RESUMO
Interleukin 37 (IL-37) and IL-1R8 (SIGIRR or TIR8) are anti-inflammatory orphan members of the IL-1 ligand family and IL-1 receptor family, respectively. Here we demonstrate formation and function of the endogenous ligand-receptor complex IL-37-IL-1R8-IL-18Rα. The tripartite complex assembled rapidly on the surface of peripheral blood mononuclear cells upon stimulation with lipopolysaccharide. Silencing of IL-1R8 or IL-18Rα impaired the anti-inflammatory activity of IL-37. Whereas mice with transgenic expression of IL-37 (IL-37tg mice) with intact IL-1R8 were protected from endotoxemia, IL-1R8-deficient IL-37tg mice were not. Proteomic and transcriptomic investigations revealed that IL-37 used IL-1R8 to harness the anti-inflammatory properties of the signaling molecules Mer, PTEN, STAT3 and p62(dok) and to inhibit the kinases Fyn and TAK1 and the transcription factor NF-κB, as well as mitogen-activated protein kinases. Furthermore, IL-37-IL-1R8 exerted a pseudo-starvational effect on the metabolic checkpoint kinase mTOR. IL-37 thus bound to IL-18Rα and exploited IL-1R8 to activate a multifaceted intracellular anti-inflammatory program.
Assuntos
Subunidade alfa de Receptor de Interleucina-18/imunologia , Interleucina-1/imunologia , Leucócitos Mononucleares/imunologia , Receptores de Interleucina-1/imunologia , Transdução de Sinais/imunologia , Animais , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-1/genética , Subunidade alfa de Receptor de Interleucina-18/antagonistas & inibidores , Subunidade alfa de Receptor de Interleucina-18/genética , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/patologia , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/imunologia , Camundongos , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/imunologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/imunologia , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/imunologia , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/deficiência , Receptores de Interleucina-1/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/imunologia , c-Mer Tirosina QuinaseRESUMO
The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Meanwhile, increased demand for testing has led to a shortage of reagents and supplies and compromised the performance of diagnostic laboratories in many countries. Both the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate the interpretation of the test results, especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in diagnostics as well as in research labs using a low-cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infections and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for the quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.
Assuntos
Técnicas de Laboratório Clínico/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Betacoronavirus/genética , Teste para COVID-19 , Células CACO-2 , Chlorocebus aethiops , Técnicas de Laboratório Clínico/economia , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/economia , Proteínas M de Coronavírus , Custos e Análise de Custo , Humanos , RNA Viral/química , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , SARS-CoV-2 , Sensibilidade e Especificidade , Células Vero , Proteínas da Matriz Viral/genéticaRESUMO
As for many opportunistic pathogens, the virulence potential of Listeria monocytogenes is highly heterogeneous between isolates and correlated, to some extent, with phylogeny and gene repertoires. In sharp contrast with copious data on intraspecies genome diversity, little is known about transcriptome diversity despite the role of complex genetic regulation in pathogenicity. The current study implemented RNA sequencing to characterize the transcriptome profiles of 33 isolates under optimal in vitro growth conditions. Transcript levels of conserved single-copy genes were comprehensively explored from several perspectives, including phylogeny, in silico-predicted virulence category based on epidemiological multilocus sequence typing (MLST) data, and in vivo virulence phenotype assessed in Galleria mellonella Comparing baseline transcriptomes between isolates was intrinsically more complex than standard genome comparison because of the inherent plasticity of gene expression in response to environmental conditions. We show that the relevance of correlation analyses and their statistical power can be enhanced by using principal-component analysis to remove the first level of irrelevant, highly coordinated changes linked to growth phase. Our results highlight the major contribution of transcription factors with key roles in virulence to the diversity of transcriptomes. Divergence in the basal transcript levels of a substantial fraction of the transcriptome was observed between lineages I and II, echoing previously reported epidemiological differences. Correlation analysis with in vivo virulence identified numerous sugar metabolism-related genes, suggesting that specific pathways might play roles in the onset of infection in G. mellonellaIMPORTANCEListeria monocytogenes is a multifaceted bacterium able to proliferate in a wide range of environments from soil to mammalian host cells. The accumulated genomic data underscore the contribution of intraspecies variations in gene repertoire to differential adaptation strategies between strains, including infection and stress resistance. It seems very likely that the fine-tuning of the transcriptional regulatory network is also a key component of the phenotypic diversity, albeit more difficult to investigate than genome content. Some studies reported incongruity in the basal transcriptome between isolates, suggesting a putative relationship with phenotypes, but small isolate numbers hampered proper correlation analyses with respect to their characteristics. The present study is the embodiment of the promising approach that consists of analyzing correlations between transcriptomes and various isolate characteristics. Statistically significant correlations were found with phylogenetic groups, epidemiological evidence of virulence potential, and virulence in Galleria mellonella larvae used as an in vivo model.
