Objective: To examine the causal association and potential mechanisms between bone mineral density in different age groups and primary malignant bone tumor based on two sample Mendelian randomization (MR), so as to provide a reference for the prevention and treatment of primary malignant bone tumor. Methods: The genome-wide association study (GWAS) of bone mineral density was obtained from the GEFOS database,which included 66 628 subjects divided into five age groups (0-15, 15-30, 30-45, 45-60, and >60 years) based on the phases of human bone development. The GWAS of primary malignant bone tumor was sourced from the FinnGen database, including 648 cases and 378 749 controls. Using bone mineral density of five age groups as the exposure and primary malignant bone tumor as the outcome, an MR analysis was performed with the inverse-variance weighted (IVW) method. Sensitivity analysis were conducted using Cochran's Q test, MR-Egger regression, MR-PRESSO test and MR Steiger test. The potential mechanisms underlying the causal association between bone density and primary malignant bone tumors were explored using Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Results: The MR analysis results showed that there was a negative causal association between bone density and primary malignant bone tumors in the 30-45 age group (OR=0.301, 95%CI: 0.126-0.721). No statistically significant associations between bone density and primary malignant bone tumors were found in the 0-15, 15-30, 45-60, and >60 age groups (all P>0.05). Sensitivity analysis did not detect heterogeneity, pleiotropy (all P>0.05) and reverse causality. KEGG enrichment analysis revealed that genes highly associated with bone density and primary malignant bone tumors were enriched in the mTOR signaling pathway and the Wnt signaling pathway, among which Low Density lipoprotein Receptor Related protein 5 and Wnt Family Member 16 are key regulatory genes. Conclusion The decrease in bone mineral density among individuals aged 30-45 may increase the risk of primary malignant bone tumors through the mTOR signaling pathway and the Wnt signaling pathway.

Acylcarnitines are metabolic intermediates of fatty acids and branched-chain amino acids having vital biofunctions and pathophysiological significances.Here,we developed a high-throughput method for quantifying hundreds of acylcarnitines in one run using ultrahigh performance liquid chromatography and tandem mass spectrometry(UPLC-MS/MS).This enabled simultaneous quantification of 1136 acyl-carnitines(C0-C26)within 10-min with good sensitivity(limit of detection＜0.7 fmol),linearity(cor-relation coefficient＞0.992),accuracy(relative error＜20％),precision(coefficient of variation(CV),CV＜15％),stability(CV＜15％),and inter-technician consistency(CV＜20％,n=6).We also established a quantitative structure-retention relationship(goodness of fit＞0.998)for predicting retention time(tR)of acylcarnitines with no standards and built a database of their multiple reaction monitoring parameters(tR,ion-pairs,and collision energy).Furthermore,we quantified 514 acylcarnitines in human plasma and urine,mouse kidney,liver,heart,lung,and muscle.This provides a rapid method for quantifying acyl-carnitines in multiple biological matrices.

[Objective] To explore the expression of Nectin-4 in invasive bladder urothelial carcinoma (BUC) tissue and its clinical significance, so as to provide reference for clinical diagnosis and treatment of BUC. [Methods] Nectin-4 expression in 60 cases of invasive BUC and 40 cases of chronic inflammation of bladder mucosa was detected with immunohistochemical staining (IHC) and RNAscope.The results of the two methods were analyzed and compared, and the relationship between the two methods and the clinicopathological characteristics of invasive BUC was discussed.The correlation between the protein expression of Nectin-4 in BUC tissues, human epidermal growth factor receptor 2 (Her-2) and programmed death factor ligand 1 (PD-L1) was analyzed. [Results] The positive protein expression rates of Nectin-4 detected by IHC were 78.33%(47/60) and 17.50% (7/40) in the invasive BUC group and inflammatory group, respectively, while the positive mRNA expression rates of Nectin-4 detected by RNAscope were 83.33% (50/60) and 12.50% (5/40), respectively.The Kappa values of Nectin-4 in the invasive BUC group and inflammatory group were 0.732 and 0.610, respectively, with general consistency.The protein expression of Nectin-4 in invasive BUC was correlated with muscular invasion, histological grade, vascular thrombus, lymph node metastasis and clinical stage (P<0.05). The mRNA expression of Nectin-4 in invasive BUC was correlated with max tumor diameter, muscular invasion, histological grade, vascular thrombus, lymph node metastasis and clinical stage (P<0.05). The high expression of Nectin-4 in invasive BUC was positively correlated with the expression of Her-2 (P=0.002), but not with the expression of PD-L1 (P>0.05). [Conclusion] Nectin-4 is highly expressed in invasive BUC, and is usually associated with the pathological parameters of poor prognosis.Detection of Nectin-4 expression will help to guide clinical diagnosis and treatment.

