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1.
Cell Rep ; 30(12): 4052-4064.e7, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32209468

RESUMO

Hypoxia-inducible factor-1 alpha (HIF-1α) is considered a global regulator of cellular metabolism and innate immune cell functions. Intracellular pathogens such as Leishmania have been reported to manipulate host cell metabolism. Herein, we demonstrate that myeloid cells from myeloid-restricted HIF-1α-deficient mice and individuals with loss-of-function HIF1A gene polymorphisms are more susceptible to L. donovani infection through increased lipogenesis. Absence of HIF-1α leads to a defect in BNIP3 expression, resulting in the activation of mTOR and nuclear translocation of SREBP-1c. We observed the induction of lipogenic gene transcripts, such as FASN, and lipid accumulation in infected HIF-1α-/- macrophages. L. donovani-infected HIF-1α-deficient mice develop hypertriglyceridemia and lipid accumulation in splenic and hepatic myeloid cells. Most importantly, our data demonstrate that manipulating FASN or SREBP-1c using pharmacological inhibitors significantly reduced parasite burden. As such, genetic deficiency of HIF-1α is associated with increased lipid accumulation, which results in impaired host-protective anti-leishmanial functions of myeloid cells.

2.
PLoS Negl Trop Dis ; 14(3): e0008125, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32214337

RESUMO

BACKGROUND: The disturbance of host metabolic pathways by Leishmania parasites has crucial consequences for the activation status of immune cells and the outcome of infection. Glutamine has been described as an immunomodulatory amino acid, yet its role during Leishmania infection is still unknown. METHODS: We performed transcriptomics in uninfected and L. donovani-infected macrophages 6 hours post-infection. Glutamine quantification by HPLC was assessed in the supernatant of macrophages throughout the infection course. For experimental L. donovani infections, mice were infected with 1.0 x 108 stationary L. donovani promastigotes. Glutaminase (GLS) chemical inhibition was performed using BPTES and glutamine was administered throughout infection. For combined therapy experiment, a daily administration of miltefosine and glutamine was performed by oral gavage. Parasite burden was determined using a Taqman-based assay. Immune cell phenotyping and cytotoxicity were performed in splenic cells using flow cytometry. FINDINGS: We show that glutamine is essential for the control of L. donovani infection. Transcriptomic analysis of L. donovani-infected macrophages demonstrated an upregulation of genes involved in glutamine metabolism. Pharmacological inhibition of glutaminolysis significantly increased the susceptibility to infection, accompanied by an increased recruitment of anti-inflammatory myeloid cells and impaired T cell responses. Remarkably, the supplementation of glutamine to mice infected with L. donovani during miltefosine treatment potentiates parasite clearance through the development of a more effective anti-Leishmania adaptive immune response. CONCLUSIONS: Our data indicates that dietary glutamine supplementation may act as a promising adjuvant for the treatment of visceral leishmaniasis.


Assuntos
Antiprotozoários/administração & dosagem , Suplementos Nutricionais , Glutamina/administração & dosagem , Fatores Imunológicos/administração & dosagem , Leishmaniose Visceral/terapia , Fosforilcolina/análogos & derivados , Animais , Modelos Animais de Doenças , Feminino , Humanos , Macrófagos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Carga Parasitária , Fosforilcolina/administração & dosagem , Subpopulações de Linfócitos T/imunologia , Resultado do Tratamento
3.
J Dairy Sci ; 103(1): 16-29, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31677838

RESUMO

MicroRNAs (miRNAs) are small gene-regulatory noncoding RNA that are highly enriched in cow milk. They are encapsulated in different extracellular vesicle (EV) subsets that protect them from the extracellular milieu and the harsh conditions of the gastrointestinal tract during digestion. Here, we isolated pellets enriched in 4 different EV subsets, via differential ultracentrifugation of commercial cow milk: 12,000 × g (P12K), 35,000 × g (P35K), 70,000 × g (P70K), and 100,000 × g (P100K). Small RNA sequencing (sRNA-Seq) analyses revealed an unprecedented level of diversity in the complete miRNA repertoire and features of unfractionated cow milk and derived EV subsets. Although 5 miRNA sequences represented more than 50% of all miRNAs, milk EV exhibited heterogeneous content of miRNAs and isomeric variants (termed isomiR): P100K EV were enriched in reference miRNA sequences, and P12K and P35K EV in related isomiR. Incubation of milk EV with human cultured HeLa cells led to cellular enrichment in miRNA miR-223, which was concomitant with decreased expression of a reporter gene placed under the control of miR-223, thereby demonstrating the functionality of miR-223. These results suggest that cow milk EV may transfer their miRNAs to human cells and regulate recipient cell gene expression programming in a manner as complex as that of their miRNA transcriptome. The biological activity and relevance of the different milk EV subsets and bioactive mediators, including small noncoding RNA, in health and disease, warrants further investigation.