Assuntos
Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Mariposas/microbiologia , Transcriptoma , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Genoma Bacteriano/genética , Genômica , Humanos , Larva/microbiologia , Listeria monocytogenes/classificação , Listeria monocytogenes/isolamento & purificação , Tipagem de Sequências Multilocus , Fenótipo , Filogenia , Regulon , Virulência/genética , Sequenciamento Completo do GenomaRESUMO
BACKGROUND: The European spurge hawkmoth, Hyles euphorbiae (Lepidoptera, Sphingidae), has been intensively studied as a model organism for insect chemical ecology, cold hardiness and evolution of species delineation. To understand species isolation mechanisms at a molecular level, this study aims at determining genetic factors underlying two adaptive ecological trait candidates, phorbol ester (TPA) detoxification and seasonal cold acclimation. METHOD: A draft transcriptome of H. euphorbiae was generated using Illumina sequencing, providing the first genomic resource for the hawkmoth subfamily Macroglossinae. RNA expression levels in tissues of experimental TPA feeding larvae and cooled pupae was compared to levels in control larvae and pupae using 26 bp RNA sequence tag libraries (DeepSuperSAGE). Differential gene expression was assessed by homology searches of the tags in the transcriptome. RESULTS: In total, 389 and 605 differentially expressed transcripts for detoxification and cold hardiness, respectively, could be identified and annotated with proteins. The majority (22 of 28) of differentially expressed detox transcripts of the four 'drug metabolism' enzyme groups (cytochrome P450 (CYP), carboxylesterases (CES), glutathione S-transferases (GST) and lipases) are up-regulated. Triacylglycerol lipase was significantly over proportionally annotated among up-regulated detox transcripts. We record several up-regulated lipases, GSTe2, two CESs, CYP9A21, CYP6BD6 and CYP9A17 as candidate genes for further H. euphorbiae TPA detoxification analyses. Differential gene expression of the cold acclimation treatment is marked by metabolic depression with enriched Gene Ontology terms among down-regulated transcripts almost exclusively comprising metabolism, aerobic respiration and dissimilative functions. Down-regulated transcripts include energy expensive respiratory proteins like NADH dehydrogenase, cytochrome oxidase and ATP synthase. Gene expression patterns show shifts in carbohydrate metabolism towards cryoprotectant production. The Glycolysis enzymes, G1Pase, A1e, Gpi and an Akr isoform are up-regulated. Glycerol, an osmolyte which lowers the body liquid supercooling point, appears to be the predominant polyol cryoprotectant in H. euphorbiae diapause pupae. Several protein candidates involved in glucose, glycerol, myo-inositol and potentially sorbitol and trehalose synthesis were identified. CONCLUSIONS: A majority of differently expressed transcripts unique for either detoxification or cold hardiness indicates highly specialized functional adaptation which may have evolved from general cell metabolism and stress response.The transcriptome and extracted candidate biomarkers provide a basis for further gene expression studies of physiological processes and adaptive traits in H. euphorbiae.
RESUMO
Human monocytes are subdivided into classical, intermediate, and nonclassical subsets, but there is no unequivocal strategy to dissect the latter 2 cell types. We show herein that the cell surface marker 6-sulfo LacNAc (slan) can define slan-positive CD14(+)CD16(++) nonclassical monocytes and slan-negative CD14(++)CD16(+) intermediate monocytes. Gene expression profiling confirms that slan-negative intermediate monocytes show highest expression levels of major histocompatibility complex class II genes, whereas a differential ubiquitin signature is a novel feature of the slan approach. In unsupervised hierarchical clustering, the slan-positive nonclassical monocytes cluster with monocytes and are clearly distinct from CD1c(+) dendritic cells. In clinical studies, we show a selective increase of the slan-negative intermediate monocytes to >100 cells per microliter in patients with sarcoidosis and a fivefold depletion of the slan-positive monocytes in patients with hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS), which is caused by macrophage colony-stimulating factor (M-CSF) receptor mutations. These data demonstrate that the slan-based definition of CD16-positive monocyte subsets is informative in molecular studies and in clinical settings.
