Objective To identify determinants affecting the implementation of outpatient mutual aid policy and propose evidence-based optimization strategies.Methods By utilizing the Horn-Mitton policy implementation framework,this article sys-tematically examined implementation barriers through multi-dimensional analysis.Results The implementation process was con-strained by multiple interacting factors:overly broad policy objectives,insufficient adoption of digital health technologies,shorta-ges of health insurance professionals,policy fragmentation due to lack of horizontal communication within organizations,an in-complete fiscal compensation mechanism,imbalance of cohesive policies,and limited public acceptance.These factors collective-ly hindered the advancement of outpatient mutual aid policy.Conclusion In order to improve the implementation efficacy,it is urgent to build robust digital monitoring platforms equipped with regulatory systems,establish multi-dimensional fiscal compensa-tion mechanisms,cooperate with a variety of policies to promote coordination,explore reform plans for employee health insurance payment fairness,unify the reimbursement level orientations,and construct differentiated reimbursement policy systems.

Objective To identify determinants affecting the implementation of outpatient mutual aid policy and propose evidence-based optimization strategies.Methods By utilizing the Horn-Mitton policy implementation framework,this article sys-tematically examined implementation barriers through multi-dimensional analysis.Results The implementation process was con-strained by multiple interacting factors:overly broad policy objectives,insufficient adoption of digital health technologies,shorta-ges of health insurance professionals,policy fragmentation due to lack of horizontal communication within organizations,an in-complete fiscal compensation mechanism,imbalance of cohesive policies,and limited public acceptance.These factors collective-ly hindered the advancement of outpatient mutual aid policy.Conclusion In order to improve the implementation efficacy,it is urgent to build robust digital monitoring platforms equipped with regulatory systems,establish multi-dimensional fiscal compensa-tion mechanisms,cooperate with a variety of policies to promote coordination,explore reform plans for employee health insurance payment fairness,unify the reimbursement level orientations,and construct differentiated reimbursement policy systems.

In order to obtain a recombinant fusion protein of porcine β-defensin-2 and interferon αwith broad-spectrum antibacterial and antiviral activities at the same time,the genes encoding both proteins were fused in series and inserted into the pPICZαA vector.This construct was then elec-trotransferred into Pichia pastoris KM71H cells to construct an engineered strain.Following methanol-induced expression,the recombinant protein was concentrated and isolated.The cytotox-icity of the protein was assessed using the MTT assay and a porcine red blood cell hemolysis test.Subsequently,the in vitro antibacterial and antiviral activities of the recombinant fusion protein were evaluated.The results showed that the engineered strain pPICZαA-PBD2-IFNα-KM71H of Pichia pastoris was successfully obtained,and the fusion protein PBD2-IFN-α was obtained by concentrating and purifying the fermentation broth after fermentation induction and expression.Its concentration was 1.116 g/L and molecular weight was 25 kDa.When the concentration of fusion protein was lower than 4-4 g/L,it had no obvious toxicity to PK-15 cells and porcine red blood cells.The diameter of the inhibition zone produced by the fusion protein on the mixed plate of Escherichia coli and Staphylococcus aureus was(15.0±0.9)mm,which had obvious antibacterial activity.The antiviral activity of the fusion protein against VSV in PK-15 cells was 8.89 × 105 U/mL measured by Reed-Muench method.This study laid a theoretical foundation for further develo-ping the recombinant fusion protein as an antibacterial and antiviral product.

In order to obtain a recombinant fusion protein of porcine β-defensin-2 and interferon αwith broad-spectrum antibacterial and antiviral activities at the same time,the genes encoding both proteins were fused in series and inserted into the pPICZαA vector.This construct was then elec-trotransferred into Pichia pastoris KM71H cells to construct an engineered strain.Following methanol-induced expression,the recombinant protein was concentrated and isolated.The cytotox-icity of the protein was assessed using the MTT assay and a porcine red blood cell hemolysis test.Subsequently,the in vitro antibacterial and antiviral activities of the recombinant fusion protein were evaluated.The results showed that the engineered strain pPICZαA-PBD2-IFNα-KM71H of Pichia pastoris was successfully obtained,and the fusion protein PBD2-IFN-α was obtained by concentrating and purifying the fermentation broth after fermentation induction and expression.Its concentration was 1.116 g/L and molecular weight was 25 kDa.When the concentration of fusion protein was lower than 4-4 g/L,it had no obvious toxicity to PK-15 cells and porcine red blood cells.The diameter of the inhibition zone produced by the fusion protein on the mixed plate of Escherichia coli and Staphylococcus aureus was(15.0±0.9)mm,which had obvious antibacterial activity.The antiviral activity of the fusion protein against VSV in PK-15 cells was 8.89 × 105 U/mL measured by Reed-Muench method.This study laid a theoretical foundation for further develo-ping the recombinant fusion protein as an antibacterial and antiviral product.

