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1.
Brief Bioinform ; 24(6)2023 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-37870287

RESUMO

Computational reproducibility is a simple premise in theory, but is difficult to achieve in practice. Building upon past efforts and proposals to maximize reproducibility and rigor in bioinformatics, we present a framework called the five pillars of reproducible computational research. These include (1) literate programming, (2) code version control and sharing, (3) compute environment control, (4) persistent data sharing and (5) documentation. These practices will ensure that computational research work can be reproduced quickly and easily, long into the future. This guide is designed for bioinformatics data analysts and bioinformaticians in training, but should be relevant to other domains of study.


Assuntos
Biologia Computacional , Disseminação de Informação , Reprodutibilidade dos Testes , Software
2.
Int J Mol Sci ; 25(13)2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-39000495

RESUMO

Patients with asthma experience elevated rates of mental illness. However, the molecular links underlying such lung-brain crosstalk remain ambiguous. Hypothalamic dysfunction is observed in many psychiatric disorders, particularly those with an inflammatory component due to many hypothalamic regions being unprotected by the blood-brain barrier. To gain a better insight into such neuropsychiatric sequelae, this study investigated gene expression differences in the hypothalamus following lung inflammation (asthma) induction in mice, using RNA transcriptome profiling. BALB/c mice were challenged with either bacterial lipopolysaccharide (LPS, E. coli) or ovalbumin (OVA) allergens or saline control (n = 7 per group), and lung inflammation was confirmed via histological examination of postmortem lung tissue. The majority of the hypothalamus was micro-dissected, and total RNA was extracted for sequencing. Differential expression analysis identified 31 statistically significant single genes (false discovery rate FDR5%) altered in expression following LPS exposure compared to controls; however, none were significantly changed following OVA treatment, suggesting a milder hypothalamic response. When gene sets were examined, 48 were upregulated and 8 were downregulated in both asthma groups relative to controls. REACTOME enrichment analysis suggests these gene sets are involved in signal transduction metabolism, immune response and neuroplasticity. Interestingly, we identified five altered gene sets directly associated with neurotransmitter signaling. Intriguingly, many of these altered gene sets can influence mental health and or/neuroinflammation in humans. These findings help characterize the links between asthma-induced lung inflammation and the brain and may assist in identifying relevant pathways and therapeutic targets for future intervention.


Assuntos
Asma , Modelos Animais de Doenças , Hipotálamo , Lipopolissacarídeos , Pulmão , Camundongos Endogâmicos BALB C , Transcriptoma , Animais , Asma/genética , Asma/metabolismo , Asma/patologia , Hipotálamo/metabolismo , Camundongos , Pulmão/metabolismo , Pulmão/patologia , Ovalbumina , Perfilação da Expressão Gênica , Feminino , Regulação da Expressão Gênica
3.
PLoS Comput Biol ; 18(3): e1009935, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35263338

RESUMO

Gene set enrichment tests (a.k.a. functional enrichment analysis) are among the most frequently used methods in computational biology. Despite this popularity, there are concerns that these methods are being applied incorrectly and the results of some peer-reviewed publications are unreliable. These problems include the use of inappropriate background gene lists, lack of false discovery rate correction and lack of methodological detail. To ascertain the frequency of these issues in the literature, we performed a screen of 186 open-access research articles describing functional enrichment results. We find that 95% of analyses using over-representation tests did not implement an appropriate background gene list or did not describe this in the methods. Failure to perform p-value correction for multiple tests was identified in 43% of analyses. Many studies lacked detail in the methods section about the tools and gene sets used. An extension of this survey showed that these problems are not associated with journal or article level bibliometrics. Using seven independent RNA-seq datasets, we show misuse of enrichment tools alters results substantially. In conclusion, most published functional enrichment studies suffered from one or more major flaws, highlighting the need for stronger standards for enrichment analysis.


Assuntos
Biologia Computacional , Biologia Computacional/métodos , RNA-Seq
4.
Cell Mol Life Sci ; 79(11): 579, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36319916

