Gallstone disease is a common disease of the biliary system， which is jointly affected by environmental and genetic factors. With the change in dietary patterns， there has been a continuous increase in the the proportion of patients with cholesterol gallstones （CGS）， which is a common type of gallstone disease. Studies have shown that genetic factors play a crucial role in the formation of CGS. The formation of CGS mainly depends on two key links of the supersaturation of cholesterol in bile and the presence of mucin that promotes the nucleation of cholesterol crystals. These two processes involve the regulation of multiple proteins. Variations in certain genes can affect the efficiency of cholesterol transport and even change the rate of crystallization and nucleation， thereby influencing the formation of gallstones. This article mainly discusses the key genes associated with cholesterol transport and crystal nucleation， as well as the impact of their mutations on the formation of CGS， in order to gain a deeper understanding of the genetic mechanism of CGS and provide new targets for future clinical treatment.

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 analyze the association between plasma circulating tumor DNA(ctDNA)TP53 mutation status and survival outcomes in breast cancer patients.Methods PubMed,Embase,and the Cochrane Library were searched for relevant literature published between 2000 and 2025,examining the impact of plasma ctDNA TP53 mutations on survival outcomes in breast cancer patients,including overall survival(OS),progression-free survival(PFS),or disease-free survival(DFS).Two researchers independently screened the literature according to predefined inclusion and exclusion criteria and extracted relevant data.The Newcastle-Ottawa scale and Cochrane Risk of Bias Assessment Tool were used to evaluate study quality.Meta-analysis,publication bias assessment,and sensitivity analysis were performed using Review Manager 5.3 and STATA 18.0 software.Results A total of 14 studies(13 cohort studies and 1 randomized controlled trial)in-volving 3521 breast cancer patients were included,among whom 921 had TP53 mutations.All studies were as-sessed as high-quality or low-risk.The random-effects model demonstrated that TP53 mutations were significantly associated with poorer OS(I2=77%,HR=1.82,95%CI:1.15-2.88,P=0.010),PFS(I2=63%,HR=1.68,95%CI:1.30-2.17,P＜0.001),and DFS(I2=85%,HR=1.98,95%CI:1.05-3.75,P=0.040).Funnel plots indicated no significant publication bias,and sensitivity analysis confirmed the stabil-ity and reliability of the results.Subgroup analyses based on study design,breast cancer stage and molecular subtype revealed that TP53 mutations were associated with worse prognosis in prospective studies(OS:HR=2.32,95%CI:1.84-2.92,P＜0.001;PFS:HR=1.83,95%CI:1.47-2.27,P＜0.001),metastatic/ad-vanced breast cancer(OS:HR=2.03,95%CI:1.44-2.87,P＜0.001;PFS:HR=1.90,95%CI:1.57-2.31,P＜0.001),and hormone receptor-positive and human epidermal growth factor receptor 2-negative(HR+HER2-)patients(OS:HR=2.11,95%CI:1.56-2.85,P＜0.001;PFS:HR=1.94,95%CI:1.62-2.33,P＜0.001).Among different treatment regimens,patients with TP53 mutations receiving trastuzumab combined with paclitaxel exhibited relatively better prognosis(PFS:HR=0.08,95%CI:0.02-0.30,P＜0.001).Conclusion Plasma ctDNA TP53 mutations are closely associated with survival outcomes in breast cancer patients and may serve as a potential predictor of poor prognosis,providing support for clinical manage-ment and prognostic assessment.

To investigate the association between obesity-related metabolic indices and the risk of knee osteoarthritis(KOA) in middle-aged and older Chinese adults(≥45 years) using data from the China Health and Retirement Longitudinal Study(CHARLS).

