Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

Under the background of deepening the reform of medical and health system,the reform of public hos-pital salary system reform faces the dual challenges of realizing the value of knowledge and maintaining public wel-fare.Based on the public value theory,it constructs a synergistic analytical framework of"knowledge value and pub-lic welfare"to reveal the internal logic and realization path of public hospital pay system reform.Through the mecha-nisms of policy design reconstruction,assessment system innovation,remuneration structure optimization and multi-dimensional collaborative governance,the current systemic obstacles such as incentive alienation,misalign-ment of assessment,and insufficient financial compensation can be effectively solved.It is necessary to build a pub-lic value target,construct a synergistic system of"constraints and incentives",and activate the main motivation of medical staff to form a closed loop of"target-setting-mechanism-driven-practice-feedback"value cycle.It pro-vides a theoretical innovation path for the reform of public hospital remuneration system,and has important reference value for balancing the efficiency and fairness of medical services.

Objective To explore and analyze the incentive changes before and after the implementation of the annual salary system in public hospitals to provide guidance for the implementation.Methods Using the principal-agent theory,construct the structure and model of government,hospitals and doctors,and analyze the salary incentive before and after the implementation of the annual salary system.Results Former doctors"seek profit"and hospital"income orientation"conflict with the government public welfare goals;after implementation,doctors transfer"public welfare"and hospital and government goals seek"public welfare coordination",but there are also new problems such as insufficient incentives.Conclusion If public hospitals successfully implement the annual salary system,they should adopt fine parameter design,supporting system and dynamic equilibrium mechanism.

Under the background of deepening the reform of medical and health system,the reform of public hos-pital salary system reform faces the dual challenges of realizing the value of knowledge and maintaining public wel-fare.Based on the public value theory,it constructs a synergistic analytical framework of"knowledge value and pub-lic welfare"to reveal the internal logic and realization path of public hospital pay system reform.Through the mecha-nisms of policy design reconstruction,assessment system innovation,remuneration structure optimization and multi-dimensional collaborative governance,the current systemic obstacles such as incentive alienation,misalign-ment of assessment,and insufficient financial compensation can be effectively solved.It is necessary to build a pub-lic value target,construct a synergistic system of"constraints and incentives",and activate the main motivation of medical staff to form a closed loop of"target-setting-mechanism-driven-practice-feedback"value cycle.It pro-vides a theoretical innovation path for the reform of public hospital remuneration system,and has important reference value for balancing the efficiency and fairness of medical services.

Objective To explore and analyze the incentive changes before and after the implementation of the annual salary system in public hospitals to provide guidance for the implementation.Methods Using the principal-agent theory,construct the structure and model of government,hospitals and doctors,and analyze the salary incentive before and after the implementation of the annual salary system.Results Former doctors"seek profit"and hospital"income orientation"conflict with the government public welfare goals;after implementation,doctors transfer"public welfare"and hospital and government goals seek"public welfare coordination",but there are also new problems such as insufficient incentives.Conclusion If public hospitals successfully implement the annual salary system,they should adopt fine parameter design,supporting system and dynamic equilibrium mechanism.

Objective:To establish a risk prediction model for the occurrence of low 1 min Apgar scores in extremely premature infants (EPIs).Methods:From January 2017 to December 2021, EPIs delivered at our hospital were retrospectively analyzed and randomly assigned into training set group and validation set group in a 7∶3 ratio. 17 clinical indicators were selected as predictive variables and low Apgar scores after birth as outcome variables. Lasso regression and multi-factor logistic regression were used within the training set group to select the final predictors for the final model, and the calibration, distinguishability and clinical decision making curves of the final model were evaluated in the validation set group.Results:A total of 169 EPIs were enrolled, including 117 in the training set group and 52 in the validation set group. 4 indicators including gender, fetal distress, assisted conception and delivery time were selected as the final predictors in the final model. Both the training set group and the validation set group had good calibration curves. The area under the receiver operating characteristic curve (AUC) of the prediction model was 0.731, the sensitivity was 72.2%, the specificity was 60.5% and the AUC of the external validation curve was 0.704. The clinical decision making curve showed that the model had a greater benefit in predicting the occurrence of low Apgar score in EPIs within the threshold of 2% to 75%.Conclusions:The clinical prediction model established in this study has good distinguishability, calibration and clinical accessibility and can be used as a reference tool to predict low Apgar scores in EPIs.