RESUMO

Sulforaphane has been investigated in human pathologies and preclinical models of airway diseases. To provide further mechanistic insights, we explored L-sulforaphane (LSF) in the ovalbumin (OVA)-induced chronic allergic airways murine model, with key hallmarks of asthma. Histological analysis indicated that LSF prevented or reversed OVA-induced epithelial thickening, collagen deposition, goblet cell metaplasia, and inflammation. Well-known antioxidant and anti-inflammatory mechanisms contribute to the beneficial effects of LSF. Fourier transform infrared microspectroscopy revealed altered composition of macromolecules, following OVA sensitization, which were restored by LSF. RNA sequencing in human peripheral blood mononuclear cells highlighted the anti-inflammatory signature of LSF. Findings indicated that LSF may alter gene expression via an epigenetic mechanism which involves regulation of protein acetylation status. LSF resulted in histone and α-tubulin hyperacetylation in vivo, and cellular and enzymatic assays indicated decreased expression and modest histone deacetylase (HDAC) inhibition activity, in comparison with the well-known pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). Molecular modeling confirmed interaction of LSF and LSF metabolites with the catalytic domain of metal-dependent HDAC enzymes. More generally, this study confirmed known mechanisms and identified potential epigenetic pathways accounting for the protective effects and provide support for the potential clinical utility of LSF in allergic airways disease.


Assuntos
Antioxidantes , Hipersensibilidade , Camundongos , Humanos , Animais , Leucócitos Mononucleares , Ovalbumina , Epigênese Genética , Anti-Inflamatórios
5.
Circulation ; 143(16): 1614-1628, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33682422

RESUMO

BACKGROUND: Despite in-depth knowledge of the molecular mechanisms controlling embryonic heart development, little is known about the signals governing postnatal maturation of the human heart. METHODS: Single-nucleus RNA sequencing of 54 140 nuclei from 9 human donors was used to profile transcriptional changes in diverse cardiac cell types during maturation from fetal stages to adulthood. Bulk RNA sequencing and the Assay for Transposase-Accessible Chromatin using sequencing were used to further validate transcriptional changes and to profile alterations in the chromatin accessibility landscape in purified cardiomyocyte nuclei from 21 human donors. Functional validation studies of sex steroids implicated in cardiac maturation were performed in human pluripotent stem cell-derived cardiac organoids and mice. RESULTS: Our data identify the progesterone receptor as a key mediator of sex-dependent transcriptional programs during cardiomyocyte maturation. Functional validation studies in human cardiac organoids and mice demonstrate that the progesterone receptor drives sex-specific metabolic programs and maturation of cardiac contractile properties. CONCLUSIONS: These data provide a blueprint for understanding human heart maturation in both sexes and reveal an important role for the progesterone receptor in human heart development.


Assuntos
Coração/fisiopatologia , Receptores de Progesterona/metabolismo , Feminino , Humanos , Masculino , Fatores Sexuais
6.
PLoS Comput Biol ; 17(7): e1008984, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34329294

RESUMO

Erroneous conversion of gene names into other dates and other data types has been a frustration for computational biologists for years. We hypothesized that such errors in supplementary files might diminish after a report in 2016 highlighting the extent of the problem. To assess this, we performed a scan of supplementary files published in PubMed Central from 2014 to 2020. Overall, gene name errors continued to accumulate unabated in the period after 2016. An improved scanning software we developed identified gene name errors in 30.9% (3,436/11,117) of articles with supplementary Excel gene lists; a figure significantly higher than previously estimated. This is due to gene names being converted not just to dates and floating-point numbers, but also to internal date format (five-digit numbers). These findings further reinforce that spreadsheets are ill-suited to use with large genomic data.


Assuntos
Biologia Computacional/normas , Genes/genética , Anotação de Sequência Molecular/normas , Humanos , PubMed , Software , Terminologia como Assunto
7.
Int J Mol Sci ; 23(20)2022 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-36293464

RESUMO

The lack of effective treatments for mitochondrial disease has seen the development of new approaches, including those that stimulate mitochondrial biogenesis to boost ATP production. Here, we examined the effects of deoxyribonucleosides (dNs) on mitochondrial biogenesis and function in Short chain enoyl-CoA hydratase 1 (ECHS1) 'knockout' (KO) cells, which exhibit combined defects in both oxidative phosphorylation (OXPHOS) and mitochondrial fatty acid ß-oxidation (FAO). DNs treatment increased mitochondrial DNA (mtDNA) copy number and the expression of mtDNA-encoded transcripts in both CONTROL (CON) and ECHS1 KO cells. DNs treatment also altered global nuclear gene expression, with key gene sets including 'respiratory electron transport' and 'formation of ATP by chemiosmotic coupling' increased in both CON and ECHS1 KO cells. Genes involved in OXPHOS complex I biogenesis were also upregulated in both CON and ECHS1 KO cells following dNs treatment, with a corresponding increase in the steady-state levels of holocomplex I in ECHS1 KO cells. Steady-state levels of OXPHOS complex V, and the CIII2/CIV and CI/CIII2/CIV supercomplexes, were also increased by dNs treatment in ECHS1 KO cells. Importantly, treatment with dNs increased both basal and maximal mitochondrial oxygen consumption in ECHS1 KO cells when metabolizing either glucose or the fatty acid palmitoyl-L-carnitine. These findings highlight the ability of dNs to improve overall mitochondrial respiratory function, via the stimulation mitochondrial biogenesis, in the face of combined defects in OXPHOS and FAO due to ECHS1 deficiency.


