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1.
Thorax ; 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31699806

RESUMO

BACKGROUND: Antibiotic resistance is a major global threat. We hypothesised that the chronic obstructive pulmonary disease (COPD) airway is a reservoir of antimicrobial resistance genes (ARGs) that associate with microbiome-specific COPD subgroups. OBJECTIVE: To determine the resistance gene profiles in respiratory samples from COPD patients and healthy volunteers. METHODS: Quantitative PCR targeting 279 specific ARGs was used to profile the resistomes in sputum from subjects with COPD at stable, exacerbation and recovery visits (n=55; COPD-BEAT study), healthy controls with (n=7) or without (n=22) exposure to antibiotics in the preceding 12 months (EXCEED study) and in bronchial brush samples from COPD (n=8) and healthy controls (n=7) (EvA study). RESULTS: ARG mean (SEM) prevalence was greater in stable COPD samples (35.2 (1.6)) than in healthy controls (27.6 (1.7); p=0.004) and correlated with total bacterial abundance (r2=0.23; p<0.001). Prevalence of ARG positive signals in individuals was not related to COPD symptoms, lung function or their changes at exacerbation. In the COPD subgroups designated High γProteobacteria and High Firmicutes, ARG prevalence was not different at stable state but significantly declined from stable through exacerbation to recovery in the former (p=0.011) without changes in total bacterial abundance. The ARG patterns were similar in COPD versus health, COPD microbiome-subgroups and between sputum and bronchoscopic samples independent of antibiotic exposure in the last 12 months. CONCLUSIONS: ARGs are highly prevalent in sputum, broadly in proportion to bacterial abundance in both healthy and COPD subjects. Thus, COPD appears to be an ARG reservoir due to high levels of bacterial colonisation.

2.
G3 (Bethesda) ; 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31645421

RESUMO

The Eastern woodchuck (Marmota monax) has been extensively used in research of chronic hepatitis B and liver cancer because its infection with the woodchuck hepatitis virus closely resembles a human hepatitis B virus infection. Development of novel immunotherapeutic approaches requires genetic information on immune pathway genes in this animal model. The woodchuck genome was assembled with a combination of high-coverage whole-genome shotgun sequencing of Illumina paired-end, mate-pair libraries and fosmid pool sequencing. The result is a 2.63 Gigabase (Gb) assembly with a contig N50 of 74.5 kilobases (kb), scaffold N50 of 892 kb, and genome completeness of 99.2%. RNA sequencing (RNA-seq) from seven different tissues aided in the annotation of 30,873 protein-coding genes, which in turn encode 41,826 unique protein products. More than 90% of the genes have been functionally annotated, with 82% of them containing open reading frames. This genome sequence and its annotation will enable further research in chronic hepatitis B and hepatocellular carcinoma and contribute to the understanding of immunological responses in the woodchuck.

4.
Allergy ; 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31506971

RESUMO

BACKGROUND: Whether the clinical or pathophysiologic significance of the "treatable trait" high blood eosinophil count in COPD is the same as for asthma remains controversial. We sought to determine the relationship between the blood eosinophil count, clinical characteristics and gene expression from bronchial brushings in COPD and asthma. METHODS: Subjects were recruited into a COPD (emphysema versus airway disease [EvA]) or asthma cohort (Unbiased BIOmarkers in PREDiction of respiratory disease outcomes, U-BIOPRED). We determined gene expression using RNAseq in EvA (n = 283) and Affymetrix microarrays in U-BIOPRED (n = 85). We ran linear regression analysis of the bronchial brushings transcriptional signal versus blood eosinophil counts as well as differential expression using a blood eosinophil > 200 cells/µL as a cut-off. The false discovery rate was controlled at 1% (with continuous values) and 5% (with dichotomized values). RESULTS: There were no differences in age, gender, lung function, exercise capacity and quantitative computed tomography between eosinophilic versus noneosinophilic COPD cases. Total serum IgE was increased in eosinophilic asthma and COPD. In EvA, there were 12 genes with a statistically significant positive association with the linear blood eosinophil count, whereas in U-BIOPRED, 1197 genes showed significant associations (266 positive and 931 negative). The transcriptome showed little overlap between genes and pathways associated with blood eosinophil counts in asthma versus COPD. Only CST1 was common to eosinophilic asthma and COPD and was replicated in independent cohorts. CONCLUSION: Despite shared "treatable traits" between asthma and COPD, the molecular mechanisms underlying these clinical entities are predominately different.

