Objective To heterologously express and purify the small ubiquitin-like modifier(SUMO)tag fused organo-phosphorus hydrolase mutant H257Y/L303T(YT),namely SUMO-YT,and evaluate its hydrolytic capacity against ethyl paraoxon and soman.Methods The SUMO tag encoding gene was constructed at the N-terminus of the YT encoding gene with a linker sequence via enzyme digestion and ligation before SUMO-YT was expressed in Escherichia coli.SUMO-YT and YT were purified through ammonium sulfate precipitation and affinity chromatography to obtain high-purity target proteins.The activity and kinetic parameters of the recombinant enzymes were examined using ethyl paraoxon and soman as substrates.Results The system for expression and purification of recombinant enzymes was established,yielding SUMO-YT and YT,and the former exhibited more significantly enhanced hydrolytic efficiency than the latter,with catalytic rates 11-fold higher for paraoxon and 4.4-fold higher for soman.At 37 ℃ and pH 7.2,SUMO-YT reduced the inhibition rate of acetylcholinesterase(AChE)by soman from 100％to 45.7％within 3 minutes,whereas YT reduced it to no more than 80％.Conclusion The high-activity recombinant SUMO-YT is prepared.SUMO tag fusion can significantly enhance the hydrolytic capacity of YT against ethyl paraoxon and soman.

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Objective:To analyze the risk factors for fat liquefaction after surgery for pediatric appendiceal abscess and to construct and validate a predictive model for post-operative fat liquefaction.Methods:The training set collected clinical data from 60 children who developed fat liquefaction after appendiceal abscess surgery and 60 children who did not develop fat liquefaction for comparison.The validation set collected data from 97 children during the same period ac-cording to the same criteria to test the model's efficacy.LASSO logistic regression was used to screen potential diag-nostic factors,and a Logistic regression model was employed for univariate analysis.Collinearity diagnostic tests were conducted among the risk factors.The stepwise regression method using the training dataset was applied to evaluate the importance of each risk factor for fat liquefaction.A multivariate Cox proportional hazards model was used to calcu-late the concordance index of the risk factors in both the training and validation sets.A predictive model was constructed using Logistic regression,and the clinical value of the predictive model was evaluated using ROC curves,calibration curves,and decision curves.Results:Compared with the non-liquefaction group,the liquefaction group had higher val-ues in terms of body weight,heart rate,diameter of the tender mass in the right lower abdomen,degree of appendiceal thickening,body temperature upon admission,fasting plasma glucose(FPG)level,surgical duration,intraoperative blood loss,wound healing time,length of hospital stay,volume of abdominal drainage fluid,and infection status(P<0.05).LASSO logistic regression analysis identified potential factors including body temperature upon admission,heart rate,body weight,diameter of the tender mass in the right lower abdomen,degree of appendiceal thickening,FPG level,sur-gical duration,and intraoperative blood loss.Univariate analysis revealed that body weight,diameter of the tender mass in the right lower abdomen,FPG level,surgical duration,and intraoperative blood loss were influencing factors for fat lique-faction after appendectomy(P<0.05).Body weight and FPG factors were mutually independent with no multicollinearity,while there was multicollinearity among the diameter of the tender mass in the right lower abdomen,surgical duration,and intraoperative blood loss.The risk model combining the diameter of the tender mass in the right lower abdomen,in-traoperative blood loss,FPG,surgical duration,and body weight had the highest concordance index in both the training and validation sets,with values of 0.811 and 0.814,respectively.The Logistic regression model established the predictive model as Logit(P)=-1.136+0.664×(diameter of the tender mass in the right lower abdomen)+0.449×(surgical duration)+0.622×(intraoperative blood loss)+0.200×(body weight)+0.578×(FPG).The area under the ROC curve was 0.920(95%CI:0.869～0.942),with a sensitivity of 93.14%and a specificity of 85.73%.The calibration curve of the predictive model showed good fit with the ideal curve.The predictive model had a high net benefit.Conclusion:Body weight,diameter of the tender mass in the right lower abdomen,FPG level,surgical duration,and intraoperative blood loss are risk factors for fat liquefaction after appendectomy and have good predictive value for the occurrence of fat liquefaction.

