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 establish a biotin-avidin system time-resolved fluorescence immunoassay(BAS-TRFIA)for detecting serum soluble fms-like tyrosine kinase-1(sFlt-1)and evaluate its performance.Methods A BAS-TRFIA was established to quantitatively determine the concentration of sFlt-1 in the serum of pregnant women,which based on the microplate was coated with streptavidin.The monoclonal antibody to capture sFlt-1 was labeled by biotin,and the detection of sFlt-1 monoclonal antibody was labeled by europium.The performance indicators such as lower limit of detection,biological limit of detection,functional sensitivity,precision,linearity,interference test,cross-reaction test,and high dose hook effect of the method were evaluated.A total of 106 remaining serum samples from pregnant women with no hemolysis,jaundice and lipemia at more than 9 weeks were detected by BAS-TRFIA and electrochemiluminescence for methodological comparison study,and the correlation of the comparison test results was analyzed by linear regression.Results The optimal reaction time of the sample was 90 min.The lower limit of detection was 1.00pg/ml.The biological limit of detection was 10.00pg/ml,and the functional sensitivity was 10.00pg/ml.The intra-assay CV and inter-assay CV were both within 5%,and the linear range was 20.00 to 40 000.00pg/ml.The relative bias of the detection results of the 17 interfering samples with interfering substances added to the low-concentration and high-concentration quality controls and the basic samples was within-4.94%～4.24%.The high dose hook effect was not found in sFlt-1 samples up to 150 000pg/ml.When the concentration of sFlt-1 in the sample was 105.40～40 972.00pg/ml,the linear regression equation of BAS-TRFIA and electrochemiluminescence(ECL)detection results was Y=1.086 7X+17.946(r=0.994 4,t=96.26,P＜0.05).Conclusion The quantitative detection of sFlt-1 by BAS-TRFIA has high sensitivity,good precision,wide linear range,strong anti-interference ability,and good correlation with the detection results of reference methods,which is valuable for clinical application.

Objective To develop a reagent for detecting of 17α-hydroxyprogesterone(17α-OHP)in dried blood spots on filter paper fixed on vascular stent by Auto TRFIA-4 automatic fluorescence immunoanalyzer and evaluate its performance.Methods The microwell plate was coated with the sheep anti-rabbit IgG antibody as microwell reaction plate,the rabbit anti-human 17α-OHP antibody was diluted as intermediate antibody,and the 17α-OHP-bovine serum albumin conjugate was labeled by europium as europium marker.The concentration of 17α-OHP in dried blood spots on filter paper fixed on blood spots stent was quantitatively detected by Auto TRFIA-4 automatic fluorescence immunoanalyzer.The analysis sensitivity,accuracy,linearity,precision,specificity and stability were evaluated,and whether they met the requirements of the formulated industry standards were evaluated.A total of 227 neonatal heel blood filter paper samples from newborns who were born 72 hours after birth and within 7 days and fully breastfeeding were selected for reagent comparison test.The consistency was analyzed by χ2 test,Kappa test,t test,linear correlation analysis,regression analysis,Bland-Altman method analysis and predictive bias analysis of medical decision level,P<0.05 indicated statistically significant difference.Results The optimal coating concentration of sheep anti-rabbit IgG antibody was 3 μg/ml.The optimal dilution ratio of rabbit anti-human 17α-OHP antibody was 1∶1 500.The optimal dilution ratio of 17α-OHP europium marker mother liquor was 1∶2 500.The limit of blank,limit of detection,limit of quantification was 0.75,1.08 and 1.99 nmol/L,respectively.The relative deviations of the standard check test were within±15.00%,and the average recovery rate was 92.36%.The linear correlation coefficient was 0.997 1 in the range of 2.00 to 300.00nmo/L.The intra-assay and inter-assay coefficients of variation were all within 10.00%.The cross-reactivity rates of 100.00 ng/ml progesterone,17α-hydroxypreg nenolone and 11-deoxycortisol were within 0.089%to 0.64%.The performance of stability test met the requirements.The total coincidence rate was 100%compared with the results of contrast reagent.The quantitative results were highly correlated with the contrast reagent(r=0.999 4,tr=452.02,P<0.05).Conclusion The self-developed reagent has the advantages of high sensitivity,good accuracy,wide linear range,good precision,high specificity and good stability,which meets the requirements of the formulated industry standards,and has high correlation and consistency with the result of contrast reagent,which meets the needs of clinical detection.