5.
Nat Rev Genet ; 20(11): 693-701, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31455890

RESUMO

Human genomics is undergoing a step change from being a predominantly research-driven activity to one driven through health care as many countries in Europe now have nascent precision medicine programmes. To maximize the value of the genomic data generated, these data will need to be shared between institutions and across countries. In recognition of this challenge, 21 European countries recently signed a declaration to transnationally share data on at least 1 million human genomes by 2022. In this Roadmap, we identify the challenges of data sharing across borders and demonstrate that European research infrastructures are well-positioned to support the rapid implementation of widespread genomic data access.

6.
Sci Rep ; 9(1): 12367, 2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31451731

RESUMO

Patient-derived 3D cell culture systems are currently advancing cancer research since they potentiate the molecular analysis of tissue-like properties and drug response under well-defined conditions. However, our understanding of the relationship between the heterogeneity of morphological phenotypes and the underlying transcriptome is still limited. To address this issue, we here introduce "pheno-seq" to directly link visual features of 3D cell culture systems with profiling their transcriptome. As prototypic applications breast and colorectal cancer (CRC) spheroids were analyzed by pheno-seq. We identified characteristic gene expression signatures of epithelial-to-mesenchymal transition that are associated with invasive growth behavior of clonal breast cancer spheroids. Furthermore, we linked long-term proliferative capacity in a patient-derived model of CRC to a lowly abundant PROX1-positive cancer stem cell subtype. We anticipate that the ability to integrate transcriptome analysis and morphological patho-phenotypes of cancer cells will provide novel insight on the molecular origins of intratumor heterogeneity.

7.
Nat Commun ; 10(1): 1749, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30988298

RESUMO

Global loss of DNA methylation and CpG island (CGI) hypermethylation are key epigenomic aberrations in cancer. Global loss manifests itself in partially methylated domains (PMDs) which extend up to megabases. However, the distribution of PMDs within and between tumor types, and their effects on key functional genomic elements including CGIs are poorly defined. We comprehensively show that loss of methylation in PMDs occurs in a large fraction of the genome and represents the prime source of DNA methylation variation. PMDs are hypervariable in methylation level, size and distribution, and display elevated mutation rates. They impose intermediate DNA methylation levels incognizant of functional genomic elements including CGIs, underpinning a CGI methylator phenotype (CIMP). Repression effects on tumor suppressor genes are negligible as they are generally excluded from PMDs. The genomic distribution of PMDs reports tissue-of-origin and may represent tissue-specific silent regions which tolerate instability at the epigenetic, transcriptomic and genetic level.


Assuntos
Neoplasias da Mama/genética , Ilhas de CpG , Metilação de DNA , Epigênese Genética , Feminino , Humanos , Modelos Logísticos
8.
Elife ; 82019 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-30860479

RESUMO

Forced transcription factor expression can transdifferentiate somatic cells into other specialised cell types or reprogram them into induced pluripotent stem cells (iPSCs) with variable efficiency. To better understand the heterogeneity of these processes, we used single-cell RNA sequencing to follow the transdifferentation of murine pre-B cells into macrophages as well as their reprogramming into iPSCs. Even in these highly efficient systems, there was substantial variation in the speed and path of fate conversion. We predicted and validated that these differences are inversely coupled and arise in the starting cell population, with Mychigh large pre-BII cells transdifferentiating slowly but reprogramming efficiently and Myclow small pre-BII cells transdifferentiating rapidly but failing to reprogram. Strikingly, differences in Myc activity predict the efficiency of reprogramming across a wide range of somatic cell types. These results illustrate how single cell expression and computational analyses can identify the origins of heterogeneity in cell fate conversion processes.

