As a biocatalyst, laccase has been widely studied and applied in the papermaking industry. However, the low catalytic efficiency and poor stability of natural laccase limit its application in the pulping process. To develop the laccase with high activity and strong tolerance, we carried out directed evolution for modification of the laccase derived from Bacillus pumilus and screened out the mutants F282L/F306L and Q275P from the random mutant library by high-throughput screening. The specific activities of F282L/F306L and Q275P were 280.87 U/mg and 453.94 U/mg, respectively, which were 1.42 times and 2.30 times that of the wild-type laccase. Q275P demonstrated significantly improved thermal stability, with the relative activity 20% higher than that of the wild-type laccase after incubation at 40 ℃, 50 ℃, and 70 ℃ for 4 h. F282L/F306L and Q275P showed greater tolerance to metal ions and organic solvents than the wild-type laccase. The K_m value of the wild-type laccase was 374.97 μmo/L, and those of F282L/F306L and Q275P were reduced to 318.96 μmo/L and 360.71 μmo/L, respectively, which suggested that the substrate affinity of laccase was improved after mutation. The k_cat values of F282L/F306L and Q275P for the substrate ABTS were 574.00 s^-1 and 898.03 s^-1, respectively, which were 1.1 times and 1.7 times that of the wild-type laccase, indicating the improved catalytic efficiency. Q275P demonstrated better performance than the wild-type laccase in pulping, as manifested by the reduction of 0.82 in the Kappa number and the increases of 2.00% ISO, 7.8%, and 7.2% in whiteness, tensile index, and breaking length, respectively. This work lays a foundation for improving the adaptation of laccase to the environment of the papermaking industry.
Laccase/chemistry* ; Directed Molecular Evolution ; Enzyme Stability ; Bacillus pumilus/genetics* ; Mutation ; Biocatalysis ; Catalysis

Objective:To investigate the application value of photon-counting detector CT (PCD-CT) in preoperative identification of critical anatomical structures and surgical assessment in pancreatic cancer, and to compare its performance with conventional energy-integrating detector CT (EID-CT) in delineating tumor margins, vascular structures, and neural anatomy.Methods:This single-center retrospective matched case-control study included 25 patients with pathologically confirmed pancreatic ductal adenocarcinoma who underwent PCD-CT enhanced scanning and curative surgery at Peking Union Medical College Hospital between February and June 2025 (PCD-CT group). These patients were matched in a 1∶2 ratio to 50 patients who underwent EID-CT between January 2016 and December 2024 and subsequently received curative surgery (EID-CT group). Tumor boundary clarity, vascular visualization scores, and neural structure visibility were subjectively evaluated using the Likert scoring system. The assessed vessels included the celiac artery, common hepatic artery, superior mesenteric artery, splenic artery, portal vein, superior mesenteric vein, splenic vein, and pancreaticoduodenal arterial arcade. Imaging-based assessment of structural involvement was compared with intraoperative findings and pathological results to calculate diagnostic accuracy. Surgeons rated the usefulness of PCD-CT images for identifying key structures and determining resectability using a 5-point Likert scale. The Mann-Whitney U test was used for group comparisons of subjective scores, and categorical data were analyzed using the χ2 test or Fisher exact test. Results:The PCD-CT group showed significantly higher scores for tumor boundary clarity, vascular visualization, and neural structure detectability than those of the EID-CT group (all P<0.05). The accuracy of assessing superior mesenteric vein involvement was 96.0% (24/25) in the PCD-CT group and 72.0% (36/50) in the EID-CT group, with a significant difference ( χ2=6.00, P=0.014). Postoperative surgeon evaluations indicated that PCD-CT provided substantial assistance for both key structure identification [5 (5, 5)] and resectability assessment [5 (4, 5)]. Conclusion:PCD-CT demonstrates superior performance over EID-CT in preoperative delineation of tumor margins, vascular structures, and neural anatomy and in the assessment of structural involvement in pancreatic cancer. It provides valuable anatomical information to support preoperative evaluation and surgical decision-making.