Objective To identify the key influencing factors and role mechanisms of large-scale medical equip-ment allocation decision-making in public hospitals,and to provide a reference basis for optimizing resource alloca-tion strategies.Methods Through searching the literature related to large-scale medical equipment decision-making in public hospitals and combing the policies,the system of decision-making influencing factors was initially screened.The Delphi method and decision-making trial and evaluation laboratory method were used to obtain the key influencing factors of equipment allocation decision-making and to determine the interaction effects among the factors.Results A total of seven cause factors and nine effect factors were screened.Among them,cost-benefit analysis,equipment technical performance,and evaluation of similar equipment were the top three key drivers;leadership emphasis,equipment market price,and equipment supporting facilities were the top three key con-straints.Conclusion Priority be given to strengthening the management of driving factors,mainly by conducting a full-cycle assessment of cost-effectiveness,paying attention to the clinical value output of equipment technical performance,and monitoring regionally similar equipment.A dynamic response mechanism for constraints should be established,mainly to strengthen leadership decision-making,implement a centralized procurement price nego-tiation model and improve the pre-assessment system for ancillary facilities.

Objective To investigate the protective effect of Ghrelin on traumatic brain injury(TBI)in rats based on the HO-1/PGC-1αsignaling pathway.Methods Thirty SPF male rats were randomly divided into sham,TBI,and Ghrelin groups,with 10 rats in each group.A TBI rat model was established using the Feeney free-fall impingement method.The Ghrelin group was injected by caudal vein at a dose of 20 μg/kg 30 min after modeling,while the sham group was not impinged.After 72 h of modeling,the brain tissues of the rats were col-lected,and the brain water content was measured in order to analyze the severity of brain edema.HE staining was used to observe patho-logical changes in brain tissue.The levels of the oxidative stress factors MDA,SOD,and GSH-Px were determined using ELISA.TUNEL staining was used to detect the apoptosis of the brain cells,and the expression levels of Bcl-2,Bax,caspase-3 and caspase-9 in the brain tissues were detected by Western blotting.Mitochondrial reactive oxygen species(mtROS)in the brain tissues were detected by immuno-fluorescence.Mitochondrial function indicators,including mitochondrial mitogen Mfn 1/2,nuclear respiration factor 1(NRF1),and mito-chondrial transcription factor A(TFAM)were detected by Western blotting.The expression levels of HO-1 and PGC-1α in the brain tis-sues of rats in each group were detected by Western blotting.Twenty TBI model rats treated with Ghrelin were divided into Ghrelin+sh-NC and Ghrelin+sh-HMOX1 group with 10 rats in each group.Rats were treated with Ghrelin and injected with knock-down control(adenovirus 2.5 × 109 pfu)or knock-down HMOX1 adenovirus(2.5 × 109 pfu)via tail vein.Western blotting was used to detect the expressions of HO-1,PGC-1 α,Bcl-2,Bax,caspase-3 and caspase-9 in the brain tissues of the two groups.The levels of MDA,SOD and GSH-Px in brain tissue of two groups were detected by ELISA.Results Compared with the sham group,the pathological injury and brain edema in TBI group were aggravated,the number of brain cell apoptosis increased,the levels of oxidative stress factors SOD and GSH-Px decreased,the level of MDA increased,the level of mtROS in brain tissue decreased,the expressions of Bax,caspase-3 and caspase-9 increased,and the expressions of Mfn1/2,NRF1,TFAM,HO-1 and PGC-1α decreased(P＜0.05).Compared with TBI group,the pathological damage of brain tissue in Ghrelin group was improved,the brain edema was alleviated,the number of brain cell apoptosis was reduced,the levels of oxidative stress factors SOD and GSH-Px were increased,the level of MDA was decreased,the mtROS in brain tissue was decreased,the expression of Bcl-2 protein was increased,the expressions of Bax,caspase-3 and caspase-9 protein were decreased,and the expressions of Mfn1/2,NRF1,TFAM,HO-1 and PGC-1α were decreased(P＜0.05).Compared with Ghrelin+sh-NC group,the expressions of Bax,caspase-3 and caspase-9,MDA in brain tissue of Ghrelin+sh-HMOX1 group increased,while the levels of SOD and GSH-Px decreased(P＜0.05).Conclusion Chrelin has protective effect on TBI in rats,and can inhibit brain tissue injury and apoptosis in rats.Its mechanism may be achieved by regulating mitochondrial oxidative stress through HO-1/PGC-1α signaling pathway.

