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1.
Sci Immunol ; 4(41)2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31784499

RESUMO

Genetic etiologies of chronic mucocutaneous candidiasis (CMC) disrupt human IL-17A/F-dependent immunity at mucosal surfaces, whereas those of connective tissue disorders (CTDs) often impair the TGF-ß-dependent homeostasis of connective tissues. The signaling pathways involved are incompletely understood. We report a three-generation family with an autosomal dominant (AD) combination of CMC and a previously undescribed form of CTD that clinically overlaps with Ehlers-Danlos syndrome (EDS). The patients are heterozygous for a private splice-site variant of MAPK8, the gene encoding c-Jun N-terminal kinase 1 (JNK1), a component of the MAPK signaling pathway. This variant is loss-of-expression and loss-of-function in the patients' fibroblasts, which display AD JNK1 deficiency by haploinsufficiency. These cells have impaired, but not abolished, responses to IL-17A and IL-17F. Moreover, the development of the patients' TH17 cells was impaired ex vivo and in vitro, probably due to the involvement of JNK1 in the TGF-ß-responsive pathway and further accounting for the patients' CMC. Consistently, the patients' fibroblasts displayed impaired JNK1- and c-Jun/ATF-2-dependent induction of key extracellular matrix (ECM) components and regulators, but not of EDS-causing gene products, in response to TGF-ß. Furthermore, they displayed a transcriptional pattern in response to TGF-ß different from that of fibroblasts from patients with Loeys-Dietz syndrome caused by mutations of TGFBR2 or SMAD3, further accounting for the patients' complex and unusual CTD phenotype. This experiment of nature indicates that the integrity of the human JNK1-dependent MAPK signaling pathway is essential for IL-17A- and IL-17F-dependent mucocutaneous immunity to Candida and for the TGF-ß-dependent homeostasis of connective tissues.

2.
Proc Natl Acad Sci U S A ; 116(33): 16463-16472, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31346092

RESUMO

Heterozygous in-frame mutations in coding regions of human STAT3 underlie the only known autosomal dominant form of hyper IgE syndrome (AD HIES). About 5% of familial cases remain unexplained. The mutant proteins are loss-of-function and dominant-negative when tested following overproduction in recipient cells. However, the production of mutant proteins has not been detected and quantified in the cells of heterozygous patients. We report a deep intronic heterozygous STAT3 mutation, c.1282-89C>T, in 7 relatives with AD HIES. This mutation creates a new exon in the STAT3 complementary DNA, which, when overexpressed, generates a mutant STAT3 protein (D427ins17) that is loss-of-function and dominant-negative in terms of tyrosine phosphorylation, DNA binding, and transcriptional activity. In immortalized B cells from these patients, the D427ins17 protein was 2 kDa larger and 4-fold less abundant than wild-type STAT3, on mass spectrometry. The patients' primary B and T lymphocytes responded poorly to STAT3-dependent cytokines. These findings are reminiscent of the impaired responses of leukocytes from other patients with AD HIES due to typical STAT3 coding mutations, providing further evidence for the dominance of the mutant intronic allele. These findings highlight the importance of sequencing STAT3 introns in patients with HIES without candidate variants in coding regions and essential splice sites. They also show that AD HIES-causing STAT3 mutant alleles can be dominant-negative even if the encoded protein is produced in significantly smaller amounts than wild-type STAT3.

3.
J Exp Med ; 215(10): 2567-2585, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30143481

RESUMO

Life-threatening pulmonary influenza can be caused by inborn errors of type I and III IFN immunity. We report a 5-yr-old child with severe pulmonary influenza at 2 yr. She is homozygous for a loss-of-function IRF9 allele. Her cells activate gamma-activated factor (GAF) STAT1 homodimers but not IFN-stimulated gene factor 3 (ISGF3) trimers (STAT1/STAT2/IRF9) in response to IFN-α2b. The transcriptome induced by IFN-α2b in the patient's cells is much narrower than that of control cells; however, induction of a subset of IFN-stimulated gene transcripts remains detectable. In vitro, the patient's cells do not control three respiratory viruses, influenza A virus (IAV), parainfluenza virus (PIV), and respiratory syncytial virus (RSV). These phenotypes are rescued by wild-type IRF9, whereas silencing IRF9 expression in control cells increases viral replication. However, the child has controlled various common viruses in vivo, including respiratory viruses other than IAV. Our findings show that human IRF9- and ISGF3-dependent type I and III IFN responsive pathways are essential for controlling IAV.


