Objective: To propose an improved method for constructing the internal quality control (IQC) framework for ELISA assays and validate its efficacy by statistically analyzing IQC data from nine blood center laboratories. Methods: 1) IQC data was collected from nine blood centers and analyzed using a domestic HBsAg ELISA detection kit as an example. 2) Differences between IQC values across batches within Blood Center 1 were assessed. 3) Statistical analyses were performed on batch usage, number of batches used, days of use, number of QC points, batch-specific means, and coefficients of variation (CV) across all nine centers. 4) Using the improved construction method for IQC framework, provisional and permanent frames were established for batches within Blood Center 1 and Blood Center 9, followed by outlier determination. Results: 1) Statistically significant differences were observed in IQC data between batches within Blood Center 1 (P<0.01). It is recommended that both the control material/reagents and the control chart framework be replaced simultaneously. 2) There were substantial differences among 9 blood centers regarding the control material/reagent lot numbers used, the number of QC runs per batch, and the QC values for identical lots. Therefore, individual laboratories should establish their own IQC chart frameworks. 3) The improved IQC framework construction method for ELISA assays is as follows: provisional frames are established via frame-shifting, using the pre-experimental mean and cumulative coefficient of variation (CV) from the preceding batch. For batches used >20 days with >20 QC points, permanent frames are constructed by aggregating in-control data accumulated over ≥20 days with ≥20 points to calculate cumulative mean and standard deviation. The provisional and permanent frames constructed by this method identified all 26 extreme outliers across Blood Centers 1 and 9 as out-of-control. Among the 218 general outliers, 10 were classified as normal by the provisional frames, while the remainder were designated as warnings or out-of-control. This method effectively monitors assay stability. Conclusion: Based on the statistical analysis of IQC practices across blood centers of varying scales, combined with the inherent characteristics of ELISA assays and the batch-to-batch instability of reagents/QC materials, it is recommended to reconstruct QC charts upon lot changes. The proposed method—utilizing frame-shifting for provisional frames and establishing permanent frames based on cumulative data—is applicable to blood center laboratories of differing sizes and effectively monitors the stability of the ELISA assay process.

Genetically engineered mice are important tools for elucidating gene function and disease mechanisms and for advancing drug development. With the growing demand in biomedical research for complex genetic modifications, rapid model generation, and high-throughput functional analysis, technologies for generating genetically engineered mice continue to evolve. This review systematically summarizes the evolution and applications of these technologies, with a focus on conventional approaches, including early transgenic technologies and embryonic stem cell (ESC)-based homologous recombination for gene targeting, as well as the use of random mutagenesis approaches, such as N-ethyl-N-nitrosourea (ENU) mutagenesis and transposon insertion, in forward genetic screening. It further discusses the principles and characteristics of programmable nucleases, including zinc finger nucleases, transcription activator-like effector nucleases, and CRISPR/Cas systems, as well as their applications in generating genetically engineered mice. With advances in genome editing, Cas9-derived systems, base editing, prime editing, and RNA editing further improve editing precision, expand the repertoire of genetic modifications and precise regulatory approaches, and provide additional options for generating genetically engineered mice. This review also compares the advantages, limitations, and suitability of different technologies for specific applications and summarizes quality-control strategies addressing practical issues that may affect model reliability, including off-target effects, mosaicism, genetic background, and genetic stability. Finally, future directions in technologies for generating genetically engineered mice are discussed to inform the generation and application of related models.

