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1.
Sci Data ; 7(1): 399, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33203859

RESUMO

The PacBio® HiFi sequencing method yields highly accurate long-read sequencing datasets with read lengths averaging 10-25 kb and accuracies greater than 99.5%. These accurate long reads can be used to improve results for complex applications such as single nucleotide and structural variant detection, genome assembly, assembly of difficult polyploid or highly repetitive genomes, and assembly of metagenomes. Currently, there is a need for sample data sets to both evaluate the benefits of these long accurate reads as well as for development of bioinformatic tools including genome assemblers, variant callers, and haplotyping algorithms. We present deep coverage HiFi datasets for five complex samples including the two inbred model genomes Mus musculus and Zea mays, as well as two complex genomes, octoploid Fragaria × ananassa and the diploid anuran Rana muscosa. Additionally, we release sequence data from a mock metagenome community. The datasets reported here can be used without restriction to develop new algorithms and explore complex genome structure and evolution. Data were generated on the PacBio Sequel II System.

2.
Microbiol Resour Announc ; 9(24)2020 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-32527783

RESUMO

Lymphatic filariasis affects ∼120 million people and can result in elephantiasis and hydrocele. Here, we report the nearly complete genome sequence of the best-studied causative agent of lymphatic filariasis, Brugia malayi The assembly contains four autosomes, an X chromosome, and only eight gaps but lacks a contiguous sequence for the known Y chromosome.

4.
Nat Commun ; 11(1): 1964, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32327641

RESUMO

Sex determination mechanisms often differ even between related species yet the evolution of sex chromosomes remains poorly understood in all but a few model organisms. Some nematodes such as Caenorhabditis elegans have an XO sex determination system while others, such as the filarial parasite Brugia malayi, have an XY mechanism. We present a complete B. malayi genome assembly and define Nigon elements shared with C. elegans, which we then map to the genomes of other filarial species and more distantly related nematodes. We find a remarkable plasticity in sex chromosome evolution with several distinct cases of neo-X and neo-Y formation, X-added regions, and conversion of autosomes to sex chromosomes from which we propose a model of chromosome evolution across different nematode clades. The phylum Nematoda offers a new and innovative system for gaining a deeper understanding of sex chromosome evolution.


Assuntos
Evolução Molecular , Nematoides/genética , Infecções por Nematoides/parasitologia , Cromossomos Sexuais/genética , Animais , Brugia Malayi/genética , Caenorhabditis elegans/genética , Mapeamento Cromossômico , Feminino , Regulação da Expressão Gênica , Genoma Helmíntico/genética , Humanos , Masculino , Nematoides/classificação , Sequências Repetitivas de Ácido Nucleico/genética , Processos de Determinação Sexual/genética
5.
PLoS One ; 15(3): e0228789, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32160188

RESUMO

Large expansions of microsatellite DNA cause several neurological diseases. In Spinocerebellar ataxia type 10 (SCA10), the repeat interruptions change disease phenotype; an (ATTCC)n or a (ATCCT)n/(ATCCC)n interruption within the (ATTCT)n repeat is associated with the robust phenotype of ataxia and epilepsy while mostly pure (ATTCT)n may have reduced penetrance. Large repeat expansions of SCA10, and many other microsatellite expansions, can exceed 10,000 base pairs (bp) in size. Conventional next generation sequencing (NGS) technologies are ineffective in determining internal sequence contents or size of these expanded repeats. Using repeat primed PCR (RP-PCR) in conjunction with a high-sensitivity pulsed-field capillary electrophoresis fragment analyzer (FEMTO-Pulse, Agilent, Santa Clara, CA) (RP-FEMTO hereafter), we successfully determined sequence content of large expansion repeats in genomic DNA of SCA10 patients and transformed yeast artificial chromosomes containing SCA10 repeats. This RP-FEMTO is a simple and economical methodology which could complement emerging NGS for very long sequence reads such as Single Molecule, Real-Time (SMRT) and nanopore sequencing technologies.