Assuntos
Alelos , Homozigoto , Influenza Humana , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/deficiência , Orthomyxoviridae/imunologia , Pneumonia Viral , Feminino , Humanos , Lactente , Influenza Humana/genética , Influenza Humana/imunologia , Influenza Humana/patologia , Interferon alfa-2/genética , Interferon alfa-2/imunologia , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/imunologia , Pneumonia Viral/genética , Pneumonia Viral/imunologia , Pneumonia Viral/patologia
4.
Sci Immunol ; 3(24)2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29907691

RESUMO

Heterozygosity for human signal transducer and activator of transcription 3 (STAT3) dominant-negative (DN) mutations underlies an autosomal dominant form of hyper-immunoglobulin E syndrome (HIES). We describe patients with an autosomal recessive form of HIES due to loss-of-function mutations of a previously uncharacterized gene, ZNF341 ZNF341 is a transcription factor that resides in the nucleus, where it binds a specific DNA motif present in various genes, including the STAT3 promoter. The patients' cells have low basal levels of STAT3 mRNA and protein. The autoinduction of STAT3 production, activation, and function by STAT3-activating cytokines is strongly impaired. Like patients with STAT3 DN mutations, ZNF341-deficient patients lack T helper 17 (TH17) cells, have an excess of TH2 cells, and have low memory B cells due to the tight dependence of STAT3 activity on ZNF341 in lymphocytes. Their milder extra-hematopoietic manifestations and stronger inflammatory responses reflect the lower ZNF341 dependence of STAT3 activity in other cell types. Human ZNF341 is essential for the STAT3 transcription-dependent autoinduction and sustained activity of STAT3.


Assuntos
Regulação da Expressão Gênica/imunologia , Síndrome de Job/genética , Fator de Transcrição STAT3/genética , Fatores de Transcrição/genética , Transcrição Genética/imunologia , Adolescente , Adulto , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Núcleo Celular/metabolismo , Consanguinidade , Citocinas/imunologia , Citocinas/metabolismo , Éxons/genética , Feminino , Genes Recessivos/genética , Genes Recessivos/imunologia , Homozigoto , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Síndrome de Job/sangue , Síndrome de Job/imunologia , Mutação com Perda de Função , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Linhagem , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT3/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Sequenciamento Completo do Exoma , Adulto Jovem , Dedos de Zinco/genética
5.
Gen Comp Endocrinol ; 252: 79-87, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28736226

RESUMO

Cytochrome P450 aromatase catalyzes conversion of C19 androgens to C18 estrogens and is critical for normal reproduction in female vertebrates. Fadrozole is a model aromatase inhibitor that has been shown to suppress estrogen production in the ovaries of fish. However, little is known about the early impacts of aromatase inhibition on steroid production and gene expression in fish. Adult female fathead minnows (Pimephales promelas) were exposed via water to 0, 5, or 50µg fadrozole/L for a time-course of 0.5, 1, 2, 4, and 6h, or 0 or 50µg fadrozole/L for a time-course of 6, 12, and 24h. We examined ex vivo ovarian 17ß-estradiol (E2) and testosterone (T) production, and plasma E2 concentrations from each study. Expression profiles of genes known or hypothesized to be impacted by fadrozole including aromatase (cytochrome P450 [cyp] 19a1a), steriodogenic acute regulatory protein (star), cytochrome P450 side-chain cleavage (cyp11a), cytochrome P450 17 alpha hydroxylase/17,20 lyase (cyp17), and follicle stimulating hormone receptor (fshr) were measured in the ovaries by quantitative real-time polymerase chain reaction (QPCR). In addition, broader ovarian gene expression was examined using a 15k fathead minnow microarray. The 5µg/L exposure significantly reduced ex vivo E2 production by 6h. In the 50µg/L treatment, ex vivo E2 production was significantly reduced after just 2h of exposure and remained depressed at all time-points examined through 24h. Plasma E2 concentrations were significantly reduced as early as 4h after initiation of exposure to either 5 or 50µg fadrozole/L and remained depressed throughout 24h in the 50µg/L exposure. Ex vivo T concentrations remained unchanged throughout the time-course. Expression of transcripts involved in steroidogenesis increased within the first 24h suggesting rapid induction of a mechanism to compensate for fadrozole inhibition of aromatase. Microarray results also showed fadrozole exposure caused concentration- and time-dependent changes in gene expression profiles in many HPG-axis pathways as early as 4h. This study provides insights into the very rapid effects of aromatase inhibition on steroidogenic processes in fish.


