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

ObjectiveTo analyze the reported cases of infectious diseases across different tiers of public medical and healthcare institutions in Minhang District, Shanghai from 2013 to 2023, to investigate the status and changes in reported infectious diseases in this district from a temporal, etiological, and demographic perspectives, so as to provide a scientific basis for the construction of a hierarchica early-warning surveillance system under the grading diagnosis and treatment model in medical institutions, as well as for optimizing sentinel surveillance at facilities of different levels. MethodsA retrospective analysis was performed using surveillance data from the China Disease Prevention and Control Information System in Minhang District from 2013 to 2023. Reported infectious diseases were categorized into three categories based on transmission routes: respiratory infectious diseases, intestinal infectious diseases, and sexually transmitted and blood borne infectious diseases. According to the implementation phase of the grading diagnosis and treatment policy, the research time was divided into four time periods: 2013‒2016, 2017‒2019, 2020‒2022, and 2023. The distribution and temporal changes of reported cases of infectious diseases were compared across community health service centers (CHCs), secondary hospitals, tertiary grade-A hospitals and tertiary grade-B hospitals. Chi-square test was used for univariate analysis of differences in the number of reported cases. Quantitative data with normal distribution were analyzed using parametric tests, otherwise, Kruskal⁃Wallis H tests were used. ResultsThe proportions of total reported cases of infectious diseases in medical institutions at all levels in Minhang District, Shanghai from 2013 to 2023 were 10.66% in CHCs, 9.10% in secondary hospitals, 64.95% in tertiary grade-B hospitals, and 15.29% in tertiary grade-A hospitals, with an overall decline and then rebound trend in the reported cases. After the implementation of grading diagnosis and treatment policy, the number of reported cases in CHCs and secondary hospitals showed a trend of first decreasing and then increasing, while that in tertiary grade-B hospitals showed a steady decreasing trend and that in tertiary grade-A hospitals showed an increasing trend. In terms of the research periods divided above, a total of 10 392 cases were reported in 2013‒2016 (70.34% from tertiary grade-B hospitals and 12.59% from CHCs), including 2 922 cases of respiratory infectious diseases, 1 241 cases of intestinal infectious diseases, and 6 229 cases of sexually transmitted and blood-borne infectious diseases. Between 2017 and 2019, a total of 6 967 cases were reported (73.49% from tertiary grade-B hospitals and 11.84% from tertiary grade-A hospitals), including 2 983 cases of respiratory infectious diseases, 279 cases of intestinal infectious diseases, and 3 705 cases of sexually transmitted and blood-borne infectious diseases. Between 2020 and 2022, a total of 4 599 cases were reported (69.92% from tertiary grade-B hospitals and 24.57% from tertiary grade-A hospitals), including 1 627 cases of respiratory infectious diseases, 123 cases of intestinal infectious diseases, and 2 849 cases of sexually transmitted and blood-borne infectious diseases. In 2023, a total of 4 648 cases were reported (35.20% from tertiary grade-B hospitals and 27.50% from tertiary grade-A hospitals), including 3 165 cases of respiratory infectious diseases, 69 cases of intestinal infectious diseases, and 1 414 cases of sexually transmitted and blood-borne infectious diseases. The proportion of reported cases from tertiary grade-B hospitals was the highest in all the four research periods, but exhibited an obvious decrease in 2023. The differences in the reported cases of infectious diseases with different transmission routes among medical institutions at all levels were statistically significant (χ²=3 225.628, P<0.05). The differences in the mean age of patients among medical institutions at all levels were statistically significant (H=1 325.927, P<0.05). ConclusionThere are significant differences in the number of reported cases of infectious disease in the medical institutions at different levels. Tertiary grade-B hospitals have historically dominated the number of reported cases, but its share has declined recently. Whereas, CHCs and tertiary grade-A hospitals have played an increasingly important role in the surveillance and early warning of respiratory and intestinal infectious diseases. Therefore, it is recommended to leverage the strengths of grading diagnosis and treatment to establish targeted sentinel sites and deploy specialized teams tailored to the epidemiological characteristics of specific disease categories.

