This study was aimed at developing an in vitro fluorescent substrate assay for the activity of leucyl aminopeptid-ase(LAP)from Echinococcus multilocularis and comparing it with the chemical chromogenic substrate enzyme activity assay.Through the establishment of reaction conditions for the fluorescent substrate-based in vitro enzyme activity assay,we com-pared the differences between the fluorescent substrate L-Leucine-7-amido-4-methylocoumarin(Leu-AMC)and the chemical chromogenic substrate L-Leucine-4-nitroanilide(Leu-pNA)through molecular docking,inhibition rates,and precision measures.Molecular docking revealed that the fluorescent substrate Leu-AMC had higher affinity for the protein than the chemical chromogenic substrate Leu-pNA.Through analysis of the effects of varying reaction conditions on fluorescence intensi-ty,we optimized the fluorescent substrate enzyme activity assay to demonstrate favorable performance at a reaction temperature of 37℃,a pH of 9.0,a protein concentration of 800 nmol/L,and a reaction duration of 60 minutes.Leu-AMC exhibited significant and distinct responses at a 5 μmol/L substrate concentration,under varying substrate conditions.The fluo-rescent substrate assay demonstrated more significant intergroup differences than the chemical chromogenic substrate assay when various inhibitors were added.This study established a fluorescence-based enzyme activity assay for leucyl aminopeptidase from Echinococcus multilocularis by using Leu-AMC as the substrate;this method demonstrated a more significant intergroup difference and sensitivity than the chemical chromogenic substrate assay.

Ganoderic acid is a class of lanostane-type triterpenoids found in Ganoderma species, and is one of the most important pharmacologically active components in G. lucidum, exhibiting antioxidant, anti-neuropsychiatric, anti-tumor, and immune-enhancing properties. The content of ganoderic acid in G. lucidum is very low, and the traditional extraction process is complex, yielding minimal amounts at high cost. The biosynthetic pathway of G. lucidum triterpenoids(GLTs), including the synthesis of different structural forms of ganoderic acid from lanosterol, as well as the molecular regulatory mechanisms involving key regulatory enzyme genes and their functions, are not yet fully understood. With the continuous development of synthetic biology technologies, there has been a deeper understanding of the biosynthesis and metabolic regulation pathways of ganoderic acid and its derivatives at the molecular level. Research has explored the key regulatory enzyme genes related to ganoderic acid biosynthesis and their functions. Moreover, through the optimization of synthetic biology and culture conditions, large-scale production and preparation of GLTs at the cellular level have been achieved. This paper reviews and analyzes the latest research progress on the biosynthesis pathways and metabolic regulation of GLTs, focusing on the configuration of ganoderic acid and its derivatives, the biosynthetic pathways, key enzyme genes, transcription factors related to ganoderic acid biosynthesis, signal transduction mechanisms, and factors affecting triterpenoid biotransformation. This review is expected to provide a theoretical basis and technical reference for improving the efficient production of triterpenoid pharmacological components and the exploitation and utilization of G. lucidum resources.
Triterpenes/chemistry* ; Reishi/chemistry* ; Biosynthetic Pathways ; Lanosterol

Objective To explore the factors that influence major adverse cardiovascular events(MACE)in atrial fibrillation(AF)patients undergoing radiofrequency ablation(RFA),as well as to compare the prognosis of patients with advanced AF to that of the general population.Methods We prospectively recruited AF patients who underwent RFA treatment at Peking University Third Hospital between January 2021 and March 2023.General patient data were collected through the hospital's inpatient system,and MACE were tracked through outpatient visits and telephone follow-ups.Patients were categorized into three age groups:Group 1(under 65 years),Group 2(65 to 75 years),and Group 3(over 75 years).In this study,MACE was defined as include cardiovascular death,all-cause death,readmission for heart failure,acute coronary syndrome(ACS),grade 2 or higher bleeding and stroke.Results A total of 431 patients were included in this study,with an average age of(66.17±12.22)years.Among these patients,259 were male(60.09％),and the mean of CHA2DS2-VASc score was(1.79±1.30).The median follow-up period was 16.0(11.3,21.3)months,during which 28(6.50％)patients experienced MACE,with ACS and stroke being the most common events.Variables were selected using LASSO regression,and a LASSO-Cox regression model was constructed.Age(HR 1.06,95％CI 1.02-1.10,P=0.006)and hypertrophic cardiomyopathy(HR 3.70,95％CI 1.27-8.68,P=0.008)were identified as independent predictors of MACE after RFA for AF.Subgroup analysis revealed that patients under 65 had significantly better prognoses compared to older AF patients(P=0.030 compared with group 2;P=0.021 compared with group 3).Conclusions Age and hypertrophic cardiomyopathy are independent risk factors for MACE in AF patients undergoing RFA.The prognosis for younger patients is better than that for older patients,while the prognosis for advanced patients is comparable to that of patients aged 65-75 years.

