Objective To investigate the killing effect of the recombinant oncolytic influenza virus OvFlu-GM-CSF,constructed using reverse genetics(RG)technology,in combination with chemotherapy drug,gemcitabine(GEM),against pancreatic cancer cells.Methods The recombinant oncolytic virus OvFlu-GM-CSF was successfully rescued using RG technology in our previous study.The virus was then comprehensively characterized through chicken red blood cell hemagglutination assay,transmission electron microscopy,and viral replication assay.CCK-8 assay was utilized to determine the impact of OvFlu-GM-CSF viruses[multiplicities of infection(MOI):0,0.1,1.0,3.0]on the survival rate of pancreatic cancer cell lines(Panc02,PANC-1,SW1990,BxPC-3)and normal pancreatic ductal epithelial cells(HPDE6-C7)after treatment for 24,48 or 72 h.Using a subcutaneous tumor-bearing mouse model of pancreatic cancer,36 female C57BL/6 mice(6 weeks old)were randomly divided into PBS group,recombinant oncolytic virus group,GEM group,and the combined treatment group,with 9 mice in each group.PBS(100 μL/animal)or OvFlu-GM-CSF virus(1× 107PFU/100 μL)was given to the mice of the corresponding groups through intratumoral injection,while GEM(100 mg/kg)was injected intraperitoneally,once per 3 days,for totally 9 times.Changes in tumor volume and survival rate were monitored.Multi-immunofluorescence staining was employed to analyze T cell infiltration and proliferation in the tumor tissues.HE staining was performed to observe the pathological changes in major organs(heart,liver,lungs,kidneys and brain),and the serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were measured to evaluate the safety of the recombinant oncolytic virus.Results The recombinant oncolytic influenza virus OvFlu-GM-CSF has a hemagglutination titer of 28,typical morphological features of influenza virus,and can selectively replicate within pancreatic cancer cells.At the cellular level,the viruses demonstrated a significant selective cytotoxic effect on Panc02,PANC-1,SW1990,and BxPC-3 cells under the conditions of 48 h post-infection and MOI=3.0,when compared to 48 h post-infection and MOI=0(P＜0.01).The cell survival rate was gradually decreased with the increase in MOI value and the extension of infection time(P＜0.01),but the viruses showed no significant effect on normal pancreatic ductal epithelial cells(HPDE6-C7).In the pancreatic cancer tumor-bearing mouse model,the combined treatment of the viruses+GEM significantly reduced the tumor volume than simple virus treatment and simple GEM treatment(P＜0.01),and enhanced the infiltration of T cells in the tumor tissues.No obvious pathological changes were observed in the above-mentioned major organs.Additionally,there were no significant differences in the serum levels of ALT and AST in the OvFlu-GM-CSF group,GEM group,and OvFlu-GM-CSF+GEM group compared to the PBS group.Conclusion RS technology-constructed recombinant oncolytic influenza virus OvFlu-GM-CSF,when combined with the chemotherapeutic agent GEM,enhances the cytotoxic efficacy against pancreatic cancer cells and effectively activates the host's anti-tumor immune response.

Objective:To investigate teachers' evaluation and willingness and the influencing factors regarding the adoption of the flipped classroom teaching mode in undergraduate pediatrics education.Methods:From December 2022 to December 2024, a questionnaire survey was conducted among the teachers who were responsible for teaching Child Health and Disease（Pediatrics) to the eight-year clinical medicine students at Peking University Health Science Center. Their views, evaluation, and willingness of implementing the flipped classroom teaching mode were investigated. Logistic regression analysis was performed using SPSS 26.0 software to explore the factors influencing teachers' willingness to adopt the flipped classroom teaching mode.Results:A total of 102 questionnaires were collected. Among the teachers, 20.59%（ n=21) believed that the teaching effect of the flipped classroom was better than that of the traditional class, 58.82%（ n=60) considered its effectiveness comparable, and 20.59%（ n=21) found it less effective. The most influential factors affecting the effectiveness of flipped classroom were students' self-learning with online videos [(4.39±0.73) points], student participation in the flipped classrooms [(4.26±0.72) points], the adequacy of teachers' pre-class preparation [(4.18±0.65) points], and the suitability of the teaching content for the flipped classroom [(4.11±0.76) points]. Teachers believed that the flipped classroom significantly enhanced students' autonomous learning ability [(4.11±0.63) points], clinical thinking [(4.04±0.58) points], and expression skills [(3.80±0.61) points]. Additionally, 78.43%（ n=80) of the teachers expressed willingness to continue participating in flipped classroom teaching. Factors influencing teachers' willingness to adopt the flipped classroom included gender, satisfaction with students' classroom participation, and personal experience with the effectiveness of the flipped classroom（ P<0.05). Conclusions:The flipped classroom teaching mode is well-accepted by teachers. Students' classroom participation affects teachers' willingness to continue using the flipped classroom teaching mode. In the future, the content of flipped classroom should be arranged individually according to specific teaching objectives to increase students' classroom participation and promote the cultivation of students' ability.

