Objective To develop GTKO (α-1,3-galactosyltransferase gene-knockout, GTKO)/hCD55 (human CD55) gene-edited xenotransplant donor pigs and verify their function. Methods In this study, CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated nuclease 9), PiggyBac transposon technology and somatic cell nuclear transfer technology were used to construct GTKO/hCD55 gene-edited Diannan miniature pigs. The phenotype and function of GTKO/hCD55 pigs were analyzed by Sanger sequencing, real-time fluorescence quantitative PCR, flow cytometry, immunofluorescence, bisulfite sequencing, antigen-antibody binding assays, and complement-dependent cytotoxicity assays. Results After transfection of PX458 and PiggyBac gene editing vectors into wild-type fetal pig fibroblasts, 48 single-cell colonies were obtained through puromycin drug screening. Two single-cell colonies were selected for somatic cell nuclear transfer, resulting in two fetal pigs at 33 days of gestation. The GGTA1(α-1,3-galactosyltransferase) genotypes of fetal pig F01 were -17 bp and wild type (WT), while the GGTA1 genotypes of fetal pig F02 were -26 bp/+2 bp and -3 bp. The hCD55 mRNA expression levels of both fetal pigs were significantly higher than those of WT pigs (P＜0.01). The fetal pig F02 was selected as the donor cell source for recloning, 11 surviving piglets were obtained, all identified as GTKO/hCD55 gene-edited pigs. These pigs showed absence of α-Gal antigen expression, but weak or no expression of hCD55 was observed. Methylation analysis of the hCD55 gene's CpG island showed hypermethylation in kidney tissue lacking hCD55 expression, whereas it was not methylated or partially methylated in kidney tissue expressing hCD55. Moreover, codon optimization of the CpG island of the hCD55 gene to reduce CG content could achieve stable expression of the hCD55 gene. In addition, antigen-antibody binding experiment showed that the amount of human IgM binding to GTKO/hCD55 gene-edited pig fibroblasts was significantly lower than that of WT pigs (P＜0.01). Complement-dependent cytotoxicity experiment showed that the survival rate of fibroblasts in GTKO/hCD55 pigs was significantly higher than that in WT pigs (P＜0.01). Conclusion This study demonstrates the successful generation of GTKO/hCD55 gene-edited xenotransplant donor pigs. Methylation-induced gene silencing of the hCD55 gene can be effectively avoided by reducing the CG content of the CpG island through codon optimization. This study provides a reference for the development of xenotransplant donor pigs and guides subsequent research on xenotransplantation.

Objective To investigate the sputum metabolic profiles of patients with occupational coal workers' pneumoconiosis (CWP) by an untargeted metabolomics method, and to identify relevant differential metabolic pathways and potential biomarkers. Methods A total of 12 male patients with stage Ⅰ CWP were selected as the CWP group, and 16 healthy male individuals were selected as the control group, using a judgmental sampling method. Sputum metabolites of individuals in both groups were detected to perform non-targeted metabolomic analysis using the ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Differential metabolites (DMs) and their pathways were screened using principal component analysis, partial least squares discriminant analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Potential biomarkers were analyzed and identified via the receiver operating characteristic curve (ROC). Results There were apparent metabolic alterations observed in sputum of CWP patients compared with healthy controls. In the positive ion mode, a total of 42 DMs were identified in sputum from CWP patients, including 19 downregulated and 23 upregulated metabolites. In the negative ion mode, a total of 25 DMs were identified in sputum from CWP patients, including 16 downregulated and 9 upregulated metabolites. KEGG enrichment analysis of sputum from CWP patients showed that seven DMs pathways were enriched in ABC transporters, histidine metabolism, phenylalanine metabolism, arachidonic acid metabolism, linoleic acid metabolism, purine metabolism, and oxidative phosphorylation, involving 26 DMs. ROC analysis indicated that 16(R)-hydroxyarachidonic acid, pyrophosphate, and 2-hydroxyphenylacetate of these 26 DMs may serve as potential biomarkers for CWP. Conclusion Sputum metabolomic profiles were altered in CWP patients compared with healthy controls. The potential biomarkers of CWP prevention and treatment are 16(R)-hydroxyarachidonic acid, pyrophosphate, and 2-hydroxyphenylacetate.

