BACKGROUND:Overexpression ofα-synuclein can induce cell apoptosis.RNA interference(RNAi) may block specific gene function and cause gene silencing. OBJECTIVE:To construct a specific and effective RNAi plasmid for the...BACKGROUND:Overexpression ofα-synuclein can induce cell apoptosis.RNA interference(RNAi) may block specific gene function and cause gene silencing. OBJECTIVE:To construct a specific and effective RNAi plasmid for theα-synuclein gene and investigate if RNAi can block apoptosis in HEK293 cells,induced by overexpression of wild-typeα-synuclein. DESIGN,TIME AND SETTING:A contrast experiment based on genetically engineered cytobiology was performed at the State Key Lab of Medical Genetics of China,Xiangya Medical College of Central South University,between October 2004 and October 2008. MATERIALS:HEK293 cells and pBSHH1 plasmid were provided by the State Key Lab of Medical Genetics of China;OligDNA sequence by Sagon Bioengineering Company,Shanghai; Lipofectamine 2000 by Invitrogen,USA;α-synuclein monoclonal antibody,Hoechst 33258,and MTT; by Sigma,USA;Horseradish peroxidase-coupled goat anti-rat IgG by KPL,USA;FACSan flow cytometry by BD,USA. METHODS:Four target sites were used to construct hairpin RNA pBSHH1 vectors-pSYNi-1, pSYNi-2,pSYNi-3 and pSYNi-4-which were cloned in the pBSHH1 plasmid.HEK293 cells were transfected using Lipofectamine 2000.In addition,a non-transfect group and a negative plasmid transfect group were established.The cultured HEK293 cells were processed as follows: transfection of blank plasmid(blank control group),transfection ofα-synuclein-pEGFP and RNAi negative vector(negative control group),and transfection ofα-synuclein-pEGFP and pSYNi-1 (transfection group).Cells in all groups were transfected with Lipofectamine 2000 for 48 hours. MAIN OUTCOME MEASURES:Expression ofα-synuclein mRNA and protein were detected by RT-PCR and Western blot.Cell morphology was observed under an inverted fluorescence microscope;cell viability was measured using MTT method;and cell apoptosis was determined with Annexin V-PE flow cytometry. RESULTS:α-synuclein mRNA and protein expressions were significantly decreased in the pSYNi-1 group when compared with the non-transfect and negative plasmid transfect groups(P<0.05).The expressions were partially decreased in the pSYNi-2 group,but there was no significant difference in the pSYNi-3 and pSYNi-4 groups.Hoechst staining indicated that cell nuclei were enlarged in the negative control group,coloring was not uniform,and chromatin was accumulated and appeared spot-like.The nucleus coloring was uniform in the transfection group compared to negative control group.Cell viability in the negative control group was significantly lower than blank control group with cell apoptosis being significantly increased(P<0.05).In comparison with negative control group, cell viability was significantly increased in the transfection group and cell apoptosis was significantly decreased(P<0.05). CONCLUSION:pSYNi-1 can inhibitα-synuclein gene expression and block apoptosis of HEK293 cells induced by overexpression of wild-typeα-synuclein.展开更多
Sensor networks can consist of large number of sensors. Often, sensors networks use low cost units and thus a subject to malfunctions that can bring the system to inconsistent states. After deployment, the system can ...Sensor networks can consist of large number of sensors. Often, sensors networks use low cost units and thus a subject to malfunctions that can bring the system to inconsistent states. After deployment, the system can be situated in places that are hard to reach and therefore manual reboot operations are undesirable and even unfeasible. Therefore, it is imperative to consider the eventual recovery of arbitrary fault when designing sensor networks. Dijkstra’s algorithm is an important foundation of self-managing computer system and fault-tolerance computing system in distributed systems, since it allows a distributed system to recover from arbitrary starting state within a finite time. The arbitrary starting state ca model arbitrary failure (as long as the code segment stays correct).Another key advantage of Dijkstra’s asynchronous algorithm is that no global clock is needed. This project tests an implementation of Dijkstra’s algorithm using snapshotting techniques that we developed in an earlier work. These sensors can initiate from any state but they come into a consistent one after several cycles of running. We demonstrate the usefulness of our testing technique.展开更多