Objective To investigate the therapeutic effect of combination treatment of transcatheter arterial chemoembolization(TACE),micro wave ablation(MW A)and targeted immunotherapy for hepatocellular carcinoma(HCC).Methods The clinical data of HCC patients,who were admitted to the Affiliated Central Hospital of Jiangnan University of China to receive TACE from August 2016 to August 2023,were retrospectively analyzed.Based on the inclusion criteria,a total of 252 patients were included in this study.According to the therapeutic scheme,the patients were divided into combination group(receiving TACE,MWA,targeted therapy and immunotherapy,n=41),and control group(receiving TACE alone,n=211).The overall survival(OS)and progression-free survival(PFS)of the two groups were statistically analyzed.After propensity score matching at 1∶1 ratio,the clinical effects of the two groups were analyzed.According to different data,t-test,test,Kaplan-Meier curve,logarithmic rank test,Cox regression analysis,or Cox proportional risk model analysis was used to make statistical analysis.Results Multivariate regression analysis showed that tumor number,maximum diameter,invasion of large vessels,tumor capsule,AFP level,glutamic oxalacetic transaminase,BCLC stage and Child-Pugh stage were the independent risk factors for patient survival.Imaging response and combination treatment were the protective factors for survival.After propensity score matching,a total of 37 pairs of patients were obtained,and the baseline data were comparable between the groups.The differences in OS and PFS between the two groups were statistically significant.Combination treatment of TACE,MWA,targeted therapy and immunotherapy could significantly prolong OS and PFS in HCC patients.Conclusion Compared with TACE alone,combination use of TACE,MWA,targeted therapy and immunotherapy is a more effective therapeutic scheme for HCC,it can remarkably and effectively improve OS and PFS in HCC patients.

Purpose To explore the feasibility and clinical value of deep learning-based image reconstruction technology in 5.0T MRI for nasopharyngeal carcinoma.Materials and Methods A prospective study was conducted on 50 newly diagnosed nasopharyngeal carcinoma patients from August to December 2024 at Sun Yat-sen University Cancer Center.5.0T MRI was performed to scan the nasopharynx region.Routine scanning protocols included transverse T2WI,transverse T1WI,transverse contrast-enhanced T1WI and coronal fat-suppressed contrast-enhanced T1WI sequences.Based on these standard scanning protocols,DeepRecon deep learning reconstruction technology with different levels(grade 1-5)was applied,generating a total of 24 sets of images.Qualitative evaluation employed a Likert scale(5-point system)for subjective scoring on lesion detection,lesion edge clarity,artifacts and overall image quality.Quantitative evaluation was performed using the signal-to-noise ratio and contrast-to-noise ratio to objectively assess the quality of the 24 image sets.Differences in qualitative and quantitative indicators between different groups were compared,while the Kappa coefficient was used to analyze the consistency of subjective evaluations by two radiologists.Results In the qualitative assessment of 24 image sets from four MRI sequences(with and without DeepRecon reconstruction),DeepRecon images(grade 2-4)significantly outperformed traditional images in all features except for artifact reduction(Z=-12.11--6.23,all P<0.001).Images reconstructed at DeepRecon grade 3 had the highest overall score and the best image quality.Furthermore,compared with traditional images,DeepRecon images(grade 2-5)demonstrated significantly improved signal-to-noise ratio for both lesions and the lateral pterygoid muscle(t=-15.67--3.44,Z=-6.09--4.63,all P<0.01).In addition,in the transverse T2WI,transverse contrast-enhanced T1WI and coronal fat-suppressed contrast-enhanced T1WI images with DeepRecon reconstruction(grade 2-5),the contrast-to-noise ratio(lesion/lateral pterygoid muscle)also showed significant improvement compared to traditional images(t=-12.71--3.19,Z=-6.08--4.47,all P<0.001).The inter-observer agreement for the overall subjective quality score between the two radiologists was good(Kappa=0.75-0.82,all P<0.01).Conclusion DeepRecon deep learning reconstruction technology significantly increases the signal-to-noise ratio and resolution of traditional magnetic resonance images of nasopharyngeal cancer,improving image clarity and bringing more possibilities for the advancement of imaging diagnosis.

