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 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 investigate the role and mechanism of suramin(Sur)in acetaminophen(APAP)-induced acute liver injury in mice.Methods 8-10 weeks old C57BL/6J mice were randomly divided into the APAP group and APAP+Sur group(20 mg/kg suramin was injected 1 h before).After 18 hrs of fasting,400 mg/kg APAP was injected intraperitoneally to establish a mouse model of acute liver failure and the survival rate was recorded.An acute liver injury model of mice was established via intraperitoneal injection of 300 mg/kg APAP(other conditions remained unchanged).A control group was also established,with liver tissues and serum collected at 0,2,and 12 hours post-APAP treatment.ELISA and CBA techniques were adopted to detect the release of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in serum and the secretion of inflammatory factors.H&E staining and immunohistochemistry were used to detect liver tissue necrosis and inflammatory cell infiltration.DCFA-DH and ELISA techniques were used to detect the levels of reactive oxygen species(ROS),malondialdehyde(MDA)and glutathione(GSH)in liver tissues.Western blotting was employed to assess the activation of the JNK signaling pathway in liver tissues.Results Suramin treatment improved the survival rate of APAP-induced mice,reduced the release of transaminases and inflammatory factors in serum,and alleviated APAP-induced liver cell necrosis and inflammatory cell infiltration in the liver.Suramin treatment delayed APAP-induced GSH depletion in the liver,reduced MDA and ROS levels,and inhibited JNK pathway activation.Conclusion This study has confirmed the protective effect of suramin against acetaminophen-induced acute liver injury in mice.The mechanism is potentially related to oxidative stress and inflammation.

Objective To investigate the correlation between high-resolution computed tomograohy(HRCT)features and thera-peutic effect in patient with non-tuberculous mycobacteria pulmonary disease(NTM-PD).Methods A total of 225 NTM-PD patients were divided into curative group and non-curative group according to the efficacy criteria,and the clinical and HRCT features between the two groups were compared.Correlation analysis and logistic regression analysis were used to screen the risk factors for potential poor efficacy,and their predictive value was evaluated by the receiver operating characteristic(ROC)curve.Results The proportion of cavities,the number of nodules and bronchiectasis involving lung lobes in the non-curative group were significantly higher than those in the curative group,while the body mass index(BMI)was significantly lower than that in the curative group.The distribution of non-tuberculous mycobacteria(NTM)strains,cavities,the number of nodules involving lung lobes,and the number of bronchiecta-sis involving lung lobes were positively correlated with poor efficacy,while BMI was negatively correlated with poor efficacy(P＜0.05).The distribution of NTM strains,the number of nodules and bronchiectasis involving lung lobes were independent risk factors for poor efficacy(P＜0.05).Cavities,the number of nodules involving lung lobes,and the number of bronchiectasis involving lung lobes alone or in combination had high efficacy in predicting poor efficacy,with the highest prediction value of the three combined model[area under the curve(AUC)=0.819;95％confidence interval(CI)0.762-0.876].Conclusion The combined model based on cavities,the number of nodules involving lung lobes,and the number of bronchiectasis involving lung lobes has the highest effi-ciency in predicting poor efficacy in NTM-PD patients.

Objective:To study effects of different pretreatment methods on the detection of pesticide residues in Angelicae Sinensis Radix,ChrysanthemI Flos,Lych Fructus,Astragali Radix and Lonicerae Japonicae Flos.Methods:The samples were treated with QuEChERS method and high-speed homogenization combined with hydro-phile-lipophile balance(HLB)solid-phase extraction method,and the residual amounts of carbofuran,3-carboxyl-carbofuran,phorate,phorate sulfone,phorate sulfoxide,and methyl isoflurophos were simultaneously determined using UHPLC-MS/MS.With a gradient elutionof 0.1％formic acid solution containing 5 mmol·L-1 ammonium formate and t 0.1％formic acid solution containing 5 mmol·L-1 ammonium formate-acetonitrile(volume ratio of 5:95),Ultra-performance liquid chromatography column Agilent Poroshell 120 SB-C18(2.1 mm × 100 mm,2.7μm)was used at 35 ℃ and the electrospray ion source was scanned in the positive ion mode and detected in the multiple reaction monitoring mode in mass spectrometry.Results:The deviation of the results measured by QuECh-ERS method and HLB solid-phase extraction method was between 9.09％-55.56％.Conclusion:In the selection of the pretreatment method for the detection of pesticide residues in traditional Chinese medicine,it is recommen-ded to take the measurement data of positive samples as the evaluation index and basis,and choose the method with higher measurement value and high extraction efficiency.

