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The signals driving the adaptation of type 2 dendritic cells (DC2s) to diverse peripheral environments remain mostly undefined. We show that differentiation of CD11blo migratory DC2s-a DC2 population unique to the dermis-required IL-13 signaling dependent on the transcription factors STAT6 and KLF4, whereas DC2s in lung and small intestine were STAT6-independent. Similarly, human DC2s in skin expressed an IL-4 and IL-13 gene signature that was not found in blood, spleen and lung DCs. In mice, IL-13 was secreted homeostatically by dermal innate lymphoid cells and was independent of microbiota, TSLP or IL-33. In the absence of IL-13 signaling, dermal DC2s were stable in number but remained CD11bhi and showed defective activation in response to allergens, with diminished ability to support the development of IL-4+GATA3+ helper T cells (TH), whereas antifungal IL-17+RORγt+ TH cells were increased. Therefore, homeostatic IL-13 fosters a noninflammatory skin environment that supports allergic sensitization.
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Comunicação Celular , Diferenciação Celular , Interleucina-13/metabolismo , Células de Langerhans/metabolismo , Pele/metabolismo , Células Th17/metabolismo , Células Th2/metabolismo , Alérgenos/farmacologia , Animais , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Células Cultivadas , Bases de Dados Genéticas , Humanos , Interleucina-13/genética , Células de Langerhans/efeitos dos fármacos , Células de Langerhans/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais , Pele/citologia , Pele/efeitos dos fármacos , Pele/imunologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , TranscriptomaRESUMO
Multiphenotype genome-wide association studies (GWAS) may reveal pleiotropic genes, which would remain undetected using single phenotype analyses. Analysis of large pedigrees offers the added advantage of more accurately assessing trait heritability, which can help prioritise genetically influenced phenotypes for GWAS analysis. In this study we performed a principal component analysis (PCA), heritability (h2) estimation and pedigree-based GWAS of 37 cardiovascular disease -related phenotypes in 330 related individuals forming a large pedigree from the Norfolk Island genetic isolate. PCA revealed 13 components explaining >75% of the total variance. Nine components yielded statistically significant h2 values ranging from 0.22 to 0.54 (P<0.05). The most heritable component was loaded with 7 phenotypic measures reflecting metabolic and renal dysfunction. A GWAS of this composite phenotype revealed statistically significant associations for 3 adjacent SNPs on chromosome 1p22.2 (P<1x10-8). These SNPs form a 42kb haplotype block and explain 11% of the genetic variance for this renal function phenotype. Replication analysis of the tagging SNP (rs1396315) in an independent US cohort supports the association (P = 0.000011). Blood transcript analysis showed 35 genes were associated with rs1396315 (P<0.05). Gene set enrichment analysis of these genes revealed the most enriched pathway was purine metabolism (P = 0.0015). Overall, our findings provide convincing evidence for a major pleiotropic effect locus on chromosome 1p22.2 influencing risk of renal dysfunction via purine metabolism pathways in the Norfolk Island population. Further studies are now warranted to interrogate the functional relevance of this locus in terms of renal pathology and cardiovascular disease risk.
