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.

Aim To evaluate the role of the glucose transporter protein 1(GLUT1)-dependent glycolytic in the attenuation of oxygen-glucose deprivation-reoxygen-ation(OGD/R)injury in HK-2 cells by dexmedetomi-dine(Dex).Methods C57/BL6 mice were random-ly divided into three groups(n=6),namely,sham operation group(Sham group),renal ischemia reper-fusion group(I/R group)and Dex group(I/R+Dex group).Serum creatinine(Cr)and urea nitrogen(BUN)were measured,while the levels of key glyco-lytic enzymes HK2,PFKFB3 and GLUT1 were meas-ured.HK-2 cells were cultured and randomised into seven groups(n=6),which was treated with OGD/R,overexpression or interference with GLUT1,Dex and glycolysis inhibitor 2-DG.CCK-8 and LDH activi-ty were used to detect cellular damage.Glycolysis lev-els were detected by lactate and ECAR.The inflamma-tory level was reflected by qRT-PCR for IL-6 and TNF-α.qRT-PCR and Western blot were performed to de-tect the levels of GLUT1,HK2,and PFKFB3.Results Dex significantly ameliorated kidney injury and HK-2 cell injury(P＜0.05).Dex inhibited the OGD/R-induced rise in lactate and extracellular acidification rate(ECAR),as evidenced by suppression of the ex-pression of GLUT1,HK2 and PFKFB3(P＜0.05).In vitro experiments showed that GLUT1 knockdown sig-nificantly improved OGD/R-induced cellular damage.Lactate,ECAR,glycolysis-related mRNAs and pro-teins were inhibited by GLUT1 knockdown(P＜0.05).Significantly,there were no significant differ-ences in above indexes after Dex treatment based on GLUT1 knockdown.Overexpression of GLUT1 abroga-ted the protective effects of Dex,while reversing the inhibitory effects of Dex on the expression of GLUT1,HK2,and PFKFB3(P＜0.05).Conclusions Dexmedetomidine attenuates OGD/R induced injury in HK-2 cells by inhibiting GLUT1-dependent glycolysis.

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.

Aim To evaluate the role of the glucose transporter protein 1(GLUT1)-dependent glycolytic in the attenuation of oxygen-glucose deprivation-reoxygen-ation(OGD/R)injury in HK-2 cells by dexmedetomi-dine(Dex).Methods C57/BL6 mice were random-ly divided into three groups(n=6),namely,sham operation group(Sham group),renal ischemia reper-fusion group(I/R group)and Dex group(I/R+Dex group).Serum creatinine(Cr)and urea nitrogen(BUN)were measured,while the levels of key glyco-lytic enzymes HK2,PFKFB3 and GLUT1 were meas-ured.HK-2 cells were cultured and randomised into seven groups(n=6),which was treated with OGD/R,overexpression or interference with GLUT1,Dex and glycolysis inhibitor 2-DG.CCK-8 and LDH activi-ty were used to detect cellular damage.Glycolysis lev-els were detected by lactate and ECAR.The inflamma-tory level was reflected by qRT-PCR for IL-6 and TNF-α.qRT-PCR and Western blot were performed to de-tect the levels of GLUT1,HK2,and PFKFB3.Results Dex significantly ameliorated kidney injury and HK-2 cell injury(P＜0.05).Dex inhibited the OGD/R-induced rise in lactate and extracellular acidification rate(ECAR),as evidenced by suppression of the ex-pression of GLUT1,HK2 and PFKFB3(P＜0.05).In vitro experiments showed that GLUT1 knockdown sig-nificantly improved OGD/R-induced cellular damage.Lactate,ECAR,glycolysis-related mRNAs and pro-teins were inhibited by GLUT1 knockdown(P＜0.05).Significantly,there were no significant differ-ences in above indexes after Dex treatment based on GLUT1 knockdown.Overexpression of GLUT1 abroga-ted the protective effects of Dex,while reversing the inhibitory effects of Dex on the expression of GLUT1,HK2,and PFKFB3(P＜0.05).Conclusions Dexmedetomidine attenuates OGD/R induced injury in HK-2 cells by inhibiting GLUT1-dependent glycolysis.