Focal epilepsy accounts for 60% of all forms of epilepsy, but the pathogenic mechanism is not well understood. In this study,three novel mutations in NPRL3(nitrogen permease regulator-like 3), c.937_945del, c.1514dup ...Focal epilepsy accounts for 60% of all forms of epilepsy, but the pathogenic mechanism is not well understood. In this study,three novel mutations in NPRL3(nitrogen permease regulator-like 3), c.937_945del, c.1514dup C and 6,706-bp genomic DNA(g DNA) deletion, were identified in three families with focal epilepsy by linkage analysis, whole exome sequencing(WES) and Sanger sequencing. NPRL3 protein is a component of the GATOR1 complex, a major inhibitor of m TOR signaling. These mutations led to truncation of the NPRL3 protein and hampered the binding between NPRL3 and DEPDC5, which is another component of the GATOR1 complex. Consequently, the mutant proteins enhanced m TOR signaling in cultured cells, possibly due to impaired inhibition of m TORC1 by GATOR1. Knockdown of nprl3 in Drosophila resulted in epilepsy-like behavior and abnormal synaptic development. Taken together, these findings expand the genotypic spectrum of NPRL3-associated focal epilepsy and provide further insight into how NPRL3 mutations lead to epilepsy.展开更多
Non-coding variants in the human genome significantly influence human traits and complex diseases via their regulation and modification effects.Hence,an increasing number of computational methods are developed to pred...Non-coding variants in the human genome significantly influence human traits and complex diseases via their regulation and modification effects.Hence,an increasing number of computational methods are developed to predict the effects of variants in human non-coding sequences.However,it is difficult for inexperienced users to select appropriate computational methods from dozens of available methods.To solve this issue,we assessed 12 performance metrics of 24 methods on four independent non-coding variant benchmark datasets:(1)rare germline variants from clinical relevant sequence variants(ClinVar),(2)rare somatic variants from Catalogue Of Somatic Mutations In Cancer(COSMIC),(3)common regulatory variants from curated expression quantitative trait locus(eQTL)data,and(4)disease-associated common variants from curated genomewide association studies(GWAS).All 24 tested methods performed differently under various conditions,indicating varying strengths and weaknesses under different scenarios.Importantly,the performance of existing methods was acceptable for rare germline variants from ClinVar with the area under the receiver operating characteristic curve(AUROC)of 0.4481–0.8033 and poor for rare somatic variants from COSMIC(AUROC=0.4984–0.7131),common regulatory variants from curated eQTL data(AUROC=0.4837–0.6472),and disease-associated common variants from curated GWAS(AUROC=0.4766–0.5188).We also compared the prediction performance of 24 methods for non-coding de novo mutations in autism spectrum disorder,and found that the combined annotation-dependent depletion(CADD)and context-dependent tolerance score(CDTS)methods showed better performance.Summarily,we assessed the performance of 24 computational methods under diverse scenarios,providing preliminary advice for proper tool selection and guiding the development of new techniques in interpreting non-coding variants.展开更多
In vitro studies have established the prevalent theory that the mitochondrial kinase PINK1 protects neurodegeneration by removing damaged mitochondria in Parkinson's disease(PD).However,difficulty in detecting end...In vitro studies have established the prevalent theory that the mitochondrial kinase PINK1 protects neurodegeneration by removing damaged mitochondria in Parkinson's disease(PD).However,difficulty in detecting endogenous PINK1 protein in rodent brains and cell lines has prevented the rigorous investigation of the in vivo role of PINK1.Here we report that PINK1 kinase form is selectively expressed in the human and monkey brains.CRISPR/Cas9-mediated deficiency of PINK1 causes similar neurodegeneration in the brains of fetal and adult monkeys as well as cultured monkey neurons without affecting mitochondrial protein expression and morphology.Importantly,PINK1 mutations in the primate brain and human cells reduce protein phosphorylation that is important for neuronal function and survival.Our findings suggest that PINK1 kinase activity rather than its mitochondrial function is essential for the neuronal survival in the primate brains and that its kinase dysfunction could be involved in the pathogenesis of PD.展开更多
