RESUMO
Fipronil is a commonly used insecticide that has been shown to have environmental and human health risks. The current standard methods of detection for fipronil and its metabolites, such as GC-MS, are time consuming and labor intensive. In this study, a variant of systematic evolution of ligands by exponential enrichment (SELEX), was utilized to identify the first single-stranded DNA (ssDNA) molecular recognition element (MRE) that binds to fipronil with high affinity (Kd = 48 ± 8 nM). The selected MRE displayed low cross binding activity on various environmentally relevant, structurally unrelated herbicides and pesticides, in addition to broad-spectrum binding activity on major metabolites of fipronil and a structurally similar pesticide in prepared river samples. Additionally, a proof-of-principle fluorescent detection assay was developed by using the selected ssDNA MRE as a signal-reporting element, with a limit of detection of 105 nM in a prepared river water sample.
Assuntos
Aptâmeros de Nucleotídeos/química , Bioensaio , DNA de Cadeia Simples/química , Inseticidas/isolamento & purificação , Pirazóis/isolamento & purificação , Poluentes Químicos da Água/isolamento & purificação , Aptâmeros de Nucleotídeos/síntese química , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , DNA de Cadeia Simples/síntese química , Água Doce/química , Limite de Detecção , Conformação de Ácido Nucleico , Reprodutibilidade dos Testes , Técnica de Seleção de AptâmerosRESUMO
Soluble epoxide hydrolase (sEH) converts epoxyeicosatrienoic acids that are endothelium-derived hyperpolarizing factors into less active dihydroxyeicosatrienoic acids. Previously, we reported a decrease in adenosine A1 receptor (A1AR) protein levels in sEH knockout (sEH-/-) and an increase in sEH and A1AR protein levels in A2AAR-/- mice. Additionally, KATP channels are involved in adenosine receptor (AR)-dependent vascular relaxation. Thus, we hypothesize that a potential relationship may exist among sEH over-expression, A1AR upregulation, inactivation of KATP channels, and increased in vascular tone. We performed DMT myograph muscle tension measurements and western blot analysis in isolated mouse mesenteric arteries (MAs) from wild-type (WT) and endothelial over-expression of sEH (Tie2-sEH Tr) mice. Our data revealed that NECA (a non-selective adenosine receptors agonist)-induced relaxation was significantly reduced in Tie2-sEH Tr mice, and CCPA (A1AR agonist)-induced contraction was increased in Tie2-sEH Tr mice. A1AR-dependent contraction in Tie2-sEH Tr mice was significantly attenuated by pharmacological inhibition of CYP4A (HET0016, 10 µM), PKCα (GO6976, 1 µM), and ERK1/2 (PD58059, 1 µM). Our western blot analysis revealed significantly higher basal protein expression of CYP4A, A1AR, and reduced p-ERK in MAs of Tie2-sEH Tr mice. Notably, pinacidil (KATP channel opener)-induced relaxation was also significantly reduced in MAs of Tie2-sEH Tr mice. Furthermore, KATP channel-dependent relaxation in MAs was enhanced by inhibition of PKCα and ERK1/2 in WT but not Tie2-sEH Tr mice. In conclusion, our data suggest that over-expression of sEH enhances A1AR-dependent contraction and reduces KATP channel-dependent relaxation in MAs. These results suggest a possible interaction between sEH, A1AR, and KATP channels in regulating vascular tone.
Assuntos
Células Endoteliais/metabolismo , Epóxido Hidrolases/biossíntese , Canais KATP/metabolismo , Artérias Mesentéricas/enzimologia , Receptor A1 de Adenosina/metabolismo , Vasoconstrição , Agonistas do Receptor A1 de Adenosina/farmacologia , Animais , Citocromo P-450 CYP4A/antagonistas & inibidores , Citocromo P-450 CYP4A/genética , Citocromo P-450 CYP4A/metabolismo , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/genética , Canais KATP/genética , Camundongos , Camundongos Transgênicos , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , Receptor A1 de Adenosina/genética , Receptor TIE-2/genética , Receptor TIE-2/metabolismoRESUMO
Alpha toxin is one of the major virulence factors secreted by Staphylococcus aureus, a bacterium that is responsible for a wide variety of infections in both community and hospital settings. Due to the prevalence of S. aureus related infections and the emergence of methicillin-resistant S. aureus, rapid and accurate diagnosis of S. aureus infections is crucial in benefiting patient health outcomes. In this study, a rigorous Systematic Evolution of Ligands by Exponential Enrichment (SELEX) variant previously developed by our laboratory was utilized to select a single-stranded DNA molecular recognition element (MRE) targeting alpha toxin with high affinity and specificity. At the end of the 12-round selection, the selected MRE had an equilibrium dissociation constant (Kd) of 93.7 ± 7.0 nM. Additionally, a modified sandwich enzyme-linked immunosorbent assay (ELISA) was developed by using the selected ssDNA MRE as the toxin-capturing element and a sensitive detection of 200 nM alpha toxin in undiluted human serum samples was achieved.