ObjectivePancreatic ductal adenocarcinoma (PDAC) exhibits a limited response to current treatments due to its dense fibrotic stroma and highly immunosuppressive tumor microenvironment. In recent years, advancements in cellular immunotherapy, particularly chimeric antigen receptor macrophage (CAR-M) therapy, have offered new hope for pancreatic cancer treatment. Although CAR-M therapy demonstrates dual potential in directly killing tumor cells and remodeling the immune microenvironment, it still faces challenges such as complex in vitro preparation processes and low in vivo targeting and delivery efficiency. Therefore, developing strategies for efficient and targeted in vivo delivery of CAR genes has become crucial for overcoming current therapeutic limitations. This study aims to develop an orally administrable nano-gene delivery system for the targeted delivery of CAR genes to pancreatic tumor sites. MethodsCore nano-gene particles (PNP/pCAR) were constructed by loading plasmid DNA encoding CAR (pCAR) with cationic polypeptides (PNP). Subsequently, PNP/pCAR was surface-modified with β-glucan to prepare the targeted nanoparticles (βGlus-PNP/pCAR). The loading efficiency of PNP for pCAR was quantitatively assessed by gel retardation assay. The particle size, Zeta potential, morphology, and storage stability of PNP/pCAR were characterized using a Malvern particle size analyzer and transmission electron microscopy. At the cellular level, RAW 264.7 macrophages were selected. The cytotoxicity of PNP/pCAR was evaluated using the CCK-8 assay. The cellular uptake efficiency and lysosomal escape ability of the nanoparticles were assessed via flow cytometry and confocal microscopy. Transfection efficiency was quantitatively evaluated by detecting the expression of the reporter gene GFP using flow cytometry. At the in vivo level, an orthotopic pancreatic cancer mouse model was established. Cy7-labeled βGlus-PNP/pCAR nanoparticles were administered orally, and the fluorescence distribution in mice was dynamically monitored at 1, 2, 4, 8, and 16 h post-administration using a small animal in vivo imaging system. Forty-eight hours after oral gavage, the mice were euthanized, and pancreatic tumor tissues were collected for further analysis of intratumoral fluorescence signals using the imaging system. Additionally, βGlus-PNP/pCAR-GFP nanoparticles loaded with the reporter gene (GFP) were administered orally. Forty-eight hours post-administration, pancreatic tumor tissues were harvested to prepare frozen sections, and GFP expression was observed and analyzed under a fluorescence microscope. ResultsThe PNP carrier exhibited a high loading capacity for pCAR. The successfully prepared PNP/pCAR nanoparticles were regular spheres with a hydrodynamic diameter of approximately (120±10) nm and a Zeta potential of about +(6±1) mV. They maintained good structural stability after incubation in PBS buffer for 7 d. Cell experiments demonstrated that PNP/pCAR exhibited no significant cytotoxicity in RAW 264.7 cells while being efficiently internalized and effectively escaping lysosomal degradation. The transfection positive rate of PNP/pCAR-GFP in RAW 264.7 cells reached (25±3)%, surpassing that of Lipofectamine 2000-loaded pCAR-GFP (Lipo/pCAR-GFP), which was (20±1)%.In vivo experiments revealed that, compared to unmodified PNP/pCAR, βGlus-PNP/pCAR exhibited strongerin situ pancreatic tumor targeting ability after oral administration. Furthermore, oral administration of βGlus-PNP/pCAR-GFP resulted in significant GFP protein expression detectable within pancreatic tumor tissues. ConclusionThis study successfully constructed and validated an orally administrable, pancreatic cancer-targeting polypeptide-based nano-gene delivery system. It provides an important technological foundation in delivery systems and experimental basis for the subsequent development of in situ CAR-M-based therapeutic strategies for pancreatic cancer.