Assuntos
Ataxina-10/genética , Eletroforese Capilar/métodos , Repetições de Microssatélites/genética , Ataxias Espinocerebelares/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Expansão das Repetições de DNA/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo
6.
J Colloid Interface Sci ; 561: 83-92, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31812869

RESUMO

While Co3O4 represents one of the most promising catalysts for soot oxidation, conventional Co3O4 nanoparticles (NPs) tend to aggregate, losing their activities. Herein, an alternative approach is proposed for preparing three-dimensional nanostructured Co3O4 (NSCo) using the hierarchically-structured Co-based Metal Organic Frameworks as a precursor. Specifically, ZIF-67 is chosen as the precursor as ZIF-67 can be conveniently synthesized with high yields and it can be easily converted to NSCo via calcination. The resulting NSCo exhibits a unique morphology which enables NSCo to possess more porosities and surface areas than the typical Co3O4 NPs. Consequently, NSCo shows a much higher catalytic activity than the typical Co3O4 NPs for soot oxidation because of superior textural properties of NSCo. Besides, when the soot oxidation by the typical Co3O4 NPs produced a significant amount of unwanted CO, soot can be completely combusted into CO2 using NSCo. In comparison with other reported Co-related catalysts, NSCo also achieves a higher soot oxidation efficiency (100% conversion) at lower temperatures with Tig of 331 °C. NSCo can be reused over many continuous cycles and still retains its catalytic activities. These features validate that NSCo is an easy-to-prepare 3D nanostructured Co3O4 catalyst, which possesses advantageous capabilities for soot oxidation at lower temperatures.

7.
Nat Med ; 25(6): 1012-1021, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31142849

RESUMO

The incidence of preterm birth exceeds 10% worldwide. There are significant disparities in the frequency of preterm birth among populations within countries, and women of African ancestry disproportionately bear the burden of risk in the United States. In the present study, we report a community resource that includes 'omics' data from approximately 12,000 samples as part of the integrative Human Microbiome Project. Longitudinal analyses of 16S ribosomal RNA, metagenomic, metatranscriptomic and cytokine profiles from 45 preterm and 90 term birth controls identified harbingers of preterm birth in this cohort of women predominantly of African ancestry. Women who delivered preterm exhibited significantly lower vaginal levels of Lactobacillus crispatus and higher levels of BVAB1, Sneathia amnii, TM7-H1, a group of Prevotella species and nine additional taxa. The first representative genomes of BVAB1 and TM7-H1 are described. Preterm-birth-associated taxa were correlated with proinflammatory cytokines in vaginal fluid. These findings highlight new opportunities for assessment of the risk of preterm birth.


Assuntos
Microbiota , Nascimento Prematuro/microbiologia , Vagina/microbiologia , Adulto , Afro-Americanos , Biodiversidade , Estudos de Coortes , Citocinas/metabolismo , Feminino , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Recém-Nascido , Mediadores da Inflamação/metabolismo , Estudos Longitudinais , Metagenômica , Microbiota/genética , Microbiota/imunologia , Nascimento Prematuro/etiologia , Nascimento Prematuro/imunologia , Fatores de Risco , Estados Unidos , Vagina/imunologia , Adulto Jovem
8.
Genet Med ; 21(9): 2092-2102, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30733599

RESUMO

PURPOSE: To demonstrate the utility of an amplification-free long-read sequencing method to characterize the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (CTG18.1). METHODS: We applied an amplification-free method, utilizing the CRISPR/Cas9 system, in combination with PacBio single-molecule real-time (SMRT) long-read sequencing, to study CTG18.1. FECD patient samples displaying a diverse range of CTG18.1 allele lengths and zygosity status (n = 11) were analyzed. A robust data analysis pipeline was developed to effectively filter, align, and interrogate CTG18.1-specific reads. All results were compared with conventional polymerase chain reaction (PCR)-based fragment analysis. RESULTS: CRISPR-guided SMRT sequencing of CTG18.1 provided accurate genotyping information for all samples and phasing was possible for 18/22 alleles sequenced. Repeat length instability was observed for all expanded (≥50 repeats) phased CTG18.1 alleles analyzed. Furthermore, higher levels of repeat instability were associated with increased CTG18.1 allele length (mode length ≥91 repeats) indicating that expanded alleles behave dynamically. CONCLUSION: CRISPR-guided SMRT sequencing of CTG18.1 has revealed novel insights into CTG18.1 length instability. Furthermore, this study provides a framework to improve the molecular diagnostic accuracy for CTG18.1-mediated FECD, which we anticipate will become increasingly important as gene-directed therapies are developed for this common age-related and sight threatening disease.


