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1.
Mol Cell ; 64(1): 51-64, 2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27546791

RESUMO

The tumor suppressor protein 53BP1, a pivotal regulator of DNA double-strand break (DSB) repair, was first identified as a p53-interacting protein over two decades ago. However, its direct contributions to p53-dependent cellular activities remain undefined. Here, we reveal that 53BP1 stimulates genome-wide p53-dependent gene transactivation and repression events in response to ionizing radiation (IR) and synthetic p53 activation. 53BP1-dependent p53 modulation requires both auto-oligomerization and tandem-BRCT domain-mediated bivalent interactions with p53 and the ubiquitin-specific protease USP28. Loss of these activities results in inefficient p53-dependent cell-cycle checkpoint and exit responses. Furthermore, we demonstrate 53BP1-USP28 cooperation to be essential for normal p53-promoter element interactions and gene transactivation-associated events, yet dispensable for 53BP1-dependent DSB repair regulation. Collectively, our data provide a mechanistic explanation for 53BP1-p53 cooperation in controlling anti-tumorigenic cell-fate decisions and reveal these activities to be distinct and separable from 53BP1's regulation of DNA double-strand break repair pathway choice.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Ubiquitina Tiolesterase/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Proteína 9 Associada à CRISPR , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/genética , Endonucleases/metabolismo , Raios gama , Edição de Genes , Regulação da Expressão Gênica , Humanos , Células MCF-7 , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/metabolismo
2.
Malar J ; 18(1): 418, 2019 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-31835999

RESUMO

BACKGROUND: Transcriptional profiling of the human immune response to malaria has been used to identify diagnostic markers, understand the pathogenicity of severe disease and dissect the mechanisms of naturally acquired immunity (NAI). However, interpreting this body of work is difficult given considerable variation in study design, definition of disease, patient selection and methodology employed. This work details a comprehensive review of gene expression profiling (GEP) of the human immune response to malaria to determine how this technology has been applied to date, instances where this has advanced understanding of NAI and the extent of variability in methodology between studies to allow informed comparison of data and interpretation of results. METHODS: Datasets from the gene expression omnibus (GEO) including the search terms; 'plasmodium' or 'malaria' or 'sporozoite' or 'merozoite' or 'gametocyte' and 'Homo sapiens' were identified and publications analysed. Datasets of gene expression changes in relation to malaria vaccines were excluded. RESULTS: Twenty-three GEO datasets and 25 related publications were included in the final review. All datasets related to Plasmodium falciparum infection, except two that related to Plasmodium vivax infection. The majority of datasets included samples from individuals infected with malaria 'naturally' in the field (n = 13, 57%), however some related to controlled human malaria infection (CHMI) studies (n = 6, 26%), or cells stimulated with Plasmodium in vitro (n = 6, 26%). The majority of studies examined gene expression changes relating to the blood stage of the parasite. Significant heterogeneity between datasets was identified in terms of study design, sample type, platform used and method of analysis. Seven datasets specifically investigated transcriptional changes associated with NAI to malaria, with evidence supporting suppression of the innate pro-inflammatory response as an important mechanism for this in the majority of these studies. However, further interpretation of this body of work was limited by heterogeneity between studies and small sample sizes. CONCLUSIONS: GEP in malaria is a potentially powerful tool, but to date studies have been hypothesis generating with small sample sizes and widely varying methodology. As CHMI studies are increasingly performed in endemic settings, there will be growing opportunity to use GEP to understand detailed time-course changes in host response and understand in greater detail the mechanisms of NAI.