Objective To construct a predictive model for the survival of transplant kidneys after kidney transplantation. Methods The clinical data of 366 kidney transplant recipients and donors were retrospectively analyzed, and the recipients were divided into low-risk group (n=101), medium-risk group (n=189), and high-risk group (n=76) based on the kidney donor profile index (KDPI). Each group was further divided into Remuzzi score ≤3 group and Remuzzi score >3 group based on time-zero biopsy Remuzzi scores. Kaplan-Meier method was used to analyze the survival of transplant kidneys. Univariate and multivariate Cox regression analyses were performed to identify risk factors affecting long-term survival after kidney transplantation. A predictive model for transplant kidney survival was established and a nomogram was drawn. The predictive performance of the model was evaluated using the receiver operating characteristic (ROC) curve and the area under the curve (AUC). Results The median KDPI was 65%, and the median Remuzzi score was 3. The 5-year survival rate of transplant kidneys was 83.5%. Kaplan-Meier survival curves showed that in the KDPI medium-risk and KDPI high-risk groups, the subgroup with lower Remuzzi score had a higher survival rates of transplant kidneys than the subgroup with higher Remuzzi score. Univariate and multivariate Cox regression analyses showed that KDPI, Remuzzi score, and donor’s age were independent risk factors for transplant kidney loss (all P<0.05). The ROC curve showed that the AUC of the nomogram prediction model established based on independent risk factors for the 1, 3 and 5-year survival rates of transplant kidneys were 0.91, 0.93 and 0.94 for the training set, and 0.89, 0.85 and 0.88 for the validation set. Calibration curves shows good consistency between the training and validation sets of the model. Conclusions The nomogram predictive model based on KDPI, time-zero biopsy Remuzzi score and donor’s age has good predictive value for transplant kidney survival.

Objective:To investigate the application value of photon-counting detector CT (PCD-CT) in preoperative identification of critical anatomical structures and surgical assessment in pancreatic cancer, and to compare its performance with conventional energy-integrating detector CT (EID-CT) in delineating tumor margins, vascular structures, and neural anatomy.Methods:This single-center retrospective matched case-control study included 25 patients with pathologically confirmed pancreatic ductal adenocarcinoma who underwent PCD-CT enhanced scanning and curative surgery at Peking Union Medical College Hospital between February and June 2025 (PCD-CT group). These patients were matched in a 1∶2 ratio to 50 patients who underwent EID-CT between January 2016 and December 2024 and subsequently received curative surgery (EID-CT group). Tumor boundary clarity, vascular visualization scores, and neural structure visibility were subjectively evaluated using the Likert scoring system. The assessed vessels included the celiac artery, common hepatic artery, superior mesenteric artery, splenic artery, portal vein, superior mesenteric vein, splenic vein, and pancreaticoduodenal arterial arcade. Imaging-based assessment of structural involvement was compared with intraoperative findings and pathological results to calculate diagnostic accuracy. Surgeons rated the usefulness of PCD-CT images for identifying key structures and determining resectability using a 5-point Likert scale. The Mann-Whitney U test was used for group comparisons of subjective scores, and categorical data were analyzed using the χ2 test or Fisher exact test. Results:The PCD-CT group showed significantly higher scores for tumor boundary clarity, vascular visualization, and neural structure detectability than those of the EID-CT group (all P<0.05). The accuracy of assessing superior mesenteric vein involvement was 96.0% (24/25) in the PCD-CT group and 72.0% (36/50) in the EID-CT group, with a significant difference ( χ2=6.00, P=0.014). Postoperative surgeon evaluations indicated that PCD-CT provided substantial assistance for both key structure identification [5 (5, 5)] and resectability assessment [5 (4, 5)]. Conclusion:PCD-CT demonstrates superior performance over EID-CT in preoperative delineation of tumor margins, vascular structures, and neural anatomy and in the assessment of structural involvement in pancreatic cancer. It provides valuable anatomical information to support preoperative evaluation and surgical decision-making.

