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 investigate whether there is a nonlinear relationship and threshold effect between the number of daily steps counts and fat loss in obese children and adolescents. Methods: Forty eight obese children and adolescents were randomly recruited to undergo 4 week closed exercise training during July to August 2021. Actigraphy GT3X+ was used to monitor the daily steps, and fat loss effect was evaluated by changes of body composition indicators before and after the intervention. Paired samples t test was used to compare the indicators before and after the intervention; the dose response relationship between daily steps counts and fat loss was analyzed by linear regression and piecewise regression, and the nonlinear relationship was analyzed by restricted cubic spline (RCS). Results: Body fat mass, percentage of body fat, lean body mass and skeletal muscle mass decreased (27.50± 7.33 )kg, (36.17±5.59)%, (47.55±6.48)kg, (26.14±3.84)kg, respectively compared with those before intervention[(31.97± 7.82 )kg，(39.06±5.12)%，(49.08±6.93)kg，(27.08±4.15)kg] ( t=21.04, 13.32, 7.65, 8.35, P <0.05). There was a significant nonlinear doseresponse relationship between daily steps counts and the change of these indicators ( P <0.05). After adjusting for age, sex and baseline BMI, each 1 000 step increase in daily walking number, BMI increased by 0.44(95% CI =0.03-0.84)kg and body fat percentage by 0.61%(95% CI =0.17%-1.04%). After adjusting for age, sex, and baseline BMI, each 1 000 step increased in daily number of steps occurred when the daily number of steps ranged from 8 300 to 11 400, lean body weight increased by 0.58(95% CI =0.11-1.04)kg, skeletal muscle mass increased by 0.29(95% CI =0.03-0.54)kg. Conclusion There is a non linear dose response relationship between daily steps and fat loss in obese children and adolescents, for optimal fat loss efficiency, daily steps to 8 300-11 400 appropriate.

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.

Objective:To investigate the correlation between the changes of oral bacterial flora during head and neck radiotherapy and radiation-induced oral mucositis (ROM).Methods:The oral bacterial samples of patients with nasopharyngeal carcinoma and accompanying family members were obtained before and at the end of radiotherapy and subjected to high-throughput sequencing. C57BL/6 mice were used to establish the ROM models. On the 9 th day after radiotherapy, oral bacterial samples were collected in the radiotherapy group and the negative control group. On the 3 rd, 5 th, 7 th, and 9 th days post-radiotherapy, the tongue tissues were obtained from another batch of mice in the negative control and radiotherapy groups. Inflammatory factors were detected with PCR and HE staining was performed. Results:The oral bacterial diversity of patients after radiotherapy significantly differed from that of patients before radiotherapy and their accompanying family members before and after radiotherapy in Observed species, Chao1, Simpson index (all P<0.05). There was a significant difference in Shannon index between the severe and mild ROM patients ( P=0.036). LEfSe analysis showed that patients with severe ROM had higher levels of g_ Streptococcus and f_ Streptococcus, and lower levels of f_ Familyxl, g_ Gemini and o_ Bacillus. The Simpson index and PCoA results in the oral bacterial samples significantly differed between the negative control and radiotherapy groups (all P<0.05). Conclusions:Radiotherapy can disrupt the balance of bacterial flora. The dysregulated oral bacterial flora is closely associated with the aggravation of ROM.

ObjectiveTo study the current status and development trend of research on the executive function in overweight or obesity， and to grasp the research hotspots in this field. MethodsA total of 1 321 literatures relevant to the executive function in overweight or obesity collected in Web of Science Core Collection database from 2010 to 2021 were selected. CiteSpace and VOSviewer were used to generate knowledge graphs for visualization analysis， then the number of articles issued， countries/regions， institutions and the co-occurrence， clustering and burst of keywords were analyzed. Results①In terms of the trend of the number of articles issued from 2010 to 2021， the annual quantity of published articles about the executive function in overweight or obesity presented a rapid growth over 2010-2014， then entered into a slow growth stage over 2014-2017， and emerged a rapid growth over 2017-2021. ②From the perspective of countries / regions， a total of 64 countries and regions participated in the research. The largest number of articles issued in this field was the USA， which had the most frequent cooperation and exchanges with other countries and regions， and exerted the greatest academic influence， occupying the core position of this research field. ③From the perspective of research institutions， a total of 1 627 institutions participated in this research field， among which the Illinois University published the most papers and closely cooperated with many productive institutions， forming a research group with a certain scale. ④From the perspective of keywords， the research content mainly involved dietary behavior research， prevention and intervention research， risk factor assessment， and analysis of the characteristics of different groups. ConclusionResearches on executive function in overweight or obesity have been developing rapidly， attracting international attention and covering a wide range of areas.