Background:Amyotrophic lateral sclerosis(ALS)is a devastating progressive neurodegenerative disease that affects neurons in the central nervous system and the spinal cord.As in many other neurodegenerative disorders,t...Background:Amyotrophic lateral sclerosis(ALS)is a devastating progressive neurodegenerative disease that affects neurons in the central nervous system and the spinal cord.As in many other neurodegenerative disorders,the genetic risk factors and pathogenesis of ALS involve dysregulation of cytoskeleton and neuronal transport.Notably,sen-sory and motor neuron diseases such as hereditary sensory and autonomic neuropathy type 2(HSAN2)and spastic paraplegia 30(SPG30)share several causative genes with ALS,as well as having common clinical phenotypes.KIF1A encodes a kinesin 3 motor that transports presynaptic vesicle precursors(SVPs)and dense core vesicles and has been reported as a causative gene for HSAN2 and SPG30.Methods:Here,we analyzed whole-exome sequencing data from 941 patients with ALS to investigate the genetic association of KIF1A with ALS.Results:We identified rare damage variants(RDVs)in the KIF1A gene associated with ALS and delineated the clini-cal characteristics of ALS patients with KIF1A RDVs.Clinically,these patients tended to exhibit sensory disturbance.Interestingly,the majority of these variants are located at the C-terminal cargo-binding region of the KIF1A protein.Functional examination revealed that the ALS-associated KIF1A variants located in the C-terminal region preferentially enhanced the binding of SVPs containing RAB3A,VAMP2,and synaptophysin.Expression of several disease-related KIF1A mutants in cultured mouse cortical neurons led to enhanced colocalization of RAB3A or VAMP2 with the KIF1A motor.Conclusions:Our study highlighted the importance of KIF1A motor-mediated transport in the pathogenesis of ALS,indicating KIF1A as an important player in the oligogenic scenario of ALS.展开更多
Background:Genetic variants of dopaminergic transcription factor-encoding genes are suggested to be Parkinson’s disease(PD)risk factors;however,no comprehensive analyses of these genes in patients with PD have been u...Background:Genetic variants of dopaminergic transcription factor-encoding genes are suggested to be Parkinson’s disease(PD)risk factors;however,no comprehensive analyses of these genes in patients with PD have been undertaken.Therefore,we aimed to genetically analyze 16 dopaminergic transcription factor genes in Chinese patients with PD.Methods:Whole-exome sequencing(WES)was performed using a Chinese cohort comprising 1917 unrelated patients with familial or sporadic early-onset PD and 1652 controls.Additionally,whole-genome sequencing(WGS)was performed using another Chinese cohort comprising 1962 unrelated patients with sporadic late-onset PD and 1279 controls.Results:We detected 308 rare and 208 rare protein-altering variants in the WES and WGS cohorts,respectively.Gene-based association analyses of rare variants suggested that MSX1 is enriched in sporadic late-onset PD.However,the significance did not pass the Bonferroni correction.Meanwhile,72 and 1730 common variants were found in the WES and WGS cohorts,respectively.Unfortunately,single-variant logistic association analyses did not identify significant associations between common variants and PD.Conclusions:Variants of 16 typical dopaminergic transcription factors might not be major genetic risk factors for PD in Chinese patients.However,we highlight the complexity of PD and the need for extensive research elucidating its etiology.展开更多