Assuntos
Distrofia Endotelial de Fuchs/genética , Predisposição Genética para Doença , Fator de Transcrição 4/genética , Expansão das Repetições de Trinucleotídeos/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Sistemas CRISPR-Cas/genética , Feminino , Distrofia Endotelial de Fuchs/patologia , Genótipo , Humanos , Íntrons/genética , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNA , Imagem Individual de Molécula , Repetições de Trinucleotídeos/genética
10.
Hum Mutat ; 39(9): 1262-1272, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29932473

RESUMO

Amplification of DNA is required as a mandatory step during library preparation in most targeted sequencing protocols. This can be a critical limitation when targeting regions that are highly repetitive or with extreme guanine-cytosine (GC) content, including repeat expansions associated with human disease. Here, we used an amplification-free protocol for targeted enrichment utilizing the CRISPR/Cas9 system (No-Amp Targeted sequencing) in combination with single molecule, real-time (SMRT) sequencing for studying repeat elements in the huntingtin (HTT) gene, where an expanded CAG repeat is causative for Huntington disease. We also developed a robust data analysis pipeline for repeat element analysis that is independent of alignment of reads to a reference genome. The method was applied to 11 diagnostic blood samples, and for all 22 alleles the resulting CAG repeat count agreed with previous results based on fragment analysis. The amplification-free protocol also allowed for studying somatic variability of repeat elements in our samples, without the interference of PCR stutter. In summary, with No-Amp Targeted sequencing in combination with our analysis pipeline, we could accurately study repeat elements that are difficult to investigate using PCR-based methods.


Assuntos
Genoma Humano/genética , Proteína Huntingtina/genética , Doença de Huntington/genética , Expansão das Repetições de Trinucleotídeos/genética , Alelos , Ataxina-10/genética , Proteína C9orf72/genética , Sistemas CRISPR-Cas/genética , Proteína do X Frágil de Retardo Mental/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Doença de Huntington/patologia , RNA Guia/genética , Análise de Sequência de DNA
11.
Sci Rep ; 8(1): 8924, 2018 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-29895987

RESUMO

We sequenced the Hyposidra talaca NPV (HytaNPV) double stranded circular DNA genome using PacBio single molecule sequencing technology. We found that the HytaNPV genome is 139,089 bp long with a GC content of 39.6%. It encodes 141 open reading frames (ORFs) including the 37 baculovirus core genes, 25 genes conserved among lepidopteran baculoviruses, 72 genes known in baculovirus, and 7 genes unique to the HytaNPV genome. It is a group II alphabaculovirus that codes for the F protein and lacks the gp64 gene found in group I alphabaculovirus viruses. Using RNA-seq, we confirmed the expression of the ORFs identified in the HytaNPV genome. Phylogenetic analysis showed HytaNPV to be closest to BusuNPV, SujuNPV and EcobNPV that infect other tea pests, Buzura suppressaria, Sucra jujuba, and Ectropis oblique, respectively. We identified repeat elements and a conserved non-coding baculovirus element in the genome. Analysis of the putative promoter sequences identified motif consistent with the temporal expression of the genes observed in the RNA-seq data.


Assuntos
Genoma Viral/genética , Mariposas/virologia , Nucleopoliedrovírus/genética , Transcriptoma/genética , Sequenciamento Completo do Genoma/métodos , Sequência de Aminoácidos , Animais , Sequência de Bases , Genes Virais/genética , Larva/virologia , Nucleopoliedrovírus/classificação , Nucleopoliedrovírus/fisiologia , Fases de Leitura Aberta/genética , Filogenia , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico
12.
NPJ Parkinsons Dis ; 3: 27, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28890930

