Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 9 de 9
Filtrar
1.
BMC Microbiol ; 18(1): 110, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30189859

RESUMO

BACKGROUND: The effects of gut microbiota on human traits are expected to be small to moderate and adding the complexity of the human diseases, microbiome research demands big sample sizes. Fecal samples for such studies are mostly self-collected by participants at home. This imposes an extra level of complexity as sample collection and storage can be challenging. Effective, low-burden collection and storage methods allowing fecal samples to be transported properly and ensuring optimal quality and quantity of bacterial DNA for upstream analyses are necessary. Moreover, accurate assessment of the microbiome composition also depends on bacterial DNA extraction method. The aim of this study was to evaluate the reliability and efficiency of the OMNIgene•GUT kit as a participant-fecal friendly collection method (storage at room temperature for 24 h (O24h) or 7 days (O7d)) in comparison to the standard collection method (Fresh, storage at 4 °C for less than 24 h) in terms of amount of variability and information content accounting for two common DNA extraction methods. RESULTS: Fourteen fecal samples were collected from healthy individuals (7 males, 7 females). Collection and storage methods did not differ significantly in terms of DNA concentration and Shannon diversity index. Phylum relative abundance showed significant differences for Bacteroidetes, Actinobacteria and Cyanobacteria. The differences were observed between control (Fresh) and O24h methods, but not between Fresh and O7d. These differences were not seen when performing bacterial DNA quantification based on three bacterial groups: Bacteroides spp., Bifidobacterium spp. and Clostridium cluster IV, which represent three major phyla: Bacteroidetes, Actinobacteria and Firmicutes respectively. The two DNA extraction methods differ in terms of DNA quantity, quality, bacterial diversity and bacterial relative abundance. Furthermore, principal component analysis revealed differences in microbial structure, which are driven by the DNA extraction methods more than the collection/storage methods. CONCLUSION: Our results have highlighted the potential of using the OMNIgene•GUT kit for collection and storage at ambient temperature, which is convenient for studies aiming to collect large samples by giving participants the possibility to send samples by post. Importantly, we revealed that the choice of DNA extraction method have an impact on the microbiome profiling.


Assuntos
Bactérias/isolamento & purificação , Fezes/microbiologia , Microbioma Gastrointestinal , Manejo de Espécimes/métodos , Bactérias/classificação , Bactérias/genética , DNA Bacteriano/genética , Feminino , Humanos , Masculino , RNA Ribossômico 16S/genética , Tamanho da Amostra
2.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1862(10 Pt A): 1056-1067, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28733267

RESUMO

Angiopoietin-like 4 (ANGPTL4) raises plasma triglyceride levels by inhibiting lipoprotein lipase. A set of compounds that are able to reduce plasma triglyceride levels are bile acids (BA). Because BA have been shown to decrease ANGPTL4 secretion by intestinal cells, we hypothesized that BA lower plasma triglycerides (partly) via ANGPTL4. To test that hypothesis, wild-type and Angptl4-/- mice were fed chow supplemented with taurocholic acid (TCA) for seven days. TCA supplementation effectively lowered plasma triglycerides in wild-type and Angptl4-/- mice, indicating that ANGPTL4 is not required for plasma triglyceride-lowering by BA. Intriguingly, however, plasma and hepatic BA concentrations were significantly lower in TCA-supplemented Angptl4-/- mice than in TCA-supplemented wild-type mice. These changes in the Angptl4-/- mice were accompanied by lower BA levels in ileal scrapings and decreased expression of FXR-target genes in the ileum, including the BA transporter Slc10a2. By contrast, faecal excretion of specifically primary BA was higher in the Angptl4-/- mice, suggesting that loss of ANGPTL4 impairs intestinal BA absorption. Since the gut microbiota converts primary BA into secondary BA, elevated excretion of primary BA in Angptl4-/- mice may reflect differences in gut microbial composition and/or functionality. Indeed, colonic microbial composition was markedly different between Angptl4-/- and wild-type mice. Suppression of the gut bacteria using antibiotics abolished differences in plasma, hepatic, and faecal BA levels between TCA-supplemented Angptl4-/- and wild-type mice. In conclusion, 1) ANGPTL4 is not involved in the triglyceride-lowering effect of BA; 2) ANGPTL4 promotes BA absorption during TCA supplementation via a mechanism dependent on the gut microbiota.


