Programmed cell death protein 1(PD-1)and its ligand,programmed cell death 1 ligand 1(PD-L1),represent a pair of prototypical immune checkpoint that plays a critical role in tumor immune evasion.However,the development of targeted therapeutics against these two proteins is limited by their low levels and high glycosylation modifications in eukaryotic cells.In this study,we designed two func-tional but truncated variants of PD-1(PD-133-150)and PD-L1(PD-L1 19-239);and subcloned them into eukaryotic expression vectors using golden gate assembly technology.Using these vectors,we achieved high level yields of these two proteins in transiently-transfected HEK-293T cells.After one-step affinity purification,the yields of PD-133-150 and PD-L119 239 proteins reached 5 mg and 3 mg per liter of cell cul-ture medium,with over 95％purity.Using biolayer interferometry and flow cytometry analysis,we deter-mined the binding kinetics,equilibrium constants,and the cellular binding activities of these proteins.Compared with the PD-1 extracellular domain expressed in insect or E.coli cells,the PD-133-150 purified from HEK-293T cells has a 24-fold and a 50-fold increase in its binding affinity to PD-L1.In addition,the dissociation rate of the binding decreased to less than l/400th of the original rate.Thus,we speculate that N-glycosylation could modulate the PD-1/PD-L1 interactions.Together,we established an effective eukaryotic expression and purification platform for functional characterization of PD-133150/PD-L119239 in-teractions,thereby providing high-quality molecular tools for PD-1/PD-L1 antibody screening and im-mune checkpoint research.

AIM:To investigate the effects of macrophage galactose-type lectin 1(Mgl1)gene deletion on he-matopoietic stem/progenitor cells(HSPCs)under steady-state conditions and inflammation.METHODS:Mice were di-vided into a control group(wild-type)and an experimental group(Mgl1 gene-deleted).Flow cytometry was used to ana-lyze the proportions of various hematopoietic cell lineages in the peripheral blood and bone marrow of both groups,assess-ing the impact of Mgl1 gene deletion on steady-state hematopoiesis(n=3～4).Transplantation and colony-forming assays were utilized to evaluate the effects of Mgl1 gene deletion onthe repopulation capacity and colony-forming ability of HSPCs(n=5).The LPS-induced inflammation model was employed to examine the effects of Mgl1 gene deletion on the inflamma-tory response of HSPCs both in vitro and in vivo(n=5～8).Western blot and RT-qPCR were conducted to analyze the alter-ations in signaling pathways regulated by Mgl1 in the inflammatory response of HSPCs(n=3).RESULTS:(1)Mgl1 gene deletion had no significant effecton steady-state hematopoiesis(P>0.05).(2)Mgl1 gene deletion promoted inflam-mation-induced cell differentiation of HSPCs(P<0.01).(3)Mgl1 gene deletion accelerated the exhaustion of HSPCs un-der prolonged inflammatory conditions(P<0.01).(4)Mgl1 was found to regulate the inflammatory response of HSPCs via the NF-κB signaling pathway.CONCLUSION:Mgl1 gene deletion enhances the inflammatory response of HSPCs via the NF-κB signaling pathway.

Purpose To evaluate the predictive value of multifunctional parameters of single photon emission computed tomography gated myocardial perfusion imaging(SPECT G-MPI)for major adverse cardiovascular events(MACE)in chronic kidney disease(CKD)with abnormal stress myocardial perfusion.Materials and Methods A total of 99 patients diagnosed with CKD from June 2017 to March 2024 who underwent stress and rest G-MPI indicating abnormal myocardial perfusion in Beijing Anzhen Hospital,Capital Medical University.The American Heart Association 17-segment 5-point method and PHASE software were used to obtain the left ventricular myocardial perfusion,functional and synchronization parameters.According to the occurrence of MACE,the patients were divided into MACE group and non-MACE group.Cox regression was used to analyze the predictors related to MACE.The receiver operator characteristic curve was used to analyze the performance of predictors,the survival curves were obtained by the Kaplan-Meier method,Log-rank test was used to compare the differences in different groups.Results Finally,we enrolled 99 CKD patients with abnormal stress myocardial perfusion.35 patients(35.35%)developed MACE during the follow-up period.Cox regression analysis showed that stress phase bandwidth(SPBW)(HR=1.015,95%CI 1.002-1.028)and sum difference score(SDS)(HR=1.105,95%CI 1.008-1.211)were independent risk factors for predicting MACE(both P<0.05).The optimal cut-off value of SPBW and SDS for predicting MACE were 69° and 6 points,the area under the curve was 0.801 and 0.778,respectively.The incidence of MACE in the SPBW≥69° group and SDS≥6 points group was higher than that in SPBW<69° group and SDS<6 points group(66.6%vs.13.2%,53.3%vs.20.4%,both P<0.05).Conclusion SPECT G-MPI multifunctional parameters can be used to predict the prognosis of CKD patients with abnormal stress myocardial perfusion.SPBW and SDS are independent risk factors for MACE in these patients.