BACKGROUND: Preclinical studies have indicated that Angong Niuhuang Pills (ANP) reduce cerebral infarct and edema volumes. This study aimed to investigate whether ANP safely reduces cerebral infarct and edema volumes in patients with moderate to severe acute ischemic stroke. METHODS: This randomized, double-blind, placebo-controlled pilot trial included patients with acute ischemic stroke with National Institutes of Health Stroke Scale (NIHSS) scores ranging from 10 to 20 in 17 centers in China between April 2021 and July 2022. Patients were allocated within 36 h after onset via block randomization to receive ANP or placebo (3 g/day for 5 days). The primary outcomes were changes in cerebral infarct and edema volumes after 14 days of treatment. The primary safety outcome was severe adverse events (SAEs) for 90 days. RESULTS: There were 57 and 60 patients finally included in the ANP and placebo groups, respectively for modified intention-to-treat analysis. The median age was 66.0 years, and the median NIHSS score at baseline was 12.0. The changes in cerebral infarct volume at day 14 were 0.3 mL and 0.4 mL in the ANP and placebo groups, respectively (median difference: -7.1 mL; interquartile range [IQR]: -18.3 to 2.3 mL, P = 0.30). The changes in cerebral edema volume of the ANP and placebo groups on day 14 were 11.4 mL and 4.0 mL, respectively ( median difference: 3.0 mL, IQR: -1.3 to 9.9 mL, P = 0.15). The rates of SAE within 90 days were similar in the ANP (3/57, 5%) and placebo (7/60, 12%) groups ( P = 0.36). Changes in serum mercury and arsenic concentrations were comparable. In patients with large artery atherosclerosis, ANP reduced the cerebral infarct volume at 14 days (median difference: -12.3 mL; IQR: -27.7 to -0.3 mL, P = 0.03). CONCLUSIONS: ANP showed a similar safety profile to placebo and non-significant tendency to reduce cerebral infarct volume in patients with moderate-to-severe stroke. Further studies are warranted to assess the efficacy of ANP in reducing cerebral infarcts and improving clinical prognosis. TRAIL REGISTRATION Clinicaltrials.gov , No. NCT04475328.
Aged ; Female ; Humans ; Male ; Middle Aged ; Double-Blind Method ; Drugs, Chinese Herbal/adverse effects* ; Ischemic Stroke/drug therapy* ; Pilot Projects ; Stroke/drug therapy* ; Treatment Outcome

Gallbladder cancer is the most common malignant tumor of the biliary system,characterized by insidious growth,rapid progression,and skip metastasis.The 5-year overall survival rate is only 5%.Although targeted immunotherapies have emerged in recent years,they are still in the exploratory phase for the treatment of gallbladder cancer,and radical surgical resection remains the only potentially curative treatment.The main goal of surgery is to completely remove the tumor and perform systematic lymph node dissection to reduce the risk of recurrence.Thanks to advances in surgical techniques and continuous optimization of perioperative management,postoperative complications,and hospital mortality in patients undergoing extended radical surgery for gallbladder cancer have decreased.However,the complex mechanisms of gallbladder cancer spread and metastasis make it challenging to determine the optimal scope of surgical resection.Clinical guidelines from different countries and hepatobiliary surgery centers often vary on surgical options,especially regarding the reasonable extent of liver resection,the scope of lymph node dissection and its impact on prognosis,and whether routine extrahepatic bile duct resection should be performed.There remains significant controversy in these areas.Future extensive prospective cohort studies are still needed to provide more evidence-based medical data for the surgical treatment of gallbladder cancer.When designing a surgical plan,surgeons must consider preoperative examination results,intraoperative findings,and pathological evaluation of frozen sections.A balance must be struck between radical treatment and safety and effectiveness.The most appropriate surgical method for each patient should be chosen while also emphasizing establishing a multidisciplinary collaborative system.Based on accurate tumor staging,postoperative adjuvant therapies should be integrated to continue making progress in improving patient prognosis.

Objective To establish a biotin-avidin system time-resolved fluorescence immunoassay(BAS-TRFIA)for detecting serum soluble fms-like tyrosine kinase-1(sFlt-1)and evaluate its performance.Methods A BAS-TRFIA was established to quantitatively determine the concentration of sFlt-1 in the serum of pregnant women,which based on the microplate was coated with streptavidin.The monoclonal antibody to capture sFlt-1 was labeled by biotin,and the detection of sFlt-1 monoclonal antibody was labeled by europium.The performance indicators such as lower limit of detection,biological limit of detection,functional sensitivity,precision,linearity,interference test,cross-reaction test,and high dose hook effect of the method were evaluated.A total of 106 remaining serum samples from pregnant women with no hemolysis,jaundice and lipemia at more than 9 weeks were detected by BAS-TRFIA and electrochemiluminescence for methodological comparison study,and the correlation of the comparison test results was analyzed by linear regression.Results The optimal reaction time of the sample was 90 min.The lower limit of detection was 1.00pg/ml.The biological limit of detection was 10.00pg/ml,and the functional sensitivity was 10.00pg/ml.The intra-assay CV and inter-assay CV were both within 5%,and the linear range was 20.00 to 40 000.00pg/ml.The relative bias of the detection results of the 17 interfering samples with interfering substances added to the low-concentration and high-concentration quality controls and the basic samples was within-4.94%～4.24%.The high dose hook effect was not found in sFlt-1 samples up to 150 000pg/ml.When the concentration of sFlt-1 in the sample was 105.40～40 972.00pg/ml,the linear regression equation of BAS-TRFIA and electrochemiluminescence(ECL)detection results was Y=1.086 7X+17.946(r=0.994 4,t=96.26,P＜0.05).Conclusion The quantitative detection of sFlt-1 by BAS-TRFIA has high sensitivity,good precision,wide linear range,strong anti-interference ability,and good correlation with the detection results of reference methods,which is valuable for clinical application.

