OBJECTIVE To screen and obtain nanobodies with neutralizing activity against granulo-cyte-macrophage colony stimulating factor(GM-CSF)to contribute to investigations into the mecha-nism of inflammation interventions and the development new drugs.METHODS Recombinant human GM-CSF was subcutaneously injected to immunize camels.Peripheral blood was collected after five immunizations,and mononuclear cells were isolated.Total mRNA was extracted,and the variable domains of the heavy chain of heavy-chain antibody(VHH,also called nanobody)genes were obtained by PCR amplification after reverse transcription.The genes were cloned into the pADSCFV-S phage display vector and electrotransformed into TG1 competent cells to construct a nanobody immune library that was screened with recombinant human GM-CSF immobilized on a solid phase.The VHH genes specifi-cally binding to human GM-CSF were cloned into the pABG eukaryotic expression vector before VHH-Fc samples were prepared by using the human embryonic kidney 293 fibroblast expression system.The binding activity of candidate VHH-Fc molecules to GM-CSF was investigated through ELISA response curves,and binding colorimetric values with different antigens were detected to determine their specificity.The binding affinity between VHH-Fc candidates and GM-CSF was measured using biolayer interferom-etry(BLI).The inhibition rate curve of VHH-Fc candidates on GM-CSF was detected through cell prolif-eration assays to determine its neutralization activity.The Uncle system was used to investigate its thermal stability.100 μg of VHH-Fc was injected into mice via the tail vein,and the serum concentration of VHH-Fc was quantitatively detected by ELISA to examine its pharmacokinetic curve in mice.RESULTS The camel serum titer of anti-human GM-CSF antibodies was higher than 1:800 000 after the fifth immuni-zation,and the capacity of the constructed nanobody library was about 5.55×107.Following the screening process,five candidate VHH-Fc molecules specifically binding to human GM-CSF were obtained.Among these,22N10 effectively neutralized the cell proliferation-promoting activity of GM-CSF(the IC50 value was 17.23 nmol·L-1).Subsequent studies revealed that 22N10 interacted with human GM-CSF with an affinity of 1.97×10-8 mol·L-1,blocked the binding of GM-CSF to its receptor CSF2Rα,exhibited good thermal stability(Tm1=59.2℃),and showed favorable metabolic characteristics in mice.CONCLU-SION A new candidate nanobody molecule 22N10 targeting human GM-CSF is obtained which is expected to facilitate the drug development and mechanism investigations.

Objective:To investigate the impact of postoperative complications on adverse outcomes following curative-intent resection for gallbladder cancer (GBC).Methods:The multi-center real-world study was conducted. The clinicopathological data of 629 patients with GBC, who were admitted to 14 medical centers including The First Affiliated Hospital of Army Medical University from the national multicenter database of Biliary Surgery Group of Elite Group of Chinese Journal of Digestive Surgery, from April 2020 to April 2024 were collected. There were 225 males and 404 females, aged (64±10)years. Patients underwent open curative-intent resection for GBC. Observation indicators: (1)surgery, postoperative complica-tions and adverse outcomes; (2) analysis of risk factors affecting postoperative adverse outcomes in patients and population attributable fraction (PAF). Missing data in predictor variables were addressed using multiple imputation with chained equations, while cases with missing outcome variables were addressed using the "multiple imputation then deletion (MID)" strategy. The severity of multicollinearity among independent variables was assessed using the variance inflation factor (VIF) test. Multivariable possion regression models with log link and robust error variance were construc-ted incorporating restricted cubic splines (3 knots) to address nonlinear relationships in continuous variables, calculating adjusted relative risk ( RR) with corresponding 95% confidence interval ( CI). Adjusted PAF was calculated for each imputed dataset using the AF package of R software, with subsequent pooling performed according to Rubin's rules. Results:(1) Surgery, postoperative complications and adverse outcomes. All 629 patients underwent curative-intent resection for GBC, of which 143 cases