Assuntos
Proteína 4 Semelhante a Angiopoietina/metabolismo , Ácidos e Sais Biliares/metabolismo , Suplementos Nutricionais , Microbioma Gastrointestinal/fisiologia , Absorção Intestinal/efeitos dos fármacos , Ácido Taurocólico , Proteína 4 Semelhante a Angiopoietina/genética , Animais , Ácidos e Sais Biliares/genética , Absorção Intestinal/genética , Camundongos , Camundongos Knockout , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/genética , Simportadores/metabolismo , Ácido Taurocólico/farmacocinética , Ácido Taurocólico/farmacologia , Triglicerídeos/sangue
3.
BMC Genomics ; 14: 337, 2013 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-23682854

RESUMO

BACKGROUND: Metabolic homeostasis in mammals critically depends on the regulation of fasting-induced genes by CREB in the liver. Previous genome-wide analysis has shown that only a small percentage of CREB target genes are induced in response to fasting-associated signaling pathways. The precise molecular mechanisms by which CREB specifically targets these genes in response to alternating hormonal cues remain to be elucidated. RESULTS: We performed chromatin immunoprecipitation coupled to high-throughput sequencing of CREB in livers from both fasted and re-fed mice. In order to quantitatively compare the extent of CREB-DNA interactions genome-wide between these two physiological conditions we developed a novel, robust analysis method, termed the 'single sample independence' (SSI) test that greatly reduced the number of false-positive peaks. We found that CREB remains constitutively bound to its target genes in the liver regardless of the metabolic state. Integration of the CREB cistrome with expression microarrays of fasted and re-fed mouse livers and ChIP-seq data for additional transcription factors revealed that the gene expression switches between the two metabolic states are associated with co-localization of additional transcription factors at CREB sites. CONCLUSIONS: Our results support a model in which CREB is constitutively bound to thousands of target genes, and combinatorial interactions between DNA-binding factors are necessary to achieve the specific transcriptional response of the liver to fasting. Furthermore, our genome-wide analysis identifies thousands of novel CREB target genes in liver, and suggests a previously unknown role for CREB in regulating ER stress genes in response to nutrient influx.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ingestão de Alimentos , Jejum/metabolismo , Genômica , Fígado/metabolismo , Animais , Sequência de Bases , Imunoprecipitação da Cromatina , DNA/metabolismo , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Transcrição Gênica
4.
PLoS One ; 16(11): e0258960, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34758050

RESUMO

During aging of human skin, a number of intrinsic and extrinsic factors cause the alteration of the skin's structure, function and cutaneous physiology. Many studies have investigated the influence of the skin microbiome on these alterations, but the molecular mechanisms that dictate the interplay between these factors and the skin microbiome are still not fully understood. To obtain more insight into the connection between the skin microbiome and the human physiological processes involved in skin aging, we performed a systematic study on interconnected pathways of human and bacterial metabolic processes that are known to play a role in skin aging. The bacterial genes in these pathways were subsequently used to create Hidden Markov Models (HMMs), which were applied to screen for presence of defined functionalities in both genomic and metagenomic datasets of skin-associated bacteria. These models were further applied on 16S rRNA gene sequencing data from skin microbiota samples derived from female volunteers of two different age groups (25-28 years ('young') and 59-68 years ('old')). The results show that the main bacterial pathways associated with aging skin are those involved in the production of pigmentation intermediates, fatty acids and ceramides. This study furthermore provides evidence for a relation between skin aging and bacterial enzymes involved in protein glycation. Taken together, the results and insights described in this paper provide new leads for intervening with bacterial processes that are associated with aging of human skin.


Assuntos
Metagenoma , Metagenômica/métodos , Microbiota/genética , Envelhecimento da Pele/genética , Pele/microbiologia , Adulto , Idoso , Bactérias/genética , Bactérias/metabolismo , Ceramidas/metabolismo , Ácidos Graxos/metabolismo , Feminino , Genes Bacterianos , Voluntários Saudáveis , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Cadeias de Markov , Pessoa de Meia-Idade , RNA Ribossômico 16S/genética , Transdução de Sinais/genética , Pele/metabolismo , Pigmentação da Pele/genética
5.
Sci Rep ; 9(1): 19834, 2019 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-31882601