Purpose To explore the feasibility and clinical value of deep learning-based image reconstruction technology in 5.0T MRI for nasopharyngeal carcinoma.Materials and Methods A prospective study was conducted on 50 newly diagnosed nasopharyngeal carcinoma patients from August to December 2024 at Sun Yat-sen University Cancer Center.5.0T MRI was performed to scan the nasopharynx region.Routine scanning protocols included transverse T2WI,transverse T1WI,transverse contrast-enhanced T1WI and coronal fat-suppressed contrast-enhanced T1WI sequences.Based on these standard scanning protocols,DeepRecon deep learning reconstruction technology with different levels(grade 1-5)was applied,generating a total of 24 sets of images.Qualitative evaluation employed a Likert scale(5-point system)for subjective scoring on lesion detection,lesion edge clarity,artifacts and overall image quality.Quantitative evaluation was performed using the signal-to-noise ratio and contrast-to-noise ratio to objectively assess the quality of the 24 image sets.Differences in qualitative and quantitative indicators between different groups were compared,while the Kappa coefficient was used to analyze the consistency of subjective evaluations by two radiologists.Results In the qualitative assessment of 24 image sets from four MRI sequences(with and without DeepRecon reconstruction),DeepRecon images(grade 2-4)significantly outperformed traditional images in all features except for artifact reduction(Z=-12.11--6.23,all P<0.001).Images reconstructed at DeepRecon grade 3 had the highest overall score and the best image quality.Furthermore,compared with traditional images,DeepRecon images(grade 2-5)demonstrated significantly improved signal-to-noise ratio for both lesions and the lateral pterygoid muscle(t=-15.67--3.44,Z=-6.09--4.63,all P<0.01).In addition,in the transverse T2WI,transverse contrast-enhanced T1WI and coronal fat-suppressed contrast-enhanced T1WI images with DeepRecon reconstruction(grade 2-5),the contrast-to-noise ratio(lesion/lateral pterygoid muscle)also showed significant improvement compared to traditional images(t=-12.71--3.19,Z=-6.08--4.47,all P<0.001).The inter-observer agreement for the overall subjective quality score between the two radiologists was good(Kappa=0.75-0.82,all P<0.01).Conclusion DeepRecon deep learning reconstruction technology significantly increases the signal-to-noise ratio and resolution of traditional magnetic resonance images of nasopharyngeal cancer,improving image clarity and bringing more possibilities for the advancement of imaging diagnosis.

Objective:To investigate teachers' evaluation and willingness and the influencing factors regarding the adoption of the flipped classroom teaching mode in undergraduate pediatrics education.Methods:From December 2022 to December 2024, a questionnaire survey was conducted among the teachers who were responsible for teaching Child Health and Disease（Pediatrics) to the eight-year clinical medicine students at Peking University Health Science Center. Their views, evaluation, and willingness of implementing the flipped classroom teaching mode were investigated. Logistic regression analysis was performed using SPSS 26.0 software to explore the factors influencing teachers' willingness to adopt the flipped classroom teaching mode.Results:A total of 102 questionnaires were collected. Among the teachers, 20.59%（ n=21) believed that the teaching effect of the flipped classroom was better than that of the traditional class, 58.82%（ n=60) considered its effectiveness comparable, and 20.59%（ n=21) found it less effective. The most influential factors affecting the effectiveness of flipped classroom were students' self-learning with online videos [(4.39±0.73) points], student participation in the flipped classrooms [(4.26±0.72) points], the adequacy of teachers' pre-class preparation [(4.18±0.65) points], and the suitability of the teaching content for the flipped classroom [(4.11±0.76) points]. Teachers believed that the flipped classroom significantly enhanced students' autonomous learning ability [(4.11±0.63) points], clinical thinking [(4.04±0.58) points], and expression skills [(3.80±0.61) points]. Additionally, 78.43%（ n=80) of the teachers expressed willingness to continue participating in flipped classroom teaching. Factors influencing teachers' willingness to adopt the flipped classroom included gender, satisfaction with students' classroom participation, and personal experience with the effectiveness of the flipped classroom（ P<0.05). Conclusions:The flipped classroom teaching mode is well-accepted by teachers. Students' classroom participation affects teachers' willingness to continue using the flipped classroom teaching mode. In the future, the content of flipped classroom should be arranged individually according to specific teaching objectives to increase students' classroom participation and promote the cultivation of students' ability.

