RESUMO
Parnassius bremeri (P. bremeri), a member of the genus Snow Apollo in the swallowtail family (Papilionidae), is a high alpine butterfly that lives in Russia, Korea, and China. It is an endangered wildlife (Class I) in South Korea and is a globally endangered species. The lack of transcriptomic and genomic resources of P. bremeri significantly hinders the study of its population genetics and conservation. The detailed information of the developmental stage-specific gene expression patterns of P. bremeri is of great demand for its conservation. However, the molecular mechanism underlying the metamorphic development of P. bremeri is still unknown. In the present study, the differentially expressed genes (DEGs) across the metamorphic developmental stages were compared using high-throughput transcriptome sequencing. We identified a total of 72,161 DEGs from eight comparisons. GO enrichment analysis showed that a range of DEGs were responsible for cuticle development and the melanin biosynthetic pathway during larval development. Pathway analysis suggested that the signaling pathways, such as the Wnt signaling pathway, hedgehog signaling pathway and Notch signaling pathway, are regulated during the developmental stages of P. bremeri. Furthermore, sensory receptors were also activated, especially during the larval to adult transition stage. Collectively, the results of this study provide a preliminary foundation and understanding of the molecular mechanism in their transcriptomes for further research on the metamorphic development of P. bremeri.
Assuntos
Borboletas , Animais , Borboletas/genética , Perfilação da Expressão Gênica , Proteínas Hedgehog/genética , Melaninas/genética , TranscriptomaRESUMO
Aryl hydrocarbon receptor (AhR) activation by environmental agents and microbial metabolites is potentially implicated in a series of skin diseases. Hence, it would be very important to identify natural compounds that could inhibit the AhR activation by ligands of microbial origin as 6-formylindolo[3,2-b]carbazole (FICZ), indirubin (IND) and pityriazepin (PZ) or the prototype ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Five different dry Rosmarinus officinalis L. extracts (ROEs) were assayed for their activities as antagonists of AhR ligand binding with guinea pig cytosol in the presence of [3H]TCDD. The methanolic ROE was further assayed towards CYP1A1 mRNA induction using RT-PCR in human keratinocytes against TCDD, FICZ, PZ, and IND. The isolated metabolites, carnosic acid, carnosol, 7-O-methyl-epi-rosmanol, 4',7-O-dimethylapigenin, and betulinic acid, were assayed for their agonist and antagonist activity in the presence and absence of TCDD using the gel retardation assay (GRA). All assayed ROE extracts showed similar dose-dependent activities with almost complete inhibition of AhR activation by TCDD at 100 ppm. The methanol ROE at 10 ppm showed 99%, 50%, 90%, and 85% inhibition against TCDD, FICZ, IND, and PZ, respectively, in human keratinocytes. Most assayed metabolites exhibited dose-dependent antagonist activity. ROEs inhibit AhR activation by TCDD and by the Malassezia metabolites FICZ, PZ, and IND. Hence, ROE could be useful for the prevention or treatment of skin diseases mediated by activation of AhR.
Assuntos
Dibenzodioxinas Policloradas , Rosmarinus , Neoplasias Cutâneas , Animais , Citocromo P-450 CYP1A1/metabolismo , Cobaias , Humanos , Queratinócitos/metabolismo , Ligantes , Extratos Vegetais/metabolismo , Extratos Vegetais/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Rosmarinus/metabolismo , Neoplasias Cutâneas/metabolismoRESUMO
Diverse organic compounds, many derived from consumer products, are found in sewage sludge worldwide. Understanding which of these poses the most significant environmental threat following land application can be investigated through a variety of predictive and cell-based toxicological techniques. Nontargeted analysis using high-resolution mass spectrometry with predictive estrogenic activity modeling was performed on sewage sludge samples from 12 wastewater treatment plants in California. Diisobutyl phthalate and dextrorphan were predicted to exhibit estrogenic activity and identified in >75% of sludge samples, signifying their universal presence and persistence. Additionally, the application of an estrogen-responsive cell bioassay revealed reductions in agonistic activity during mesophilic and thermophilic treatment but significant increases in antagonism during thermophilic treatment, which warrants further research. Ten nontarget features were identified (metoprolol, fenofibric acid, erythrohydrobupropion, oleic acid, mestranol, 4'-chlorobiphenyl-2,3-diol, medrysone, scillarenin, sudan I, and N,O-didesmethyltramadol) in treatment set samples and are considered to have influenced the in vitro estrogenic activity observed. The combination of predictive and in vitro estrogenicity with nontargeted analysis has led to confirmation of 12 estrogen-active contaminants in California sewage sludge and has highlighted the importance of evaluating both agonistic and antagonistic responses when evaluating the bioactivity of complex samples.