Assuntos
Expressão Gênica/imunologia , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Malária/imunologia , Plasmodium/imunologia , Perfilação da Expressão Gênica , Humanos
3.
Clin Immunol ; 160(2): 301-14, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26122175

RESUMO

Common Variable Immunodeficiency Disorders (CVIDs) are the most prevalent cause of primary antibody failure. CVIDs are highly variable and a genetic causes have been identified in <5% of patients. Here, we performed whole genome sequencing (WGS) of 34 CVID patients (94% sporadic) and combined them with transcriptomic profiling (RNA-sequencing of B cells) from three patients and three healthy controls. We identified variants in CVID disease genes TNFRSF13B, TNFRSF13C, LRBA and NLRP12 and enrichment of variants in known and novel disease pathways. The pathways identified include B-cell receptor signalling, non-homologous end-joining, regulation of apoptosis, T cell regulation and ICOS signalling. Our data confirm the polygenic nature of CVID and suggest individual-specific aetiologies in many cases. Together our data show that WGS in combination with RNA-sequencing allows for a better understanding of CVIDs and the identification of novel disease associated pathways.


Assuntos
Linfócitos B/metabolismo , Imunodeficiência de Variável Comum/genética , Genoma/genética , RNA Mensageiro/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Adulto , Receptor do Fator Ativador de Células B/genética , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Pessoa de Meia-Idade , Herança Multifatorial , Análise de Sequência de DNA , Análise de Sequência de RNA , Proteína Transmembrana Ativadora e Interagente do CAML/genética , Adulto Jovem
4.
Blood ; 122(12): 2052-61, 2013 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-23929855

RESUMO

Patients with the dedicator of cytokinesis 8 (DOCK8) immunodeficiency syndrome suffer from recurrent viral and bacterial infections, hyper-immunoglobulin E levels, eczema, and greater susceptibility to cancer. Because natural killer T (NKT) cells have been implicated in these diseases, we asked if these cells were affected by DOCK8 deficiency. Using a mouse model, we found that DOCK8 deficiency resulted in impaired NKT cell development, principally affecting the formation and survival of long-lived, differentiated NKT cells. In the thymus, DOCK8-deficient mice lack a terminally differentiated subset of NK1.1(+) NKT cells expressing the integrin CD103, whereas in the liver, DOCK8-deficient NKT cells express reduced levels of the prosurvival factor B-cell lymphoma 2 and the integrin lymphocyte function-associated antigen 1. Although the initial NKT cell response to antigen is intact in the absence of DOCK8, their ongoing proliferative and cytokine responses are impaired. Importantly, a similar defect in NKT cell numbers was detected in DOCK8-deficient humans, highlighting the relevance of the mouse model. In conclusion, our data demonstrate that DOCK8 is required for the development and survival of mature NKT cells, consistent with the idea that DOCK8 mediates survival signals within a specialized niche. Accordingly, impaired NKT cell numbers and function are likely to contribute to the susceptibility of DOCK8-deficient patients to recurrent infections and malignant disease.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Células T Matadoras Naturais/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos Ly/metabolismo , Sobrevivência Celular/genética , Análise por Conglomerados , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Imunofenotipagem , Cadeias alfa de Integrinas/metabolismo , Fígado/imunologia , Fígado/metabolismo , Contagem de Linfócitos , Camundongos , Camundongos Knockout , Subfamília B de Receptores Semelhantes a Lectina de Células NK/metabolismo , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Timo/imunologia , Timo/metabolismo
5.
Genome Res ; 21(7): 1042-54, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21628452

RESUMO

The human major histocompatibility complex (MHC) on chromosome 6p21 is a paradigm for genomics, showing remarkable polymorphism and striking association with immune and non-immune diseases. The complex genomic landscape of the MHC, notably strong linkage disequilibrium, has made resolving causal variants very challenging. A promising approach is to investigate gene expression levels considered as tractable intermediate phenotypes in mapping complex diseases. However, how transcription varies across the MHC, notably relative to specific haplotypes, remains unknown. Here, using an original hybrid tiling and splice junction microarray that includes alternate allele probes, we draw the first high-resolution strand-specific transcription map for three common MHC haplotypes (HLA-A1-B8-Cw7-DR3, HLA-A3-B7-Cw7-DR15, and HLA-A26-B18-Cw5-DR3-DQ2) strongly associated with autoimmune diseases including type 1 diabetes, systemic lupus erythematosus, and multiple sclerosis. We find that haplotype-specific differences in gene expression are common across the MHC, affecting 96 genes (46.4%), most significantly the zing finger protein gene ZFP57. Differentially expressed probes are correlated with polymorphisms between haplotypes, consistent with cis effects that we directly demonstrate for ZFP57 in a cohort of healthy volunteers (P = 1.2 × 10(-14)). We establish that alternative splicing is significantly more frequent in the MHC than genome-wide (72.5% vs. 62.1% of genes, P ≤ 1 × 10(-4)) and shows marked haplotypic differences. We also unmask novel and abundant intergenic transcription involving 31% of transcribed blocks identified. Our study reveals that the renowned MHC polymorphism also manifests as transcript diversity, and our novel haplotype-based approach marks a new step toward identification of regulatory variants involved in the control of MHC-associated phenotypes and diseases.