Objective To analyze the ultrasonographic features of secondary squamous cell carcinoma of the thyroid(SSCC-T)and evaluate the diagnostic value of ultrasound.Methods A retrospective analysis was conducted on clinical and ultrasonographic data from 12 patients with pathologically confirmed SSCC-T treated at Beijing Friendship Hospital,Capital Medical University between January 2016 and January 2025.Evaluated parameters included lesion size,echogenicity,edge,vascularity,calcification,and cervical lymph node metastasis.Descriptive statistical analysis was performed to analyze the ultrasonographic features of SSCC-T,and Fisher's exact test was conducted to analyze the correlation between different ultrasound classifications and thyroid dysfunction.Results The 12 patients showed the following ultrasound classifications:nodular type(50.0%,6/12),diffuse type(33.3%,4/12),and mixed type(16.7%,2/12).All diffuse-type patients exhibited a characteristic cord-like hypoechoic pattern.Cervical lymph node metastasis was observed in all the patients,with 75.0%(9/12)showing lymph nodes >2 cm in maximum diameter.Thyroid dysfunction occurred in 66.7%(8/12)of patients,including 2 patients with dynamic shifts from hyperthyroidism to hypothyroidism.Diffuse and mixed types were associated with hypothyroidism(P=0.038).Conclusions SSCC-T demonstrates specific ultrasonographic features,particularly the cord-like hypoechoic pattern in the diffuse type.For patients with squamous cell carcinoma,regular ultrasound examinations of the thyroid and cervical lymph nodes combined with changes in thyroid function are conducive to the timely detection of thyroid metastasis of squamous cell carcinoma.
Humans ; Retrospective Studies ; Ultrasonography ; Carcinoma, Squamous Cell/diagnostic imaging* ; Thyroid Neoplasms/diagnostic imaging* ; Lymphatic Metastasis ; Male ; Female ; Middle Aged ; Thyroid Gland/diagnostic imaging* ; Adult

Objective To investigate the efficacy and safety of domestic RECO flow restoration device in endovascular treatment of acute ischemic stroke(AIS)patients in a real-world setting.Methods From January 2019 to June 2021,consecutive patients with acute intracranial vessel occlusion treated with RECO device from 8 stroke centers in Jiangsu Province were analyzed retrospectively.Baseline and clinical data were collected,including age,gender,baseline modified Rankin scale(mRS)scores,National Institutes of Health stroke scale(NIHSS)score on admission,trial of Org 10172 in acute stroke treatment(TOAST)classification,responsible location of occlusion,wake-up stroke,and medical history(hypertension,diabetes mellitus,hyperlipidemia,atrial fibrillation,coronary heart disease,peripheral arterial disease),smoking,and alcohol consumption.Furthermore,surgical parameters were collected,including time indicators(intervals between symptom onset,admission,puncture and recanalization),preoperative intravenous thrombolysis,general anesthesia,combination of aspiration,number of passes,first pass effect(modified thrombolysis in cerebral infarction[mTICI]grade 3 achieved with s single stent-based thrombectomy),rescue therapy(including thrombectomy with other stents,intra-arterial urokinase thrombolysis,intra-arterial application of tirofiban,emergency angioplasty[stent implantation,balloon dilatation,balloon dilatation combined with stent implantation]),balloon guided catheter and NIHSS score at discharge.The effectiveness indicators included immediate successful recanalization after the operation(mTICI grade ≥ 2b),complete recanalization(mTICI grade 3),and a good prognosis at 90 d after the operation(mRS score ≤2).The safety indicators included symptomatic intracranial hemorrhage(sICH)within 24 h after the operation,device-related intraoperative complications(such as thrombus escape,iatrogenic dissection,bleeding at the surgical site),and all-cause mortality within 90 days after the operation.All patients were divided into the anterior circulation occlusion group and the posterior circulation occlusion group according to the location of the responsible occluded vessel,and the effectiveness and safety indexes were analyzed.Results A total of 366 patients with AIS caused by intracranial vessel occlusion who underwent mechanical thrombectomy with the domestic RECO thrombectomy stent were included.The average age was(69±11)years,with 54 patients(14.8％)over 80 years old and 221 patients(60.4％)male.There were 296 patients in the anterior circulation occlusion group and 70 patients in the posterior circulation occlusion group.(1)Immediate successful recanalization(mTICI grade≥2b)was achieved in 337 patients(92.1％),and complete recanalization was achieved in 282 patients(77.0％)immediately after the operation.The average number of thrombectomies was(1.9±1.0)times,and 141 patients(38.5％)achieved recanalization on the first attempt.9 patients(2.5％)used other stents during the operation,and 65 patients(17.8％)underwent emergency angioplasty.The incidence of sICH within 24 h after the operation was 10.9％(40/366),the good prognosis rate at 90 d after the operation was 53.6％(196/366),and the all-cause mortality rate at 90 d after the operation was 23.0％(84/366).The incidence of device-related intraoperative complications was 3.6％(13/366).The median time from arterial puncture to recanalization was 75(52,110)min,and the median time from onset to recanalization was 370(280,488)min.(2)The good prognosis rates at 90 d after the operation in the anterior and posterior circulation occlusion groups were 55.1％(163/296)and 47.1％(33/70),respectively.The immediate successful recanalization rates were 93.2％(276/296)and 87.1％(61/70),respectively.The immediate complete recanalization rates were 79.1％(234/296)and 68.6％(48/70),respectively.The incidence of sICH within 24h after the operation was 12.2％(36/296)and 5.7％(4/70),respectively.The incidence of device-related intraoperative complications was 3.7％(11/296)and 2.9％(2/70),respectively.The all-cause mortality rate within 90 d after the operation was 21.6％(64/296)and 28.6％(20/70),respectively.There were no statistically significant differences between the two groups in the above aspects(all P＞0.05).Conclusions The application of the domestic RECO thrombectomy stent in the treatment of AIS caused by intracranial vessel occlusion can effectively and safely achieve vascular recanalization.The results of this study still need to be further verified by prospective controlled studies.