had postoperative complications, including 68 cases of intra-abdominal ascites, 39 cases of pulmonary infection, 21 cases of bile leakage, 12 cases of intra-abdominal hemorrhage, 11 cases of liver failure, 10 cases of pan-creatic fistula, 10 cases of wound infection, 10 cases of gastroparesis, 7 cases of cholangitis, 7 cases of sepsis. The same patient could have more than one kind of complication. Of 629 patients, there were 19 cases of postoperative 90-day death and 11 cases of missing data, 42 cases with post-operative 90-day reoperation and 7 cases with missing data, 44 cases with postoperative 90-day readmission and 3 cases with missing data, 155 cases with prolonged postoperative hospital stay and 3 cases with missing data. (2) Analysis of risk factors affecting the postoperative adverse outcomes in patients and PAF. Results of multivariate analysis showed that pulmonary infection and liver failure were independent risk factors for postoperative 90-day mortality ( RR=3.74, 12.15, 95% CI as 1.18-11.83, 1.98-74.48, P<0.05). Pulmonary infection demons-trated the highest PAF as 4.61% (95% CI as 3.94%-5.28%, P<0.05). Intra-abdominal ascites, pulmonary infection, bile leakage, and intra-abdominal hemorrhage were independent risk factors for post-operative 90-day reoperation ( RR=4.80, 3.62, 3.46, 4.99, 95% CI as 2.49-9.26, 1.42-9.21, 1.34-8.92, 1.55-16.06, P<0.05). Intra-abdominal ascites demonstrated the highest PAF as 8.65% (95% CI as 8.22%-9.08%, P<0.05). Intra-abdominal ascites, bile leakage, and liver failure were independent risk factors for postoperative 90-day readmission ( RR=6.20, 3.33, 14.33, 95% CI as 3.21-11.95, 1.33-8.35, 3.72-55.28, P<0.05). Intra-abdominal ascites demonstrated the highest PAF as 9.11% (95% CI as 8.85%-9.37%, P<0.05). Intra-abdominal ascites, pulmonary infection, bile leakage, liver failure, and wound infection were independent risk factors for prolonged postoperative hospital stay ( RR=2.29, 2.21, 2.26, 2.14, 3.35, 95% CI as 1.63-3.23, 1.41-3.46, 1.32-3.86, 1.11-4.13, 1.70-6.60, P<0.05). Intra-abdominal ascites demonstrated the highest PAF as 6.03% (95% CI as 5.71%-6.35%, P<0.05). Conclusion:Pulmonary infection is the most significant risk factor for postoperative 90-day mortality after curative-intent resection for GBC, while intra-abdominal ascites is the most significant risk factor for postoperative 90-day reoperation, postoperative 90-day readmission, and prolonged postoperative hospital stay.

Objective To explore the prognosis-related genes of lung adenocarcinoma(LUAD)and establish a prognostic prediction model for LUAD.Methods The differentially expressed genes of LUAD tissues and adjacent normal tissues in the GSE43458,GSE7670,and GSE140797 datasets were screened.The protein-protein interaction(PPI)network analysis,gene ontology(GO)function,Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway enrichment analyses,least absolute shrinkage and selection operator for prognosis coefficient screening,disease-specific survival analysis,Cox regression analysis,and gene correlation analysis were performed.Independent prognostic genes of LUAD were screened from the differential genes,and a prognostic prediction model of LUAD was established.The expression of independent prognostic genes was analyzed,and the predictive model was evaluated by receiver operating characteristic(ROC)curve.The GSE68465 data set was used as an external validation set to verify the predictive model.Results There were 197 up-regulated differential genes and 77 down-regulated differential genes in LUAD and adjacent normal tissues common to the three datasets.Through stepwise screening,two genes,IL7R and SLC2A1,were identified as independent prognostic factors for LUAD.IL7R was an independent protective factor,while the SLC2A1 was an independent risk factor.A prediction model for curve was built with IL7R and SLC2A1.The prediction model for LUAD constructed with IL7R and SLC2A1 is as follows:Risk score=(-0.694)×expression level of IL7R+0.763×expression level of SLC2A1.Conclusion This study screened out IL7R as an independent prognostic protective factor for LUDA,and SLC2A1 as an independent prognostic risk factor for LUAD.The LUDA prediction model constructed based on these two genes has good predictive ability and generalization ability,which can provide references for the research and clinical individualized treatment of LUAD.