Assuntos
Perfilação da Expressão Gênica/métodos , Variação Genética , Haplótipos , Complexo Principal de Histocompatibilidade , Alelos , Processamento Alternativo , Células Cultivadas , Mapeamento Cromossômico , Diabetes Mellitus Tipo 1/genética , Predisposição Genética para Doença , Antígenos HLA-A/genética , Antígenos HLA-A/metabolismo , Humanos , Desequilíbrio de Ligação , Lúpus Eritematoso Sistêmico/genética , Esclerose Múltipla/genética , Análise de Sequência com Séries de Oligonucleotídeos , Locos de Características Quantitativas , Análise de Sequência de DNA , Transcrição Gênica
6.
PLoS Genet ; 7(7): e1002145, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21765815

RESUMO

Forkhead-box protein P2 is a transcription factor that has been associated with intriguing aspects of cognitive function in humans, non-human mammals, and song-learning birds. Heterozygous mutations of the human FOXP2 gene cause a monogenic speech and language disorder. Reduced functional dosage of the mouse version (Foxp2) causes deficient cortico-striatal synaptic plasticity and impairs motor-skill learning. Moreover, the songbird orthologue appears critically important for vocal learning. Across diverse vertebrate species, this well-conserved transcription factor is highly expressed in the developing and adult central nervous system. Very little is known about the mechanisms regulated by Foxp2 during brain development. We used an integrated functional genomics strategy to robustly define Foxp2-dependent pathways, both direct and indirect targets, in the embryonic brain. Specifically, we performed genome-wide in vivo ChIP-chip screens for Foxp2-binding and thereby identified a set of 264 high-confidence neural targets under strict, empirically derived significance thresholds. The findings, coupled to expression profiling and in situ hybridization of brain tissue from wild-type and mutant mouse embryos, strongly highlighted gene networks linked to neurite development. We followed up our genomics data with functional experiments, showing that Foxp2 impacts on neurite outgrowth in primary neurons and in neuronal cell models. Our data indicate that Foxp2 modulates neuronal network formation, by directly and indirectly regulating mRNAs involved in the development and plasticity of neuronal connections.


Assuntos
Encéfalo/embriologia , Fatores de Transcrição Forkhead/genética , Redes Reguladoras de Genes , Neuritos/metabolismo , Proteínas Repressoras/genética , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Corpo Estriado/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Cultura Primária de Células , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
7.
Brief Bioinform ; 12(6): 634-44, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21498550

RESUMO

The use of microarray technology to measure gene expression on a genome-wide scale has been well established for more than a decade. Methods to process and analyse the vast quantity of expression data generated by a typical microarray experiment are similarly well-established. The Affymetrix Exon 1.0 ST array is a relatively new type of array, which has the capability to assess expression at the individual exon level. This allows a more comprehensive analysis of the transcriptome, and in particular enables the study of alternative splicing, a gene regulation mechanism important in both normal conditions and in diseases. Some aspects of exon array data analysis are shared with those for standard gene expression data but others present new challenges that have required development of novel tools. Here, I will introduce the exon array and present a detailed example tutorial for analysis of data generated using this platform.