RESUMO

Large, non-coding pentanucleotide repeat expansions of ATTCT in intron 9 of the ATXN10 gene typically cause progressive spinocerebellar ataxia with or without seizures and present neuropathologically with Purkinje cell loss resulting in symmetrical cerebellar atrophy. These ATXN10 repeat expansions can be interrupted by sequence motifs which have been attributed to seizures and are likely to act as genetic modifiers. We identified a Mexican kindred with multiple affected family members with ATXN10 expansions. Four affected family members showed clinical features of spinocerebellar ataxia type 10 (SCA10). However, one affected individual presented with early-onset levodopa-responsive parkinsonism, and one family member carried a large repeat ATXN10 expansion, but was clinically unaffected. To characterize the ATXN10 repeat, we used a novel technology of single-molecule real-time (SMRT) sequencing and CRISPR/Cas9-based capture. We sequenced the entire span of ~5.3-7.0 kb repeat expansions. The Parkinson's patient carried an ATXN10 expansion with no repeat interruption motifs as well as an unaffected sister. In the siblings with typical SCA10, we found a repeat pattern of ATTCC repeat motifs that have not been associated with seizures previously. Our data suggest that the absence of repeat interruptions is likely a genetic modifier for the clinical presentation of l-Dopa responsive parkinsonism, whereas repeat interruption motifs contribute clinically to epilepsy. Repeat interruptions are important genetic modifiers of the clinical phenotype in SCA10. Advanced sequencing techniques now allow to better characterize the underlying genetic architecture for determining accurate phenotype-genotype correlations.

13.
mBio ; 7(1): e01948-15, 2016 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-26861018

RESUMO

UNLABELLED: Deep metagenomic shotgun sequencing has emerged as a powerful tool to interrogate composition and function of complex microbial communities. Computational approaches to assemble genome fragments have been demonstrated to be an effective tool for de novo reconstruction of genomes from these communities. However, the resultant "genomes" are typically fragmented and incomplete due to the limited ability of short-read sequence data to assemble complex or low-coverage regions. Here, we use single-molecule, real-time (SMRT) sequencing to reconstruct a high-quality, closed genome of a previously uncharacterized Corynebacterium simulans and its companion bacteriophage from a skin metagenomic sample. Considerable improvement in assembly quality occurs in hybrid approaches incorporating short-read data, with even relatively small amounts of long-read data being sufficient to improve metagenome reconstruction. Using short-read data to evaluate strain variation of this C. simulans in its skin community at single-nucleotide resolution, we observed a dominant C. simulans strain with moderate allelic heterozygosity throughout the population. We demonstrate the utility of SMRT sequencing and hybrid approaches in metagenome quantitation, reconstruction, and annotation. IMPORTANCE: The species comprising a microbial community are often difficult to deconvolute due to technical limitations inherent to most short-read sequencing technologies. Here, we leverage new advances in sequencing technology, single-molecule sequencing, to significantly improve reconstruction of a complex human skin microbial community. With this long-read technology, we were able to reconstruct and annotate a closed, high-quality genome of a previously uncharacterized skin species. We demonstrate that hybrid approaches with short-read technology are sufficiently powerful to reconstruct even single-nucleotide polymorphism level variation of species in this a community.


Assuntos
Bacteriófagos/genética , Corynebacterium/genética , Corynebacterium/virologia , Metagenômica/métodos , Microbiota , Pele/microbiologia , Bacteriófagos/isolamento & purificação , Corynebacterium/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Dados de Sequência Molecular , Análise de Sequência de DNA/métodos
14.
PLoS One ; 10(4): e0123639, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25860355

RESUMO

The methylation of DNA bases plays an important role in numerous biological processes including development, gene expression, and DNA replication. Salmonella is an important foodborne pathogen, and methylation in Salmonella is implicated in virulence. Using single molecule real-time (SMRT) DNA-sequencing, we sequenced and assembled the complete genomes of eleven Salmonella enterica isolates from nine different serovars, and analysed the whole-genome methylation patterns of each genome. We describe 16 distinct N6-methyladenine (m6A) methylated motifs, one N4-methylcytosine (m4C) motif, and one combined m6A-m4C motif. Eight of these motifs are novel, i.e., they have not been previously described. We also identified the methyltransferases (MTases) associated with 13 of the motifs. Some motifs are conserved across all Salmonella serovars tested, while others were found only in a subset of serovars. Eight of the nine serovars contained a unique methylated motif that was not found in any other serovar (most of these motifs were part of Type I restriction modification systems), indicating the high diversity of methylation patterns present in Salmonella.