Objective To rescue a recombinant oncolytic influenza virus chimeric with IL-21 and evaluate its inhibitory effects and safety against hepatocell ular carcinoma(HCC)both in vitro and in vivo,and to explore the mechanism by which this virus enhances antitumor effects when combined with anti-programmed cell death protein 1(anti-PD-1)antibody.Methods The IL-21 gene fragment was inserted into the nonstructural protein(NS)sequence of the influenza virus PR8 using reverse genetics(RG)technology to rescue the recombinant oncolytic influenza virus rOV-IL-21-NS.The virus titer and virulence were determined using the 50％tissue culture infectious dose(TCID50)and hemagglutination assays.The successful insertion of the exogenous gene into the NS sequence was verified using RT-qPCR,gel electrophoresis,and sequencing analysis.Viral morphology and size were observed with transmission electron microscopy.The impact of the virus on the viability of hepatocellular carcinoma cells was assessed with CCK-8 assay.A subcutaneous tumor model of HCC was established in 45 female C57BL/6 mice(8 weeks old,weighing 16～20 g),and then the mice were randomly assigned into PBS,PR8,anti-PD-1,rOV-IL-21-NS,and the rOV-IL-21-NS+anti-PD-1 treatment groups,with 9 mice in each group,to evaluate the anti-tumor effects of monotherapy versus combination therapy.Flow cytometry was conducted to assess the regulatory effects of monotherapy and combination therapy on the tumor immune microenvironment.Results RG technology successfully rescued the recombinant oncolytic influenza virus rOV-IL-21-NS.Sequencing confirmed the successful insertion of IL-21 into the target sequence,and the obtained virus could be stably propagated,with its sixth passage reaching a hemagglutination titer of 211,and a viral titer of 6 Log10(TCID50/mL).Oncolytic virus rOV-IL-21-NS,at a multiplicity of infection(MOI)of 3,selectively reduced the viability of HCC cells without significantly affecting normal liver cells.Compared to the control group,the combination of rOV-IL-21-NS and anti-PD-1 antibodies significantly inhibited tumor growth(P＜0.001)and increased the proportions of CD4+T and CD8+T cells in the spleen tissue of the mouse model of subcutaneous HCC tumor(P＜0.001).Conclusion The recombinant oncolytic influenza virus rOV-IL-21-NS chimeric with IL-21 can effectively and safely exert targeted killing to HCC cells,enhance T cell activation by synergizing with anti-PD-1 antibodies,and improve the immune microenvironment.

Background: Insulin resistance (IR) is the key pathological basis of many metabolic disorders. Lack of asialoglycoprotein receptor 1 (ASGR1) decreased the serum lipid levels and reduced the risk of coronary artery disease. However, whether ASGR1 also participates in the regulatory network of insulin sensitivity and glucose metabolism remains unknown. Methods: The constructed ASGR1 knockout mice and ASGR1-/- HepG2 cell lines were used to establish the animal model of metabolic syndrome and the IR cell model by high-fat diet (HFD) or drug induction, respectively. Then we evaluated the glucose metabolism and insulin signaling in vivo and in vitro. Results: ASGR1 deficiency ameliorated systemic IR in mice fed with HFD, evidenced by improved insulin intolerance, serum insulin, and homeostasis model assessment of IR index, mainly contributed from increased insulin signaling in the liver, but not in muscle or adipose tissues. Meanwhile, the insulin signal transduction was significantly enhanced in ASGR1-/- HepG2 cells. By transcriptome analyses and comparison, those differentially expressed genes between ASGR1 null and wild type were enriched in the insulin signal pathway, particularly in phosphoinositide 3-kinase-AKT signaling. Notably, ASGR1 deficiency significantly reduced hepatic gluconeogenesis and glycogenolysis. Conclusion The ASGR1 deficiency was consequentially linked with improved hepatic insulin sensitivity under metabolic stress, hepatic IR was the core factor of systemic IR, and overcoming hepatic IR significantly relieved the systemic IR. It suggests that ASGR1 is a potential intervention target for improving systemic IR in metabolic disorders.