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Doenças Cardiovasculares/genética , Pleiotropia Genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Doenças Cardiovasculares/patologia , Feminino , Haplótipos , Humanos , Masculino , Melanesia , Fenótipo , Polimorfismo de Nucleotídeo Único , Análise de Componente PrincipalRESUMO
PURPOSE: Primary open-angle glaucoma (POAG) refers to a group of heterogeneous diseases involving optic nerve damage. Two well-established risk factors for POAG are elevated intraocular pressure (IOP) and a thinner central corneal thickness (CCT). These endophenotypes exhibit a high degree of heritability across populations. Large-scale genome-wide association studies (GWASs) of outbred populations have robustly implicated several susceptibility gene variants for both IOP and CCT. Despite this progress, a substantial amount of genetic variance remains unexplained. Population-specific variants that might be rare in outbred populations may also influence POAG endophenotypes. The Norfolk Island population is a founder-effect genetic isolate that has been well characterized for POAG endophenotypes. This population is therefore a suitable candidate for mapping new variants that influence these complex traits. METHODS: Three hundred and thirty participants from the Norfolk Island Eye Study (NIES) core pedigree provided DNA. Ocular measurements of CCT and IOP were also taken for analysis. Heritability analyses and genome-wide linkage analyses of short tandem repeats (STRs) were conducted using SOLAR. Pedigree-based GWASs of single-nucleotide polymorphisms (SNPs) were performed using the GenABEL software. RESULTS: CCT was the most heritable endophenotype in this cohort (h2 = 0.77, p = 6×10-6), while IOP showed a heritability of 0.39 (p = 0.008). A genome-wide linkage analysis of these POAG phenotypes identified a maximum logarithm of the odds (LOD) score of 1.9 for CCT on chromosome 20 (p = 0.0016) and 1.3 for IOP on chromosome 15 (p = 0.0072). The GWAS results revealed a study-wise significant association for IOP at rs790357, which is located within DLG2 on chr11q14.1 (p = 1.02×10-7). DLG2 is involved in neuronal signaling and development, and while it has not previously been associated with IOP, it has been associated with myopia. An analysis of 12 known SNPs for IOP showed that rs12419342 in RAPSN on chromosome 11 was nominally associated in Norfolk Island (NI; p = 0.0021). For CCT, an analysis of 26 known SNPs showed rs9938149 in BANP-ZNF469 on chromosome 16 was nominally associated in NI (p = 0.002). CONCLUSIONS: These study results indicate that CCT and IOP exhibit a substantial degree of heritability in the NI pedigree, indicating a genetic component. A genome-wide linkage analysis of POAG endophenotypes did not reveal any major effect loci, but the GWASs did implicate several known loci, as well as a potential new locus in DLG2, suggesting a role for neuronal signaling in development in IOP and perhaps POAG. These results also highlight the need to target rarer variants via whole genome sequencing in this genetic isolate.
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Endofenótipos , Efeito Fundador , Predisposição Genética para Doença , Glaucoma de Ângulo Aberto/genética , Polimorfismo de Nucleotídeo Único , Adulto , Feminino , Estudo de Associação Genômica Ampla , Técnicas de Genotipagem , Humanos , Pressão Intraocular , Masculino , Melanesia , Linhagem , Tonometria OcularRESUMO
Variation in the human monoamine oxidase A (MAO-A) gene can influence neurotransmittor levels and is thought to have a role in many behavioral traits. The genetic architecture of MAO-A is known to vary across different geographic subgroups. Previous studies have reported evidence for positive selection within the MAO-A gene region in seven ethnic groups: Pygmy, Aboriginal Taiwanese, Chinese, Japanese, Mexican and Russian. Polynesian populations have not been tested and repeated founder effects due to the island-hopping voyages of Polynesians across the South Pacific suggest a unique demographic history exists at the MAO-A gene, perhaps including selective effects. To explore this, we genotyped 13 key single-nucleotide polymorphisms (SNPs) spanning MAO-A gene as well as the functional polymorphism (MAO-A-uVNTR) in 47 unrelated Maori individuals. A comparison of genetic variation between Maori and non-Maori groups found a substantial reduction in genetic diversity at the MAO-A gene locus and an increase in the frequency of the most common MAO-A gene variant in the Maori group. Results of this study support previous findings and also point toward a 5-SNP haplotype that may have been influenced by selective effects in the Maori population. Full-sequence data for MAO-A in a large cohort are now required to conclusively determine whether MAO-A has undergone positive selection in Polynesians. Overall, these new data describe a unique demographic history for the MAO-A gene in the Maori population and will be helpful for studies wishing to investigate MAO-A as a candidate gene for influencing behavioral traits in the Polynesians.
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Emigração e Imigração , Repetições Minissatélites/genética , Monoaminoxidase/genética , Mutação , Havaiano Nativo ou Outro Ilhéu do Pacífico/genética , Polimorfismo de Nucleotídeo Único/genética , Feminino , Genótipo , Haplótipos , Humanos , Masculino , Nova ZelândiaRESUMO
The power of high-dimensional reduction techniques using multiparameter images has been demonstrated across a variety of different publications. Recently, we published an end-to-end low-cost GUI-based protocol for performing histocytometric spatial analysis on images derived from the most common microscope image formats. However, this protocol is limited by the normalized marker intensity outputs and the difficulty in processing images of highly aggregated and/or exceptionally heterogenous cell populations. Here we present the basic protocols required to construct an advanced histocytometric data file using only freeware. This data file is compatible with images containing cell nuclei clusters that are difficult to segment, and results in histocytometry files retaining the original marker intensity values of the microscopic images they were derived from. This is especially useful in cells that are phenotyped based on relative marker expression levels. Histocytometry data files produced by these protocols are compatible with high-dimensional reduction analysis using marker intensity data, such as tSNEs. This methodology is showcased using stitched microscopic images of murine lymph nodes, complex organs with highly aggregated heterogenous cell populations, that are typically difficult to segment. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Image preprocessing and generation of nuclei marker probability maps Basic Protocol 2: Cell segmentation using ilastik-derived probability maps Basic Protocol 3: Generation of histocytometric .fcs files.
