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Genetic screens in mammalian cells commonly focus on loss-of-function approaches. To evaluate the phenotypic consequences of extra gene copies, we used bulk segregant analysis (BSA) of radiation hybrid (RH) cells. We constructed six pools of RH cells, each consisting of â¼2500 independent clones, and placed the pools under selection in media with or without paclitaxel. Low pass sequencing identified 859 growth loci, 38 paclitaxel loci, 62 interaction loci, and three loci for mitochondrial abundance at genome-wide significance. Resolution was measured as â¼30 kb, close to single-gene. Divergent properties were displayed by the RH-BSA growth genes compared to those from loss-of-function screens, refuting the balance hypothesis. In addition, enhanced retention of human centromeres in the RH pools suggests a new approach to functional dissection of these chromosomal elements. Pooled analysis of RH cells showed high power and resolution and should be a useful addition to the mammalian genetic toolkit.
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Processos de Crescimento Celular/genética , Mapeamento de Híbridos Radioativos/métodos , Animais , Centrômero , Cricetinae , DNA , Doença/genética , Loci Gênicos , Células HEK293 , Humanos , Mitocôndrias , Mycoplasma/isolamento & purificação , Paclitaxel/farmacologiaRESUMO
Cocaine self-administration is a complexly determined trait, with a substantial proportion of individual differences being determined by genetic variation. However, the relevant genetic variants that drive heritable differences in cocaine use remain undiscovered. Cocaine intravenous self-administration (IVSA) procedures in laboratory animals provide opportunities to prospectively investigate neurogenetic influences on the acquisition of voluntary cocaine use. Here, we provide information on cocaine (or saline-as a control) IVSA in 84 members of the hybrid mouse diversity panel (HMDP), an array of genetically distinct classical or recombinant inbred strains. We found cocaine IVSA to be substantially heritable in this population, with strain-level intake ranging for near 0 to >25 mg/kg/session. Though saline IVSA was also found to be heritable, a modest genetic correlation between cocaine and saline IVSA indicates that operant responding for the cocaine reinforcer was influenced, at least in part, by unique genetic variants. Genome-wide association studies (GWAS) of infusions earned in cocaine and saline groups revealed significant quantitative trait loci (QTL) on Chromosomes 3 and 14 for cocaine, but not saline, IVSA. Positional candidates were further prioritized through use of bulk RNA sequencing data that revealed genes with cis-eQTL and genetic correlation to number of infusions. Additionally, these data identify reference strains with extreme cocaine IVSA phenotypes, revealing them as polygenic models of risk and resilience to cocaine reinforcement. This work is part of an ongoing effort to characterize genetic variation that moderates cocaine IVSA that may, in turn, provide a more comprehensive understanding of cocaine risk genetics and neurobiology.
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Transtornos Relacionados ao Uso de Cocaína , Cocaína , Animais , Cocaína/farmacologia , Estudo de Associação Genômica Ampla , Camundongos , Fenótipo , AutoadministraçãoRESUMO
Feline leukemia virus (FeLV) is horizontally transmitted among cats and causes a variety of hematopoietic disorders. Five subgroups of FeLV, A to D and T, each with distinct receptor usages, have been described. Recently, we identified a new FeLV Env (TG35-2) gene from a pseudotyped virus that does not belong to any known subgroup. FeLV-A is the primary virus from which other subgroups have emerged via mutation or recombination of the subgroup A env gene. Retrovirus entry into cells is mediated by the interaction of envelope protein (Env) with specific cell surface receptors. Here, phenotypic screening of a human/hamster radiation hybrid panel identified SLC19A1, a feline reduced folate carrier (RFC) and potential receptor for TG35-2-phenotypic virus. RFC is a multipass transmembrane protein. Feline and human RFC cDNAs conferred susceptibility to TG35-2-pseudotyped virus when introduced into nonpermissive