Assuntos
Enoil-CoA Hidratase , Biogênese de Organelas , Enoil-CoA Hidratase/genética , Enoil-CoA Hidratase/metabolismo , DNA Mitocondrial/genética , Ácidos Graxos/metabolismo , Glucose , Carnitina , Desoxirribonucleosídeos , Trifosfato de Adenosina
8.
Circulation ; 141(17): 1393-1403, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32093510

RESUMO

BACKGROUND: High blood pressure (BP) continues to be a major, poorly controlled but modifiable risk factor for cardiovascular death. Among key Western lifestyle factors, a diet poor in fiber is associated with prevalence of high BP. The impact of lack of prebiotic fiber and the associated mechanisms that lead to higher BP are unknown. Here we show that lack of prebiotic dietary fiber leads to the development of a hypertensinogenic gut microbiota, hypertension and its complications, and demonstrate a role for G-protein coupled-receptors (GPCRs) that sense gut metabolites. METHODS: One hundred seventy-nine mice including C57BL/6J, gnotobiotic C57BL/6J, and knockout strains for GPR41, GPR43, GPR109A, and GPR43/109A were included. C57BL/6J mice were implanted with minipumps containing saline or a slow-pressor dose of angiotensin II (0.25 mg·kg-1·d-1). Mice were fed diets lacking prebiotic fiber with or without addition of gut metabolites called short-chain fatty acids ([SCFA)] produced during fermentation of prebiotic fiber in the large intestine), or high prebiotic fiber diets. Cardiac histology and function, BP, sodium and potassium excretion, gut microbiome, flow cytometry, catecholamines and methylation-wide changes were determined. RESULTS: Lack of prebiotic fiber predisposed mice to hypertension in the presence of a mild hypertensive stimulus, with resultant pathological cardiac remodeling. Transfer of a hypertensinogenic microbiota to gnotobiotic mice recapitulated the prebiotic-deprived hypertensive phenotype, including cardiac manifestations. Reintroduction of SCFAs to fiber-depleted mice had protective effects on the development of hypertension, cardiac hypertrophy, and fibrosis. The cardioprotective effect of SCFAs were mediated via the cognate SCFA receptors GPR43/GPR109A, and modulated L-3,4-dihydroxyphenylalanine levels and the abundance of T regulatory cells regulated by DNA methylation. CONCLUSIONS: The detrimental effects of low fiber Westernized diets may underlie hypertension, through deficient SCFA production and GPR43/109A signaling. Maintaining a healthy, SCFA-producing microbiota is important for cardiovascular health.


Assuntos
Fibras na Dieta/deficiência , Ácidos Graxos Voláteis/metabolismo , Microbioma Gastrointestinal , Hipertensão , Mucosa Intestinal , Prebióticos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Animais , Hipertensão/genética , Hipertensão/metabolismo , Hipertensão/microbiologia , Hipertensão/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Knockout , Receptores Acoplados a Proteínas G/genética
9.
BMC Genomics ; 21(1): 447, 2020 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-32600408

RESUMO

BACKGROUND: Inference of biological pathway activity via gene set enrichment analysis is frequently used in the interpretation of clinical and other omics data. With the proliferation of new omics profiling approaches and ever-growing size of data sets generated, there is a lack of tools available to perform and visualise gene set enrichments in analyses involving multiple contrasts. RESULTS: To address this, we developed mitch, an R package for multi-contrast gene set enrichment analysis. It uses a rank-MANOVA statistical approach to identify sets of genes that exhibit joint enrichment across multiple contrasts. Its unique visualisation features enable the exploration of enrichments in up to 20 contrasts. We demonstrate the utility of mitch with case studies spanning multi-contrast RNA expression profiling, integrative multi-omics, tool benchmarking and single-cell RNA sequencing. Using simulated data we show that mitch has similar accuracy to state of the art tools for single-contrast enrichment analysis, and superior accuracy in identifying multi-contrast enrichments. CONCLUSION: mitch is a versatile tool for rapidly and accurately identifying and visualising gene set enrichments in multi-contrast omics data. Mitch is available from Bioconductor ( https://bioconductor.org/packages/mitch ).