OBJECTIVE To explore the effects of esmolol on cardiac function， inflammatory factors and serum microRNA （miR）-29a and miR-129-5p in patients with acute anterior ST-segment elevation myocardial infarction after percutaneous coronary intervention （PCI）. METHODS A total of 120 patients with acute anterior ST-segment elevation myocardial infarction undergoing PCI in our hospital from April 2021 to June 2023 were selected and divided into control group （60 cases） and study group （60 cases） according to the random number table method. The control group was given conventional treatment， and the study group was additionally given Esmolol hydrochloride injection based on the control group for one week. The levels of cardiac function indexes （left ventricular ejection fraction， left ventricular end-systolic volume index， left ventricular end-diastolic diameter， peak ejection fraction， cardiac output）， inflammatory factors （C-reactive protein， myeloperoxidase， interleukin-6， brain natriuretic peptide， homocysteine） myocardial enzyme indexes （creatine kinase isoenzyme， β2-microglobulin， cardiac troponin Ⅰ）， and serum expression of miR-29a and miR-129-5p were observed in two groups， and the occurrence of ADR was recorded. RESULTS After one week of treatment， left ventricular ejection fraction， peak ejection fraction， cardiac output， and the expression of miR-129-5p in two groups was significantly higher than before treatment （P＜0.05）， while left ventricular end-systolic volume index， left ventricular end-diastolic diameter， inflammatory factors and myocardial enzyme index levels， and the expression of miR-29a were significantly lower than before treatment （P＜ 0.05）. The study group was significantly better than the control group （except for the creatine kinase-MB） （P＜0.05）. There was no statistically significant difference in the incidence of symptomatic hypotension， symptomatic bradycardia， cardiogenic shock， and arrhythmia between two groups （P＞0.05）. CONCLUSIONS Esmolol can improve cardiac function， reduce inflammatory factors， lessen myocardial injury， and regulate serum expressions of miR-29a and miR-129-5p in patients with acute anterior ST- segment elevation myocardial infarction after PCI， with good safety.

Objective To investigate the correlation between serum leptin level and body mass index(BMI)in in-fants with cyanosis congenital heart disease,and the relationship between leptin and Ob gene receptor(Ob-R)and hypoxia-inducible factor 1α(HIF-1α)in myocardium.Methods A total of 52 children under 6 months of age with congenital heart disease who underwent surgical treatment in the Department of Congenital Heart Surgery,Fuwai Hospital from January 2019 to October 2020 were included in this study.According to the arterial partial pressure of oxygen(PaO2)of 90 mmHg,they were divided into cyanotic group(n=30)and acyanotic group(n=22).Their height and weight were collected to calculate BMI.The serum leptin level was measured by ELISA.The ex-pressions of HIF-1α and Ob-R in myocardial tissue were detected by RT-PCR and Western blot.In animal mod-el,SD rats were divided into normoxia group and hypoxia intervention group,which were subjected to continuous hypoxia(10％ O2)for 4 weeks.The hypoxia intervention group received intraperitoneal injection of HIF-1α in-hibitor digoxin(2 mg/kg)daily from the 14 th to 21st day of hypoxia,respectively.The body weight of rats was recorded,and the expressions of HIF-1α and Ob-R were detected by RT-qPCR and Western blot.Results Com-pared with the acyanosis group,the cyanosis group had a significantly lower BMI(P<0.05)and a lower leptin/BMI ratio(leptin/BMI)(P<0.05).Spearman correlation analysis confirmed that serum leptin in the circulatory system was positively correlated with BMI(P<0.05).In the cyanosis group,the expression of Ob-R increased with the upregulation of HIF-1α,showing a positive correlation.In animal model,with the down-regulation of HIF-1α expression in digoxin injection,the Ob-R level was significantly lower than that in the control group(P<0.05),the trend of weight loss was significantly inhibited(P<0.05).The right ventricular hypertrophy in-dex was significantly lower than that in the control group(P<0.05).Conclusions HIF-1α regulates the expres-sion of Ob-R in myocardial tissue,and the mechanism of its association with leptin and Ob-R may help to find new therapeutic target for improving the prognosis of infants with congenital heart disease.

Objective: To investigate the incidence of contact dermatitis among workers in cemented carbide production enterprises. Methods: From October 1997 to October 2017, an occupational epidemiological survey was conducted on a large-scale cemented carbide production enterprise, and occupational health examinations were conducted for employees. 152 people were exposed to hard metal dust (hard metal raw material dust and alloy dust) . The employees in the work group were contact groups, and 142 employees in the non-dusting operation of the company were in the control group. A detailed retrospective survey of hard metal production workers with contact dermatitis history in the two groups was conducted to analyze the risk factors of contact dermatitis exposure to hard metal dust. Results: The incidence of allergic diseases in the exposed group was significantly higher than that in the control group. The difference was statistically significant (χ²=23.793, P<0.05) . The incidence of contact dermatitis in the exposed group was significantly higher than that in the control group. The difference was statistically significant (χ²=24.659, P<0.05) ; the changes of contact dermatitis in the contact group were mainly allergic contact dermatitis, and some showed irritative contact dermatitis; the operator had respiratory symptoms (including work-related nasal congestion, cough, wheezing) , difficulty breathing may be the influencing factors of contact dermatitis (RR=2.60, 95%CI: 1.10-6.20, P<0.05) . Conclusion Hard metal alloy enterprises are exposed to hard metal dust. The incidence of contact dermatitis is high in workers, and the occurrence of contact dermatitis may be associated with those with respiratory symptoms.

Objective: o explore the problems encountered in the application of GBZ/T 237-2011 edition of "Diagnosis of Chronic Obstructive Pulmonary Disease Caused by Occupational Stimulating Chemicals" , and to provide reference for the revision of the new standard. Methods: Delphi expert survey method was used to consult experts on the reasonableness of six primary indicators (scope of application, diagnostic principles, diagnostic indicators, treatment principles, diagnostic grading, appendix A) and their corresponding secondary indicators in the Diagnosis of Chronic Obstructive Pulmonary Disease (COPD) caused by Occupational Stimulant Chemicals. Results: The results of the first round of Delphi method showed that the reasonable mean of the diagnostic criteria about long-term occupational history, smoking history and clinical diagnostic criteria was less than 7, and the coefficient of variation was greater than 0.25, indicating that the experts disagreed with the scope of application of the original criteria; the average of smoking history and 3-year working age were 1.78 and 3.43 (less than 7) respectively, which indicated that the experts agreed that the two diagnostic indexes were not conducive to the practical work of occupational disease clinicians; among the diagnostic indicators, smoking history and smoking volume were taken into account in a comprehensive way, and the average values were 7.61 and 7.61 (greater than 7) , respectively. the coefficient of variation was less than 0.25, indicating that the experts had concentrated their opinions and agreed that such indicators could be considered into the diagnostic index system. Conclusion "Diagnosis of Chronic Obstructive Pulmonary Disease Caused by Occupational Stimulating Chemicals" should be improved by expanding the scope of occupational irritant gases. Smoking problems can be considered in combination with clinical practice, and occupational exposure history can be considered to reduce appropriately.

Objective: To investigate the functional classification of differentially expressed genes in manganese-poisoned rats and related metabolic pathways, and to provide a reference for the study of the mechanism of manganese poisoning and gene regulation in the prevention and treatment of manganese poisoning. Methods: Six healthy specific pathogen-free male Sprague-Dawley rats were randomly divided into control group and experimental group according to body weight, with 3 rats in each group. Rats in the experimental group were injected intraperitoneally with MnCl₂·4H₂O (25 mg/kg) at 0.2 ml/100 g once every 48 h, and the control group was injected with phosphate-buffered saline at the same dose. After one month of exposure, the rats were anesthetized and then sacrificed by cardiac puncture blood collection. The striatum was isolated on ice, and RNA was extracted to establish a DNA data library. Whole genome sequencing was used to identify the differentially expressed genes in the rats with manganese poisoning. Gene Ontology functional enrichment analysis and pathway enrichment analysis were performed to investigate the possible metabolic pathways in which the differentially expressed genes may participate. Results: A total of 18439 genes were detected in the striatum of rats, and 17 differentially expressed genes were screened out. Among them, 10 genes were up-regulated, and 7 genes were down-regulated. According to gene function analysis, 164 functional branches and 26 metabolic pathways with high gene enrichment were screened out. The genes were enriched in synaptic signaling, signal transduction, etc., especially behavioral function. The metabolic pathways with high gene enrichment were endocytosis pathway, PI3K-Akt pathway, and neuroactive ligand-receptor interaction pathway, in which the PI3K-Akt pathway had enrichment of the same differentially expressed gene (29 517) as the FoxO signaling pathway and mTOR signaling pathway, and the neuroactive ligand-receptor interaction pathway had enrichment of the same differentially expressed gene (24 415) as the glutamatergic synaptic pathway. Conclusion The differentially expressed genes in manganese-poisoned rats may influence the susceptibility to manganese poisoning through the PI3K-Akt pathway, mTOR metabolic pathway, or FoxO metabolic pathway, and may be involved in behavioral changes.

Objective The aim of this study was to investigate the differences of the cell ultrastucture of normal mouse hatched blastocysts and their dormant ones cultured in vitro after freezing-thawing, and to explore whether the dor-mant embryos have a better anti-freezing shock property than the normal hatched mouse embryos .Methods By transmis-sion electron microscopy , the ultrastructure of these two types of mouse embryos was observed and analyzed .Results By comparative analysis of their ultrastructure , the results showed that the dormant embryos before freezing are being austerity and with lower energy metabolism at a ‘ground state ’ .After freezing-thawing and culture , their cellular structure seemed to be similar to that of the normal embryos cultured in vitro before freezing.However, after freezing-thawing and culture, the number of mitochondria decreased , the nuclei were loose , and their heterochromatin also increased .Conclusions From the ultrastructural observation , compared with the normal mouse hatched embryos , the cellular state of dormant mouse em-bryos after freezing-thawing is more favorable for material storage and energy metabolism , thus, indicating that they have a better anti-freezing property than normal hatched embryos .

Objective:To identify the HLA-A0201 restricted CTL epitopes derived from hepatitis B virus X protein predicted by computer program and general principles in vitro.Methods:HBx gene sequences of Hepatitis B virus genotypes B/C and serotypes adw/adr,with the highest frequencies in Chinese,were computed and analyzed by screening service offered by Internet combined with peptide supermotif,extended motif and quantitative motif prediction.Four most ideal nine-peptides(HBx1,HBx2,HBx3,and HBx4)were selected as candidate peptides.Using flow cytometry,the fluorescence index of both control and experimental groups were detected and the 4 nine-peptides were evaluated with T2 binding assay and DC50 assay.Results:The nine-peptides VLCLRPVGA(HBx1),CLFKDWEEL(HBx2),VLHKRTLGL(HBx3)and HLSLRGLPV(HBx4)were selected as candidate targets.Among the 4 candidate peptides,HBx2 showed higher HLA-A0201 affinity and HBx2,HBx4 showed better stability.Conclusion:Our study indicates that CLFKDWEEL might be a potential HLA-A0201 restricted CTL epitope from hepatitis B virus X protein;further study is needed for verification of its immunity in vivo.

Objective: To clone porcine IL-4 cDNA and observe its adjuvanticity of vaccine against T. solium cysticerco-sis. Methods:The cDNA of porcine IL-4 was amplified by RT-PCR, which had 5' AUG initiatory codon with optimized trans-lational initiation. After sequencing,the cDNA was intergrated into expression vector pcDNA and transient expression was performed. Then newborn pigs were immunized with IL-4 expression vector and protective antigen DNA vaccine against T. solium cysticercosis. Results:The obtained sequence of porcine IL-4 cDNA was the same as reported. IL-4 and protective antigen could effectively promote the humoral response. Conclusion:An efficient expression plasmid containing porcine IL-4 cDNA is constructed. Its adjuvanticity of DNA vaccine against T. solium cysticercosis is preliminarily conformed.