Benzylisoquinoline alkaloids (BIAs) are a structurally diverse group of plant metabolites renowned for their pharmacological properties. However, sustainable sources for these compounds remain limited. Consequently, researchers are focusing on elucidating BIA biosynthetic pathways and genes to explore alternative sources using synthetic biology approaches. CYP80B, a family of cytochrome P450 (CYP450) enzymes, plays a crucial role in BIA biosynthesis. Previously reported CYP80Bs are known to catalyze the 3'-hydroxylation of (S)-N-methylcoclaurine, with the N-methyl group essential for catalytic activity. In this study, we successfully cloned a full-length CYP80B gene (StCYP80B) from Stephania tetrandra (S. tetrandra) and identified its function using a yeast heterologous expression system. Both in vivo yeast feeding and in vitro enzyme analysis demonstrated that StCYP80B could catalyze N-methylcoclaurine and coclaurine into their respective 3'-hydroxylated products. Notably, StCYP80B exhibited an expanded substrate selectivity compared to previously reported wild-type CYP80Bs, as it did not require an N-methyl group for hydroxylase activity. Furthermore, StCYP80B displayed a clear preference for the (S)-configuration. Co-expression of StCYP80B with the CYP450 reductases (CPRs, StCPR1, and StCPR2), also cloned from S. tetrandra, significantly enhanced the catalytic activity towards (S)-coclaurine. Site-directed mutagenesis of StCYP80B revealed that the residue H205 is crucial for coclaurine catalysis. Additionally, StCYP80B exhibited tissue-specific expression in plants. This study provides new genetic resources for the biosynthesis of BIAs and further elucidates their synthetic pathway in natural plant systems.
Cytochrome P-450 Enzyme System/chemistry* ; Benzylisoquinolines/chemistry* ; Hydroxylation ; Plant Proteins/chemistry* ; Alkaloids/metabolism* ; Stephania tetrandra/genetics*

Objective To explore the application value of MR MAGiC sequence reconstructed based on deep learning reconstruction(DLR)for localizing epileptogenic focus of intractable epilepsy.Methods Fifty intractable epilepsy patients(epilepsy group)and 20 healthy controls(control group)were retrospectively collected.Brain MR MAGiC sequence was scanned,then traditional reconstruction(inverse Fourier transform)and DLR algorithm were performed to obtain MAGiC and MAGiC DLR images,respectively.The corresponding quantitative parameter maps were generated after post-processing,including T1 mapping,T2 mapping and proton density(PD)mapping.The subjective scores of noise,artifacts,structural clarity and overall quality were compared between two kinds of quantitative parameter images.The asymmetry index(AI)of quantitative parameters(T1,T2 and PD values)between the affected side and the contralateral side in epilepsy group,the contralateral side or the suspected epileptogenic focus in epilepsy group and HC group were calculated.Then epileptogenic focus were localized based on performance of MAGiC and MAGiC DLR corresponding quantitative parameter maps combining obtained AI of quantitative parameter values.According to surgical results,follow-up review,electroencephalogram or PET results,the accuracy of MAGiC and MAGiC DLR images for localizing epileptogenic focus was calculated.Results Compared with MAGiC quantitative parametric maps,noise of MAGiC DLR quantitative parametric maps reduced,while subjective scores of structure clarity and overall quality improved(all P＜0.05).The accuracy of MAGiC and MAGiC DLR images for localizing epileptogenic focus was 82.00％(41/50)and 88.00％(44/50),respectively.Conclusion MR MAGiC sequence based on DLR was beneficial for improving accuracy of localizing epileptogenic focus in intractable epilepsy.

Objective To investigate the detection conditions,risk factors and preventive measures of colorectal polyps in health examination population undergoing painless colonoscopy.Methods A total of 2 680 people who underwent painless colonoscopy at the Health Management Center of the First Affiliated Hospital of Naval Medical University from January to December 2023 were enrolled in this retrospective study.They were assigned into polyp group and non-polyp group according the examination results.The general information of the two groups was collected to explore the risk factors of colorectal polyps,so as to provide reference for the prevention strategy of colorectal polyps.Results The colorectal polyps were detected in 1 223 people(45.63%),including 633 cases(51.76%)of adenomatous polyps,587 cases(48.00%)of non-adenomatous polyps,and 3 cases(0.25%)of colorectal cancer.Logistic regression analysis indicated that male,smoking history,alcohol drinking history,age 40-59 years old,hyperlipidemia,and CRP>10 mg/L were risk factors of colorectal polyps(P<0.05).Conclusion There is a high detection rate of colorectal polyps in physical examination population.Men,aged 40-59 years old,hyperlipidemia,smoking history,alcohol drinking history,and CRP>10 mg/L are high risk factors of colorectal polyps,which should be paid more attention for the prevention and monitoring of colorectal polyps.