Assuntos
Vesículas Extracelulares/química , MicroRNAs/síntese química , Transcriptoma/fisiologia , Ultracentrifugação/veterinária , Animais , Bovinos , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Feminino , Regulação da Expressão Gênica , Células HeLa , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Leite/metabolismo , Análise de Sequência de RNA
4.
NPJ Breast Cancer ; 5: 38, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31700994

RESUMO

Breast cancer is a common disease partially caused by genetic risk factors. Germline pathogenic variants in DNA repair genes BRCA1, BRCA2, PALB2, ATM, and CHEK2 are associated with breast cancer risk. FANCM, which encodes for a DNA translocase, has been proposed as a breast cancer predisposition gene, with greater effects for the ER-negative and triple-negative breast cancer (TNBC) subtypes. We tested the three recurrent protein-truncating variants FANCM:p.Arg658*, p.Gln1701*, and p.Arg1931* for association with breast cancer risk in 67,112 cases, 53,766 controls, and 26,662 carriers of pathogenic variants of BRCA1 or BRCA2. These three variants were also studied functionally by measuring survival and chromosome fragility in FANCM -/- patient-derived immortalized fibroblasts treated with diepoxybutane or olaparib. We observed that FANCM:p.Arg658* was associated with increased risk of ER-negative disease and TNBC (OR = 2.44, P = 0.034 and OR = 3.79; P = 0.009, respectively). In a country-restricted analysis, we confirmed the associations detected for FANCM:p.Arg658* and found that also FANCM:p.Arg1931* was associated with ER-negative breast cancer risk (OR = 1.96; P = 0.006). The functional results indicated that all three variants were deleterious affecting cell survival and chromosome stability with FANCM:p.Arg658* causing more severe phenotypes. In conclusion, we confirmed that the two rare FANCM deleterious variants p.Arg658* and p.Arg1931* are risk factors for ER-negative and TNBC subtypes. Overall our data suggest that the effect of truncating variants on breast cancer risk may depend on their position in the gene. Cell sensitivity to olaparib exposure, identifies a possible therapeutic option to treat FANCM-associated tumors.

5.
Data Brief ; 25: 104372, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31485472

RESUMO

Prostate cancer (PCa) is the most frequent cancer in North American men and PCa cells rely on the androgen receptor (AR) for growth and survival. To understand the effect of AR in cancer cells, we have treated LNCaP and LAPC4 cells, two immortalized human PCa cells in vitro, with the synthetic androgen R1881 and then performed RNA-seq analyses. High quality sequencing data have been analyzed using our bioinformatic pipeline which consists of FastQC for quality controls, Trimmomatic for trimming, and Kallisto for pseudoalignment to the transcriptome. Differentially expressed genes were identified using DESeq2 after adjustment for false-discovery rate (FDR q values < 0.05) and Relative Log Expression (RLE) normalization. Gene Set Enrichment Analysis (GSEA) was also performed to identify biological pathways significantly modulated by androgens. GSEA analyses identified the androgen signaling pathway, as well as several metabolic pathways, as significantly enriched following androgen stimulation. These analyses highlight the most significant metabolic pathways up-regulated following AR activation. Raw and processed RNA-seq data were deposited and made publicly available on the Gene Expression Omnibus (GEO; GSE128749). These data have been incorporated in a recent article describing the functions of AR as a master regulator of PCa cell metabolism. For more details about interpretation of these results, please refer to "Functional genomics studies reveal the androgen receptor as a master regulator of cellular energy metabolism in prostate cancer" by Gonthier et al. (doi: 10.1016/j.jsbmb.2019.04.016).

6.
Cell Rep ; 28(9): 2443-2454.e4, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31461657

RESUMO

In the ovary, follicular growth and maturation are complicated processes that involve a series of morphological and physiological changes in oocytes and somatic cells leading to ovulation and luteinization, essential processes for fertility. Given the complexity of ovulation, characterization of genome-wide regulatory elements is essential to understand the mechanisms governing the expression of specific genes in the rapidly differentiating follicle. We therefore employed a systems biology approach to determine global transcriptional mechanisms during the early stages of the ovulatory process. We demonstrate that, following the hormonal signal that initiates ovulation, granulosa cells undergo major modification of distal regulatory elements, which coincides with cistrome reprogramming of the indispensable orphan nuclear receptor liver receptor homolog-1 (LRH-1). This cistromic reorganization correlates with the extensive changes in gene expression in granulosa cells leading to ovulation. Together, our study yields a highly detailed transcriptional map delineating ovarian cell differentiation during the initiation of ovulation.