Assuntos
Éxons , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Software , Perfilação da Expressão Gênica/métodos , Transcriptoma
8.
Endocr Relat Cancer ; 29(10): 557-568, 2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-35900839

RESUMO

Multiple endocrine neoplasia type 1 (MEN1), caused by mutations in the MEN1 gene encoding menin, is an autosomal dominant disorder characterised by the combined occurrence of parathyroid, pituitary and pancreatic neuroendocrine tumours (NETs). Development of these tumours is associated with wide variations in their severity, order and ages (from <5 to >80 years), requiring life-long screening. To improve tumour surveillance and quality of life, better circulating biomarkers, particularly for pancreatic NETs that are associated with higher mortality, are required. We, therefore, examined the expression of circulating miRNA in the serum of MEN1 patients. Initial profiling analysis followed by qRT-PCR validation studies identified miR-3156-5p to be significantly downregulated (-1.3 to 5.8-fold, P < 0.05-0.0005) in nine MEN1 patients, compared to matched unaffected relatives. MEN1 knock-down experiments in BON-1 human pancreatic NET cells resulted in reduced MEN1 (49%, P < 0.05), menin (54%, P < 0.05) and miR-3156-5p expression (20%, P < 0.005), compared to control-treated cells, suggesting that miR-3156-5p downregulation is a consequence of loss of MEN1 expression. In silico analysis identified mortality factor 4-like 2 (MOR4FL2) as a potential target of miR-3156-5p, and in vitro functional studies in BON-1 cells transfected with either miR-3156-5p mimic or inhibitors showed that the miR-3156-5p mimic significantly reduced MORF4L2 protein expression (46%, P < 0.005), while miR-3156-5p inhibitor significantly increased MORF4L2 expression (1.5-fold, P < 0.05), compared to control-treated cells, thereby confirming that miR-3156-5p regulates MORF4L2 expression. Thus, the inverse relationship between miR-3156-5p and MORF4L2 expression represents a potential serum biomarker that could facilitate the detection of NET occurrence in MEN1 patients.


Assuntos
MicroRNAs , Neoplasia Endócrina Múltipla Tipo 1 , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Humanos , MicroRNAs/genética , Pessoa de Meia-Idade , Neoplasia Endócrina Múltipla Tipo 1/patologia , Mutação , Qualidade de Vida , Fatores de Transcrição/genética , Adulto Jovem
9.
Am J Respir Crit Care Med ; 181(12): 1367-75, 2010 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-20194811

RESUMO

RATIONALE: Approximately 60 to 70% of patients with pulmonary sarcoidosis have disease that resolves spontaneously; the rest follow a chronic course with varying levels of fibrosis. It is unclear why some patients progress and if treatment affects outcome. OBJECTIVES: To determine differential gene expression profile in lungs of patients with self-limiting sarcoidosis compared to those with progressive-fibrotic disease, and to analyze the biological relevance of these differentially expressed genes. METHODS: We examined microarray expression of 26,626 genes in transbronchial biopsies of granulomatous areas in lungs of patients with active but self-limiting (n = 8) versus those with active, progressive (+/- fibrotic) pulmonary disease (n = 7). MEASUREMENTS AND MAIN RESULTS: Three hundred thirty-four genes were differentially expressed between the two groups (P < 0.01, Bayesian moderated t test). Gene Set Enrichment Analysis showed over-representation of gene-sets (defined by Gene Ontology) related to host immune activation, proliferation, and defense, among genes up-regulated in the progressive-fibrotic group (FDR q < 0.0001 for the top 43 gene sets), and a marked enrichment of, and similarity in gene expression profiles between, progressive-fibrotic sarcoidosis and hypersensitivity pneumonitis (HP), (q < 0.001), but not idiopathic pulmonary fibrosis (IPF). CONCLUSIONS: The findings suggest that patients with progressive/fibrotic pulmonary sarcoidosis have intense immune activity related to host defense in their lungs, with processes more similar to HP than IPF. The study also demonstrates that transbronchial lung biopsy samples can provide good-quality RNA for gene expression profiling, supporting its potential use as a prognostic classifier for pulmonary sarcoidosis.