OBJECTIVE: The present study evaluated the effects of deep acupuncture at Weizhong acupoint (BL40) on bladder function and brain activity in a rat model of overactive bladder (OAB), and investigated the possible mechanisms around the acupuncture area that initiate the effects of acupuncture. METHODS: Adult female Sprague-Dawley rats were randomly divided into six groups, comprising a control group, model group, group treated with deep acupuncture at BL40, group treated with shallow acupuncture at BL40, group treated with acupuncture at non-acupoint next to BL40, and group treated with acupuncture at Xuanzhong (GB39). Urodynamic evaluation was used to observe the urination, and functional magnetic resonance imaging was used to observe the brain activation. The mechanism of acupuncture at BL40 in regulating bladder function was explored by toluidine blue staining and enzyme-linked immunosorbent assay, and the mechanism was verified by stabilizing mast cells (MCs) or blocking tibial nerve. RESULTS: Deep acupuncture at BL40 significantly increased the intercontraction interval in OAB rats and enhanced the mean amplitude of low frequency fluctuation of primary motor cortex (M1), periaquaductal gray matter (PAG), and pontine micturition center (PMC). It also increased the zero-lag functional connectivity between M1 and PAG and between PAG and PMC. Shallow acupuncture at BL40 and acupuncture at non-acupoint or GB39 had no effect on these indexes. Further studies suggested that deep acupuncture at BL40 increased the number and degranulation rate of MCs as well as the contents of 5-hydroxytryptamine, substance P, and histamine in the tissues around BL40. Blocking the tibial nerve by lidocaine injection or inhibiting MC degranulation by sodium cromoglycate injection obstructed the effects of acupuncture on restoring urinary function and modulating brain activation in OAB rats. CONCLUSION Deep acupuncture at BL40 may be more effective for inhibiting OAB by promoting degranulation of MCs around the acupoint and stimulating tibial nerve, thereby regulating the activation of the brain area that controls the lower urinary tract. Please cite this article as: Liu X, Zhang CY, Du XY, Li SS, Wang YQ, Zheng Y, Deng HZ, Fang XQ, Li JY, Wang ZQ, Xu SF, Mi YQ. Acupuncture at Weizhong (BL40) attenuates acetic acid-induced overactive bladder in rats by regulating brain neural activity through the modulation of mast cells and tibial nerves. J Integr Med. 2025; 23(1): 46-55.
Animals ; Urinary Bladder, Overactive/physiopathology* ; Mast Cells/physiology* ; Rats, Sprague-Dawley ; Female ; Acupuncture Therapy ; Acupuncture Points ; Rats ; Brain/physiopathology* ; Tibial Nerve/physiopathology* ; Acetic Acid ; Urinary Bladder/physiopathology*

Objective:To evaluate the clinical predictive value of SPECT myocardial perfusion imaging (MPI) in the occurrence of cardiac death in patients with chronic kidney disease (CKD).Methods:A retrospective follow-up was performed for 160 patients (109 males, 51 females; age: 68.5(61.0, 74.0) years) who underwent MPI in Beijing Anzhen Hospital, Capital Medical University between June 2017 and March 2024. The 17-segment 5-point method was used for image analysis to obtain the left ventricular myocardial perfusion and functional parameters. The patients were followed up for cardiac death, and divided into death group and survival group. Clinical data of those 2 groups were compare by χ2 test, the independent-sample t test or Mann-Whitney U test. Cox proportional hazards regression analysis was used to analyze the predictors related to cardiac death. The ROC curve was used to analyze the performance of predictors. Survival curves were obtained by the Kaplan-Meier method, and log-rank test was performed to compare the difference between 2 groups. Results:The follow-up time of 160 patients with CKD was 26.0(10.0, 46.5) months. Of 160 patients, 17 died and 143 survived. There were statistically significant differences in body mass index (BMI), previous myocardial infarction, previous revascularization, hypersensitive C-reactive protein (hs-CRP), positive MPI, left ventricular end-diastolic volume (EDV), end-systolic volume (ESV), ejection fraction (EF), summed stress score (SSS), summed rest score (SRS) and summed difference score (SDS) between the death group and the survival group ( χ2 values: 4.58-16.13, t values: -2.34, -3.97, Z values: from -2.81 to 5.02, all P<0.05). Multivariate Cox regression analysis showed that SSS (hazard ratio ( HR)=1.153, 95% CI: 1.062-1.252, P=0.001) and hs-CRP ( HR=1.031, 95% CI: 1.004-1.058, P=0.023) were independent risk factors for cardiac death in patients with CKD. The optimal cut-off value of SSS for predicting cardiac death in those patients was determined to be 8 with the AUC of 0.815, and the incidence of cardiac death in the SSS ≥8 group was significantly higher than that in the SSS<8 group (33.3%(12/36) vs 4.0%(5/124); χ2 = 25.44, P<0.001). Conclusion:MPI is an important imaging method for the evaluation of cardiac death in patients with CKD, SSS and hs-CRP are important risk factors in predicting cardiac death in those patients.

