Background:This study aims to identify distinct cellular subtypes within brain tissue using single-cell transcriptomic analysis,focusing on specific biomarkers that differentiate cell types and the effects of traditio...Background:This study aims to identify distinct cellular subtypes within brain tissue using single-cell transcriptomic analysis,focusing on specific biomarkers that differentiate cell types and the effects of traditional and exercise therapy.Methods:Four samples were analyzed:older control(OC),older exercise(OE),younger control(YC),and younger exercise(YE).Single-cell RNA sequencing was used to distinguish cellular subtypes through their biomarker profiles.Data visualization included violin and t-SNE plots to illustrate biomarker expression across cell clusters such as oligodendrocytes,microglia,and astrocytes.Additionally,BV2 cells were exposed to amyloid-beta fragments to simulate Alzheimer’s disease,assessing the impact of exercise-induced cellular responses.Results:Distinct cellular subtypes were identified:oligodendrocytes(MBP,St18),microglia(Dock8),and astrocytes(Aqp4,Gpc5).Sample OE was predominantly oligodendrocytes,while YE had more astrocytes,inhibitory neurons,and Canal-Retzius cells.YC showed a significant presence of Olfm3+ganglion neurons.ZEB1 gene knockout revealed changes in SMAD family gene expression,which regulate ferroptosis.Oxidative stress levels were also evaluated.Conclusion:This profiling enhances our understanding of brain cellular functions and interactions,potentially informing targeted therapies in neurological research.Exercise may influence brain cell immune responses and cell death pathways by regulating specific gene expressions,offering new insights for treating neuroinflammation and degeneration.展开更多
Background:Bladder cancer prognosis remains suboptimal despite advancements in research.Current molecular subtyping methods are resource-intensive,highlighting the need for efficient,cost-effective approaches to predi...Background:Bladder cancer prognosis remains suboptimal despite advancements in research.Current molecular subtyping methods are resource-intensive,highlighting the need for efficient,cost-effective approaches to predict BCa molecular subtypes.Method:We developed a predictive model for BCa molecular subtypes using machine learning(ML)and pathomics derived from Hematoxylin-Eosin stained pathological slides.A cohort of 353 patients from TCGA was employed,and image features were extracted for analysis.Pathomic signatures were constructed using the LASSO Cox regression algorithm,and a pathomic-clinical nomogram was developed and validated in training and testing cohorts.Results:Seventy distinct image features were identified from 150 pathomic signatures.The model demonstrated robust predictive ability,with AUCs of 0.833 and 0.822 in the training and validation cohorts,respectively.The addition of pathomic score,N stage,and M stage improved the model’s discrimination,achieving AUCs of 0.877 and 0.794 in the training and validation cohorts.Limitations include the lack of an external validation cohort.Conclusion:Our ML-based pathomics model shows promise in predicting BCa molecular subtypes and has the potential to enhance prognosis prediction and inform treatment strategies,marking a significant step towards precision medicine for BCa.展开更多
Objective:Circadian rhythm disruption(CRD)is a risk factor that correlates with poor prognosis across multiple tumor types,including hepatocellular carcinoma(HCC).However,its mechanism remains unclear.This study aimed...Objective:Circadian rhythm disruption(CRD)is a risk factor that correlates with poor prognosis across multiple tumor types,including hepatocellular carcinoma(HCC).However,its mechanism remains unclear.This study aimed to define HCC subtypes based on CRD and explore their individual heterogeneity.Methods:To quantify CRD,the HCC CRD score(HCCcrds)was developed.Using machine learning algorithms,we identified CRD module genes and defined CRD-related HCC subtypes in The Cancer Genome Atlas liver HCC cohort(n=369),and the robustness of this method was validated.Furthermore,we used bioinformatics tools to investigate the cellular heterogeneity across these CRD subtypes.Results:We defined three distinct HCC subtypes that exhibit significant heterogeneity in prognosis.The CRD-related subtype with high HCCcrds was significantly correlated with