Assuntos
Biologia Computacional/métodos , Análise de Célula Única/métodos , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Análise de Sequência de RNA , Software
10.
Am J Physiol Heart Circ Physiol ; 316(1): H45-H60, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30387702

RESUMO

Dilated cardiomyopathy (DCM) is a major cause of heart failure without effective therapy. Fibrogenesis plays a key role in the development of DCM, but little is known of the expression of the profibrotic factor galectin-3 (Gal-3) and its role in DCM pathophysiology. In a mouse DCM model with transgenic (TG) overexpression of mammalian sterile 20-like kinase 1 (Mst1), we studied Gal-3 expression and effects of the Gal-3 inhibitor modified citrus pectin (MCP) or Gal-3 gene knockout (KO). Gal-3 deletion in TG mice (TG/KO) was achieved by crossbreeding Mst1-TG mice with Gal-3 KO mice. The DCM phenotype was assessed by echocardiography and micromanometry. Cardiac expression of Gal-3 and fibrosis were determined. The cardiac transcriptome was profiled by RNA sequencing. Mst1-TG mice at 3-8 mo of age exhibited upregulated expression of Gal-3 by ~40-fold. TG mice had dilatation of cardiac chambers, suppressed left ventricular (LV) ejection fraction, poor LV contractility and relaxation, a threefold increase in LV collagen content, and upregulated fibrotic genes. Four-month treatment with MCP showed no beneficial effects. Gal-3 deletion in Mst1-TG mice attenuated chamber dilatation, organ congestion, and fibrogenesis. RNA sequencing identified profound disturbances by Mst1 overexpression in the cardiac transcriptome, which largely remained in TG/KO hearts. Gal-3 deletion in Mst1-TG mice, however, partially reversed the dysregulated transcriptional signaling involving extracellular matrix remodeling and collagen formation. We conclude that cardiac Mst1 activation leads to marked Gal-3 upregulation and transcriptome disturbances in the heart. Gal-3 deficiency attenuated cardiac remodeling and fibrotic signaling. NEW & NOTEWORTHY We found in a transgenic mouse dilated cardiomyopathy (DCM) model a pronounced upregulation of galectin-3 in cardiomyocytes. Galectin-3 gene deletion reduced cardiac fibrosis and fibrotic gene profiles and ameliorated cardiac remodeling and dysfunction. These benefits of galectin-3 deletion were in contrast to the lack of effect of treatment with the galectin-3 inhibitor modified citrus pectin. Our study suggests that suppression of galectin-3 mRNA expression could be used to treat DCM with high cardiac galectin-3 content.


Assuntos
Cardiomiopatia Dilatada/metabolismo , Galectina 3/genética , Fator de Crescimento de Hepatócito/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Remodelação Ventricular , Animais , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Colágeno/genética , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibrose , Galectina 3/metabolismo , Fator de Crescimento de Hepatócito/genética , Camundongos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais
11.
Epilepsia ; 60(6): 1091-1103, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31074842

RESUMO

OBJECTIVES: Focal cortical dysplasia (FCD) is a major cause of drug-resistant focal epilepsy in children, and the clinicopathological classification remains a challenging issue in daily practice. With the recent progress in DNA methylation-based classification of human brain tumors we examined whether genomic DNA methylation and gene expression analysis can be used to also distinguish human FCD subtypes. METHODS: DNA methylomes and transcriptomes were generated from massive parallel sequencing in 15 surgical FCD specimens, matched with 5 epilepsy and 6 nonepilepsy controls. RESULTS: Differential hierarchical cluster analysis of DNA methylation distinguished major FCD subtypes (ie, Ia, IIa, and IIb) from patients with temporal lobe epilepsy patients and nonepileptic controls. Targeted panel sequencing identified a novel likely pathogenic variant in DEPDC5 in a patient with FCD type IIa. However, no enrichment of differential DNA methylation or gene expression was observed in mechanistic target of rapamycin (mTOR) pathway-related genes. SIGNIFICANCE: Our studies extend the evidence for disease-specific methylation signatures toward focal epilepsies in favor of an integrated clinicopathologic and molecular classification system of FCD subtypes incorporating genomic methylation.