OBJECTIVE To screen and obtain nanobodies with neutralizing activity against granulo-cyte-macrophage colony stimulating factor(GM-CSF)to contribute to investigations into the mecha-nism of inflammation interventions and the development new drugs.METHODS Recombinant human GM-CSF was subcutaneously injected to immunize camels.Peripheral blood was collected after five immunizations,and mononuclear cells were isolated.Total mRNA was extracted,and the variable domains of the heavy chain of heavy-chain antibody(VHH,also called nanobody)genes were obtained by PCR amplification after reverse transcription.The genes were cloned into the pADSCFV-S phage display vector and electrotransformed into TG1 competent cells to construct a nanobody immune library that was screened with recombinant human GM-CSF immobilized on a solid phase.The VHH genes specifi-cally binding to human GM-CSF were cloned into the pABG eukaryotic expression vector before VHH-Fc samples were prepared by using the human embryonic kidney 293 fibroblast expression system.The binding activity of candidate VHH-Fc molecules to GM-CSF was investigated through ELISA response curves,and binding colorimetric values with different antigens were detected to determine their specificity.The binding affinity between VHH-Fc candidates and GM-CSF was measured using biolayer interferom-etry(BLI).The inhibition rate curve of VHH-Fc candidates on GM-CSF was detected through cell prolif-eration assays to determine its neutralization activity.The Uncle system was used to investigate its thermal stability.100 μg of VHH-Fc was injected into mice via the tail vein,and the serum concentration of VHH-Fc was quantitatively detected by ELISA to examine its pharmacokinetic curve in mice.RESULTS The camel serum titer of anti-human GM-CSF antibodies was higher than 1:800 000 after the fifth immuni-zation,and the capacity of the constructed nanobody library was about 5.55×107.Following the screening process,five candidate VHH-Fc molecules specifically binding to human GM-CSF were obtained.Among these,22N10 effectively neutralized the cell proliferation-promoting activity of GM-CSF(the IC50 value was 17.23 nmol·L-1).Subsequent studies revealed that 22N10 interacted with human GM-CSF with an affinity of 1.97×10-8 mol·L-1,blocked the binding of GM-CSF to its receptor CSF2Rα,exhibited good thermal stability(Tm1=59.2℃),and showed favorable metabolic characteristics in mice.CONCLU-SION A new candidate nanobody molecule 22N10 targeting human GM-CSF is obtained which is expected to facilitate the drug development and mechanism investigations.