  Objective  To investigate the predictive value of albumin-bilirubin (ALBI) score combined with liver function index and carcinoembryonic antigen (CEA) for liver metastasis of colorectal cancer.  Methods  We retrospectively analyzed the clinical data of patients with colorectal cancer who underwent surgical treatment in the Second Hospital & Clinical Medical Hospital, Lanzhou University from January 2016 to July 2021 and were followed up for 24 months. According to the follow-up results, the enrolled patients were divided into liver metastasis group and non-liver metastasis group, and were randomly divided into modeling group and validation group by a ratio of 2∶1. The risk factors of liver metastasis in the patients with colorectal cancer were analyzed. Lasso-Logistic regression was used to construct the prediction model. Bootstrap method was used for internal verification. Receiver operating characteristic curve, calibration curve and clinical decision curve were used to evaluate the reliability of the prediction model. Finally, a nomogram was drawn to show the prediction results.  Results  A total of 195 patients who met the inclusion and exclusion criteria were enrolled, including 130 in the modeling group and 65 in the validation group. Through Lasso regression variable screening and Logistic regression analysis, the results showed that ALBI score(OR=8.062, 95% CI: 2.545-25.540), alanine transaminase (ALT) (OR=1.037, 95% CI: 1.004-1.071) and CEA (OR=1.025, 95% CI: 1.008-1.043) were independent predictors of liver metastasis in colorectal cancer. The area under curve (AUC) of the combined prediction of liver metastasis of colorectal cancer in the modeling group was 0.921, the sensitivity was 78%, the specificity was 95%, the C-index was 0.921, the H-L fitting curve χ²=0.851, P=0.654, and the slope of the calibration curve was close to 1, suggesting that the accuracy of the model was high, and the DCA curve showed that the model had good clinical application value. For the data of the modeling group, the Bootstrap method was used for internal verification of 1000 resamplings. The accuracy was 0.869, the kappa consistency was 0.709, and the AUC was 0.913. When ALBI score, ALT and CEA were used to diagnose liver metastasis of colorectal cancer alone, the AUC of CEA was the largest (0.897), and the combination of the three had the highest efficacy in the diagnosis of liver metastasis of colorectal cancer. In the validation group, the AUC, sensitivity, specificity, C-index of the combined prediction of liver metastasis of colorectal cancer were 0.918, 85.0%, 95.6%, 0.918, respectively, and H-L fitting curve χ²=0.586, P=0.746.  Conclusions  ALBI score, ALT and CEA have certain predictive value for liver metastasis of colorectal cancer. The combined diagnosis of liver metastasis of colorectal cancer has high efficacy, and the risk prediction model constructed has a good predictive effect.

OBJECTIVE To observe the ther apeutic efficacy and safety of albumin-bound paclitaxel combined with oxaliplatin in the first-line treatment of advanced gastric cancer （GC）. METHODS A total of 90 advanced unresectable GC patients were randomized into observation group and control group ，with 45 patients in each group. Control group was given oxaliplatin combined with tegafur ，and observation group was given albumin-bound paclitaxel combined with oxaliplatin. A chemotherapy cycle last for 21 days，and both groups received 6 cycles of chemotherapy ，and then the follow-up observation was conducted every 2 weeks. Short-term efficacy （after 2 chemotherapy cycles ），mid-term efficacy （after 6 chemotherapy cycles ），tumor marker，long-term efficacy （after follow-up ），quality of life ，the occurrence of ADRs were compared between 2 groups. RESULTS After 2 cycles of treatment ，objective remission rate （ORR）of observation group was significantly higher than that of control group （60.00％ vs. 37.78％，P＜0.05）；there was no statistical significance in disease control rate between the two groups （P＞0.05）. After 6 chemotherapy cycles ，ORR of observation group was significantly higher than that of control group （55.56％ vs. 31.11％，P＜0.05）；there was no statistical significance in disease control rate between the two groups （P＞0.05）；the levels of tumor markers [carcinoembryonic antigen ， carbohydrate antigen （CA）724， CA199， CA242] in observation group were significantly lower than those of control group （P＜0.05）. After the follow-up period ，the median progression-free survival [ 8.78 （95％CI：6.94-11.01）months] and the median overall survival [ 13.02（95％CI：12.78-15.62）months] of observation group were significantly longer than those [ 6.99（95％CI：3.67-10.88）months and 10.42（95％CI：8.72-13.22）months] of control group （P＜ 0.05）. The score of body dimension in the observation group was significantly higher than that in the control group （P＜0.05）. Except for peripheral neurotoxicity ，there was no significant difference in the incidence of ADR between 2 groups（P＞0.05）. CONCLUSIONS Albumin-bound paclitaxel combined with oxaliplatin has a good effect and safety in the first-line treatment of advanced unresectable GC.

【Objective】 To analyze the implementation and problems in the process of the first policy of serious illness benefits for voluntary blood donors in China, therefore provide reference for other provinces and cities to formulate care policies for voluntary blood donors. 【Methods】 The number of blood donors who received serious illness benefits and the proportion to the total number of blood donors in that year were obtained by Excel, and their basic demographic information, illness and reasons for failing to receive benefits were analyzed. 【Results】 From 2015 to 2019, a total of 198 blood donors applied for serious illness benefits, and 159 received benefits (638 000 yuan), accounting for 0.017% of the total number of voluntary blood donors in Nanjing. The average age of the recipients was 52.66, with the proportion of males (63.5%) higher than that of females (36.5%). 52.8% (the highest proportion) got benefits of 5 000 yuan. 39 applicants failed to obtain serious illness benefits, among which 27 applicants failed during 2017 to 2018 due to unidentifiable funding source. But after Nanjing Blood Donation Regulations clarified the funding sources, the implementation of serious illness benefits policy was continued in 2018. 【Conclusion】 The implementation of serious illness benefits for voluntary blood donors presented the Nanjing municipal government's care for voluntary blood donors. The legalization of this policy is conducive to the continuity and stability of policy implementation. Relevant departments and blood centers should continue to strengthen the policy publicity and make sure the policy is implemented effectively.

【Objective】 To establish a prognostic model of fatty acid metabolism related genes for predicting the prognosis of renal clear cell carcinoma. 【Methods】 The differentially expressed fatty acid metabolism-related genes in renal clear cell carcinoma samples and normal samples in TCGA database were screened by R language software. The Cox proportional hazard regression model was used to select and establish a multigene prognostic model and the prognostic score was calculated. Patients were divided into high-risk group and low-risk group according to the median prognostic score. Kaplan-Meier survival curve was used to analyze the difference in two groups. The clinical pathological factors and prognostic score factors were included in the Cox regression model to analyze the factors affecting the survival of patients with renal clear cell carcinoma. ROC receiver operating curve analysis was used to evaluate the accuracy of the prognostic prediction model. The prognostic model of fatty acid metabolism-related genes and their correlation with clinical factors were analyzed. GSEA enrichment analysis analyzed the differences of gene sets in risk groups. 【Results】 A total of 4 differential genes (CPT1B, HADH, CYP4A11, and ACADSB) were selected to establish a prognostic model for genes related to fatty acid metabolism in renal cell carcinoma. The prognostic risk score (RS) formula is as follows: RS=0.490×CPT1B-0.428×HADH-0.11 × CYP4A11-0.372 × ACADSB. Kaplan-Meier survival analysis confirmed that the overall survival rate of patients with low-risk prognostic score was significantly higher in patients with overall renal clear cell carcinoma, and the difference was statistically significant (P<0.001). Cox regression analysis showed that the prognostic model of genes related to age and fatty acid metabolism is an independent influencing factor for the prognosis of patients with renal clear cell carcinoma (P<0.01). The 5 years’ AUC of the renal clear cell carcinoma ROC curve of the renal cancer fatty acid metabolism related gene model was 0.802. GSEA analysis showed that the difference of 81 gene sets in the low-risk group was statistically significant (P<0.05). 【Conclusion】 The prognostic model of renal cancer fatty acid metabolism-related genes can be used to predict the prognosis of patients with renal clear cell carcinoma, which is conducive to further guide clinical treatment.

【Objective】 To explore and verify the mechanism of curcumin’s inhibition of the proliferation of renal clear cell carcinoma （RCCC） based on network pharmacology and bioinformatics. 【Methods】 We screened common target genes of RCCC and curcumin from PharmMapper and GeneCards databases. We used TCGA database data analysis to screen out common target genes which not only differentially expressed between RCCC tissue samples and normal tissue samples but also affected prognosis. We also used STRING platform to construct curcumin-RCCC targets interaction network， used R software to perform GO biological process analysis and KEGG pathway enrichment analysis based on the above-mentioned screening target proteins. After curcumin and/or active oxygen inhibitor N-acetyl-L-cysteine （NAC） were incubated in renal cancer 786-O and ACHN cells， CCK8 was used to detect the effects of different concentrations of curcumin on cell proliferation and cell viability. Reactive oxygen detection kit （DCFH-DA） was used to detect the level of intracellular reactive oxygen species， and malondialdehyde （MDA） determination kit （TBA method） to detect intracellular malondialdehyde changes. 【Results】 PharmMapper website and GeneCards database screened out 109 common targets of curcumin and RCCC. TCGA database data analysis screened out 37 differentially expressed genes （DEGs） that might affect the overall survival of patients. The core target proteins of curcumin screened out by protein-protein interaction （PPI） that inhibited the biological behavior of RCCC mainly involved CASP3， EGFR， CHEK1， HSP90AA1， and AR. GO enrichment analysis identified 213 items， mainly including reactive oxygen species metabolic process， response to steroid hormones， fibrinolysis and other biologically active processes. KEGG enrichment analysis identified 24 items， which were mainly related to pyruvate metabolism， glycolysis/gluconeogenesis， FoxO signaling pathway， colorectal cancer， tyrosine metabolism， IL-17 signaling pathway， apoptosis and other signaling pathways. Curcumin reduced the cell viability of 786-O and ACHN in a time- and dose-dependent manner （P<0.05）. After curcumin was incubated with kidney cancer cells， the level of reactive oxygen species and MDA increased significantly （P<0.05）. The addition of NAC reversed the effect of curcumin on the cell viability of 786-O and ACHN cells （P<0.05）. 【Conclusion】 Curcumin may participate in the oxidative stress pathway to inhibit the proliferation of renal cell carcinoma.

Objective To analyze the chemical compounds of Shenqi Dihuang decoction by the ultraperformance liquid chromatography coupled with linear quadrupole ion trap-orbitrap mass spectrometry (UPLC-LTQ-Orbitrap-MS). Methods Warters ACQUITY UPLC HSS T3 (2.1 mm ×100 mm, 1.8 μm) was used as chromatographic column with mobile phase: 0.1% formic acid water (A)-0.1% formic acid acetonitrile (B) with gradient elution, and flow rate was 0.3 ml/min. Electrospray ion source (ESI) and an electrostatic field orbital ion trap mass analyzer were adopted, which was used to collect mass spectrometry fragment information with positive and negative ion modes, by comparing with the relative retention time of the reference substance. In addition, the fragment information of the mass spectrum was used to identify the compounds. The accurate identification of the chemical components in Shenqi Dihuang decoction was confirmed with literature. Results The study found that UPLC-LTQ-Orbitrap-MS technology could be used to identify 62 chemical components, including 13 aromatic acids, 9 flavonoids, 8 saponins, and 5 aromatic amines, 3 keto acids, 2 phenols, 1 aromatic quinone and other ingredients in Shenqi Dihuang decoction. Conclusion The identification analysis method in this study was efficient and accurate, which could be applied to the identification and analysis of chemical components in Shenqi Dihuang decoction and provided the important experimental data for the research on the material basis and mechanism.