Assuntos
Amino Açúcares/análise , Monócitos/classificação , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptores de IgG/análise , Antígenos CD1/análise , Células Dendríticas/química , Feminino , Citometria de Fluxo , Proteínas Ligadas por GPI/análise , Perfilação da Expressão Gênica , Genes MHC da Classe II , Estudo de Associação Genômica Ampla , Glicoproteínas/análise , Antígenos HLA-D/análise , Humanos , Separação Imunomagnética , Leucoencefalopatias/genética , Leucoencefalopatias/imunologia , Leucoencefalopatias/patologia , Receptores de Lipopolissacarídeos/análise , Masculino , Pessoa de Meia-Idade , Monócitos/química , Monócitos/imunologia , Mutação Puntual , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoidose/imunologia , Sarcoidose/patologia , Adulto JovemRESUMO
KEY MESSAGE: Molecular markers including a potential resistance gene co-segregating with the LpPg1 stem rust resistance locus in perennial ryegrass were identified by massive analysis of cDNA ends (MACE) transcriptome profiling. Stem rust caused by Puccinia graminis subsp. graminicola is a severe fungal disease in the forage crop perennial ryegrass and other grasses. The previously identified LpPg1 locus confers efficient resistance against the pathogen. The aim of this study was to identify candidate genes involved in rust resistance and to use them as a resource for the development of molecular markers for LpPg1. To identify such candidates, bulked segregant analysis was combined with NGS-based massive analysis of cDNA ends (MACE) transcriptome profiling. Total RNA was isolated from bulks of infected and non-infected leaf segments from susceptible and resistant genotypes of a full-sibling mapping population and their respective parental lines and MACE was performed. Bioinformatic analysis detected 330 resistance-specific SNPs in 178 transcripts and 341 transcripts that were exclusively expressed in the resistant bulk. The sequences of many of these transcripts were homologous to genes in distinct regions of chromosomes one and four of the model grass Brachypodium distachyon. Of these, 30 were genetically mapped to a 50.8 cM spanning region surrounding the LpPg1 locus. One candidate NBS-LRR gene co-segregated with the resistance locus. Quantitative analysis of gene expression suggests that LpPg1 mediates an efficient resistance mechanism characterized by early recognition of the pathogen, fast defense signaling and rapid induction of antifungal proteins. We demonstrate here that MACE is a cost-efficient, fast and reliable tool that detects polymorphisms for genetic mapping of candidate resistance genes and simultaneously reveals deep insight into the molecular and genetic base of resistance.
Assuntos
Resistência à Doença/genética , Genes de Plantas , Lolium/genética , Doenças das Plantas/genética , Análise de Sequência de DNA/métodos , Basidiomycota , Biologia Computacional , DNA Complementar/genética , DNA de Plantas/genética , Marcadores Genéticos , Lolium/microbiologia , Doenças das Plantas/microbiologia , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND: Treatment of iron deficiency with intravenous (i.v.) iron is a first-line strategy to improve anaemia of chronic kidney disease. Previous in vitro experiments demonstrated that different i.v. iron preparations inhibit differentiation of haematopoietic stem cells to monocytes, but their effect on monocyte differentiation to macrophages and mature dendritic cells (mDCs) has not been assessed. We investigated substance-specific effects of iron sucrose (IS), sodium ferric gluconate (SFG), ferric carboxymaltose (FCM) and iron isomaltoside 1000 (IIM) on monocytic differentiation to M1/M2 macrophages and mDCs. METHODS: Via flow cytometry and microRNA (miRNA) expression analysis, we morphologically and functionally characterized monocyte differentiation to M1/M2 macrophages and mDCs after monocyte stimulation with IS, SFG, FCM and IIM (0.133, 0.266 and 0.533 mg/mL, respectively). To assess potential clinical implications, we compared monocytic phagocytosis capacity in dialysis patients who received either 500 mg IS or IIM. RESULTS: Phenotypically, IS and SFG dysregulated the expression of macrophage (e.g. CD40, CD163) and mDC (e.g. CD1c, CD141) surface markers. Functionally, IS and SFG impaired macrophage phagocytosis capacity. Phenotypic and functional alterations were less pronounced with FCM, and virtually absent with IIM. In miRNA expression analysis of mDCs, IS dysregulated miRNAs such as miR-146b-5p and miR-155-5p, which are linked to Toll-like receptor and mitogen-activated protein kinase signalling pathways. In vivo, IS reduced monocytic phagocytosis capacity within 1 h after infusion, while IIM did not. CONCLUSIONS: This study demonstrates that less stable i.v. iron preparations specifically affect monocyte differentiation towards macrophages and mDCs.