Assuntos
Expressão Gênica/genética , Pulmão/patologia , Sarcoidose Pulmonar/genética , Sarcoidose Pulmonar/patologia , Adulto , Idoso , Biópsia , Brônquios/patologia , Progressão da Doença , Feminino , Fibroblastos , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Regulação para Cima/genética
10.
BMC Genomics ; 11: 96, 2010 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-20141636

RESUMO

BACKGROUND: Readily accessible samples such as peripheral blood or cell lines are increasingly being used in large cohorts to characterise gene expression differences between a patient group and healthy controls. However, cell and RNA isolation procedures and the variety of cell types that make up whole blood can affect gene expression measurements. We therefore systematically investigated global gene expression profiles in peripheral blood from six individuals collected during two visits by comparing five of the following cell and RNA isolation methods: whole blood (PAXgene), peripheral blood mononuclear cells (PBMCs), lymphoblastoid cell lines (LCLs), CD19 and CD20 specific B-cell subsets. RESULTS: Gene expression measurements were clearly discriminated by isolation method although the reproducibility was high for all methods (range rho = 0.90-1.00). The PAXgene samples showed a decrease in the number of expressed genes (P < 1*10(-16)) with higher variability (P < 1*10(-16)) compared to the other methods. Differentially expressed probes between PAXgene and PBMCs were correlated with the number of monocytes, lymphocytes, neutrophils or erythrocytes. The correlations (rho = 0.83; rho = 0.79) of the expression levels of detected probes between LCLs and B-cell subsets were much lower compared to the two B-cell isolation methods (rho = 0.98). Gene ontology analysis of detected genes showed that genes involved in inflammatory responses are enriched in B-cells CD19 and CD20 whereas genes involved in alcohol metabolic process and the cell cycle were enriched in LCLs. CONCLUSION: Gene expression profiles in blood-based samples are strongly dependent on the predominant constituent cell type(s) and RNA isolation method. It is crucial to understand the differences and variability of gene expression measurements between cell and RNA isolation procedures, and their relevance to disease processes, before application in large clinical studies.


Assuntos
Subpopulações de Linfócitos B/metabolismo , Perfilação da Expressão Gênica/métodos , Leucócitos Mononucleares/metabolismo , RNA/sangue , Linhagem Celular , Análise por Conglomerados , Humanos , RNA/isolamento & purificação , Reprodutibilidade dos Testes
11.
PLoS One ; 8(12): e82825, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24324835

RESUMO

Nutritional factors play important roles in the etiology of obesity, type 2 diabetes mellitus and their complications through genotype x environment interactions. We have characterised molecular adaptation to high fat diet (HFD) feeding in inbred mouse strains widely used in genetic and physiological studies. We carried out physiological tests, plasma lipid assays, obesity measures, liver histology, hepatic lipid measurements and liver genome-wide gene transcription profiling in C57BL/6J and BALB/c mice fed either a control or a high fat diet. The two strains showed marked susceptibility (C57BL/6J) and relative resistance (BALB/c) to HFD-induced insulin resistance and non alcoholic fatty liver disease (NAFLD). Global gene set enrichment analysis (GSEA) of transcriptome data identified consistent patterns of expression of key genes (Srebf1, Stard4, Pnpla2, Ccnd1) and molecular pathways in the two strains, which may underlie homeostatic adaptations to dietary fat. Differential regulation of pathways, including the proteasome, the ubiquitin mediated proteolysis and PPAR signalling in fat fed C57BL/6J and BALB/c suggests that altered expression of underlying diet-responsive genes may be involved in contrasting nutrigenomic predisposition and resistance to insulin resistance and NAFLD in these models. Collectively, these data, which further demonstrate the impact of gene x environment interactions on gene expression regulations, contribute to improved knowledge of natural and pathogenic adaptive genomic regulations and molecular mechanisms associated with genetically determined susceptibility and resistance to metabolic diseases.