Objective To collect the serum samples of patients with nonalcoholic fatty liver disease (NAFLD), and to investigate the changes in serum metabolic biomarkers before and after lifestyle intervention. Methods A total of 23 patients who were diagnosed with NAFLD in Department of Gastroenterology and Inpatient Department, Putuo District Central Hospital of Shanghai, from January 2019 to January 2020 were enrolled, and all patients received the intervention with aerobic exercise and equicaloric low-carbohydrate high-protein diet. A total of 13 healthy volunteers who underwent physical examination in Physical Examination Center were enrolled as control group. For the patients with NAFLD, basic information was collected before and after intervention, blood samples were collected twice to measure liver function, blood glucose, and blood lipids, and part of serum was used for serum metabolomics analysis. The serum samples were analyzed by ultra-performance liquid chromatography/tandem high-resolution mass spectrometry. The data collected were processed in Compound Discover, and then principal component analysis (PCA) and orthogonal partial least squares discriminant analysis were used to establish the profile of differentially expressed blood metabolites between patients and healthy people and perform the enrichment analysis of differentially expressed metabolic pathways. The independent samples t -test was used for comparison of normally distributed continuous data between two groups, and the Wilcoxon non-parametric test was used for comparison of non-normally distributed continuous data between two groups. Results After lifestyle intervention, the patients had significant reductions in body mass index ( P < 0.01), body weight ( P < 0.01), and serum biochemical parameters alkaline phosphatase, albumin, gamma-glutamyl transpeptidase, and alanine aminotransferase (all P < 0.05), as well as a significant reduction in total protein ( P < 0.01), while there were no significant improvements in cholinesterase, aspartate aminotransferase, and glucose. As for the four items for blood lipids, there was a significant reduction in triglyceride ( P < 0.01), while there were no significant improvements in high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and total cholesterol. The metabolomics analysis showed that 33 serum metabolites changed significantly after lifestyle intervention. In addition, PCA results showed that after intervention, the level of metabolites in patients tended to be normal. The signaling pathway analysis showed that exercise and diet mainly affected the pathways of bile acid, unsaturated fatty acid synthesis, and phenylalanine metabolism. Conclusion Lifestyle intervention can achieve varying degrees of reduction in the body weight of patients with NAFLD, improve serum biochemical parameters, and regulate the abnormal metabolic pathway in patients with NAFLD, which has important clinical value and significance for guiding clinicians to formulate reasonable diet and exercise strategies for patients with NAFLD and prevent the progression of NAFLD.

Objective:To investigate the risk factors for Gleason score upgrading after radical prostatectomy in clinical low-risk prostate cancer patients aged≥65 years.Methods:A total of 485 clinical low-risk prostate cancer patients aged≥65 years at five centers of the national multi-center PC-follow database from January 2015 to March 2019 were retrospectively analyzed.Data including age at diagnosis, prostate-specific antigen(PSA), MRI prostate imaging, puncture Gleason score, operation method, puncture method, positive incision margin and capsule penetration were collected.Differences in Gleason scores before and after operation were compared, and the risk factors for Gleason score upgrading after radical resection were evaluated by univariate and multivariate Logistic regression analysis.Results:Of 485 patients with a puncture Gleason score of 3+ 3=6, 261(53.8%)cases had postoperative pathological upgrading, in whom 228(87.4%)cases had Gleason score upgrading of 7, 22(8.4%)had Gleason score upgrading of 8, and 11(4.2%)had Gleason score upgrading of 9 or more.The rate of Gleason score upgrading was elevated with increased preoperative PSA levels, positive pelvic MRI, and higher positive rates of puncture biopsy.The incidences of postoperative capsule penetration(27.2% vs.12.5%, P<0.001)and positive incision margin(25.2% vs.17.4%, P=0.036)had statistically significant differences between the pathologically upgraded group and the pathologically non-upgraded group.Multivariate analysis showed that preoperative PSA level, percentage of positive puncture biopsies, biopsy Gleason score and pelvic MRI were independent predictors of prostate cancer. Conclusions:For clinical low-risk prostate cancer patients aged≥65 years with high risk factors for Gleason score upgrading, repeated biopsies should be carried out when necessary and the treatment plan should be adjusted accordingly.