Assuntos
Anemia Ferropriva/tratamento farmacológico , Diferenciação Celular/efeitos dos fármacos , Células Dendríticas/citologia , Compostos de Ferro/administração & dosagem , Macrófagos/citologia , Monócitos/citologia , Anemia Ferropriva/imunologia , Anemia Ferropriva/patologia , Estudos de Casos e Controles , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Dissacarídeos/administração & dosagem , Dissacarídeos/farmacologia , Compostos Férricos/administração & dosagem , Compostos Férricos/farmacologia , Óxido de Ferro Sacarado , Ácido Glucárico/administração & dosagem , Ácido Glucárico/farmacologia , Hematínicos/administração & dosagem , Hematínicos/farmacologia , Humanos , Injeções Intravenosas , Compostos de Ferro/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Maltose/administração & dosagem , Maltose/análogos & derivados , Maltose/farmacologia , MicroRNAs/genética , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fagocitose/efeitos dos fármacosRESUMO
BACKGROUND: The interaction of eukaryotic host and prokaryotic pathogen cells is linked to specific changes in the cellular proteome, and consequently to infection-related gene expression patterns of the involved cells. To simultaneously assess the transcriptomes of both organisms during their interaction we developed dual 3'Seq, a tag-based sequencing protocol that allows for exact quantification of differentially expressed transcripts in interacting pro- and eukaryotic cells without prior fixation or physical disruption of the interaction. RESULTS: Human epithelial cells were infected with Salmonella enterica Typhimurium as a model system for invasion of the intestinal epithelium, and the transcriptional response of the infected host cells together with the differential expression of invading and intracellular pathogen cells was determined by dual 3'Seq coupled with the next-generation sequencing-based transcriptome profiling technique deepSuperSAGE (deep Serial Analysis of Gene Expression). Annotation to reference transcriptomes comprising the operon structure of the employed S. enterica Typhimurium strain allowed for in silico separation of the interacting cells including quantification of polycistronic RNAs. Eighty-nine percent of the known loci are found to be transcribed in prokaryotic cells prior or subsequent to infection of the host, while 75% of all protein-coding loci are represented in the polyadenylated transcriptomes of human host cells. CONCLUSIONS: Dual 3'Seq was alternatively coupled to MACE (Massive Analysis of cDNA ends) to assess the advantages and drawbacks of a library preparation procedure that allows for sequencing of longer fragments. Additionally, the identified expression patterns of both organisms were validated by qRT-PCR using three independent biological replicates, which confirmed that RELB along with NFKB1 and NFKB2 are involved in the initial immune response of epithelial cells after infection with S. enterica Typhimurium.
Assuntos
Interações Hospedeiro-Patógeno/genética , Salmonelose Animal/genética , Salmonella typhi/genética , Transcriptoma/genética , Animais , Sequência de Bases , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Salmonelose Animal/microbiologia , Salmonella typhi/patogenicidadeRESUMO
BACKGROUND: The unprecedented role of sncRNAs in the regulation of pollen biogenesis on both transcriptional and epigenetic levels has been experimentally proven. However, little is known about their global regulation, especially under stress conditions. We used tomato pollen in order to identify pollen stage-specific sncRNAs and their target mRNAs. We further deployed elevated temperatures to discern stress responsive sncRNAs. For this purpose high throughput sncRNA-sequencing as well as Massive Analysis of cDNA Ends (MACE) were performed for three-replicated sncRNAs libraries derived from tomato tetrad, post-meiotic, and mature pollen under control and heat stress conditions. RESULTS: Using the omiRas analysis pipeline we identified known and predicted novel miRNAs as well as sncRNAs from other classes, responsive or not to heat. Differential expression analysis revealed that post-meiotic and mature pollen react most strongly by regulation of the expression of coding and non-coding genomic regions in response to heat. To gain insight to the function of these miRNAs, we predicted targets and annotated them to Gene Ontology terms. This approach revealed that most of them belong to protein binding, transcription, and Serine/Threonine kinase activity GO categories. Beside miRNAs, we observed differential expression of both tRNAs and snoRNAs in tetrad, post-meiotic, and mature pollen when comparing normal and heat stress conditions. CONCLUSIONS: Thus, we describe a global spectrum of sncRNAs expressed in pollen as well as unveiled those which are regulated at specific time-points during pollen biogenesis. We integrated the small RNAs into the regulatory network of tomato heat stress response in pollen.
