Objective To establish a rat model of Alzheimer's disease (AD) expressing human triple mutant amyloid precursor protein (APP) in the hippocampus, and to provide a model for the study of disease mechanisms and drug development. Methods Twenty-four 12-week-old SPF-grade female SD rats were randomly divided into a blank control group, a virus control group and an experimental group, with eight rats in each group; among them, the experimental group received a stereotaxic injection of adeno-associated virus (AAV) carrying the human triple mutant APP and NanoLuc luciferase genes into the hippocampus. In vivo imaging was used to observe viral expression in the brains of rats in each group, the novel object recognition test was used to assess the recognition memory of the rats in each group, real-time fluorescent quantitative PCR was used to detect the expression level of the APP gene, HE staining was used to examine the brain histopathology, Nissl staining was used to assess the hippocampal lesions, and immunohistochemistry was used to detect the deposition of amyloid β-protein (Aβ). Results In vivo imaging showed that reporter fluorescence was detected in the brains of rats in both experimental and virus control groups. Fluorescence quantitative PCR showed that the expression level of the APP gene was significantly increased in the brains of rats in the experimental group (P<0.01). Novel object recognition test revealed that the recognition memory of rats in the experimental group was significantly reduced compared with that of the blank control group (P<0.01). Six months after recombinant AAV virus infection, HE staining and Nissl staining of brain tissues showed that the number of neurons and Nissl bodies in the CA1 region of the hippocampus in the experimental group was reduced and disorganized; immuno-histochemistry testing of the CA1 region of the hippocampus and the pyramidal cell layer of the experimental group revealed prominent brown deposits, indicating Aβ protein deposition. Conclusion The rat model successfully established by stereotaxic injection and AAV-mediated delivery of human triple mutant APP gene exhibits typical AD features, providing a valuable animal model for studying AD pathology and developing drug therapies targeting Aβ protein deposition.

Objective To establish a rat model of Alzheimer's disease (AD) expressing human triple mutant amyloid precursor protein (APP) in the hippocampus, and to provide a model for the study of disease mechanisms and drug development. Methods Twenty-four 12-week-old SPF-grade female SD rats were randomly divided into a blank control group, a virus control group and an experimental group, with eight rats in each group; among them, the experimental group received a stereotaxic injection of adeno-associated virus (AAV) carrying the human triple mutant APP and NanoLuc luciferase genes into the hippocampus. In vivo imaging was used to observe viral expression in the brains of rats in each group, the novel object recognition test was used to assess the recognition memory of the rats in each group, real-time fluorescent quantitative PCR was used to detect the expression level of the APP gene, HE staining was used to examine the brain histopathology, Nissl staining was used to assess the hippocampal lesions, and immunohistochemistry was used to detect the deposition of amyloid β-protein (Aβ). Results In vivo imaging showed that reporter fluorescence was detected in the brains of rats in both experimental and virus control groups. Fluorescence quantitative PCR showed that the expression level of the APP gene was significantly increased in the brains of rats in the experimental group (P<0.01). Novel object recognition test revealed that the recognition memory of rats in the experimental group was significantly reduced compared with that of the blank control group (P<0.01). Six months after recombinant AAV virus infection, HE staining and Nissl staining of brain tissues showed that the number of neurons and Nissl bodies in the CA1 region of the hippocampus in the experimental group was reduced and disorganized; immuno-histochemistry testing of the CA1 region of the hippocampus and the pyramidal cell layer of the experimental group revealed prominent brown deposits, indicating Aβ protein deposition. Conclusion The rat model successfully established by stereotaxic injection and AAV-mediated delivery of human triple mutant APP gene exhibits typical AD features, providing a valuable animal model for studying AD pathology and developing drug therapies targeting Aβ protein deposition.