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Corantes , Processamento de Imagem Assistida por Computador , Animais , Núcleo Celular , Processamento de Imagem Assistida por Computador/métodos , Camundongos , Microscopia , RegistrosRESUMO
The ability of cancer cells to adjust their metabolism in response to environmental changes is a well-recognized hallmark of cancer. Diverse cancer and non-cancer cells within tumors compete for metabolic resources. Metabolic demands change frequently during tumor initiation, progression and metastasis, challenging our quest to better understand tumor biology and develop novel therapeutics. Vascularization, physical constraints, immune responses and genetic instability promote tumor evolution resulting in immune evasion, opportunities to breach basement membrane barriers and spread through the circulation and lymphatics. In addition, the unfolded protein response linked to the ubiquitin proteasome system is a key player in addressing stoichiometric imbalances between nuclear and mitochondrially-encoded protein subunits of respiratory complexes, and nuclear-encoded mitochondrial ribosomal protein subunits. While progressive genetic changes, some of which affect metabolic adaptability, contribute to tumorigenesis and metastasis through clonal expansion, epigenetic changes are also important and more dynamic in nature. Understanding the role of stromal and immune cells in the tumor microenvironment in remodeling cancer cell energy metabolism has become an increasingly important area of research. In this perspective, we discuss the adaptations made by cancer cells to balance mitochondrial and glycolytic energy metabolism. We discuss how hypoxia and nutrient limitations affect reductive and oxidative stress through changes in mitochondrial electron transport activity. We propose that integrated responses to cellular stress in cancer cells are central to metabolic flexibility in general and bioenergetic adaptability in particular and are paramount in tumor progression and metastasis.
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BACKGROUND: the thrifty gene hypothesis posits that, in populations that experienced periods of feast and famine, natural selection favoured individuals carrying thrifty alleles that promote the storage of fat and energy. Polynesians likely experienced long periods of cold stress and starvation during their settlement of the Pacific and today have high rates of obesity and type 2 diabetes (T2DM), possibly due to past positive selection for thrifty alleles. Alternatively, T2DM risk alleles may simply have drifted to high frequency in Polynesians. To identify thrifty alleles in Polynesians, we previously examined evidence of positive selection on T2DM-associated SNPs and identified a T2DM risk allele at unusually high frequency in Polynesians. We suggested that the risk allele of the Gly482Ser variant in the PPARGC1A gene was driven to high frequency in Polynesians by positive selection and therefore possibly represented a thrifty allele in the Pacific. METHODS: here we examine whether PPARGC1A is a thrifty gene in Pacific populations by testing for an association between Gly482Ser genotypes and BMI in two Pacific populations (Maori and Tongans) and by evaluating the frequency of the risk allele of the Gly482Ser variant in a sample of worldwide populations. RESULTS: we find that the Gly482Ser variant is associated with BMI in Tongans but not in Maori. In a sample of 58 populations worldwide, we also show that the 482Ser risk allele reaches its highest frequency in the Pacific. CONCLUSION: the association between Gly482Ser genotypes and BMI in Tongans together with the worldwide frequency distribution of the Gly482Ser risk allele suggests that PPARGC1A remains a candidate thrifty gene in Pacific populations.