Assuntos
Dieta Hiperlipídica , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Nutrigenômica , Obesidade/complicações , Obesidade/etiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Adaptação Fisiológica , Animais , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glucose/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica , Reprodutibilidade dos Testes , Transdução de Sinais , Transcriptoma
12.
BMC Med Genomics ; 2: 28, 2009 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-19457239

RESUMO

BACKGROUND: Many critical maturational processes take place in the human brain during postnatal development. In particular, the prefrontal cortex does not reach maturation until late adolescence and this stage is associated with substantial white matter volume increases. Patients with schizophrenia and other major psychiatric disorders tend to first present with overt symptoms during late adolescence/early adulthood and it has been proposed that this developmental stage represents a "window of vulnerability". METHODS: In this study we used whole genome microarrays to measure gene expression in post mortem prefrontal cortex tissue from human individuals ranging in age from 0 to 49 years. To identify genes specifically altered in the late adolescent period, we applied a template matching procedure. Genes were identified which showed a significant correlation to a template showing a peak of expression between ages 15 and 25. RESULTS: Approximately 2000 genes displayed an expression pattern that was significantly correlated (positively or negatively) with the template. In the majority of cases, these genes in fact reached a plateau during adolescence with only subtle changes thereafter. These include a number of genes previously associated with schizophrenia including the susceptibility gene neuregulin 1 (NRG1). Functional profiling revealed peak expression in late adolescence for genes associated with energy metabolism and protein and lipid synthesis, together with decreases for genes involved in glutamate and neuropeptide signalling and neuronal development/plasticity. Strikingly, eight myelin-related genes previously found decreased in schizophrenia brain tissue showed a peak in their expression levels in late adolescence, while the single myelin gene reported increased in patients with schizophrenia was decreased in late adolescence. CONCLUSION: The observed changes imply that molecular mechanisms critical for adolescent brain development are disturbed in schizophrenia patients.

13.
BMC Med Genomics ; 2: 54, 2009 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-19689793

RESUMO

BACKGROUND: MicroRNAs (miRNAs) are non-coding RNA molecules involved in post-transcriptional control of gene expression of a wide number of genes, including those involved in glucose homeostasis. Type 2 diabetes (T2D) is characterized by hyperglycaemia and defects in insulin secretion and action at target tissues. We sought to establish differences in global miRNA expression in two insulin-target tissues from inbred rats of spontaneously diabetic and normoglycaemic strains. METHODS: We used a miRNA microarray platform to measure global miRNA expression in two insulin-target tissues: liver and adipose tissue from inbred rats of spontaneously diabetic (Goto-Kakizaki [GK]) and normoglycaemic (Brown-Norway [BN]) strains which are extensively used in genetic studies of T2D. MiRNA data were integrated with gene expression data from the same rats to investigate how differentially expressed miRNAs affect the expression of predicted target gene transcripts. RESULTS: The expression of 170 miRNAs was measured in liver and adipose tissue of GK and BN rats. Based on a p-value for differential expression between GK and BN, the most significant change in expression was observed for miR-125a in liver (FC = 5.61, P = 0.001, Padjusted = 0.10); this overexpression was validated using quantitative RT-PCR (FC = 13.15, P = 0.0005). MiR-125a also showed over-expression in the GK vs. BN analysis within adipose tissue (FC = 1.97, P = 0.078, Padjusted = 0.99), as did the previously reported miR-29a (FC = 1.51, P = 0.05, Padjusted = 0.99). In-silico tools assessing the biological role of predicted miR-125a target genes suggest an over-representation of genes involved in the MAPK signaling pathway. Gene expression analysis identified 1308 genes with significantly different expression between GK and BN rats (Padjusted < 0.05): 233 in liver and 1075 in adipose tissue. Pathways related to glucose and lipid metabolism were significantly over-represented among these genes. Enrichment analysis suggested that differentially expressed genes in GK compared to BN included more predicted miR-125a target genes than would be expected by chance in adipose tissue (FDR = 0.006 for up-regulated genes; FDR = 0.036 for down-regulated genes) but not in liver (FDR = 0.074 for up-regulated genes; FDR = 0.248 for down-regulated genes). CONCLUSION: MiR-125a is over-expressed in liver in hyperglycaemic GK rats relative to normoglycaemic BN rats, and our array data also suggest miR-125a is over-expressed in adipose tissue. We demonstrate the use of in-silico tools to provide the basis for further investigation of the potential role of miR-125a in T2D. In particular, the enrichment of predicted miR-125a target genes among differentially expressed genes has identified likely target genes and indicates that integrating global miRNA and mRNA expression data may give further insights into miRNA-mediated regulation of gene expression.