cells but did not render these cells permissive to other FeLV subgroups or feline endogenous retrovirus. Moreover, human cells with genomic deletion of RFC were nonpermissive for TG35-2-pseudotyped virus infection, but the introduction of feline and human cDNAs rendered them permissive. Mutation analysis of FeLV Env demonstrated that amino acid substitutions within variable region A altered the specificity of the Env-receptor interaction. We isolated and reconstructed the full-length infectious TG35-2-phenotypic provirus from a naturally FeLV-infected cat, from which the FeLV Env (TG35-2) gene was previously isolated, and compared the replication of the virus in hematopoietic cell lines with that of FeLV-A 61E by measuring the viral RNA copy numbers. These results provide a tool for further investigation of FeLV infectious disease.IMPORTANCE Feline leukemia virus (FeLV) is a member of the genus Gammaretrovirus, which causes malignant diseases in cats. The most prevalent FeLV among cats is FeLV subgroup A (FeLV-A), and specific binding of FeLV-A Env to its viral receptor, thiamine transporter feTHTR1, is the first step of infection. In infected cats, novel variants of FeLV with altered receptor specificity for viral entry have emerged by mutation or recombination of the env gene. A novel FeLV variant arose from a subtle mutation of FeLV-A Env, which altered the specific interaction of the virus with its receptor. RFC, a folate transporter, is a potential receptor for the novel FeLV variant. The perturbation of specific retrovirus-receptor interactions under selective pressure by the host results in the emergence of novel viruses.
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Genes env/genética , Vírus da Leucemia Felina/genética , Receptores Virais/genética , Proteína Carregadora de Folato Reduzido/genética , Proteínas do Envelope Viral/genética , Internalização do Vírus , Sequência de Aminoácidos , Animais , Gatos , Linhagem Celular , Cricetinae , Retrovirus Endógenos/metabolismo , Produtos do Gene env/genética , Células HeLa , Humanos , Vírus da Leucemia Felina/metabolismo , Leucemia Felina/virologia , Filogenia , Provírus , RNA Viral/genética , Receptores Virais/metabolismo , Proteína Carregadora de Folato Reduzido/classificação , Proteína Carregadora de Folato Reduzido/metabolismo , Alinhamento de Sequência , Replicação ViralRESUMO
OBJECTIVE: To evaluate the various methods and outcomes of post-traumatic partial auricle wound reconstruction, and to review the benefit to the patient's quality of life and their psychological improvement after the operation. METHODS: The prospective study included patients who suffered from post-traumatic partial auricular wounds. The defects were repaired using various techniques including simple local cutaneous advancement flaps, tube flaps, cartilage framework using conchal and costal cartilage with local skin flap cover. RESULTS: A total of 18 patients were included, with a male predominance (sex ratio: 7:2), mean age with standard deviation of 31.66 ±9.27 years. Causes included road traffic accident (RTA), assault, human and insect bite and avulsion injuries. Injuries were sustained in the upper third of the auricle (n=8); middle third (n=5), lower third (n=3), and upper two-thirds (n=2). Out of the 18 patients, wound were repaired using post auricular mastoid skin flap (n=7); local superior and inferior chondrocutaneous flap (n=3); costal cartilage as a cartilage framework (n=4), temporoparietal fascia used to cover the costal cartilage graft (n=1), and conchal cartilage as a cartilage framework (n=3). In our study 13/18 patients were 'highly satisfied' with the aesthetic outcome, 3/18 were 'moderately satisfied', and 2/18 were 'slightly satisfied'. None were dissatisfied by the postoperative result. In terms of objective assessment, patient outcome in two patients was graded 'good' while the outcome of remaining patients (n=16) was graded as 'excellent'. CONCLUSIONS: The use of skin flaps in the post-auricular region and the mastoid region associated with or without cartilage framework yields good cosmetic and functional result. The various techniques used for ear reconstruction yielded 100% satisfactory results in terms of functional outcome as well as boosting the confidence of patients.
