Hemoglobin A1c (HbA1c) has a unique structure that makes monoclonal antibody (mAb) preparation challenging. This study aims to develop a method for preparing HbA1c mAbs and establish a fluorescent immunochromatographic assay (FICA) for rapid detection of HbA1c. Three glycosylated peptides were synthesized and used to prepare complete antigens, which were identified by dot enzyme-linked immunosorbent assay (Dot-ELISA) and ultraviolet absorption spectroscopy. The complete antigens and natural HbA1c were used for cross-immunization of mice, and the optimal complete antigen was selected. The mouse with the highest serum titer was chosen for mAb preparation. The purity and specificity of the mAbs were verified, and a FICA method was developed. The optimal complete antigen, with a titer of 1:512 000, was successfully prepared and selected. Fusion with splenocytes resulted in four specific HbA1c antibodies (purity > 90%). The best antibody exhibited a binding constant (Ka) of 1.67×10¹⁰ L/mol with the antigen. Based on this antibody, a FICA method was successfully established, capable of producing results within 15 min. The method demonstrated a good linear range (3%-13% HbA1c, y=0.071 3x+0.005 6, R²=0.993 7), recovery rates of 98%-102%, precision < 10.00%, and no nonspecific reactions. Clinical testing of 210 samples showed positive agreement of 96.36%, negative agreement of 97.00%, and overall agreement of 96.68%. The receiver operating characteristic (ROC) curve analysis yielded an area under curve (AUC) of 0.980 9 [95% confidence interval (CI): 0.961 0-1.000 0], with high consistency verified in multicenter studies. We successfully developed a key technique for preparing HbA1c monoclonal antibodies and established a FICA method for rapid detection of HbA1c. It will provide an efficient and convenient detection method for the early diagnosis and long-term management of diabetes and its complications.
Antibodies, Monoclonal/biosynthesis* ; Animals ; Mice ; Glycated Hemoglobin/immunology* ; Mice, Inbred BALB C ; Humans ; Antibody Specificity ; Chromatography, Affinity/methods* ; Enzyme-Linked Immunosorbent Assay/methods* ; Female

Aiming at the problems of tongue diagnosis instruments relying on manual operation,uneven illumination,color distortion and lack of dynamic information,an automated operation black box tongue diagnosis instrument based on machine vision is proposed.This instrument utilizes machine vision technology and image processing algorithms to achieve automatic positioning and automatic acquisition of tongue manifestations.High-resolution camera technology enables the acquisition of 3-second dynamic videos and high-quality static images.The performance test results show that,based on the dual-camera geometric projection calibration algorithm,the average time for tongue recognition is less than 0.15 seconds.The diffuse reflection light source improves the gray uniformity to 76.74%,and the maximum color difference in color reproduction degree is 19,which is better than the industry standard value of 25.The developed tongue diagnosis instrument will promote the objective,efficient and precise acquisition of tongue diagnosis in traditional Chinese medicine.

Objective To explore the application value of intravoxel incoherent motion(IVIM)imaging parameters in the differential diagnosis of benign and malignant breast nodules and their correlation with the expression of Ki-67 receptor in breast cancer.Methods The data of 41 female patients who completed 3.0 T breast magnetic resonance imaging(MRI)with complete surgical pathology results in the Second Hospital of Jiaxing City from October 2022 to January 2025 were analysed and evaluated.Conventional images and diffusion-weighted images with 11 b values were collected.The IVIM imaging parameters of real diffusion coefficient(D),perfusion related diffusion coefficient(D*)and perfusion fraction(f)were measured and calculated in the region of interest of each lesion.The receiver operating characteristic curve were plotted to quantify the differential diagnostic efficacy of each parameter of IVIM imaging in benign and malignant breast nodules.The differences of parameters between benign and malignant breast nodules and between the groups with different expression levels of Ki-67 receptor in breast cancer were analysed,and the correlation between each parameter and the expression level of Ki-67 in breast cancer was counted.Results The D value of benign breast nodules group(benign groups)was significantly higher than that of malignant breast nodules group(malignant groups),and the D*value was significantly lower than that of malignant group,and the differences between benign group and malignant group were statistically significant(t=-4.773,t=2.063,P＜0.05);Thefvalue of benign group was slightly lower than that of malignant group,and the differences between two groups were not statistically significant(t=0.035,P＞0.05).Among the parameters of IVIM imaging,D value had the best differential diagnostic efficacy for benign and malignant breast nodules,with area under the curve(AUC)of 0.870(95％CI:0.755-0.985)and a specificity of 75.0％;D*value had the second best differential diagnostic efficacy after D value,with an AUC of 0.789(95％CI:0.658-0.920),but it had the highest sensitivity of 88.2％;And the differential diagnosis efficiency off value was the worst,much less than D and D*values.The D value in the high Ki-67 expression group of breast cancer was lower than that in the low expression group,while the D* and f values in the high expression group were higher than that in the low expression group,and the differences of each imaging parameter of IVIM were not statistically significant between two groups(t=-2.617,t=2.169,t=0.647,P＞0.05).There was a significant negative correlation between the expression of the Ki-67 receptor and the D value(r=-0.615,P＜0.05),and no significant correlation was seen with either D *or f value(r=0.223,r-0.031,P＞0.05).Conclusion The D and D*values of IVIM imaging parameters have great clinical value in the differential diagnosis of benign and malignant breast nodules.