Assuntos
Metilação de DNA/genética , Malformações do Desenvolvimento Cortical/genética , Adolescente , Adulto , Criança , Pré-Escolar , Análise por Conglomerados , DNA/genética , Epilepsias Parciais/classificação , Epilepsias Parciais/genética , Feminino , Perfilação da Expressão Gênica , Genoma Humano , Humanos , Lactente , Masculino , Malformações do Desenvolvimento Cortical/classificação , Malformações do Desenvolvimento Cortical/diagnóstico por imagem , Pessoa de Meia-Idade , RNA Mensageiro/genética , Serina-Treonina Quinases TOR/genética , Bancos de Tecidos , Tomografia Computadorizada de Emissão de Fóton Único , Tomografia Computadorizada por Raios X , Transcriptoma , Adulto Jovem
12.
J Am Soc Nephrol ; 29(5): 1437-1448, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29490938

RESUMO

Background The failure of spontaneous resolution underlies chronic inflammatory conditions, including microvascular complications of diabetes such as diabetic kidney disease. The identification of endogenously generated molecules that promote the physiologic resolution of inflammation suggests that these bioactions may have therapeutic potential in the context of chronic inflammation. Lipoxins (LXs) are lipid mediators that promote the resolution of inflammation.Methods We investigated the potential of LXA4 and a synthetic LX analog (Benzo-LXA4) as therapeutics in a murine model of diabetic kidney disease, ApoE-/- mice treated with streptozotocin.Results Intraperitoneal injection of LXs attenuated the development of diabetes-induced albuminuria, mesangial expansion, and collagen deposition. Notably, LXs administered 10 weeks after disease onset also attenuated established kidney disease, with evidence of preserved kidney function. Kidney transcriptome profiling defined a diabetic signature (725 genes; false discovery rate P≤0.05). Comparison of this murine gene signature with that of human diabetic kidney disease identified shared renal proinflammatory/profibrotic signals (TNF-α, IL-1ß, NF-κB). In diabetic mice, we identified 20 and 51 transcripts regulated by LXA4 and Benzo-LXA4, respectively, and pathway analysis identified established (TGF-ß1, PDGF, TNF-α, NF-κB) and novel (early growth response-1 [EGR-1]) networks activated in diabetes and regulated by LXs. In cultured human renal epithelial cells, treatment with LXs attenuated TNF-α-driven Egr-1 activation, and Egr-1 depletion prevented cellular responses to TGF-ß1 and TNF-αConclusions These data demonstrate that LXs can reverse established diabetic complications and support a therapeutic paradigm to promote the resolution of inflammation.


Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/genética , Proteína 1 de Resposta de Crescimento Precoce/genética , Lipoxinas/uso terapêutico , Albuminúria/etiologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Colágeno/metabolismo , Diabetes Mellitus Experimental , Nefropatias Diabéticas/complicações , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Mesângio Glomerular/patologia , Humanos , Injeções Intraperitoneais , Lipoxinas/farmacologia , Masculino , Camundongos Knockout para ApoE , NF-kappa B/genética , Fator de Crescimento Derivado de Plaquetas/genética , Transcriptoma , Fator de Crescimento Transformador beta1/genética , Fator de Necrose Tumoral alfa/genética
13.
Circulation ; 136(12): 1123-1139, 2017 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-28733351

RESUMO

BACKGROUND: The inability of the adult mammalian heart to regenerate following injury represents a major barrier in cardiovascular medicine. In contrast, the neonatal mammalian heart retains a transient capacity for regeneration, which is lost shortly after birth. Defining the molecular mechanisms that govern regenerative capacity in the neonatal period remains a central goal in cardiac biology. Here, we assemble a transcriptomic framework of multiple cardiac cell populations during postnatal development and following injury, which enables comparative analyses of the regenerative (neonatal) versus nonregenerative (adult) state for the first time. METHODS: Cardiomyocytes, fibroblasts, leukocytes, and endothelial cells from infarcted and noninfarcted neonatal (P1) and adult (P56) mouse hearts were isolated by enzymatic dissociation and fluorescence-activated cell sorting at day 3 following surgery. RNA sequencing was performed on these cell populations to generate the transcriptome of the major cardiac cell populations during cardiac development, repair, and regeneration. To complement our transcriptomic data, we also surveyed the epigenetic landscape of cardiomyocytes during postnatal maturation by performing deep sequencing of accessible chromatin regions by using the Assay for Transposase-Accessible Chromatin from purified mouse cardiomyocyte nuclei (P1, P14, and P56). RESULTS: Profiling of cardiomyocyte and nonmyocyte transcriptional programs uncovered several injury-responsive genes across regenerative and nonregenerative time points. However, the majority of transcriptional changes in all cardiac cell types resulted from developmental maturation from neonatal stages to adulthood rather than activation of a distinct regeneration-specific gene program. Furthermore, adult leukocytes and fibroblasts were characterized by the expression of a proliferative gene expression network following infarction, which mirrored the neonatal state. In contrast, cardiomyocytes failed to reactivate the neonatal proliferative network following infarction, which was associated with loss of chromatin accessibility around cell cycle genes during postnatal maturation. CONCLUSIONS: This work provides a comprehensive framework and transcriptional resource of multiple cardiac cell populations during cardiac development, repair, and regeneration. Our findings define a regulatory program underpinning the neonatal regenerative state and identify alterations in the chromatin landscape that could limit reinduction of the regenerative program in adult cardiomyocytes.