worse prognosis,higher pathological grade,and advanced clinical stages,while the CRD-related subtype with low HCCcrds had better clinical outcomes.We also identified novel biomarkers for each subtype,such as nicotinamide nmethyltransferase and myristoylated alanine-rich protein kinase C substrate-like 1.Conclusion:We classify the HCC patients into three distinct groups based on circadian rhythm and identify their specific biomarkers.Within these groups greater HCCcrds was associated with worse prognosis.This approach has the potential to improve prediction of an individual’s prognosis,guide precision treatments,and assist clinical decision making for HCC patients.展开更多
Objectives:Triple-negative breast cancer(TNBC)presents a major treatment challenge due to its aggressive behavior.The dysfunction of the Golgi apparatus(GA)contributes to the development of various cancers.This study ...Objectives:Triple-negative breast cancer(TNBC)presents a major treatment challenge due to its aggressive behavior.The dysfunction of the Golgi apparatus(GA)contributes to the development of various cancers.This study aimed to utilize GA-related genes(GARGs)to forecast the prognosis and immune profile of TNBC.Methods:The data were downloaded from The Cancer Genome Atlas(TCGA)database,including 175 TNBC and 99 healthy samples.The differentially expressed GARGs(DEGARGs)were analyzed using the TCGA biolinks package.The patients with TNBC were classified into two clusters utilizing the ConsensusClusterPlus package according to prognosis-related DEGARGs,followed by comparing the differences in prognosis and immune infiltration between the two clusters.Next,LASSO and stepwise Cox regression were applied to establish a GARGs signature to forecast the TNBC prognosis.The association of the GARGs signature with immune infiltrates and drug sensitivity was further explored.Results:In total,430 DEGARGs were identified between TNBC and healthy samples,among which 20 were related to TNBC prognosis.Two GARG-related molecular clusters associated with different survival times and immune heterogeneity were identified.A risk model for TNBC was established based on six GARGs,and the high-risk(HR)group exhibited a poor prognosis.The HR group demonstrated a distinctly high M2 macrophage infiltration and low M1 macrophage infiltration,which contributed to an immunosuppressive tumor microenvironment and thus led to poor prognosis of the HR group.Immune dysfunction scores and programmed cell death ligand 1(PD-L1)expression were substantially elevated in the HR group.The HR group showed increased sensitivity to anticancer drugs,such as cisplatin.Conclusion:Our findings suggest that GARGs are involved in the pathogenesis of TNBC and provide new insights into prognostic prediction.The identified clusters and GARGs signatures have the potential to guide individualized therapy.展开更多
Sonic Hedgehog Medulloblastoma(SHH-MB)is one of the four primary molecular subgroups of Medulloblastoma.It is estimated to be responsible for nearly one-third of allMB cases.Using transcriptomic and DNA methylation pr...Sonic Hedgehog Medulloblastoma(SHH-MB)is one of the four primary molecular subgroups of Medulloblastoma.It is estimated to be responsible for nearly one-third of allMB cases.Using transcriptomic and DNA methylation profiling techniques,new developments in this field determined four molecular subtypes for SHH-MB.SHH-MB subtypes show distinct DNAmethylation patterns that allow their discrimination fromoverlapping subtypes and predict clinical outcomes.Class overlapping occurs when two or more classes share common features,making it difficult to distinguish them as separate.Using the DNA methylation dataset,a novel classification technique is presented to address the issue of overlapping SHH-MBsubtypes.Penalizedmultinomial regression(PMR),Tomek links(TL),and singular value decomposition(SVD)were all smoothly integrated into a single framework.SVD and group lasso improve computational efficiency,address the problem of high-dimensional datasets,and clarify class distinctions by removing redundant or irrelevant features that might lead to class overlap.As a method to eliminate the issues of decision boundary overlap and class imbalance in the classification task,TL enhances dataset balance and increases the clarity of decision boundaries through the elimination of overlapping samples.Using fivefold