation of Ikkβ in fibroblasts led to progressive ROS accumulation and TGFβ activation, and ultimately accelerated cell migration, fibroblast-myofibroblast transformation and senescence. Mechanistically, the basal IKKβ activity was required for anti-oxidant gene expression and redox homeostasis. Lacking this activity, IKKβ-null cells showed ROS accumulation and activation of stress-sensitive transcription factor AP-1/c-Jun. AP-1/c-Jun activation led to up-regulation of the Tgfβ2 promoter, which in turn further potentiated intracellular ROS through the induction of NADPH oxidase (NOX). These data suggest that by blocking the autocrine amplification of a ROS-TGFβ loop IKKβ plays a crucial role in the prevention of fibroblast-myofibroblast transformation and senescence.
Adenoviridae ; genetics ; Animals ; Autocrine Communication ; physiology ; Cell Line ; Cell Movement ; Cellular Senescence ; Genetic Vectors ; genetics ; metabolism ; I-kappa B Kinase ; deficiency ; genetics ; metabolism ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Mice ; Myofibroblasts ; cytology ; metabolism ; NADPH Oxidases ; metabolism ; Oxidative Stress ; Promoter Regions, Genetic ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; Superoxide Dismutase ; genetics ; metabolism ; Transcription Factor AP-1 ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Up-Regulation

Objective To prepare a recombinant pneumococcal surface protein A clade 4 ( PspA4) and to analyze its immunogenicity.Methods The gene encoding PspA4 protein was synthesized and inserted into pET-20b to construct the recombinant expression plasmid.The transformed E.coli strains carrying expression plasmid were induced to express PspA4 protein.ELISA was performed to analyze the ti-ters of PspA4-specific IgG in a mouse model.Results The recombinant PspA4 protein of high purity ( 90%) was successfully prepared.The titers of PspA4-specific antibody in mice received PspA4 immuniza-tion were 106 times higher than those of the blank control group, suggesting that the expressed PspA4 protein had the advantage of high immunogenicity.Conclusion This study suggested that the PspA4 protein might be used as one of the candidate protein for the development of pneumovax and laid a foundation for further in-vestigation on pneumococcal protein based vaccine.

Objective To study the influence of donor GFR on the early renal function in recipients undergoing living donor transplantation.Methods A total of 172 living donor transplant recipients in our kidney transplantation center from 2006 to 2011 were enrolled into this study.Among them,166 were genetically related (96.5％),while 6 were genetically unrelated (spouses in 5 and other in 1).The predonation GFR was measured by isotope clearance (99mTC-DTPA with few exceptions).The range of donor GFR was 62 to 148 ml/min.The recipients were classified into two groups according to donor graft GFR level (GFR≤45 ml/min,n=76; GFR＞45 ml/min,n =96).The predonation dialysis,cold and warm ischemia time,antibody induction,immunosuppressive regimens and HLA mismatch were not significantly different between two groups.Results There were no significant differences in the incidence of postoperative acute rejection and delay graft function (DGF).The postoperative Scr of GFR＞45 ml/min group in 1 week,1 month,3 months and 1 year was lower compared with the GFR ≤45 ml/min group,and only the difference of Scr in 1 week was significantly different (P＜0.05).A repeated-measure ANOVA revealed no significant differences were found in Scr variation of two groups during the first year after transplantation.Conclusions Predonation GFR of the donor has effect on the Scr of postoperative Ⅰ week of recipients,not on the Scr within a year.Recipients with graft GFR＞45 ml/min have lower Scr levels.

Objective To investigate the impact on the endothelial cell function and renal blood flow through effective treatment for elderly patients with hypertension. Methods 64 cases of elderly hypertensive patients over 70years old with effective treatment, according to pre-treatment blood pressure, were divided into two groups: hyptension level-2 group(30cases) and hypertension level-3 group(34cases), and 30 cases of elderly people with health physical examination during the same period were setlected as the control group. Endothelial cell function was detected, including the endothelin-1 (ET-1)、 nitrous oxide (NO), thromboxane B2 (TXB2)、 6-keto-prostaglandinF1α (6-K-PDGF1α) in plasma. Renal blood flow was explored by color doppler ultrasonic instrument ,involving peak velocity in systole(PSV) and lowest velocity in end-diastole(EDV) of renal arteries, segmental arteries and interlobar arteries.After indicators had reached the standard 1 month in the hypertension level-2 and hypertension leve1-3 patients with effective antihypertensive therapy, the difference of ET-1, NO, TXB2,6-k-PGF1o and PSV, EDV among the three groups were compared. Results The differences were statistically significant in ET-1, NO, TXB2,6-k-PGF1α, PSV and EDV before treatment among three groups (P ＜ 0.05). After blood pressure treated had reached the standard 1 month,indicators were no statistically meaningful difference among the three groups (P ＞ 0.05). Conclusion After effective clinical treatments, the endothelial cell function and renal blood flow of the elderly hypertensive patients can be improved. At the same time, to delay renal damage in elderly patients with hypertension provide a theoretical basis.