基金the National "863" High-Technology Research and Development Program Grant,No. 2004AA2270402002BA711A07+3 种基金the National "Tenth-Five Year" Science and Technology Program,No. 2004BA720A03the National Natural Science Foundation of China,No. 30370515the Natural Science Foundation of Hainan Province,No. 806119807080
文摘BACKGROUND:Overexpression ofα-synuclein can induce cell apoptosis.RNA interference(RNAi) may block specific gene function and cause gene silencing. OBJECTIVE:To construct a specific and effective RNAi plasmid for theα-synuclein gene and investigate if RNAi can block apoptosis in HEK293 cells,induced by overexpression of wild-typeα-synuclein. DESIGN,TIME AND SETTING:A contrast experiment based on genetically engineered cytobiology was performed at the State Key Lab of Medical Genetics of China,Xiangya Medical College of Central South University,between October 2004 and October 2008. MATERIALS:HEK293 cells and pBSHH1 plasmid were provided by the State Key Lab of Medical Genetics of China;OligDNA sequence by Sagon Bioengineering Company,Shanghai; Lipofectamine 2000 by Invitrogen,USA;α-synuclein monoclonal antibody,Hoechst 33258,and MTT; by Sigma,USA;Horseradish peroxidase-coupled goat anti-rat IgG by KPL,USA;FACSan flow cytometry by BD,USA. METHODS:Four target sites were used to construct hairpin RNA pBSHH1 vectors-pSYNi-1, pSYNi-2,pSYNi-3 and pSYNi-4-which were cloned in the pBSHH1 plasmid.HEK293 cells were transfected using Lipofectamine 2000.In addition,a non-transfect group and a negative plasmid transfect group were established.The cultured HEK293 cells were processed as follows: transfection of blank plasmid(blank control group),transfection ofα-synuclein-pEGFP and RNAi negative vector(negative control group),and transfection ofα-synuclein-pEGFP and pSYNi-1 (transfection group).Cells in all groups were transfected with Lipofectamine 2000 for 48 hours. MAIN OUTCOME MEASURES:Expression ofα-synuclein mRNA and protein were detected by RT-PCR and Western blot.Cell morphology was observed under an inverted fluorescence microscope;cell viability was measured using MTT method;and cell apoptosis was determined with Annexin V-PE flow cytometry. RESULTS:α-synuclein mRNA and protein expressions were significantly decreased in the pSYNi-1 group when compared with the non-transfect and negative plasmid transfect groups(P<0.05).The expressions were partially decreased in the pSYNi-2 group,but there was no significant difference in the pSYNi-3 and pSYNi-4 groups.Hoechst staining indicated that cell nuclei were enlarged in the negative control group,coloring was not uniform,and chromatin was accumulated and appeared spot-like.The nucleus coloring was uniform in the transfection group compared to negative control group.Cell viability in the negative control group was significantly lower than blank control group with cell apoptosis being significantly increased(P<0.05).In comparison with negative control group, cell viability was significantly increased in the transfection group and cell apoptosis was significantly decreased(P<0.05). CONCLUSION:pSYNi-1 can inhibitα-synuclein gene expression and block apoptosis of HEK293 cells induced by overexpression of wild-typeα-synuclein.
文摘Sensor networks can consist of large number of sensors. Often, sensors networks use low cost units and thus a subject to malfunctions that can bring the system to inconsistent states. After deployment, the system can be situated in places that are hard to reach and therefore manual reboot operations are undesirable and even unfeasible. Therefore, it is imperative to consider the eventual recovery of arbitrary fault when designing sensor networks. Dijkstra’s algorithm is an important foundation of self-managing computer system and fault-tolerance computing system in distributed systems, since it allows a distributed system to recover from arbitrary starting state within a finite time. The arbitrary starting state ca model arbitrary failure (as long as the code segment stays correct).Another key advantage of Dijkstra’s asynchronous algorithm is that no global clock is needed. This project tests an implementation of Dijkstra’s algorithm using snapshotting techniques that we developed in an earlier work. These sensors can initiate from any state but they come into a consistent one after several cycles of running. We demonstrate the usefulness of our testing technique.