cross-validation,our proposed method(TL-SVDPMR)achieved a remarkable overall accuracy of almost 95%in the classification of SHH-MB molecular subtypes.The results demonstrate the strong performance of the proposed classification model among the various SHH-MB subtypes given a high average of the area under the curve(AUC)values.Additionally,the statistical significance test indicates that TL-SVDPMR is more accurate than both SVM and random forest algorithms in classifying the overlapping SHH-MB subtypes,highlighting its importance for precision medicine applications.Our findings emphasized the success of combining SVD,TL,and PMRtechniques to improve the classification performance for biomedical applications with many features and overlapping subtypes.展开更多
1文献来源CHEN H,DENG C,GAO J,et al.Integrative spatial analysis reveals tumor heterogeneity and immune colony niche related to clinical outcomes in small cell lung cancer[J].Cancer Cell,2025,43(3):519−536.e5.doi:10.10...1文献来源CHEN H,DENG C,GAO J,et al.Integrative spatial analysis reveals tumor heterogeneity and immune colony niche related to clinical outcomes in small cell lung cancer[J].Cancer Cell,2025,43(3):519−536.e5.doi:10.1016/j.ccell.2025.01.012.展开更多
[Objective] The study aimed to investigate the genetic variation characters of entire sequences between two H9N2 subtype avian influenza virus strains and other reference strains.[Method] The entire sequences of 8 gen...[Objective] The study aimed to investigate the genetic variation characters of entire sequences between two H9N2 subtype avian influenza virus strains and other reference strains.[Method] The entire sequences of 8 genes were obtained by using RT-PCR,and these sequences were analyzed with that of six H9N2 subtype avian influenza isolates in homology comparison and genetic evolution relation.[Result] The results showed that the nucleotide sequence of entire gene of the strain shared 91.1%-95.4% homology with other seven reference strains,and PG08 shared the highest homology 91.3% with C/BJ/1/94;ZD06 shared the highest homology 92.3% with D/HK/Y280/97.HA cleavage sites of two H9N2 subtype avian influenza virus isolated strains were PARSSR/GLF,typical of mildly pathogenic avian influenza virus.[Conclusion] Phylogenetic tree for entire gene of eight strains showed that the genetic relationship was the closest between ZD06 and C/Pak/2/99 strains,which belonged to the Eurasian lineage;PG08 shared the highest homology 91.3% with ZD06,it may be the product of gene rearrangements of other sub-lines.展开更多
In order to compare the potential selectivity of R-（-）-DM-phencynonate hydrochloride with its racemate （±）-DM- phencynonate hydrochloride on acetylcholine muscarinic receptor subtypes, the five human acetylch...In order to compare the potential selectivity of R-（-）-DM-phencynonate hydrochloride with its racemate （±）-DM- phencynonate hydrochloride on acetylcholine muscarinic receptor subtypes, the five human acetylcholine muscarinic receptor subtypes （M1- M5） （CHO-hml-5R） were cloned and expressed in Chinese hamster ovary （CHO-K1） cell line. The specific mRNAs of the five acetylcholine muscarinic receptor subtypes were detected by the reverse transcription-polymerase chain reaction （RT-PCR） method, demonstrating the definite expression of muscarinic receptor subtype genes （CHO-hml-5R）. The affinity and saturability of different muscarinic receptor subtypes to [^3H] N-methylscopolamine （[^3H]-NMS） were obtained by radioligand binding assay. Equilibrium binding assay revealed that the maximum binding capacity of [^3H]-NMS （Bmax value） to CHO-hml-5R were 40.22±3.23, 24.53±4.11, 29.65±2.65, 25.41±2.46, 32.78±4.81 pmol/mg·protein, respectively. Kd values of [^3H]-NMS to muscarinic receptors M1 to M5 were 0.97±0.22, 1.16±0.14, 0.99±0.06, 0.56±0.08, 1.12±0.06 nM, respectively. R-（-）-DM- phencynonate hydrochloride was found to block the M4 receptor with a much higher potency （pD2 = 7.48） than those displayed on M1 （pD2 = 6.20）, M2 （pD2 = 5.99）, M3 （pD2 = 5.99） and M5 （pD2 = 6.70） subtypes. However, for （±）-DM-phencynonate hydrochloride, no significant subtype receptor selectivity was found. Both （±）-DM- and R-（-）-DM-phencynonate hydrochloride showed allosteric effects on muscarinic receptors, the Hill coefficient （nH） of five receptor subtypes was less than 1, respectively. The results revealed that R-（-）-DM-phencynonate hydrochloride showed selectivity torwards M4 subtype, and there were allosteric effects for both R-（-）-DM-phencynonate hydrochloride and （±）-DM-phencynonate hydrochloride on muscarinic receptors.展开更多