9.
Sci Rep ; 9(1): 3656, 2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30842494

RESUMO

Non-alcoholic fatty liver disease (NAFLD) is often associated with obesity and type 2 diabetes. To disentangle etiological relationships between these conditions and identify genetically-determined metabolites involved in NAFLD processes, we mapped 1H nuclear magnetic resonance (NMR) metabolomic and disease-related phenotypes in a mouse F2 cross derived from strains showing resistance (BALB/c) and increased susceptibility (129S6) to these diseases. Quantitative trait locus (QTL) analysis based on single nucleotide polymorphism (SNP) genotypes identified diet responsive QTLs in F2 mice fed control or high fat diet (HFD). In HFD fed F2 mice we mapped on chromosome 18 a QTL regulating liver micro- and macrovesicular steatosis and inflammation, independently from glucose intolerance and adiposity, which was linked to chromosome 4. Linkage analysis of liver metabolomic profiling data identified a QTL for octopamine, which co-localised with the QTL for liver histopathology in the cross. Functional relationship between these two QTLs was validated in vivo in mice chronically treated with octopamine, which exhibited reduction in liver histopathology and metabolic benefits, underlining its role as a mechanistic biomarker of fatty liver with potential therapeutic applications.

10.
Nucleic Acids Res ; 47(6): 2778-2792, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30799488

RESUMO

The concept of tissue-specific gene expression posits that lineage-determining transcription factors (LDTFs) determine the open chromatin profile of a cell via collaborative binding, providing molecular beacons to signal-dependent transcription factors (SDTFs). However, the guiding principles of LDTF binding, chromatin accessibility and enhancer activity have not yet been systematically evaluated. We sought to study these features of the macrophage genome by the combination of experimental (ChIP-seq, ATAC-seq and GRO-seq) and computational approaches. We show that Random Forest and Support Vector Regression machine learning methods can accurately predict chromatin accessibility using the binding patterns of the LDTF PU.1 and four other key TFs of macrophages (IRF8, JUNB, CEBPA and RUNX1). Any of these TFs alone were not sufficient to predict open chromatin, indicating that TF binding is widespread at closed or weakly opened chromatin regions. Analysis of the PU.1 cistrome revealed that two-thirds of PU.1 binding occurs at low accessible chromatin. We termed these sites labelled regulatory elements (LREs), which may represent a dormant state of a future enhancer and contribute to macrophage cellular plasticity. Collectively, our work demonstrates the existence of LREs occupied by various key TFs, regulating specific gene expression programs triggered by divergent macrophage polarizing stimuli.

11.
Sci Rep ; 9(1): 975, 2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30700761

RESUMO

Chronic lymphocytic leukaemia is the most prevalent leukaemia in Western countries. It is an incurable disease characterized by a highly variable clinical course. Chronic lymphocytic leukaemia is an ideal model for studying clonal heterogeneity and dynamics during cancer progression, response to therapy and/or relapse because the disease usually develops over several years. Here we report an analysis by deep sequencing of sequential samples taken at different times from the affected organs of two patients with 12- and 7-year disease courses, respectively. One of the patients followed a linear pattern of clonal evolution, acquiring and selecting new mutations in response to salvage therapy and/or allogeneic transplantation, while the other suffered loss of cellular tumoral clones during progression and histological transformation.

12.
Cell Rep ; 26(4): 1059-1069.e6, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30673601

RESUMO

Global investigation of histone marks in acute myeloid leukemia (AML) remains limited. Analyses of 38 AML samples through integrated transcriptional and chromatin mark analysis exposes 2 major subtypes. One subtype is dominated by patients with NPM1 mutations or MLL-fusion genes, shows activation of the regulatory pathways involving HOX-family genes as targets, and displays high self-renewal capacity and stemness. The second subtype is enriched for RUNX1 or spliceosome mutations, suggesting potential interplay between the 2 aberrations, and mainly depends on IRF family regulators. Cellular consequences in prognosis predict a relatively worse outcome for the first subtype. Our integrated profiling establishes a rich resource to probe AML subtypes on the basis of expression and chromatin data.

13.
Nat Commun ; 10(1): 4, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30602775

RESUMO

Mammalian Y chromosomes are often neglected from genomic analysis. Due to their inherent assembly difficulties, high repeat content, and large ampliconic regions, only a handful of species have their Y chromosome properly characterized. To date, just a single human reference quality Y chromosome, of European ancestry, is available due to a lack of accessible methodology. To facilitate the assembly of such complicated genomic territory, we developed a novel strategy to sequence native, unamplified flow sorted DNA on a MinION nanopore sequencing device. Our approach yields a highly continuous assembly of the first human Y chromosome of African origin. It constitutes a significant improvement over comparable previous methods, increasing continuity by more than 800%. Sequencing native DNA also allows to take advantage of the nanopore signal data to detect epigenetic modifications in situ. This approach is in theory generalizable to any species simplifying the assembly of extremely large and repetitive genomes.