Objective To construct myeloid-specific Spi1 gene knockout mice and analyze their genotypes,so as to provide animal model basis for the study of pathological mechanism of diseases and drug targets.Methods Ac-cording to the principle of CRISPR/Cas9 technology and Cre/LoxP system,sgRNA and Donor vectors were de-signed and constructed.The transcript of Exon 2(Exon 2)was used as the knockout region,and Loxp elements were placed on both sides of Exon 2.Cas9 protein,sgRNA and Donor vector were mixed and microinjected into the fertilized eggs of C57BL/6J mice,the fertilized eggs were transplanted into the uterus of C57BL/6J pregnant female mice,and F0 generation was obtained after 19～20 days.Positive F0 mice were mated with C57BL/6J mice to ob-tain stable F1 Spi1flox/+mice.Spi1flox/+mice of F1 generation were selfed to obtain Spi1flox/flox mice.Spi1flox/flox mated with Lyz2-Cre+mice to obtain Spi1flox/+/Lyz2-Cre+mice,and then mated with Spi1flox/flox,the Spi1flox/flox/Lyz2-Cre+mice were myeloid-specific Spi1 gene knockout(KO)mice.Spi1flox/flox/Lyz2-cre-mice were used as wild-type(WT)mice.DNA of WT and KO mice was extracted,and the genotypes were identified by agarose gel electro-phoresis after PCR amplification.Western blot was used to detect the expression of spleen focus forming virus provi-ral integration oncogene,Spi-1/purine rich box-1(PU.1)in immune cells of WT and KO mice.Results The results of PCR identification showed that the genotype of mice with only 220 bp amplified by flox primer was Spi1flox/flox homozygote,and the genotype of mice with 700 bp amplified by Lyz2-Cre primer was Lyz2-Cre+.Western blot showed that compared with WT group,the protein PU.1 was not expressed in bone marrow-derived macropha-ges(BMDMs)and peritoneal macrophages(PM)in KO group(P<0.01).There was no significant difference of statistics in the expression level of PU.1 in T cells between KO mice and WT mice.The results of PCR and West-ern blot showed that myeloid-specific Spi1 KO mice were successfully constructed.Conclusion The myeloid-spe-cific Spi1 gene KO mice are successfully constructed and identified,which provides animal model basis for further revealing the potential mechanism of PU.1 inimmune regulation.

Objective To breed and identify the T lymphocyte-conditional Spi1 knockout mice for the further in-vestgation of the specific role of Spi1-encoded protein PU.1.Methods The Lck-Cre mice were mated with Spi1flox/flox mice to obtain Lck-Cre×Spi1flox/flox mice(T lymphocyte-specific Spi1 knockout mice),and the genotype was determined by polymerase chain reaction(PCR)and agarose gel electrophoresis.Magnetic beads were used to sort out the splenic T lymphocytes,and the knockdown efficiency of PU.1 in T cells was detected by Western blot,quantitative real-time PCR(qPCR)and flow cytometry.Results The Lck-Cre×Spi1flox/flox mouse genotype was stably inherited.Compared with Spi1flox/flox mice,the expression level of PU.1 was significantly reduced in splenic T cells of Lck-Cre×Spi1flox/flox mice.Conclusion In this study,the T lymphocyte-specific Spi1 knockout mice was successfully constructed by applying Cre/LoxP system and CRISPR/Cas9 technology,which provided a reliable an-imal model for the subsequent experiments of the specific role of PU.1 in T cell-related diseases.