ObjectivePancreatic ductal adenocarcinoma (PDAC) exhibits a limited response to current treatments due to its dense fibrotic stroma and highly immunosuppressive tumor microenvironment. In recent years, advancements in cellular immunotherapy, particularly chimeric antigen receptor macrophage (CAR-M) therapy, have offered new hope for pancreatic cancer treatment. Although CAR-M therapy demonstrates dual potential in directly killing tumor cells and remodeling the immune microenvironment, it still faces challenges such as complex in vitro preparation processes and low in vivo targeting and delivery efficiency. Therefore, developing strategies for efficient and targeted in vivo delivery of CAR genes has become crucial for overcoming current therapeutic limitations. This study aims to develop an orally administrable nano-gene delivery system for the targeted delivery of CAR genes to pancreatic tumor sites. MethodsCore nano-gene particles (PNP/pCAR) were constructed by loading plasmid DNA encoding CAR (pCAR) with cationic polypeptides (PNP). Subsequently, PNP/pCAR was surface-modified with β-glucan to prepare the targeted nanoparticles (βGlus-PNP/pCAR). The loading efficiency of PNP for pCAR was quantitatively assessed by gel retardation assay. The particle size, Zeta potential, morphology, and storage stability of PNP/pCAR were characterized using a Malvern particle size analyzer and transmission electron microscopy. At the cellular level, RAW 264.7 macrophages were selected. The cytotoxicity of PNP/pCAR was evaluated using the CCK-8 assay. The cellular uptake efficiency and lysosomal escape ability of the nanoparticles were assessed via flow cytometry and confocal microscopy. Transfection efficiency was quantitatively evaluated by detecting the expression of the reporter gene GFP using flow cytometry. At the in vivo level, an orthotopic pancreatic cancer mouse model was established. Cy7-labeled βGlus-PNP/pCAR nanoparticles were administered orally, and the fluorescence distribution in mice was dynamically monitored at 1, 2, 4, 8, and 16 h post-administration using a small animal in vivo imaging system. Forty-eight hours after oral gavage, the mice were euthanized, and pancreatic tumor tissues were collected for further analysis of intratumoral fluorescence signals using the imaging system. Additionally, βGlus-PNP/pCAR-GFP nanoparticles loaded with the reporter gene (GFP) were administered orally. Forty-eight hours post-administration, pancreatic tumor tissues were harvested to prepare frozen sections, and GFP expression was observed and analyzed under a fluorescence microscope. ResultsThe PNP carrier exhibited a high loading capacity for pCAR. The successfully prepared PNP/pCAR nanoparticles were regular spheres with a hydrodynamic diameter of approximately (120±10) nm and a Zeta potential of about +(6±1) mV. They maintained good structural stability after incubation in PBS buffer for 7 d. Cell experiments demonstrated that PNP/pCAR exhibited no significant cytotoxicity in RAW 264.7 cells while being efficiently internalized and effectively escaping lysosomal degradation. The transfection positive rate of PNP/pCAR-GFP in RAW 264.7 cells reached (25±3)%, surpassing that of Lipofectamine 2000-loaded pCAR-GFP (Lipo/pCAR-GFP), which was (20±1)%.In vivo experiments revealed that, compared to unmodified PNP/pCAR, βGlus-PNP/pCAR exhibited strongerin situ pancreatic tumor targeting ability after oral administration. Furthermore, oral administration of βGlus-PNP/pCAR-GFP resulted in significant GFP protein expression detectable within pancreatic tumor tissues. ConclusionThis study successfully constructed and validated an orally administrable, pancreatic cancer-targeting polypeptide-based nano-gene delivery system. It provides an important technological foundation in delivery systems and experimental basis for the subsequent development of in situ CAR-M-based therapeutic strategies for pancreatic cancer.