[ Objective] The study aimed to lay a foundation for the further studies on function mechanism of NS1 protein in the interspecies transmission of waterfowl influenza virus. [Method] Using the serologic assay and the s...[ Objective] The study aimed to lay a foundation for the further studies on function mechanism of NS1 protein in the interspecies transmission of waterfowl influenza virus. [Method] Using the serologic assay and the specific RT-PCR method, some strains of H9 subtype waterfowl influenza virus were isolated from the 12 to 20 day-old muscovy duck flocks without any clinical symptoms in different areas of Guangdong Province. Four of these strains, including A/duck/ZQ/303/2007（H9N2） （A3 for short）, A/Duck/FJ/301/2007 （H9N2） （C1 for short）, A/Duck/NH/306/2007（H9N2） （ D6 for short）, A/duck/SS/402/2007（H9N2） （ E2 for short）, and a strain named A/duck/ZC/2007（H9N2） （L1 for short） from a muscovy duck died of avian influenza virus （AIV）, were used for NSl gene cloning and sequencing. Subsequently, the obtained NSl gene sequences were compared with other NS1 sequences registered in GenBank, and the phylogenetic analysis was also conducted. [Result] When compared with the H9N2 AIV NS1 sequences in GenBank, the NSl genes of the four AIV strains A3, C1, 136 and E2 displayed homologies ranging from 99% to 100% at nucleotide level, and 95% to 100% at amino acid level; while the NSl gene of L1 strain displayed homology ranging from 94% to 97% at nucleotide level, and 93% to 98% at amino acid level. The phylogenetic tree demonstrated that A3, C1, D6 and E2 were highly resemblant, and L1 was closest to AY66473 （chicken, 2003）. By comparison with the NS1 gene sequences of L1, AF523514 （duck）, AY664743 （chicken） and EF155262.1 （quail） using DNAstar, A3, C1, D6 and E.2 presented nucleotide variations at site 21 （ R→Q）, 70, 71 （ KE→EG）, 86 （ A→S）, 124 （V→M） and 225 （ S→N）, and amino acid variations at site 21,70, 71 and 86 in dsRNA- dependent protein kinase （PKR） binding domain of NSl gene, which induced the evident variations of antigenic determinant and surface proba- bility plot of NS1 protein. [ Conclusion] This study suggested that the amino acid sequence variation in PKR binding domain of NS1 protein had something to do with the virus pathogenicity.展开更多
文摘Background:This study aims to identify distinct cellular subtypes within brain tissue using single-cell transcriptomic analysis,focusing on specific biomarkers that differentiate cell types and the effects of traditional and exercise therapy.Methods:Four samples were analyzed:older control(OC),older exercise(OE),younger control(YC),and younger exercise(YE).Single-cell RNA sequencing was used to distinguish cellular subtypes through their biomarker profiles.Data visualization included violin and t-SNE plots to illustrate biomarker expression across cell clusters such as oligodendrocytes,microglia,and astrocytes.Additionally,BV2 cells were exposed to amyloid-beta fragments to simulate Alzheimer’s disease,assessing the impact of exercise-induced cellular responses.Results:Distinct cellular subtypes were identified:oligodendrocytes(MBP,St18),microglia(Dock8),and astrocytes(Aqp4,Gpc5).Sample OE was predominantly oligodendrocytes,while YE had more astrocytes,inhibitory neurons,and Canal-Retzius cells.YC showed a significant presence of Olfm3+ganglion neurons.ZEB1 gene knockout revealed changes in SMAD family gene expression,which regulate ferroptosis.Oxidative stress levels were also evaluated.Conclusion:This profiling enhances our understanding of brain cellular functions and interactions,potentially informing targeted therapies in neurological research.Exercise may influence brain cell immune responses and cell death pathways by regulating specific gene expressions,offering new insights for treating neuroinflammation and degeneration.
基金supported by the Guangzhou Municipal Basic Research Program Jointly Funded by City,University,and Enterprise Special Project(2024A03J0907)the Natural Science Foundation of Guangdong Province(2024A1515013201)+1 种基金the National Natural Science Foundation of China(82203720,82203188,82002682,81972731,81773026,81972383)the Science and Technology Project of Zhongshan Municipality(No.2024B1032).