RESUMO

We present TaxPhlAn, a new method and bioinformatics pipeline for design and analysis of single-locus sequence typing (SLST) markers to type and profile bacteria beyond the species-level in a complex microbial community background. TaxPhlAn can be applied to any group of phylogenetically-related bacteria, provided reference genomes are available. As TaxPhlAn requires the SLST targets identified to fit the phylogenetic pattern as determined through comprehensive evolutionary reconstruction of input genomes, TaxPhlAn allows for the identification and phylogenetic inference of new biodiversity. Here, we present a clinically relevant case study of high-resolution Staphylococcus profiling on skin of atopic dermatitis (AD) patients. We demonstrate that SLST enables profiling of cutaneous Staphylococcus members at (sub)species level and provides higher resolution than current 16S-based techniques. With the higher discriminative ability provided by our approach, we further show that the presence of Staphylococcus capitis on the skin together with Staphylococcus aureus associates with AD disease.


Assuntos
Bactérias/genética , Técnicas de Tipagem Bacteriana/métodos , Biologia Computacional/métodos , Genes Bacterianos/genética , Microbiota/genética , Bactérias/classificação , Dermatite Atópica/microbiologia , Feminino , Humanos , Masculino , Filogenia , Pele/microbiologia , Pele/patologia , Especificidade da Espécie , Infecções Estafilocócicas/microbiologia , Staphylococcus/classificação , Staphylococcus/genética , Staphylococcus/fisiologia , Fluxo de Trabalho
6.
Cell Mol Gastroenterol Hepatol ; 2(6): 783-795, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28090567

RESUMO

BACKGROUND & AIMS: The continuously self-renewing mammalian intestinal epithelium, with high cellular turnover, depends on adequate protein synthesis for its proliferative capacity. RNA polymerase III activity is closely related to cellular growth and proliferation. Here, we studied the role of Polr3b, a large RNA polymerase III subunit, in the mammalian intestinal epithelium. METHODS: We derived mice with an intestinal epithelium-specific hypomorphic mutation of the Polr3b gene, using VillinCre-mediated gene ablation. Phenotypic consequences of the Polr3b mutation on the intestinal epithelium in mice were assessed using histological and molecular methodologies, including genetic lineage tracing. RESULTS: The Polr3b mutation severely reduced survival and growth in mice during the first postnatal week, the period when the expansion of the intestinal epithelium, and thus the requirement for protein synthesis, are highest. The neonatal intestinal epithelium of Polr3bloxP/loxP;VillinCre mice was characterized by areas with reduced proliferation, abnormal epithelial architecture, loss of Wnt signaling and a dramatic increase in apoptotic cells in crypts. Genetic lineage tracing using Polr3bLoxP/LoxP;Rosa26-lox-stop-lox-YFP;VillinCre mice demonstrated that in surviving mutant mice, Polr3b-deficient dying crypts were progressively replaced by 'Cre-escaper' cells that had retained wild type Polr3b function. In addition, enteroids cultured from Polr3bloxP/loxP;VillinCre mice show reduced proliferative activity and increased apoptosis. CONCLUSIONS: We provide evidence for an essential role of the Pol III subunit Polr3b in orchestrating the maintenance of the intestinal crypt during early postnatal development in mice.

7.
mBio ; 5(4)2014 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-25118238

RESUMO

UNLABELLED: The gut microbiota is essential for numerous aspects of human health. However, the underlying mechanisms of many host-microbiota interactions remain unclear. The aim of this study was to characterize effects of the microbiota on host epithelium using a novel ex vivo model based on mouse ileal organoids. We have explored the transcriptional response of organoids upon exposure to short-chain fatty acids (SCFAs) and products generated by two abundant microbiota constituents, Akkermansia muciniphila and Faecalibacterium prausnitzii. We observed that A. muciniphila metabolites affect various transcription factors and genes involved in cellular lipid metabolism and growth, supporting previous in vivo findings. Contrastingly, F. prausnitzii products exerted only weak effects on host transcription. Additionally, A. muciniphila and its metabolite propionate modulated expression of Fiaf, Gpr43, histone deacetylases (HDACs), and peroxisome proliferator-activated receptor gamma (Pparγ), important regulators of transcription factor regulation, cell cycle control, lipolysis, and satiety. This work illustrates that specific bacteria and their metabolites differentially modulate epithelial transcription in mouse organoids. We demonstrate that intestinal organoids provide a novel and powerful ex vivo model for host-microbiome interaction studies. IMPORTANCE: We investigated the influence of the gut microbiota and microbially produced short-chain fatty acids (SCFAs) on gut functioning. Many commensal bacteria in the gut produce SCFAs, particularly butyrate, acetate, and propionate, which have been demonstrated to reduce the risk of gastrointestinal disorders. Organoids-small crypt-villus structures grown from ileal intestinal stem cells-were exposed to SCFAs and two specific gut bacteria. Akkermansia muciniphila, found in the intestinal mucus, was recently shown to have a favorable effect on the disrupted metabolism associated with obesity. Faecalibacterium prausnitzii is a commensal gut bacterium, the absence of which may be associated with Crohn's disease. We showed that in our model, A. muciniphila induces stronger effects on the host than F. prausnitzii. We observed that A. muciniphila and propionate affect the expression of genes involved in host lipid metabolism and epigenetic activation or silencing of gene expression. We demonstrated that organoids provide a powerful tool for host-microbe interaction studies.