Objective: To compare the effects of immediate versus delayed post-space preparation on the bond strength of fiber posts and the fracture resistance of roots after root canal obturation using iRoot SP and the single-cone technique, and to provide an experimental basis for the selection of the timing of clinical post-space preparation. Methods: This study was approved by the Medical Ethics Committee of the institution, and written informed consent was obtained from all participants. Seventy-two extracted human single-rooted premolars were randomly divided into two groups (n = 36 each): the immediate post-space preparation group (post-space preparation performed immediately after root canal obturation) and the delayed post-space preparation group (post-space preparation performed 1 week after root canal obturation). Cleanliness of the root canal walls was assessed with a dental microscope after preparation in both groups; the ultrastructural characteristics of the dentinal surface were examined via scanning electron microscopy; the fracture resistance of roots was measured with root fracture resistance tests; the bond strength of the fiber posts was measured with the thin-slice push-out test; and the bond failure modes were observed and analyzed under a stereomicroscope. Results: Under dental microscopy, the dentin surface of the immediate post-space preparation group appeared cleaner than that of the delayed post-space preparation group. Scanning electron microscopy revealed that most dentinal tubules remained open in the immediate post-space preparation group, while the tubules in the delayed post-space preparation group showed incomplete patency. The fracture resistance of the immediate post-space preparation group was (2 872 ± 241.5) N and that of the delayed post-space preparation group was (2 934 ± 353.1) N, with no statistically significant difference (t = -0.328, P = 0.751). The push-out bond strength of the immediate post-space preparation group (10.310 ± 2.907) MPa was significantly higher than that of the delayed post-space preparation group (7.917 ± 2.429) MPa, with a statistically significant difference (t = 4.457, P < 0.001). Analysis of failure modes showed that adhesive-to-dental interface failure was the predominant mode in both groups. Conclusion Following root canal obturation with iRoot SP using the single-cone technique, immediate post-space preparation yields superior fiber post bond strength compared to delayed post-space preparation, without compromising root fracture resistance.

The CDH23 gene is a pathogenic mutant gene of the USH1D subtype in Usher syndrome.In this study,two wild-type Cdh23 full-length plasmids(～16 kb)with different promoters were construc-ted,and fluorinated polyethylene imine(FPEI)was used as a delivery vector to transfect the house ear institute-organ of corti 1(HEI-OC1)and the optimal expression plasmid was obtained by evaluating the transfection efficiency in vitro.Firstly,the results of the synthesis of FPEI were analyzed using Fourier transform infrared absorption spectroscopy to prove the successful synthesis of FPEI.After that,the plas-mid encapsulation ability of FPEI and the surface potential and hydration diameter of the formed comple-xes were characterized by agarose gel blocking assay,Zeta potential assay,and dynamic light scattering assay.It was found that FPEI had good plasmid encapsulation ability,and the FPEI plasmid complexes were all positively charged at high mass ratio,with the distribution of particle sizes in the range of 100-300 nm.The low cytotoxicity and high transfection efficiency of FPEI in HEI-OC1 cells were verified by Cell Counting Kit-8(CCK-8)and flow cytometry.Comparing FPEI with Lipofectamine 3000 and differ-ent quality PEI(25K,40K)transfection reagents,the transfection efficiency of FPEI was found to be significantly better than that of the traditional transfection reagents.Quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot results showed that the CAG promoter was better than the CMV promoter,which could be used as the optimal expression plasmid for the subsequent in vivo experi-ments.In addition,it was verified by cellular immunofluorescence that CDH23 was mainly distributed in the cytoplasm after overexpression.The above results demonstrated that FPEI can be used as an efficient delivery vector for in vitro overexpression of large genes represented by Cdh23,which provides an impor-tant experimental basis for subsequent in vivo gene therapy of USH1D syndrome.

Objective To analyze the prevalence of latent tuberculosis infection(LTBI)among relevant healthcare workers(HCWs)in designated tuberculosis medical institutions(MIs)in Yantai City,and explore its influencing factors.Methods The cluster random sampling method was adopted to select two county-and district-level desig-nated tuberculosis MIs.All HCWs underwent questionnaire survey and creation tuberculin skin test(C-TST)at the same time,and the influencing factors for LTBI were analyzed.Results A total of 215 HCWs from designated tu-berculosis MIs were included for analysis,37 were diagnosed with LTBI,with an infection rate of 17.21％(95％CI:12.42％-22.93％).Multivariate logistic regression analysis showed that clinicians(OR=3.19,95％CI:1.05-9.69),laboratory technician(OR=5.90,95％CI:1.21-28.77),working years≥10 years(OR=3.31,95％CI:1.39-7.90),and tuberculosis history of family members(OR=6.49,95％CI:1.01-41.46)were independent risk factors for LTBI.Conclusion The infection risk of clinicians and laboratory technicians who directly contact with tuberculosis patients or Mycobacterium tuberculosis is higher than that of other HCWs,and is related to the length of working years.It is suggested that healthcare-associated infection control measures should be streng-thened,and tuberculosis active screening should be carried out regularly for HCWs in key departments.