文摘Background:Bladder cancer prognosis remains suboptimal despite advancements in research.Current molecular subtyping methods are resource-intensive,highlighting the need for efficient,cost-effective approaches to predict BCa molecular subtypes.Method:We developed a predictive model for BCa molecular subtypes using machine learning(ML)and pathomics derived from Hematoxylin-Eosin stained pathological slides.A cohort of 353 patients from TCGA was employed,and image features were extracted for analysis.Pathomic signatures were constructed using the LASSO Cox regression algorithm,and a pathomic-clinical nomogram was developed and validated in training and testing cohorts.Results:Seventy distinct image features were identified from 150 pathomic signatures.The model demonstrated robust predictive ability,with AUCs of 0.833 and 0.822 in the training and validation cohorts,respectively.The addition of pathomic score,N stage,and M stage improved the model’s discrimination,achieving AUCs of 0.877 and 0.794 in the training and validation cohorts.Limitations include the lack of an external validation cohort.Conclusion:Our ML-based pathomics model shows promise in predicting BCa molecular subtypes and has the potential to enhance prognosis prediction and inform treatment strategies,marking a significant step towards precision medicine for BCa.
基金supported by Tianjian advanced biomedical laboratory key research and development projectHenan Province Natural Science Foundation(grant number:242300421283)+1 种基金Henan Province Science and Technology Research and Development(grant number:242102311176)Henan Province medical science and technology research project(grant number:SBGJ202403038)。
文摘Objective:Circadian rhythm disruption(CRD)is a risk factor that correlates with poor prognosis across multiple tumor types,including hepatocellular carcinoma(HCC).However,its mechanism remains unclear.This study aimed to define HCC subtypes based on CRD and explore their individual heterogeneity.Methods:To quantify CRD,the HCC CRD score(HCCcrds)was developed.Using machine learning algorithms,we identified CRD module genes and defined CRD-related HCC subtypes in The Cancer Genome Atlas liver HCC cohort(n=369),and the robustness of this method was validated.Furthermore,we used bioinformatics tools to investigate the cellular heterogeneity across these CRD subtypes.Results:We defined three distinct HCC subtypes that exhibit significant heterogeneity in prognosis.The CRD-related subtype with high HCCcrds was significantly correlated with worse prognosis,higher pathological grade,and advanced clinical stages,while the CRD-related subtype with low HCCcrds had better clinical outcomes.We also identified novel biomarkers for each subtype,such as nicotinamide nmethyltransferase and myristoylated alanine-rich protein kinase C substrate-like 1.Conclusion:We classify the HCC patients into three distinct groups based on circadian rhythm and identify their specific biomarkers.Within these groups greater HCCcrds was associated with worse prognosis.This approach has the potential to improve prediction of an individual’s prognosis,guide precision treatments,and assist clinical decision making for HCC patients.
文摘Objectives:Triple-negative breast cancer(TNBC)presents a major treatment challenge due to its aggressive behavior.The dysfunction of the Golgi apparatus(GA)contributes to the development of various cancers.This study aimed to utilize GA-related genes(GARGs)to forecast the prognosis and immune profile of TNBC.Methods:The data were downloaded from The Cancer Genome Atlas(TCGA)database,including 175 TNBC and 99 healthy samples.The differentially expressed GARGs(DEGARGs)were analyzed using the TCGA biolinks package.The patients with TNBC were classified into two clusters utilizing the ConsensusClusterPlus package according to prognosis-related DEGARGs,followed by comparing the differences in prognosis and immune infiltration between the two clusters.Next,LASSO and stepwise Cox regression were applied to establish a GARGs signature to forecast the TNBC prognosis.The association of the GARGs signature with immune infiltrates and drug sensitivity was further explored.Results:In total,430 DEGARGs were identified between TNBC and healthy samples,among which 20 were related to TNBC prognosis.Two GARG-related molecular clusters associated with different survival times and immune heterogeneity were identified.A risk model for TNBC was established based on six GARGs,and the high-risk(HR)group exhibited a poor prognosis.The HR group demonstrated a distinctly high M2 macrophage infiltration and low M1 macrophage infiltration,which contributed to an immunosuppressive tumor microenvironment and thus led to poor prognosis of the HR group.Immune dysfunction scores and programmed cell death ligand 1(PD-L1)expression were substantially elevated in the HR group.The HR group showed increased sensitivity to anticancer drugs,such as cisplatin.Conclusion:Our findings suggest that GARGs are involved in the pathogenesis of TNBC and provide new insights into prognostic prediction.The identified clusters and GARGs signatures have the potential to guide individualized therapy.