Objective:To analyze the risk factors for fat liquefaction after surgery for pediatric appendiceal abscess and to construct and validate a predictive model for post-operative fat liquefaction.Methods:The training set collected clinical data from 60 children who developed fat liquefaction after appendiceal abscess surgery and 60 children who did not develop fat liquefaction for comparison.The validation set collected data from 97 children during the same period ac-cording to the same criteria to test the model's efficacy.LASSO logistic regression was used to screen potential diag-nostic factors,and a Logistic regression model was employed for univariate analysis.Collinearity diagnostic tests were conducted among the risk factors.The stepwise regression method using the training dataset was applied to evaluate the importance of each risk factor for fat liquefaction.A multivariate Cox proportional hazards model was used to calcu-late the concordance index of the risk factors in both the training and validation sets.A predictive model was constructed using Logistic regression,and the clinical value of the predictive model was evaluated using ROC curves,calibration curves,and decision curves.Results:Compared with the non-liquefaction group,the liquefaction group had higher val-ues in terms of body weight,heart rate,diameter of the tender mass in the right lower abdomen,degree of appendiceal thickening,body temperature upon admission,fasting plasma glucose(FPG)level,surgical duration,intraoperative blood loss,wound healing time,length of hospital stay,volume of abdominal drainage fluid,and infection status(P<0.05).LASSO logistic regression analysis identified potential factors including body temperature upon admission,heart rate,body weight,diameter of the tender mass in the right lower abdomen,degree of appendiceal thickening,FPG level,sur-gical duration,and intraoperative blood loss.Univariate analysis revealed that body weight,diameter of the tender mass in the right lower abdomen,FPG level,surgical duration,and intraoperative blood loss were influencing factors for fat lique-faction after appendectomy(P<0.05).Body weight and FPG factors were mutually independent with no multicollinearity,while there was multicollinearity among the diameter of the tender mass in the right lower abdomen,surgical duration,and intraoperative blood loss.The risk model combining the diameter of the tender mass in the right lower abdomen,in-traoperative blood loss,FPG,surgical duration,and body weight had the highest concordance index in both the training and validation sets,with values of 0.811 and 0.814,respectively.The Logistic regression model established the predictive model as Logit(P)=-1.136+0.664×(diameter of the tender mass in the right lower abdomen)+0.449×(surgical duration)+0.622×(intraoperative blood loss)+0.200×(body weight)+0.578×(FPG).The area under the ROC curve was 0.920(95%CI:0.869～0.942),with a sensitivity of 93.14%and a specificity of 85.73%.The calibration curve of the predictive model showed good fit with the ideal curve.The predictive model had a high net benefit.Conclusion:Body weight,diameter of the tender mass in the right lower abdomen,FPG level,surgical duration,and intraoperative blood loss are risk factors for fat liquefaction after appendectomy and have good predictive value for the occurrence of fat liquefaction.

Objective To investigate the protective effect of Ghrelin on traumatic brain injury(TBI)in rats based on the HO-1/PGC-1αsignaling pathway.Methods Thirty SPF male rats were randomly divided into sham,TBI,and Ghrelin groups,with 10 rats in each group.A TBI rat model was established using the Feeney free-fall impingement method.The Ghrelin group was injected by caudal vein at a dose of 20 μg/kg 30 min after modeling,while the sham group was not impinged.After 72 h of modeling,the brain tissues of the rats were col-lected,and the brain water content was measured in order to analyze the severity of brain edema.HE staining was used to observe patho-logical changes in brain tissue.The levels of the oxidative stress factors MDA,SOD,and GSH-Px were determined using ELISA.TUNEL staining was used to detect the apoptosis of the brain cells,and the expression levels of Bcl-2,Bax,caspase-3 and caspase-9 in the brain tissues were detected by Western blotting.Mitochondrial reactive oxygen species(mtROS)in the brain tissues were detected by immuno-fluorescence.Mitochondrial function indicators,including mitochondrial mitogen Mfn 1/2,nuclear respiration factor 1(NRF1),and mito-chondrial transcription factor A(TFAM)were detected by Western blotting.The expression levels of HO-1 and PGC-1α in the brain tis-sues of rats in each group were detected by Western blotting.Twenty TBI model rats treated with Ghrelin were divided into Ghrelin+sh-NC and Ghrelin+sh-HMOX1 group with 10 rats in each group.Rats were treated with Ghrelin and injected with knock-down control(adenovirus 2.5 × 109 pfu)or knock-down HMOX1 adenovirus(2.5 × 109 pfu)via tail vein.Western blotting was used to detect