Objective To compare the effects of single antiplatelet therapy(SAPT)versus dual antiplatelet therapy(DAPT)on bleeding and ischemic events in patients undergoing transcatheter aortic valve replacement(TAVR)without long-term anticoagulation indications.Methods This randomized controlled trial included 90 post-TAVR patients without anticoagulation indications,who were allocated to the SAPT group(n=46,aspirin 100 mg/d)or DAPT group(n=44,aspirin 100 mg/d+clopidogrel 75 mg/d for 3 months,followed by aspirin monotherapy).Maximum aggregation rates of platelets induced by arachidonic acid(MARAA)and adenosine diphosphate(MARADP)were measured 1,3,6,and 12 months postoperatively.Bleeding and ischemic events were recorded during the follow-up visits.Results The SAPT group exhibited significantly higher MARAA and MARADP scores at 1 and 3 months,and higher MARAA scores at 6 months compared to the DAPT group(P<0.05).At the 12-month follow-up,the SAPT group had a significantly lower inci-dence of bleeding events compared to the DAPT group(13.0%vs.31.8%,P=0.043).No statistically significant difference was observed in ischemic events between the groups(15.2%vs.11.4%,P=0.759).Conclusion For TAVR patients without anticoagulation indica-tions,SAPT significantly reduced the 1-year bleeding risk compared to DAPT,without increasing ischemic events.These findings support the safety and efficacy of SAPT after TAVR.

OBJECTIVE To investigate the anti-inflammatory mechanisms of demethylzeylasteral(Dem)in ulcerative colitis(UC)and collagen-induced arthritis(CIA),focusing on its regulation of Th17 cell differentiation and associated signaling pathways.METHODS UC was induced in C57BL/6 mice using dextran sulfate sodium(DSS),and Dem was administered by gavage at low(1 mg·kg-1)or high(2 mg·kg-1)doses.Disease severity was assessed by body weight loss,colon length,and stool consisten-cy.Serum cytokines(TNF-α,IL-1β,IL-6)were quantified by ELISA,and Th17 cell ratio in mesenteric lymph nodes was deter-mined by flow cytometry.The anti-inflammatory efficacy of Dem was further validated using a CIA mouse model.The efficacy of Dem was further verified in the CIA model,and the expression of JAK2 and STAT3 was intervened by siRNA to investigate its mechanism of action in Th17 differentiation.RESULTS Dem-treated mice showed reduced weight loss and colon shortening,and decreases in ser-um TNF-α,IL-1β,and IL-6 levels(P<0.01)and Th17 cell proportion(P<0.01).Western blot and siRNA assays showed that Dem significantly inhibited the differentiation and activation of Th17 cells by suppressing the phosphorylation of the JAK2-STAT3 path-way.Dem also significantly alleviated arthritis symptoms and related markers in the CIA model,confirming its anti-inflammatory effects.CONCLUSION Dem improves UC and rheumatoid arthritis by downregulating the JAK2-STAT3 signaling pathway,inhibi-ting Th17 cell differentiation and pro-inflammatory cytokine expression,suggesting its potential therapeutic value in immune-related diseases.

Objective To compare the effects of single antiplatelet therapy(SAPT)versus dual antiplatelet therapy(DAPT)on bleeding and ischemic events in patients undergoing transcatheter aortic valve replacement(TAVR)without long-term anticoagulation indications.Methods This randomized controlled trial included 90 post-TAVR patients without anticoagulation indications,who were allocated to the SAPT group(n=46,aspirin 100 mg/d)or DAPT group(n=44,aspirin 100 mg/d+clopidogrel 75 mg/d for 3 months,followed by aspirin monotherapy).Maximum aggregation rates of platelets induced by arachidonic acid(MARAA)and adenosine diphosphate(MARADP)were measured 1,3,6,and 12 months postoperatively.Bleeding and ischemic events were recorded during the follow-up visits.Results The SAPT group exhibited significantly higher MARAA and MARADP scores at 1 and 3 months,and higher MARAA scores at 6 months compared to the DAPT group(P<0.05).At the 12-month follow-up,the SAPT group had a significantly lower inci-dence of bleeding events compared to the DAPT group(13.0%vs.31.8%,P=0.043).No statistically significant difference was observed in ischemic events between the groups(15.2%vs.11.4%,P=0.759).Conclusion For TAVR patients without anticoagulation indica-tions,SAPT significantly reduced the 1-year bleeding risk compared to DAPT,without increasing ischemic events.These findings support the safety and efficacy of SAPT after TAVR.