Purpose To evaluate the predictive value of multifunctional parameters of single photon emission computed tomography gated myocardial perfusion imaging(SPECT G-MPI)for major adverse cardiovascular events(MACE)in chronic kidney disease(CKD)with abnormal stress myocardial perfusion.Materials and Methods A total of 99 patients diagnosed with CKD from June 2017 to March 2024 who underwent stress and rest G-MPI indicating abnormal myocardial perfusion in Beijing Anzhen Hospital,Capital Medical University.The American Heart Association 17-segment 5-point method and PHASE software were used to obtain the left ventricular myocardial perfusion,functional and synchronization parameters.According to the occurrence of MACE,the patients were divided into MACE group and non-MACE group.Cox regression was used to analyze the predictors related to MACE.The receiver operator characteristic curve was used to analyze the performance of predictors,the survival curves were obtained by the Kaplan-Meier method,Log-rank test was used to compare the differences in different groups.Results Finally,we enrolled 99 CKD patients with abnormal stress myocardial perfusion.35 patients(35.35%)developed MACE during the follow-up period.Cox regression analysis showed that stress phase bandwidth(SPBW)(HR=1.015,95%CI 1.002-1.028)and sum difference score(SDS)(HR=1.105,95%CI 1.008-1.211)were independent risk factors for predicting MACE(both P<0.05).The optimal cut-off value of SPBW and SDS for predicting MACE were 69° and 6 points,the area under the curve was 0.801 and 0.778,respectively.The incidence of MACE in the SPBW≥69° group and SDS≥6 points group was higher than that in SPBW<69° group and SDS<6 points group(66.6%vs.13.2%,53.3%vs.20.4%,both P<0.05).Conclusion SPECT G-MPI multifunctional parameters can be used to predict the prognosis of CKD patients with abnormal stress myocardial perfusion.SPBW and SDS are independent risk factors for MACE in these patients.

Objective:To evaluate the clinical predictive value of SPECT myocardial perfusion imaging (MPI) in the occurrence of cardiac death in patients with chronic kidney disease (CKD).Methods:A retrospective follow-up was performed for 160 patients (109 males, 51 females; age: 68.5(61.0, 74.0) years) who underwent MPI in Beijing Anzhen Hospital, Capital Medical University between June 2017 and March 2024. The 17-segment 5-point method was used for image analysis to obtain the left ventricular myocardial perfusion and functional parameters. The patients were followed up for cardiac death, and divided into death group and survival group. Clinical data of those 2 groups were compare by χ2 test, the independent-sample t test or Mann-Whitney U test. Cox proportional hazards regression analysis was used to analyze the predictors related to cardiac death. The ROC curve was used to analyze the performance of predictors. Survival curves were obtained by the Kaplan-Meier method, and log-rank test was performed to compare the difference between 2 groups. Results:The follow-up time of 160 patients with CKD was 26.0(10.0, 46.5) months. Of 160 patients, 17 died and 143 survived. There were statistically significant differences in body mass index (BMI), previous myocardial infarction, previous revascularization, hypersensitive C-reactive protein (hs-CRP), positive MPI, left ventricular end-diastolic volume (EDV), end-systolic volume (ESV), ejection fraction (EF), summed stress score (SSS), summed rest score (SRS) and summed difference score (SDS) between the death group and the survival group ( χ2 values: 4.58-16.13, t values: -2.34, -3.97, Z values: from -2.81 to 5.02, all P<0.05). Multivariate Cox regression analysis showed that SSS (hazard ratio ( HR)=1.153, 95% CI: 1.062-1.252, P=0.001) and hs-CRP ( HR=1.031, 95% CI: 1.004-1.058, P=0.023) were independent risk factors for cardiac death in patients with CKD. The optimal cut-off value of SSS for predicting cardiac death in those patients was determined to be 8 with the AUC of 0.815, and the incidence of cardiac death in the SSS ≥8 group was significantly higher than that in the SSS<8 group (33.3%(12/36) vs 4.0%(5/124); χ2 = 25.44, P<0.001). Conclusion:MPI is an important imaging method for the evaluation of cardiac death in patients with CKD, SSS and hs-CRP are important risk factors in predicting cardiac death in those patients.