the expressions of HO-1,PGC-1 α,Bcl-2,Bax,caspase-3 and caspase-9 in the brain tissues of the two groups.The levels of MDA,SOD and GSH-Px in brain tissue of two groups were detected by ELISA.Results Compared with the sham group,the pathological injury and brain edema in TBI group were aggravated,the number of brain cell apoptosis increased,the levels of oxidative stress factors SOD and GSH-Px decreased,the level of MDA increased,the level of mtROS in brain tissue decreased,the expressions of Bax,caspase-3 and caspase-9 increased,and the expressions of Mfn1/2,NRF1,TFAM,HO-1 and PGC-1α decreased(P＜0.05).Compared with TBI group,the pathological damage of brain tissue in Ghrelin group was improved,the brain edema was alleviated,the number of brain cell apoptosis was reduced,the levels of oxidative stress factors SOD and GSH-Px were increased,the level of MDA was decreased,the mtROS in brain tissue was decreased,the expression of Bcl-2 protein was increased,the expressions of Bax,caspase-3 and caspase-9 protein were decreased,and the expressions of Mfn1/2,NRF1,TFAM,HO-1 and PGC-1α were decreased(P＜0.05).Compared with Ghrelin+sh-NC group,the expressions of Bax,caspase-3 and caspase-9,MDA in brain tissue of Ghrelin+sh-HMOX1 group increased,while the levels of SOD and GSH-Px decreased(P＜0.05).Conclusion Chrelin has protective effect on TBI in rats,and can inhibit brain tissue injury and apoptosis in rats.Its mechanism may be achieved by regulating mitochondrial oxidative stress through HO-1/PGC-1α signaling pathway.

Objective To identify the key influencing factors and role mechanisms of large-scale medical equip-ment allocation decision-making in public hospitals,and to provide a reference basis for optimizing resource alloca-tion strategies.Methods Through searching the literature related to large-scale medical equipment decision-making in public hospitals and combing the policies,the system of decision-making influencing factors was initially screened.The Delphi method and decision-making trial and evaluation laboratory method were used to obtain the key influencing factors of equipment allocation decision-making and to determine the interaction effects among the factors.Results A total of seven cause factors and nine effect factors were screened.Among them,cost-benefit analysis,equipment technical performance,and evaluation of similar equipment were the top three key drivers;leadership emphasis,equipment market price,and equipment supporting facilities were the top three key con-straints.Conclusion Priority be given to strengthening the management of driving factors,mainly by conducting a full-cycle assessment of cost-effectiveness,paying attention to the clinical value output of equipment technical performance,and monitoring regionally similar equipment.A dynamic response mechanism for constraints should be established,mainly to strengthen leadership decision-making,implement a centralized procurement price nego-tiation model and improve the pre-assessment system for ancillary facilities.

OBJECTIVE To heterologously express and purify bioscavenger organophosphorus hydrolase(OPH),and evaluate its ability to resist ethyl paraoxon poisoning in vivo.METHODS The Escherichia coli expression strain of OPH was constructed and purified by Ni-column affinity chroma-tography and gel filtration chromatography,and the purified product was identified by mass spectrometry.The enzyme activity and kinetic constants(Km,Vmax,kcat and kcat/Km)were measured using ethyl paraoxon as the substrate.Twelve SD rats were randomly divided into the experimental group and control group.The experimental group was given 1 mg·kg-1 of OPH solution by iv administration while the control group was given the same volume of normal saline.After administration,the two groups were immedi-ately sc administration with 2×LD50 ethyl paraoxon(0.86 mg·kg-1).The state of the rats was observed and the poisoning symptoms were scored.The survival rats were given 2×LD50 ethyl paraoxon every 24 h,and the survival curve and symptom score chart were drawn according to survival and poisoning symp-toms of the rats to evaluate the anti-organophosphorus poisoning ability of OPH.RESULTS The expres-sion strain of OPH in E.coli was successfully constructed.After two-step purification,a single band of OPH was obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis,indicating that OPH had high purity,and the prepared protein sequence was consistent with the target protein.For OPH,Km=7.5×10-5 mol·L-1,Vmax=2.2×10-7 mol·L-1·s-1,kcat=158.4 s-1,kcat/Km=2.1×106 L·mol-1·s-1.The rats in the control group showed obvious poisoning symptoms after being given 2×LD50 ethyl paraoxon,and all rats died with in 15 min.The rats in the experimental group did not show poisoning symptoms after the first exposure,and the poisoning symptoms gradually deepened after continuous exposure until all the rats died on the 4th day.CONCLUSION OPH with high purity is successfully prepared in this study,and OPH could effectively resist ethyl paraoxon poisoning in rats.