Assuntos
Metilação de DNA , Epigenômica , Genoma Bacteriano , Salmonella enterica/genética , Sequência de Bases , Perfilação da Expressão Gênica , Metiltransferases/genética , Motivos de Nucleotídeos
15.
Genome Announc ; 2(6)2014 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-25540345

RESUMO

Klebsiella pneumoniae is an important nosocomial pathogen, and multidrug-resistant strains have become a worldwide concern. Here, we report the complete genome of a K. pneumoniae isolate with chromosomally integrated blaKPC genes and a colibactin synthesis locus.

16.
Sci Transl Med ; 6(254): 254ra126, 2014 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-25232178

RESUMO

Public health officials have raised concerns that plasmid transfer between Enterobacteriaceae species may spread resistance to carbapenems, an antibiotic class of last resort, thereby rendering common health care-associated infections nearly impossible to treat. To determine the diversity of carbapenemase-encoding plasmids and assess their mobility among bacterial species, we performed comprehensive surveillance and genomic sequencing of carbapenem-resistant Enterobacteriaceae in the National Institutes of Health (NIH) Clinical Center patient population and hospital environment. We isolated a repertoire of carbapenemase-encoding Enterobacteriaceae, including multiple strains of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Enterobacter cloacae, Citrobacter freundii, and Pantoea species. Long-read genome sequencing with full end-to-end assembly revealed that these organisms carry the carbapenem resistance genes on a wide array of plasmids. K. pneumoniae and E. cloacae isolated simultaneously from a single patient harbored two different carbapenemase-encoding plasmids, indicating that plasmid transfer between organisms was unlikely within this patient. We did, however, find evidence of horizontal transfer of carbapenemase-encoding plasmids between K. pneumoniae, E. cloacae, and C. freundii in the hospital environment. Our data, including full plasmid identification, challenge assumptions about horizontal gene transfer events within patients and identify possible connections between patients and the hospital environment. In addition, we identified a new carbapenemase-encoding plasmid of potentially high clinical impact carried by K. pneumoniae, E. coli, E. cloacae, and Pantoea species, in unrelated patients and in the hospital environment.


Assuntos
Proteínas de Bactérias/biossíntese , Infecção Hospitalar , Enterobacteriaceae/enzimologia , Plasmídeos , beta-Lactamases/biossíntese , Enterobacteriaceae/classificação , Enterobacteriaceae/genética , Hospitais Públicos , Humanos , National Institutes of Health (U.S.) , Vigilância da População , Reação em Cadeia da Polimerase em Tempo Real , Estados Unidos
17.
Antimicrob Agents Chemother ; 58(10): 5947-53, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25070096

RESUMO

The whole-genome sequence of a carbapenem-resistant Klebsiella pneumoniae strain, PittNDM01, which coproduces NDM-1 and OXA-232 carbapenemases, was determined in this study. The use of single-molecule, real-time (SMRT) sequencing provided a closed genome in a single sequencing run. K. pneumoniae PittNDM01 has a single chromosome of 5,348,284 bp and four plasmids: pPKPN1 (283,371 bp), pPKPN2 (103,694 bp), pPKPN3 (70,814 bp), and pPKPN4 (6,141 bp). The contents of the chromosome were similar to that of the K. pneumoniae reference genome strain MGH 78578, with the exception of a large inversion spanning 23.3% of the chromosome. In contrast, three of the four plasmids are unique. The plasmid pPKPN1, an IncHI1B-like plasmid, carries the blaNDM-1, armA, and qnrB1 genes, along with tellurium and mercury resistance operons. blaNDM-1 is carried on a unique structure in which Tn125 is further bracketed by IS26 downstream of a class 1 integron. The IncFIA-like plasmid pPKPN3 also carries an array of resistance elements, including blaCTX-M-15 and a mercury resistance operon. The ColE-type plasmid pPKPN4 carrying blaOXA-232 is identical to a plasmid previously reported from France. SMRT sequencing was useful in resolving the complex bacterial genomic structures in the de novo assemblies.