Objective To explore the factors that influence major adverse cardiovascular events(MACE)in atrial fibrillation(AF)patients undergoing radiofrequency ablation(RFA),as well as to compare the prognosis of patients with advanced AF to that of the general population.Methods We prospectively recruited AF patients who underwent RFA treatment at Peking University Third Hospital between January 2021 and March 2023.General patient data were collected through the hospital's inpatient system,and MACE were tracked through outpatient visits and telephone follow-ups.Patients were categorized into three age groups:Group 1(under 65 years),Group 2(65 to 75 years),and Group 3(over 75 years).In this study,MACE was defined as include cardiovascular death,all-cause death,readmission for heart failure,acute coronary syndrome(ACS),grade 2 or higher bleeding and stroke.Results A total of 431 patients were included in this study,with an average age of(66.17±12.22)years.Among these patients,259 were male(60.09％),and the mean of CHA2DS2-VASc score was(1.79±1.30).The median follow-up period was 16.0(11.3,21.3)months,during which 28(6.50％)patients experienced MACE,with ACS and stroke being the most common events.Variables were selected using LASSO regression,and a LASSO-Cox regression model was constructed.Age(HR 1.06,95％CI 1.02-1.10,P=0.006)and hypertrophic cardiomyopathy(HR 3.70,95％CI 1.27-8.68,P=0.008)were identified as independent predictors of MACE after RFA for AF.Subgroup analysis revealed that patients under 65 had significantly better prognoses compared to older AF patients(P=0.030 compared with group 2;P=0.021 compared with group 3).Conclusions Age and hypertrophic cardiomyopathy are independent risk factors for MACE in AF patients undergoing RFA.The prognosis for younger patients is better than that for older patients,while the prognosis for advanced patients is comparable to that of patients aged 65-75 years.

This study was aimed at developing an in vitro fluorescent substrate assay for the activity of leucyl aminopeptid-ase(LAP)from Echinococcus multilocularis and comparing it with the chemical chromogenic substrate enzyme activity assay.Through the establishment of reaction conditions for the fluorescent substrate-based in vitro enzyme activity assay,we com-pared the differences between the fluorescent substrate L-Leucine-7-amido-4-methylocoumarin(Leu-AMC)and the chemical chromogenic substrate L-Leucine-4-nitroanilide(Leu-pNA)through molecular docking,inhibition rates,and precision measures.Molecular docking revealed that the fluorescent substrate Leu-AMC had higher affinity for the protein than the chemical chromogenic substrate Leu-pNA.Through analysis of the effects of varying reaction conditions on fluorescence intensi-ty,we optimized the fluorescent substrate enzyme activity assay to demonstrate favorable performance at a reaction temperature of 37℃,a pH of 9.0,a protein concentration of 800 nmol/L,and a reaction duration of 60 minutes.Leu-AMC exhibited significant and distinct responses at a 5 μmol/L substrate concentration,under varying substrate conditions.The fluo-rescent substrate assay demonstrated more significant intergroup differences than the chemical chromogenic substrate assay when various inhibitors were added.This study established a fluorescence-based enzyme activity assay for leucyl aminopeptidase from Echinococcus multilocularis by using Leu-AMC as the substrate;this method demonstrated a more significant intergroup difference and sensitivity than the chemical chromogenic substrate assay.