Objective To develop a reagent for detecting of 17α-hydroxyprogesterone(17α-OHP)in dried blood spots on filter paper fixed on vascular stent by Auto TRFIA-4 automatic fluorescence immunoanalyzer and evaluate its performance.Methods The microwell plate was coated with the sheep anti-rabbit IgG antibody as microwell reaction plate,the rabbit anti-human 17α-OHP antibody was diluted as intermediate antibody,and the 17α-OHP-bovine serum albumin conjugate was labeled by europium as europium marker.The concentration of 17α-OHP in dried blood spots on filter paper fixed on blood spots stent was quantitatively detected by Auto TRFIA-4 automatic fluorescence immunoanalyzer.The analysis sensitivity,accuracy,linearity,precision,specificity and stability were evaluated,and whether they met the requirements of the formulated industry standards were evaluated.A total of 227 neonatal heel blood filter paper samples from newborns who were born 72 hours after birth and within 7 days and fully breastfeeding were selected for reagent comparison test.The consistency was analyzed by χ2 test,Kappa test,t test,linear correlation analysis,regression analysis,Bland-Altman method analysis and predictive bias analysis of medical decision level,P<0.05 indicated statistically significant difference.Results The optimal coating concentration of sheep anti-rabbit IgG antibody was 3 μg/ml.The optimal dilution ratio of rabbit anti-human 17α-OHP antibody was 1∶1 500.The optimal dilution ratio of 17α-OHP europium marker mother liquor was 1∶2 500.The limit of blank,limit of detection,limit of quantification was 0.75,1.08 and 1.99 nmol/L,respectively.The relative deviations of the standard check test were within±15.00%,and the average recovery rate was 92.36%.The linear correlation coefficient was 0.997 1 in the range of 2.00 to 300.00nmo/L.The intra-assay and inter-assay coefficients of variation were all within 10.00%.The cross-reactivity rates of 100.00 ng/ml progesterone,17α-hydroxypreg nenolone and 11-deoxycortisol were within 0.089%to 0.64%.The performance of stability test met the requirements.The total coincidence rate was 100%compared with the results of contrast reagent.The quantitative results were highly correlated with the contrast reagent(r=0.999 4,tr=452.02,P<0.05).Conclusion The self-developed reagent has the advantages of high sensitivity,good accuracy,wide linear range,good precision,high specificity and good stability,which meets the requirements of the formulated industry standards,and has high correlation and consistency with the result of contrast reagent,which meets the needs of clinical detection.

Gallbladder cancer is the most common malignant tumor of the biliary system,characterized by insidious growth,rapid progression,and skip metastasis.The 5-year overall survival rate is only 5%.Although targeted immunotherapies have emerged in recent years,they are still in the exploratory phase for the treatment of gallbladder cancer,and radical surgical resection remains the only potentially curative treatment.The main goal of surgery is to completely remove the tumor and perform systematic lymph node dissection to reduce the risk of recurrence.Thanks to advances in surgical techniques and continuous optimization of perioperative management,postoperative complications,and hospital mortality in patients undergoing extended radical surgery for gallbladder cancer have decreased.However,the complex mechanisms of gallbladder cancer spread and metastasis make it challenging to determine the optimal scope of surgical resection.Clinical guidelines from different countries and hepatobiliary surgery centers often vary on surgical options,especially regarding the reasonable extent of liver resection,the scope of lymph node dissection and its impact on prognosis,and whether routine extrahepatic bile duct resection should be performed.There remains significant controversy in these areas.Future extensive prospective cohort studies are still needed to provide more evidence-based medical data for the surgical treatment of gallbladder cancer.When designing a surgical plan,surgeons must consider preoperative examination results,intraoperative findings,and pathological evaluation of frozen sections.A balance must be struck between radical treatment and safety and effectiveness.The most appropriate surgical method for each patient should be chosen while also emphasizing establishing a multidisciplinary collaborative system.Based on accurate tumor staging,postoperative adjuvant therapies should be integrated to continue making progress in improving patient prognosis.