INTRODUCTION: This study aimed to elucidate the cognitive profile of patients with mild cognitive impairment with Lewy bodies (MCI-LB) and to compare it to that of patients with mild cognitive impairment due to Alzheimer's disease (MCI-AD). METHODS: Subjects older than 60 years with probable MCI-LB (n = 60) or MCI-AD (n = 60) were recruited. All patients were tested with Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) to assess their global cognitive profile. RESULTS: The MCI-AD and MCI-LB patients did not differ in total MMSE and MoCA scores. However, some sub-items in MMSE and MoCA were shown to be screening markers for differentiating MCI-LB from MCI-AD. In the visuoconstructive test, the total score and hands subitem score in the clock-drawing test were significantly lower in MCI-LB than in MCI-AD. As for the executive function, the 'animal fluency test', 'repeat digits backward test' and 'take paper by your right hand' in MMSE all showed lower scores in MCI-LB compared with MCI-AD. As for memory, 'velvet' and 'church' in MoCA and 'ball' and 'national flag' in MMSE had lower scores in MCI-AD than in MCI-LB. CONCLUSION This study presents the cognitive profile of patients with MCI-LB. In line with the literature on Dementia with Lewy bodies, our results showed lower performance on tests for visuoconstructive and executive function, whereas memory remained relatively spared in the early period.
Humans ; Cognitive Dysfunction ; Alzheimer Disease/diagnosis* ; Neuropsychological Tests ; Cognition

This study was performed to explore the differential genes and functions of double-negative T cells in peripheral blood of patients with colorectal cancer.Two colorectal cancer patients and two healthy physical examiners were selected,and peripheral blood double-negative T cells were firstly sorted by flow cytometry,and then sequencing data were obtained using single cell full-length transcriptome(SMART-seq2)sequencing technology to screen differentially expressed genes.The screened differentially expressed genes were subjected to Gene Ontology Enrichment(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.The STRING database was used to construct the protein interaction network and identify key genes by Cytoscape software;RT-qPCR was used to verify the differential expression genes in DNT cells.Compared with healthy subjects,there were 1 276 peripheral blood double-negative T-cell differential genes in colorectal cancer patients,including 141 up-regulated genes and 1 135 down-regulated genes.GO analysis showed that the differential genes were mainly involved in biological functions such as methylation,metabolic processes and transferase activity;KEGG pathway analysis showed that the differential genes were mainly involved in signaling pathways such as autophagy,P53 signaling pathway and phosphatidylinositol metabolism.The protein interaction network contains 1 154 nodes and 1 022 edges,in addition,10 hub genes were identified:PIK3C3,WIPI1,ATG101,PIK3R4,DDX10,RBM28,SDAD1,ATG16L1,UVRAG,ATG7.RT-qPCR validated 10 differentially expressed genes,of which 7 differentially expressed genes showed trends consistent with sequencing results,and 3 genes showed expression inconsistent with sequencing results.DNT cells may be involved in the development of colorectal cancer through methylation,P53 signaling pathway and autophagy,and at the same time,DNT cells may inhibit the development of colorectal cancer through the regulation of genes.This study provides a theoretical basis for further investigation of the function of DNT cells in malignant tumors.

Objective:To investigate the clinical effect of dapagliflozin combined with metformin on type 2 diabetes mellitus (T2DM).Methods:A total of 100 patients with T2DM who received treatment in The Second People's Hospital of Hefei from June 2019 to May 2021 were included in this study. They were randomly divided into a control group ( n = 50) and an experimental group ( n = 50). The control group was treated with metformin, and the experimental group was treated with dagglitazin combined with metformin. All patients were treated for 3 months. Blood glucose index, blood lipid level, and the incidence of adverse reactions were compared between the two groups. Results:After treatment, fasting blood glucose, 2-hour post-prandial blood glucose, and glycosylated hemoglobin in the experimental group were (5.56 ± 0.37) mmol/L, (8.32 ± 0.23) mmol/L, and (6.17 ± 0.26)% respectively, which were significantly lower than (6.96 ± 0.48) mmol/L, (9.58 ± 0.39) mmol/L, and (7.27 ± 0.26)% respectively in the control group ( t = 3.59, 6.92, 5.03, all P < 0.05). The total cholesterol and triglyceride in the experimental group were (3.58 ± 0.53) mmol/L and (1.25±0.26) mmol/L, respectively, which were significantly lower than (4.94 ± 0.58) mmol/L and (1.93 ± 0.18) mmol/L in the control group ( t = 3.16, 4.25, both P < 0.05). There was no significant difference in the incidence of adverse reactions between the two groups ( P > 0.05). Conclusion:Dapagliflozin combined with metformin can effectively control blood glucose and blood lipid in T2DM patients without increasing adverse reactions.