Assuntos
Pólen/genética , Pequeno RNA não Traduzido/genética , Solanum lycopersicum/genéticaRESUMO
BACKGROUND: Previous studies identified microRNAs (miRNAs) and messenger RNAs with significantly different expression between normal pancreas and pancreatic cancer (PDAC) tissues. Due to technological limitations of microarrays and real-time PCR systems these studies focused on a fixed set of targets. Expression of other RNA classes such as long intergenic non-coding RNAs or sno-derived RNAs has rarely been examined in pancreatic cancer. Here, we analysed the coding and non-coding transcriptome of six PDAC and five control tissues using next-generation sequencing. RESULTS: Besides the confirmation of several deregulated mRNAs and miRNAs, miRNAs without previous implication in PDAC were detected: miR-802, miR-2114 or miR-561. SnoRNA-derived RNAs (e.g. sno-HBII-296B) and piR-017061, a piwi-interacting RNA, were found to be differentially expressed between PDAC and control tissues. In silico target analysis of miR-802 revealed potential binding sites in the 3' UTR of TCF4, encoding a transcription factor that controls Wnt signalling genes. Overexpression of miR-802 in MiaPaCa pancreatic cancer cells reduced TCF4 protein levels. Using Massive Analysis of cDNA Ends (MACE) we identified differential expression of 43 lincRNAs, long intergenic non-coding RNAs, e.g. LINC00261 and LINC00152 as well as several natural antisense transcripts like HNF1A-AS1 and AFAP1-AS1. Differential expression was confirmed by qPCR on the mRNA/miRNA/lincRNA level and by immunohistochemistry on the protein level. CONCLUSIONS: Here, we report a novel lncRNA, sncRNA and mRNA signature of PDAC. In silico prediction of ncRNA targets allowed for assigning potential functions to differentially regulated RNAs.
Assuntos
Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/genética , Neoplasias Pancreáticas/genética , RNA Longo não Codificante/genética , RNA Interferente Pequeno/genética , Células Acinares/metabolismo , Células Acinares/patologia , Sequência de Bases , Estudos de Casos e Controles , Simulação por Computador , Regulação para Baixo/genética , Redes Reguladoras de Genes , Humanos , Imuno-Histoquímica , MicroRNAs/metabolismo , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Regulação para Cima/genéticaRESUMO
KEY MESSAGE: A novel and highly effective source of anthracnose resistance in narrow-leafed lupin was identified. Resistance was shown to be governed by a single dominant locus. Molecular markers have been developed, which can be used for selecting resistant genotypes in lupin breeding. A screening for anthracnose resistance of a set of plant genetic resources of narrow-leafed lupin (Lupinus angustifolius L.) identified the breeding line Bo7212 as being highly resistant to anthracnose (Colletotrichum lupini). Segregation analysis indicated that the resistance of Bo7212 is inherited by a single dominant locus. The corresponding resistance gene was given the designation LanrBo. Previously published molecular anchor markers allowed us to locate LanrBo on linkage group NLL-11 of narrow-leafed lupin. Using information from RNAseq data obtained with inoculated resistant vs. susceptible lupin entries as well as EST-sequence information from the model genome Lotus japonicus, additional SNP and EST markers linked to LanrBo were derived. A bracket of two LanrBo-flanking markers allows for precise marker-assisted selection of the novel resistance gene in narrow-leafed lupin breeding programs.