Objective To conduct a scoping review of the application of gamification programs in exercise rehabilitation for knee osteoarthritis,examining the application carriers,game content,outcome indicators,measurement tools,and application effects of gamification programs,to provide references for future practice and related research in this field.Methods The search was conducted in PubMed,Web of Science,Cochrane Library,Scopus,CIN AHL,China National Knowledge Infrastructure,CNKI,and Wanfang Data from their inception to December 28,2024.The included studies were summarized and analyzed.Results A total of 28 studies were included in the review.The game carriers encompassed virtual reality technology,sensor devices,and mobile applications.The game content covered interactive motion games,aerobic exercises,and goal-motivated games.Outcome indicators included functional performance,physical activity,pain,psychological and health status,and user experience.Gamification programs were found to effectively enhance user engagement,improve physical function,reduce negative emotions,and improve quality of life.However,there remains controversy regarding their efficacy in pain relief.Conclusion Gamification programs have shown positive effects in exercise rehabilitation for knee osteoarthritis.Future efforts should focus on developing gamification programs that are culturally appropriate for China,creating"digital therapeutics",continuously updating systems,conducting economic evaluations,and ensuring digital equity to enhance patients'rehabilitation experiences and improve health outcomes.

To prepare a recombinant adeno-associated virus(rAAV9-ZsGreen1-EsASP)capable of expressing the aspartic protease protein of Eimeria stiedai(E.stiedai),and explore its in vitro ac-tivity.The EsASP gene was amplified by PCR,and recombinant adeno-associated viral vector pAAV-IRES-ZsGreen1 was combined with the EsASP gene using homologous recombination to construct the pAAV-ZsGreen1-EsASP expression plasmid;pAAV-ZsGreen1-EsASP expression plasmid,pHelper,and pAAV-RC9 plasmids were cotransfected into HEK-293T cells by liposomal transfection to package and produce recombinant adeno-associated virus rAAV9-ZsGreen1-EsASP capable of expressing EsASP protein.rAAV9-ZsGreen1-EsASP was purified using chloroform treatment-PEG/NaCl precipitation-chloroform extraction method,the purity of the virus was iden-tified by silver staining,the virus morphology was observed by TEM,and virus titer was detected by qRT-PCR;the purified recombinant virus was further infected into HEK-293T cells,and EsASP expression was detected by observing green fluorescent protein ZsGreen1 and Western blot method.The results indicated that double enzyme digestion and DNA sequencing confirmed that the EsASP gene had been successfully constructed into the pAAV-IRES-ZsGreen1 expression plas-mid.The expression of green fluorescent protein in HEK-293T cells suggested that co-transfection was successful.Western blot results of cell protein preparation showed that EsASP protein was successfully expressed;The purified recombinant viral capsid proteins show three distinct bands(VP1-3).The purified rAAV9-ZsGreen1-EsASP was uniform in size,around 20 nm,and had a titer higher than 1.0 × 1012 vg/mL;Green fluorescent protein expression was observed after infection of HEK-293T cells with the recombinant virus,and EsASP expression was detected by Western blot.The results suggest that rAAV9-ZsGreen1-EsASP with in vitro infectious activity was suc-cessfully obtained,providing a material basis for the development of a novel vaccine against Eimer-ia stiedai.