Assuntos
Montagem e Desmontagem da Cromatina , Folículo Ovariano/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Feminino , Células da Granulosa/citologia , Células da Granulosa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Motivos de Nucleotídeos , Folículo Ovariano/citologia , Ovulação
7.
J Hum Genet ; 54(3): 152-61, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19197335

RESUMO

Breast cancer is a heterogeneous disease displaying some degree of familial clustering. Highly penetrant breast cancer susceptibility genes represent approximately 20-25% of the familial aggregation of breast cancer. A significant proportion of this familial aggregation of breast cancer is thus yet to be explained by other breast cancer susceptibility genes. Given the high susceptibility conferred by the two major breast cancer predisposition genes, BRCA1 and BRCA2 and the implication of these genes in many key cellular processes, assessment of genes encoding BRCA1-interacting proteins as plausible breast cancer candidate genes is thus attractive. In this study, four genes encoding BRCA1-interacting proteins were analyzed in a cohort of 96 breast cancer individuals from high-risk non-BRCA1/BRCA2 French Canadian families. Although no deleterious truncating germline mutations or aberrant spliced mRNA species were identified, a total of 10, 4, 11 and 6 variants were found in the AURKA, BAP1, BARD1 and DHX9 genes, respectively. The allele frequency of each variant was further ascertained in a cohort of 98 healthy French Canadian unrelated women and a difference in allele frequency was observed for one BARD1 variant based on single-marker analysis. Haplotype estimation, haplotype blocks and tagging SNPs identification were then performed for each gene, providing a valuable tool for further searches of common disease-associated variants in these genes and therefore further analyses on these genes in larger cohorts is warranted in the search of low-to-moderate penetrance breast cancer susceptibility alleles.


Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Grupo com Ancestrais do Continente Europeu/genética , Predisposição Genética para Doença , Variação Genética , Proteínas de Neoplasias/genética , Aurora Quinase A , Aurora Quinases , Sequência de Bases , Canadá , Estudos de Casos e Controles , RNA Helicases DEAD-box/genética , Família , Feminino , França/etnologia , Haplótipos , Heterozigoto , Humanos , Desequilíbrio de Ligação/genética , Masculino , Linhagem , Polimorfismo de Nucleotídeo Único/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Ubiquitina-Proteína Ligases/genética
8.
Fam Cancer ; 6(4): 483-90, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17636424

RESUMO

Cowden syndrome is a disease associated with an increase in breast cancer susceptibility. Alleles in PTEN and other breast cancer susceptibility genes would be responsible for approximately 25% of the familial component of breast cancer risk, BRCA1 and BRCA2 being the two major genes responsible for this inherited risk. In order to evaluate the proportion of high-risk French Canadian non-BRCA1/BRCA2 breast/ovarian cancer families potentially harboring a PTEN germline mutation, the whole coding and flanking intronic sequences were analyzed in a series of 98 breast cancer cases. Although no germline mutation has been identified in the coding region, our study led to the identification of four intronic variants. Further investigations were performed to analyze the effect of these variants, alone and/or in combination, on splicing and PTEN protein levels. Despite suggestive evidence emerging from in silico analyses, the presence of these intronic variants do not seem to alter RNA splicing or PTEN protein levels. In addition, as loss of PTEN or part of it has been reported, Western blot analysis has also been performed. No major deletion could be identified in our cohort. Therefore, assuming a Poisson distribution for the frequency of deleterious mutation in our cohort, if the frequency of such deleterious mutation was 2%, we would have had a 90% or greater chance of observing at least one such mutation. These results suggest that PTEN germline mutations are rare and are unlikely to account for a significant proportion of familial breast cancer cases in the French Canadian population.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Predisposição Genética para Doença/genética , Mutação em Linhagem Germinativa/genética , PTEN Fosfo-Hidrolase/genética , Animais , Sequência de Bases , Neoplasias da Mama/etnologia , Canadá/etnologia , França/etnologia , Predisposição Genética para Doença/etnologia , Humanos , Dados de Sequência Molecular , Ribonucleases/metabolismo , Fatores de Risco , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico
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