PAC1 is the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) preferring receptor, which belongs to class B G protein-coupled receptors (GPCR) family. PAC1 mediates the most effects of PACAP as neurotransmitter, neuroregulator and neuroprotectant, while its high expression has close relationship with some physiological and pathological processes such as nerve-injury and tumor. To further understand the function of PAC1, a cell line that expressed inducible PAC1 was constructed to achieve Doxycycline (Dox) dependent expression of PAC1 in CHO (Chinese hamster ovary) cell using the improved Tet (tetracycline)-on Advanced System. First, the PAC1-EYFP fusion gene composed of PAC1 gene and gene encoding EYFP (enhanced yellow fluorescent protein) was sub-cloned to the tetracycline response element pTRE-Tight vector to construct the recombinant vector pEYFP-PAC1-EYFP by double enzyme digestion. Second, the tetracycline regulation components pTet-On advanced vector and the response element pTRE-PAC1-EYFP vector were both introduced into CHO cells successively and the positive clones were screened with G418 and hygromycin respectively. Third, the controlled expression of PAC1-EYFP in CHO was induced by tetracycline analogues Dox in different concentrations and the different levels of receptor PAC1-EYFP were detected. The results of fluorescence analysis and western blotting show that the cell strain with Dox dependent expression of PAC1-EYFP named PAC1-Tet-CHO was obtained. Moreover, in PAC1-Tet-CHO cells the expression of PAC1-EYFP was induced by Dox in a dose-dependent manner. The inducible expression of PAC1 still was stable after sub-culturing for more than 10 passages. It was also found by MTT assay that the higher expression level of PAC1 endowed the cells with higher proliferative viabilities. The construction of controlled expression system of PAC1 will lay a foundation for the further research on PAC1 profiles.
Animals ; Blotting, Western ; CHO Cells ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Genetic Vectors ; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I ; biosynthesis

Objective To compare the effects of telmisartan and (or) amlodipine on the reversal left ventricular re-modeling in two-kidney one clip hypertensive rats. Methods A total of 50 healthy male SD rats were randomly divided into 5 groups (n=10):two-kidney one clip renal hypertensive (2KIC) model group, sham group, telmisartan (10 mg/kg) group, am-lodipine (2.5 mg/kg) group and telmisartan (10 mg/kg)+amlodipine(2.5 mg/kg) group. The model of two-kidney one clip re-nal hypertensive rats was established. The tail arterial blood pressure was detected once a week. After 20 weeks, rats were sacrificed and specimens were collected. The left ventricular mass index (LVMI) was assessed. The myocardial ultrastructur-al changes were observed by electron microscope. Values of plasma renin activity (PRA), angiotensionⅡ(AngⅡ) and atrial natriuretic peptide (ANP) were measured by enzyme linked immunosorbent assay (ELISA).Results Compared with sham group, the levels of systolic blood pressure (SBP), LVMI, PRA, AngⅡand ANP were significantly higher in 2KIC group (P＜0.01). Compared with 2KIC group, values of SBP, LVMI, PRA and ANP were significantly lower in telmisartan group and am-lodipine group (P＜0.01), but the value of AngⅡwas significantly higher (P＜0.01). The levels of SBP, LVMI, AngⅡand ANP were significantly lower in combined medication group than those of single drug medication group (P＜0.01). There was no significant difference in the plasma PRA level between those groups (P>0.05). Results of myocardial electron microsco-py showed that the left ventricular remodeling was significantly improved in combined treatment group. Conclusion Telmisartan and amlodipine can effectively improve the left ventricular remodeling induced by hypertension. There was more effective therapy using both medications together.