Objective:To investigate the impact of postoperative complications on adverse outcomes following curative-intent resection for gallbladder cancer (GBC).Methods:The multi-center real-world study was conducted. The clinicopathological data of 629 patients with GBC, who were admitted to 14 medical centers including The First Affiliated Hospital of Army Medical University from the national multicenter database of Biliary Surgery Group of Elite Group of Chinese Journal of Digestive Surgery, from April 2020 to April 2024 were collected. There were 225 males and 404 females, aged (64±10)years. Patients underwent open curative-intent resection for GBC. Observation indicators: (1)surgery, postoperative complica-tions and adverse outcomes; (2) analysis of risk factors affecting postoperative adverse outcomes in patients and population attributable fraction (PAF). Missing data in predictor variables were addressed using multiple imputation with chained equations, while cases with missing outcome variables were addressed using the "multiple imputation then deletion (MID)" strategy. The severity of multicollinearity among independent variables was assessed using the variance inflation factor (VIF) test. Multivariable possion regression models with log link and robust error variance were construc-ted incorporating restricted cubic splines (3 knots) to address nonlinear relationships in continuous variables, calculating adjusted relative risk ( RR) with corresponding 95% confidence interval ( CI). Adjusted PAF was calculated for each imputed dataset using the AF package of R software, with subsequent pooling performed according to Rubin's rules. Results:(1) Surgery, postoperative complications and adverse outcomes. All 629 patients underwent curative-intent resection for GBC, of which 143 cases had postoperative complications, including 68 cases of intra-abdominal ascites, 39 cases of pulmonary infection, 21 cases of bile leakage, 12 cases of intra-abdominal hemorrhage, 11 cases of liver failure, 10 cases of pan-creatic fistula, 10 cases of wound infection, 10 cases of gastroparesis, 7 cases of cholangitis, 7 cases of sepsis. The same patient could have more than one kind of complication. Of 629 patients, there were 19 cases of postoperative 90-day death and 11 cases of missing data, 42 cases with post-operative 90-day reoperation and 7 cases with missing data, 44 cases with postoperative 90-day readmission and 3 cases with missing data, 155 cases with prolonged postoperative hospital stay and 3 cases with missing data. (2) Analysis of risk factors affecting the postoperative adverse outcomes in patients and PAF. Results of multivariate analysis showed that pulmonary infection and liver failure were independent risk factors for postoperative 90-day mortality ( RR=3.74, 12.15, 95% CI as 1.18-11.83, 1.98-74.48, P<0.05). Pulmonary infection demons-trated the highest PAF as 4.61% (95% CI as 3.94%-5.28%, P<0.05). Intra-abdominal ascites, pulmonary infection, bile leakage, and intra-abdominal hemorrhage were independent risk factors for post-operative 90-day reoperation ( RR=4.80, 3.62, 3.46, 4.99, 95% CI as 2.49-9.26, 1.42-9.21, 1.34-8.92, 1.55-16.06, P<0.05). Intra-abdominal ascites demonstrated the highest PAF as 8.65% (95% CI as 8.22%-9.08%, P<0.05). Intra-abdominal ascites, bile leakage, and liver failure were independent risk factors for postoperative 90-day readmission ( RR=6.20, 3.33, 14.33, 95% CI as 3.21-11.95, 1.33-8.35, 3.72-55.28, P<0.05). Intra-abdominal ascites demonstrated the highest PAF as 9.11% (95% CI as 8.85%-9.37%, P<0.05). Intra-abdominal ascites, pulmonary infection, bile leakage, liver failure, and wound infection were independent risk factors for prolonged postoperative hospital stay ( RR=2.29, 2.21, 2.26, 2.14, 3.35, 95% CI as 1.63-3.23, 1.41-3.46, 1.32-3.86, 1.11-4.13, 1.70-6.60, P<0.05). Intra-abdominal ascites demonstrated the highest PAF as 6.03% (95% CI as 5.71%-6.35%, P<0.05). Conclusion:Pulmonary infection is the most significant risk factor for postoperative 90-day mortality after curative-intent resection for GBC, while intra-abdominal ascites is the most significant risk factor for postoperative 90-day reoperation, postoperative 90-day readmission, and prolonged postoperative hospital stay.

