RESUMO
BACKGROUND: High-quality malaria diagnosis is essential for effective treatment and clinical disease management. Microscopy and rapid diagnostic tests are the conventional methods performed as first-line malaria diagnostics in non-endemic countries. However, these methods lack the characteristic to detect very low parasitaemia, and accurate identification of the Plasmodium species can be difficult. This study evaluated the performance of the MC004 melting curve-based qPCR for the diagnosis of malaria in routine clinical practice in non-endemic setting. METHODS AND RESULTS: Whole blood samples were collected from 304 patients with clinical suspicion of malaria and analysed by both the MC004 assay and conventional diagnostics. Two discrepancies were found between the MC004 assay and microscopy. Repeated microscopic analysis confirmed the qPCR results. Comparison of the parasitaemia of nineteen Plasmodium falciparum samples determined by both microscopy and qPCR showed the potential of the MC004 assay to estimate the parasite load of P. falciparum. Eight Plasmodium infected patients were followed after anti-malarial treatment by the MC004 assay and microscopy. The MC004 assay still detected Plasmodium DNA although no parasites were seen with microscopy in post-treatment samples. The rapid decline in Plasmodium DNA showed the potential for therapy-monitoring. CONCLUSION: Implementation of the MC004 assay in non-endemic clinical setting improved the diagnosis of malaria. The MC004 assay demonstrated superior Plasmodium species identification, the ability to indicate the Plasmodium parasite load, and can potentially detect submicroscopic Plasmodium infections.
Assuntos
Malária Falciparum , Malária , Plasmodium , Humanos , Malária Falciparum/diagnóstico , Malária Falciparum/parasitologia , Malária/diagnóstico , Malária/parasitologia , Plasmodium falciparum/genética , Microscopia/métodos , Parasitemia/diagnóstico , Parasitemia/parasitologia , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: The entry of PCR-based techniques into malaria diagnostics has improved the sensitivity and specificity of the detection of Plasmodium infections. It has been shown that humans are regularly infected by at least six different Plasmodium species. The MC004 real-time PCR assay for malaria diagnosis is a novel single-tube assay that has been developed for the purpose of simultaneously detecting all Plasmodium species known to infect humans, and discrimination between Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale wallikeri, Plasmodium ovale curtisi, Plasmodium knowlesi (including differentiation of three strains) and Plasmodium cynomolgi (including differentiation of three strains). Detection and identification of Plasmodium species relies on molecular beacon probe-based melting curve analysis. In addition, this assay might be used to quantify the parasitaemia of at least P. falciparum by calculating the level of parasitaemia directly from the Cq-value. METHODS: The samples used in this study comprised reference samples, patient samples, and synthetic controls. The following analytical performance characteristics of the MC004 assay were determined: analytical specificity, limit of detection, the ability to detect mixed infections, and the potential to determine the level of parasitaemia of P. falciparum, including assessment of within-run and between-run precisions. RESULTS: No false positive or false negative results were observed. The limit of detection of P. falciparum was 1 × 10-3 IU/mL (WHO standard). Mixed infections with P. falciparum and non-falciparum species were correctly identified. A calibration curve could be established to quantify the parasitaemia of at least P. falciparum. The within-run and between-run precisions were less than 20% CV at the tested parasitaemia levels of 0.09%, 0.16%, 2.15% and 27.27%. CONCLUSION: Based upon the analytical performance characteristics that were determined, the MC004 assay showed performance suitable for use in clinical settings, as well as epidemiological studies.
Assuntos
Malária/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Humanos , Plasmodium falciparum/genética , Plasmodium ovale/genética , Plasmodium vivax/genética , Sensibilidade e EspecificidadeRESUMO
During embryonic development, maintenance of cell identity and lineage commitment requires the Polycomb-group PRC2 complex, which catalyzes histone H3 lysine 27 trimethylation (H3K27me3). However, the developmental origins of this regulation are unknown. Here we show that H3K27me3 enrichment increases from blastula stages onward in embryos of the Western clawed frog (Xenopus tropicalis) within constrained domains strictly defined by sequence. Strikingly, although PRC2 also binds widely to active enhancers, H3K27me3 is only deposited at a small subset of these sites. Using a Support Vector Machine algorithm, these sequences can be predicted accurately on the basis of DNA sequence alone, with a sequence signature conserved between humans, frogs, and fish. These regions correspond to the subset of blastula-stage DNA methylation-free domains that are depleted for activating promoter motifs, and enriched for motifs of developmental factors. These results imply a genetic-default model in which a preexisting absence of DNA methylation is the major determinant of H3K27 methylation when not opposed by transcriptional activation. The sequence and motif signatures reveal the hierarchical and genetically inheritable features of epigenetic cross-talk that impose constraints on Polycomb regulation and guide H3K27 methylation during the exit of pluripotency.