Human naïve pluripotent stem cells (PSCs) hold great promise for embryonic development studies. Existing induction and culture strategies for these cells, heavily dependent on MEK inhibitors, lead to widespread DNA hypomethylation, aberrant imprinting loss, and genomic instability during extended culture. Here, employing high-content analysis alongside a bifluorescence reporter system indicative of human naïve pluripotency, we screened over 1,600 chemicals and identified seven promising candidates. From these, we developed four optimized media-LAY, LADY, LUDY, and LKPY-that effectively induce and sustain PSCs in the naïve state. Notably, cells reset or cultured in these media, especially in the LAY system, demonstrate improved genome-wide DNA methylation status closely resembling that of pre-implantation counterparts, with partially restored imprinting and significantly enhanced genomic stability. Overall, our study contributes advancements to naïve pluripotency induction and long-term maintenance, providing insights for further applications of naïve PSCs.
Humans ; DNA Methylation/drug effects* ; Genomic Instability ; Pluripotent Stem Cells/metabolism* ; Cell Culture Techniques/methods* ; Cells, Cultured

Senecavirus A (SVA) is a major viral pathogen causing disease in pigs, and effective monitoring of SVA infection is critical for disease control. In this study, we aimed to develop a reliable ELISA method for rapidly detecting neutralizing antibodies against SVA. We used HEK293F cells to express an SVA-specific porcine Fab antibody and verified the biological activity of the Fab antibody by indirect ELISA, immunofluorescence assay, virus neutralization test, and Western blotting. The Fab antibody was biotinylated and used as a competitive antibody to establish a competitive ELISA (C-ELISA) for detecting neutralizing antibodies against SVA. We then evaluated the C-ELISA in terms of sensitivity, specificity, repeatability, and result agreement rate with the VNT. The results showed that we successfully prepared an SVA-specific porcine Fab antibody, which showed high affinity for SVA. We named this antibody 1M33Fab and designated it as Bio-1M33Fab after biotin labeling. The assay conditions were optimized as follows: the coating concentration of SVA particles being 1 μg/mL, the working concentration of Bio-1M33Fab being 0.5 μg/mL, the optimal serum dilution of 1:10, and the optimal dilution of enzyme-labeled avidin being 1:30 000. At a percent inhibition (PI) of 47%, the assay demonstrated the highest sensitivity (96.88%) and specificity (100%), with no cross-reactivity observed with the positive sera of major porcine viral diseases. The intra-assay coefficient of variation ranged from 1.12% to 7.34%, while the inter-assay coefficient of variation ranged from 1.10% to 8.97%, indicating good repeatability. In the detection of 224 clinical pig serum samples, C-ELISA and VNT showed a result agreement rate of 93.75%. In conclusion, we successfully develop a C-ELISA method for detecting neutralizing antibodies against SVA by using a porcine-derived Fab antibody, which lays a foundation for the development of detection kits.
Animals ; Swine ; Antibodies, Neutralizing/immunology* ; Enzyme-Linked Immunosorbent Assay/methods* ; Immunoglobulin Fab Fragments/immunology* ; Antibodies, Viral/immunology* ; Picornaviridae/immunology* ; Humans ; HEK293 Cells ; Swine Diseases/diagnosis* ; Picornaviridae Infections/diagnosis*