Objective To explore the prognosis-related genes of lung adenocarcinoma(LUAD)and establish a prognostic prediction model for LUAD.Methods The differentially expressed genes of LUAD tissues and adjacent normal tissues in the GSE43458,GSE7670,and GSE140797 datasets were screened.The protein-protein interaction(PPI)network analysis,gene ontology(GO)function,Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway enrichment analyses,least absolute shrinkage and selection operator for prognosis coefficient screening,disease-specific survival analysis,Cox regression analysis,and gene correlation analysis were performed.Independent prognostic genes of LUAD were screened from the differential genes,and a prognostic prediction model of LUAD was established.The expression of independent prognostic genes was analyzed,and the predictive model was evaluated by receiver operating characteristic(ROC)curve.The GSE68465 data set was used as an external validation set to verify the predictive model.Results There were 197 up-regulated differential genes and 77 down-regulated differential genes in LUAD and adjacent normal tissues common to the three datasets.Through stepwise screening,two genes,IL7R and SLC2A1,were identified as independent prognostic factors for LUAD.IL7R was an independent protective factor,while the SLC2A1 was an independent risk factor.A prediction model for curve was built with IL7R and SLC2A1.The prediction model for LUAD constructed with IL7R and SLC2A1 is as follows:Risk score=(-0.694)×expression level of IL7R+0.763×expression level of SLC2A1.Conclusion This study screened out IL7R as an independent prognostic protective factor for LUDA,and SLC2A1 as an independent prognostic risk factor for LUAD.The LUDA prediction model constructed based on these two genes has good predictive ability and generalization ability,which can provide references for the research and clinical individualized treatment of LUAD.

Objective:To compare the mid-term clinical effects of AngioJet rheolytic thrombectomy assisted catheter-directed thrombolysis (ART+CDT) with catheter-directed thrombolysis (CDT) in the treatment of acute deep venous thrombosis of lower extremities.Methods:Ninety-one patients admitted to the Department from Jan 2016 to Dec 2017 were placed with inferior vena cava filters and divided into ART+CDT group (30 cases)and CDT group (61 cases). Total urokinase dosge, thrombolytic time, operative cost, length of hospital stay, detumescence rate, thrombus clearance rate, cumulative patency rate of lower limb veins, Villalta score at 2 years and 5 years, thrombosis recurrence rate and chronic venous insufficiency quality of life questionnaire were compared between the two groups.Results:The success rate of surgery was 100% in both groups, there was no mortality. There were significant differences in the short-term postoperative outcomes between the two groups in terms of total dosage of urokinase, thrombolysis time, total cost of surgery, length of hospital stay, detumescence rate, venous patency scores before and after treatment, and venous patency rate (all P<0.05). For the mid- and long-term postoperative outcomes of 2 and 5 years, there were no significant differences in the incidence of PTS, recurrence rate of thrombus, chronic venous function scale, and cumulative patency rate at 2 years (all P>0.05). Conclusions:ART+CDT has a significant advantage over CDT alone in terms of early efficacy and early reopening of blood flow in patients. Both ART+CDT and CDT have a low incidence of PTS and a low recurrence rate of thrombus in the mid-term follow-up, and both have satisfactory performance in the mid- and long-term efficacy of interventional treatment of deep venous thrombosis of lower limbs.

Currently,there is an urgent need to develop new antimicrobial drugs or materials to treat drug-resistant bacterial infectious diseases.The reactive oxygen species(ROS)responsive nano-drug delivery systems hold great significance for bacterial infection treatment.This paper reviewed polymeric,inorganic,and composite ROS-responsive nano-drug delivery systems.It discussed synergistic treatment using these systems with photothermal therapy,photodynamic therapy,sonodynamic therapy,and gas therapy.It outlines new trends in these systems for bacterial infectious disease treatment.