Objective To investigate the effect of Lumbricus protein on the phenotypic transformation of corporal cavernosum smooth muscle cells(CCSMC)and erectile function in diabetic erectile dysfunction(DMED)rats.Methods Sixty male SD rats with normal erectile function were randomly divided into a blank group,a model group,a Sildelafil group(5 mg·kg-1),and a Lumbricus protein low-,medium-,and high-dose group(45,90,and 180 mg·kg-1),with 10 rats in each group.The diabetic rat model was established by intraperitoneal injection of Streptozotocin(STZ,50 mg·kg-1)combined with high-fat feed feeding;after 8 weeks,the DMED rat model was prepared by neck injection of Apomorphine(APO,100 μg·kg-1).After successful modeling,the rats were administered with a dose of Apomorphine by gavage once a day for 4 weeks.The blood glucose levels and body mass of rats in each group were measured before modeling,on the third day of modeling,and after 4 weeks of drug administration.The intracavernous pressure(ICP)and carotid artery pressure(MAP)were measured by multi-channel physiological recorder,and the ICP/MAP ratio was calculated.The expressions of contractile markers α-smooth muscle actin(α-SMA),smooth muscle myosin heavy chain(SMMHC)and synthetic markers Collagen I and osteopontin(OPN)in corpus cavernosum were detected by immunohistochemistry.The mRNA expression levels of α-SMA,SMMHC and Collagen I in corpus cavernosum were detected by RT-PCR.The protein expression levels of α-SMA,Desmin,Collagen I and OPN in corpus cavernosum were detected by Western Blot.Results Compared with the blank group,the blood glucose levels of the rats in the model group were significantly increased on the third day of modeling and after 4 weeks of administration(P＜0.01),and the body mass was significantly decreased after 4 weeks of administration(P＜0.01).ICP and ICP/MAP ratio were significantly decreased(P＜0.01).The protein expression levels of α-SMA,SMMHC and Desmin in penile corpus cavernosum were significantly decreased(P＜0.01),and the protein expression levels of Collagen I and OPN were significantly increased(P＜0.01).The mRNA expression levels of α-SMA and SMMHC in corpus cavernosum were significantly decreased(P＜0.01),and the mRNA expression level of Collagen I was significantly increased(P＜0.01).Compared with the model group,there was no significant change in blood glucose and body mass of rats in the administration group(P＞0.05).ICP and ICP/MAP ratio were significantly increased(P＜0.01).The expression levels of α-SMA,SMMHC and Desmin in corpus cavernosum were significantly increased(P＜0.01),while the expression levels of Collagen I and OPN were significantly decreased(P＜0.01).The mRNA expression levels of α-SMA and SMMHC in corpus cavernosum were significantly increased(P＜0.01),and the mRNA expression level of Collagen I was significantly decreased(P＜0.01).Conclusion Lumbricus protein can improve the erectile function of DMED rats,and its mechanism may be related to the inhibition of CCSMC from'contractile'to'synthetic(proliferative)'transformation.

Objective To investigate the effect of dioscin on uric acid(UA)-induced oxidative stress injury of human renal tubular epithelial cells(HK-2)and its molecular mechanism.Methods HK-2 cells were cultured and divided into four groups:blank group(normal group),model group(uric acid-stimulation modeling),condition control group(UA+DMSO)and dioscin group(UA+dioscin).Oxidative stress injury model was induced by UA in HK-2 cells.Cells viability was detected by CCK-8.ROS level was detected by flow cytometry.Real-time PCR was used to detect the expressions of glycogen synthase kinase 3β(GSK3β),nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase 1(HO-1)at mRNA level,and Western Blot was used to detect the expressions of phosphorylated glycogen synthesis kinase 3β(p-GSK3β),GSK3β,Nrf2 and HO-1 at protein level.Results After stimulation by UA,HK-2 cells viability was obviously decreased,and ROS level was significantly increased(all P＜0.001).When treated with dioscin,HK-2 cells viability was obviously increased,and the ROS level of HK-2 cells was significantly decreased(all P＜0.001).The expressions of Nrf2 and HO-1 decreased at the protein and mRNA levels after stimulation with UA.But the expressions of Nrf2 and HO-1 significantly increased after treated with dioscin(all P＜0.001).Compared with the blank group,the p-GSK3β/GSK3β ratio in the model group decreased significantly at the protein level,but the p-GSK3β/GSK3β ratio increased after treated with dioscin(all P＜0.001).Conclusion Dioscin can alleviate UA-induced oxidative stress injury in HK-2 cells.The mechanism might be that dioscin can promote phosphorylation of GSK3β,and activate Nrf2/HO-1 pathway.