Assuntos
Perfilação da Expressão Gênica , Coração/fisiologia , Transcriptoma , Animais , Animais Recém-Nascidos , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes , Leucócitos/citologia , Leucócitos/metabolismo , Camundongos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , RNA/isolamento & purificação , RNA/metabolismo , Regeneração/fisiologia , Análise de Sequência de RNA , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
Circulation ; 135(10): 964-977, 2017 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-27927713

RESUMO

BACKGROUND: Dietary intake of fruit and vegetables is associated with lower incidence of hypertension, but the mechanisms involved have not been elucidated. Here, we evaluated the effect of a high-fiber diet and supplementation with the short-chain fatty acid acetate on the gut microbiota and the prevention of cardiovascular disease. METHODS: Gut microbiome, cardiorenal structure/function, and blood pressure were examined in sham and mineralocorticoid excess-treated mice with a control diet, high-fiber diet, or acetate supplementation. We also determined the renal and cardiac transcriptome of mice treated with the different diets. RESULTS: We found that high consumption of fiber modified the gut microbiota populations and increased the abundance of acetate-producing bacteria independently of mineralocorticoid excess. Both fiber and acetate decreased gut dysbiosis, measured by the ratio of Firmicutes to Bacteroidetes, and increased the prevalence of Bacteroides acidifaciens. Compared with mineralocorticoid-excess mice fed a control diet, both high-fiber diet and acetate supplementation significantly reduced systolic and diastolic blood pressures, cardiac fibrosis, and left ventricular hypertrophy. Acetate had similar effects and markedly reduced renal fibrosis. Transcriptome analyses showed that the protective effects of high fiber and acetate were accompanied by the downregulation of cardiac and renal Egr1, a master cardiovascular regulator involved in cardiac hypertrophy, cardiorenal fibrosis, and inflammation. We also observed the upregulation of a network of genes involved in circadian rhythm in both tissues and downregulation of the renin-angiotensin system in the kidney and mitogen-activated protein kinase signaling in the heart. CONCLUSIONS: A diet high in fiber led to changes in the gut microbiota that played a protective role in the development of cardiovascular disease. The favorable effects of fiber may be explained by the generation and distribution of one of the main metabolites of the gut microbiota, the short-chain fatty acid acetate. Acetate effected several molecular changes associated with improved cardiovascular health and function.


Assuntos
Acetato de Desoxicorticosterona/farmacologia , Fibras na Dieta/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Hipertensão/prevenção & controle , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Pressão Sanguínea/efeitos dos fármacos , Acetato de Desoxicorticosterona/uso terapêutico , Fibras na Dieta/uso terapêutico , Suplementos Nutricionais , Modelos Animais de Doenças , Fibrose , Trato Gastrointestinal/microbiologia , Hipertensão/patologia , Hipertensão/veterinária , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miocárdio/patologia , Tamanho do Órgão/efeitos dos fármacos , Análise de Componente Principal , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Transcriptoma/efeitos dos fármacos
15.
RNA ; 22(8): 1120-38, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27284164

RESUMO

Genomic alignment of small RNA (smRNA) sequences such as microRNAs poses considerable challenges due to their short length (∼21 nucleotides [nt]) as well as the large size and complexity of plant and animal genomes. While several tools have been developed for high-throughput mapping of longer mRNA-seq reads (>30 nt), there are few that are specifically designed for mapping of smRNA reads including microRNAs. The accuracy of these mappers has not been systematically determined in the case of smRNA-seq. In addition, it is unknown whether these aligners accurately map smRNA reads containing sequence errors and polymorphisms. By using simulated read sets, we determine the alignment sensitivity and accuracy of 16 short-read mappers and quantify their robustness to mismatches, indels, and nontemplated nucleotide additions. These were explored in the context of a plant genome (Oryza sativa, ∼500 Mbp) and a mammalian genome (Homo sapiens, ∼3.1 Gbp). Analysis of simulated and real smRNA-seq data demonstrates that mapper selection impacts differential expression results and interpretation. These results will inform on best practice for smRNA mapping and enable more accurate smRNA detection and quantification of expression and RNA editing.