Objective To explore the related risk factors of hypokalemia in elderly patients with acute cerebral hemorrhage(ACH),and construct a risk prediction model based on logistic regres-sion.Methods A total of 190 elderly ACH patients treated in Foshan Hospital of Traditional Chi-nese Medicine from June 2022 to May 2024 were enrolled as study objects,and were divided into hypokalemic group(potassium＜3.5 mmol/L,n=51)and normal group(potassium 3.5-5.5 mmol/L,n=139)according to whether hypokalemia occurred.Logistic regression model was used to analyze the risk factors of hypokalemia in the elderly ACH patients.Based on the identified risk factors,a comprehensive index model was constructed.ROC curve was drawn to analyze the diag-nostic value of the index for occurrence of hypokalemia in the patients.Results Larger female ra-tio,higher NIHSS score at admission,elevated urea nitrogen and blood creatinine at admission,and higher glomerular filtration rate(GFR)≤60 ml/min,and ratio of using hydrochlorothiazide＞20 mg/d were observed in the hypokalemic group than the normal group(P＜0.01).Univariate logistic regression analysis showed that female,NIHSS score at admission,urea nitrogen at ad-mission,serum creatinine at admission,GFR ≤60 ml/min,and hydrochlorothiazide dose＞20 mg/d were risk factors for hypokalemia in the ACH patients(P＜0.05,P＜0.01).Multivariate lo-gistic regression analysis indicated that female,NIHSS score at admission,GFR ≤60 ml/min,and hydrochlorothiazide dose＞20 mg/d were independent risk factors for hypokalemia in the elderly ACH patients(OR=6.393,95％CI:2.138-19.112,P=0.001;OR=3.123,95％CI:2.161-4.513,P=0.000;OR=3.327,95％CI:1.137-9.736,P=0.028;OR=3.111,95％CI:1.083-8.933,P=0.035).ROC curve analysis revealed that the AUC values of female,NIHSS score at admission,GFR,hydrochlorothiazide dose and comprehensive index in predicting hypokalemia in elderly ACH patients were 0.621,0.897,0.601,0.613 and 0.857,with a sensitivity of 52.90％,76.50％,49.00％,54.90％and 72.50％,and a specificity of 71.20％,88.50％,71.20％,67.60％and 87.80％,respectively.Conclusion Female,NIHSS score at admission,GFR ≤60 ml/min,and hydrochlorothiazide dose＞20 mg/d may affect the occurrence of hypokalemia in elderly ACH pa-tients,and our comprehensive index model based on these risk factors has high performance in predicting the occurrence of hypokalemia in elderly ACH patients.

OBJECTIVE: To investigate the effectiveness of joystick technique assisted closed reduction and cannulated screw fixation in the treatment of femoral neck fracture. METHODS: Seventy-four patients with fresh femoral neck fractures who met the selection criteria between April 2017 and December 2018 were selected and divided into observation group (36 cases with closed reduction assisted by joystick technique) and control group (38 cases with closed manual reduction). There was no significant difference in gender, age, fracture side, cause of injury, Garden classification, Pauwels classification, time from injury to operation, and complications (except for hypertension) between the two groups ( P>0.05). The operation time, intraoperative infusion volume, complications, and femoral neck shortening were recorded and compared between the two groups. Garden reduction index was used to evaluate the effect of fracture reduction, and score of fracture reduction (SFR) was designed and was used to evaluate the subtle reduction effect of joystick technique. RESULTS: The operation was successfully completed in both groups. There was no significant difference in operation time and intraoperative infusion volume between the two groups ( P>0.05). All patients were followed up 17-38 months, with an average of 27.7 months. Two patients in the observation group received joint replacement due to failure of internal fixation during the follow-up, and the other patients had fracture healing. Within 1 week after operation, the Garden reduxtion index of the observation group was better than the control group; the SFR score of the observation group was also higher than that of the control group; the proportion of femoral neck shortening within 1 week after operation and at 1 year after operation in the observation group were lower than those in the control group. The differences of the above indexes between the two groups were significant ( P<0.05). CONCLUSION The joystick technique can improve the effectiveness of closed reduction of femoral neck fractures and reduce the incidence of femoral neck shortening. The designed SFR score can directly and objectively evaluate the reduction effect of femoral neck fracture.
Humans ; Treatment Outcome ; Bone Screws ; Femoral Neck Fractures/surgery* ; Plastic Surgery Procedures ; Fracture Fixation, Internal/methods* ; Retrospective Studies