BACKGROUND: MiR-148b-3p is an important microRNA that has been reported to be significantly related to various types of cancer, but its role in lung adenocarcinoma remains elusive. The purpose of this study is to detect the expression level of miR-148b-3p in lung adenocarcinoma specimens, and to analyze its correlation with the clinicopathological features as well as the prognosis of patients with lung adenocarcinoma. METHODS: A total of 123 tumor specimens from lung adenocarcinoma patients who underwent surgical resection in our department from January 2011 to December 2012 were collected. The expression of miR-148b-3p was detected by quantitative real-time PCR (qRT-PCR), and its correlation with clinicopathological features of patients with lung adenocarcinoma was analyzed. Multivariate Cox proportional hazard models were used to analyze independent predictors of overall survival in patients with lung adenocarcinoma. The overall survival (OS) of patients in miR-148b-3p high expression group and miR-148b-3p low expression group were estimated by means of the Kaplan-Meier method and were compared using the Log-rank test method. RESULTS: Of the 123 patients with lung adenocarcinoma, 71 were in miR-148b-3p high expression group and 52 in low expression group. MiR-148b-3p was significantly associated with tumor grade (P=0.001) and tumor size (P=0.007), but not with age, gender, smoking history, history of alcohol, tumor thrombus, pleural invasion, node status or metastasis status. Multivariate Cox proportional hazard model analysis showed that tumor size (P=0.032), node status (P=0.005) and miR-148b-3p expression level (P=0.047) were significant independent predictors of overall survival of patients with lung adenocarcinoma. Kaplan-Meier survival analysis showed that the overall survival of patients with high expression of miR-148b-3p was significantly better than that of patients with low expression (P=0.010). CONCLUSIONS MiR-148b-3p was significantly associated with tumor grade and tumor size in lung adenocarcinoma, and served as an independent predictor of overall survival of patients with lung adenocarcinoma. The overall survival of patients with high expression level of miR-148b-3p was significantly better than that of patients with low expression.
Adenocarcinoma of Lung ; diagnosis ; genetics ; pathology ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Kaplan-Meier Estimate ; Male ; MicroRNAs ; genetics ; Middle Aged ; Neoplasm Grading ; Prognosis ; Tumor Burden

To investigate whether postoperative therapy can bring survival benefits to patients with locally advanced esophageal squamous cell carcinoma who have received neoadjuvant chemotherapy with TP regimen. Methods     We retrospectively reviewed clinical data of 115 patients with locally advanced esophageal squamous cell carcinoma who received neoadjuvant chemotherapy with TP regimen and underwent esophagectomy in our hospital from January 2007 through December 2016. Patients were divided into two groups including a non-receiving treatment group (54 patients with 47 males and 7 females) and a receiving treatment group (61 patients with 52 males and 9 females). There were 31 patients with postoperative chemotherapy, 14 with postoperative radiotherapy, and 16 with postoperative chemotherapy and radiotherapy in the receiving treatment group. Results     In the non-receiving treatment group, the 5-year median disease free survival (DFS) rate was 54.7%, and the 5-year overall survival (OS) rate was 55.3%. In the receiving treatment group, the median DFS was 46.0 months (95% CI 22.9–69.1), the 5-year DFS rate was 42.3%; and the   median OS was 68.0 months (95% CI 33.0–103.0), the 5-year OS rate was 51.3%. Furthermore, there was no statistical difference between the two groups with regards to DFS (P=0.641) or OS (P=0.757) using Kaplan-Meier method. Besides, in each subgroup, the results of Cox proportional hazard model analysis showed postoperative treatment did not improve survival (P>0.05, respectively). Conclusion     Postoperative treatment does not bring survival benefits to patients with esophageal squamous cell carcinoma who have received neoadjuvant chemotherapy with TP regimen.

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*