Objective:To study the effects and mechanisms of juglone on the proliferation and apoptosis of acute myeloid leukemia(AML)cells.Methods:Juglone and AML targets were collected from public databases,and the intersecting target clusters were taken for functional enrichment analysis to explore the potential mechanism of juglone in the treatment of AML.Then wet experiments were performed to verify.AML cell lines including KG-1a,MV-411,THP-1 and MOLM-13 were treated with different concentrations of juglone for 24 h.MTT assay was used to detect cell viability and determine the IC50,and the most sensitive cell line was screened for subsequent experiments.Flow cytometry was used to detect the apoptosis of cells treated with different concentrations of juglone.Western blot was performed to check the expression of relevant proteins.Results:Eleven targets were obtained as potential targets for juglone in the treatment of AML,and the top ten significantly enriched pathways were intrinsic pathway of apoptosis,programmed cell death,cytochrome c-mediated apoptotic response,apoptosis,apoptotic factor-mediated response,regulated necrosis,cytokine signaling in immune system,signaling by interleukins,oncogene induced senescence,and signal transduction.The cell viability of KG-1a,MV-411,THP-1 and MOLM-13 was decreased with increasing juglone concentration after 24 h of juglone treatment(r=-0.992,-0.886,-0.956,-0.910).Among them,MOLM-13 was the most sensitive to juglone.The results of flow cytometry showed that the apoptosis rate of MOLM-13 tended to significantly increase with the increasing concentration of juglone(r=0.99).At the same time point,p-RIPK1/RIPK1,p-RIPK3/RIPK3,and p-MLKL/MLK were decreased in each juglone concentration group compared with control group.Conclusion:Juglone inhibits the viability of KG-1a,MV-411,THP-1 and MOLM-13 cells,and induces apoptosis of MOLM-13 cells,the mechanism of which may be related to the inhibition of RIPK1/RIPK3/MLKL signaling pathway.