Objective:To establish a high-performance liquid chromatography(HPLC)method for the determina-tion of related substances in buprenorphine active pharmaceutical ingredient(API)using advanced ACD/Auto-Chrom method development software for comprehensive parameter simulation and design.Methods:An Agilent ZORBAX Eclipse Plus C18 column(4.6 mm × 150 mm,3.5 μm)was used with a mobile phase consisting of 40 mmol·L-1 potassium dihydrogen phosphate solution and acetonitrile in a gradient elution mode.The flow rate was set at 1.3 mL·min-1,the column temperature was maintained at 35 ℃,the detection wavelength was 240 nm,and the injection volume was 5 μL.Results:The impurities A,B,D,E,F,G,H,I,and J in buprenorphine were effectively separated from the main component.The linear ranges were 0.33-83.73,0.20-78.74,0.20-40.28,0.22-43.31,0.32-78.98,0.13-63.74,0.51-101.54,0.22-43.72,and 0.40-80.37 μg·mL-1,respectively.The limits of detection(LOD)were 0.10,0.06,0.06,0.06,0.09,0.04,0.15,0.07,and 0.12 μg·mL-1,respectively,while the limits of quantification(LOQ)were 0.33,0.20,0.20,0.22,0.32,0.13,0.51,0.22,and 0.40 μg·mL-1,respectively.The accuracy,precision,and robustness of the method met the required standards.Conclusion:This method is suitable for the determi-nation and quality control of related substances such as impurities A,B,D,E,F,G,H,I,and J in buprenorphine API.

Objective:To establish a high-performance liquid chromatography(HPLC)method for the determina-tion of related substances in buprenorphine active pharmaceutical ingredient(API)using advanced ACD/Auto-Chrom method development software for comprehensive parameter simulation and design.Methods:An Agilent ZORBAX Eclipse Plus C18 column(4.6 mm × 150 mm,3.5 μm)was used with a mobile phase consisting of 40 mmol·L-1 potassium dihydrogen phosphate solution and acetonitrile in a gradient elution mode.The flow rate was set at 1.3 mL·min-1,the column temperature was maintained at 35 ℃,the detection wavelength was 240 nm,and the injection volume was 5 μL.Results:The impurities A,B,D,E,F,G,H,I,and J in buprenorphine were effectively separated from the main component.The linear ranges were 0.33-83.73,0.20-78.74,0.20-40.28,0.22-43.31,0.32-78.98,0.13-63.74,0.51-101.54,0.22-43.72,and 0.40-80.37 μg·mL-1,respectively.The limits of detection(LOD)were 0.10,0.06,0.06,0.06,0.09,0.04,0.15,0.07,and 0.12 μg·mL-1,respectively,while the limits of quantification(LOQ)were 0.33,0.20,0.20,0.22,0.32,0.13,0.51,0.22,and 0.40 μg·mL-1,respectively.The accuracy,precision,and robustness of the method met the required standards.Conclusion:This method is suitable for the determi-nation and quality control of related substances such as impurities A,B,D,E,F,G,H,I,and J in buprenorphine API.

Objective To explore the application value of MR MAGiC sequence reconstructed based on deep learning reconstruction(DLR)for localizing epileptogenic focus of intractable epilepsy.Methods Fifty intractable epilepsy patients(epilepsy group)and 20 healthy controls(control group)were retrospectively collected.Brain MR MAGiC sequence was scanned,then traditional reconstruction(inverse Fourier transform)and DLR algorithm were performed to obtain MAGiC and MAGiC DLR images,respectively.The corresponding quantitative parameter maps were generated after post-processing,including T1 mapping,T2 mapping and proton density(PD)mapping.The subjective scores of noise,artifacts,structural clarity and overall quality were compared between two kinds of quantitative parameter images.The asymmetry index(AI)of quantitative parameters(T1,T2 and PD values)between the affected side and the contralateral side in epilepsy group,the contralateral side or the suspected epileptogenic focus in epilepsy group and HC group were calculated.Then epileptogenic focus were localized based on performance of MAGiC and MAGiC DLR corresponding quantitative parameter maps combining obtained AI of quantitative parameter values.According to surgical results,follow-up review,electroencephalogram or PET results,the accuracy of MAGiC and MAGiC DLR images for localizing epileptogenic focus was calculated.Results Compared with MAGiC quantitative parametric maps,noise of MAGiC DLR quantitative parametric maps reduced,while subjective scores of structure clarity and overall quality improved(all P＜0.05).The accuracy of MAGiC and MAGiC DLR images for localizing epileptogenic focus was 82.00％(41/50)and 88.00％(44/50),respectively.Conclusion MR MAGiC sequence based on DLR was beneficial for improving accuracy of localizing epileptogenic focus in intractable epilepsy.