[Purpose]To analyze the prevalence characteristics and disease burden of lung cancer in Hebei cancer registration areas from 2012 to 2020.[Methods]Lung cancer data were collected from the Hebei Provincial Cancer Registry from 2012 to 2020.The crude incidence/mortality rates,age-standardized incidence/mortality rates by Chinese standard population(ASIRC/ASMRC)and by world standard population(ASIRW/ASMRW)were calculated.The Joinpoint model was used to calculate the annual percentage change(APC)and average annual percentage change(AAPC).Years of life lost(YLL)and years lived with disability(YLD)and the disability-adjusted life years(DALY)were calculated.[Results]From 2012 to 2020,the ASIRW of lung cancer in Hebei can-cer registration areas was 33.13/105,44.56/105 for men and 22.54/105 for women,respectively;the incidence rates of urban and rural areas were 29.05/105 and 33.52/105,respectively.The incidence rates increased with ages,reaching a peak in the age group of 80～84 years old.There was a de-creasing trend in the ASIRW of lung cancer(AAPC=-3.99％,P＜0.05).From 2012 to 2020,the ASMRW of lung cancer was 25.80/105,36.56/105 for men and 15.96/105 for women,respectively;the mortality rates of urban and rural areas were 25.14/105 and 26.12/105,respectively.The mor-tality rates increase with ages,reaching a peak in the age group of 85 and above years old.There was a decreasing trend in the mortality of lung cancer(AAPC=-4.65％,P＜0.001)from 2012 to 2020.The DALY of lung cancer in Hebei Province from 2012 to 2020 was 484 194 person-years,with male accounting for 66.77％,female accounted for 33.23％,the DALY rate of lung cancer was 3.31‰,of which 35.57％in urban areas and 64.43％in rural areas.[Conclusion]Lung can-cer incidence and mortality rate in Hebei cancer registration areas from 2012 to 2020 showed a decreasing trend.The disease burden is gradually increasing with age in middle-aged and el-derly population.

Objective:To explore the changes of brain excitation/inhibition balance and gray matter volume (GMV) and their correlations with clinical features in benign childhood epilepsy with centrotemporal spikes (BECTS).Methods:A cross-sectional study was performed; 83 BECTS children enrolled from Department of Diagnostic Radiology, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University from January 2015 to January 2024 were selected as BECTS group. During the same period, 101 age- and gender-matched healthy children were recruited as healthy control group through advertisements in local primary schools. Data of conventional MRI and resting-state functional MRI (rs-fMRI) of the two groups were collected. Whole brain GMV was analyzed by voxel-based morphometry (VBM), and Hurst index was calculated based on time series data of blood oxygen level dependent (BOLD) signal of rs-fMRI. Correlations of GMV and Hurst index with disease duration and onset age in children with BECTS were explored by Pearson correlation analysis.Results:Compared with the healthy control group, the BECTS group had significantly increased GMV and decreased Hurst index in the bilateral Rolandic region ( P<0.05). Pearson correlation analysis showed that in the BECTS group, GMV in bilateral Rolandic region was negatively correlated with onset age ( r=-0.267, P=0.015) and positively correlated with disease course ( r=0.267, P=0.015); Hurst index in bilateral Rolandic region was positively correlated with onset age ( r=0.323, P=0.003) and negatively correlated with disease course ( r=-0.240, P=0.029); Hurst index was negatively correlated with GMV in bilateral Rolandic region ( r=-0.328, P=0.003). Conclusion:BECTS children have excitation/inhibition imbalance in epilepsy-related regions and cortical structural delay, and both of them are related to onset age and disease course.