Assuntos
Poluentes Químicos da Água , Purificação da Água , Estrogênios , Estrona/análise , Esgotos , Poluentes Químicos da Água/análiseRESUMO
Urban wildfires may generate numerous unidentified chemicals of toxicity concern. Ash samples were collected from burned residences and from an undeveloped upwind reference site, following the Tubbs fire in Sonoma County, California. The solvent extracts of ash samples were analyzed using GC- and LC-high-resolution mass spectrometry (HRMS) and using a suite of in vitro bioassays for their bioactivity toward nuclear receptors [aryl hydrocarbon receptor (AhR), estrogen receptor (ER), and androgen receptor (AR)], their influence on the expression of genetic markers of stress and inflammation [interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2)], and xenobiotic metabolism [cytochrome P4501A1 (CYP1A1)]. Genetic markers (CYP1A1, IL-8, and COX-2) and AhR activity were significantly higher with wildfire samples than in solvent controls, whereas AR and ER activities generally were unaffected or reduced. The bioassay responses of samples from residential areas were not significantly different from the samples from the reference site despite differing chemical compositions. Suspect and nontarget screening was conducted to identify the chemicals responsible for elevated bioactivity using the multiple streams of HRMS data and open-source data analysis workflows. For the bioassay endpoint with the largest available database of pure compound results (AhR), nontarget features statistically related to whole sample bioassay response using Spearman's rank-order correlation coefficients or elastic net regression were significantly more likely (by 10 and 15 times, respectively) to be known AhR agonists than the overall population of compounds tentatively identified by nontarget analysis. The findings suggest that a combination of nontarget analysis, in vitro bioassays, and statistical analysis can identify bioactive compounds in complex mixtures.
Assuntos
Poluentes Químicos da Água , Incêndios Florestais , Animais , Bioensaio , Linhagem Celular Tumoral , Humanos , Espectrometria de Massas , Camundongos , Receptores de Hidrocarboneto Arílico , Receptores de Estrogênio , Poluentes Químicos da Água/análiseRESUMO
In this study, an AhR-responsive reporter-gene cell-based bioassay CALUX was used to assess the biological potency of dioxins and dioxin-like PCBs (dl-PCBs) in top soil samples collected from a former airbase (A-So) and remote regions from urban and agricultural areas in Thua Thien Hue, Vietnam. In top soil collected from A-So airbase, Bioanalytical EQuivalent (BEQ) concentrations of up to 2700 pg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) per g dry weight (pg BEQ-TCDD g-1 dw) were assessed. Interestingly, while BEQ values for dl-PCBs were found to be up to 13 pg BEQ-TCDD g-1 dw, the dl-PCB activity was not detected in all the hotspot sample extracts. In contrasts, BEQ values for dioxin like compounds from remote regions were much lower and occasionally below the quantification limits of the method. The BEQ activities obtained in this study have a similar trend to the WHO-TEQ results for the samples collected in the A-So airbase. However, BEQ values were higher than those of TEQ, probably reflecting the presence of additional AhR ligands and/or possible non-additive interactions in the sample mixture. This study confirms that after more than 60 years, a strong residual pollution of PCDD/Fs remains on this former air base following the use and storage of Agent Orange during the Vietnam War, raising a health risk for populations exposed in this area because livestock animals graze there.