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Orelha Externa/cirurgia , Satisfação do Paciente , Procedimentos de Cirurgia Plástica/métodos , Qualidade de Vida/psicologia , Retalhos Cirúrgicos/transplante , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Resultado do Tratamento , Adulto JovemRESUMO
To identify addiction genes, we evaluate intravenous self-administration of cocaine or saline in 84 inbred and recombinant inbred mouse strains over 10 days. We integrate the behavior data with brain RNA-seq data from 41 strains. The self-administration of cocaine and that of saline are genetically distinct. We maximize power to map loci for cocaine intake by using a linear mixed model to account for this longitudinal phenotype while correcting for population structure. A total of 15 unique significant loci are identified in the genome-wide association study. A transcriptome-wide association study highlights the Trpv2 ion channel as a key locus for cocaine self-administration as well as identifying 17 additional genes, including Arhgef26, Slc18b1, and Slco5a1. We find numerous instances where alternate splice site selection or RNA editing altered transcript abundance. Our work emphasizes the importance of Trpv2, an ionotropic cannabinoid receptor, for the response to cocaine.
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Transtornos Relacionados ao Uso de Cocaína , Cocaína , Camundongos , Animais , Cocaína/farmacologia , Estudo de Associação Genômica Ampla , Encéfalo , Administração Intravenosa , Camundongos Endogâmicos C57BLRESUMO
Exposure to pesticides in humans increases the risk of Parkinson's disease (PD), but the mechanisms remain poorly understood. To elucidate these pathways, we dosed C57BL/6J mice with a combination of the pesticides maneb and paraquat. Behavioral analysis revealed motor deficits consistent with PD. Single-cell RNA sequencing of substantia nigra pars compacta revealed both cell-type-specific genes and genes expressed differentially between pesticide and control, including Fam241b, Emx2os, Bivm, Gm1439, Prdm15, and Rai2. Neurons had the largest number of significant differentially expressed genes, but comparable numbers were found in astrocytes and less so in oligodendrocytes. In addition, network analysis revealed enrichment in functions related to the extracellular matrix. These findings emphasize the importance of support cells in pesticide-induced PD and refocus our attention away from neurons as the sole agent of this disorder.
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BACKGROUND: There is only a limited understanding of the relation between copy number and expression for mammalian genes. We fine mapped cis and trans regulatory loci due to copy number change for essentially all genes using a human-hamster radiation hybrid (RH) panel. These loci are called copy number expression quantitative trait loci (ceQTLs). RESULTS: Unexpected findings from a previous study of a mouse-hamster RH panel were replicated. These findings included decreased expression as a result of increased copy number for 30% of genes and an attenuated relationship between expression and copy number on the X chromosome suggesting an Xist independent form of dosage compensation. In a separate glioblastoma dataset, we found conservation of genes in which dosage was negatively correlated with gene expression. These genes were enriched in signaling and receptor activities. The observation of attenuated X-linked gene expression in response to increased gene number was also replicated in the glioblastoma dataset. Of 523 gene deserts of size > 600 kb in the human RH panel, 325 contained trans ceQTLs with -log10 P > 4.1. Recently discovered genes, ultra conserved regions, noncoding RNAs and microRNAs explained only a small fraction of the results, suggesting a substantial portion of gene deserts harbor as yet unidentified functional elements. CONCLUSION: Radiation hybrids are a useful tool for high resolution mapping of cis and trans loci capable of affecting gene expression due to copy number change. Analysis of two independent radiation hybrid panels show agreement in their findings and may serve as a discovery source for novel regulatory loci in noncoding regions of the genome.
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Variações do Número de Cópias de DNA , Genoma , Animais , Cromossomos Humanos X , Cricetinae , Mecanismo Genético de Compensação de Dose , Perfilação da Expressão Gênica , Ligação Genética , Glioblastoma/genética , Humanos , Células Híbridas , Camundongos , Locos de Características QuantitativasRESUMO
Comprehensive maps of genetic interactions in mammalian cells are daunting to construct because of the large number of potential interactions, ~ 2 × 108 for protein coding genes. We previously used co-inheritance of distant genes from published radiation hybrid (RH) datasets to identify genetic interactions. However, it was necessary to combine six legacy datasets from four species to obtain adequate statistical power. Mapping resolution was also limited by the low density PCR genotyping. Here, we employ shallow sequencing of nascent human RH clones as an economical approach to constructing interaction maps. In this initial study, 15 clones were analyzed, enabling construction of a network with 225 genes and 2,359 interactions (FDR < 0.05). Despite its small size, the network showed significant overlap with the previous RH network and with a protein-protein interaction network. Consumables were â²$50 per clone, showing that affordable, high quality genetic interaction maps are feasible in mammalian cells.
