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1.
BMC Biol ; 22(1): 30, 2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38317114

RESUMO

BACKGROUND: Despite the increasing number of epigenomic studies in plants, little is known about the forces that shape the methylome in long-lived woody perennials. The Lombardy poplar offers an ideal opportunity to investigate the impact of the individual environmental history of trees on the methylome. RESULTS: We present the results of three interconnected experiments on Lombardy poplar. In the first experiment, we investigated methylome variability during a growing season and across vegetatively reproduced generations. We found that ramets collected over Europe and raised in common conditions have stable methylomes in symmetrical CG-contexts. In contrast, seasonal dynamics occurred in methylation patterns in CHH context. In the second experiment, we investigated whether methylome patterns of plants grown in a non-parental environment correlate with the parental climate. We did not observe a biological relevant pattern that significantly correlates with the parental climate. Finally, we investigated whether the parental environment has persistent carry-over effects on the vegetative offspring's phenotype. We combined new bud set observations of three consecutive growing seasons with former published bud set data. Using a linear mixed effects analysis, we found a statistically significant but weak short-term, parental carry-over effect on the timing of bud set. However, this effect was negligible compared to the direct effects of the offspring environment. CONCLUSIONS: Genome-wide cytosine methylation patterns in symmetrical CG-context are stable in Lombardy poplar and appear to be mainly the result of random processes. In this widespread poplar clone, methylation patterns in CG-context can be used as biomarkers to infer a common ancestor and thus to investigate the recent environmental history of a specific Lombardy poplar. The Lombardy poplar shows high phenotypic plasticity in a novel environment which enabled this clonal tree to adapt and survive all over the temperate regions of the world.


Assuntos
Adaptação Fisiológica , Epigenoma , Fenótipo , Estações do Ano , Metilação de DNA
2.
Clin Immunol ; 256: 109791, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37769787

RESUMO

Uncontrolled severe chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with elevated levels of type 2 inflammatory cytokines and raised immunoglobulin concentrations in nasal polyp tissue. By using single-cell RNA sequencing, transcriptomics, surface proteomics, and T cell and B cell receptor sequencing, we found the predominant cell types in nasal polyps were shifted from epithelial and mesenchymal cells to inflammatory cells compared to nasal mucosa from healthy controls. Broad expansions of CD4 T effector memory cells, CD4 tissue-resident memory T cells, CD8 T effector memory cells and all subtypes of B cells in nasal polyp tissues. The T and B cell receptor repertoires were skewed in NP. This study highlights the deviated immune response and remodeling mechanisms that contribute to the pathogenesis of uncontrolled severe CRSwNP. CLINICAL IMPLICATIONS: We identified differences in the cellular compositions, transcriptomes, proteomes, and deviations in the immune profiles of T cell and B cell receptors as well as alterations in the intercellular communications in uncontrolled severe CRSwNP patients versus healthy controls, which might help to define potential therapeutic targets in the future.


Assuntos
Pólipos Nasais , Rinite , Sinusite , Humanos , Rinite/metabolismo , Pólipos Nasais/patologia , Multiômica , Mucosa Nasal/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Doença Crônica
3.
Vet Res ; 51(1): 62, 2020 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-32381076