基金funded by the Deanship of Graduate Studies and Scientific Research at Jouf University under grant No.(DGSSR-2024-02-01137).
文摘Sonic Hedgehog Medulloblastoma(SHH-MB)is one of the four primary molecular subgroups of Medulloblastoma.It is estimated to be responsible for nearly one-third of allMB cases.Using transcriptomic and DNA methylation profiling techniques,new developments in this field determined four molecular subtypes for SHH-MB.SHH-MB subtypes show distinct DNAmethylation patterns that allow their discrimination fromoverlapping subtypes and predict clinical outcomes.Class overlapping occurs when two or more classes share common features,making it difficult to distinguish them as separate.Using the DNA methylation dataset,a novel classification technique is presented to address the issue of overlapping SHH-MBsubtypes.Penalizedmultinomial regression(PMR),Tomek links(TL),and singular value decomposition(SVD)were all smoothly integrated into a single framework.SVD and group lasso improve computational efficiency,address the problem of high-dimensional datasets,and clarify class distinctions by removing redundant or irrelevant features that might lead to class overlap.As a method to eliminate the issues of decision boundary overlap and class imbalance in the classification task,TL enhances dataset balance and increases the clarity of decision boundaries through the elimination of overlapping samples.Using fivefold cross-validation,our proposed method(TL-SVDPMR)achieved a remarkable overall accuracy of almost 95%in the classification of SHH-MB molecular subtypes.The results demonstrate the strong performance of the proposed classification model among the various SHH-MB subtypes given a high average of the area under the curve(AUC)values.Additionally,the statistical significance test indicates that TL-SVDPMR is more accurate than both SVM and random forest algorithms in classifying the overlapping SHH-MB subtypes,highlighting its importance for precision medicine applications.Our findings emphasized the success of combining SVD,TL,and PMRtechniques to improve the classification performance for biomedical applications with many features and overlapping subtypes.
文摘1文献来源CHEN H,DENG C,GAO J,et al.Integrative spatial analysis reveals tumor heterogeneity and immune colony niche related to clinical outcomes in small cell lung cancer[J].Cancer Cell,2025,43(3):519−536.e5.doi:10.1016/j.ccell.2025.01.012.
基金Supported by a Sub-project of 973 Program of China(2005CB523001)~~
文摘[Objective] The study aimed to investigate the genetic variation characters of entire sequences between two H9N2 subtype avian influenza virus strains and other reference strains.[Method] The entire sequences of 8 genes were obtained by using RT-PCR,and these sequences were analyzed with that of six H9N2 subtype avian influenza isolates in homology comparison and genetic evolution relation.[Result] The results showed that the nucleotide sequence of entire gene of the strain shared 91.1%-95.4% homology with other seven reference strains,and PG08 shared the highest homology 91.3% with C/BJ/1/94;ZD06 shared the highest homology 92.3% with D/HK/Y280/97.HA cleavage sites of two H9N2 subtype avian influenza virus isolated strains were PARSSR/GLF,typical of mildly pathogenic avian influenza virus.[Conclusion] Phylogenetic tree for entire gene of eight strains showed that the genetic relationship was the closest between ZD06 and C/Pak/2/99 strains,which belonged to the Eurasian lineage;PG08 shared the highest homology 91.3% with ZD06,it may be the product of gene rearrangements of other sub-lines.