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Índice de Massa Corporal , Predisposição Genética para Doença/genética , Proteínas de Choque Térmico/genética , Fatores de Transcrição/genética , Adulto , Idoso , Estudos de Coortes , Diabetes Mellitus Tipo 2/genética , Feminino , Frequência do Gene , Estudos de Associação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Tonga/epidemiologiaRESUMO
The MinION is a portable DNA sequencer that allows real time sequencing at low capital cost investment. We assessed accuracy and cost-effectivess of the MinION for genetic diagnostic testing of known SCN1A mutations that cause Dravet Syndrome (DS). DNA samples (n = 7) from DS patients previously shown to carry SCN1A mutations via Ion Torrent and Sanger sequencing were sequenced using the MinION. SCN1A amplicons for 8 exons were sequenced using the MinION with 1D chemistry on an R9.4 flow cell. All known missense mutations were detected in all samples showing 100 % concordance with results from other methods. However, the MinION failed to detect the insertions/deletions (INDELs) present in these patients. Nevertheless, these results indicate that MinION is a cost-effective platform for use as an initial screening step in the detection of nucleotide substitution mutations in in SCN1A, especially in under-resourced laboratories or hospitals. Further improvements are required to reliably detect INDELS in this gene.
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Epilepsias Mioclônicas , Epilepsia , Canal de Sódio Disparado por Voltagem NAV1.1/análise , Análise Custo-Benefício , Epilepsias Mioclônicas/diagnóstico , Epilepsias Mioclônicas/genética , Epilepsia/diagnóstico , Epilepsia/genética , Síndromes Epilépticas , Humanos , Mutação/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Espasmos Infantis , TecnologiaRESUMO
Tumor cells without mitochondrial (mt) DNA (ρ0 cells) are auxotrophic for uridine, and their growth is supported by pyruvate. While ATP synthesis in ρ0 cells relies on glycolysis, they fail to form tumors unless they acquire mitochondria from stromal cells. Mitochondrial acquisition restores respiration that is essential for de novo pyrimidine biosynthesis and for mitochondrial ATP production. The physiological processes that underpin intercellular mitochondrial transfer to tumor cells lacking mtDNA and the metabolic remodeling and restored tumorigenic properties of cells that acquire mitochondria are not well understood. Here, we investigated the changes in mitochondrial and nuclear gene expression that accompany mtDNA deletion and acquisition in metastatic murine 4T1 breast cancer cells. Loss of mitochondrial gene expression in 4T1ρ0 cells was restored in cells recovered from subcutaneous tumors that grew from 4T1ρ0 cells following acquisition of mtDNA from host cells. In contrast, the expression of most nuclear genes that encode respiratory complex subunits and mitochondrial ribosomal subunits was not greatly affected by loss of mtDNA, indicating ineffective mitochondria-to-nucleus communication systems for these nuclear genes. Further, analysis of nuclear genes whose expression was compromised in 4T1ρ0 cells showed that immune- and stress-related genes were the most highly differentially expressed, representing over 70% of those with greater than 16-fold higher expression in 4T1 compared with 4T1ρ0 cells. The monocyte recruiting chemokine, Ccl2, and Psmb8, a subunit of the immunoproteasome that generates MHCI-binding peptides, were the most highly differentially expressed. Early monocyte/macrophage recruitment into the tumor mass was compromised in 4T1ρ0 cells but recovered before mtDNA could be detected. Taken together, our results show that mitochondrial acquisition by tumor cells without mtDNA results in bioenergetic remodeling and re-expression of genes involved in immune function and stress adaptation.
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Colorectal cancer is a major contributor to death and disease worldwide. The ApcMin mouse is a widely used model of intestinal neoplasia, as it carries a mutation also found in human colorectal cancers. However, the method most commonly used to quantify tumour burden in these mice is manual adenoma counting, which is time consuming and poorly suited to standardization across different laboratories. We describe a method to produce suitable photographs of the small intestine of ApcMin mice, process them with an ImageJ macro, FeatureCounter, which automatically locates image features potentially corresponding to adenomas, and a machine learning pipeline to identify and quantify them. Compared to a manual method, the specificity (or True Negative Rate, TNR) and sensitivity (or True Positive Rate, TPR) of this method in detecting adenomas are similarly high at about 80% and 87%, respectively. Importantly, total adenoma area measures derived from the automatically-called tumours were just as capable of distinguishing high-burden from low-burden mice as those established manually. Overall, our strategy is quicker, helps control experimenter bias, and yields a greater wealth of information about each tumour, thus providing a convenient route to getting consistent and reliable results from a study.