OBJECTIVE: To assess the independent and combined effects of air pollutants, meteorological factors, and greenspace exposure on new tuberculosis (TB) cases. METHODS: TB case data from Shanghai (2013-2018) were obtained from the Shanghai Center for Disease Control and Prevention. Environmental data on air pollutants, meteorological variables, and greenspace exposure were obtained from the National Tibetan Plateau Data Center. We employed a distributed-lag nonlinear model to assess the effects of these environmental factors on TB cases. RESULTS: Increased TB risk was linked to PM _2.5, PM ₁₀, and rainfall, whereas NO ₂, SO ₂, and air pressure were associated with a reduced risk. Specifically, the strongest cumulative effects occurred at various lags: PM _2.5 ( RR = 1.166, 95% CI: 1.026-1.325) at 0-19 weeks; PM ₁₀ ( RR = 1.167, 95% CI: 1.028-1.324) at 0-18 weeks; NO ₂ ( RR = 0.968, 95% CI: 0.938-0.999) at 0-1 weeks; SO ₂ ( RR = 0.945, 95% CI: 0.894-0.999) at 0-2 weeks; air pressure ( RR = 0.604, 95% CI: 0.447-0.816) at 0-8 weeks; and rainfall ( RR = 1.404, 95% CI: 1.076-1.833) at 0-22 weeks. Green space exposure did not significantly impact TB cases. Additionally, low temperatures amplified the effect of PM _2.5 on TB. CONCLUSION Exposure to PM _2.5, PM ₁₀, and rainfall increased the risk of TB, highlighting the need to address air pollutants for the prevention of TB in Shanghai.
China/epidemiology* ; Humans ; Air Pollutants/analysis* ; Tuberculosis/epidemiology* ; Incidence ; Meteorological Concepts ; Particulate Matter/adverse effects* ; Environmental Exposure ; Male ; Female ; Adult ; Air Pollution ; Middle Aged

OBJECTIVE To investigate the anti-inflammatory mechanisms of demethylzeylasteral(Dem)in ulcerative colitis(UC)and collagen-induced arthritis(CIA),focusing on its regulation of Th17 cell differentiation and associated signaling pathways.METHODS UC was induced in C57BL/6 mice using dextran sulfate sodium(DSS),and Dem was administered by gavage at low(1 mg·kg-1)or high(2 mg·kg-1)doses.Disease severity was assessed by body weight loss,colon length,and stool consisten-cy.Serum cytokines(TNF-α,IL-1β,IL-6)were quantified by ELISA,and Th17 cell ratio in mesenteric lymph nodes was deter-mined by flow cytometry.The anti-inflammatory efficacy of Dem was further validated using a CIA mouse model.The efficacy of Dem was further verified in the CIA model,and the expression of JAK2 and STAT3 was intervened by siRNA to investigate its mechanism of action in Th17 differentiation.RESULTS Dem-treated mice showed reduced weight loss and colon shortening,and decreases in ser-um TNF-α,IL-1β,and IL-6 levels(P<0.01)and Th17 cell proportion(P<0.01).Western blot and siRNA assays showed that Dem significantly inhibited the differentiation and activation of Th17 cells by suppressing the phosphorylation of the JAK2-STAT3 path-way.Dem also significantly alleviated arthritis symptoms and related markers in the CIA model,confirming its anti-inflammatory effects.CONCLUSION Dem improves UC and rheumatoid arthritis by downregulating the JAK2-STAT3 signaling pathway,inhibi-ting Th17 cell differentiation and pro-inflammatory cytokine expression,suggesting its potential therapeutic value in immune-related diseases.

Apurinic/apyrimidinic endonuclease 1(APE 1)is a multifunctional protein that plays important roles in DNA repair and regulation of gene expression.Because APE 1 is overexpressed in various cancers,it can serve as a cancer biomarker for aiding clinical diagnosis,guiding therapy,and monitoring prognosis.On this basis,a label-free fluorescent probe was designed based on the primer exchange reaction(PER)strategy for highly sensitive detection of APE 1 activity.In the absence of APE 1,the structure of catalytic hairpin(HP)was stable and could not form G-quadruplex.Therefore,the background fluorescence of this sensing system was very low due to the dissociation of thioflavin T(ThT).In the presence of APE 1,the apurinic/apyrimidinic(AP)site of HP was cleaved by APE 1 and a short nucleic acid fragment that acted as a primer to initiate PER was generated.After PER reaction,a large number of G-quadruplex were produced,which could specifically bind with ThT and resulted in significant increase of fluorescence signal.The combination of low background design of HP and PER amplification made this biosensor had high sensitivity with a detection limit(3σ)of 0.0008 U/mL.Furthermore,the primer sequence was directly generated by the cleavage of APE 1 without additional addition,which not only increased the specificity of the reaction,but also simplified the experiment procedure.Moreover,the use of label-free fluorescence signal reduced the cost of the experiment,and realized rapid detection of APE 1.Finally,this sensor was used to detect APE 1 in human serum samples with spiked recoveries of 91%-104%,proving great potential in study of biological enzyme.