Objective:To investigate whether the rs612529 C/T and rs11669663 G/A in VSTM1 gene are associated with leukocyte signaling inhibitory receptor-1(SIRL-1)expression and an increased risk for systemic lupus erythematosus(SLE)in a Han Chinese cohort.Methods:A total of 200 patients with SLE and 218 healthy controls(HC)were enrolled.Relevant laboratory characteris-tics of patients with SLE were also collected.Genotyping of rs612529 C/T and rs11669663 G/A were performed by Sanger sequencing technology.SIRL-1 expression was assessed in peripheral blood neutrophils and monocytes was detected by flow cytometry.Levels of autoantibodies associated with SLE were detected by ELISA.Results:In both SLE group and HC,the C allele of rs612529 was asso-ciated with a decreased expression level of SIRL-1 on monocytes,with a gradual increased in SIRL-1 protein level from the CC over the CT to the TT genotype.C allele of rs612529 was associated with higher serum anti-dsDNA antibody titers in patients with SLE(P<0.05).In the case of rs11669663 G/A,no significant association of genotypes with SLE susceptibility was detected.Conclusion:VSTM1 rs612529 C/T may contribute to SLE disease activity and regulate SIRL-1 expression on monocytes in the Han Chinese cohort.

The CDH23 gene is a pathogenic mutant gene of the USH1D subtype in Usher syndrome.In this study,two wild-type Cdh23 full-length plasmids(～16 kb)with different promoters were construc-ted,and fluorinated polyethylene imine(FPEI)was used as a delivery vector to transfect the house ear institute-organ of corti 1(HEI-OC1)and the optimal expression plasmid was obtained by evaluating the transfection efficiency in vitro.Firstly,the results of the synthesis of FPEI were analyzed using Fourier transform infrared absorption spectroscopy to prove the successful synthesis of FPEI.After that,the plas-mid encapsulation ability of FPEI and the surface potential and hydration diameter of the formed comple-xes were characterized by agarose gel blocking assay,Zeta potential assay,and dynamic light scattering assay.It was found that FPEI had good plasmid encapsulation ability,and the FPEI plasmid complexes were all positively charged at high mass ratio,with the distribution of particle sizes in the range of 100-300 nm.The low cytotoxicity and high transfection efficiency of FPEI in HEI-OC1 cells were verified by Cell Counting Kit-8(CCK-8)and flow cytometry.Comparing FPEI with Lipofectamine 3000 and differ-ent quality PEI(25K,40K)transfection reagents,the transfection efficiency of FPEI was found to be significantly better than that of the traditional transfection reagents.Quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot results showed that the CAG promoter was better than the CMV promoter,which could be used as the optimal expression plasmid for the subsequent in vivo experi-ments.In addition,it was verified by cellular immunofluorescence that CDH23 was mainly distributed in the cytoplasm after overexpression.The above results demonstrated that FPEI can be used as an efficient delivery vector for in vitro overexpression of large genes represented by Cdh23,which provides an impor-tant experimental basis for subsequent in vivo gene therapy of USH1D syndrome.

Objective To analyze the prevalence of latent tuberculosis infection(LTBI)among relevant healthcare workers(HCWs)in designated tuberculosis medical institutions(MIs)in Yantai City,and explore its influencing factors.Methods The cluster random sampling method was adopted to select two county-and district-level desig-nated tuberculosis MIs.All HCWs underwent questionnaire survey and creation tuberculin skin test(C-TST)at the same time,and the influencing factors for LTBI were analyzed.Results A total of 215 HCWs from designated tu-berculosis MIs were included for analysis,37 were diagnosed with LTBI,with an infection rate of 17.21％(95％CI:12.42％-22.93％).Multivariate logistic regression analysis showed that clinicians(OR=3.19,95％CI:1.05-9.69),laboratory technician(OR=5.90,95％CI:1.21-28.77),working years≥10 years(OR=3.31,95％CI:1.39-7.90),and tuberculosis history of family members(OR=6.49,95％CI:1.01-41.46)were independent risk factors for LTBI.Conclusion The infection risk of clinicians and laboratory technicians who directly contact with tuberculosis patients or Mycobacterium tuberculosis is higher than that of other HCWs,and is related to the length of working years.It is suggested that healthcare-associated infection control measures should be streng-thened,and tuberculosis active screening should be carried out regularly for HCWs in key departments.