Assuntos
Inibidores da Aromatase/farmacologia , Cyprinidae/genética , Fadrozol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Ovário/metabolismo , Esteroides/biossíntese , Animais , Cyprinidae/sangue , Cyprinidae/metabolismo , Estradiol/sangue , Feminino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Testosterona/sangue , Transcriptoma/genética
6.
Environ Sci Technol ; 51(15): 8701-8712, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28651047

RESUMO

We examined whether contaminants present in surface waters could be prioritized for further assessment by linking the presence of specific chemicals to gene expression changes in exposed fish. Fathead minnows were deployed in cages for 2, 4, or 8 days at three locations near two different wastewater treatment plant discharge sites in the Saint Louis Bay, Duluth, MN and one upstream reference site. The biological impact of 51 chemicals detected in the surface water of 133 targeted chemicals was determined using biochemical endpoints, exposure activity ratios for biological and estrogenic responses, known chemical:gene interactions from biological pathways and knowledge bases, and analysis of the covariance of ovary gene expression with surface water chemistry. Thirty-two chemicals were significantly linked by covariance with expressed genes. No estrogenic impact on biochemical endpoints was observed in male or female minnows. However, bisphenol A (BPA) was identified by chemical:gene covariation as the most impactful estrogenic chemical across all exposure sites. This was consistent with identification of estrogenic effects on gene expression, high BPA exposure activity ratios across all test sites, and historical analysis of the study area. Gene expression analysis also indicated the presence of nontargeted chemicals including chemotherapeutics consistent with a local hospital waste stream. Overall impacts on gene expression appeared to be related to changes in treatment plant function during rain events. This approach appears useful in examining the impacts of complex mixtures on fish and offers a potential route in linking chemical exposure to adverse outcomes that may reduce population sustainability.


Assuntos
Cyprinidae/genética , Águas Residuárias , Poluentes Químicos da Água/toxicidade , Animais , Monitoramento Ambiental , Estrona , Feminino , Masculino , Testes de Mutagenicidade , Medição de Risco
7.
Mol Cell Endocrinol ; 439: 431-443, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-27717743

RESUMO

Glucocorticoids are important therapeutic compounds for acute lymphoblastic leukemia (ALL). AKT1 or the protein kinase B is frequently activated in ALL, and contributes to the development of glucocorticoid resistance. We examined impact of AKT1 on glucocorticoid receptor (GR)-induced transcriptional activity in cooperation with phospho-serine/threonine-binding protein 14-3-3. AKT1 has two distinct actions on GR transcriptional activity, one through segregation of GR in the cytoplasm by phosphorylating GR at Ser-134 and subsequent association of 14-3-3, and the other through direct modulation of GR transcriptional activity in the nucleus. For the latter, AKT1 and 14-3-3 are attracted to DNA-bound GR, accompanied by AKT1-dependent p300 phosphorylation, H3S10 phosphorylation and H3K14 acetylation at the DNA site. These two actions of AKT1 regulate distinct sets of glucocorticoid-responsive genes. Our results suggest that specific inhibition of the AKT1/14-3-3 activity on the cytoplasmic retention of GR may be a promising target for treating glucocorticoid resistance observed in ALL.


Assuntos
Proteínas 14-3-3/metabolismo , Biomarcadores Tumorais/metabolismo , Exorribonucleases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Glucocorticoides/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteína p300 Associada a E1A/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Células HCT116 , Código das Histonas , Humanos , Células Jurkat , Vírus do Tumor Mamário do Camundongo/genética , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Regiões Promotoras Genéticas , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Isoformas de Proteínas/metabolismo , Receptores de Glucocorticoides/química , Elementos de Resposta/genética , Serina/genética , Transcrição Genética/efeitos dos fármacos
8.
Oncotarget ; 7(46): 75854-75864, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27716616

RESUMO

It has been shown previously that cancer cells with an activated oncogenic pathway, including Met activation, require Ran for growth and survival.Here, we show that knockdown of Ran leads to a reduction of Met receptor expression in several breast and lung cancer cell lines. This, in turn suppressed HGF expression and the Met-mediated activation of the Akt pathway, as well as cell adhesion, migration, and invasion. In a cell line model where Met amplification has previously been shown to contribute to gefitinib resistance, Ran knockdown sensitized cells to gefitinib-mediated inhibition of Akt and ERK1/2 phosphorylation and consequently reduced cell proliferation. We further demonstrate that Met reduction-mediated by knockdown of Ran, occurs at the post-transcriptional level, probably via a matrix metalloproteinase. Moreover, the level of immunoreactive Ran and Met are positively associated in human breast cancer specimens, suggesting that a high level of Ran may be a pre-requisite for Met overexpression. Interestingly, a high level of immunoreactive Ran dictates the prognostic significance of Met, indicating that the co-overexpression of Met and Ran may be associated with cancer progression and could be used in combination as a prognostic indicator.