基金supported by the National Natural Science Foundation of China (32270663, 31871262, U20A20355,32022035)Shanghai Municipal Science and Technology Major Project(2018SHZDZX05)the Ministry of Science and Technology of China STI2030-Major Projects (2021ZD0203202)。
文摘Focal epilepsy accounts for 60% of all forms of epilepsy, but the pathogenic mechanism is not well understood. In this study,three novel mutations in NPRL3(nitrogen permease regulator-like 3), c.937_945del, c.1514dup C and 6,706-bp genomic DNA(g DNA) deletion, were identified in three families with focal epilepsy by linkage analysis, whole exome sequencing(WES) and Sanger sequencing. NPRL3 protein is a component of the GATOR1 complex, a major inhibitor of m TOR signaling. These mutations led to truncation of the NPRL3 protein and hampered the binding between NPRL3 and DEPDC5, which is another component of the GATOR1 complex. Consequently, the mutant proteins enhanced m TOR signaling in cultured cells, possibly due to impaired inhibition of m TORC1 by GATOR1. Knockdown of nprl3 in Drosophila resulted in epilepsy-like behavior and abnormal synaptic development. Taken together, these findings expand the genotypic spectrum of NPRL3-associated focal epilepsy and provide further insight into how NPRL3 mutations lead to epilepsy.
基金supported by the National Natural Science Foundation of China(Grant No.81801133 to JL)the Young Elite Scientist Sponsorship Program by China Association for Science and Technology(Grant No.2018QNRC001 to JL)+2 种基金the Innovation-Driven Project of Central South University,China(Grant No.20180033040004 to JL)the Natural Science Foundation for Young Scientists of Hunan Province,China(Grant No.2019JJ50974 to GZ)the Natural Science Foundation of Hunan Province for outstanding Young Scholars,China(Grant No.2020JJ3059 to JL).
文摘Non-coding variants in the human genome significantly influence human traits and complex diseases via their regulation and modification effects.Hence,an increasing number of computational methods are developed to predict the effects of variants in human non-coding sequences.However,it is difficult for inexperienced users to select appropriate computational methods from dozens of available methods.To solve this issue,we assessed 12 performance metrics of 24 methods on four independent non-coding variant benchmark datasets:(1)rare germline variants from clinical relevant sequence variants(ClinVar),(2)rare somatic variants from Catalogue Of Somatic Mutations In Cancer(COSMIC),(3)common regulatory variants from curated expression quantitative trait locus(eQTL)data,and(4)disease-associated common variants from curated genomewide association studies(GWAS).All 24 tested methods performed differently under various conditions,indicating varying strengths and weaknesses under different scenarios.Importantly,the performance of existing methods was acceptable for rare germline variants from ClinVar with the area under the receiver operating characteristic curve(AUROC)of 0.4481–0.8033 and poor for rare somatic variants from COSMIC(AUROC=0.4984–0.7131),common regulatory variants from curated eQTL data(AUROC=0.4837–0.6472),and disease-associated common variants from curated GWAS(AUROC=0.4766–0.5188).We also compared the prediction performance of 24 methods for non-coding de novo mutations in autism spectrum disorder,and found that the combined annotation-dependent depletion(CADD)and context-dependent tolerance score(CDTS)methods showed better performance.Summarily,we assessed the performance of 24 computational methods under diverse scenarios,providing preliminary advice for proper tool selection and guiding the development of new techniques in interpreting non-coding variants.
文摘In vitro studies have established the prevalent theory that the mitochondrial kinase PINK1 protects neurodegeneration by removing damaged mitochondria in Parkinson's disease(PD).However,difficulty in detecting endogenous PINK1 protein in rodent brains and cell lines has prevented the rigorous investigation of the in vivo role of PINK1.Here we report that PINK1 kinase form is selectively expressed in the human and monkey brains.CRISPR/Cas9-mediated deficiency of PINK1 causes similar neurodegeneration in the brains of fetal and adult monkeys as well as cultured monkey neurons without affecting mitochondrial protein expression and morphology.Importantly,PINK1 mutations in the primate brain and human cells reduce protein phosphorylation that is important for neuronal function and survival.Our findings suggest that PINK1 kinase activity rather than its mitochondrial function is essential for the neuronal survival in the primate brains and that its kinase dysfunction could be involved in the pathogenesis of PD.