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Adenoma/diagnóstico , Genes APC , Processamento de Imagem Assistida por Computador , Animais , Automação , Peso Corporal , Análise Discriminante , Estudos de Viabilidade , Feminino , Intestino Delgado/diagnóstico por imagem , Intestino Delgado/patologia , Masculino , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Reprodutibilidade dos Testes , Baço/patologia , Carga TumoralRESUMO
In many species, the offspring of related parents suffer reduced reproductive success, a phenomenon known as inbreeding depression. In humans, the importance of this effect has remained unclear, partly because reproduction between close relatives is both rare and frequently associated with confounding social factors. Here, using genomic inbreeding coefficients (FROH) for >1.4 million individuals, we show that FROH is significantly associated (p < 0.0005) with apparently deleterious changes in 32 out of 100 traits analysed. These changes are associated with runs of homozygosity (ROH), but not with common variant homozygosity, suggesting that genetic variants associated with inbreeding depression are predominantly rare. The effect on fertility is striking: FROH equivalent to the offspring of first cousins is associated with a 55% decrease [95% CI 44-66%] in the odds of having children. Finally, the effects of FROH are confirmed within full-sibling pairs, where the variation in FROH is independent of all environmental confounding.
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Tamanho Corporal/genética , Cognição , Consanguinidade , Fertilidade/genética , Nível de Saúde , Depressão por Endogamia/genética , Assunção de Riscos , Alelos , Haplótipos , Homozigoto , HumanosRESUMO
Background: We investigated the molecular etiology of a young male proband with confirmed immunodeficiency of unknown cause, presenting with recurrent bacterial and Varicella zoster viral infections in childhood and persistent lymphopenia into early adulthood. Aim: To identify causative functional genetic variants related to an undiagnosed primary immunodeficiency. Method: Whole genome microarray copy number variant (CNV) analysis was performed on the proband followed by whole exome sequencing (WES) and trio analysis of the proband and family members. A >4 kbp deletion identified by repeated CNV analysis of exome sequencing data along with three damaging missense single nucleotide variants were validated by Sanger sequencing in all family members. Confirmation of the causative role of the candidate gene was performed by qPCR and Western Blot analyses on the proband, family members and a healthy control. Results: CNV identified our previously reported interleukin 25 amplification in the proband; however, the variant was not validated to be a candidate gene for immunodeficiency. WES trio analysis, data filtering and in silico prediction identified a novel, damaging (SIFT: 0; Polyphen 1; Grantham score: 101) and disease-causing (MutationTaster) single base mutation in the X chromosome (c.511C > T p.Arg171Trp) MSN gene not identified in the UCSC Genome Browser database. The mutation was validated by Sanger sequencing, confirming the proband was hemizygous X-linked recessive (-/T) at this locus and inherited the affected T allele from his non-symptomatic carrier mother (C/T), with other family members (father, sister) confirmed to be wild type (C/C). Western Blot analysis demonstrated an absence of moesin protein in lymphocytes derived from the proband, compared with normal expression in lymphocytes derived from the healthy control, father and mother. qPCR identified significantly lower MSN mRNA transcript expression in the proband compared to an age- and sex-matched healthy control subject in whole blood (p = 0.02), and lymphocytes (p = 0.01). These results confirmed moesin deficiency in the proband, directly causative of his immunodeficient phenotype. Conclusion: These findings confirm X-linked moesin-associated immunodeficiency in a proband previously undiagnosed up to 24 years of age. This study also highlights the utility of WES for the diagnosis of rare or novel forms of primary immunodeficiency disease.
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Sequenciamento do Exoma/métodos , Genótipo , Linfopenia/genética , Proteínas dos Microfilamentos/genética , Deleção de Sequência/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Adulto , Análise Mutacional de DNA , Frequência do Gene , Estudos de Associação Genética , Humanos , Masculino , Linhagem , Adulto JovemRESUMO
Nippostrongylus brasiliensis, a nematode parasite of rodents, has a parasitic life cycle that is an extremely useful model for the study of human hookworm infection, particularly in regards to the induced immune response. The current reference genome for this parasite is highly fragmented with minimal annotation, but new advances in long-read sequencing suggest that a more complete and annotated assembly should be an achievable goal. We de-novo assembled a single contig mitochondrial genome from N. brasiliensis using MinION R9 nanopore data. The assembly was error-corrected using existing Illumina HiSeq reads, and annotated in full (i.e. gene boundary definitions without substantial gaps) by comparing with annotated genomes from similar parasite relatives. The mitochondrial genome has also been annotated with a preliminary electrical consensus sequence, using raw signal data generated from a Nanopore R9 flow cell.