14.
J Proteome Res ; 8(7): 3284-97, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19400588

RESUMO

Haloperidol and olanzapine are widely used antipsychotic drugs in the treatment of schizophrenia and other psychotic disorders. Despite extensive research efforts within the biopharmaceutical industry and academia, the exact molecular mechanisms of their action remain largely unknown. Since the response of patients to existing medications can be variable and often includes severe side effects, it is critical to increase our knowledge on their mechanism of action to guide clinical usage and new drug development. In this study, we have employed the label-free liquid chromatography tandem mass spectrometry (LC-MSE) to identify differentially expressed proteins in rat frontal cortex following subchronic treatment with haloperidol or olanzapine. Subcellular fractionation was performed to increased proteomic coverage and provided insight into the subcellular location involved in the mechanism of drug action. LC-MSE profiling identified 531 and 741 annotated proteins in fractions I (cytoplasmic-) and II (membrane enriched-) in two drug treatments. Fifty-nine of these proteins were altered significantly by haloperidol treatment, 74 by olanzapine and 21 were common to both treatments. Pathway analysis revealed that both drugs altered similar classes of proteins associated with cellular assembly/organization, nervous system development/function (particularly presynaptic function) and neurological disorders, which indicate a common mechanism of action. The top affected canonical signaling pathways differed between the two treatments. The haloperidol data set showed a stronger association with Huntington's disease signaling, while olanzapine treatment showed stronger effects on glycolysis/gluconeogenesis. This could either relate to a difference in clinical efficacy or side effect profile of the two compounds. The results were consistent with the findings reported previously by targeted studies, demonstrating the validity of this approach. However, we have also identified many novel proteins which have not been found previously to be associated with these drugs. Further study of these proteins could provide new insights into the etiology of the disease or the mechanism of antipsychotic medications.


Assuntos
Antipsicóticos/farmacologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Lobo Frontal/efeitos dos fármacos , Animais , Benzodiazepinas/farmacologia , Cromatografia Líquida/métodos , Detergentes/farmacologia , Haloperidol/farmacologia , Masculino , Espectrometria de Massas/métodos , Sistema Nervoso/efeitos dos fármacos , Olanzapina , Proteômica/métodos , Ratos , Ratos Wistar , Transmissão Sináptica
15.
Genome Biol ; 9(8): R124, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18681948

RESUMO

BACKGROUND: Despite decades of research, the molecular changes responsible for the evolution of human cognitive abilities remain unknown. Comparative evolutionary studies provide detailed information about DNA sequence and mRNA expression differences between humans and other primates but, in the absence of other information, it has proved very difficult to identify molecular pathways relevant to human cognition. RESULTS: Here, we compare changes in gene expression and metabolite concentrations in the human brain and compare them to the changes seen in a disorder known to affect human cognitive abilities, schizophrenia. We find that both genes and metabolites relating to energy metabolism and energy-expensive brain functions are altered in schizophrenia and, at the same time, appear to have changed rapidly during recent human evolution, probably as a result of positive selection. CONCLUSION: Our findings, along with several previous studies, suggest that the evolution of human cognitive abilities was accompanied by adaptive changes in brain metabolism, potentially pushing the human brain to the limit of its metabolic capabilities.


Assuntos
Encéfalo/metabolismo , Esquizofrenia/metabolismo , Adulto , Idoso , Animais , Evolução Biológica , Cognição/fisiologia , Metabolismo Energético/genética , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Macaca mulatta/genética , Macaca mulatta/metabolismo , Masculino , Pessoa de Meia-Idade , Ressonância Magnética Nuclear Biomolecular , Análise de Sequência com Séries de Oligonucleotídeos , Pan troglodytes/genética , Pan troglodytes/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Esquizofrenia/genética
16.
J Proteome Res ; 6(1): 141-9, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17203958