Objective To investigate the efficacy and safety of domestic RECO flow restoration device in endovascular treatment of acute ischemic stroke(AIS)patients in a real-world setting.Methods From January 2019 to June 2021,consecutive patients with acute intracranial vessel occlusion treated with RECO device from 8 stroke centers in Jiangsu Province were analyzed retrospectively.Baseline and clinical data were collected,including age,gender,baseline modified Rankin scale(mRS)scores,National Institutes of Health stroke scale(NIHSS)score on admission,trial of Org 10172 in acute stroke treatment(TOAST)classification,responsible location of occlusion,wake-up stroke,and medical history(hypertension,diabetes mellitus,hyperlipidemia,atrial fibrillation,coronary heart disease,peripheral arterial disease),smoking,and alcohol consumption.Furthermore,surgical parameters were collected,including time indicators(intervals between symptom onset,admission,puncture and recanalization),preoperative intravenous thrombolysis,general anesthesia,combination of aspiration,number of passes,first pass effect(modified thrombolysis in cerebral infarction[mTICI]grade 3 achieved with s single stent-based thrombectomy),rescue therapy(including thrombectomy with other stents,intra-arterial urokinase thrombolysis,intra-arterial application of tirofiban,emergency angioplasty[stent implantation,balloon dilatation,balloon dilatation combined with stent implantation]),balloon guided catheter and NIHSS score at discharge.The effectiveness indicators included immediate successful recanalization after the operation(mTICI grade ≥ 2b),complete recanalization(mTICI grade 3),and a good prognosis at 90 d after the operation(mRS score ≤2).The safety indicators included symptomatic intracranial hemorrhage(sICH)within 24 h after the operation,device-related intraoperative complications(such as thrombus escape,iatrogenic dissection,bleeding at the surgical site),and all-cause mortality within 90 days after the operation.All patients were divided into the anterior circulation occlusion group and the posterior circulation occlusion group according to the location of the responsible occluded vessel,and the effectiveness and safety indexes were analyzed.Results A total of 366 patients with AIS caused by intracranial vessel occlusion who underwent mechanical thrombectomy with the domestic RECO thrombectomy stent were included.The average age was(69±11)years,with 54 patients(14.8％)over 80 years old and 221 patients(60.4％)male.There were 296 patients in the anterior circulation occlusion group and 70 patients in the posterior circulation occlusion group.(1)Immediate successful recanalization(mTICI grade≥2b)was achieved in 337 patients(92.1％),and complete recanalization was achieved in 282 patients(77.0％)immediately after the operation.The average number of thrombectomies was(1.9±1.0)times,and 141 patients(38.5％)achieved recanalization on the first attempt.9 patients(2.5％)used other stents during the operation,and 65 patients(17.8％)underwent emergency angioplasty.The incidence of sICH within 24 h after the operation was 10.9％(40/366),the good prognosis rate at 90 d after the operation was 53.6％(196/366),and the all-cause mortality rate at 90 d after the operation was 23.0％(84/366).The incidence of device-related intraoperative complications was 3.6％(13/366).The median time from arterial puncture to recanalization was 75(52,110)min,and the median time from onset to recanalization was 370(280,488)min.(2)The good prognosis rates at 90 d after the operation in the anterior and posterior circulation occlusion groups were 55.1％(163/296)and 47.1％(33/70),respectively.The immediate successful recanalization rates were 93.2％(276/296)and 87.1％(61/70),respectively.The immediate complete recanalization rates were 79.1％(234/296)and 68.6％(48/70),respectively.The incidence of sICH within 24h after the operation was 12.2％(36/296)and 5.7％(4/70),respectively.The incidence of device-related intraoperative complications was 3.7％(11/296)and 2.9％(2/70),respectively.The all-cause mortality rate within 90 d after the operation was 21.6％(64/296)and 28.6％(20/70),respectively.There were no statistically significant differences between the two groups in the above aspects(all P＞0.05).Conclusions The application of the domestic RECO thrombectomy stent in the treatment of AIS caused by intracranial vessel occlusion can effectively and safely achieve vascular recanalization.The results of this study still need to be further verified by prospective controlled studies.

Objective To investigate the therapeutic effects and mechanism of enalapril maleate tablet on doxorubicin-induced heart failure rats based on mitogen-activated protein kinase(MAPK)signaling pathway.Methods Eleven of the 40 male SD rats were randomly selected as the normal group(equivalent to 0.9％NaCl),and the remaining 29 were prepared with intraperitoneal injection of 3 mg·kg-1·w-1 doxorubicin to prepare heart failure model.After successful modeling,they were randomly divided into model group(n=15 cases)and experimental group(n=14 cases).Experimental group was given 1.8 mg·kg-1·d-1 enalapril maleate suspension for gavage;normal and model groups were given the same amount of 0.9％NaCl by gavage.After 8 weeks,the rats were subjected to cardiac ultrasound,the left ventricular ejection fractions(LVEF)of each group were recorded,the serum myocardial injury index level was detected by enzyme-linked immunosorbent assay,and the expression levels of mRNA and protein related to the MAPK signaling pathway were detected by real-time quantitative polymerase chain reaction and Western blot.Results The LVEF values of control,model and experimental groups were(77.85±3.34％)％,(41.39±2.87％)％and(60.10±6.53％)％;serum brain natriuretic peptide contents were(219.30±10.59),(333.90±61.19)and(260.00±16.10)pg·mL-1;the relative expression levels of Mapk8ip2 were 1.00±0.01,2.60±0.12 and 2.00±0.08;the relative expression levels of Mapk8ip3 were 1.00±0.00,6.77±1.04 and 3.66±0.54;the relative expression levels of Mapk1 were 1.00±0.00,4.40±0.14 and 2.71±0.24;the relative expression levels of Mapk3 were 1.00±0.01,7.83±0.34 and 2.71±0.24;the relative expression levels of P38-MAPK were 1.00±0.05,1.14±0.02 and 1.02±0.03;the relative expression levels of extracellular regulated protein kinase 1/2 protein were 1.00±0.07,1.49±0.03 and 1.16±0.10;the relative expression levels of c-Jun N-terminal kinase 1/2 protein were 1.00±0.03,1.65±0.19 and 1.14±0.01,respectively.Compared with the model group,the differences of above indexes in the normal and experimental groups were statistically significant(all P＜0.01).Conclusion Enalapril maleate tablets have therapeutic effects on rats with heart failure,and the mechanism may be achieved by regulating the MAPK signaling pathway.