Assuntos
Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais , Proteína ran de Ligação ao GTP/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Neoplasias/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Transdução de Sinais/efeitos dos fármacos , Proteína ran de Ligação ao GTP/genética
9.
Epigenomics ; 8(9): 1227-38, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27529370

RESUMO

AIM: To assess the correlation between KDM6B and estrogen receptor ß (ERß) expression in malignant pleural mesothelioma (MPM). MATERIALS & METHODS: We evaluated gene expression by in silico analysis of microarray data, real-time PCR and western blot in MPM tumors and cell lines. RESULTS & CONCLUSION: We report a strong positive correlation between the expression of KDM6B and ERß in MPM tumors and cell lines. We describe that, in hypoxia, the HIF2α-KDM6B axis induces an epithelioid morphology and ERß expression in biphasic MPM cells with estrogen receptor-negative phenotype. Reduced histone H3K27 tri-methylation confirms KDM6B activity under hypoxic conditions. Importantly, cells treated during reoxygenation with the selective ERß agonist, KB9520, maintain ERß expression and the less aggressive phenotype acquired in hypoxia.


Assuntos
Epigênese Genética , Receptor beta de Estrogênio/genética , Histona Desmetilases com o Domínio Jumonji/genética , Neoplasias Pulmonares/genética , Mesotelioma/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Receptor beta de Estrogênio/metabolismo , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , Metilação , Processamento de Proteína Pós-Traducional
10.
BMC Genomics ; 15: 591, 2014 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-25016412

RESUMO

BACKGROUND: Corals represent symbiotic meta-organisms that require harmonization among the coral animal, photosynthetic zooxanthellae and associated microbes to survive environmental stresses. We investigated integrated-responses among coral and zooxanthellae in the scleractinian coral Acropora formosa in response to an emerging marine pollutant, the munitions constituent, 1,3,5-trinitro-1,3,5 triazine (RDX; 5 day exposures to 0 (control), 0.5, 0.9, 1.8, 3.7, and 7.2 mg/L, measured in seawater). RESULTS: RDX accumulated readily in coral soft tissues with bioconcentration factors ranging from 1.1 to 1.5. Next-generation sequencing of a normalized meta-transcriptomic library developed for the eukaryotic components of the A. formosa coral holobiont was leveraged to conduct microarray-based global transcript expression analysis of integrated coral/zooxanthellae responses to the RDX exposure. Total differentially expressed transcripts (DET) increased with increasing RDX exposure concentrations as did the proportion of zooxanthellae DET relative to the coral animal. Transcriptional responses in the coral demonstrated higher sensitivity to RDX compared to zooxanthellae where increased expression of gene transcripts coding xenobiotic detoxification mechanisms (i.e. cytochrome P450 and UDP glucuronosyltransferase 2 family) were initiated at the lowest exposure concentration. Increased expression of these detoxification mechanisms was sustained at higher RDX concentrations as well as production of a physical barrier to exposure through a 40% increase in mucocyte density at the maximum RDX exposure. At and above the 1.8 mg/L exposure concentration, DET coding for genes involved in central energy metabolism, including photosynthesis, glycolysis and electron-transport functions, were decreased in zooxanthellae although preliminary data indicated that zooxanthellae densities were not affected. In contrast, significantly increased transcript expression for genes involved in cellular energy production including glycolysis and electron-transport pathways was observed in the coral animal. CONCLUSIONS: Transcriptional network analysis for central energy metabolism demonstrated highly correlated responses to RDX among the coral animal and zooxanthellae indicative of potential compensatory responses to lost photosynthetic potential within the holobiont. These observations underscore the potential for complex integrated responses to RDX exposure among species comprising the coral holobiont and highlight the need to understand holobiont-species interactions to accurately assess pollutant impacts.