OBJECTIVE Aiming at the current vacancy of large language models(LLMs)in TCM evaluation,a TCM benchmark dataset is designed and constructed to comprehensively and objectively evaluate the mastery and reasoning performance of LLMs in TCM knowledge,providing scientific and reliable basis for optimizing the performance of LLMs in the field of TCM.METHODS This benchmark includes 29 506 questions across 13 subjects,with data collected from standardized TCM exams and textbooks.Three gen-eral-purpose models(GPT-3.5,ChatGLM3,Baichuan)and five Chinese medical models(PULSE,BenTsao,HuatuoGPT2,Bian-Que2,ShenNong)were evaluated with answer prediction and answer reasoning tasks.The evaluation results were quantitatively as-sessed using metrics including accuracy,F1 score,BLEU,and Rouge.RESULTS For the answer prediction task,Baichuan had the highest accuracy of 36.07%in single-choice questions,while ChatGLM3 achieved the highest accuracy of 18.96%and F1 score of 76.31%in multiple-choice questions.For the answer reasoning experiment,Baichuan scored highest on BLEU-1 with 24.71,while ChatGLM3 achieved the highest Rouge-1 score of 44.64.CONCLUSION In this study,general LLMs performed slightly better than Chinese medical LLMs.Meanwhile,all models'accuracy on choice questions remained below 60%,reflecting the significant challen-ges and room for improvement that LLMs still face in the field of TCM.

Objective:To screen the differentially expressed genes(DEGs)under high phosphate-induced calcification in the vascular smooth muscle cells(VSMCs)by mRNA high-throughput sequencing technology,and to analyze the key genes and signaling pathways involved in the VSMCs calcification.Methods:The human VSMCs were divided into control group and model group.The cells in model group was exposed to the high-phosphate medium,while the cells in control group were cultured in DMEM supplemented with 10%fetal bovine serum under the same conditions.The VSMCs in two groups,stably transfected,were cultured for 12 d.The morphology of the cells in two groups were observed and photographed under inverted microscope.The DEGs were selected by Hisat2 software,and Gene Ontology(GO)functional and Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway enrichment analysis were performed by Stringtie software from three aspects,such as biological processes(BP),molecular functions(MF),and cellular components(CC).The calcification of the cells in two groups was observed by Von Kossa staining method.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to analyze the expression levels of alkaline phosphatase(ALP),bone morphogenetic protein 2(BMP2),alpha-smooth muscle actin(α-SMA),tumor protein 53(Tp53),glutathione peroxidase 4(GPX4),ferritin light chain 1(Ftl1),and glycosylphosphatidylinositol-specific phospholipase D1(GPLD1)mRNA in the cells in two groups.Results:Compared with control group,there were 2 524 DEGs in the cells in model group,and there were 1 368 upregulated DEGs and 1 156 downregulated DEGs.Clustering of DEGs between the cells in two groups was distinct.The GO functional and KEGG pathway enrichment analysis results showed that the upregulated DEGs were primarily involved in regulating the microtubule cytoskeleton,cell polarity,protein localization,and cell cycle regulation among BPs;in constructing cell membrane,microtubule organization,chromosomes,and kinetochore among CCs;and functioning in phosphatidylinositol phosphate,Rho GTPase protein binding,transmembrane transport,and protein kinase regulatory activity among MFs.Downregulated DEGs were mainly involved in cytoplasmic translation,protein membrane localization,mRNA metabolism,and protein endoplasmic reticulum localization among BPs;in forming ribosome subunits,cell membrane,and autophagy among CCs;and functioning in single-stranded DNA,ribonucleoprotein complex,growth factor binding,regulating protein kinase activity,and catalytic activity among MFs.Seven signaling pathways were significantly enriched in upregulated genes,most notably in the biosynthesis of glycosylphosphatidylinositol(GPI)anchors;whereas 18 signaling pathways were significantly enriched in the downregulated genes,most notably in ferroptosis.The RT-qPCR results showed that compared with control group,the expression levels of GPX4,Ftl1,and Tp53 mRNA in the cells in model group were significantly decreased(P<0.01),while the expression level of GPLD1 mRNA was significantly increased(P<0.01);compared with control group,the expression level of α-SMA mRNA in the cells in model group was significantly decreased(P<0.01),and the expression levels of ALP and BMP2 mRNA were significantly increased(P<0.01).Conclusion:The VSMCs underwent calcification and normal cells exhibit the DEGs.The key signaling pathways in the calcification induced by high phosphate in the VSMCs include ferroptosis and GPI anchor biosynthesis,mediated primarily through GPX4,Ftl1,Tp53,and GPLD1.