基金National Natural Science Foundation of China (Grant No. 30672445)
文摘In order to compare the potential selectivity of R-（-）-DM-phencynonate hydrochloride with its racemate （±）-DM- phencynonate hydrochloride on acetylcholine muscarinic receptor subtypes, the five human acetylcholine muscarinic receptor subtypes （M1- M5） （CHO-hml-5R） were cloned and expressed in Chinese hamster ovary （CHO-K1） cell line. The specific mRNAs of the five acetylcholine muscarinic receptor subtypes were detected by the reverse transcription-polymerase chain reaction （RT-PCR） method, demonstrating the definite expression of muscarinic receptor subtype genes （CHO-hml-5R）. The affinity and saturability of different muscarinic receptor subtypes to [^3H] N-methylscopolamine （[^3H]-NMS） were obtained by radioligand binding assay. Equilibrium binding assay revealed that the maximum binding capacity of [^3H]-NMS （Bmax value） to CHO-hml-5R were 40.22±3.23, 24.53±4.11, 29.65±2.65, 25.41±2.46, 32.78±4.81 pmol/mg·protein, respectively. Kd values of [^3H]-NMS to muscarinic receptors M1 to M5 were 0.97±0.22, 1.16±0.14, 0.99±0.06, 0.56±0.08, 1.12±0.06 nM, respectively. R-（-）-DM- phencynonate hydrochloride was found to block the M4 receptor with a much higher potency （pD2 = 7.48） than those displayed on M1 （pD2 = 6.20）, M2 （pD2 = 5.99）, M3 （pD2 = 5.99） and M5 （pD2 = 6.70） subtypes. However, for （±）-DM-phencynonate hydrochloride, no significant subtype receptor selectivity was found. Both （±）-DM- and R-（-）-DM-phencynonate hydrochloride showed allosteric effects on muscarinic receptors, the Hill coefficient （nH） of five receptor subtypes was less than 1, respectively. The results revealed that R-（-）-DM-phencynonate hydrochloride showed selectivity torwards M4 subtype, and there were allosteric effects for both R-（-）-DM-phencynonate hydrochloride and （±）-DM-phencynonate hydrochloride on muscarinic receptors.
基金Supported by Key Specific Program for Science and Technology of Guangdong Province (2008B020700003 A2007A020400006)~~
文摘[ Objective] The study aimed to lay a foundation for the further studies on function mechanism of NS1 protein in the interspecies transmission of waterfowl influenza virus. [Method] Using the serologic assay and the specific RT-PCR method, some strains of H9 subtype waterfowl influenza virus were isolated from the 12 to 20 day-old muscovy duck flocks without any clinical symptoms in different areas of Guangdong Province. Four of these strains, including A/duck/ZQ/303/2007（H9N2） （A3 for short）, A/Duck/FJ/301/2007 （H9N2） （C1 for short）, A/Duck/NH/306/2007（H9N2） （ D6 for short）, A/duck/SS/402/2007（H9N2） （ E2 for short）, and a strain named A/duck/ZC/2007（H9N2） （L1 for short） from a muscovy duck died of avian influenza virus （AIV）, were used for NSl gene cloning and sequencing. Subsequently, the obtained NSl gene sequences were compared with other NS1 sequences registered in GenBank, and the phylogenetic analysis was also conducted. [Result] When compared with the H9N2 AIV NS1 sequences in GenBank, the NSl genes of the four AIV strains A3, C1, 136 and E2 displayed homologies ranging from 99% to 100% at nucleotide level, and 95% to 100% at amino acid level; while the NSl gene of L1 strain displayed homology ranging from 94% to 97% at nucleotide level, and 93% to 98% at amino acid level. The phylogenetic tree demonstrated that A3, C1, D6 and E2 were highly resemblant, and L1 was closest to AY66473 （chicken, 2003）. By comparison with the NS1 gene sequences of L1, AF523514 （duck）, AY664743 （chicken） and EF155262.1 （quail） using DNAstar, A3, C1, D6 and E.2 presented nucleotide variations at site 21 （ R→Q）, 70, 71 （ KE→EG）, 86 （ A→S）, 124 （V→M） and 225 （ S→N）, and amino acid variations at site 21,70, 71 and 86 in dsRNA- dependent protein kinase （PKR） binding domain of NSl gene, which induced the evident variations of antigenic determinant and surface proba- bility plot of NS1 protein. [ Conclusion] This study suggested that the amino acid sequence variation in PKR binding domain of NS1 protein had something to do with the virus pathogenicity.