Viral myocarditis is a myocardial disease resulting from various viral infections.Due to the com-plexity of its pathogenesis,effective prevention and treatment options are currently lacking.Establishing appropriate ex-perimental models is crucial for studying the pathogenic mechanisms,disease progression,early diagnosis,and the devel-opment of new drugs and therapies for viral myocarditis.This article reviews the construction methods,research advance-ments,and applications of common experimental models of viral myocarditis,which range from cell models to small ani-mal models,including mice,hamsters,and rabbits,as well as larger animals such as pigs and non-human primates.Ad-ditionally,we summarize and discuss future research directions,providing a theoretical foundation and technological guid-ance for the prevention and treatment of viral myocarditis in clinical settings.

Objectives:To investigate the enhancement of tumor penetration and photodynamic therapy (PDT) efficacy in triple-negative breast cancer by hyaluronidase (HAase) using a novel pH-responsive IR780-loaded photosensitive micelle.Methods:The pH-responsive IR780-loaded photosensitive micelles were prepared using the nanoprecipitation method, and their morphology, size, and encapsulation efficiency were characterized. The in vitro stability and pH-responsive drug release of the micelles were also evaluated. The cytotoxicity of the micelles on triple-negative breast cancer cells (MDA-MB-231) was assessed using a cell counting kit. A nude mouse breast cancer model was established, and HAase was injected intratumorally 24 hours before intravenous injection of the photosensitive micelles. The effect of HAase on the biodistribution and tumor uptake of the micelles was detected using small animal in vivo imaging. CD31 and HIF-1α immunofluorescence staining were performed to investigate the mechanism of HAase-enhanced tumor penetration. The body weight and tumor volume of the mice were measured, and necrosis and apoptosis of tumor tissues were assessed using HE staining and TUNEL staining, respectively. Results:Transmission electron microscopy showed that the micelles had a uniform particle size of approximately 60-70 nm, with a hydrated particle size of (98.03±0.22) nm. The IR780 encapsulation efficiency was 74.15%, with a drug loading content of 2.07%. After 7 days at 4 ℃, there was no significant change in hydrated particle size ( P=0.062). The 24-hour release rates of the micelles in PBS at pH 7.4 and 6.5 were (2.41±0.21)% and (43.69±2.09)%, respectively, showing a significant difference ( P＜0.000 1). The cytotoxicity assay revealed that the cell viability in the micelles group without light exposure was significantly higer than that in the micelles group under light exposure [(97.00±5.38)% vs. (53.27±9.00)%, P=0.000 2]. The micelles were able to target and accumulate in the tumor tissue, and this accumulation increased significantly with HAase treatment. CD31 and HIF-1α immunofluorescence staining indicated that the CD31 signal was enhanced [(0.27±0.05)% vs. (4.57±0.27)%, P＜0.000 1] and the HIF-1α signal was reduced [(5.14±0.38)% vs. (0.08±0.04)%, P＜0.000 1] in the HAase-treated group compared to that in the micelle-only group. After 11 days of treatment with HAase combined with photosensitive micelles, there was no statistically significant difference in mouse body weight ( P＞0.05). However, the tumor volume inhibition rate in the HAase-micelle-mediated PDT group was significantly higher than that in the micelle-mediated PDT group [(87.66±6.37)% vs. (25.34±12.63)%, P=0.002]. Histological staining showed a significant increase in tumor cell necrosis and apoptosis in the HAase-micelle-mediated PDT group. Conclusion:HAase enhances the deep tumor penetration and targeted accumulation of pH-responsive IR780-loaded photosensitive micelles, significantly improves the efficacy of photodynamic therapy in triple-negative breast cancer.