AIM:To investigate the protective ef-fect of osthole(Ost)on APAP-induced liver injury in mice and its molecular mechanism.METHODS:We established the APAP-induced liver injury model in mice,and Ost was used to intervene.The expres-sion of AST,ALT,SOD,ROS,MDA,LDH,GSH-PX in mice plasma were detected by biochemical meth-od.HE staining was used to observe the changes of liver tissue structure.Immunofluorescence assay was used to detect the expression of Tif1γ and Smad4 in liver tissue.The mRNA expression of IL-1β,IL-6,TNF-α,Smad4,and Tif1γ were detected by qRT-PCR.Western blot was applied to assess the protein expression of Smad2/3 and pSmad2/3 in liver tissue.RESULTS:Compared with the control group,the liver structure destruction and hepato-cyte death was increased,ALT,AST,ROS,MDA and LDH were increased,while SOD and GSH-PX were decreased,and the mRNA expressions of IL-1β,IL-6 and TNF-α were increased in the model group.Compared with the model group,the Ost interven-tion group had improved liver structure and de-creased liver cell death;decreased ALT,AST,ROS,MDA and LDH,increased SOD and GSH-PX,and de-creased expression of IL-1β,IL-6 and TNF-α mRNA.Compared with the control group,liver tissues of model mice showed increased expression of pS-mad2/3,Smad4 protein and Smad4 mRNA,and de-creased Tif1γ protein and mRNA.Compared with the model group,the liver tissues of the Ost inter-vention group showed decreased expression of pS-mad2/3,Smad4 protein and Smad4 mRNA,and in-creased expression of Tif1γ protein and mRNA.CONCLUSION:Ost can improve liver function,re-duce oxidative stress and inflammatory reaction,and protect hepatocyte damage induced by APAP in mice,which may be related to the up-regulation of Tif1γ and inhibition of TGF-β1/Smad signaling pathway.

Objective:To explore the changes of brain excitation/inhibition balance and gray matter volume (GMV) and their correlations with clinical features in benign childhood epilepsy with centrotemporal spikes (BECTS).Methods:A cross-sectional study was performed; 83 BECTS children enrolled from Department of Diagnostic Radiology, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University from January 2015 to January 2024 were selected as BECTS group. During the same period, 101 age- and gender-matched healthy children were recruited as healthy control group through advertisements in local primary schools. Data of conventional MRI and resting-state functional MRI (rs-fMRI) of the two groups were collected. Whole brain GMV was analyzed by voxel-based morphometry (VBM), and Hurst index was calculated based on time series data of blood oxygen level dependent (BOLD) signal of rs-fMRI. Correlations of GMV and Hurst index with disease duration and onset age in children with BECTS were explored by Pearson correlation analysis.Results:Compared with the healthy control group, the BECTS group had significantly increased GMV and decreased Hurst index in the bilateral Rolandic region ( P<0.05). Pearson correlation analysis showed that in the BECTS group, GMV in bilateral Rolandic region was negatively correlated with onset age ( r=-0.267, P=0.015) and positively correlated with disease course ( r=0.267, P=0.015); Hurst index in bilateral Rolandic region was positively correlated with onset age ( r=0.323, P=0.003) and negatively correlated with disease course ( r=-0.240, P=0.029); Hurst index was negatively correlated with GMV in bilateral Rolandic region ( r=-0.328, P=0.003). Conclusion:BECTS children have excitation/inhibition imbalance in epilepsy-related regions and cortical structural delay, and both of them are related to onset age and disease course.