Assuntos
MicroRNAs/genética , Alinhamento de Sequência , Humanos , RNA de Plantas/genética
16.
Nucleic Acids Res ; 44(19): 9206-9217, 2016 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-27439711

RESUMO

Histone methylation by lysine methyltransferase enzymes regulate the expression of genes implicated in lineage specificity and cellular differentiation. While it is known that Set7 catalyzes mono-methylation of histone and non-histone proteins, the functional importance of this enzyme in stem cell differentiation remains poorly understood. We show Set7 expression is increased during mouse embryonic stem cell (mESC) differentiation and is regulated by the pluripotency factors, Oct4 and Sox2. Transcriptional network analyses reveal smooth muscle (SM) associated genes are subject to Set7-mediated regulation. Furthermore, pharmacological inhibition of Set7 activity confirms this regulation. We observe Set7-mediated modification of serum response factor (SRF) and mono-methylation of histone H4 lysine 4 (H3K4me1) regulate gene expression. We conclude the broad substrate specificity of Set7 serves to control key transcriptional networks in embryonic stem cells.


Assuntos
Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Histona-Lisina N-Metiltransferase/metabolismo , Transcrição Gênica , Animais , Ataxina-1/metabolismo , Biomarcadores , Diferenciação Celular/genética , Linhagem Celular , Análise por Conglomerados , Células-Tronco Embrionárias/citologia , Ativação Enzimática , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Humanos , Camundongos , Modelos Biológicos , Fator 3 de Transcrição de Octâmero/metabolismo , Fenótipo , Fatores de Transcrição SOXB1/metabolismo
17.
Genome Res ; 24(8): 1271-84, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24732587

RESUMO

HDAC inhibitors can regulate gene expression by post-translational modification of histone as well as nonhistone proteins. Often studied at single loci, increased histone acetylation is the paradigmatic mechanism of action. However, little is known of the extent of genome-wide changes in cells stimulated by the hydroxamic acids, TSA and SAHA. In this article, we map vascular chromatin modifications including histone H3 acetylation of lysine 9 and 14 (H3K9/14ac) using chromatin immunoprecipitation (ChIP) coupled with massive parallel sequencing (ChIP-seq). Since acetylation-mediated gene expression is often associated with modification of other lysine residues, we also examined H3K4me3 and H3K9me3 as well as changes in CpG methylation (CpG-seq). RNA sequencing indicates the differential expression of ∼30% of genes, with almost equal numbers being up- and down-regulated. We observed broad deacetylation and gene expression changes conferred by TSA and SAHA mediated by the loss of EP300/CREBBP binding at multiple gene promoters. This study provides an important framework for HDAC inhibitor function in vascular biology and a comprehensive description of genome-wide deacetylation by pharmacological HDAC inhibition.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Histonas/metabolismo , Ácidos Hidroxâmicos/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Acetilação , Animais , Anti-Inflamatórios/farmacologia , Aorta/citologia , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genoma Humano , Humanos , Masculino , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/metabolismo , Transcriptoma , Vorinostat
18.
FASEB J ; 29(4): 1329-43, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25491312

RESUMO

Relatively little is known about the epigenetic control mechanisms that guide postnatal organ maturation. The goal of this study was to determine whether DNA methylation plays an important role in guiding transcriptional changes during the first 2 wk of mouse heart development, which is an important period for cardiomyocyte maturation, loss of proliferative capacity and loss of regenerative potential. Gene expression profiling (RNA-seq) and genome-wide sequencing of methylated DNA (MBD-seq) identified dynamic changes in the cardiac methylome during postnatal development [2545 differentially methylated regions (DMRs) from P1 to P14 in the mouse]. The vast majority (~80%) of DMRs were hypermethylated between P1 and P14, and these hypermethylated regions were associated with transcriptional shut down of important developmental signaling pathways, including Hedgehog, bone morphogenetic protein, TGF-ß, fibroblast growth factor, and Wnt/ß-catenin signaling. Postnatal inhibition of DNA methylation with 5-aza-2'-deoxycytidine induced a marked increase (~3-fold) in cardiomyocyte proliferation and ~50% reduction in the percentage of binucleated cardiomyocytes compared with saline-treated controls. This study provides novel evidence for widespread alterations in DNA methylation during postnatal heart maturation and suggests that cardiomyocyte cell cycle arrest during the neonatal period is subject to regulation by DNA methylation.