Assuntos
Proteínas de Bactérias/metabolismo , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Genoma Bacteriano/genética , Klebsiella pneumoniae/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Óperon/genética , Plasmídeos/genética , beta-Lactamases/genética
18.
Genome Announc ; 2(2)2014 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-24699952

RESUMO

Bacteria belonging to the phylum Gemmatimonadetes are found in a wide variety of environments and are particularly abundant in soils. Here, we present the complete genome sequence and methylation pattern of the newly described Gemmatirosa kalamazoonensis type strain.

19.
J Nanobiotechnology ; 11: 8, 2013 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-23552456

RESUMO

BACKGROUND: Zero-mode waveguides (ZMWs) are photonic nanostructures that create highly confined optical observation volumes, thereby allowing single-molecule-resolved biophysical studies at relatively high concentrations of fluorescent molecules. This principle has been successfully applied in single-molecule, real-time (SMRT®) DNA sequencing for the detection of DNA sequences and DNA base modifications. In contrast, RNA sequencing methods cannot provide sequence and RNA base modifications concurrently as they rely on complementary DNA (cDNA) synthesis by reverse transcription followed by sequencing of cDNA. Thus, information on RNA modifications is lost during the process of cDNA synthesis. RESULTS: Here we describe an application of SMRT technology to follow the activity of reverse transcriptase enzymes synthesizing cDNA on thousands of single RNA templates simultaneously in real time with single nucleotide turnover resolution using arrays of ZMWs. This method thereby obtains information from the RNA template directly. The analysis of the kinetics of the reverse transcriptase can be used to identify RNA base modifications, shown by example for N6-methyladenine (m6A) in oligonucleotides and in a specific mRNA extracted from total cellular mRNA. Furthermore, the real-time reverse transcriptase dynamics informs about RNA secondary structure and its rearrangements, as demonstrated on a ribosomal RNA and an mRNA template. CONCLUSIONS: Our results highlight the feasibility of studying RNA modifications and RNA structural rearrangements in ZMWs in real time. In addition, they suggest that technology can be developed for direct RNA sequencing provided that the reverse transcriptase is optimized to resolve homonucleotide stretches in RNA.


Assuntos
Nanotecnologia/métodos , RNA Mensageiro/análise , Transcrição Reversa , DNA Complementar/análise , DNA Complementar/genética , Rearranjo Gênico , Cinética , Nanoestruturas/química , Nucleotídeos/química , RNA Mensageiro/química , RNA Mensageiro/genética , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Análise de Sequência de DNA/métodos
20.
J Biomed Sci ; 16: 12, 2009 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-19272177

RESUMO

BACKGROUND: Vascular endothelial cells (ECs) constantly experience fluid shear stresses generated by blood flow. Laminar flow is known to produce atheroprotective effects on ECs. Nrf2 is a transcription factor that is essential for the antioxidant response element (ARE)-mediated induction of genes such as heme-oxygenase 1 (HO-1). We previously showed that fluid shear stress increases intracellular reactive oxygen species (ROS) in ECs. Moreover, oxidants are known to stimulate Nrf2. We thus examined the regulation of Nrf2 in cultured human ECs by shear stress. RESULTS: Exposure of human umbilical vein endothelial cells (HUVECs) to laminar shear stress (12 dyne/cm2) induced Nrf2 nuclear translocation, which was inhibited by a phosphatidylinositol 3-kinase (PI3K) inhibitor, a protein kinase C (PKC) inhibitor, and an antioxidant agent N-acetyl cysteine (NAC), but not by other protein kinase inhibitors. Therefore, PI3K, PKC, and ROS are involved in the signaling pathway that leads to the shear-induced nuclear translocation of Nrf2. We also found that shear stress increased the ARE-binding activity of Nrf2 and the downstream expression of HO-1. CONCLUSION: Our data suggest that the atheroprotective effect of laminar flow is partially attributed to Nrf2 activation which results in ARE-mediated gene transcriptions, such as HO-1 expression, that are beneficial to the cardiovascular system.


Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Células Endoteliais/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Mecânico , Núcleo Celular/metabolismo , Células Cultivadas , Células Endoteliais/citologia , Inibidores Enzimáticos/metabolismo , Regulação da Expressão Gênica , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Fator 2 Relacionado a NF-E2/genética , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Oxidantes/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Resistência ao Cisalhamento
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