Assuntos
Agente Laranja , Monitoramento Ambiental , Poluentes do Solo/análise , Animais , Benzofuranos , Bioensaio/métodos , Dibenzofuranos , Dioxinas/toxicidade , Genes Reporter , Bifenilos Policlorados/análise , Dibenzodioxinas Policloradas , Solo , Poluentes do Solo/toxicidade , VietnãRESUMO
BACKGROUND: Bacterial fruit blotch (BFB), a disease caused by Acidovorax citrulli, results in significant economic losses in melon. The causal QTLs and genes for resistance to this disease have yet to be identified. Resistance (R)-genes play vital roles in resistance to plant diseases. Since the complete genome sequence of melon is available and genome-wide identification of R-genes has been performed for this important crop, comprehensive expression profiling may lead to the identification of putative candidate genes that function in the response to BFB. RESULTS: We identified melon accessions that are resistant and susceptible to BFB through repeated bioassays and characterized all 70 R-genes in melon, including their gene structures, chromosomal locations, domain organizations, motif distributions, and syntenic relationships. Several disease resistance-related domains were identified, including NBS, TIR, LRR, CC, RLK, and DUF domains, and the genes were categorized based on the domains of their encoded proteins. In addition, we profiled the expression patterns of the genes in melon accessions with contrasting levels of BFB resistance at 12 h, 1 d, 3 d, and 6 d after inoculation with A. citrulli. Six R-genes exhibited consistent expression patterns (MELO3C023441, MELO3C016529, MELO3C022157, MELO3C022146, MELO3C025518, and MELO3C004303), with higher expression levels in the resistant vs. susceptible accession. CONCLUSION: We identified six putative candidate R-genes against BFB in melon. Upon functional validation, these genes could be targeted for manipulation via breeding and biotechnological approaches to improve BFB resistance in melon in the future.
Assuntos
Comamonadaceae/patogenicidade , Cucurbitaceae/genética , Resistência à Doença/genética , Genes de Plantas , Doenças das Plantas/genética , Cucurbitaceae/microbiologia , Frutas , Doenças das Plantas/microbiologiaRESUMO
Disease tolerance is the ability of the host to reduce the effect of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (endotoxin tolerance). We found that a first exposure of mice to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long-term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR-complex-associated Src kinase activity promoted IDO1 phosphorylation and signalling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in Gram-negative and Gram-positive infections, pointing to a role for AhR in contributing to host fitness.
Assuntos
Resistência à Doença/genética , Resistência à Doença/imunologia , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Resistência à Doença/efeitos dos fármacos , Endotoxemia/genética , Endotoxemia/imunologia , Endotoxemia/metabolismo , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Inflamação/enzimologia , Inflamação/genética , Inflamação/metabolismo , Cinurenina/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Fosforilação , Receptores de Hidrocarboneto Arílico/genética , Transdução de Sinais , Triptofano Oxigenase/metabolismo , Quinases da Família src/metabolismoRESUMO
The aryl hydrocarbon receptor (AHR) plays pleiotropic roles in the development and physiology of vertebrates in conjunction with xenobiotic and endogenous ligands. It is best known for mediating the toxic effects of dioxin-like pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). While most vertebrates possess at least one AHR that binds TCDD tightly, amphibian AHRs bind TCDD with very low affinity. Previous analyses of AHRs from Xenopus laevis (a frog; order Anura) and Ambystoma mexicanum (a salamander; order Caudata) identified three amino acid residues in the ligand-binding domain (LBD) that underlie low-affinity binding. In X. laevis AHR1ß, these are A354, A370, and N325. Here we extend the analysis of amphibian AHRs to the caecilian Gymnopis multiplicata, representing the remaining extant amphibian order, Gymnophiona. G. multiplicata AHR groups with the monophyletic vertebrate AHR/AHR1 clade. The LBD includes all three signature residues of low TCDD affinity, and a structural homology model suggests that its architecture closely resembles those of other amphibians. In transactivation assays, the EC50 for reporter gene induction by TCDD was 17.17 nM, comparable to X. laevis AhR1ß (26.23 nM) and Ambystoma AHR (34.09 nM) and dramatically higher than mouse AhR (0.13 nM), a trend generally reflected in direct measures of TCDD binding. These shared properties distinguish amphibian AHRs from the high-affinity proteins typical of both vertebrate groups that diverged earlier (teleost fish) and those that appeared more recently (other tetrapods). These findings suggest the hypothesis that AHRs with low TCDD affinity represent a characteristic that evolved in a common ancestor of all three extant amphibian groups.
Assuntos
Ambystoma mexicanum/metabolismo , Dibenzodioxinas Policloradas/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Xenopus laevis/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Ligantes , Filogenia , Dibenzodioxinas Policloradas/química , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/genética , Homologia de SequênciaRESUMO
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological and toxicological effects of an AhR lacking the entire PASB structurally diverse chemicals, including halogenated aromatic hydrocarbons. Ligand-dependent transformation of the AhR into its DNA binding form involves a ligand-dependent conformational change, heat shock protein 90 (hsp90), dissociation from the AhR complex and AhR dimerization with the AhR nuclear translocator (ARNT) protein. The mechanism of AhR transformation was examined using mutational approaches and stabilization of the AhR:hsp90 complex with sodium molybdate. Insertion of a single mutation (F281A) in the hsp90-binding region of the AhR resulted in its constitutive (ligand-independent) transformation/DNA binding in vitro. Mutations of AhR residues within the Arg-Cys-rich region (R212A, R217A, R219A) and Asp371 (D371A) impaired AhR transformation without a significant effect on ligand binding. Stabilization of AhR:hsp90 binding with sodium molybdate decreased transformation/DNA binding of the wild type AhR but had no effect on constitutively active AhR mutants. Interestingly, transformation of the AhR in the presence of molybdate allowed detection of an intermediate transformation ternary complex containing hsp90, AhR, and ARNT. These results are consistent with a stepwise transformation mechanism in which binding of ARNT to the liganded AhR:hsp90 complex results in a progressive displacement of hsp90 and conversion of the AhR into its high affinity DNA binding form. The available molecular insights into the signaling mechanism of other Per-ARNT-Sim (PAS) domains and structural information on hsp90 association with other client proteins are consistent with the proposed transformation mechanism of the AhR.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Transformação Celular Neoplásica/metabolismo , DNA/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Transformação Celular Neoplásica/genética , DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Ligantes , Modelos Moleculares , Molibdênio/farmacologia , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de Hidrocarboneto Arílico/química , Relação Estrutura-AtividadeRESUMO
1,2-naphthoquinone (1,2-NQ) and 1,4-naphthoquinone (1,4-NQ) are clinically promising biologically active chemicals that have been shown to stimulate the aryl hydrocarbon receptor (AhR) signaling pathway, but whether they are direct or indirect ligands or activate the AhR in a ligand-independent manner is unknown. Given the structural diversity of AhR ligands, multiple mechanisms of AhR activation of gene expression, and species differences in AhR ligand binding and response, we examined the ability of 1,2-NQ and 1,4-NQ to bind to and activate the mouse and human AhRs using a series of in vitro AhR-specific bioassays and in silico modeling techniques. Both NQs induced AhR-dependent gene expression in mouse and human hepatoma cells, but were more potent and efficacious in human cells. 1,2-NQ and 1,4-NQ stimulated AhR transformation and DNA binding in vitro and was inhibited by AhR antagonists. Ligand binding analysis confirmed the ability of 1,2-NQ and 1,4-NQ to competitively bind to the AhR ligand binding cavity and the molecular determinants for interactions were predicted by molecular modeling methods. NQs were shown to bind distinctly differently from that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and differences were also observed between species. Mutation of amino acid residues (F289, M334, and M342) involved in critical NQ:AhR binding interactions, decreased NQ- and AhR-dependent gene expression, consistent with a role for these residues in binding and activation of the AhR by NQs. These studies provide insights into the molecular mechanism of action of NQs and contribute to the development of emerging NQ-based therapeutics.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Naftoquinonas/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Ligação Competitiva , Células COS , Linhagem Celular , Chlorocebus aethiops , Citocromo P-450 CYP1A1/genética , DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutação , Naftoquinonas/metabolismo , Dibenzodioxinas Policloradas/farmacologia , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/genética , Especificidade da EspécieRESUMO
BACKGROUND: In recent years, the BRAF inhibitor vemurafenib has been successfully established in the therapy of advanced melanoma. Despite its superior efficacy, the use of vemurafenib is limited by frequent inflammatory cutaneous adverse events that affect patients' quality of life and may lead to dose reduction or even cessation of anti-tumor therapy. To date, the molecular and cellular mechanisms of vemurafenib-induced rashes have remained largely elusive. METHODS: In this study, we deployed immunohistochemistry, RT-qPCR, flow cytometry, lymphocyte activation tests, and different cell-free protein-interaction assays. RESULTS: We here demonstrate that vemurafenib inhibits the downstream signaling of the canonical pathway of aryl hydrocarbon receptor (AhR) in vitro, thereby inducing the expression of proinflammatory cytokines (eg, TNF) and chemokines (eg, CCL5). In line with these results, we observed an impaired expression of AhR-regulated genes (eg, CYP1A1) and an upregulation of the corresponding proinflammatory genes in vivo. Moreover, results of lymphocyte activation tests showed the absence of drug-specific T cells in respective patients. CONCLUSION: Taken together, we obtained no hint of an underlying sensitization against vemurafenib but found evidence suggesting that vemurafenib enhances proinflammatory responses by inhibition of canonical AhR signaling. Our findings contribute to our understanding of the central role of the AhR in skin inflammation and may point toward a potential role for topical AhR agonists in supportive cancer care.
Assuntos
Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Inibidores de Proteínas Quinases/farmacologia , Receptores de Hidrocarboneto Arílico/agonistas , Vemurafenib/farmacologia , Idoso , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Biomarcadores , Biópsia , Estudos de Casos e Controles , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Dermatite/diagnóstico , Dermatite/etiologia , Modelos Animais de Doenças , Cobaias , Humanos , Modelos Moleculares , Conformação Proteica , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/uso terapêutico , Receptores de Hidrocarboneto Arílico/química , Relação Estrutura-Atividade , Subpopulações de Linfócitos T , Células Th1/imunologia , Células Th1/metabolismo , Vemurafenib/efeitos adversos , Vemurafenib/uso terapêuticoRESUMO
Malassezia furfur isolates from diseased skin preferentially biosynthesize compounds which are among the most active known aryl-hydrocarbon receptor (AhR) inducers, such as indirubin, tryptanthrin, indolo[3,2-b]carbazole, and 6-formylindolo[3,2-b]carbazole. In our effort to study their production from Malassezia spp., we investigated the role of indole-3-carbaldehyde (I3A), the most abundant metabolite of Malassezia when grown on tryptophan agar, as a possible starting material for the biosynthesis of the alkaloids. Treatment of I3A with H2O2 and use of catalysts like diphenyldiselenide resulted in the simultaneous one-step transformation of I3A to indirubin and tryptanthrin in good yields. The same reaction was first applied on simple indole and then on substituted indoles and indole-3-carbaldehydes, leading to a series of mono- and bisubstituted indirubins and tryptanthrins bearing halogens, alkyl, or carbomethoxy groups. Afterward, they were evaluated for their AhR agonist activity in recombinant human and mouse hepatoma cell lines containing a stably transfected AhR-response luciferase reporter gene. Among them, 3,9-dibromotryptanthrin was found to be equipotent to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as an AhR agonist, and 3-bromotryptanthrin was 10-times more potent than TCDD in the human HG2L7.5c1 cell line. In contrast, 3,9-dibromotryptanthrin and 3-bromotryptanthrin were â¼4000 and >10,000 times less potent than TCDD in the mouse H1L7.5c3 cell line, respectively, demonstrating that they are species-specific AhR agonists. Involvement of the AhR in the action of 3-bromotryptanthrin was confirmed by the ability of the AhR antagonists CH223191 and SR1 to inhibit 3-bromotryptanthrin-dependent reporter gene induction in human HG2L7.5c1 cells. In conclusion, I3A can be the starting material used by Malassezia for the production of both indirubin and tryptanthrin through an oxidation mechanism, and modification of these compounds can produce some highly potent, efficacious and species-selective AhR agonists.
Assuntos
Alcaloides/síntese química , Biomimética/métodos , Indóis/química , Malassezia/metabolismo , Quinazolinas/síntese química , Receptores de Hidrocarboneto Arílico/metabolismo , Alcaloides/química , Alcaloides/farmacologia , Peróxido de Hidrogênio/farmacologia , Indóis/síntese química , Indóis/farmacologia , Malassezia/crescimento & desenvolvimento , Estrutura Molecular , Quinazolinas/química , Quinazolinas/farmacologiaRESUMO
Acidovorax citrulli (A. citrulli) strains cause bacterial fruit blotch (BFB) in cucurbit crops and affect melon significantly. Numerous strains of the bacterium have been isolated from melon hosts globally. Strains that are aggressively virulent towards melon and diagnostic markers for detecting such strains are yet to be identified. Using a cross-inoculation assay, we demonstrated that two Korean strains of A. citrulli, NIHHS15-280 and KACC18782, are highly virulent towards melon but avirulent/mildly virulent to the other cucurbit crops. The whole genomes of three A. citrulli strains isolated from melon and three from watermelon were aligned, allowing the design of three primer sets (AcM13, AcM380, and AcM797) that are specific to melon host strains, from three pathogenesis-related genes. These primers successfully detected the target strain NIHHS15-280 in polymerase chain reaction (PCR) assays from a very low concentration of bacterial gDNA. They were also effective in detecting the target strains from artificially infected leaf, fruit, and seed washing suspensions, without requiring the extraction of bacterial DNA. This is the first report of PCR-based markers that offer reliable, sensitive, and rapid detection of strains of A. citrulli causing BFB in melon. These markers may also be useful in early disease detection in the field samples, in seed health tests, and for international quarantine purposes.
Assuntos
Comamonadaceae/isolamento & purificação , Cucurbitaceae/microbiologia , Doenças das Plantas/microbiologia , Comamonadaceae/genética , Produtos Agrícolas/microbiologia , DNA Bacteriano/análise , DNA Bacteriano/genética , Frutas/microbiologia , Genoma Bacteriano , Reação em Cadeia da PolimeraseRESUMO
Patients with chronic kidney disease (CKD) are exposed to uremic toxins and have an increased risk of cardiovascular disease. Some uremic toxins, like indoxyl sulfate, are agonists of the transcription factor aryl hydrocarbon receptor (AHR). These toxins induce a vascular procoagulant phenotype. Here we investigated AHR activation in patients with CKD and in a murine model of CKD. We performed a prospective study in 116 patients with CKD stage 3 to 5D and measured the AHR-Activating Potential of serum by bioassay. Compared to sera from healthy controls, sera from CKD patients displayed a strong AHR-Activating Potential; strongly correlated with eGFR and with the indoxyl sulfate concentration. The expression of the AHR target genes Cyp1A1 and AHRR was up-regulated in whole blood from patients with CKD. Survival analyses revealed that cardiovascular events were more frequent in CKD patients with an AHR-Activating Potential above the median. In mice with 5/6 nephrectomy, there was an increased serum AHR-Activating Potential, and an induction of Cyp1a1 mRNA in the aorta and heart, absent in AhR-/- CKD mice. After serial indoxyl sulfate injections, we observed an increase in serum AHR-AP and in expression of Cyp1a1 mRNA in aorta and heart in WT mice, but not in AhR-/- mice. Thus, the AHR pathway is activated both in patients and mice with CKD. Hence, AHR activation could be a key mechanism involved in the deleterious cardiovascular effects observed in CKD.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/sangue , Receptores de Hidrocarboneto Arílico/sangue , Insuficiência Renal Crônica/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/mortalidade , Estudos de Casos e Controles , Causas de Morte , Linhagem Celular Tumoral , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Indicã/administração & dosagem , Indicã/sangue , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Estudos Prospectivos , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/deficiência , Receptores de Hidrocarboneto Arílico/genética , Diálise Renal , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/mortalidade , Insuficiência Renal Crônica/terapia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Risco , Resultado do TratamentoRESUMO
Time Reversal (TR) is a technique that may be used to focus an acoustic signal at a particular point in space. While many variables contribute to the quality of TR focusing of sound in a particular room, the most important have been shown to be the number of sound sources, signal bandwidth, and absorption properties of the medium as noted by Ribay, de Rosny, and Fink [J. Acoust. Soc. Am. 117(5), 2866-2872 (2005)]. However, the effect of room size on TR focusing has not been explored. Using the image source method algorithm proposed by Allen and Berkley [J. Acoust. Soc. Am. 65(4), 943-950 (1979)], TR focusing was simulated in a variety of rooms with different absorption and volume properties. Experiments are also conducted in a couple rooms to verify the simulations. The peak focal amplitude, the temporal focus quality, and the spatial focus clarity are defined and calculated for each simulation. The results are used to determine the effects of absorption and room volume on TR. Less absorption increases the amplitude of the focusing and spatial clarity while decreasing temporal quality. Dissimilarly, larger volumes decrease focal amplitude and spatial clarity while increasing temporal quality.
RESUMO
Time reversal (TR) is a signal processing technique that can be used for intentional sound focusing. While it has been studied in room acoustics, the application of TR to produce a high amplitude focus of sound in a room has not yet been explored. The purpose of this study is to create a virtual source of spherical waves with TR that are of sufficient intensity to study nonlinear acoustic propagation. A parameterization study of deconvolution, one-bit, clipping, and decay compensation TR methods is performed to optimize high amplitude focusing and temporal signal focus quality. Of all TR methods studied, clipping is shown to produce the highest amplitude focal signal. An experiment utilizing eight horn loudspeakers in a reverberation chamber is done with the clipping TR method. A peak focal amplitude of 9.05 kPa (173.1 dB peak re 20 µPa) is achieved. Results from this experiment indicate that this high amplitude focusing is a nonlinear process.
RESUMO
The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that modulates gene expression following its binding and activation by structurally diverse chemicals. Species differences in AhR functionality have been observed, with the mouse AhR (mAhR) and human AhR (hAhR) exhibiting significant differences in ligand binding, coactivator recruitment, gene expression and response. While the AhR agonist indirubin (IR) is a more potent activator of hAhR-dependent gene expression than the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), it is a significantly less potent activator of the mAhR. DNA binding analysis confirmed the greater potency/efficacy of IR in stimulating transformation/DNA binding of the hAhR in vitro and domain-swapping experiments demonstrated that the enhanced response to IR was primarily due to the hAhR ligand binding domain (LBD). Site-directed mutagenesis and functional analysis studies revealed that mutation of H326 and A349 in the mAhR LBD to the corresponding residues in the hAhR LBD significantly increased the potency of IR. Since these mutations had no significant effect on ligand binding, these residues likely contribute to an enhanced efficiency of transformation/DNA binding by IR-bound hAhR. Molecular docking to mAhR LBD homology models further elucidated the different roles of the A375V mutation in TCDD and IR binding, as revealed by [³H]TCDD competitive binding results. These results demonstrate the differential binding of structurally diverse ligands within the LBD of a given AhR and confirm that amino acid differences within the LBD of AhRs contribute to significant species differences in ligand response.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Simulação por Computador , Humanos , Técnicas In Vitro , Indóis/farmacologia , Camundongos , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Dibenzodioxinas Policloradas/farmacologia , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Receptores de Hidrocarboneto Arílico/genética , Especificidade da EspécieRESUMO
A novel type of diffusive gradients in thin-film (DGT) was combined with a chemically activated luciferase gene expression bioassay (CALUX) to measure estrogens in aquatic systems. The performance of this novel method was assessed with 17ß-estradiol (E2) as the model steroid hormone, XAD 18 resin gel as the binding phase in the DGT method and VM7Luc4E2 cells (formerly BG1Luc4E2) for the Estrogen Responsive Element (ERE)-CALUX bioassay. The measured effective diffusion coefficient of E2 in agarose diffusive gel was 4.65 ± 0.37 × 10-6 cm2 s-1 at 25 °C. The detection limit of this combined DGT/ERE-CALUX method for 1 day of sampling (0.026 ± 0.003 ng L-1 of E2) is significantly lower than that obtained by spot sampling combined with GC-MS/MS or LC-MS/MS analysis (0.1-7.0 ng L-1). The method is independent of pH (5-8), ionic strength (0.001-0.5 M), and dissolved organic matter (DOM; concentrations up to 30 mg L-1). Field applications of this novel DGT in effluents of three sewage treatment plants in Beijing city (China) showed comparable results to conventional spot (grab) sampling. This study demonstrates that the combined DGT/ERE-CALUX approach is an effective and sensitive tool for in situ monitoring of estrogenic activity in waters and wastewaters.