ObjectiveTo investigate the role of sphingosine-1-phosphate （S1P） in abnormal ossification in ankylosing spondylitis （AS）， clarify the relationship between S1P and “angiogenesis-osteogenesis” coupling， and provide new strategies for AS treatment. MethodsFemoral heads from AS patients and patients undergoing routine hip replacement were collected for immunohistochemical （IHC） staining to evaluate osteogenesis and H-type vessel formation. In vitro， ELISA was used to quantify the synthesis of S1P and analyze the expression changes of S1P signaling pathway-related molecules during the osteogenic differentiation of mesenchymal stem cells derived from patients with ankylosing spondylitis （ASMSCs） and those from healthy donors （HDMSCs）， to evaluate the activation status of S1P pathway during osteogenesis. Sphingosine kinase 1 （SK1） expression was knocked down in MSCs， and the S1P receptor inhibitor FTY720 was applied to block S1P signaling. Alkaline phosphatase （ALP） activity and Alizarin Red S （ARS） quantification were used to assess the effect of S1P on ASMSCs osteogenesis. Conditioned medium from osteogenically induced MSCs was used to treat human umbilical vein endothelial cells （HUVECs） to evaluate the effect of S1P on angiogenesis. An AS mouse model （SKG mice） was treated with FTY720 or the SK1 inhibitor PF-543 citrate. IHC staining and micro-CT scanning were used to assess abnormal ossification and spinal fusion， and immunofluorescence was used to evaluate H-type vessel formation. ResultsCompared with Osteonecrosis of the Femoral Head（ONFH） patients， AS patients exhibited excessive osteogenesis and H-type vessel formation （OCN P＜0.001， CD31 P＜0.001， EMCN P＜0.001）. During osteogenic differentiation， S1P expression and secretion were significantly higher in ASMSCs than in HDMSCs （P=0.0179）. Inhibition of S1P signaling with FTY720 or SK1 knockdown significantly suppressed osteogenic differentiation （compared with ASMSC， ARS： HDMSC P=0.001 8， FTY720 P＜0.001， si-SK1 P＜0.001； ALP： HDMSC P=0.032 8， FTY720 P=0.001 6， si-SK1 P＜0.001） of ASMSCs and the angiogenesis of HUVEC（compared with ASMSC， cell-covered area， total loops， total tube length and total branch points P＜0.001）. Treatment with FTY720 or PF-543 markedly inhibited abnormal ossification and spinal fusion（compared with Curdlan， arthritis index score， P＜0.001； OCN：control P=0.002， PF-543 P=0.010 7， FTY720 P=0.015 9 ） in AS mice and reduced H-type vessel formation （CD31⁺EMCN⁺： compared with curdlan， control P＜0.001， PF-543 P=0.001 7， FTY720 P=0.002 1）. ConclusionIncreased S1P synthesis in ASMSCs promotes osteogenic differentiation via autocrine mechanisms and further enhances ossification by facilitating H-type angiogenesis. Inhibiting S1P secretion in ASMSCs significantly suppresses abnormal ossification in AS.

Objective:To explore the characteristics of endothelial function in children with incomplete Kawasaki disease (IKD) of different ages and its relationship with coronary artery lesion (CAL).Methods:A total of 200 children with IKD admitted to the Affiliated Hospital of Jining Medical University from February 2020 to May 2022 were selected as the IKD group, and another 200 healthy children who underwent physical examinations during the same period were selected as the control group. According to the age of children with IKD, infants ( n=78) were classified as those under 1 year old, infants ( n=62) were classified as those between 1-3 years old, and preschool children ( n=60) were classified as those over 3-6 years old. The endothelial function characteristics of IKD children in different age groups and normal control group children were analyzed and compared. In addition, IKD patients were divided into CAL group ( n=110) and nCAL group ( n=90) based on whether CAL was merged. The age, carotid intima-media thickness (IMT), flow-mediated-dilation (FMD), carotid stiffness index (SI), nitroglycerin-mediated dilation (NMD), tumor necrosis factor α (TNF-α), C-reactive protein (CRP), brachial artery endothelial dependent dilation function (EDD) and other clinical data were compared between the two groups. Cox proportional hazards model was used for univariate and multivariate analysis to identify the risk factors for CAL in IKD patients. A column chart prediction model was constructed and evaluated. Results:There were statistically significant differences in FMD, NMD, SI, IMT, reactive congestion index (RHI), and EDD between IKD patients and normal control group children (all P<0.05). There were statistically significant differences in FMD, NMD, SI, IMT, RHI, and EDD among three groups of IKD children of different ages (all P<0.05). There were statistically significant differences in the proportion of children aged ≤3 years, TNF-α, CRP, SI, IMT, EDD, FMD, and NMD between the nCAL and CAL groups (all P<0.05). Age ≤3 years, elevated levels of TNF-α, CRP, SI, IMT, and EDD, as well as decreased levels of FMD and NMD, were independent risk factors for CAL in children with IKD (all P<0.05). The column chart prediction model had high discrimination, and the area under the curve for predicting CAL occurrence in children with IKD was 0.868 and 0.830, respectively. Conclusions:There are significant differences in endothelial function among children with IKD of different ages, and endothelial function is closely related to the occurrence of CAL. When endothelial function is abnormal, CAL is more likely to occur.