Background Some studies have shown that PM_2.5 exposure is closely related to central nervous system diseases that lead to cognitive dysfunction and change the composition of intestinal flora. However, there are few studies on the role of intestinal flora in PM_2.5-induced depression- and anxiety-like behaviors in mice. Objective To observe the effects of PM_2.5 exposure on depression- and anxiety-like behaviors and the composition of intestinal flora in mice, and to explore the role of intestinal flora in regulating 5-hydroxytryptamine (5-HT) in depression- and anxiety-like behaviors in mice exposed to PM_2.5. Methods Eight-week-old male SPF C57BL/6J mice were randomly divided into control group (NS group), probiotic group (LGG group), PM_2.5 group (PM group), and combined exposure group (PML group), 6 mice in each group. Mice in the PM group and the PML group were exposed to PM_2.5 in a dynamic exposure cabinet for 6 h per day, 6 d a week for 7 consecutive weeks, and the PM_2.5 concentrations were approximately 8 times higher than the outdoor concentration. The LGG group and the PML group were orally administered with Lactobacillus rhamnosus while the NS group and the PM group were orally administered with the same amount of saline. Elevated plus maze test and open field test were used to detect depression and anxiety in mice. Fecal samples of mice were collected to evaluate intestinal flora abundance, diversity, and structure between groups using high-throughput sequencing of 16S rRNA. ELISA was employed to detect the levels of 5-HT in serum and hippocampus. Spearman correlation was used to analyze the correlations of differential intestinal flora with 5-HT level in hippocampus and depression- and anxiety-like behavior indicators in mice. Results The percentage of open-arm entry [M(P₂₅, P₇₅)] in the PM group was 0.0% (0.0%, 33.3%), lower than those in the NS group [47.7% (25.0%, 50.8%) ] and the PML group [46.9% (40.0%, 50.0%)], and the differences were statistically significant (P<0.05). The total travelled distance and the time spent in central area (\begin{document}$\bar x \pm s $\end{document}) in the PM group were (2.01±0.90) m and (10.31±1.99) s respectively, shorter than those of the NS group [(3.80±0.89) m, (14.47±3.07) s], the total travelled distance in the PML group [(2.73±1.12) m] was shorter than those of the NS group and the LGG group [(4.21±1.08) m], and the differences were statistically significant (P<0.05). Compared to the NS group, the Simpson index of the PM group significantly increased (P<0.05). Compared to the LGG group, the Simpson index of the PML group significantly decreased (P<0.05). The results of Beta diversity analysis showed that there were differences in the composition of intestinal flora among the four groups of mice. Compared with the NS group and the LGG group, the abundances of Erysipelotrichaceae and Dubosiella in the PM group and the PML group increased, while the abundances of Prevotellaceae_UCG-001 decreased, and the differences were statistically significant (P<0.05). In hippocampus, the level of 5-HT in the PM group [(135.02±10.31) μg·g⁻¹] was lower than those in the NS group [(178.77±43.15) μg·g⁻¹] and the LGG group [(224.85±22.98) μg·g⁻¹], and the level of 5-HT in the PML group [(161.27±15.81) μg·g⁻¹] was lower than that in the LGG group (P<0.05). 5-HT level in hippocampus was significantly positively correlated with the relative abundance of Prevotellaceae_UCG-001 (r=0.6090, P=0.012). The percentage of open-arm entry was significantly negatively correlated with the relative abundance of Dubosiella (r=−0.4630, P=0.023). Conclusion Atmospheric PM_2.5 exposure may cause depression- and anxiety-like behaviors in mice. The observed behavior dysfunction may be associated with the changes in diversity and relative abundance of intestinal flora as well as the decrease of 5-HT level. Such depression- and anxiety-like behaviors are alleviated after adding probiotics.