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 the performance evaluation index system of specialist cooperation alliance in hos-pital-community in order to scientifically evaluate the effect of the construction of the specialist cooperation alliance.Methods By searching literature related to the construction of specialized league,sort out national and local policy documents,and the"structure-process-result"model was adopted to initially construct the index system.The ex-pert letter consultation form of the indicator system was formed through the discussion of the special group,two rounds of Delphi expert consultation were carried out to determine the evaluation indicator system,and the weight coefficient of each indicator was determined by the simultaneous application of analytic hierarchy process.Results The effective recovery rates of the two rounds of questionnaire were 84.62％and 100.00％,respectively.The positive de-gree of experts was high,and the average authority degree of experts was 0.864.The importance,operability,sen-sitivity Kendall's W coefficient and x2 test value of all indexes were P＜0.05.Finally,an evaluation index system covering 9 first-level indicators and 25 second-level indicators was established.Conclusion The constructed perfor-mance evaluation index is scientific and reliable.The practical application shows that it can be used as a tool to evalu-ate the effect of integrated,continuous and collaborative health service delivery in hospital-community.

Objective To analyze the association between plasma circulating tumor DNA(ctDNA)TP53 mutation status and survival outcomes in breast cancer patients.Methods PubMed,Embase,and the Cochrane Library were searched for relevant literature published between 2000 and 2025,examining the impact of plasma ctDNA TP53 mutations on survival outcomes in breast cancer patients,including overall survival(OS),progression-free survival(PFS),or disease-free survival(DFS).Two researchers independently screened the literature according to predefined inclusion and exclusion criteria and extracted relevant data.The Newcastle-Ottawa scale and Cochrane Risk of Bias Assessment Tool were used to evaluate study quality.Meta-analysis,publication bias assessment,and sensitivity analysis were performed using Review Manager 5.3 and STATA 18.0 software.Results A total of 14 studies(13 cohort studies and 1 randomized controlled trial)in-volving 3521 breast cancer patients were included,among whom 921 had TP53 mutations.All studies were as-sessed as high-quality or low-risk.The random-effects model demonstrated that TP53 mutations were significantly associated with poorer OS(I2=77%,HR=1.82,95%CI:1.15-2.88,P=0.010),PFS(I2=63%,HR=1.68,95%CI:1.30-2.17,P＜0.001),and DFS(I2=85%,HR=1.98,95%CI:1.05-3.75,P=0.040).Funnel plots indicated no significant publication bias,and sensitivity analysis confirmed the stabil-ity and reliability of the results.Subgroup analyses based on study design,breast cancer stage and molecular subtype revealed that TP53 mutations were associated with worse prognosis in prospective studies(OS:HR=2.32,95%CI:1.84-2.92,P＜0.001;PFS:HR=1.83,95%CI:1.47-2.27,P＜0.001),metastatic/ad-vanced breast cancer(OS:HR=2.03,95%CI:1.44-2.87,P＜0.001;PFS:HR=1.90,95%CI:1.57-2.31,P＜0.001),and hormone receptor-positive and human epidermal growth factor receptor 2-negative(HR+HER2-)patients(OS:HR=2.11,95%CI:1.56-2.85,P＜0.001;PFS:HR=1.94,95%CI:1.62-2.33,P＜0.001).Among different treatment regimens,patients with TP53 mutations receiving trastuzumab combined with paclitaxel exhibited relatively better prognosis(PFS:HR=0.08,95%CI:0.02-0.30,P＜0.001).Conclusion Plasma ctDNA TP53 mutations are closely associated with survival outcomes in breast cancer patients and may serve as a potential predictor of poor prognosis,providing support for clinical manage-ment and prognostic assessment.

Objective To construct the performance evaluation index system of specialist cooperation alliance in hos-pital-community in order to scientifically evaluate the effect of the construction of the specialist cooperation alliance.Methods By searching literature related to the construction of specialized league,sort out national and local policy documents,and the"structure-process-result"model was adopted to initially construct the index system.The ex-pert letter consultation form of the indicator system was formed through the discussion of the special group,two rounds of Delphi expert consultation were carried out to determine the evaluation indicator system,and the weight coefficient of each indicator was determined by the simultaneous application of analytic hierarchy process.Results The effective recovery rates of the two rounds of questionnaire were 84.62％and 100.00％,respectively.The positive de-gree of experts was high,and the average authority degree of experts was 0.864.The importance,operability,sen-sitivity Kendall's W coefficient and x2 test value of all indexes were P＜0.05.Finally,an evaluation index system covering 9 first-level indicators and 25 second-level indicators was established.Conclusion The constructed perfor-mance evaluation index is scientific and reliable.The practical application shows that it can be used as a tool to evalu-ate the effect of integrated,continuous and collaborative health service delivery in hospital-community.