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BACKGROUND: Migraine is a common neurological disorder which affects a large proportion of the population. The Norfolk Island population is a genetically isolated population and is an ideal discovery cohort for genetic variants involved in complex disease susceptibility given the reduced genetic and environmental heterogeneity. Given that the majority of proteins responsible for mitochondrial function are nuclear encoded, this study aimed to investigate the role of Nuclear Encoded Mitochondrial Protein (NEMP) genes in relation to migraine susceptibility. METHODS: A gene-centric association analysis of NEMP genes was undertaken in the most related individuals (n = 315) within the genetically isolated Norfolk Island population. The discovery phase included genes with three or more SNP associations (P < 0.005), which were investigated further in a replication phase using an unrelated migraine case-control cohort (544 patients and 584 controls). RESULTS: The discovery phase of the study implicated SNPs in 5 NEMP genes to be associated with migraine susceptibility (P < 0.005). Replication analysis validated some of these implicated genes with SNPs in three NEMP genes shown to be associated with migraine in the replication cohort. These were CSNK1G3 (P = 0.00037), ELOVL6 (P = 0.00035) and SARDH (P = 0.00081), which are involved in phosphorylation, fatty acid metabolism, and oxidative demethylation, respectively. CONCLUSION: Here we provide evidence that variation in NEMP genes is associated with migraine susceptibility. This study provides evidence for a link between mitochondrial function and migraine susceptibility.
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Neisseria meningitidis (meningococcus) can cause meningococcal disease, a rapidly progressing and often fatal disease that can occur in previously healthy children. Meningococci are found in healthy carriers, where they reside in the nasopharynx as commensals. While carriage is relatively common, invasive disease, associated with hypervirulent strains, is a comparatively rare event. The basis of increased virulence in some strains is not well understood. New Zealand suffered a protracted meningococcal disease epidemic, from 1991 to 2008. During this time, a household carriage study was carried out in Auckland: household contacts of index meningococcal disease patients were swabbed for isolation of carriage strains. In many households, healthy carriers harbored strains identical, as determined by laboratory typing, to the ones infecting the associated patient. We carried out more-detailed analyses of carriage and disease isolates from a select number of households. We found that isolates, although indistinguishable by laboratory typing methods and likely closely related, had many differences. We identified multiple genome variants and transcriptional differences between isolates. These studies enabled the identification of two new phase-variable genes. We also found that several carriage strains had lost their type IV pili and that this loss correlated with reduced tumor necrosis factor alpha (TNF-α) expression when cultured with epithelial cells. While nonpiliated meningococcal isolates have been previously found in carriage strains, this is the first evidence of an association between type IV pili from meningococci and a proinflammatory epithelial response. We also identified potentially important metabolic differences between carriage and disease isolates, including the sulfate assimilation pathway. IMPORTANCENeisseria meningitidis causes meningococcal disease but is frequently carried in the throats of healthy individuals; the factors that determine whether invasive disease develops are not completely understood. We carried out detailed studies of isolates, collected from patients and their household contacts, to identify differences between commensal throat isolates and those that caused invasive disease. Though isolates were identical by laboratory typing methods, we uncovered many differences in their genomes, in gene expression, and in their interactions with host cells. In particular, we found that several carriage isolates had lost their type IV pili, a surprising finding since pili are often described as essential for colonization. However, loss of type IV pili correlated with reduced secretion of a proinflammatory cytokine, TNF-α, when meningococci were cocultured with human bronchial epithelial cells; hence, the loss of pili could provide an advantage to meningococci, by resulting in a dampened localized host immune response.
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The dendritic cell signals required for the in vivo priming of IL-4-producing T cells are unknown. We used RNA sequencing to characterize DCs from skin LN of mice exposed to two different Th2 stimuli: the helminth parasite Nippostrongylus brasiliensis (Nb) and the contact sensitizer dibutyl phthalate (DBP)-FITC. Both Nb and DBP-FITC induced extensive transcriptional changes that involved multiple DC subsets. Surprisingly, these transcriptional changes were highly distinct in the two models, with only a small number of genes being similarly regulated in both conditions. Pathway analysis of expressed genes identified no shared pathways between Nb and DBP-FITC, but revealed a type-I IFN (IFN-I) signature unique to DCs from Nb-primed mice. Blocking the IFN-I receptor at the time of Nb treatment had little effect on DC migration and antigen transport to the LN, but inhibited the up-regulation of IFN-I-induced markers on DCs and effectively blunted Th2 development. In contrast, the response to DBP-FITC was not affected by IFN-I receptor blockade, a finding consistent with the known dependence of this response on the innate cytokine TSLP. Thus, the priming of Th2 responses is associated with distinct transcriptional signatures in DCs in vivo, reflecting the diverse environments in which Th2 immune responses are initiated.