Assuntos
Blástula/metabolismo , Núcleo Celular/genética , Gástrula/metabolismo , Histonas/metabolismo , Complexo Repressor Polycomb 2/fisiologia , Proteínas de Xenopus/genética , Xenopus/embriologia , Animais , Sequência de Bases , Sequência Conservada , Metilação de DNA , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Processamento de Proteína Pós-Traducional , Máquina de Vetores de Suporte , Xenopus/genética , Xenopus/metabolismoRESUMO
Zinc (Zn) is an essential nutrient for plants, with a crucial role as a cofactor for many enzymes. Approximately one-third of the global arable land area is Zn deficient, leading to reduced crop yield and quality. To improve crop tolerance to Zn deficiency, it is important to understand the mechanisms plants have adopted to tolerate suboptimal Zn supply. In this study, physiological and molecular aspects of traits related to Zn deficiency tolerance were examined in a panel of 19 Arabidopsis thaliana accessions. Accessions showed a larger variation for shoot biomass than for Zn concentration, indicating that they have different requirements for their minimal Zn concentration required for growth. Accessions with a higher tolerance to Zn deficiency showed an increased expression of the Zn deficiency-responsive genes ZIP4 and IRT3 in comparison with Zn deficiency-sensitive accessions. Changes in the shoot ionome, as a result of the Zn treatment of the plants, were used to build a multinomial logistic regression model able to distinguish plants regarding their Zn nutritional status. This set of biomarkers, reflecting the A. thaliana response to Zn deficiency and Zn deficiency tolerance, can be useful for future studies aiming to improve the performance and Zn status of crop plants grown under suboptimal Zn concentrations.
Assuntos
Arabidopsis/fisiologia , Biomassa , Expressão Gênica , Zinco/deficiência , Arabidopsis/genética , Biomarcadores/metabolismo , Variação Genética , Íons/metabolismo , Brotos de Planta/metabolismoRESUMO
Transcription initiation involves the recruitment of basal transcription factors to the core promoter. A variety of core promoter elements exists; however for most of these motifs, the distribution across species is unknown. Here we report on the comparison of human and amphibian promoter sequences. We have used oligo-capping in combination with deep sequencing to determine transcription start sites in Xenopus tropicalis. To systematically predict regulatory elements, we have developed a de novo motif finding pipeline using an ensemble of computational tools. A comprehensive comparison of human and amphibian promoter sequences revealed both similarities and differences in core promoter architecture. Some of the differences stem from a highly divergent nucleotide composition of Xenopus and human promoters. Whereas the distribution of some core promoter motifs is conserved independently of species-specific nucleotide bias, the frequency of another class of motifs correlates with the single nucleotide frequencies. This class includes the well-known TATA box and SP1 motifs, which are more abundant in Xenopus and human promoters, respectively. While these motifs are enriched above the local nucleotide background in both organisms, their frequency varies in step with this background. These differences are likely adaptive as these motifs can recruit TFIID to either CpG island or sharply initiating promoters. Our results highlight both the conserved and diverged aspects of vertebrate transcription, most notably showing co-opted motif usage to recruit the transcriptional machinery to promoters with diverging nucleotide composition. This shows how sweeping changes in nucleotide composition are compatible with highly conserved mechanisms of transcription initiation.
Assuntos
Sequência Conservada , Transcrição Gênica , Adaptação Biológica , Animais , Sequência de Bases , Ilhas de CpG , Feminino , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Homologia de Sequência do Ácido Nucleico , TATA Box , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/metabolismo , Sítio de Iniciação de Transcrição , XenopusRESUMO
The tumor suppressor p53 is a sequence-specific transcription factor, which regulates the expression of target genes involved in different stress responses. To understand p53's essential transcriptional functions, unbiased analysis of its DNA-binding repertoire is pivotal. In a genome-wide tiling ChIP-on-chip approach, we have identified and characterized 1546 binding sites of p53 upon Actinomycin D treatment. Among those binding sites were known as well as novel p53 target sites, which included regulatory regions of potentially novel transcripts. Using this collection of genome-wide binding sites, a new high-confidence algorithm was developed, p53scan, to identify the p53 consensus-binding motif. Strikingly, this motif was present in the majority of all bound sequences with 83% of all binding sites containing the motif. In the surrounding sequences of the binding sites, several motifs for potential regulatory cobinders were identified. Finally, we show that the majority of the genome-wide p53 target sites can also be bound by overexpressed p63 and p73 in vivo, suggesting that they can possibly play an important role at p53 binding sites. This emphasizes the possible interplay of p53 and its family members in the context of target gene binding. Our study greatly expands the known, experimentally validated p53 binding site repertoire and serves as a valuable knowledgebase for future research.