Objective:To investigate the clinical characteristics of psoriasis and status quo of medical care for patients in county areas of China.Methods:This study was a cross-sectional investigation. Based on the “Qianxian Wuyin” Project (a national project for upgrating ability for psoriasis care at county level), an online questionnaire survey was conducted in the dermatology departments of 459 county hospitals in 404 pilot administrative counties across China from February to June 2023. The questionnaire included demographic information of patients (gender, ethnicity, age, place of residence, education, marital status), and clinical characteristics of psoriasis (disease course, type, comorbidities, body surface area (BSA) and previous treatment. The Dermatology Life Quality Index (DLQI) and Psoriasis Area and Severity Index (PASI) were applied for assessing the quality of life and disease severity, and completed by patients or guardian and doctors, respectively.Results:A total of 16 935 patients completed the questionnaire. The age of patients was 1-102(44.17±11.58)years, and 71.0% (12 036/16 935) were 30-59 years old. The ratio of male to female was 2.21∶1; 24.3%(4 117/16 935) of patients had high school education; there were 9 940 patients(58.7%) with previous or current smoking and/or alcohol use; 42.8%(7 218/16 855) of patients had a disease course of 1-5 years. There were 15 630 patients(92.3%) with DLQI≥10, 8 346 patients(49.7%) with PASI≥10, 15 017 patients(89.2%) with BSA≥10%. The plaque type was the most common disease type ( n=14 965, 88.7%), and spotting type ranked the second ( n=1 141, 6.8%). The most common initial site was the trunk ( n=12 309, 72.9%). Among the comorbidities, hypertension was the most common one ( n=1 681, 10.0%). There were 7 650 reports of treatment response to conventional topical drug therapy and 3 112 reports of treatment response to systemic drug therapy, with 6 269 (81.9%) and 2 493 (80.1%) reporting poor or no response, respectively. Conclusions:The survey shows that in the county areas of China, the majority of psoriasis patients are severe patients with short course of disease, plaque type is the most common type, and hypertension is the most common comorbidity; and the conventional treatment is less effective for most patients.

Objective To explore the effects of drug therapy combined with multi-dimensional psychological intervention on the insomnia of shift medical staff in"sunshine hospital"mode,and to provide a reference for improving the physical and mental health of shift medical staff.Methods A total of 140 cases of medical staff with insomnia in shifts in Wuhan were included and divided into study group and control group by random number table method,70 cases in each group.The control group was given drug treatment,and the study group was treated combined with multi-dimensional psychological intervention.based on"sunshine hospital"model.The scale and questionnaire of PSQI,ISI,ESS and FAS were used to evaluate sleep status and HAMD,HAMA,PHQ-9 and SAS were used to evaluate psychological status,at baseline,visit 1(baseline+intervention for 1 month)and visit 2(baseline+intervention for 3 months),respectively.Results(1)Sleep status:There were differences in 7 dimensions of PSQI score,ISI,ESS,and FAS score between the study group and the control group at different periods(P<0.05);Among them,the PSQI scores of the two groups showed significant difference between the two groups at interview 2(P<0.05);There were significant differences in sleep quality,sleep efficiency scores,ESS and FAS between the two groups at visit 1 and visit 2(P<0.05).(2)Psychological status:HAMD,HAMA,PHQ-9 and SAS scores of the study group and the control group were different in different periods(P<0.05).At interview 2,there were significant differences in HAMA and SAS scores between the two groups(P<0.05).Conclusion Based on the"sunshine hospital"model of drug therapy combined with multi-dimensional psychological intervention,it can significantly improve sleepiness,fatigue symptoms,sleep quality,sleep efficiency,and anxiety and depression of first-line shift medical staff in the short term,and significantly alleviate overall insomnia and anxiety in the long term.