Assuntos
Colletotrichum , Resistência à Doença/genética , Lupinus/genética , Doenças das Plantas/genética , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Mapeamento Cromossômico , DNA de Plantas/genética , Genes de Plantas , Marcadores Genéticos , Lupinus/microbiologia , Repetições de Microssatélites , Fenótipo , Melhoramento Vegetal , Doenças das Plantas/microbiologia , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND: Grass pea (Lathyrus sativus L.) is a valuable resource for potentially durable partial resistance to rust. To gain insight into the resistance mechanism and identify potential resistance genes, we generated the first comprehensive transcriptome assemblies from control and Uromyces pisi inoculated leafs of a susceptible and a partially rust-resistant grass pea genotype by RNA-seq. RESULTS: 134,914 contigs, shared by both libraries, were used to analyse their differential expression in response to rust infection. Functional annotation grouped 60.4% of the contigs present in plant databases (37.8% of total) to 33 main functional categories, being "protein", "RNA", "signalling", "transport" and "stress" the most represented. Transcription profiles revealed considerable differences in regulation of major phytohormone signalling pathways: whereas Salicylic and Abscisic Acid pathways were up-regulated in the resistant genotype, Jasmonate and Ethylene pathways were down-regulated in the susceptible one. As potential Resistance-genes we identified a mildew resistance locus O (MLO)-like gene, and MLO-related transcripts. Also, several pathogenesis-related genes were up-regulated in the resistant and exclusively down regulated in the susceptible genotype. Pathogen effectors identified in both inoculated libraries, as e.g. the rust Rtp1 transcript, may be responsible for the down-regulation of defence-related transcripts. The two genotypes contained 4,892 polymorphic contigs with SNPs unevenly distributed between different functional categories. Protein degradation (29.7%) and signalling receptor kinases (8.2%) were the most diverged, illustrating evolutionary adaptation of grass pea to the host/pathogens arms race. CONCLUSIONS: The vast array of novel, resistance-related genomic information we present here provides a highly valuable resource for future smart breeding approaches in this hitherto under-researched, valuable legume crop.
Assuntos
Resistência à Doença/genética , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Lathyrus/fisiologia , Transcriptoma , Alelos , Basidiomycota/fisiologia , Genótipo , Lathyrus/microbiologia , Anotação de Sequência Molecular , Doenças das Plantas , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Estresse FisiológicoRESUMO
SUMMARY: Small RNA deep sequencing is widely used to characterize non-coding RNAs (ncRNAs) differentially expressed between two conditions, e.g. healthy and diseased individuals and to reveal insights into molecular mechanisms underlying condition-specific phenotypic traits. The ncRNAome is composed of a multitude of RNAs, such as transfer RNA, small nucleolar RNA and microRNA (miRNA), to name few. Here we present omiRas, a Web server for the annotation, comparison and visualization of interaction networks of ncRNAs derived from next-generation sequencing experiments of two different conditions. The Web tool allows the user to submit raw sequencing data and results are presented as: (i) static annotation results including length distribution, mapping statistics, alignments and quantification tables for each library as well as lists of differentially expressed ncRNAs between conditions and (ii) an interactive network visualization of user-selected miRNAs and their target genes based on the combination of several miRNA-mRNA interaction databases. AVAILABILITY AND IMPLEMENTATION: The omiRas Web server is implemented in Python, PostgreSQL, R and can be accessed at: http://tools.genxpro.net/omiras/.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/genética , RNA Nucleolar Pequeno/genética , Animais , Biblioteca Gênica , Humanos , Internet , Análise de Sequência de RNA/métodos , SoftwareRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0145919.].
RESUMO
Plasma membrane accumulation of phosphorylated mixed lineage kinase domain-like (MLKL) is a hallmark of necroptosis, leading to membrane rupture and inflammatory cell death. Pro-death functions of MLKL are tightly controlled by several checkpoints, including phosphorylation. Endo- and exocytosis limit MLKL membrane accumulation and counteract necroptosis, but the exact mechanisms remain poorly understood. Here, we identify linear ubiquitin chain assembly complex (LUBAC)-mediated M1 poly-ubiquitination (poly-Ub) as novel checkpoint for necroptosis regulation downstream of activated MLKL in cells of human origin. Loss of LUBAC activity inhibits tumor necrosis factor α (TNFα)-mediated necroptosis, not by affecting necroptotic signaling, but by preventing membrane accumulation of activated MLKL. Finally, we confirm LUBAC-dependent activation of necroptosis in primary human pancreatic organoids. Our findings identify LUBAC as novel regulator of necroptosis which promotes MLKL membrane accumulation in human cells and pioneer primary human organoids to model necroptosis in near-physiological settings.