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We performed an unbiased experimental search for enhancers and silencers in a 153-kb region containing the human apolipoprotein (APO) E/C1/C4/C2 gene cluster using shotgun cloning into a luciferase vector. A continuum of transcriptional effect sizes was observed, possibly explaining the limited success of bioinformatics in identifying regulatory regions. We identified nine statistically significant enhancers and five silencers functional in either liver or astrocyte cells, including two previously known enhancers. Only two of the fourteen elements contained conserved noncoding sequences. Within the coding sequence of the APOE gene we identified an enhancer for the E4 allele associated with Alzheimer's disease, but not E3. The single nucleotide polymorphism (SNP) causing the E4/E3 amino acid substitution was responsible for these variations, potentially explaining the higher expression levels of E4. Our results suggest a wider variety of mammalian transcriptional regulatory sequences than is currently recognized and that these may include coding region SNPs.
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Apolipoproteína E4/genética , Apolipoproteínas E/genética , Sequência Conservada , Elementos Facilitadores Genéticos/genética , Família Multigênica , Elementos Reguladores de Transcrição , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Apolipoproteína E3/química , Apolipoproteína E3/genética , Apolipoproteína E4/química , Astrócitos , Sequência de Bases , Linhagem Celular , Clonagem Molecular/métodos , Dosagem de Genes , Vetores Genéticos , Humanos , Fígado/citologia , Luciferases/genética , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Gene expression signatures in the mammalian brain hold the key to understanding neural development and neurological diseases. We have reconstructed two-dimensional images of gene expression for 20,000 genes in a coronal slice of the mouse brain at the level of the striatum by using microarrays in combination with voxelation at a resolution of 1 mm3. Good reliability of the microarray results were confirmed using multiple replicates, subsequent quantitative RT-PCR voxelation, mass spectrometry voxelation, and publicly available in situ hybridization data. Known and novel genes were identified with expression patterns localized to defined substructures within the brain. In addition, genes with unexpected patterns were identified, and cluster analysis identified a set of genes with a gradient of dorsal/ventral expression not restricted to known anatomical boundaries. The genome-scale maps of gene expression obtained using voxelation will be a valuable tool for the neuroscience community.
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Mapeamento Encefálico/métodos , Encéfalo/metabolismo , Perfilação da Expressão Gênica/métodos , Genoma , Microtomia/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Coleta de Tecidos e Órgãos/métodos , Animais , Regulação da Expressão Gênica , Processamento de Imagem Assistida por Computador , Camundongos , Camundongos Endogâmicos C57BL , Modelos BiológicosRESUMO
Previous studies had shown that the integration of genome wide expression profiles, in metabolic tissues, with genetic and phenotypic variance, provided valuable insight into the underlying molecular mechanisms. We used RNA-Seq to characterize hypothalamic transcriptome in 99 inbred strains of mice from the Hybrid Mouse Diversity Panel (HMDP), a reference resource population for cardiovascular and metabolic traits. We report numerous novel transcripts supported by proteomic analyses, as well as novel non coding RNAs. High resolution genetic mapping of transcript levels in HMDP, reveals both local and trans expression Quantitative Trait Loci (eQTLs) demonstrating 2 trans eQTL 'hotspots' associated with expression of hundreds of genes. We also report thousands of alternative splicing events regulated by genetic variants. Finally, comparison with about 150 metabolic and cardiovascular traits revealed many highly significant associations. Our data provide a rich resource for understanding the many physiologic functions mediated by the hypothalamus and their genetic regulation.