基金the National Key R&D Program of China(2021YFA0805200)the National Major Projects in Brain Science and Brain-like Research(2021ZD0201803 to J.W.)+7 种基金the National Natural Science Foundation of China(82171431,81671120,81300981 to J.W.,31872778 and 82171506 to Z.H.)the National Key Research and Development Program of China(#2018YFC1312003 to J.W.)the Natural Science Fund for Distinguished Young Scholars of Hunan Province,China(2020JJ2057 to J.W.)the Project Program of National Clinical Research Center for Geriatric Disorders at Xiangya Hospital(2020LNJJ13 to J.W.)Key Research and Development Programs from Hunan Province(2021DK2001 to Z.H.)the Innovative Team Program from Hunan Province(2019RS1010)the Innovation-driven Team Project from Central South University(2020CX016)the Discipline Innovative Engineering Plan(111 Program)of China(B13036).Z.H.is supported by the Hunan Hundred Talents Program for Young Outstanding Scientists.
文摘Background:Amyotrophic lateral sclerosis(ALS)is a devastating progressive neurodegenerative disease that affects neurons in the central nervous system and the spinal cord.As in many other neurodegenerative disorders,the genetic risk factors and pathogenesis of ALS involve dysregulation of cytoskeleton and neuronal transport.Notably,sen-sory and motor neuron diseases such as hereditary sensory and autonomic neuropathy type 2(HSAN2)and spastic paraplegia 30(SPG30)share several causative genes with ALS,as well as having common clinical phenotypes.KIF1A encodes a kinesin 3 motor that transports presynaptic vesicle precursors(SVPs)and dense core vesicles and has been reported as a causative gene for HSAN2 and SPG30.Methods:Here,we analyzed whole-exome sequencing data from 941 patients with ALS to investigate the genetic association of KIF1A with ALS.Results:We identified rare damage variants(RDVs)in the KIF1A gene associated with ALS and delineated the clini-cal characteristics of ALS patients with KIF1A RDVs.Clinically,these patients tended to exhibit sensory disturbance.Interestingly,the majority of these variants are located at the C-terminal cargo-binding region of the KIF1A protein.Functional examination revealed that the ALS-associated KIF1A variants located in the C-terminal region preferentially enhanced the binding of SVPs containing RAB3A,VAMP2,and synaptophysin.Expression of several disease-related KIF1A mutants in cultured mouse cortical neurons led to enhanced colocalization of RAB3A or VAMP2 with the KIF1A motor.Conclusions:Our study highlighted the importance of KIF1A motor-mediated transport in the pathogenesis of ALS,indicating KIF1A as an important player in the oligogenic scenario of ALS.
基金supported by grants from the National Natural Science Foundation of China(Nos.82071437,U20A20355,and 82101342)the Hunan Innovative Province Construction Project(No.2019SK2335)+2 种基金the Natural Science Foundations of Hunan Province(No.2021JJ31115)the National Key Research and Development Program of China(Nos.2016YFC1306000 and 2021YFC2502100)the Project Program of National Clinical Research Center for Geriatric Disorders(Xiangya Hospital)(No.2021KFJJ10)
文摘Background:Genetic variants of dopaminergic transcription factor-encoding genes are suggested to be Parkinson’s disease(PD)risk factors;however,no comprehensive analyses of these genes in patients with PD have been undertaken.Therefore,we aimed to genetically analyze 16 dopaminergic transcription factor genes in Chinese patients with PD.Methods:Whole-exome sequencing(WES)was performed using a Chinese cohort comprising 1917 unrelated patients with familial or sporadic early-onset PD and 1652 controls.Additionally,whole-genome sequencing(WGS)was performed using another Chinese cohort comprising 1962 unrelated patients with sporadic late-onset PD and 1279 controls.Results:We detected 308 rare and 208 rare protein-altering variants in the WES and WGS cohorts,respectively.Gene-based association analyses of rare variants suggested that MSX1 is enriched in sporadic late-onset PD.However,the significance did not pass the Bonferroni correction.Meanwhile,72 and 1730 common variants were found in the WES and WGS cohorts,respectively.Unfortunately,single-variant logistic association analyses did not identify significant associations between common variants and PD.Conclusions:Variants of 16 typical dopaminergic transcription factors might not be major genetic risk factors for PD in Chinese patients.However,we highlight the complexity of PD and the need for extensive research elucidating its etiology.