RESUMO

The porcine Helicobacter suis and canine-feline H. heilmannii are gastric Helicobacter species with zoonotic potential. However, little is known about the pathogenesis of human infections with these Helicobacter species. To gain more insight into the interactions of both zoonotic Helicobacter species with human gastric epithelial cells, we investigated bacterial genes that are differentially expressed in a H. suis and H. heilmannii strain after adhesion to the human gastric epithelial cell line MKN7. In vitro Helicobacter-MKN7 binding assays were performed to obtain bacterial RNA for sequencing analysis. H. suis and H. heilmannii bacteria attached to the gastric epithelial cells (i.e. cases) as well as unbound bacteria (i.e. controls) were isolated, after which prokaryotic RNA was purified and sequenced. Differentially expressed genes were identified using the DESeq2 package and SARTools pipeline in R. A list of 134 (83 up-regulated and 51 down-regulated) and 143 (60 up-regulated and 83 down-regulated) differentially expressed genes (padj ≤ 0.01; fold change ≥ 2) were identified for the adherent H. suis and H. heilmannii strains, respectively. According to BLASTp analyses, only 2 genes were commonly up-regulated and 4 genes commonly down-regulated in both pathogens. Differentially expressed genes of the H. suis and H. heilmannii strains belonged to multiple functional classes, indicating that adhesion of both strains to human gastric epithelial cells evokes pleiotropic adaptive responses. Our results suggest that distinct pathways are involved in human gastric colonization of H. suis and H. heilmannii. Further research is needed to elucidate the clinical significance of these findings.


Assuntos
Aderência Bacteriana , Regulação Bacteriana da Expressão Gênica , Helicobacter heilmannii/fisiologia , Transcriptoma , Linhagem Celular , Células Epiteliais , Expressão Gênica , Helicobacter heilmannii/classificação , Helicobacter heilmannii/genética , Humanos , Estômago
4.
Int J Mol Sci ; 19(8)2018 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-30110965

RESUMO

Plasmonic nanoparticles for drug delivery have attracted increasing interest over the last few years. Their localized surface plasmon resonance causes photothermal effects on laser irradiation, which allows for delivering drugs in a spatio-temporally controlled manner. Here, we explore the use of gold nanoparticles (AuNP) as carriers for pDNA in combination with pulsed laser irradiation to induce endosomal escape, which is currently considered to be one of the major bottlenecks in macromolecular drug delivery on the intracellular level. In particular, we evaluate nanocomplexes composed of JetPEI (polyethylenimine)pDNA and 10 nm AuNP, which do not exhibit endosomal escape by themselves. After incubating HeLa cells with these complexes, we evaluated endosomal escape and transfection efficiency using low- and high-energy laser pulses. At low laser energy heat is produced by the nanocomplexes, while, at higher laser energy, explosive vapour nanobubbles (VNB) are formed. We investigated the ability of heat transfer and VNB formation to induce endosomal escape and we examine the integrity of pDNA cargo after inducing both photothermal effects. We conclude that JetPEI/pDNA/AuNP complexes are unable to induce meaningful transfection efficiencies because laser treatment causes either dysfunctionality of the cargo when VNB are formed or forms too small pores in the endosomal membrane to allow pDNA to escape in case of heating. We conclude that laser-induced VNB is the most suitable to induce effective pDNA endosomal escape, but a different nanocomplex structure will be required to keep the pDNA intact.


Assuntos
Endossomos/metabolismo , Ouro , Hipertermia Induzida , Terapia com Luz de Baixa Intensidade , Nanopartículas Metálicas , Neoplasias/terapia , Polietilenoimina , Transfecção/métodos , DNA/genética , DNA/farmacologia , Endossomos/genética , Endossomos/patologia , Ouro/química , Ouro/farmacologia , Células HeLa , Humanos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Neoplasias/genética , Neoplasias/metabolismo , Polietilenoimina/química , Polietilenoimina/farmacologia
5.
Nucleic Acids Res ; 43(5): e29, 2015 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-25510491

RESUMO

An increasing amount of studies integrate mRNA sequencing data into MS-based proteomics to complement the translation product search space. However, several factors, including extensive regulation of mRNA translation and the need for three- or six-frame-translation, impede the use of mRNA-seq data for the construction of a protein sequence search database. With that in mind, we developed the PROTEOFORMER tool that automatically processes data of the recently developed ribosome profiling method (sequencing of ribosome-protected mRNA fragments), resulting in genome-wide visualization of ribosome occupancy. Our tool also includes a translation initiation site calling algorithm allowing the delineation of the open reading frames (ORFs) of all translation products. A complete protein synthesis-based sequence database can thus be compiled for mass spectrometry-based identification. This approach increases the overall protein identification rates with 3% and 11% (improved and new identifications) for human and mouse, respectively, and enables proteome-wide detection of 5'-extended proteoforms, upstream ORF translation and near-cognate translation start sites. The PROTEOFORMER tool is available as a stand-alone pipeline and has been implemented in the galaxy framework for ease of use.