Assuntos
Antozoários/genética , Dinoflagelados/genética , Transcriptoma/efeitos dos fármacos , Triazinas/farmacologia , Poluentes Químicos da Água/farmacologia , Animais , Antozoários/efeitos dos fármacos , Antozoários/metabolismo , Dinoflagelados/efeitos dos fármacos , Dinoflagelados/metabolismo , Anotação de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Fisiológico , Simbiose
11.
Environ Sci Technol ; 48(8): 4546-55, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24684273

RESUMO

Nanoparticles are of concern because of widespread use, but it is unclear if metal nanoparticles cause effects directly or indirectly. We explored whether polyvinylpyrrolidone-coated silver nanoparticles (PVP-AgNPs) cause effects through intact nanoparticles or dissolved silver. Females of the model species fathead minnow (Pimephales promelas) were exposed to either 4.8 µg/L of AgNO3 or 61.4 µg/L of PVP-AgNPs for 96h. Microarray analyses were used to identify impacted receptors and toxicity pathways in liver and brain tissues that were confirmed using in vitro mammalian assays. AgNO3 and PVP-AgNP exposed fish had common and distinct effects consistent with both intact nanoparticles and dissolved silver causing effects. PVP-AgNPs and AgNO3 both affected pathways involved in Na(+), K(+), and H(+) homeostasis and oxidative stress but different neurotoxicity pathways. In vivo effects were supported by PVP-AgNP activation of five in vitro nuclear receptor assays and inhibition of ligand binding to the dopamine receptor. AgNO3 inhibited ligand binding to adrenergic receptors α1 and α2 and cannabinoid receptor CB1, but had no effect in nuclear receptor assays. PVP-AgNPs have the potential to cause effects both through intact nanoparticles and metal ions, each interacting with different initiating events. Since the in vitro and in vivo assays examined here are commonly used in human and ecological hazard screening, this work suggests that environmental health assessments should consider effects of intact nanoparticles in addition to dissolved metals.


Assuntos
Cyprinidae/metabolismo , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Análise por Conglomerados , Ensaios Enzimáticos , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Povidona/toxicidade , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptores Citoplasmáticos e Nucleares/metabolismo , Nitrato de Prata/toxicidade , Fatores de Transcrição/metabolismo
12.
Gen Comp Endocrinol ; 203: 193-202, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24704562

RESUMO

Aromatase, a member of the cytochrome P450 superfamily, is a key enzyme in estradiol synthesis that catalyzes the aromatization of androgens into estrogens in ovaries. Here, we used an integrated approach to assess the mechanistic basis of the direct effects of aromatase inhibition, as well as adaptation and recovery processes in fish. We exposed female fathead minnows (Pimephales promelas) via the water to 30 µg/L of a model aromatase inhibitor, fadrozole, during 8 days (exposure phase). Fish were then held in clean water for 8 more days (recovery phase). Samples were collected at 1, 2, 4, and 8 days of both the exposure and the recovery phases. Transcriptomics, metabolomics, and network inference were used to understand changes and infer connections at the transcript and metabolite level in the ovary. Apical endpoints directly indicative of endocrine function, such as plasma estradiol, testosterone, and vitellogenin levels were also measured. An integrated analysis of the data revealed changes in gene expression consistent with increased testosterone in fadrozole-exposed ovaries. Metabolites such as glycogen and taurine were strongly correlated with increased testosterone levels. Comparison of in vivo and ex vivo steroidogenesis data suggested the accumulation of steroidogenic enzymes, including aromatase, as a mechanism to compensate for aromatase inhibition.


Assuntos
Inibidores da Aromatase/farmacologia , Aromatase/metabolismo , Cyprinidae/genética , Cyprinidae/metabolismo , Fadrozol/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Androgênios/sangue , Animais , Estradiol/sangue , Feminino , Glicogênio/sangue , Humanos , Masculino , Metabolômica , Taurina/sangue , Testosterona/sangue , Transcriptoma/efeitos dos fármacos , Vitelogeninas/sangue
13.
Environ Sci Technol ; 47(16): 9424-33, 2013 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-23898970

RESUMO

A hormetic response is characterized by an opposite effect in small and large doses of chemical exposure, often resulting in seemingly beneficial effects at low doses. Here, we examined the potential mechanisms underlying the hormetic response of Daphnia magna to the energetic trinitrotoluene (TNT). Daphnia magna were exposed to TNT for 21 days, and a significant increase in adult length and number of neonates was identified at low concentrations (0.002-0.22 mg/L TNT), while toxic effects were identified at high concentrations (0.97 mg/L TNT and above). Microarray analysis of D. magna exposed to 0.004, 0.12, and 1.85 mg/L TNT identified effects on lipid metabolism as a potential mechanism underlying hormetic effects. Lipidomic analysis of exposed D. magna supported the hypothesis that TNT exposure affected lipid and fatty acid metabolism, showing that hormetic effects could be related to changes in polyunsaturated fatty acids known to be involved in Daphnia growth and reproduction. Our results show that Daphnia exposed to low levels of TNT presented hormetic growth and reproduction enhancement, while higher TNT concentrations had an opposite effect. Our results also show how a systems approach can help elucidate potential mechanisms of action and adverse outcomes.


Assuntos
Daphnia/efeitos dos fármacos , Hormese , Trinitrotolueno/administração & dosagem , Animais , Tamanho Corporal/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Metabolismo dos Lipídeos/efeitos dos fármacos , Reprodução/efeitos dos fármacos , Transcriptoma
14.
Environ Sci Technol ; 47(16): 9434-43, 2013 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-23855649

RESUMO

The molecular mechanisms explaining hormetic effects of selective serotonin reuptake inhibitors (SSRIs) and 4-nonylphenol in Daphnia magna reproduction were studied in juveniles and adults. Transcriptome analyses showed changes in mRNA levels for 1796 genes in juveniles and 1214 genes in adults (out of 15000 total probes) exposed to two SSRIs (fluoxetine and fluvoxamine) or to 4-nonylphenol. Functional annotation of affected genes was improved by assuming the annotations of putatively homologous Drosophila genes. Self-organizing map analysis and partial least-square regression coupled with selectivity ratio procedures analyses allowed to define groups of genes with specific responses to the different treatments. Differentially expressed genes were analyzed for functional enrichment using Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes databases. Serotonin metabolism, neuronal developmental processes, and carbohydrates and lipid metabolism functional categories appeared as selectively affected by SSRI treatment, whereas 4-nonylphenol deregulated genes from the carbohydrate metabolism and the ecdysone regulatory pathway. These changes in functional and metabolic pathways are consistent with previously reported SSRIs and 4-nonylphenol hormetic effects in D. magna, including a decrease in reserve carbohydrates and an increase in respiratory metabolism.


Assuntos
Daphnia/metabolismo , Hormese , Fenótipo , Animais , Feminino , Fluoxetina/administração & dosagem , Fluvoxamina/administração & dosagem , Análise de Sequência com Séries de Oligonucleotídeos , Fenóis/administração & dosagem , Inibidores de Captação de Serotonina/administração & dosagem , Transcriptoma
15.
Environ Sci Technol ; 46(14): 7790-8, 2012 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-22697906

RESUMO

Interspecies uncertainty factors in ecological risk assessment provide conservative estimates of risk where limited or no toxicity data is available. We quantitatively examined the validity of interspecies uncertainty factors by comparing the responses of zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) to the energetic compound 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), a known neurotoxicant. Relative toxicity was measured through transcriptional, morphological, and behavioral end points in zebrafish and fathead minnow fry exposed for 96 h to RDX concentrations ranging from 0.9 to 27.7 mg/L. Spinal deformities and lethality occurred at 1.8 and 3.5 mg/L RDX respectively for fathead minnow and at 13.8 and 27.7 mg/L for zebrafish, indicating that zebrafish have an 8-fold greater tolerance for RDX than fathead minnow fry. The number and magnitude of differentially expressed transcripts increased with increasing RDX concentration for both species. Differentially expressed genes were enriched in functions related to neurological disease, oxidative-stress, acute-phase response, vitamin/mineral metabolism and skeletal/muscular disorders. Decreased expression of collagen-coding transcripts were associated with spinal deformity and likely involved in sensitivity to RDX. Our work provides a mechanistic explanation for species-specific sensitivity to RDX where zebrafish responded at lower concentrations with greater numbers of functions related to RDX tolerance than fathead minnow. While the 10-fold interspecies uncertainty factor does provide a reasonable cross-species estimate of toxicity in the present study, the observation that the responses between ZF and FHM are markedly different does initiate a call for concern regarding establishment of broad ecotoxicological conclusions based on model species such as zebrafish.


Assuntos
Cyprinidae/metabolismo , Ecotoxicologia/métodos , Biologia de Sistemas/métodos , Triazinas/toxicidade , Peixe-Zebra/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Bioensaio , Colágeno/genética , Colágeno/metabolismo , Cyprinidae/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Software , Especificidade da Espécie , Coluna Vertebral/anormalidades , Coluna Vertebral/efeitos dos fármacos , Análise de Sobrevida , Natação/fisiologia , Peixe-Zebra/genética
16.
Ecotoxicology ; 20(3): 580-94, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21516383

RESUMO

At military training sites, a variety of pollutants such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), may contaminate the area originating from used munitions. Studies investigating the mechanism of toxicity of RDX have shown that it affects the central nervous system causing seizures in humans and animals. Environmental pollutants such as RDX have the potential to affect many different species, therefore it is important to establish how phylogenetically distant species may respond to these types of emerging pollutants. In this paper, we have used a transcriptional network approach to compare and contrast the neurotoxic effects of RDX among five phylogenetically disparate species: rat (Sprague-Dawley), Northern bobwhite quail (Colinus virginianus), fathead minnow (Pimephales promelas), earthworm (Eisenia fetida), and coral (Acropora formosa). Pathway enrichment analysis indicated a conservation of RDX impacts on pathways related to neuronal function in rat, Northern bobwhite quail, fathead minnows and earthworm, but not in coral. As evolutionary distance increased common responses decreased with impacts on energy and metabolism dominating effects in coral. A neurotransmission related transcriptional network based on whole rat brain responses to RDX exposure was used to identify functionally related modules of genes, components of which were conserved across species depending upon evolutionary distance. Overall, the meta-analysis using genomic data of the effects of RDX on several species suggested a common and conserved mode of action of the chemical throughout phylogenetically remote organisms.


Assuntos
Evolução Biológica , Poluentes Ambientais/toxicidade , Substâncias Explosivas/toxicidade , Triazinas/toxicidade , Animais , Antozoários/efeitos dos fármacos , Cyprinidae , Feminino , Neurônios/fisiologia , Oligoquetos/efeitos dos fármacos , Filogenia , Codorniz , Ratos , Receptores de GABA-A/genética , Transmissão Sináptica , Toxicogenética
17.
Environ Toxicol Chem ; 30(1): 22-38, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20963852

RESUMO

The toxicological effects of many stressors are mediated through unknown, or incompletely characterized, mechanisms of action. The application of reverse engineering complex interaction networks from high dimensional omics data (gene, protein, metabolic, signaling) can be used to overcome these limitations. This approach was used to characterize adverse outcome pathways (AOPs) for chemicals that disrupt the hypothalamus-pituitary-gonadal endocrine axis in fathead minnows (FHM, Pimephales promelas). Gene expression changes in FHM ovaries in response to seven different chemicals, over different times, doses, and in vivo versus in vitro conditions, were captured in a large data set of 868 arrays. Potential AOPs of the antiandrogen flutamide were examined using two mutual information-based methods to infer gene regulatory networks and potential AOPs. Representative networks from these studies were used to predict network paths from stressor to adverse outcome as candidate AOPs. The relationship of individual chemicals to an adverse outcome can be determined by following perturbations through the network in response to chemical treatment, thus leading to the nodes associated with the adverse outcome. Identification of candidate pathways allows for formation of testable hypotheses about key biological processes, biomarkers, or alternative endpoints that can be used to monitor an AOP. Finally, the unique challenges facing the application of this approach in ecotoxicology were identified and a road map for the utilization of these tools presented.


Assuntos
Monitoramento Ambiental/métodos , Testes de Toxicidade/métodos , Poluentes Químicos da Água/toxicidade , Animais , Cyprinidae/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Modelos Biológicos , Modelos Químicos , Ovário/efeitos dos fármacos , Ovário/metabolismo
18.
Aquat Toxicol ; 101(1): 135-45, 2011 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-20965580