RESUMO

The molecular disease mechanisms associated with schizophrenia remain largely unknown. Although primarily considered a disorder of the brain, there is evidence of a peripheral component to schizophrenia. In this study, we investigated liver tissue and red blood cells (RBC) from schizophrenia patients and controls using 2-D DIGE proteomic analysis. Fourteen proteins were significantly altered in liver samples from schizophrenia patients (n = 15) compared to healthy controls (n = 15). Analysis of the schizophrenia RBC proteome revealed 8 proteins significantly altered in samples from schizophrenia patients (13 antipsychotic-treated and 7 drug-naïve) compared to controls (n = 20). Six of the altered proteins in the liver and four of the altered RBC proteins are related to oxidative stress. These results corroborate our earlier findings obtained from post-mortem brain studies and substantiate our hypothesis that metabolic alterations leading to oxidative stress are linked to the schizophrenia disease process. Our results also suggest that at least some of the pathological processes associated with the schizophrenia disease process can be traced in peripheral tissue. If peripheral cells can be used as a disease surrogate, promising new investigative avenues could be explored.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Eritrócitos/metabolismo , Fígado/metabolismo , Estresse Oxidativo , Esquizofrenia/metabolismo , Adulto , Antipsicóticos/farmacologia , Feminino , Hemoglobinas/metabolismo , Humanos , Focalização Isoelétrica , Masculino , Pessoa de Meia-Idade , Proteoma , Proteômica/métodos
17.
PLoS One ; 2(8): e692, 2007 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-17668069

RESUMO

Despite decades of research into the aetiology and pathophysiology of schizophrenia, our understanding of this devastating disorder remains incomplete, with adverse consequences for both diagnosis and treatment. Here we investigate whether differences between patients and controls can be observed in peripheral patient tissue, with a view of establishing a means for dynamic investigations into cell function. In vitro stimulation of peripheral blood CD3+ pan T cells with anti-CD3 (clone OKT3) was used to investigate disease-associated cell responses. T cells from both medicated (n = 39), unmedicated (n = 6) and minimally medicated (n = 5) schizophrenia patients were found to have significantly lower proliferative responses to stimulation, compared to well-matched controls (n = 32). Expression of CD3 and TCR (T cell receptor) alphabeta chains was equivalent between patients and controls, ensuring equal stimulation with anti-CD3, and there was no significant difference in the proportions of CD4+ and CD8+ T cells between samples (n = 12). Lower T cell proliferation in schizophrenia patients was not found to result from deficient early tyrosine phosphorylation signalling or lower IL-2 (interleukin-2) production, as these parameters were similar between patients and controls, as was the expression of CD25, the IL-2 receptor alpha chain. Analysis of CD45 isoforms, however, revealed that patients had a significantly greater percentage of CD8+ and CD4+ CD45RA+ cells before stimulation and significantly higher fluorescence intensity of CD45RA on CD4+ and CD8+ cells before and after stimulation. There was significantly higher expression of CD45 RB on both CD4+ and CD8+ unstimulated cells, with a trend towards lower numbers of CD45RO+ T cells in patient blood. Gene expression analysis in freshly isolated T cells from six minimally treated or first onset patients and six controls was carried out using human whole-genome CodeLink microarrays to identify functional pathways that may affect the ability of patient cells to respond to stimulation. Functional profiling showed prominent transcript changes in categories pertaining to cell cycle machinery, intracellular signalling, oxidative stress and metabolism. Intriguingly, chromosomal location analysis of genes significantly altered between schizophrenia and controls revealed clusters at 1p36, 1q42 and 6p22, which have previously been identified as strong susceptibility loci for schizophrenia.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Esquizofrenia/imunologia , Esquizofrenia/fisiopatologia , Subpopulações de Linfócitos T/imunologia , Animais , Antipsicóticos/uso terapêutico , Complexo CD3/genética , Complexo CD3/imunologia , Proliferação de Células , Mapeamento Cromossômico , Feminino , Perfilação da Expressão Gênica , Humanos , Interleucina-2/genética , Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/imunologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Esquizofrenia/tratamento farmacológico , Transdução de Sinais/imunologia
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