Assuntos
Biologia Computacional/métodos , Espectrometria de Massas/métodos , Proteoma/metabolismo , Proteômica/métodos , Ribossomos/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Bases de Dados de Proteínas , Genoma/genética , Células HCT116 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Biossíntese de Proteínas/genética , Proteoma/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Ribossomos/genética , Homologia de Sequência de Aminoácidos
6.
Proteomics ; 14(23-24): 2688-98, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25156699

RESUMO

Next-generation transcriptome sequencing is increasingly integrated with MS to enhance MS-based protein and peptide identification. Recently, a breakthrough in transcriptome analysis was achieved with the development of ribosome profiling (ribo-seq). This technology is based on the deep sequencing of ribosome-protected mRNA fragments, thereby enabling the direct observation of in vivo protein synthesis at the transcript level. In order to explore the impact of a ribo-seq-derived protein sequence search space on MS/MS spectrum identification, we performed a comprehensive proteome study on a human cancer cell line, using both shotgun and N-terminal proteomics, next to ribosome profiling, which was used to delineate (alternative) translational reading frames. By including protein-level evidence of sample-specific genetic variation and alternative translation, this strategy improved the identification score of 69 proteins and identified 22 new proteins in the shotgun experiment. Furthermore, we discovered 18 new alternative translation start sites in the N-terminal proteomics data and observed a correlation between the quantitative measures of ribo-seq and shotgun proteomics with a Pearson correlation coefficient ranging from 0.483 to 0.664. Overall, this study demonstrated the benefits of ribosome profiling for MS-based protein and peptide identification and we believe this approach could develop into a common practice for next-generation proteomics.


Assuntos
Biologia Computacional/métodos , Proteínas/metabolismo , Proteômica/métodos , Ribossomos/metabolismo , Células HCT116 , Humanos , Biossíntese de Proteínas/genética , Proteínas/genética , Espectrometria de Massas em Tandem
7.
Genet Med ; 14(6): 576-85, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22261762

RESUMO

PURPOSE: Leber congenital amaurosis (LCA) is a rare congenital retinal dystrophy associated with 16 genes. Recent breakthroughs in LCA gene therapy offer the first prospect of treating inherited blindness, which requires an unequivocal and early molecular diagnosis. While present genetic tests do not address this due to a tremendous genetic heterogeneity, massively parallel sequencing (MPS) strategies might bring a solution. Here, we developed a comprehensive molecular test for LCA based on targeted MPS of all exons of 16 known LCA genes. METHODS: We designed a unique and flexible workflow for targeted resequencing of all 236 exons from 16 LCA genes based on quantitative PCR (qPCR) amplicon ligation, shearing, and parallel sequencing of multiple patients on a single lane of a short-read sequencer. Twenty-two prescreened LCA patients were included, five of whom had a known molecular cause. RESULTS: Validation of 107 variations was performed as proof of concept. In addition, the causal genetic defect and a single heterozygous mutation were identified in 3 and 5, respectively, of 17 patients without previously identified mutations. CONCLUSION: We propose a novel targeted MPS-based approach that is suitable for accurate, fast, and cost-effective early molecular testing in LCA, and easily applicable in other genetic disorders.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Amaurose Congênita de Leber/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Proteínas Adaptadoras de Transdução de Sinal , Antígenos de Neoplasias/genética , Biomarcadores/análise , Cegueira/congênito , Cegueira/genética , Proteínas de Transporte/genética , Estudos de Casos e Controles , Proteínas de Ciclo Celular , Criança , Pré-Escolar , Consanguinidade , Proteínas do Citoesqueleto , Éxons/genética , Proteínas do Olho/genética , Heterogeneidade Genética , Guanilato Ciclase/genética , Heterozigoto , Proteínas de Homeodomínio/genética , Humanos , Amaurose Congênita de Leber/genética , Proteínas de Membrana/genética , Mutação , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Receptores de Superfície Celular/genética , Transativadores/genética , Estudos de Validação como Assunto , cis-trans-Isomerases/genética
8.
Cell Rep ; 36(6): 109500, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34380029