Objective To investigate the correlation between the expression level of glypican-3(GPC3)and the immune therapy response in clinical liver cancer patients.Methods Clinical data of 232 liver cancer immunotherapy response/tolerance group patients from January 2019 to May 2023 at the General Hospital of the People's Liberation Army were collected,and the correlation between GPC3 expression levels and the efficacy of immunotherapy in liver cancer patients was statistically analyzed;TCGA database validation of immune cell infiltration in GPC3 high and low expression groups of liver cancer patients;Further,real-time fluorescence quantitative PCR(qPCR)and immunohistochemical staining methods were used to detect the expression of GPC3 and immune cell infiltration in paired tissues of liver cancer immunotherapy response/tolerance group patients,and multi-color immunofluorescence was applied to detect the expression of GPC3 and related molecules.Results Clinical evidence shows that the GPC3 positive group of liver cancer patients has a low response rate to immunotherapy.Univariate/multivariate analysis results indicate that GPC3 is an independent risk factor for tumor recurrence after liver cancer immunotherapy;The analysis of the TCGA database revealed that high expression of GPC3 in liver cancer tissue leads to increased infiltration of regulatory T cells(Tregs);Paired tissue testing of liver cancer patients and adjacent tissues revealed that the immunotherapy effect was worse in the GPC3 high expression group,and Tregs cell infiltration increased,consistent with the results of database analysis.The TCGA database analysis results showed that GPC3 was positively correlated with CCL20 and its ligand CCR6,and the multi-color immunohistochemistry results were consistent with the database analysis results.Conclusion GPC3 is highly expressed in tumor tissues of liver cancer patients and is positively correlated with immune therapy tolerance in liver cancer.GPC3 regulates Tregs cell infiltration through the CCL20-CCR6 signaling axis and is expected to serve as a biomarker for predicting the efficacy of immune therapy in clinical liver cancer patients.

Objective To investigate the correlation between the expression level of glypican-3(GPC3)and the immune therapy response in clinical liver cancer patients.Methods Clinical data of 232 liver cancer immunotherapy response/tolerance group patients from January 2019 to May 2023 at the General Hospital of the People's Liberation Army were collected,and the correlation between GPC3 expression levels and the efficacy of immunotherapy in liver cancer patients was statistically analyzed;TCGA database validation of immune cell infiltration in GPC3 high and low expression groups of liver cancer patients;Further,real-time fluorescence quantitative PCR(qPCR)and immunohistochemical staining methods were used to detect the expression of GPC3 and immune cell infiltration in paired tissues of liver cancer immunotherapy response/tolerance group patients,and multi-color immunofluorescence was applied to detect the expression of GPC3 and related molecules.Results Clinical evidence shows that the GPC3 positive group of liver cancer patients has a low response rate to immunotherapy.Univariate/multivariate analysis results indicate that GPC3 is an independent risk factor for tumor recurrence after liver cancer immunotherapy;The analysis of the TCGA database revealed that high expression of GPC3 in liver cancer tissue leads to increased infiltration of regulatory T cells(Tregs);Paired tissue testing of liver cancer patients and adjacent tissues revealed that the immunotherapy effect was worse in the GPC3 high expression group,and Tregs cell infiltration increased,consistent with the results of database analysis.The TCGA database analysis results showed that GPC3 was positively correlated with CCL20 and its ligand CCR6,and the multi-color immunohistochemistry results were consistent with the database analysis results.Conclusion GPC3 is highly expressed in tumor tissues of liver cancer patients and is positively correlated with immune therapy tolerance in liver cancer.GPC3 regulates Tregs cell infiltration through the CCL20-CCR6 signaling axis and is expected to serve as a biomarker for predicting the efficacy of immune therapy in clinical liver cancer patients.