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Hipotálamo/fisiologia , Locos de Características Quantitativas , Transcriptoma , Processamento Alternativo , Animais , Doenças Cardiovasculares/genética , Mapeamento Cromossômico , Estudos de Associação Genética , Doenças Metabólicas/genética , Camundongos , Proteoma/análise , RNA não Traduzido/análise , Análise de Sequência de RNARESUMO
Gene expression tomography, or GET, is a new method to increase the speed of three-dimensional (3-D) gene expression analysis in the brain. The name is evocative of the method's dual foundations in high-throughput gene expression analysis and computerized tomographic image reconstruction, familiar from techniques such as positron emission tomography (PET) and X-ray computerized tomography (CT). In GET, brain slices are taken using a cryostat in conjunction with axial rotation about independent axes to create a series of "views" of the brain. Gene expression information obtained from the axially rotated views can then be used to recreate 3-D gene expression patterns. GET was used to successfully reconstruct images of tyrosine hydroxylase gene expression in the mouse brain, using both RNase protection and real-time quantitative reverse transcription PCR (QRT-PCR). A Monte-Carlo analysis confirmed the good quality of the GET image reconstruction. By speeding acquisition of gene expression patterns, GET may help improve our understanding of the genomics of the brain in both health and disease.
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Perfilação da Expressão Gênica/métodos , Tomografia Computadorizada de Emissão/métodos , Tomografia Computadorizada por Raios X/métodos , Animais , Mapeamento Encefálico/métodos , Linhagem Celular , Processamento de Imagem Assistida por Computador/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/metabolismo , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
Voxelation allows high-throughput acquisition of multiple volumetric images of brain gene expression, similar to those obtained from biomedical imaging systems. To obtain these images, the method employs analysis of spatially registered voxels (cubes). For creation of high-resolution maps using voxelation, relatively small voxel sizes are necessary and instruments will be required for semiautomated harvesting of such voxels. Here, we describe two devices that allow spatially registered harvesting of voxels from the human and rodent brain, giving linear resolutions of 3.3 and 1 mm, respectively. Gene expression patterns obtained using these devices showed good agreement with known expression patterns. The voxelation instruments and their future iterations represent a valuable approach to the genome scale acquisition of gene expression patterns in the human and rodent brain.
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Encéfalo/fisiologia , Perfilação da Expressão Gênica/métodos , Expressão Gênica , Imageamento Tridimensional , Tomografia Computadorizada de Emissão , Animais , Mapeamento Encefálico , Primers do DNA , Humanos , Processamento de Imagem Assistida por Computador , Camundongos , Camundongos Endogâmicos C57BL , Sondas de Oligonucleotídeos , RNA Mensageiro/biossíntese , Ratos , Ratos Long-Evans , Receptores de Dopamina D2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Antígenos Thy-1/genéticaRESUMO
We sought exonic transcriptional regulatory elements by shotgun cloning human cDNA fragments into luciferase reporter vectors and measuring the resulting expression levels in liver cells. We uncovered seven regulatory elements within coding regions and three within 3' untranslated regions (UTRs). Two of the putative regulatory elements were enhancers and eight were silencers. The regulatory elements were generally but not consistently evolutionarily conserved and also showed a trend toward decreased population diversity. Furthermore, the exonic regulatory elements were enriched in known transcription factor binding sites (TFBSs) and were associated with several histone modifications and transcriptionally relevant chromatin. Evidence was obtained for bidirectional cis-regulation of a coding region element within a tubulin gene, TUBA1B, by the transcription factors PPARA and RORA. We estimate that hundreds of exonic transcriptional regulatory elements exist, an unexpected finding that highlights a surprising multi-functionality of sequences in the human genome.