RESUMO

Loss of function of adenosine deaminase acting on double-stranded RNA (dsRNA)-1 (ADAR1) causes the severe autoinflammatory disease Aicardi-Goutières syndrome (AGS). ADAR1 converts adenosines into inosines within dsRNA. This process called A-to-I editing masks self-dsRNA from detection by the antiviral dsRNA sensor MDA5. ADAR1 binds to dsRNA in both the canonical A-form and the poorly defined Z conformation (Z-RNA). Mutations in the Z-RNA-binding Zα domain of ADAR1 are common in patients with AGS. How loss of ADAR1/Z-RNA interaction contributes to disease development is unknown. We demonstrate that abrogated binding of ADAR1 to Z-RNA leads to reduced A-to-I editing of dsRNA structures formed by base pairing of inversely oriented short interspersed nuclear elements. Preventing ADAR1 binding to Z-RNA triggers an MDA5/MAVS-mediated type I interferon response and leads to the development of lethal autoinflammation in mice. This shows that the interaction between ADAR1 and Z-RNA restricts sensing of self-dsRNA and prevents AGS development.


Assuntos
Adenosina Desaminase/metabolismo , Imunidade , Helicase IFIH1 Induzida por Interferon/metabolismo , Edição de RNA/genética , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Desaminase/genética , Animais , Animais Recém-Nascidos , Linhagem Celular , Células HEK293 , Hematopoese , Heterozigoto , Humanos , Inflamação/patologia , Interferon Tipo I/metabolismo , Camundongos Endogâmicos C57BL , Mutação/genética , Ligação Proteica , Proteínas de Ligação a RNA/genética , Elementos Nucleotídeos Curtos e Dispersos/genética
9.
Front Cell Dev Biol ; 9: 583555, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33816458

RESUMO

Song learning in zebra finches (Taeniopygia guttata) is a prototypical example of a complex learned behavior, yet knowledge of the underlying molecular processes is limited. Therefore, we characterized transcriptomic (RNA-sequencing) and epigenomic (RRBS, reduced representation bisulfite sequencing; immunofluorescence) dynamics in matched zebra finch telencephalon samples of both sexes from 1 day post hatching (1 dph) to adulthood, spanning the critical period for song learning (20 and 65 dph). We identified extensive transcriptional neurodevelopmental changes during postnatal telencephalon development. DNA methylation was very low, yet increased over time, particularly in song control nuclei. Only a small fraction of the massive differential expression in the developing zebra finch telencephalon could be explained by differential CpG and CpH DNA methylation. However, a strong association between DNA methylation and age-dependent gene expression was found for various transcription factors (i.e., OTX2, AR, and FOS) involved in neurodevelopment. Incomplete dosage compensation, independent of DNA methylation, was found to be largely responsible for sexually dimorphic gene expression, with dosage compensation increasing throughout life. In conclusion, our results indicate that DNA methylation regulates neurodevelopmental gene expression dynamics through steering transcription factor activity, but does not explain sexually dimorphic gene expression patterns in zebra finch telencephalon.