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Células Dendríticas/imunologia , Pele/imunologia , Células Th2/imunologia , Animais , Imunoglobulinas/fisiologia , Interferon Tipo I/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Nippostrongylus/imunologia , Receptor de Interferon alfa e beta/fisiologia , Receptores de Citocinas/fisiologia , Transcrição GênicaRESUMO
BACKGROUND: Long-read sequencing is rapidly evolving and reshaping the suite of opportunities for genomic analysis. For the MinION in particular, as both the platform and chemistry develop, the user community requires reference data to set performance expectations and maximally exploit third-generation sequencing. We performed an analysis of MinION data derived from whole genome sequencing of Escherichiacoli K-12 using the R9.0 chemistry, comparing the results with the older R7.3 chemistry. METHODS: We computed the error-rate estimates for insertions, deletions, and mismatches in MinION reads. RESULTS: Run-time characteristics of the flow cell and run scripts for R9.0 were similar to those observed for R7.3 chemistry, but with an 8-fold increase in bases per second (from 30 bps in R7.3 and SQK-MAP005 library preparation, to 250 bps in R9.0) processed by individual nanopores, and less drop-off in yield over time. The 2-dimensional ("2D") N50 read length was unchanged from the prior chemistry. Using the proportion of alignable reads as a measure of base-call accuracy, 99.9% of "pass" template reads from 1-dimensional ("1D") experiments were mappable and ~97% from 2D experiments. The median identity of reads was ~89% for 1D and ~94% for 2D experiments. The total error rate (miscall + insertion + deletion ) decreased for 2D "pass" reads from 9.1% in R7.3 to 7.5% in R9.0 and for template "pass" reads from 26.7% in R7.3 to 14.5% in R9.0. CONCLUSIONS: These Phase 2 MinION experiments serve as a baseline by providing estimates for read quality, throughput, and mappability. The datasets further enable the development of bioinformatic tools tailored to the new R9.0 chemistry and the design of novel biological applications for this technology. ABBREVIATIONS: K: thousand, Kb: kilobase (one thousand base pairs), M: million, Mb: megabase (one million base pairs), Gb: gigabase (one billion base pairs).
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Influenza epidemics and pandemics have significant impacts on economies, morbidity and mortality worldwide. The ability to rapidly and accurately sequence influenza viruses is instrumental in the prevention and mitigation of influenza. All eight influenza genes from an influenza A virus were amplified by PCR simultaneously and then subjected to sequencing on a MinION nanopore sequencer. A complete influenza virus genome was obtained that shared greater than 99% identity with sequence data obtained from Illumina MiSeq and traditional Sanger-sequencing. The laboratory infrastructure and computing resources used to perform this experiment on the MinION nanopore sequencer would be available in most molecular laboratories around the world. Using this system, the concept of portability, and thus sequencing influenza viruses in the clinic or field is now tenable.
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The advent of a miniaturized DNA sequencing device with a high-throughput contextual sequencing capability embodies the next generation of large scale sequencing tools. The MinION™ Access Programme (MAP) was initiated by Oxford Nanopore Technologies™ in April 2014, giving public access to their USB-attached miniature sequencing device. The MinION Analysis and Reference Consortium (MARC) was formed by a subset of MAP participants, with the aim of evaluating and providing standard protocols and reference data to the community. Envisaged as a multi-phased project, this study provides the global community with the Phase 1 data from MARC, where the reproducibility of the performance of the MinION was evaluated at multiple sites. Five laboratories on two continents generated data using a control strain of Escherichia coli K-12, preparing and sequencing samples according to a revised ONT protocol. Here, we provide the details of the protocol used, along with a preliminary analysis of the characteristics of typical runs including the consistency, rate, volume and quality of data produced. Further analysis of the Phase 1 data presented here, and additional experiments in Phase 2 of E. coli from MARC are already underway to identify ways to improve and enhance MinION performance.