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Genoma Humano , Elementos Reguladores de Transcrição , Regiões 3' não Traduzidas , Sítios de Ligação , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , DNA Complementar/genética , Desoxirribonuclease I/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Fatores de Transcrição/metabolismo , Tubulina (Proteína)/genéticaRESUMO
BACKGROUND: Our understanding of the genetic basis of learning and memory remains shrouded in mystery. To explore the genetic networks governing the biology of conditional fear, we used a systems genetics approach to analyze a hybrid mouse diversity panel (HMDP) with high mapping resolution. RESULTS: A total of 27 behavioral quantitative trait loci were mapped with a false discovery rate of 5%. By integrating fear phenotypes, transcript profiling data from hippocampus and striatum and also genotype information, two gene co-expression networks correlated with context-dependent immobility were identified. We prioritized the key markers and genes in these pathways using intramodular connectivity measures and structural equation modeling. Highly connected genes in the context fear modules included Psmd6, Ube2a and Usp33, suggesting an important role for ubiquitination in learning and memory. In addition, we surveyed the architecture of brain transcript regulation and demonstrated preservation of gene co-expression modules in hippocampus and striatum, while also highlighting important differences. Rps15a, Kif3a, Stard7, 6330503K22RIK, and Plvap were among the individual genes whose transcript abundance were strongly associated with fear phenotypes. CONCLUSION: Application of our multi-faceted mapping strategy permits an increasingly detailed characterization of the genetic networks underlying behavior.
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Medo/fisiologia , Redes Reguladoras de Genes/genética , Marcadores Genéticos/genética , Modelos Biológicos , Fenótipo , Biologia de Sistemas/métodos , Animais , Corpo Estriado/metabolismo , Hipocampo/metabolismo , Aprendizagem/fisiologia , Memória/fisiologia , Camundongos , Locos de Características Quantitativas , UbiquitinaçãoRESUMO
We mapped regulatory loci for nearly all protein-coding genes in mammals using comparative genomic hybridization and expression array measurements from a panel of mouse-hamster radiation hybrid cell lines. The large number of breaks in the mouse chromosomes and the dense genotyping of the panel allowed extremely sharp mapping of loci. As the regulatory loci result from extra gene dosage, we call them copy number expression quantitative trait loci, or ceQTLs. The -2log10P support interval for the ceQTLs was <150 kb, containing an average of <2-3 genes. We identified 29,769 trans ceQTLs with -log10P > 4, including 13 hotspots each regulating >100 genes in trans. Further, this work identifies 2,761 trans ceQTLs harboring no known genes, and provides evidence for a mode of gene expression autoregulation specific to the X chromosome.
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Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genes/fisiologia , Locos de Características Quantitativas , Mapeamento de Híbridos Radioativos , Animais , Cricetinae , Mecanismo Genético de Compensação de Dose , Genoma , Genótipo , Células Híbridas , Camundongos , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Cromossomo X/genéticaRESUMO
The molecular mechanisms underlying the changes in the nigrostriatal pathway in Parkinson's disease (PD) are not completely understood. Here, we use mass spectrometry and microarrays to study the proteomic and transcriptomic changes in the striatum of two mouse models of PD, induced by the distinct neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine (METH). Proteomic analyses resulted in the identification and relative quantification of 912 proteins with two or more unique peptides and 86 proteins with significant abundance changes following neurotoxin treatment. Similarly, microarray analyses revealed 181 genes with significant changes in mRNA, following neurotoxin treatment. The combined protein and gene list provides a clearer picture of the potential mechanisms underlying neurodegeneration observed in PD. Functional analysis of this combined list revealed a number of significant categories, including mitochondrial dysfunction, oxidative stress response, and apoptosis. These results constitute one of the largest descriptive data sets integrating protein and transcript changes for these neurotoxin models with many similar end point phenotypes but distinct mechanisms.