Objective:To understand the spatiotemporal distribution of pulmonary tuberculosis (TB) in Shaanxi Province from 2015 to 2023, and provide reference for the prevention and control of pulmonary TB in Shaanxi.Methods:The registration data of etiologically positive pulmonary TB cases in Shaanxi from 2015 to 2023 were collected from the tuberculosis subsystem of Chinese Disease Control and Prevention Information System. Descriptive method was used to analyze the basic characteristics of the etiologically positive pulmonary TB cases. Linear trend χ2 test was used to analyze trends in registration rate and pathogen positive rate. Software SPSS 25.0 was used for statistical analysis. Software ArcGIS 10.8 was used for global spatial autocorrelation and hotspot analysis to explore spatial clustering of the etiologically positive pulmonary TB cases. Software SaTScan 10.0 was used for spatiotemporal scan statistics, and software ArcGIS 10.8 was used to visualize the spatiotemporal clustering. Results:A total of 64 148 cases of etiologically positive pulmonary TB were registered in Shaanxi from 2015 to 2023, with an average annual registration rate of 18.33/100 000. The registration rate and pathgen positive rate all showed upward trends from 2015 to 2023, and the differences were significant (the trend χ2=4 555.18 and 19 330.43, both P<0.001). Global spatial autocorrelation and hotspot analysis showed that the registration rate of etiologically positive pulmonary TB in Shaanxi from 2017 to 2023 showed a spatial clustering. The hotspots were mainly in Zhenba and Xixiang counties of Hanzhong, six counties (districts) of Ankang, and Yanchuan and Yanchang counties of Yan'an. The coldspots were mainly in parts of the Guanzhong area, including Baoji, Xi'an, and Xianyang. A total of 4 spatiotemporal clustering areas were explored by spatiotemporal scanning analysis (all P<0.001), in which the first-level clustering areas covered 17 counties (districts), mainly Zhenping, Ziyang, Zhenba, in southern Shaanxi from 2019 to 2022, the second-level clustering areas covered 6 counties (districts), mainly Yanchuan, Yanchang, Qingjian, in northern Shaanxi from 2018 to 2021, the third-level clustering areas covered 14 counties (districts), mainly Yanta, Chang'an, Jingyang, in Guanzhong area from 2018 to 2019, and the fourth-level clustering areas covered 10 counties (districts) from 2019 to 2021. Conclusions:The registration rate of labortory confirmed pulmonary TB cases in Shaanxi showed an upward trend, with obvious differences in spatiotemporal clustering distribution. The clustering areas were mainly in southern Shaanxi, such as Zhenba, Zhenping, Hanbin, Langao, Pingli, Xunyang, Ziyang counties, and northern Shaanxi, such as Yanchuan and Yanchang counties, as well as in capital city, Xi'an and the adjacent Guanzhong area. It is necessary to develope targeted measures according to local conditions for the improvement of pulmonary TB prevention and control strategies in Shaanxi.

Objective:To develop and validate a novel PET tracer, N-cyclohexyl-4-((2, 4-dichlorophenyl)(4-(fluoro- 18F)phenyl)methyl)piperazine-1-carboxamide ( 18F-SQKJ-2), targeting cannabinoid type 1 (CB1) receptors for diagnosing psychiatric disorders associated with fear memory. Methods:18F-SQKJ-2 was prepared using a nucleophilic substitution radiochemical synthesis method. For the CB1 receptor blocking experiment, 7 ICR mice were divided into blocking group ( n=4; rimonabant for blocking treatment) and control group 1 ( n=3; no rimonabant blocking treatment). The affinity and specificity of 18F-SQKJ-2 for CB1 receptors were analyzed based on the differences in 18F-SQKJ-2 uptake (percentage injected dose per gram of tissue, %ID/g) by various organs between two groups. The metabolic stability of 18F-SQKJ-2 in vitro was studied using animal tissue homogenates. Ten C57 mice were used to establish fear memory mouse models (fear group, n=6; control group 2, n=4), and the percentage of freezing time was compared between 2 groups. MicroPET scans were used to detect the intracranial distribution of 18F-SQKJ-2, and the relative uptake in each brain region compared to total brain uptake was calculated. Statistical analysis was conducted to compare the differences in CB1 receptor relative total brain uptake in fear-related brain regions between 2 groups. Independent-sample t test and Mann-Whitney U test were used to analyze the data. Results:18F-SQKJ-2 was successfully synthesized with a radiochemical purity ≥98.0% and a corrected radioactive yield of (12.3±6.0)%( n=4). In vitro metabolic stability experiments showed that 18F-SQKJ-2 was basically stable in the liver, blood, and brain within 60min. The CB1 receptor blocking experiment demonstrated that the uptake of 18F-SQKJ-2 in the brains of mice in blocking group was significantly lower than that in control group 1 ((0.95±0.28) vs (3.44±1.16) %ID/g; t=-3.57, P=0.023). The percentage of freezing time in fear group was significantly higher than that in control group 2 (43.28%(39.46%, 52.93%) vs 2.74%(1.52%, 4.85%); Z=-2.45, P=0.010). 18F-SQKJ-2 microPET imaging showed that the uptake of 18F-SQKJ-2 in the cerebral cortex of mice in fear group was significantly increased compared with that in control group 2 ((5.83±0.47)% vs (5.00±0.52)%; t=2.42, P=0.046). Conclusion:18F-SQKJ-2 is successfully prepared with acceptable radiochemical purity and metabolic stability, demonstrating potential for visualizing and quantifying fear memory.