Objective To analyze the potential mechanism of action of Portulaca oleracea L.in the treatment of colorectal cancer(CRC)using network pharmacology and experimental verification.Methods With reference to TCMSP and ETCM databases,the effective ingredients and targets of Chinese medicine P.oleracea were obtained,and targets related to CRC were retrieved from Genecards,Drugbank and OMIM databases,which were imported into Venny database,String database and Cytoscape platform to construct the PPI proteins.The common core targets were analyzed for GO function and KEGG pathway enrichment using Metascape database and Microbiology Letter platform,and the active ingredients and key targets were molecularly docked using Autodock and Pymol software.To construct a CRC mouse model and give P.oleracea treatment,observe the histopathological changes of its colon,and validate the key targets using qRT-PCR experiments and Western blot experiments.In addition,quercetin,luteolin,and kaempferol were used to act on HCT116 cells to observe the effects on cell proliferation and calculate the IC50,which was further verified using qRT-PCR assay and Western blot assay.Results Network pharmacological yielded 11 active ingredients and 209 targets corresponding to the target of P.oleracea.Disease targets were 2667.By analyzing the results,it was predicted that the active ingredients such as quercetin,luteolin,and kaempferol were involved in biological processes such as cellular response to lipids through key targets such as IL6,TP53,and IL1β,which were closely related to pathways in cance.Molecular docking showed that quercetin,luteolin,and kaempferol had good affinity for IL6,TP53 and IL1β.In vivo experiments confirmed that P.oleracea significantly inhibited colon tumorigenesis in mice,and inhibited the levels of IL6,IL1β inflammatory factors and increased the expression level of P53 protein.In vitro experiments showed that quercetin,luteolin and kaempferol could inhibit the proliferation of HCT116 cells to different degrees,and luteolin could inhibit the expression of IL6 and IL1β and up-regulate the expression of P53.Conclusion Through network pharmacology,in vivo and in vitro experiments,the"multi-component-multi-target-multi-pathway"effect of P.oleracea in the treatment of CRC has been confirmed,which reveals that inhibition of inflammation and delay of inflammation-cancer transformation may be one of the potential therapeutic pathways,and provides a theoretical basis and research direction for further exploration of the mechanism and clinical application of P.oleracea.

Objective To analyze the potential mechanism of action of Portulaca oleracea L.in the treatment of colorectal cancer(CRC)using network pharmacology and experimental verification.Methods With reference to TCMSP and ETCM databases,the effective ingredients and targets of Chinese medicine P.oleracea were obtained,and targets related to CRC were retrieved from Genecards,Drugbank and OMIM databases,which were imported into Venny database,String database and Cytoscape platform to construct the PPI proteins.The common core targets were analyzed for GO function and KEGG pathway enrichment using Metascape database and Microbiology Letter platform,and the active ingredients and key targets were molecularly docked using Autodock and Pymol software.To construct a CRC mouse model and give P.oleracea treatment,observe the histopathological changes of its colon,and validate the key targets using qRT-PCR experiments and Western blot experiments.In addition,quercetin,luteolin,and kaempferol were used to act on HCT116 cells to observe the effects on cell proliferation and calculate the IC50,which was further verified using qRT-PCR assay and Western blot assay.Results Network pharmacological yielded 11 active ingredients and 209 targets corresponding to the target of P.oleracea.Disease targets were 2667.By analyzing the results,it was predicted that the active ingredients such as quercetin,luteolin,and kaempferol were involved in biological processes such as cellular response to lipids through key targets such as IL6,TP53,and IL1β,which were closely related to pathways in cance.Molecular docking showed that quercetin,luteolin,and kaempferol had good affinity for IL6,TP53 and IL1β.In vivo experiments confirmed that P.oleracea significantly inhibited colon tumorigenesis in mice,and inhibited the levels of IL6,IL1β inflammatory factors and increased the expression level of P53 protein.In vitro experiments showed that quercetin,luteolin and kaempferol could inhibit the proliferation of HCT116 cells to different degrees,and luteolin could inhibit the expression of IL6 and IL1β and up-regulate the expression of P53.Conclusion Through network pharmacology,in vivo and in vitro experiments,the"multi-component-multi-target-multi-pathway"effect of P.oleracea in the treatment of CRC has been confirmed,which reveals that inhibition of inflammation and delay of inflammation-cancer transformation may be one of the potential therapeutic pathways,and provides a theoretical basis and research direction for further exploration of the mechanism and clinical application of P.oleracea.