Assuntos
Elementos Reguladores de Transcrição , Proteína Supressora de Tumor p53/metabolismo , Algoritmos , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Sequência Consenso , Proteínas de Ligação a DNA/metabolismo , Dactinomicina/farmacologia , Genômica , Humanos , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Transcrição/metabolismo , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/metabolismoRESUMO
All living organisms require a variety of essential elements for their basic biological functions. While the homeostasis of nutrients is highly intertwined, the molecular and genetic mechanisms of these dependencies remain poorly understood. Here, we report a discovery of a molecular pathway that controls phosphate (Pi) accumulation in plants under Zn deficiency. Using genome-wide association studies, we first identified allelic variation of the Lyso-PhosphatidylCholine (PC) AcylTransferase 1 (LPCAT1) gene as the key determinant of shoot Pi accumulation under Zn deficiency. We then show that regulatory variation at the LPCAT1 locus contributes significantly to this natural variation and we further demonstrate that the regulation of LPCAT1 expression involves bZIP23 TF, for which we identified a new binding site sequence. Finally, we show that in Zn deficient conditions loss of function of LPCAT1 increases the phospholipid Lyso-PhosphatidylCholine/PhosphatidylCholine ratio, the expression of the Pi transporter PHT1;1, and that this leads to shoot Pi accumulation.
Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Homeostase , Fosfatos/metabolismo , Oligoelementos/metabolismo , Zinco/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferase/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Regulação da Expressão Gênica de Plantas , Estudo de Associação Genômica Ampla , Brotos de Planta/enzimologia , Brotos de Planta/metabolismo , Ligação ProteicaRESUMO
BACKGROUND: Cytochrome P450 (CYP) isoenzymes (CYP2C and CYP2J) are involved in the production of epoxyeicosatrienoic acids, which are postulated as endothelium-derived hyperpolarizing factors (EDHFs). We hypothesized that if CYP2C11 is involved in the EDHF-mediated responses, its mRNA should be expressed in endothelial cells. We, therefore, examined the mRNA expression of CYP2C11 in endothelial cells of renal arterioles. METHODS: Laser microdissection was applied to isolate endothelial cells from the renal arterioles of 4 male and 4 female Wistar rats. As a positive control of CYP2C11 expression, hepatocytes were also dissected from these rats. RNA was isolated and real-time quantitative polymerase chain reaction (Q-PCR) analysis was applied. RESULTS: Q-PCR analysis showed that CYP2C11 mRNA was not expressed in laser microdissected endothelial cells of renal arterioles of male and female rats. CYP2C11 mRNA expression was highly abundant in hepatocytes dissected from male livers, but in female livers hardly any CYP2C11 mRNA was detected. CONCLUSION: We have shown that endothelial cells can be dissected from small renal arterioles by laser microdissection to study the mRNA expression of specific genes by Q-PCR. Using this novel tool, we demonstrated that the CYP2C11 mRNA was not expressed in the endothelial cells of renal arterioles. Therefore, we speculate that CYP2C11 does not contribute to the EDHF-mediated responses in renal arterioles.
Assuntos
Arteríolas/enzimologia , Hidrocarboneto de Aril Hidroxilases/metabolismo , Células Endoteliais/enzimologia , Rim/irrigação sanguínea , Rim/enzimologia , RNA Mensageiro/metabolismo , Esteroide 16-alfa-Hidroxilase/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Células Cultivadas , Família 2 do Citocromo P450 , Feminino , Técnicas In Vitro , Masculino , Ratos , Ratos Wistar , Esteroide 16-alfa-Hidroxilase/genéticaRESUMO
Chromatin immunoprecipitation (ChIP) is a powerful technique to study epigenetic regulation and transcription factor binding events in the nucleus. It is based on immune-affinity capture of epitopes that have been cross-linked to genomic DNA in vivo. A readout of the extent to which the epitope is associated with particular genomic regions can be obtained by quantitative PCR (ChIP-qPCR), microarray hybridization (ChIP-chip), or deep sequencing (ChIP-seq). ChIP can be used for molecular and quantitative analyses of histone modifications, transcription factors, and elongating RNA polymerase II at specific loci. It can also be applied to assess the cellular state of transcriptional activation or repression as a predictor of the cells' capabilities and potential. Another possibility is to employ ChIP to characterize genomes, as histone modifications and binding events occur at specific and highly characteristic genomic elements and locations. This chapter provides a step-by-step protocol of ChIP using early Xenopus embryos and discusses potential pitfalls and other issues relevant for successful probing of protein-genome interactions by ChIP-qPCR and ChIP-seq.