Objective:To investigate the methylation status of SDC2, PPP2R5C and ADHFE1 genes in stool and their values in the screening of colorectal cancer and precancerous lesions.Methods:From August 2020 to March 2021, 64 patients with colorectal cancer, 72 patients with adenoma, 33 patients with hyperplastic polyps and 59 healthy people were recruited from Qingdao Central Hospital Affiliated to Qingdao University, and the morning stool samples were collected from the research subjects. The genomic DNA was extracted and modified with sulfite. The methylation status of SDC2, PPP2R5C and ADHFE1 genes were detected by methylation specific polymerase chain reaction (MSP), and the fecal occult blood test (FOBT) was performed. Taking the pathological results as the gold standard, receiver operating characteristic (ROC) curve and area under the curve (AUC) were used to compare the effect of combined detection of methylation of three genes and FOBT in predicting colorectal cancer and precancerous lesions. R-Studio software was used to construct a nomogram for the prediction of colorectal cancer with combined detection of gene methylation in stool and other clinical features, and the calibration and validation were performed.Results:The positive rates of combined detection of methylation of SDC2, PPP2R5C and ADHFE1 genes in stool were higher than those of FOBT in colorectal cancer+adenoma [74.3% (101/136) vs. 47.1% (64/136), χ2 = 23.20, P = 0.001], colorectal cancer [90.6% (58/64) vs. 70.3% (45/64), χ2 = 8.91, P = 0.003] and adenoma [59.7% (43/72) vs. 26.4% (19/72), χ2 = 14.43, P = 0.002]. There was no significant difference in the positive rates in hyperplastic polyps [21.2% (7/33) vs. 6.1% (2/33), χ2 = 0.12, P = 0.125] and healthy controls [10.2% (6/59) vs. 8.5% (5/59), χ2 = 4.01, P = 1.000]. The combined detection of gene methylation was better than FOBT in the prediction of colorectal cancer + adenoma [AUC: 0.85 (95% CI 0.80-0.91) vs. 0.71 (95% CI 0.64-0.78), P < 0.05], especially in the prediction of adenoma [AUC: 0.82 (95% CI 0.74-0.89) vs 0.64 (95% CI 0.57-0.69), P < 0.001]. The sensitivity and specificity of ADHFE1 gene methylation status in predicting colorectal cancer were high (90.6% and 96.6%). In colorectal cancer patients over 50 years old, the positive rate of combined detection of gene methylation was higher than that of FOBT [90.2% (55/61) vs. 68.9% (42/61), P < 0.05]. The nomogram calibration curve for predicting colorectal cancer constructed based on the combined detection of gene methylation and each clinical feature showed a high degree of concordance between the predicted and observed diagnostic performance of colorectal cancer. Conclusions:The methylation levels of SDC2, PPP2R5C AND ADHFE1 genes in stool are increased in patients with colorectal cancer or adenoma. The combined detection of gene methylation is expected to be a non-invasive method for the screening of colorectal cancer and precancerous lesions.

Objective:To explore the effect of problem-based learning (PBL) combined with case teaching method in the three-year probationary teaching of clinical urology in three-year higher vocational colleges.Methods:A total of 45 students from Class 1, Grade 2015, and 37 students from Class 2, Grade 2016 of Ningxia Medical University were selected as study subjects. They were divided into experimental group and control group. Forty-two students in experimental group were taught with PBL combined with case teaching method. 40 students in control group were taught with traditional clinical probation teaching method. Besides, a questionnaire survey was conducted among two groups of students in the Department of urology.Results:There was no significant difference in the scores of theoretical examination between the experimental group and the control group ( P>0.05). The scores of medical history collection, physical examination, practical skills and total scores were much higher in the experimental group than in the control group. The results of the questionnaire survey showed that the teaching of the experimental group could significantly motivate students' enthusiasm and participation, as well as enhance ability of self-study, information acquisition, innovation, analysis and problem-solving and team consciousness ( P<0.05). Conclusion:The new model of PBL combined with case teaching is helpful in improving students' academic performance, probation effect and comprehensive practical ability in urology internship teaching, which is worth being popularized in clinical teaching.

OBJECTIVE： To investigate the potential effective components and mechanism of Achyranthes bidentata in the treatment of osteoporosis （OP）. METHODS： The effective components of A. bidentata were retrieved from the TCMSP database， and corresponding targets of them were collected. The targets related to OP were retrieved from DisGeNET database. TBtools 1.0 mapping software was used to draw the Wayne diagram， and screen the intersecting targets of effective components of A. bidentata and disease OP. Cytoscape 3.6.1 software and STRING database were used to construct and analyze the “drug-component- disease-target” network and protein-protein interaction （PPI） network； KEGG pathway enrichment analysis was conducted by using DAVID bioinformatics resource database. RESULTS： A total of 19 effective components were screened from A. bidentata， and there were 32 intersecting targets between effective components and disease OP. In “drug-component-disease-target” network， there were 45 nodes [1 for A. bidentata， 1 for OP， 11 for effective components （8 of the 19 effective components had no corresponding OP target）， 32 for intersecting targets] and 119 edges between nodes； quercetin， kaempferol， wogonin， baicalein and palmatine were important effective components. In PPI network， there were 31 nodes （1 of 32 intersecting targets was not associated with other proteins） and 212 edges， among which IL6， ESR1， MAPK1， IL8 and MAPK14 were the core targets of the network. There were 67 KEGG enrichment pathways， including rheumatoid arthritis， hepatitis B， Toll-like receptor signaling pathway， PI3K/Akt signaling pathway， JAK/STAT signaling pathway， NF-κB signaling pathway and so on. CONCLUSIONS： The main potential effective components of A. bidentata in the treatment of OP are quercetin， kaempferol， wogonin， baicalein and palmatine， the mechanism of which may be associated with cell differentiation and apoptosis， metabolism， inflammation reaction， etc. It     has multi-component， multi-target and multi-system chara- cteristics.