Objective To investigate the role of Toll-like receptor 4(TLR4)in hepatocyte regeneration after acet-aminophen(APAP)-induced injury in human normal liver cell(L02)and its possible mechanism.Methods L02 cells were cultured in vitro,and cell viability was detected by CCK-8 assay.The optimal concentration and duration of APAP and the concentration of TLR4 inhibitor(TAK-242)were determined.The protein expression levels of nu-clear factor-κB(NF-κB),microtubule-associated protein light chain 3(LC3),p62,receptor interacting protein kinase 1(RIP1),receptor interacting protein kinase 3(RIP3),signal transducer and activator of transcription 3(STAT3),phosphorylation of STAT3(p-STAT3),proliferating cell nuclear antigen(PCNA)and Cyclin D1 were detected by Western blot.The mRNA expression levels of TLR4,NF-κB,tumor necrosis factor-α(TNF-α),inter-leukin-6(IL-6),interleukin-1 β(IL-1 β),PCNA,Cyclin D1 and Ki67 were detected by qRT-PCR.Results Ac-cording to the results of CCK-8,L02 cells were treated with 5 mmol/L APAP for 24,36,48 h to simulate liver in-jury and regeneration model in vitro,and TAK-242 100 nmol/L was pretreated 2 ht before APAP to inhibit TLR4.Compared with the control group,the protein levels of NF-κB,RIP1,p-STAT3,PCNA,Cyclin D1 and the mRNA levels of TNF-α,IL-1 β and PCNA increased in the APAP 24 h group;the protein levels of NF-κB,RIP1,RIP3,p-STAT3,PCNA,Cyclin D1 and the mRNA levels of TLR4,NF-κB,TNF-α,IL-1 β,PCNA and Cyclin D1 in-creased in the APAP 36 h group;the protein levels of NF-κB,RIP1,p-STAT3,PCNA,Cyclin D1 and the mRNA levels of TLR4,NF-κB,TNF-α,IL-1β,IL-6,PCNA,Cyclin D1 and Ki67 increased in the APAP 48 h group.The protein levels of NF-κB,RIP1,RIP3,p-STAT3,PCNA,Cyclin D1 and the mRNA levels of TLR4,NF-κB,TNF-α,IL-1β,IL-6,PCNA,Cyclin D1,Ki67 significantly decreased in APAP+TAK-242 24 h and 48 h group than the APAP group at the same time point;the protein levels of NF-κB,PCNA and the mRNA levels of TLR4,NF-κB,TNF-α,IL-1β,IL-6,PCNA and Ki67 in APAP+TAK-242 36 h group were also significantly lower than those in APAP 36 h group.Compared with the control group,autophagy was activated in the APAP group,while autophagy was inhibited in the APAP+TAK-242 group.Conclusion TLR4 may affect the TLR4/NF-κB pathway,up-regulate the levels of inflammatory factors and autophagy,and promote hepatocyte regeneration after APAP-in-duced liver injury in L02 cells.

Objective:To understand the current status of core measures of enhanced recovery after surgery (ERAS) nursing in Anhui Province and analyze its promotion strategies, so we can provide reference for further implementation and promotion of clinical practice guidelines of ERAS.Methods:Design a Development Questionnaire of Enhanced Recovery After Surgery in Anhui Province based on the Clinical Practice Guidelines for Enhanced Recovery After Surgery in China (2021 edition) (I) and, a cross-sectional survey was conducted on surgical nurses in 132 secondary and above hospitals in Anhui Province from July to September 2023 by the convenience sampling method.Results:A total of 1 375 nurses from 13 cities in Anhui province participated in the survey. The nurses′ awareness rate of ERAS concept was 100.00%, but only 52.51% (722/1 375) of the nurses had received standardized ERAS training, and only 26.11% (359/1 375) of the nurses indicated that they had a multidisciplinary team such as medical care, anesthesia and nutrition were involved in the implementation of the ERAS. Nearly 38.33% (527/1 375) of the nurses said that the department lacked the relevant nursing quality control system, and the overall implementation rate of quality control training, development, feedback and monitoring was not high. Only 55.71% (766/1 375) of nurses reported that a quality control team has been established within their department, and only 40.44% (556/1 375) of departments conduct monthly checks on the implementation and progress of ERAS. In terms of the implementation of core measures, preoperative thromboprevention therapy, intraoperative temperature monitoring and management, postoperative multimodal analgesia were better; however, the implementation of preoperative fasting time ≤ 6 h, water ban time≤ 2 h, oral carbohydrate drinks or clean streaming drinks, oral intake of enteral nutritional supplementation when less than 60% of the normal amount was poor.Conclusions:There are still many deficiencies in the overall development of ERAS nursing in Anhui Province, and the implementation level of core measures also needs to be improved. It is necessary to combine the concept of precision nursing, strengthen the whole process management and quality control, so as to promote the clinical practice and promotion of ERAS.