Objective To establish an efficient baculovirus-insect cell expression system for the production of human immunodeficiency virus-1 ( HIV-1 ) envelope glycoprotein gp120 and to evaluate the physiochemical properties, antigenicity and immunogenicity of the recombinant protein. Methods The gene encoding HIV-1 NL4-3 gp120 was cloned into the downstream of pH promoter of the baculovirus transfer vec-tor pAcgp67B to construct the recombinant transfer vector pAc-gp120. Expression of the protein of interest was induced in baculovirus-infected High FiveTM insect cells. ELISA, analytical ultracentrifugation and size-exclusion chromatography were carried out to characterize physicochemical properties of the expressed gp120 protein. Immunogenicity of the recombinant gp120 protein was analyzed by HIV neutralization assay after im-munizing BALB/c mice with it. Results The recombinant HIV-1 gp120 protein was successfully obtained from the established insect cell expression system with a purity of more than 90％ and a mean yield of 13 mg/L in four batches. That recombinant HIV-1 gp120 protein was characterized by homogeneity in solution and possessed a good immunoreactivity to neutralizing antibodies and antisera against HIV. Immunogenicity analysis in BALB/c mice demonstrated that the recombinant gp120 protein could induce effective immune re-sponses against HIV-1 NL4-3. Conclusion A simple and scalable approach to obtain homogeneous and im-munogenic HIV-1 gp120 antigen is successfully established, which will promote further investigation of HIV vaccine candidates.

Background and objective Chronic cough after pulmonary resection is one of the most common complications,which seriously affects the quality of life of patients after surgery.Therefore,the aim of this study is to explore the risk factors of chronic cough after pulmonary resection and construct a prediction model.Methods The clinical data and postoperative cough of 499 patients who underwent pneumonectomy or pulmonary resection in The First Affiliated Hospital of University of Science and Technology of China from January 2021 to June 2023 were retrospectively analyzed.The patients were randomly divided into training set(n=348)and validation set(n=151)according to the principle of 7:3 randomization.According to whether the patients in the training set had chronic cough after surgery,they were divided into cough group and non-cough group.The Mandarin Chinese version of Leicester cough questionnare(LCQ-MC)was used to assess the severity of cough and its impact on patients'quality of life before and after surgery.The visual analog scale(VAS)and the self-designed numerical rating scale(NRS)were used to evaluate the postoperative chronic cough.Univariate and multivariate Logistic regression analysis were used to analyze the independent risk factors and construct a model.Receiver operator characteristic(ROC)curve was used to evaluate the discrimination of the model,and calibration curve was used to evaluate the consistency of the model.The clinical application value of the model was evaluated by decision curve analysis(DCA).Results Multivariate Logistic analysis screened out that preoperative forced expiratory volume in the first second/forced vital capacity(FEV1/FVC),surgical procedure,upper mediastinal lymph node dissection,subcarinal lymph node dissection,and postoperative closed tho-racic drainage time were independent risk factors for postoperative chronic cough.Based on the results of multivariate analysis,a Nomogram prediction model was constructed.The area under the ROC curve was 0.954(95％CI:0.930-0.978),and the cut-off value corresponding to the maximum Youden index was 0.171,with a sensitivity of 94.7％and a specificity of 86.6％.With a Bootstrap sample of 1000 times,the predicted risk of chronic cough after pulmonary resection by the calibration curve was highly consistent with the actual risk.DCA showed that when the preprobability of the prediction model probability was be-tween 0.1 and 0.9,patients showed a positive net benefit.Conclusion Chronic cough after pulmonary resection seriously af-fects the quality of life of patients.The visual presentation form of the Nomogram is helpful to accurately predict chronic cough after pulmonary resection and provide support for clinical decision-making.