Objective:To investigate the prognosis and the factors that influence it in patients with non-gastric gastrointestinal stromal tumors (GISTs) who are at low risk of recurrence.Methods:This was a retrospective cohort study. Clinicopathologic and prognostic data from patients with non-gastric GISTs and at low risk of recurrence (i.e., very low-risk or low-risk according to the 2008 version of the Modified NIH Risk Classification), who attended 18 medical centers in China between January 2000 and June 2023, were collected. We excluded patients with a history of prior malignancy, concurrent primary malignancy, multiple GISTs, and those who had received preoperative imatinib. The study cohort comprised 1,571 patients with GISTs, 370 (23.6%) of whom were at very low-risk and 1,201 (76.4%) at low-risk of recurrence. The cohort included 799 (50.9%) men and 772 (49.1%) women of median age 57 (16–93) years. Patients were followed up to July 2024. The prognosis and its influencing factors were analyzed. Receiver operating characteristic curves for tumor diameter and Ki67 were established, and the sensitivity, specificity, area under the curve (AUC) and optimal cut-off value with 95% confidence intervals were calculated. Propensity score matching was implemented using the 1:1 nearest neighbor matching method with a matching tolerance of 0.02.Results:With a median follow-up of 63 (12–267) months, the 5- and 10-year overall survival (OS) rates of the 1,571 patients were 99.5% and 98.0%, respectively, and the 5- and 10-year disease-free survival (DFS) rates were 96.3% and 94.4%, respectively. During postoperative follow-up, 3.8% (60/1,571) patients had disease recurrence or metastasis, comprising 0.8% (3/370) in the very low-risk group and 4.7% (57/1,201) in the low-risk group. In the low-risk group, recurrence or metastasis occurred in 5.5% (25/457) of patients with duodenal GISTs, 3.9% (25/645) of those with small intestinal GISTs, 9.2% (6/65) of those with rectal GISTs, and 10.0% (1/10) of those with colonic GISTs. Among the 60 patients with metastases, 56.7% (34/60) of the metastases were located in the abdominal cavity, 53.3% (32/60) in the liver, and 3.3% (2/60) in bone. During the follow-up period, 13 patients (0.8%) died of disease. Receiver operating characteristic curves were plotted for tumor diameter and Ki67 and assessed using the Jordon index. This showed that the difference in DFS between the two groups was statistically significant when the cutoff value for tumor diameter was 3.5 cm (AUC 0.731, 95% CI: 0.670–0.793, sensitivity 77.7%, specificity 64.1%). Furthermore, the difference in DFS between the two groups was statistically significant when the cutoff value for Ki67 was 5% (AUC 0.693, 95% CI: 0.624–0.762, sensitivity 60.7%, specificity 65.3%). Multifactorial analysis revealed that tumor diameter ≥3.5 cm, Ki67 ≥5%, and R1 resection were independent risk factors for DFS in patients with non-gastric GISTs at low risk of recurrence (all P<0.05). Furthermore, age >57 years, Ki67 ≥5%, and R1 resection were also independent risk factors for OS in patients with non-gastric GISTs at low risk of recurrence (all P<0.05). We also grouped the patients according to whether they had received postoperative adjuvant treatment with imatinib for 1 or 3 years. This yielded 137 patients in the less than 1-year group, 139 in the 1-year plus group; and 44 in both the less than 3 years and 3-years plus group. After propensity score matching for age, tumor diameter, Ki67, and resection status, the differences in survival between the two groups were not statistically significant (all P>0.05). The 10-year DFS and OS were 87.5% and 95.5%, respectively, in the group treated with imatinib for less than 1 year and 88.5% and 97.8%, respectively, in the group treated for more than 1 year. The 10-year DFS and OS were 89.6% and 92.6%, respectively, in the group treated with imatinib for less than 3 years and 88.0% and 100.0%, respectively, in the group treated with imatinib for more than 3 years. Conclusion:The overall prognosis of primary, non-gastric, low recurrence risk GISTs is relatively favorable; however, recurrences and metastases do occur. Age, tumor diameter, Ki67, and R1 resection may affect the prognosis. For some patients with low risk GISTs, administration of adjuvant therapy with imatinib for an appropriate duration may help prevent recurrence and improve survival.