AIM:To investigate the changes of retinal microcirculation after phacoemulsification and the influencing factors of visual acuity.METHODS: Retrospective analysis. A total of 264 cataract patients(264 eyes)who underwent phacoemulsification in our hospital from January 2022 to December 2022 were selected as the study objects. Patients were divided into < 0.3 group(66 eyes)and ≥0.3 group(198 eyes)according to the recovery of best corrected visual acuity(BCVA)at 3 mo after surgery. The changes of retinal microcirculation indexes were compared before and after treatment. Logistic regression and LASSO regression models were used to screen the influencing factors of postoperative BCVA. A nomogram prediction model of postoperative BCVA was constructed and verified. A restricted cubic spline Logistic regression model was established to analyze the dose-response relationship between end-diastolic velocity(EDV), peak systolic velocity(PSV)and the risk of BCVA recovery.RESULTS: At 3 mo postoperatively, EDV and PSV were significantly improved compared with those before treatment, and resistance index(RI)levels were significantly lower than those before treatment(all P<0.05). Preoperative EDV, PSV, aqueous humor cell grade, fundus lesion grade, advanced age and Emery grade were influencing factors for poor BCVA recovery after phacoemulsification in cataract patients(P<0.05). The AUC before and after validation of the nomogram model by Bootstrap method were 0.869(95%CI: 0.815-0.903)and 0.866(95%CI: 0.802-0.895), respectively. The sensitivity was 88.36% and 88.27%, and the specificity was 91.82% and 91.78%, respectively. Restricted cubic spline model analysis showed no nonlinear dose-response relationship between EDV and PSV levels and the risk of poor BCVA recovery in either male or female(P>0.05).CONCLUSION: After phacoemulsification, retinal microcirculation in cataract patients improved significantly. EDV, PSV, aqueous humor cell grade, fundus lesion grade, advanced age and Emery grade are all factors influencing poor BCVA recovery after cataract surgery.

Objective:To discuss the effect of sialic acid-binding immunoglobulin-like lectin-15(Siglec15)derived from M2 tumor-associated macrophages(M2-TAMs)on promoting the malignant biological behavior of the esophageal squamous cell carcinoma(ESCC)through bioinformatics analysis,and to validate the findings through cell experiment.Methods:The Tumor Immune Estimation Resource(TIMER)online Database was used to analyze the expression differences and immune infiltration of Siglec15 in pan-cancer and adjacent normal tissues.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of Siglec15 mRNA in M2-TAMs and ESCC EC109 and KYSE150 cells.Based on the non-contact co-culture of M2-TAMs and ESCC cells,the following groups were set up,such as EC109/KYSE150 group,EC109/KYSE150+si-NC group(transfected with si-NC sequence),and EC109/KYSE150+si-Siglec15 group(transfected with si-Siglec15#1 and si-Siglec15#2 sequences).CCK-8 method was used to detect the proliferation activities of the cells in various groups;wound healing assay was used to detect the wound healing rates of the cells in various groups;Transwell chamber assay was used to detect the numbers of migration and invasion cells in various groups;flow cytometry was used to detect the apoptotic rates of the cells in various groups.Results:The bioinformatics analysis results showed that compared with adjacent normal tissue,the expression levels of Siglec15 mRNA in pan-cancer tissues such as esophageal cancer,colon cancer,and head and neck squamous cell carcinoma tissues were increased(P＜0.05 or P＜0.01),and the expression level of Siglec15 mRNA in esophageal cancer tissue was significantly positively correlated with the infiltration of the macrophages(P＜0.05).Compared with the EC109 cells and KYSE150 cells,the expression level of Siglec15 mRNA in M2-TAMs was significantly increased(P＜0.01).There was no significant difference in the proliferation rate of the cells among EC109/KYSE150 group,EC109/KYSE150+si-NC group,and EC109/KYSE150+si-Siglec15 group(P＞0.05).Compared with EC109/KYSE150 group,after treated for 24 and 48 h,the wound healing rate of the cells in EC109/KYSE150+si-NC group was increased(P＜0.01),the numbers of migration and invasion cells were increased(P＜0.05),and the apoptotic rate was decreased(P＜0.01).Compared with EC109/KYSE150+si-NC group,the wound healing rates of the cells in EC109/KYSE150+si-Siglec15#1 group and EC109/KYSE150+si-Siglec15#2 group were decreased(P＜0.05),the numbers of migration and invasion cells were decreased(P＜0.05),and the apoptotic rates of the cells had no significant difference(P＞0.05).Conclusion:Siglec15 derived from M2-TAMs may be a key factor in promoting the migration and invasion of the ESCC cells.