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Apoptose/fisiologia , Perfilação da Expressão Gênica , Mitocôndrias/patologia , Neostriado/metabolismo , Estresse Oxidativo/fisiologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteômica , Animais , Apoptose/genética , Modelos Animais de Doenças , Dopamina/deficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Mitocôndrias/metabolismo , Neostriado/efeitos dos fármacos , Neostriado/patologia , Neurotoxinas/farmacologia , Estresse Oxidativo/genética , Doença de Parkinson/genética , Proteoma/genética , Proteoma/metabolismo , RNA/metabolismoRESUMO
We report a global proteomic approach for analyzing brain tissue and for the first time a comprehensive characterization of the whole mouse brain proteome. Preparation of the whole brain sample incorporated a highly efficient cysteinyl-peptide enrichment (CPE) technique to complement a global enzymatic digestion method. Both the global and the cysteinyl-enriched peptide samples were analyzed by SCX fractionation coupled with reversed phase LC-MS/MS analysis. A total of 48,328 different peptides were confidently identified (>98% confidence level), covering 7792 nonredundant proteins ( approximately 34% of the predicted mouse proteome). A total of 1564 and 1859 proteins were identified exclusively from the cysteinyl-peptide and the global peptide samples, respectively, corresponding to 25% and 31% improvements in proteome coverage compared to analysis of only the global peptide or cysteinyl-peptide samples. The identified proteins provide a broad representation of the mouse proteome with little bias evident due to protein pI, molecular weight, and/or cellular localization. Approximately 26% of the identified proteins with gene ontology (GO) annotations were membrane proteins, with 1447 proteins predicted to have transmembrane domains, and many of the membrane proteins were found to be involved in transport and cell signaling. The MS/MS spectrum count information for the identified proteins was used to provide a measure of relative protein abundances. The mouse brain peptide/protein database generated from this study represents the most comprehensive proteome coverage for the mammalian brain to date, and the basis for future quantitative brain proteomic studies using mouse models. The proteomic approach presented here may have broad applications for rapid proteomic analyses of various mouse models of human brain diseases.
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Encéfalo/metabolismo , Cisteína/química , Proteínas de Membrana/metabolismo , Peptídeos/química , Proteoma , Animais , Cromatografia Líquida , Espectrometria de Massas , Camundongos , Peptídeos/análiseRESUMO
Challenges associated with the efficient and effective preparation of micro- and nanoscale (micro- and nanogram) clinical specimens for proteomic applications include the unmitigated sample losses that occur during the processing steps. Herein, we describe a simple "single-tube" preparation protocol appropriate for small proteomic samples using the organic cosolvent, trifluoroethanol (TFE) that circumvents the loss of sample by facilitating both protein extraction and protein denaturation without requiring a separate cleanup step. The performance of the TFE-based method was initially evaluated by comparisons to traditional detergent-based methods on relatively large scale sample processing using human breast cancer cells and mouse brain tissue. The results demonstrated that the TFE-based protocol provided comparable results to the traditional detergent-based protocols for larger, conventionally sized proteomic samples (>100 microg protein content), based on both sample recovery and numbers of peptide/protein identifications. The effectiveness of this protocol for micro- and nanoscale sample processing was then evaluated for the extraction of proteins/peptides and shown effective for small mouse brain tissue samples (approximately 30 microg total protein content) and also for samples of approximately 5000 MCF-7 human breast cancer cells (approximately 500 ng total protein content), where the detergent-based methods were ineffective due to losses during cleanup and transfer steps.
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Nanotecnologia/métodos , Proteômica/instrumentação , Proteômica/métodos , Animais , Encéfalo/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Detergentes/farmacologia , Humanos , Masculino , Espectrometria de Massas , Camundongos , Peptídeos/química , Proteínas/química , Proteoma , Solventes , Fatores de Tempo , Trifluoretanol/químicaRESUMO
We describe a microarray design based on the concept of error-correcting codes from digital communication theory. Currently, microarrays are unable to efficiently deal with "drop-outs," when one or more spots on the array are corrupted. The resulting information loss may lead to decoding errors in which no quantitation of expression can be extracted for the corresponding genes. This issue is expected to become increasingly problematic as the number of spots on microarrays expands to accommodate the entire genome. The error-correcting approach employs multiplexing (encoding) of more than one gene onto each spot to efficiently provide robustness to drop-outs in the array. Decoding then allows fault-tolerant recovery of the expression information from individual genes. The error-correcting method is general and may have important implications for future array designs in research and diagnostics.