RESUMO

Production, usage and disposal of the munitions constituent (MC) cyclotrimethylenetrinitramine (RDX) has led to environmental releases on military facilities. The chemical attributes of RDX are conducive for leaching to surface water which may put aquatic organisms at risk of exposure. Because RDX has been observed to cause aberrant neuromuscular effects across a wide range of animal phyla, we assessed the effects of RDX on central nervous system (CNS) functions in the representative aquatic ecotoxicological model species, fathead minnow (Pimephales promelas). We developed a fathead minnow brain-tissue cDNA library enriched for transcripts differentially expressed in response to RDX and trinitrotoluene (TNT) exposure. All 4,128 cDNAs were sequenced, quality filtered and assembled yielding 2230 unique sequences and 945 significant blastx matches (E ≤10(-5)). The cDNA library was leveraged to create custom-spotted microarrays for use in transcript expression assays. The impact of RDX on transcript expression in brain tissue was examined in fathead minnows exposed to RDX at 0.625, 2.5, 5, 10mg/L or an acetone-spike control for 10 days. Overt toxicity of RDX in fathead minnow occurred only at the highest exposure concentration resulting in 50% mortality and weight loss. Conversely, Bayesian analysis of microarray data indicated significant changes in transcript expression at concentrations as low as 0.625 mg/L. In total, 154 cDNAs representing 44 unique transcripts were differentially expressed in RDX exposures, the majority of which were validated by reverse transcriptase-quantitative PCR (RT-qPCR). Investigation of molecular pathways, gene ontology (GO) and individual gene functions affected by RDX exposures indicated changes in metabolic processes involved in: oxygen transport, neurological function, calcium binding/signaling, energy metabolism, cell growth/division, oxidative stress and ubiquitination. In total, our study indicated that RDX exposure affected molecular processes critical to CNS function in fathead minnow.


Assuntos
Encéfalo/metabolismo , Cyprinidae/metabolismo , Poluentes Ambientais/toxicidade , Substâncias Explosivas/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Triazinas/toxicidade , Animais , Sequência de Bases , Teorema de Bayes , Encéfalo/efeitos dos fármacos , Biologia Computacional , Cyprinidae/genética , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Biblioteca Gênica , Análise em Microsséries , Dados de Sequência Molecular , Análise de Sequência de DNA , Trinitrotolueno/toxicidade
19.
Bioinformatics ; 24(21): 2561-3, 2008 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-18728045

RESUMO

MOTIVATION: An important contribution to the Gene Ontology (GO) project is to develop tools that facilitate the creation, maintenance and use of ontologies. Several tools have been created for communicating and using the GO project. However, a limitation with most of these tools is that they suffer from lack of a comprehensive search facility. We developed a web application, GOfetcher, with a very comprehensive search facility for the GO project and a variety of output formats for the results. GOfetcher has three different levels for searching the GO: 'Quick Search', 'Advanced Search' and 'Upload Files' for searching. The application includes a unique search option which generates gene information given a nucleotide or protein accession number which can then be used in generating GO information. The output data in GOfetcher can be saved into several different formats; including spreadsheet, comma-separated values and the extensible markup language (XML) format. The database is available at http://mcbc.usm.edu/gofetcher/.


Assuntos
Bases de Dados Genéticas , Software , Vocabulário Controlado , Sistemas de Gerenciamento de Base de Dados , Especificidade da Espécie , Interface Usuário-Computador
20.
BMC Genomics ; 9 Suppl 1: S14, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18366603

RESUMO

BACKGROUND: The budding yeast Saccharomyces cerevisiae is a eukaryotic organism with extensive genetic redundancy. Large-scale gene deletion analysis has shown that over 80% of the ~6200 predicted genes are nonessential and that the functions of 30% of all ORFs remain unclassified, implying that yeast cells can tolerate deletion of a substantial number of individual genes. For example, a class of zinc finger proteins containing C2H2 zinc fingers in tandem arrays of two or three is predicted to be transcription factors; however, seven of the thirty-one predicted genes of this class are nonessential, and their functions are poorly understood. In this study we completed a transcriptomic profiling of three mutants lacking C2H2 zinc finger proteins, ypr013cDelta,ypr015cDelta and ypr013cDeltaypr015cDelta. RESULTS: Gene expression patterns were remarkably different between wild type and the mutants. The results indicate altered expression of 79 genes in ypr013cDelta, 185 genes in ypr015cDelta and 426 genes in the double mutant when compared with that of the wild type strain. More than 80% of the alterations in the double mutants were not observed in either one of the single deletion mutants. Functional categorization based on Munich Information Center for Protein Sequences (MIPS) revealed up-regulation of genes related to transcription and down-regulation of genes involving cell rescue and defense, suggesting a decreased response to stress conditions. Genes related to cell cycle and DNA processing whose expression was affected by single or double deletions were also identified. CONCLUSION: Our results suggest that microarray analysis can define the biological roles of zinc finger proteins with unknown functions and identify target genes that are regulated by these putative transcriptional factors. These findings also suggest that both YPR013C and YPR015C have biological processes in common, in addition to their own regulatory pathways.


Assuntos
Proteínas de Ligação a DNA/deficiência , Perfilação da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Dedos de Zinco/genética , Proteínas de Ligação a DNA/genética , Mutação/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidade da Espécie
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