Objective To explore the application value of intravoxel incoherent motion(IVIM)imaging parameters in the differential diagnosis of benign and malignant breast nodules and their correlation with the expression of Ki-67 receptor in breast cancer.Methods The data of 41 female patients who completed 3.0 T breast magnetic resonance imaging(MRI)with complete surgical pathology results in the Second Hospital of Jiaxing City from October 2022 to January 2025 were analysed and evaluated.Conventional images and diffusion-weighted images with 11 b values were collected.The IVIM imaging parameters of real diffusion coefficient(D),perfusion related diffusion coefficient(D*)and perfusion fraction(f)were measured and calculated in the region of interest of each lesion.The receiver operating characteristic curve were plotted to quantify the differential diagnostic efficacy of each parameter of IVIM imaging in benign and malignant breast nodules.The differences of parameters between benign and malignant breast nodules and between the groups with different expression levels of Ki-67 receptor in breast cancer were analysed,and the correlation between each parameter and the expression level of Ki-67 in breast cancer was counted.Results The D value of benign breast nodules group(benign groups)was significantly higher than that of malignant breast nodules group(malignant groups),and the D*value was significantly lower than that of malignant group,and the differences between benign group and malignant group were statistically significant(t=-4.773,t=2.063,P＜0.05);Thefvalue of benign group was slightly lower than that of malignant group,and the differences between two groups were not statistically significant(t=0.035,P＞0.05).Among the parameters of IVIM imaging,D value had the best differential diagnostic efficacy for benign and malignant breast nodules,with area under the curve(AUC)of 0.870(95％CI:0.755-0.985)and a specificity of 75.0％;D*value had the second best differential diagnostic efficacy after D value,with an AUC of 0.789(95％CI:0.658-0.920),but it had the highest sensitivity of 88.2％;And the differential diagnosis efficiency off value was the worst,much less than D and D*values.The D value in the high Ki-67 expression group of breast cancer was lower than that in the low expression group,while the D* and f values in the high expression group were higher than that in the low expression group,and the differences of each imaging parameter of IVIM were not statistically significant between two groups(t=-2.617,t=2.169,t=0.647,P＞0.05).There was a significant negative correlation between the expression of the Ki-67 receptor and the D value(r=-0.615,P＜0.05),and no significant correlation was seen with either D *or f value(r=0.223,r-0.031,P＞0.05).Conclusion The D and D*values of IVIM imaging parameters have great clinical value in the differential diagnosis of benign and malignant breast nodules.

Objective To conduct a scoping review of the application of gamification programs in exercise rehabilitation for knee osteoarthritis,examining the application carriers,game content,outcome indicators,measurement tools,and application effects of gamification programs,to provide references for future practice and related research in this field.Methods The search was conducted in PubMed,Web of Science,Cochrane Library,Scopus,CIN AHL,China National Knowledge Infrastructure,CNKI,and Wanfang Data from their inception to December 28,2024.The included studies were summarized and analyzed.Results A total of 28 studies were included in the review.The game carriers encompassed virtual reality technology,sensor devices,and mobile applications.The game content covered interactive motion games,aerobic exercises,and goal-motivated games.Outcome indicators included functional performance,physical activity,pain,psychological and health status,and user experience.Gamification programs were found to effectively enhance user engagement,improve physical function,reduce negative emotions,and improve quality of life.However,there remains controversy regarding their efficacy in pain relief.Conclusion Gamification programs have shown positive effects in exercise rehabilitation for knee osteoarthritis.Future efforts should focus on developing gamification programs that are culturally appropriate for China,creating"digital therapeutics",continuously updating systems,conducting economic evaluations,and ensuring digital equity to enhance patients'rehabilitation experiences and improve health outcomes.