Assuntos
Grupo com Ancestrais do Continente Africano/genética , Cromossomos Humanos Y , Ilhas de CpG , Metilação de DNA , Humanos , Cariotipagem , Anotação de Sequência Molecular , Análise de Sequência de DNA
15.
Eur Urol ; 2018 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-30245085

RESUMO

Causes of high mortality of prostate cancer in men of African ancestry living in the French West Indies are still debated, between suspicions of environmental factors and genetic susceptibility. We report an integrated genomic study of 25 tumour tissues from radical prostatectomy of aggressive (defined by International Society of Urological Pathology ≥3) prostate cancer patients (10 African Caribbean and 15 French Caucasian) using single nucleotide polymorphism arrays, whole-genome sequencing, and RNA sequencing. The results show that African Caribbean tumours are characterised by a more frequent deletion at 1q41-43 encompassing the DNA repair gene PARP1, and a higher proportion of intrachromosomal rearrangements including duplications associated with CDK12 truncating mutations. Transcriptome analyses show an overexpression of genes related to androgen receptor activity in African Caribbean tumours, and of PVT1, a long non-coding RNA located at 8q24 that confirms the strong involvement of this region in prostate tumours from men of African ancestry. Patient summary: Mortality of prostate cancer is higher in African Caribbean men than in French Caucasian men. Specificities of the former could be explained by genomic events linked with key genes such as DNA damage pathway genes PARP1, CDK12, and the oncogenic long non-coding RNA gene PVT1 at the 8q24 prostate cancer susceptibility locus.

16.
Cell Rep ; 24(10): 2784-2794, 2018 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-30184510

RESUMO

Neutrophils are short-lived blood cells that play a critical role in host defense against infections. To better comprehend neutrophil functions and their regulation, we provide a complete epigenetic overview, assessing important functional features of their differentiation stages from bone marrow-residing progenitors to mature circulating cells. Integration of chromatin modifications, methylation, and transcriptome dynamics reveals an enforced regulation of differentiation, for cellular functions such as release of proteases, respiratory burst, cell cycle regulation, and apoptosis. We observe an early establishment of the cytotoxic capability, while the signaling components that activate these antimicrobial mechanisms are transcribed at later stages, outside the bone marrow, thus preventing toxic effects in the bone marrow niche. Altogether, these data reveal how the developmental dynamics of the chromatin landscape orchestrate the daily production of a large number of neutrophils required for innate host defense and provide a comprehensive overview of differentiating human neutrophils.

17.
Cell Stem Cell ; 23(5): 727-741.e9, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30220521

RESUMO

Here, we report DNA methylation and hydroxymethylation dynamics at nucleotide resolution using C/EBPα-enhanced reprogramming of B cells into induced pluripotent cells (iPSCs). We observed successive waves of hydroxymethylation at enhancers, concomitant with a decrease in DNA methylation, suggesting active demethylation. Consistent with this finding, ablation of the DNA demethylase Tet2 almost completely abolishes reprogramming. C/EBPα, Klf4, and Tfcp2l1 each interact with Tet2 and recruit the enzyme to specific DNA sites. During reprogramming, some of these sites maintain high levels of 5hmC, and enhancers and promoters of key pluripotency factors become demethylated as early as 1 day after Yamanaka factor induction. Surprisingly, methylation changes precede chromatin opening in distinct chromatin regions, including Klf4 bound sites, revealing a pioneer factor activity associated with alternation in DNA methylation. Rapid changes in hydroxymethylation similar to those in B cells were also observed during compound-accelerated reprogramming of fibroblasts into iPSCs, highlighting the generality of our observations.