Objective:To explore the feasibility and accuracy of three-dimensional (3D) modeling methods based on ultrasound imaging data for normal and abnormal fetal cardiac structures, and to construct a methodology system for 3D printing of fetal heart based on ultrasound.Methods:A total of 93 fetuses examined in Tangdu Hospital of Air Force Military Medical University from January to December 2019 were selected. Fetal echocardiography was obtained using spatio-temporal image correlation (STIC). Ninety-three hearts were 3D modeled by blood flow modeling, blood pool modeling and cavity modeling, and printed by stereolithography technique. The data measured on the 3D digital models and 3D printed solid models were compared with the corresponding fetal echocardiographic images respectively in order to evaluate the accuracy of the modeling methods.Results:The fetal cardiac blood flow models based on Doppler flow image data showed the malformation and trend of small blood vessels. The fetal cardiac structure models printed based on blood pool modeling displayed the malformation of heart and large blood vessels. Models printed based on cavity modeling method accurately displayed valve and structural defects.For 83 normal fetal hearts, the long diameters of left and right ventricles measured on echocardiography [(15.3±1.9)mm, (13.2±1.9)mm] were compared with those measured on digital models [(15.1±1.9)mm, (12.9±1.9)mm] and 3D printed models[(15.1±1.9)mm, (13.0±1.9)mm], respectively, and there were no significant differences between any two groups of them ( P>0.05). Bland-Altman showed good consistency for all measurements within and between operators. Conclusions:The three modeling methods, including blood flow modeling, blood pool modeling and cavity modeling, have their own advantages in displaying different types of fetal heart malformations. Appropriate modeling methods should be selected for 3D modeling and printing to make up for the limitations of single modeling method. The consistency between measurements on 3D models and those on echocardiography is high, and the repeatability between operators is good.

With the deepening of modern drug research,traditional computer simulation can not meet the needs of future drug design experiments.As a classic technology of standard computer simulation,molecular simulation can construct and analyze complex molecular models to study the dynamic processes of molecular motion.However,the simulation results are easy to be affected by human factors.In recent years,the integration of artificial intelligence and molecular simulation has become a new method of drug design research.Artificial intelligence technology uses big data to screen out the corresponding compounds for molecular simulation and feedback on the simulation results to the artificial intelligence system to optimize the artificial neural network.The combination of artificial intelligence and molecular simulation technology improves the efficiency of drug design research,reduces the influence of human factors on simulation results,and increases the credibility of simulation results.In this review,we summarized the progress of artificial intelligence and molecular simulation technology in drug design to provide a reference for the change from computer assisted drug design(CADD)to artificial intelligence-aided drug design(AIDD)in future pharmaceutical development.

Mammals exhibit limited heart regeneration ability, which can lead to heart failure after myocardial infarction. In contrast, zebrafish exhibit remarkable cardiac regeneration capacity. Several cell types and signaling pathways have been reported to participate in this process. However, a comprehensive analysis of how different cells and signals interact and coordinate to regulate cardiac regeneration is unavailable. We collected major cardiac cell types from zebrafish and performed high-precision single-cell transcriptome analyses during both development and post-injury regeneration. We revealed the cellular heterogeneity as well as the molecular progress of cardiomyocytes during these processes, and identified a subtype of atrial cardiomyocyte exhibiting a stem-like state which may transdifferentiate into ventricular cardiomyocytes during regeneration. Furthermore, we identified a regeneration-induced cell (RIC) population in the epicardium-derived cells (EPDC), and demonstrated Angiopoietin 4 (Angpt4) as a specific regulator of heart regeneration. angpt4 expression is specifically and transiently activated in RIC, which initiates a signaling cascade from EPDC to endocardium through the Tie2-MAPK pathway, and further induces activation of cathepsin K in cardiomyocytes through RA signaling. Loss of angpt4 leads to defects in scar tissue resolution and cardiomyocyte proliferation, while overexpression of angpt4 accelerates regeneration. Furthermore, we found that ANGPT4 could enhance proliferation of neonatal rat cardiomyocytes, and promote cardiac repair in mice after myocardial infarction, indicating that the function of Angpt4 is conserved in mammals. Our study provides a mechanistic understanding of heart regeneration at single-cell precision, identifies Angpt4 as a key regulator of cardiomyocyte proliferation and regeneration, and offers a novel therapeutic target for improved recovery after human heart injuries.
Humans ; Mice ; Rats ; Cell Proliferation ; Heart/physiology* ; Mammals ; Myocardial Infarction/metabolism* ; Myocytes, Cardiac/metabolism* ; Pericardium/metabolism* ; Single-Cell Analysis ; Zebrafish/metabolism*