Objective:To single amino acid mutation of the full human scFvs against TSLP to enhance its affinity.Methods: The specific scFvs against TSLP was screened in our previous study and here the three-dimensional structures of TSLP and anti-TSLP scFvs were simulated by Discovery Studio system,then the molecular docking was made.The amino acids of binding epitope were randomly mutated and the mutated amino acids were selected which could remarkably improve the affinity of scFvs.The primers were designed based on the sequence of mutation amino acids and the scFv sequences were mutated by the overlapping extension PCR.The DNA of mutated scFvs was ligated with the expression vector pLZ16 and transformed into E.coli DH5αF′.Then the scFvs were expressed and the scFvs with improved affinity were selected by ELISA and BIAcore.Results: The five scFvs with single amino acid mutation were screened out by DS system,which could elevate the affinity of scFvs.The mutated anti-TSLP-scFvs were amplified by PCR,which size was about 1 000 bp.The mutated scFvs with correct sequence were expressed,and the mutated scFvs with improved affinity were detected by ELISA and BIAcore.The affinity of selected mutated scFv (M4) has been about 10 times higher than the scFv nonmutation.Conclusion: The affinity of anti-TSLP-scFv has been improved successfully.

To study the inhibitory effect of gigantol on proliferation, migration and invasion of human osteosarcoma U20S cells and to explore the mechanism. Methods: After being treated with different concentrations (10, 25, 50, 75, 100, 150 µmol/L) of gigantol for 24 and 48 h, the proliferation of U20S cells was detected by CCK-8 assay. Transwell assay was used to detect the effects of 25 µmol/L and 50 µmol/L gigantol on the migration and invasion abilities of U20S cells. The lipopolysaccharide (LPS) was used to induce inflammatory reaction in U20S cells before gigantol treatment; qPCR and WB were used to detect the mRNA and protein expressions of NF-κB (p65), TNF-α, IL-6 and PRL-3, respectively. Results: Different concentrations of gigantol could all inhibit the proliferation of sarcoma U20S cells at different time (P<0.05 or P<0.01). The 25 µmol/L and 50 µmol/L of gigantol could significantly inhibit the migration and invasion of osteosarcoma U20S cells (all P<0.01); at the same time, it could inhibit the protein expressions of NF-κB, TNF-α, IL-6 and PRL-3 (P<0.05 or P<0.01). After LPS induction, the mRNA and protein expressions of NF-κB, TNF-α, IL-6 and PRL-3 in U20S cells were significantly increased (all P<0.01); however, the consequent treatment with gigantol (25 and 50 µmol/L) reversed the effects of LPS on U20S cells obviously (P<0.05 or P<0.01). Conclusion: Gigantol can inhibit the proliferation, migration and invasion of osteosarcoma U20S cells, and its mechanism may be related to the regulation of NF-κB/PRL-3 signaling pathway.

Objective:To investigate the serum levels, diagnosis and prognosis value of endothelin-1(ET-1) in children suffering from lung metastasis of osteosarcoma.Methods:A total of 84 children with osteosarcoma, 67 children with lung metastasis of osteosarcoma and 35 healthy people from January 1, 2013 to January 1, 2018 in Second People′s Hospital of Nanyang were retrospectively included.The serum level of ET-1 was measured by performing enzyme linked immunosorbent assay(ELISA) methods and the influencing factors of serum ET-1 levels in children with lung metastasis of osteosarcoma were conducted by Logistic regression analysis.The clinical value of ET-1 in the prediction of the incidence of lung metastasis in children with osteosarcoma was analyzed by receiver operating characteristic (ROC) curve.Forty-five children with lung metastasis of osteosarcoma were followed up for 18 months and the prognosis value of serum ET-1 levels in children with lung metastasis of osteosarcoma was evaluated by Kaplan-Meier survival analysis. Results:The serum ET-1 level in children with lung metastasis of osteosarcoma was 97.23 (65.13, 134.98) ng/L and significantly higher than osteosarcoma group 60.21 (43.12, 74.63) ng/L and healthy control group 34.45 (12.01, 63.03) ng/L, respectively ( Z=-5.671, -4.92, all P<0.05), with significant differences. Logistic regression analysis proved that lung bilateral involvement ( OR=3.449), numbers of lung metastases (more than 3)( OR=3.449), average diameter of lung metastases (more than 5 cm) ( OR=6.501) and extrapulmonary metastasis ( OR=4.369) were independent risk factors for elevated serum ET-1 levels in children developing lung metastasis of osteosarcoma.The predictive value of ET-1 in the incidence of lung metastasis in children with osteosarcoma was significant (area under ROC curve: 0.841). When the cut-off value was 94.27 ng/L, the sensitivity and specificity were 88.5% and 92.6%, respectively.Survival analysis revealed that higher levels of ET-1 was correlated with poor prognosis ( OR=3.287, 95% CI: 1.119-9.547). Conclusions:The serum levels of ET-1 in children with lung metastasis of osteosarcoma are significantly elevated.ET-1 is a serological marker for the differential diagnosis of lung metastasis of osteosarcoma.Moreover, the higher levels of ET-1 are correlated with poor prognosis in children with lung metastasis of osteosarcoma.

OBJECTIVE To analyze the traditional Chinese medicine （TCM） emergency management policy texts in China， reveal the characteristics， problems and improvement directions of Chinese medicine emergency management policies in China， and provide references and lessons for improving the level of Chinese medicine emergency management. METHODS Twenty-four TCM emergency management policy texts issued at the central level from 2016 to 2023 were coded and analyzed using Nvivo11 software to construct a three-dimensional analysis framework based on policy tools， stakeholders and policy strength. RESULTS In the policy tools dimension， the environmental type was the most （46.74%）， the supply type was the second （31.80%）， and the demand type was the least （21.46%）； in the stakeholder dimension， there were more healthcare institutions （40.63%） and government departments （31.25%）， and fewer healthcare workers （14.84%） and residents （13.28%）； in the policy strength dimension， the overall policy strength was poor， and the differences in effectiveness across policy instruments and stakeholders were more significant. The cross-cutting results showed that there was a certain degree of mismatch in policy instruments， stakeholders and policy strength. CONCLUSIONS The use of supply-oriented policy tools is slightly lacking， and the use of policy tools should be optimized in a coordinated manner； the distribution of stakeholders is relatively unbalanced， and synergies among stakeholders should be enhanced； the overall strength of policies is poor， and the top-level design of relevant policies should be improved.