Objective To investigate whether soluble PD-1 overexpression in 4T1 senescence tumor cells enhances the anti-tumor effect of senescence tumor cell vaccine (STCV) against breast tumor cells in a tumor-bearing mouse model. Methods 4T1 cells were treated with interferon-γ (IFN-γ) and the expression of PD-L1 was detected by flow cytometry. CCK8 assay was used to compare the cell proliferation activity between 4T1 cells and 4T1 cells infected by a lentiviral vector of sPD-1 (4T1/sPD-1 cells), and the expressions of sPD-1 mRNA and protein in 4T1/sPD-1 cells were detected using qPCR and Western blotting. The culture supernatant of 4T1/sPD-1 cells was added in 4T1 cells pre-treated with IFN-γ, and PD-1-positive 4T1 cells were detected with flow cytometry. Senescenceβ-galactosidase staining kit was used to detect the senescent 4T1 and 4T1/sPD-1 cells following exposure to X-ray radiation and Veliparib. Balb/c mice bearing subcutaneous 4T1 tumor xenografts were treated with injections of PBS, 4T1 STCV, or 4T1/sPD-1 STCV, and tumor growth was observed. Results Stimulation with IFN-γconcentration-dependently up-regulated PD-L1 expression by as much as (84.80 ± 1.03)％ in 4T1 cells (P<0.001). sPD-1 overexpression in 4T1 cells did not significantly affect the cell proliferation. Treatment of 4T1 cells with 4T1/sPD-1 cell culture supernatant significantly increased the proportion of PD-1 + cells from (6.893 ± 0.271)％ to (55.450 ± 0.555)％ (P<0.001). X-ray irradiation combined with Veliparib caused obvious senescence in 4T1 and 4T1/sPD-1 cells. In the tumor-preventing experiment, tumor formation occurred in all the mice in PBS group;28.787％of the mice in 4T1 STCV group and 55.556％in 4T1/sPD-1 STCV group showed no tumor formation. In the tumor treatment experiment, tumor formation occurred in all the mice in PBS groups while in 4T1 STCV and 4T1/sPD-1 STCV groups, 11.111％ and 38.89％ of the mice were tumor-free during the observation period, respectively. Conclusions Senescence tumor cells vaccine has anti-tumor effect against breast cancer in mice, and sPD-1 over-expression can enhance this effect of the vaccine.

Objective To investigate whether soluble PD-1 overexpression in 4T1 senescence tumor cells enhances the anti-tumor effect of senescence tumor cell vaccine (STCV) against breast tumor cells in a tumor-bearing mouse model. Methods 4T1 cells were treated with interferon-γ (IFN-γ) and the expression of PD-L1 was detected by flow cytometry. CCK8 assay was used to compare the cell proliferation activity between 4T1 cells and 4T1 cells infected by a lentiviral vector of sPD-1 (4T1/sPD-1 cells), and the expressions of sPD-1 mRNA and protein in 4T1/sPD-1 cells were detected using qPCR and Western blotting. The culture supernatant of 4T1/sPD-1 cells was added in 4T1 cells pre-treated with IFN-γ, and PD-1-positive 4T1 cells were detected with flow cytometry. Senescenceβ-galactosidase staining kit was used to detect the senescent 4T1 and 4T1/sPD-1 cells following exposure to X-ray radiation and Veliparib. Balb/c mice bearing subcutaneous 4T1 tumor xenografts were treated with injections of PBS, 4T1 STCV, or 4T1/sPD-1 STCV, and tumor growth was observed. Results Stimulation with IFN-γconcentration-dependently up-regulated PD-L1 expression by as much as (84.80 ± 1.03)％ in 4T1 cells (P<0.001). sPD-1 overexpression in 4T1 cells did not significantly affect the cell proliferation. Treatment of 4T1 cells with 4T1/sPD-1 cell culture supernatant significantly increased the proportion of PD-1 + cells from (6.893 ± 0.271)％ to (55.450 ± 0.555)％ (P<0.001). X-ray irradiation combined with Veliparib caused obvious senescence in 4T1 and 4T1/sPD-1 cells. In the tumor-preventing experiment, tumor formation occurred in all the mice in PBS group;28.787％of the mice in 4T1 STCV group and 55.556％in 4T1/sPD-1 STCV group showed no tumor formation. In the tumor treatment experiment, tumor formation occurred in all the mice in PBS groups while in 4T1 STCV and 4T1/sPD-1 STCV groups, 11.111％ and 38.89％ of the mice were tumor-free during the observation period, respectively. Conclusions Senescence tumor cells vaccine has anti-tumor effect against breast cancer in mice, and sPD-1 over-expression can enhance this effect of the vaccine.