10.
Sci Rep ; 10(1): 6285, 2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32269259

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

11.
Sci Rep ; 6: 20957, 2016 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-26864856

RESUMO

Learning and memory formation are known to require dynamic CpG (de)methylation and gene expression changes. Here, we aimed at establishing a genome-wide DNA methylation map of the zebra finch genome, a model organism in neuroscience, as well as identifying putatively epigenetically regulated genes. RNA- and MethylCap-seq experiments were performed on two zebra finch cell lines in presence or absence of 5-aza-2'-deoxycytidine induced demethylation. First, the MethylCap-seq methodology was validated in zebra finch by comparison with RRBS-generated data. To assess the influence of (variable) methylation on gene expression, RNA-seq experiments were performed as well. Comparison of RNA-seq and MethylCap-seq results showed that at least 357 of the 3,457 AZA-upregulated genes are putatively regulated by methylation in the promoter region, for which a pathway analysis showed remarkable enrichment for neurological networks. A subset of genes was validated using Exon Arrays, quantitative RT-PCR and CpG pyrosequencing on bisulfite-treated samples. To our knowledge, this study provides the first genome-wide DNA methylation map of the zebra finch genome as well as a comprehensive set of genes of which transcription is under putative methylation control.


Assuntos
Proteínas Aviárias/genética , Epigênese Genética , Tentilhões/genética , Genoma , Proteínas do Tecido Nervoso/genética , Animais , Proteínas Aviárias/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Ilhas de CpG , Metilação de DNA/efeitos dos fármacos , Decitabina , Feminino , Tentilhões/metabolismo , Redes Reguladoras de Genes , Aprendizagem/fisiologia , Masculino , Memória/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Análise de Sequência de RNA
12.
Sci Rep ; 5: 12810, 2015 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-26235384

RESUMO

By limiting sequencing to those sequences transcribed as mRNA, whole exome sequencing is a cost-efficient technique often used in disease-association studies. We developed two target enrichment designs based on the recently released annotation of the canine genome: the exome-plus design and the exome-CDS design. The exome-plus design combines the exons of the CanFam 3.1 Ensembl annotation, more recently discovered protein-coding exons and a variety of non-coding RNA regions (microRNAs, long non-coding RNAs and antisense transcripts), leading to a total size of ≈ 152 Mb. The exome-CDS was designed as a subset of the exome-plus by omitting all 3' and 5' untranslated regions. This reduced the size of the exome-CDS to ≈ 71 Mb. To test the capturing performance, four exome-plus captures were sequenced on a NextSeq 500 with each capture containing four pre-capture pooled, barcoded samples. At an average sequencing depth of 68.3x, 80% of the regions and well over 90% of the targeted base pairs were completely covered at least 5 times with high reproducibility. Based on the performance of the exome-plus, we estimated the performance of the exome-CDS. Overall, these designs provide flexible solutions for a variety of research questions and are likely to be reliable tools in disease studies.


Assuntos
Cães/genética , Exoma , Genômica/métodos , Proteínas/genética , RNA não Traduzido , Animais , Composição de Bases , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reprodutibilidade dos Testes
13.
Sci Rep ; 4: 5597, 2014 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-24998260

RESUMO

Whole exome sequencing is a technique that aims to selectively sequence all exons of protein-coding genes. A canine whole exome sequencing enrichment kit was designed based on the latest canine reference genome (build 3.1.72). Its performance was tested by sequencing 2 exome captures, each consisting of 4 pre-capture pooled, barcoded Illumina libraries on an Illumina HiSeq 2500. At an average sequencing depth of 102x, 83 to 86% of the target regions were completely sequenced with a minimum coverage of five and 90% of the reads mapped on the target regions. Additionally, it is shown that the reproducibility within and between captures is high and that pooling four samples per capture is a valid option. Overall, we have demonstrated the strong performance of this WES enrichment kit and are confident it will be a valuable tool in future disease association studies.


Assuntos
Exoma , Análise de Sequência de DNA , Animais , Cães , Sequenciamento de Nucleotídeos em Larga Escala , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
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