Assuntos
Necroptose , Proteínas Quinases , Humanos , Necrose/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Fosforilação , Morte Celular , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Apoptose/fisiologiaRESUMO
Understanding the extent of local adaptation in natural populations and the mechanisms that allow individuals to adapt to their native environment is a major avenue in molecular ecology research. Evidence for the frequent occurrence of diverging ecotypes in species that inhabit multiple ecological habitats is accumulating, but experimental approaches to understanding the biological pathways as well as the underlying genetic mechanisms are still rare. Parasites are invoked as one of the major selective forces driving evolution and are themselves dependent on the ecological conditions in a given habitat. Immunological adaptation to local parasite communities is therefore expected to be a key component of local adaptation in natural populations. Here, we use next-generation sequencing technology to compare the transcriptome-wide response of experimentally infected three-spined sticklebacks from a lake and a river population, which are known to evolve under selection by distinct parasite communities. By comparing overall gene expression levels as well as the activation of functional pathways in response to parasite exposure, we identified potential differences between the two stickleback populations at several levels. Our results suggest locally adapted patterns of gene regulation in response to parasite exposure, which may reflect different local optima in the trade-off between the benefits and the disadvantages of mounting an immune response because of quantitative differences of the local parasite communities.
Assuntos
Adaptação Fisiológica/imunologia , Ecótipo , Smegmamorpha/genética , Smegmamorpha/parasitologia , Transcriptoma , Animais , Ecossistema , Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/parasitologia , Biblioteca Gênica , Helmintíase/genética , Helmintíase/imunologia , Lagos , Carga Parasitária , Rios , Seleção Genética , Smegmamorpha/imunologiaRESUMO
Monocytes are a heterogeneous cell population with subset-specific functions and phenotypes. The differential expression of CD14 and CD16 distinguishes classical CD14(++)CD16(-), intermediate CD14(++)CD16(+), and nonclassical CD14(+)CD16(++) monocytes. Current knowledge on human monocyte heterogeneity is still incomplete: while it is increasingly acknowledged that CD14(++)CD16(+) monocytes are of outstanding significance in 2 global health issues, namely HIV-1 infection and atherosclerosis, CD14(++)CD16(+) monocytes remain the most poorly characterized subset so far. We therefore developed a method to purify the 3 monocyte subsets from human blood and analyzed their transcriptomes using SuperSAGE in combination with high-throughput sequencing. Analysis of 5 487 603 tags revealed unique identifiers of CD14(++)CD16(+) monocytes, delineating these cells from the 2 other monocyte subsets. Gene Ontology (GO) enrichment analysis suggests diverse immunologic functions, linking CD14(++)CD16(+) monocytes to Ag processing and presentation (eg, CD74, HLA-DR, IFI30, CTSB), to inflammation and monocyte activation (eg, TGFB1, AIF1, PTPN6), and to angiogenesis (eg, TIE2, CD105). In conclusion, we provide genetic evidence for a distinct role of CD14(++)CD16(+) monocytes in human immunity. After CD14(++)CD16(+) monocytes have earlier been discussed as a potential therapeutic target in inflammatory diseases, we are hopeful that our data will spur further research in the field of monocyte heterogeneity.