Assuntos
Bioensaio , Estradiol/análise , Estrogênios/análise , Água/química , Linhagem Celular , Difusão , Humanos , Concentração de Íons de Hidrogênio , Concentração OsmolarRESUMO
The Aryl hydrocarbon Receptor (AhR) is a transcription factor that mediates the biochemical response to xenobiotics and the toxic effects of a number of environmental contaminants, including dioxins. Recently, endogenous regulatory roles for the AhR in normal physiology and development have also been reported, thus extending the interest in understanding its molecular mechanisms of activation. Since dimerization with the AhR Nuclear Translocator (ARNT) protein, occurring through the Helix-Loop-Helix (HLH) and PER-ARNT-SIM (PAS) domains, is needed to convert the AhR into its transcriptionally active form, deciphering the AhR:ARNT dimerization mode would provide insights into the mechanisms of AhR transformation. Here we present homology models of the murine AhR:ARNT PAS domain dimer developed using recently available X-ray structures of other bHLH-PAS protein dimers. Due to the different reciprocal orientation and interaction surfaces in the different template dimers, two alternative models were developed for both the PAS-A and PAS-B dimers and they were characterized by combining a number of computational evaluations. Both well-established hot spot prediction methods and new approaches to analyze individual residue and residue-pairwise contributions to the MM-GBSA binding free energies were adopted to predict residues critical for dimer stabilization. On this basis, a mutagenesis strategy for both the murine AhR and ARNT proteins was designed and ligand-dependent DNA binding ability of the AhR:ARNT heterodimer mutants was evaluated. While functional analysis disfavored the HIF2α:ARNT heterodimer-based PAS-B model, most mutants derived from the CLOCK:BMAL1-based AhR:ARNT dimer models of both the PAS-A and the PAS-B dramatically decreased the levels of DNA binding, suggesting this latter model as the most suitable for describing AhR:ARNT dimerization. These novel results open new research directions focused at elucidating basic molecular mechanisms underlying the functional activity of the AhR.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Biologia Computacional/métodos , Modelos Moleculares , Domínios Proteicos , Receptores de Hidrocarboneto Arílico , Translocador Nuclear Receptor Aril Hidrocarboneto/química , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Humanos , Mutação , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismoRESUMO
The toxic effects of dioxins and related compounds (DRCs) are mediated by the aryl hydrocarbon receptor (AHR). Our previous study identified AHR1 and AHR2 genes from the red seabream (Pagrus major). Moreover, we found that AHR2 mRNA levels were notably elevated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in the early life stage of red seabream embryos, while AHR1 mRNA level was not altered. In this study, to investigate the regulatory mechanism of these AHR transcripts, we cloned and characterized 5'-flanking regions of AHR1 and AHR2 genes. Both of the 5'-flanking regions in these AHR genes contained three potential xenobiotic-responsive elements (XREs). To assess whether the 5'-flanking region is transactivated by rsAHR1 and rsAHR2 proteins, we measured the transactivation potency of the luciferase reporter plasmids containing the 5'-flanking regions by AHR1 and AHR2 proteins that were transiently co-expressed in COS-7. Only reporter plasmid (pGL4-rsAHR2-3XREs) that contained three putative XRE sites in the 5'-flanking region of AHR2 gene showed a clear TCDD dose-dependent transactivation by AHR1 and AHR2 proteins. TCDD-EC50 values for the rsAHR2-derived XRE transactivation were 1.3 and 1.4 nM for AHR1 and AHR2, respectively. These results suggest that the putative XREs of AHR2 gene have a function for AHR1- and AHR2-mediated transactivation, supporting our in ovo observation of an induction of AHR2 mRNA levels by TCDD exposure. Mutations in XREs of AHR2 gene led to a decrease in luciferase induction. Electrophoretic mobility shift assay showed that XRE1, the closest XRE from the start codon in AHR2 gene, is mainly responsible for the binding with TCDD-activated AHR. This suggests that TCDD-activated AHR1 and AHR2 up-regulate the AHR2 mRNA levels and this auto-induced AHR2 may amplify the signal transduction of its downstream targets including CYP1A in the red seabream.