Objective:To investigate the incidence and prothrombotic risk factors of postoperative venous thromboembolism(VTE) in Cushing′s disease and to further develop an assessment model to identify those at high risk of postoperative VTE events.Methods:A retrospective study was performed in 82 patients who were admitted to Huashan Hospital, Fudan University during January 2019 and January 2020 and diagnosed with Cushing′s disease. These patients underwent the evaluation about their clinical, hormonal, and coagulation parameters, as well as ultrasonography and pulmonary angio-CT when necessary. The least absolute shrinkage and selection operator(LASSO) regression analysis was used to screen independent risk factors, and a nomogram model for postsurgical VTE risk assessment in Cushing′s disease was initially established, and Bootstrap method was used for internal verification. Finally, the predictive model was evaluated for calibration and clinical applicability in the study cohort.Results:Nineteen patients(23.17%) developed VTE events, with 14 cases occurring after endoscopic transsphenoidal surgery. Compared to patients without VTE, those in the VTE group were older( P<0.001), had longer postoperative bed rest, higher rates of current infection, higher HbA 1C levels, and more severe glucose tolerance impairment(all P<0.05). Through LASSO regression analysis, two independent risk factors for postoperative VTE were identified: Age and current infection. Then a VTE risk assessment nomogram model was established to predict the patients at high risk of VTE. In the nomogram model for VTE risk assessment, the area under the receiver operating characteristic curve was 0.868(95% CI 0.787-0.949), with the calibration curve closely aligning with the ideal diagonal line and the clinical decision curve exceeding the two extreme curves. Conclusions:Advanced perioperative assessment needs to be taken to screen those with high VTE risks in patients diagnosed with Cushing′s disease. Additionally, during the perioperative period, patients with Cushing′s disease should undergo mandatory physical activity or prophylactic anticoagulant therapy.

Objective:This study aimed to investigate the clinical value of glucocorticoids in patients with neutropenic severe pneumonia at moderate to high risk according to the Pneumonia Severity Index (PSI) in patients with hematologic diseases.Methods:Clinical data were collected from 534 patients at the Fujian Medical University Union Hospital from October 2016 to December 2018. We evaluated the changes in inflammatory cytokines, treatment failure, in-hospital mortality, and other outcomes, adjusting for potential confounders through propensity score matching.Results:Patients were categorized into glucocorticoids ( n=176) and control ( n=358) groups. The glucocorticoid group demonstrated higher levels of C-reactive protein, procalcitonin, and interleukin-6, alongside higher PSI scores. The differences in comorbidities diminished, except for inflammatory cytokine levels, with a notable reduction in inflammatory cytokines observed in the glucocorticoid group, after matching 125 pairs based on propensity scores. Late treatment failure was more prevalent in the glucocorticoid group (39.2% vs 24.8%, P=0.015), but this was primarily caused by radiographic progression. The incidences of respiratory failure, mechanical ventilation, and septic shock were similar between the groups. Logistic regression analyses revealed that glucocorticoids reduced the risk of treatment failure ( OR=0.367, 95% CI 0.165-0.818, P=0.014). The 30-day in-hospital mortality rates were comparable (8.0% in glucocorticoids vs 7.2% in controls, P=0.811), with indications that glucocorticoids may exert a protective effect on mortality. The PSI score was determined as the sole independent risk factor for 30-day in-hospital mortality ( OR=1.077, 95% CI 1.032-1.123, P=0.001). No evidence indicated that glucocorticoids increased the incidence of hyperglycemia, gastrointestinal bleeding, or 30-day infection recurrence. Conclusion:Glucocorticoids reduce inflammatory cytokine levels and are potentially related to decreased treatment failure and mortality in patients with neutropenic pneumonia classified as PSI Ⅳ and Ⅴ among hematological patients.