To prepare a recombinant adeno-associated virus(rAAV9-ZsGreen1-EsASP)capable of expressing the aspartic protease protein of Eimeria stiedai(E.stiedai),and explore its in vitro ac-tivity.The EsASP gene was amplified by PCR,and recombinant adeno-associated viral vector pAAV-IRES-ZsGreen1 was combined with the EsASP gene using homologous recombination to construct the pAAV-ZsGreen1-EsASP expression plasmid;pAAV-ZsGreen1-EsASP expression plasmid,pHelper,and pAAV-RC9 plasmids were cotransfected into HEK-293T cells by liposomal transfection to package and produce recombinant adeno-associated virus rAAV9-ZsGreen1-EsASP capable of expressing EsASP protein.rAAV9-ZsGreen1-EsASP was purified using chloroform treatment-PEG/NaCl precipitation-chloroform extraction method,the purity of the virus was iden-tified by silver staining,the virus morphology was observed by TEM,and virus titer was detected by qRT-PCR;the purified recombinant virus was further infected into HEK-293T cells,and EsASP expression was detected by observing green fluorescent protein ZsGreen1 and Western blot method.The results indicated that double enzyme digestion and DNA sequencing confirmed that the EsASP gene had been successfully constructed into the pAAV-IRES-ZsGreen1 expression plas-mid.The expression of green fluorescent protein in HEK-293T cells suggested that co-transfection was successful.Western blot results of cell protein preparation showed that EsASP protein was successfully expressed;The purified recombinant viral capsid proteins show three distinct bands(VP1-3).The purified rAAV9-ZsGreen1-EsASP was uniform in size,around 20 nm,and had a titer higher than 1.0 × 1012 vg/mL;Green fluorescent protein expression was observed after infection of HEK-293T cells with the recombinant virus,and EsASP expression was detected by Western blot.The results suggest that rAAV9-ZsGreen1-EsASP with in vitro infectious activity was suc-cessfully obtained,providing a material basis for the development of a novel vaccine against Eimer-ia stiedai.

Aiming at the problems of tongue diagnosis instruments relying on manual operation,uneven illumination,color distortion and lack of dynamic information,an automated operation black box tongue diagnosis instrument based on machine vision is proposed.This instrument utilizes machine vision technology and image processing algorithms to achieve automatic positioning and automatic acquisition of tongue manifestations.High-resolution camera technology enables the acquisition of 3-second dynamic videos and high-quality static images.The performance test results show that,based on the dual-camera geometric projection calibration algorithm,the average time for tongue recognition is less than 0.15 seconds.The diffuse reflection light source improves the gray uniformity to 76.74%,and the maximum color difference in color reproduction degree is 19,which is better than the industry standard value of 25.The developed tongue diagnosis instrument will promote the objective,efficient and precise acquisition of tongue diagnosis in traditional Chinese medicine.

Objective To investigate the regulatory mechanism of apurnic/apyrimidinic endonuclease 1(APE1)in the transformation of chronic intestinal inflammation to colitis-associated colorectal cancer(CAC).Methods C64S mutant(APE1C64S)mice and APE1 wild type(APE1WT)mice were randomly divided into experimental group and control group.In vivo CAC model was established by azoxymethane(AOM)and dextran sulfate sodium salt(DSS)solution.Immunohistochemistry(IHC)and multiple IHC(mIHC)assays were used to observe the expression of APE1 and immune cell infiltration in colon tissues of each group.A mouse colon cancer cell line MC38 with stable knockdown of APE1 was constructed by lentivirus transfection,and subcutaneous tumor bearing experiments were performed in APE1WT and APE1C64S mice to confirm that tumor cell-derived APE1 caused immunosuppressive tumor microenvironment.The expression of APE1 and CXCL1[chemokine(C-X-C motif)ligand 1]and the infiltration of immune cells in tumor-bearing specimens were analyzed by IHC and mIHC assays.The tumor specimens of a 28-year-old female patient with CAC from Army Medical Center of PLA were analyzed for the expression of APE1 and CXCL1 and the infiltration of immune cells in the tumor and adjacent inflammatory tissues by IHC and mIHC assays.Results Compared with the control group and APE1WT erperimental group,APE1C64S erperimental group had significantly reduced disease activity index and tumor formation,polymorphonuclear myeloid-derived suppressor cells(PMN-MDSCs)infiltration,and CD4+and CD8+T cells(P＜0.05).No significant differences were observed in tumor growth and immune cells between APE1WT and APE 1C64S mice bearing subcutaneous tumors.However,in the tumor-bearing experiment using tumor cells with knockdown of APE1,the tumor growth was significantly lower and the number of infiltrated PMN-MDSCs was reduced,while those of CD4+and CD8+T cells were significantly increased(P＜0.05).Furthermore,high expression of APE1 and increased infiltration of PMN-MDSCs were found in the tumor tissues of the young CAC patient,and CD8+T cells were significantly reduced in the tumor tissues compared with the inflammatory tissues(P＜0.05).Conclusion APE1-redox in tumor cells can promote the infiltration of PMN-MDSCs and reduce the number of T cells,thereby forming an immunosuppressive tumor microenvironment and mediating the occurrence and development of CAC.