Objective:To assess the clinical value of digital PCR (dPCR) for the prenatal diagnosis of common fetal aneuploidies.Methods:A dPCR-based assay was developed for detecting trisomies 21, 18, and 13. A retrospective analysis was carried out on 173 amniotic fluid samples collected by the Prenatal Diagnosis Center of Taizhou Hospital between January 2017 and December 2023. By using chromosomal karyotyping as the gold standard, the diagnostic performance of the multiplex dPCR system was evaluated in a double-blind manner. This study has been approved by the Ethics Committee of Taizhou Hospital (Ethics No. K20250339).Results:Chromosomal karyotyping has identified 59 cases of trisomy 21, 5 cases of trisomy 18, 2 cases of trisomy 13, 6 cases with chromosomal structural abnormalities or mosaicisms, and 101 cases with a normal karyotype. The dPCR results ( Z-score cutoff = 4.0, CI = 99.997%) showed full concordance with karyotyping (sensitivity = 100%, specificity = 100%, Kappa = 1). Among the 6 structurally abnormal or mosaicism samples, dPCR has accurately detected 4 cases, but mis-classified 2 cases of trisomy 21 with very low-level mosaicisms (3.3%, 6.9%, respectively) as normal. Conclusion:The established multiplex dPCR system demonstrated high diagnostic accuracy for common chromosomal aneuploidies, with results available within 24 hours. It can serve as an efficient supplementary tool to conventional chromosomal karyotyping, providing reliable support for time-sensitive clinical decision-making in prenatal diagnosis.

Objective:To analyze the clinical characteristics and cytokines expression characteristics in infants with mild and severe respiratory syncytial virus (RSV) infection.Methods:From May 2023 to December 2023, plasma samples and clinical information were collected from 16 infants with RSV infection and 14 control infants. Cytek Aurora flow cytometry (Cytek, America) and Enzyme linked immunosorbent assay (ELISA) were used to detect the expression levels of 25 cytokines after mild and severe RSV infection.Results:Cough and nasal obstruction were the main clinical manifestations in infants with mild RSV infection, accompanied by polypnea, wheezing and other symptoms. The main symptoms of severe RSV infection were cough and rales, accompanied by fever and polypnea. In comparison with the control group, the expression levels of IL-2, IL-4, IL-5, IL-6, IL-9, IL-13, IL-22, TNF-α, IFN-α, IFN-β, MIP-1β, I-TAC, ENA-78, GROα, Eotaxin, and MCP-1 in the RSV infection group all exhibited an upregulation trend. Both IP-10 and MIP-3α demonstrated a downward trend in the RSV infection group; however, there was no statistically significant difference ( P>0.05). The levels of IL-10, IFN-γ, MIP-1α, and IL-8 in the RSV infection group were significantly higher than those in the control group, whereas the levels of MIG, TARC, and RANTES in the RSV infection group were significantly lower than those in the control group ( P<0.05). The levels of IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-22, IFN-β, IFN-γ, TNF-α, IL-8, I-TAC, MIP-1β, Eotaxin, and MCP-1 in the mild RSV infection group were significantly higher than those in the severe RSV infection group ( P>0.05). Among these, the levels of MIG, RANTES, TARC, MIP-3α, and ENA-78 in the mild infection group were all lower than those in the severe infection group. The expressions of ENA-78 and MIP-1α in the severe infection group were significantly higher than those in the mild infection group and also higher than those in the control group. There was no significant difference in IP-10 and GROα between the mild and severe RSV infection groups ( P>0.05). Conclusions:The differences in clinical features and cytokines between infants with mild and severe RSV infection provide important data support for the prevention and treatment of RSV infection in infants.