Programmed cell death protein 1(PD-1)and its ligand,programmed cell death 1 ligand 1(PD-L1),represent a pair of prototypical immune checkpoint that plays a critical role in tumor immune evasion.However,the development of targeted therapeutics against these two proteins is limited by their low levels and high glycosylation modifications in eukaryotic cells.In this study,we designed two func-tional but truncated variants of PD-1(PD-133-150)and PD-L1(PD-L1 19-239);and subcloned them into eukaryotic expression vectors using golden gate assembly technology.Using these vectors,we achieved high level yields of these two proteins in transiently-transfected HEK-293T cells.After one-step affinity purification,the yields of PD-133-150 and PD-L119 239 proteins reached 5 mg and 3 mg per liter of cell cul-ture medium,with over 95％purity.Using biolayer interferometry and flow cytometry analysis,we deter-mined the binding kinetics,equilibrium constants,and the cellular binding activities of these proteins.Compared with the PD-1 extracellular domain expressed in insect or E.coli cells,the PD-133-150 purified from HEK-293T cells has a 24-fold and a 50-fold increase in its binding affinity to PD-L1.In addition,the dissociation rate of the binding decreased to less than l/400th of the original rate.Thus,we speculate that N-glycosylation could modulate the PD-1/PD-L1 interactions.Together,we established an effective eukaryotic expression and purification platform for functional characterization of PD-133150/PD-L119239 in-teractions,thereby providing high-quality molecular tools for PD-1/PD-L1 antibody screening and im-mune checkpoint research.

AIM:To investigate the effects of macrophage galactose-type lectin 1(Mgl1)gene deletion on he-matopoietic stem/progenitor cells(HSPCs)under steady-state conditions and inflammation.METHODS:Mice were di-vided into a control group(wild-type)and an experimental group(Mgl1 gene-deleted).Flow cytometry was used to ana-lyze the proportions of various hematopoietic cell lineages in the peripheral blood and bone marrow of both groups,assess-ing the impact of Mgl1 gene deletion on steady-state hematopoiesis(n=3～4).Transplantation and colony-forming assays were utilized to evaluate the effects of Mgl1 gene deletion onthe repopulation capacity and colony-forming ability of HSPCs(n=5).The LPS-induced inflammation model was employed to examine the effects of Mgl1 gene deletion on the inflamma-tory response of HSPCs both in vitro and in vivo(n=5～8).Western blot and RT-qPCR were conducted to analyze the alter-ations in signaling pathways regulated by Mgl1 in the inflammatory response of HSPCs(n=3).RESULTS:(1)Mgl1 gene deletion had no significant effecton steady-state hematopoiesis(P>0.05).(2)Mgl1 gene deletion promoted inflam-mation-induced cell differentiation of HSPCs(P<0.01).(3)Mgl1 gene deletion accelerated the exhaustion of HSPCs un-der prolonged inflammatory conditions(P<0.01).(4)Mgl1 was found to regulate the inflammatory response of HSPCs via the NF-κB signaling pathway.CONCLUSION:Mgl1 gene deletion enhances the inflammatory response of HSPCs via the NF-κB signaling pathway.

Objective To observe the efficacy and safety of Morinda officinalis oligosaccharides in the continuation treatment of mild and moderate depression.Methods An open,single-arm,multi-center design was adopted in our study.Adult patients with mild and moderate depression who had received acute treatment of Morinda officinalis oligosaccharides were enrolled and continue to receive Morinda officinalis oligosaccharides capsules for 24 weeks,the dose remained unchanged during continuation treatment.The remission rate,recurrence rate,recurrence time,and the change from baseline to endpoint of Hamilton Depression Scale(HAMD),Hamilton Anxiety Scale(HAMA),Clinical Global Impression-Severity(CGI-S)and Arizona Sexual Experience Scale(ASEX)were evaluated.The incidence of treatment-related adverse events was reported.Results The scores of HAMD-17 at baseline and after treatment were 6.60±1.87 and 5.85±4.18,scores of HAMA were 6.36±3.02 and 4.93±3.09,scores of CGI-S were 1.49±0.56 and 1.29±0.81,scores of ASEX were 15.92±4.72 and 15.57±5.26,with significant difference(P＜0.05).After continuation treatment,the remission rate was 54.59％(202 cases/370 cases),and the recurrence rate was 6.49％(24 cases/370 cases),the recurrence time was(64.67±42.47)days.The incidence of treatment-related adverse events was 15.35％(64 cases/417 cases).Conclusion Morinda officinalis oligosaccharides capsules can be effectively used for the continuation treatment of mild and moderate depression,and are well tolerated and safe.