Objective:To investigate the prognosis and risk factors for children diagnosed with all types of subaortic stenosis(SAS) who developed recurrent left ventricular outflow tract obstruction after surgical treatment.Methods:The study retrospectively included patients aged 0-18 years old who underwent open heart SAS surgery at Fuwai Hospital from 2016-2019. Children with hypertrophic obstructive cardiomyopathy were excluded. Detailed operative notes, medical records and ultrasound information, and follow-ups were extracted. Recurrent SAS was defined as left ventricular outflow tract gradient 30 mmHg(1 mmHg=0.133 kPa) 1 month after SAS surgical treatment.Results:A total of 137 children were included in this study. The medium age of children at the time of SAS surgery was 4.6 years old(3 months-17.8 years old). After a median follow-up of 4.36 years(3.2-5.7 years), a total of 30 patients developed recurrent LVOTO, with a recurrence rate of 21.9%, and 7(5.1%) underwent a second surgery. Compared to the non-recurrent group, children in the recurrent group were younger at the time of surgery( P=0.0443), had a smaller body surface area( P=0.0485), and a longer length of stay( P=0.0380). In Cox analysis, when only considering preoperative variables, the independent risk factor for LVOTO recurrence were a peak left ventricular outflow tract gradient higher than 50 mmHg( HR=5.25, P=0.001), a BSA less than 0.9( HR=2.5, P=0.023), and a length of SAS 5 mm( HR=2.29, P=0.050). When both preoperative and intraoperative variables were considered, preoperative peak left ventricular outflow tract gradient 50 mmHg( HR=4.91, P=0.002) and peeling from the aortic valve( HR=3.23, P=0.010) were independent risk factors for postoperative recurrence. Conclusion:Recurrent LVOTO after SAS surgical repair is common, and regular postoperative follow-up is crucial to evaluate whether a secondary intervention is required. Regular postoperative follow-up is needed for children at high risk.

Objective:To investigate the application effect of WeChat group combined with case-based learning (CBL) in probation teaching of vascular surgery.Methods:A total of 128 intern doctors of the seven-year medical program in vascular surgery were selected and divided into experimental group (WeChat group combined with CBL teaching) and control group (CBL teaching alone), and a unified assessment method was used to evaluate the mastery degree of professional theories and clinical knowledge and assess the teaching effect of these two teaching modes in vascular surgery. SPSS 22.0 was used to perform the t-test. Results:Compared with the control group, the experimental group had significantly higher scores of theoretical examination [(86.36±7.42) vs. (84.71±6.72)] and clinical examination [(88.44±7.62) vs. (86.22±6.41)], as well as significantly higher degrees of satisfaction with learning interest, classroom climate, self-learning ability, clinical thinking ability, and teamwork spirit.Conclusion:The teaching mode of WeChat group combined with CBL can improve the effect of the probation teaching of vascular surgery and the self-learning and clinical thinking abilities of intern doctors.