Assuntos
Bactérias Gram-Positivas/metabolismo , Histonas/metabolismo , Íleo/efeitos dos fármacos , Íleo/fisiologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Metabolismo dos Lipídeos , Verrucomicrobia/metabolismo , Acetilação , Proteína 4 Semelhante a Angiopoietina , Angiopoietinas/genética , Animais , Epigênese Genética , Ácidos Graxos Voláteis/administração & dosagem , Perfilação da Expressão Gênica , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/crescimento & desenvolvimento , Histona Desacetilases/genética , Intestinos/efeitos dos fármacos , Lipólise , Camundongos , Microbiota , Organoides , PPAR gama/genética , Propionatos/metabolismo , Receptores Acoplados a Proteínas G/genética , Verrucomicrobia/química , Verrucomicrobia/crescimento & desenvolvimento
8.
Best Pract Res Clin Gastroenterol ; 27(1): 101-13, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23768556

RESUMO

It is increasingly apparent that the microbial ecosystems in the mammalian gastrointestinal tract play an intricate role in health and disease. There is a growing interest in the development of targeted strategies for modulating health through the modification of these microbiota. Ecologists are faced with the challenge of understanding the structure and function of ecosystems, the component parts of which interact with each other in complex and diffuse ways. The human gut microbiota, with its high species richness and diversity (up to 1000 bacterial species per individual) including members of all three domains of life, situated in the dynamic environment of the gastrointestinal tract, is probably among the most complex ecosystems on this planet. In order to elucidate the mechanistic foundations, and physiological significance, of beneficial or pathogenic relationships between the gut microbiota and their hosts, researchers require tractable model ecosystems that allow to recapitulate and investigate host-microbe and microbe-microbe interactions. This review discusses ex vivo gastrointestinal models systems that can be used to gain mechanistic insights into the emergent properties of the host-microbial superorganism.


Assuntos
Trato Gastrointestinal/microbiologia , Interações Hospedeiro-Patógeno/fisiologia , Metagenoma/fisiologia , Animais , Bactérias/crescimento & desenvolvimento , Ecossistema , Humanos , Interações Microbianas/fisiologia
9.
Nestle Nutr Workshop Ser Pediatr Program ; 59: 147-57; discussion 157-9, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17245097

RESUMO

Cholestatic liver disease (CLD) in children negatively affects nutritional status, growth and development, which all lead to an increased risk of morbidity and mortality. This is illustrated by the fact that the clinical outcome of children with CLD awaiting a liver transplantation is in part predicted by their nutritional status, which is integrated in the pediatric end-stage liver disease model. Preservation of the nutritional status becomes more relevant as the number of patients waiting for liver transplantation increases and the waiting time for a donor organ becomes prolonged. Nutritional strategies are available to optimize feeding of children with CLD. Patients with CLD, however, form a heterogeneous group and the clinical manifestations of their disease vary. This makes a tailor-made approach for these children crucial. Not all aspects of nutrient metabolism and absorption in children with CLD are well understood and studied. Experiments with stable isotope-labeled triglycerides and fatty acids have provided essential information about fat absorption under physiological and cholestatic conditions in animal models and humans. We expect that in the future, tests using other isotope-labeled macronutrients, i.e. carbohydrates and proteins, can be used to further assess nutritional status of children with CLD, thereby creating tailor-made nutritional therapies.


Assuntos
Fenômenos Fisiológicos da Nutrição Infantil , Colestase/terapia , Hepatopatias/terapia , Transplante de Fígado , Estado Nutricional , Criança , Ingestão de Energia/fisiologia , Metabolismo Energético/fisiologia , Feminino , Humanos , Masculino
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA