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 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.

Enterotoxigenic Escherichia coli(ETEC)is the main cause of diarrhea in piglets,which seriously threatens the development of animal husbandry.In order to effectively prevent and con-trol piglet diarrhea,the neutralizing epitopes of the screened K88(FTDYEGASVELRKPDGGT-NK),K99(NVGNGSGGANIN),987P(LAAPAENNTSQAN),F41(VMAADWTEGQPGDII)and F18(PPNAQTYPLSSGDLK)ETEC adhesins were assembled to construct the multi-epitope fusion antigen(MEFA),whose immunogenicity was verified by in silico simulations and in vitro.The computer simulation results showed that MEFA has excellent physicochemical properties and reasonable prediction results of secondary and tertiary structures and exhibits strong interaction force when docking with toll-like receptor 3,and the kinetic simulation shows that it can be stably presented.In vitro validation results showed that anti-K88,K99,987P,F18 and F41 IgY could spe-cifically recognize MEFA proteins.After immunization of laying hens with MEFA,the titers of IgY antibodies against the five ETEC were higher than those of non-specific IgY.In addition,specific anti-MEFA IgY significantly inhibited the adhesion of K88,K99,987P,F41 and F18 ETEC strains to piglet intestinal IPEC-J2 cell lines.These results indicate that MEFA has strong immunogenicity and can be used as an ideal vaccine candidate for the prevention of K88,K99,987P,F41 and F18 ETEC infection,and provide reference and technical support for the development of multivalent broad-spectrum ETEC vaccine candidates.

Enterotoxigenic Escherichia coli(ETEC)is the main cause of diarrhea in piglets,which seriously threatens the development of animal husbandry.In order to effectively prevent and con-trol piglet diarrhea,the neutralizing epitopes of the screened K88(FTDYEGASVELRKPDGGT-NK),K99(NVGNGSGGANIN),987P(LAAPAENNTSQAN),F41(VMAADWTEGQPGDII)and F18(PPNAQTYPLSSGDLK)ETEC adhesins were assembled to construct the multi-epitope fusion antigen(MEFA),whose immunogenicity was verified by in silico simulations and in vitro.The computer simulation results showed that MEFA has excellent physicochemical properties and reasonable prediction results of secondary and tertiary structures and exhibits strong interaction force when docking with toll-like receptor 3,and the kinetic simulation shows that it can be stably presented.In vitro validation results showed that anti-K88,K99,987P,F18 and F41 IgY could spe-cifically recognize MEFA proteins.After immunization of laying hens with MEFA,the titers of IgY antibodies against the five ETEC were higher than those of non-specific IgY.In addition,specific anti-MEFA IgY significantly inhibited the adhesion of K88,K99,987P,F41 and F18 ETEC strains to piglet intestinal IPEC-J2 cell lines.These results indicate that MEFA has strong immunogenicity and can be used as an ideal vaccine candidate for the prevention of K88,K99,987P,F41 and F18 ETEC infection,and provide reference and technical support for the development of multivalent broad-spectrum ETEC vaccine candidates.

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 association between plasma uric acid and hypertension and the gender difference in community-dwelling middle-aged and elderly population.Methods:A community-based cross-sectional study was conducted in Beijing Tongzhou Yongshun Community Health Service Center from June to December 2021, among residents aged 45 years or older selected by cluster sampling method. According to plasma uric acid (UA) level in quartiles, the subjects were divided into 4 groups; and stratified by gender, the subjects were further divided into subgroups. Multivariate logistic regression model was used to analyze the related factors of hypertension, and restricted cubic spline fitting logistic regression model was used to analyze the nonlinear association between uric acid and hypertension and the cut-off values of uric acid.Results:A total of 6 229 residents with the age of (63.2±7.3) years were enrolled in the study. In 1 874 male participants (30.1%), 946 participants (50.5%) had hypertension, and the uric acid level was 359 (309, 418)μmol/L; in 4 355 female participants (69.9%), 2 003 participants (46.0%) had hypertension, and the uric acid level was 306 (261, 359)μmol/L. Multivariate logistic regression analysis showed that after adjusting for factors that were statistically significant in univariate analyses or potentially clinically relevant (including age, body mass index, diabetes mellitus, coronary heart disease, cerebrovascular disease, albumin, estimated glomerular filtration rate, and cholesterol), uric acid was independently associated with hypertension ( P<0.001), for total participants the risk of hypertension in Q4 group was 1.33 times of that in Q1 group ( OR=1.33,95% CI 1.13-1.56, P=0.001); while for females the risk of hypertension in Q4 group was 1.38 times of that in Q1 group ( OR=1.38,95% CI 1.13-1.68, P=0.002), but no significant association was observed for males ( P>0.05). The results of restricted cubic spline fitting logistic regression analysis showed that there was a linear association between uric acid level and hypertension in the total population and males, and the risk of hypertension increased with uric acid level ( P<0.001 for the total population, P=0.016 for male). However, there was a non-linear association in females. When uric acid>307 μmol/L in females, the risk of hypertension increased significantly as the level of uric acid increased ( P<0.001). Conclusions:Uric acid level was independently associated with hypertension in the community-dwelling middle-aged and elderly population, and there was a gender difference in the correlation. The association was nonlinear in females and the cut-off value of uric acid in females was 307 μmol/L.

Objective:To explore the relationship between the triglyceride glucose (TyG) index and impairment of renal function in community-dwelling middle-aged and elderly population.Methods:A total of 4 988 residents aged ≥45 years undergoing health check-up in Yongshun Health Service Center from January 2016 to December 2021 were enrolled and followed up. According to the quartile of the baseline TyG index, all subjects were divided into Q1, Q2, Q3 and Q4 groups. The medical history, physical examination and laboratory test results were documented. Participants were followed up through regular health check-up until March 31, 2023. The outcomes were rapid decline of estimated glomerular filtration rate (eGFR) (a loss in eGFR>3 ml·min -1·1.73 m -2 per year) and the new-onset of chronic kidney disease (CKD) during the follow-up period. Linear regression model, multivariate logistic regression model, restricted cubic spline fitting logistic regression model and ROC curve analysis were used to analyze the association between the TyG index and the impairment of renal function. Results:Among 4 988 residents, 1 396 (28.0%) were males and the age was (59.76±6.28) years. There were 1 247 participants in Q1, Q2, Q3 and Q4 groups, respectively. After 56 months of follow-up, the incidence of rapid eGFR decline and new-onset CKD was 21.9% (1 294/4 988) and 4.0% (200/4 988), respectively. Multivariate logistic regression model analysis showed that TyG index was correlated positively with rapid eGFR decline and new-onset of CKD ( OR=1.34, 95%CI: 1.17-1.52, P<0.001, and OR=1.57, 95%CI:1.19-2.06, P=0.001). Taking group Q1 as a reference, higher levels of TyG index ( Q2, Q3 and Q4 groups) was an independent risk factor for rapid eGFR decline ( P<0.05), which has a dose-response relationship (for trend P=0.002). Compared with the lowest quartile, the adjusted OR of new-onset CKD in the highest quartile was 1.85 ( 95%CI:1.13-3.03, P=0.014). The results of restricted cubic spline fitting logistic regression analysis showed a linear association between TyG index and both outcomes (both P>0.05). The area under ROC curve ( AUC) of the TyG index for predicting the two adverse outcomes were 0.536 ( 95%CI: 0.516-0.556, P<0.001) and 0.588 ( 95%CI:0.548-0.627, P<0.001), respectively. Conclusion:The elevated levels of TyG index may be used as an independent predictor of rapid eGFR decline and new-onset CKD.

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.

Myocardial remodeling is one of the major pathogenic mechanisms in chronic heart failure, and myocardial fibrosis plays an important role in myocardial remodeling. Myocardial fibrosis is mainly due to the synthesis exceeding degradation of collagen, resulting in the accumulation of collagen. The metabolites produced during the dynamic transformation of collagen synthesis and degradation can indirectly reflect myocardial fibrosis. Due to the complexity of collagen metabolism and the large number of intermediate products, it has not been widely used as a biomarker in clinical practice. We briefly reviewed the process of collagen metabolism and its products, and focused on the clinical value of collagen metabolites (PICP, PIIINP, and MMP) in the diagnosis, risk stratification, and prognostic assessment of heart failure. It provides a reference basis for the wide application of collagen-related biomarkers in heart failure in the future.

Objective:To explore the reference interval of urinary iodine concentration(UIC)/urinary creatinine(UCr) ratio evaluating the iodine nutritional status in early pregnancy women.Methods:A reference interval of UIC/UCr ratio was determined among 5 609 early pregnant women with normal thyroid function, negative thyroid autoantibodies, and no history of diseases or taking drug that may affect thyroid function. Then we verified the reliability of this reference interval in a group of 7 514 women in early pregnancy.Results:We determined the UIC/UCr ratio of 75-149 μg/g as the reference interval. In the reference interval, thyroglobulin antibody(TgAb), thyroid peroxidase antibody(TPOAb), and thyroglobulin(Tg) were all at lower levels, and the overall distributions were approximately U-shaped. The prevalence of thyroid dysfunction, the positive rates of antibodies and the proportion of Tg>40 μg/L were the lowest within the reference interval, while higher on both sides of the interval.Conclusion:The reasonable reference interval of the UIC/UCr ratio in iodine-sufficient regions is 75-149 μg/g in early pregnerty women.