Objective To construct Csf1r-CreERT2 R26REYFP reporter gene mice and assess the efficacy of Csf1r-CreERT2-mediated enhancement of CSF1R in CD45+cells labeled with yellow fluorescein protein EYFP.Methods Csf1r-CreERT2 mice were crossbred with R26REYFP homozygous mice,and Csf1r-CreERT2R26REYFP mice were identified through PCR and Western Blot analyses.Flow cytometry was employed to evaluate CSF1R tag-efficiency in CD45+cells across different mouse tissues following tamoxifen induction.Results Csf1r-CreERT2 R26REYFP reporter gene mice were acquired.In addition,it was found that Csf1r-CreERT2-mediated EYFP could effectively mark CSF1R in various tissues of mice and CD45+cells in different locations.Compared to the R26REYF P group,the highest labeling efficiency was observed in the brain tissue(P＜0.001),the lowest in the thymus tissue(P＜0.05),and no sig-nificant difference was observed in the spleen tissue.Conclusion Adult Csf1r-CreERT2 mice and R26REYFP mice are effective ways to obtain Csf1r-CreERT2 R26REYFP induced conditional fluorescence mice.Csf1r-CreERT2 can mediate EYFP to effectively trace CSF1R in CD45+cells in different parts of mice.

Objective To investigate the feasibility of granzyme B(GB)promoter-controlled ferritin heavy chain(FTH1)reporter gene expression for monitoring the activation status of chimeric antigen receptor T cells(CAR-T)by magnetic resonance imaging(MRI).Methods Cytotoxic T lymphocytes(CTLs)were screened by Ficoll density gradient centrifugation and flow sorting.The GB promoter and FTH1 gene were ligated together with disialoganglioside 2(GD2)CAR,and lentiviral vectors were transfected into CTLs to construct GD2-CAR-T/pGB-FTH1 cells.GD2-CAR-T/pCMV-FTH1,GD2-CAR-T,and T cells served as control cells.CytoTox96@non-radioactive cytotoxicity was used to detect the killing effect of each group of cells after co-culture with human neuroblastoma cells(SK-N-SH).ELISA was employed to detect the coincubation factor as well as the amount of GB secretion.Western blotting,Prussian blue staining and cellular MRI were applied to detect the expression of the FTH1 gene after co-culture.Results CTLs were successfully obtained,and then GD2-CAR-T/pGB-FTH1,GD2-CAR-T/pCMV-FTH1 and GD2-CAR-T cells were constructed.The killing effect,co-incubation factor and GB secretion of the above 3 groups of cells were significantly higher than those of the T cells,and the level of GB expression was highest at day 1,and then decreased in order at day 3 and day 7 after co-culturing with SK-N-SH cells.The relative expression of FTH1 and iron content of the GD2-CAR-T/pGB-FTH1 cells showed the same trend as GB expression,and the MRI signals were gradually increased.There were no significant differences in the relative expression of FTH1,iron content and MRI signals in the GD2-CAR-T/pCMV-FTH1 cells at all time points.No FTH1 expression or iron aggregation was observed in the GD2-CAR-T and T cells groups.Conclusion MRI based on the FTH1 reporter gene driven by the granzyme B promoter can reflect the GB expression level and tumor killing effect of CAR-T cells,which provides a potential real-time visual means to monitor the cell activation status for CAR-T therapy.

Objective·To analyze the epidemiological characteristics of risk factors for cardiovascular diseases and malignant tumors based on the Shanghai community elderly cohort.Methods·The study subjects were selected from the Shanghai community elderly cohort established from February to August 2019,with a total of 17 948 people.The study subjects were divided into 4 groups according to self-reported presence or absence of tumors and/or cardiovascular diseases during the baseline survey:tumor-free and non-cardiovascular disease group,single cardiovascular disease group,single tumor group and tumor cardiovascular disease co-occurrence group.The differences among the four groups of subjects were collected and compared in terms of demographic characteristics and physiological indicators,daily living habits(smoking,drinking tea,drinking coffee,drinking carbonated drink,drinking alcohol,sedentary time,physical activity level and sleep quality),past medical history,psychological status(depression and anxiety)and dietary compliance.Results·Among the study subjects,60.1%of tumor patients were complicated with cardiovascular diseases.The differences among the four groups of subjects in age,gender,educational level,pre-retirement occupation,waist circumference,hip circumference and body mass index were statistically significant(all P<0.05).Compared with the tumor-free and non-cardiovascular disease group,the single cardiovascular disease group,single tumor group and tumor cardiovascular disease co-occurrence group all exhibited lower proportions of smoking and high physical activity levels(all P<0.05),and higher proportion of sedentary time exceeding 4 h/d and poor sleep quality(all P<0.05);the proportion of subjects with past medical histories including hyperlipidemia,peripheral vascular disease,endocrine system disease,respiratory system disease,urinary system disease and digestive system disease of the single cardiovascular disease group and the tumor cardiovascular disease co-occurrence group was higher(all P<0.05),and the proportion of subjects with depression and anxiety was also higher(all P<0.05).Furthermore,compared with the tumor-free and non-cardiovascular disease group,the single cardiovascular disease group had lower compliance rates of poultry,fish,fruit and liquid milk(all P<0.05).Among the four groups,only the compliance rate of vegetable intake exceeded 50%,while the compliance rates of poultry,fish,fruit,liquid milk and tubers were all below 20%.Conclusion·In the elderly population of Shanghai communities,over half of malignant tumor patients are concomitant with cardiovascular diseases.Unhealthy daily habits are prevalent among those with cardiovascular diseases,tumors and tumor-cardiovascular disease co-occurrence.The intake of many foods in the elderly of the community do not reach the levels recommended by Chinese Dietary Guidelines.

Objective:To explore the training needs for clinical core competence of master of nursing specialist (MNS) from the perspective of supervisors, providing reference for the development of future MNS clinical practice training programs.Methods:Using phenomenological research methods from qualitative research, purposive sampling was used to select 10 MNS supervisors from Jilin Province, Heilongjiang Province, Sichuan Province, and Zhejiang Province as research subjects for semi-structured interviews from May to July 2023. Colaizzi 7-step analysis method was used to extract themes.Results:Six themes were extracted, including the need to strengthen MNS ideological and political education, differences in clinical training needs and ability goals between fresh and non-fresh students, the need to enhance MNS clinical practice ability, clinical research should be a key training content, thinking ability training should be integrated throughout the entire clinical training process, and achievement transformation.Conclusions:Relevant training institutions should attach importance to the cultivation of MNS ideological and political education, specialized practical abilities, thinking abilities, clinical research, and achievement transformation abilities, distinguish the tendency of cultivating fresh and non-fresh students, and actively set up relevant courses to improve students' core competence and job competitiveness, and cultivate nursing expert talents that truly meet the needs of clinical development.

Objective:To develop a joint clinical practice teaching and assessment program tailored to the clinical training needs of master of nursing specialist (MNS) postgraduates which focuses on core competency requirements.Methods:Totally 10 MNS postgraduate supervisors were selected by convenience sampling for semi-structured interviews between May and July 2023. Subsequently, a Delphi method was employed with 22 MNS postgraduate supervisors over two rounds of consultations from October to December 2023.Results:A total of 22 experts participated in the Delphi consultations, with an effective response rate of 100.00% (22/22) in both rounds. The expert authority coefficients were 0.822 and 0.833, respectively, for the two rounds. The Kendall's W for various levels of indicators ranged from 0.097 to 0.243 and 0.159 to 0.256, respectively ( P<0.01). The final training program included five primary indicators, 10 secondary indicators, and 26 tertiary indicators. Conclusions:The development process for the joint clinical practice teaching and assessment program for MNS postgraduates is scientific and reliable. The program can serve as a reference for the clinical practice training of MNS postgraduates.