Assuntos
Embrião não Mamífero/citologia , Xenopus/genética , Animais , Anticorpos/química , Anticorpos/imunologia , Especificidade de Anticorpos , Sequência de Bases , Cromatina/genética , Cromatina/isolamento & purificação , Imunoprecipitação da Cromatina , DNA/genética , DNA/isolamento & purificação , Primers do DNA/genética , Proteínas Fetais/genética , Loci Gênicos , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/metabolismo , Metilação , Análise de Sequência com Séries de Oligonucleotídeos , Processamento de Proteína Pós-Traducional , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Sonicação , Proteínas com Domínio T/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/imunologia , Proteínas de Xenopus/metabolismoRESUMO
BACKGROUND: Chromatin immunoprecipitation combined with genome tile path microarrays or deep sequencing can be used to study genome-wide epigenetic profiles and the transcription factor binding repertoire. Although well studied in a variety of cell lines, these genome-wide profiles have so far been little explored in vertebrate embryos. PRINCIPAL FINDINGS: Here we report on two genome tile path ChIP-chip designs for interrogating the Xenopus tropicalis genome. In particular, a whole-genome microarray design was used to identify active promoters by close proximity to histone H3 lysine 4 trimethylation. A second microarray design features these experimentally derived promoter regions in addition to currently annotated 5' ends of genes. These regions truly represent promoters as shown by binding of TBP, a key transcription initiation factor. CONCLUSIONS: A whole-genome and a promoter tile path microarray design was developed. Both designs can be used to study epigenetic phenomena and transcription factor binding in developing Xenopus embryos.
Assuntos
Imunoprecipitação da Cromatina , Epigênese Genética , Genoma , Fatores de Transcrição/metabolismo , Xenopus/embriologia , Animais , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Ligação Proteica , Xenopus/genéticaRESUMO
Epigenetic mechanisms set apart the active and inactive regions in the genome of multicellular organisms to produce distinct cell fates during embryogenesis. Here, we report on the epigenetic and transcriptome genome-wide maps of gastrula-stage Xenopus tropicalis embryos using massive parallel sequencing of cDNA (RNA-seq) and DNA obtained by chromatin immunoprecipitation (ChIP-seq) of histone H3 K4 and K27 trimethylation and RNA Polymerase II (RNAPII). These maps identify promoters and transcribed regions. Strikingly, genomic regions featuring opposing histone modifications are mostly transcribed, reflecting spatially regulated expression rather than bivalency as determined by expression profile analyses, sequential ChIP, and ChIP-seq on dissected embryos. Spatial differences in H3K27me3 deposition are predictive of localized gene expression. Moreover, the appearance of H3K4me3 coincides with zygotic gene activation, whereas H3K27me3 is predominantly deposited upon subsequent spatial restriction or repression of transcriptional regulators. These results reveal a hierarchy in the spatial control of zygotic gene activation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Animais , Epigênese Genética , Gástrula/metabolismo , Genoma , Humanos , Camundongos , Modelos Biológicos , Modelos Genéticos , Nucleossomos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Tempo , Xenopus/embriologia , Xenopus laevis/embriologiaRESUMO
In addition to TATA-binding protein (TBP), a key factor for transcription initiation, the metazoan-specific TBP-like factor TLF/TRF2 and the vertebrate-specific factor TBP2/TRF3 are known to be required for transcription of specific subsets of genes. We have combined an antisense-knockdown approach with transcriptome profiling to determine the significance and biological role of TBP-independent transcription in early gastrula-stage Xenopus laevis embryos. Here, we report that, although each of the TBP family members is essential for embryonic development, relatively few genes depend on TBP in the embryo. Most of the transcripts that depend on TBP in the embryo are also expressed maternally and in adult stages, and show no functional specialization. In contrast, TLF is linked to preferential expression in embryos and shows functional specialization in catabolism. A requirement for TBP2 is linked to vertebrate-specific embryonic genes and ventral-specific expression. Therefore TBP paralogs are essential for the gene-regulatory repertoire that is directly linked to early embryogenesis.