Assuntos
Aterosclerose/imunologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Infecções por HIV/imunologia , Imunidade/genética , Inflamação/imunologia , Receptores de Lipopolissacarídeos/genética , Monócitos/imunologia , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Aterosclerose/genética , Aterosclerose/metabolismo , Proliferação de Células , Etiquetas de Sequências Expressas , Citometria de Fluxo , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Inflamação/genética , Inflamação/metabolismo , Receptores de Lipopolissacarídeos/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Monócitos/classificação , Monócitos/citologia , Monócitos/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/imunologia , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/imunologia , RNA Mensageiro/análise , RNA Mensageiro/biossínteseRESUMO
We have compared the transcriptomic profiles of microdissected live ovules at four developmental stages between a diploid sexual and diploid apomictic Boechera. We sequenced >2 million SuperSAGE tags and identified (1) heterochronic tags (n = 595) that demonstrated significantly different patterns of expression between sexual and apomictic ovules across all developmental stages, (2) stage-specific tags (n = 577) that were found in a single developmental stage and differentially expressed between the sexual and apomictic ovules, and (3) sex-specific (n = 237) and apomixis-specific (n = 1106) tags that were found in all four developmental stages but in only one reproductive mode. Most heterochronic and stage-specific tags were significantly downregulated during early apomictic ovule development, and 110 were associated with reproduction. By contrast, most late stage-specific tags were upregulated in the apomictic ovules, likely the result of increased gene copy number in apomictic (hexaploid) versus sexual (triploid) endosperm or of parthenogenesis. Finally, we show that apomixis-specific gene expression is characterized by a significant overrepresentation of transcription factor activity. We hypothesize that apomeiosis is associated with global downregulation at the megaspore mother cell stage. As the diploid apomict analyzed here is an ancient hybrid, these data are consistent with the postulated link between hybridization and asexuality and provide a hypothesis for multiple evolutionary origins of apomixis in the genus Boechera.
Assuntos
Brassicaceae/genética , Perfilação da Expressão Gênica , Óvulo Vegetal/genética , Brassicaceae/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Óvulo Vegetal/embriologia , RNA de Plantas/genética , Reprodução/genética , Fatores de Transcrição/metabolismoRESUMO
Peyronellaea pinodes is a devastating pathogen of pea crop. Quantitative trait loci (QTL) associated with resistance have been identified, as well as genes differentially expressed between resistant and susceptible pea lines. The key question is which of these many genes located into these QTLs, or differentially expressed, are the key genes that distinguish resistant from susceptible plants and could be used as markers. To identify these key genes, in the present study we applied MACE (Massive Analysis of cDNA Ends) -Seq to a whole Recombinant Inbred Line population segregating for resistance to this disease and their parental lines and identified those genes which expression was more correlated with the level of resistance. We also compared gene expression profiles between the most resistant and the most susceptible families of the RIL population. A total of 6780 transcripts were differentially expressed between the parental lines after inoculation. Of them, 803 showed the same expression pattern in the bulks formed by the most resistant and most susceptible RIL families. These genes, showing a consistent expression pattern, could be used as expression markers to distinguish resistant from susceptible plants. The analysis of these genes also discovered the crucial mechanisms acting against P. pinodes.
Assuntos
Ascomicetos , Pisum sativum , Pisum sativum/genética , Doenças das Plantas/genética , Ascomicetos/genética , Perfilação da Expressão Gênica , Transcriptoma , Resistência à Doença/genéticaRESUMO
Conduct Disorder (CD) is an impairing psychiatric disorder of childhood and adolescence characterized by aggressive and dissocial behavior. Environmental factors such as maternal smoking during pregnancy, socio-economic status, trauma, or early life stress are associated with CD. Although the number of females with CD is rising in Western societies, CD is under-researched in female cohorts. We aimed at exploring the epigenetic signature of females with CD and its relation to psychosocial and environmental risk factors. We performed HpaII sensitive genome-wide methylation sequencing of 49 CD girls and 50 matched typically developing controls and linear regression models to identify differentially methylated CpG loci (tags) and regions. Significant tags and regions were mapped to the respective genes and tested for enrichment in pathways and brain developmental processes. Finally, epigenetic signatures were tested as mediators for CD-associated risk factors. We identified a 12% increased methylation 5' of the neurite modulator SLITRK5 (FDR = 0.0046) in cases within a glucocorticoid receptor binding site. Functionally, methylation positively correlated with gene expression in lymphoblastoid cell lines. At systems-level, genes (uncorr. P < 0.01) were associated with development of neurons, neurite outgrowth or neuronal developmental processes. At gene expression level, the associated gene-networks are activated perinatally and during early childhood in neocortical regions, thalamus and striatum, and expressed in amygdala and hippocampus. Specifically, the epigenetic signatures of the gene network activated in the thalamus during early childhood correlated with the effect of parental education on CD status possibly mediating its protective effect. The differential methylation patterns identified in females with CD are likely to affect genes that are expressed in brain regions previously indicated in CD. We provide suggestive evidence that protective effects are likely mediated by epigenetic mechanisms impairing specific brain developmental networks and therefore exerting a long-term effect on neural functions in CD. Our results are exploratory and thus, further replication is needed.