Assuntos
Metilação de DNA , Coração/crescimento & desenvolvimento , Miocárdio/metabolismo , Animais , Animais Recém-Nascidos , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Pontos de Checagem do Ciclo Celular , Peptídeos Penetradores de Células , Metilação de DNA/efeitos dos fármacos , Decitabina , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Transdução de Sinais
19.
Mol Ther ; 23(3): 434-44, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25399825

RESUMO

Soluble activin type II receptors (ActRIIA/ActRIIB), via binding to diverse TGF-ß proteins, can increase muscle and bone mass, correct anemia or protect against diet-induced obesity. While exciting, these multiple actions of soluble ActRIIA/IIB limit their therapeutic potential and highlight the need for new reagents that target specific ActRIIA/IIB ligands. Here, we modified the activin A and activin B prodomains, regions required for mature growth factor synthesis, to generate specific activin antagonists. Initially, the prodomains were fused to the Fc region of mouse IgG2A antibody and, subsequently, "fastener" residues (Lys(45), Tyr(96), His(97), and Ala(98); activin A numbering) that confer latency to other TGF-ß proteins were incorporated. For the activin A prodomain, these modifications generated a reagent that potently (IC(50) 5 nmol/l) and specifically inhibited activin A signaling in vitro, and activin A-induced muscle wasting in vivo. Interestingly, the modified activin B prodomain inhibited both activin A and B signaling in vitro (IC(50) ~2 nmol/l) and in vivo, suggesting it could serve as a general activin antagonist. Importantly, unlike soluble ActRIIA/IIB, the modified prodomains did not inhibit myostatin or GDF-11 activity. To underscore the therapeutic utility of specifically antagonising activin signaling, we demonstrate that the modified activin prodomains promote significant increases in muscle mass.


Assuntos
Ativinas/metabolismo , Engenharia Genética/métodos , Músculo Esquelético/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Ativinas/antagonistas & inibidores , Ativinas/genética , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Dependovirus/genética , Regulação da Expressão Gênica , Vetores Genéticos/genética , Fatores de Diferenciação de Crescimento/genética , Fatores de Diferenciação de Crescimento/metabolismo , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Miostatina/genética , Miostatina/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Transfecção , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
20.
Mol Genet Genomics ; 290(5): 1899-910, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25893418

RESUMO

Invasive phytoplasmas wreak havoc on coconut palms worldwide, leading to high loss of income, food insecurity and extreme poverty of farmers in producing countries. Phytoplasmas as strictly biotrophic insect-transmitted bacterial pathogens instigate distinct changes in developmental processes and defence responses of the infected plants and manipulate plants to their own advantage; however, little is known about the cellular and molecular mechanisms underlying host-phytoplasma interactions. Further, phytoplasma-mediated transcriptional alterations in coconut palm genes have not yet been identified. This study evaluated the whole transcriptome profiles of naturally infected leaves of Cocos nucifera ecotype Malayan Red Dwarf in response to yellow decline phytoplasma from group 16SrXIV, using RNA-Seq technique. Transcriptomics-based analysis reported here identified genes involved in coconut innate immunity. The number of down-regulated genes in response to phytoplasma infection exceeded the number of genes up-regulated. Of the 39,873 differentially expressed unigenes, 21,860 unigenes were suppressed and 18,013 were induced following infection. Comparative analysis revealed that genes associated with defence signalling against biotic stimuli were significantly overexpressed in phytoplasma-infected leaves versus healthy coconut leaves. Genes involving cell rescue and defence, cellular transport, oxidative stress, hormone stimulus and metabolism, photosynthesis reduction, transcription and biosynthesis of secondary metabolites were differentially represented. Our transcriptome analysis unveiled a core set of genes associated with defence of coconut in response to phytoplasma attack, although several novel defence response candidate genes with unknown function have also been identified. This study constitutes valuable sequence resource for uncovering the resistance genes and/or susceptibility genes which can be used as genetic tools in disease resistance breeding.


Assuntos
Cocos/genética , Genes de Plantas , Phytoplasma/patogenicidade , Folhas de Planta/genética , Análise de Sequência de RNA , Transcriptoma , Cocos/imunologia , Cocos/microbiologia , Imunidade Inata
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