18.
Lancet Respir Med ; 6(10): 771-781, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30100403

RESUMO

BACKGROUND: Anti-programmed death-1 (PD-1) treatment for advanced non-small-cell lung cancer (NSCLC) has improved the survival of patients. However, a substantial percentage of patients do not respond to this treatment. We examined the use of DNA methylation profiles to determine the efficacy of anti-PD-1 treatment in patients recruited with current stage IV NSCLC. METHODS: In this multicentre study, we recruited adult patients from 15 hospitals in France, Spain, and Italy who had histologically proven stage IV NSCLC and had been exposed to PD-1 blockade during the course of the disease. The study structure comprised a discovery cohort to assess the correlation between epigenetic features and clinical benefit with PD-1 blockade and two validation cohorts to assess the validity of our assumptions. We first established an epigenomic profile based on a microarray DNA methylation signature (EPIMMUNE) in a discovery set of tumour samples from patients treated with nivolumab or pembrolizumab. The EPIMMUNE signature was validated in an independent set of patients. A derived DNA methylation marker was validated by a single-methylation assay in a validation cohort of patients. The main study outcomes were progression-free survival and overall survival. We used the Kaplan-Meier method to estimate progression-free and overall survival, and calculated the differences between the groups with the log-rank test. We constructed a multivariate Cox model to identify the variables independently associated with progression-free and overall survival. FINDINGS: Between June 23, 2014, and May 18, 2017, we obtained samples from 142 patients: 34 in the discovery cohort, 47 in the EPIMMUNE validation cohort, and 61 in the derived methylation marker cohort (the T-cell differentiation factor forkhead box P1 [FOXP1]). The EPIMMUNE signature in patients with stage IV NSCLC treated with anti-PD-1 agents was associated with improved progression-free survival (hazard ratio [HR] 0·010, 95% CI 3·29 × 10-4-0·0282; p=0·0067) and overall survival (0·080, 0·017-0·373; p=0·0012). The EPIMMUNE-positive signature was not associated with PD-L1 expression, the presence of CD8+ cells, or mutational load. EPIMMUNE-negative tumours were enriched in tumour-associated macrophages and neutrophils, cancer-associated fibroblasts, and senescent endothelial cells. The EPIMMUNE-positive signature was associated with improved progression-free survival in the EPIMMUNE validation cohort (0·330, 0·149-0·727; p=0·0064). The unmethylated status of FOXP1 was associated with improved progression-free survival (0·415, 0·209-0·802; p=0·0063) and overall survival (0·409, 0·220-0·780; p=0·0094) in the FOXP1 validation cohort. The EPIMMUNE signature and unmethylated FOXP1 were not associated with clinical benefit in lung tumours that did not receive immunotherapy. INTERPRETATION: Our study shows that the epigenetic milieu of NSCLC tumours indicates which patients are most likely to benefit from nivolumab or pembrolizumab treatments. The methylation status of FOXP1 could be associated with validated predictive biomarkers such as PD-L1 staining and mutational load to better select patients who will experience clinical benefit with PD-1 blockade, and its predictive value should be evaluated in prospective studies. FUNDING: "Obra Social" La Caixa, Cellex Foundation, and the Health and Science Departments of the Generalitat de Catalunya.

19.
Bioinformatics ; 2018 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-30137223

RESUMO

Motivation: DNA methylation is essential for normal embryogenesis and development in mammals and can be captured at single base pair resolution by whole genome bisulfite sequencing (WGBS). Current available analysis tools are becoming rapidly outdated as they lack sensible functionality and efficiency to handle large amounts of data now commonly created. Results: We developed gemBS, a fast high-throughput bioinformatics pipeline specifically designed for large scale BS-Seq analysis that combines a high performance BS-mapper (GEM3) and a variant caller specifically for BS-Seq data (BScall). gemBS provides genotype information and methylation estimates for all genomic cytosines in different contexts (CpG and non-CpG) and a set of quality reports for comprehensive and reproducible analysis. gemBS is highly modular and can be easily automated, while producing robust and accurate results. Availability: gemBS is released under the GNU GPLv3+ license. Source code and documentation are freely available from www.statgen.cat/gemBS. Supplementary Information: Supplementary data are available at Bioinformatics online.

20.
Nat Neurosci ; 21(8): 1139, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29875394

RESUMO

In the supplementary information PDF originally posted, there were discrepancies from the integrated supplementary information that appeared in the HTML; the former has been corrected as follows. In the legend to Supplementary Fig. 2c, "major organs of the mouse" has been changed to "major organs of the adult mouse." In the legend to Supplementary Fig. 6d,h, "At E14.5 Mbe/Mbe mutants have a smaller percentage of Brdu positive cells in bin 3" has been changed to "At E14.5 Mbe/Mbe mutants have a higher percentage of Brdu positive cells in bin 3."

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