RESUMO
The development of deoxynucleoside triphosphate (dNTP)-based drugs requires a quantitative understanding of any inhibition, activation, or hydrolysis by off-target cellular enzymes. SAMHD1 is a regulatory dNTP-triphosphohydrolase that inhibits HIV-1 replication in human myeloid cells. We describe here an enzyme-coupled assay for quantifying the activation, inhibition, and hydrolysis of dNTPs, nucleotide analogues, and nucleotide analogue inhibitors by triphosphohydrolase enzymes. The assay facilitates mechanistic studies of triphosphohydrolase enzymes and the quantification of off-target effects of nucleotide-based antiviral and chemotherapeutic agents.
Assuntos
Hidrolases Anidrido Ácido/análise , Bioensaio/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas Monoméricas de Ligação ao GTP/análise , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Aciclovir/química , Aciclovir/metabolismo , Aciclovir/farmacologia , Nucleotídeos de Adenina/química , Nucleotídeos de Adenina/farmacologia , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Arabinonucleosídeos/química , Arabinonucleosídeos/farmacologia , Catálise/efeitos dos fármacos , Clofarabina , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Relação Dose-Resposta a Droga , Ganciclovir/química , Ganciclovir/farmacologia , HIV-1 , Hidrólise , Proteína 1 com Domínio SAM e Domínio HDRESUMO
Enhanced biological phosphorus removal (EBPR) from wastewater relies on the preferential selection of active polyphosphate-accumulating organisms (PAO) in the underlying bacterial community continuum. Efficient management of the bacterial resource requires understanding of population dynamics as well as availability of bioanalytical methods for rapid and regular assessment of relative abundances of active PAOs and their glycogen-accumulating competitors (GAO). A systems approach was adopted here toward the investigation of multilevel correlations from the EBPR bioprocess to the bacterial community, metabolic, and enzymatic levels. Two anaerobic-aerobic sequencing-batch reactors were operated to enrich activated sludge in PAOs and GAOs affiliating with "Candidati Accumulibacter and Competibacter phosphates", respectively. Bacterial selection was optimized by dynamic control of the organic loading rate and the anaerobic contact time. The distinct core bacteriomes mainly comprised populations related to the classes Betaproteobacteria, Cytophagia, and Chloroflexi in the PAO enrichment and of Gammaproteobacteria, Alphaproteobacteria, Acidobacteria, and Sphingobacteria in the GAO enrichment. An anaerobic metabolic batch test based on electrical conductivity evolution and a polyphosphatase enzymatic assay were developed for rapid and low-cost assessment of the active PAO fraction and dephosphatation potential of activated sludge. Linear correlations were obtained between the PAO fraction, biomass specific rate of conductivity increase under anaerobic conditions, and polyphosphate-hydrolyzing activity of PAO/GAO mixtures. The correlations between PAO/GAO ratios, metabolic activities, and conductivity profiles were confirmed by simulations with a mathematical model developed in the aqueous geochemistry software PHREEQC.
Assuntos
Hidrolases Anidrido Ácido/metabolismo , Reatores Biológicos , Modelos Biológicos , Fósforo/isolamento & purificação , Fósforo/metabolismo , Hidrolases Anidrido Ácido/análise , Anaerobiose , Condutividade Elétrica , Microbiota , Fósforo/química , Esgotos , Biologia de SistemasRESUMO
Previous researches have demonstrated that biological phosphorus removal (BPR) from wastewater could be driven by the aerobic/extended-idle (A/EI) regime. This study further investigated temperature effects on phosphorus removal performance in six A/EI sequencing batch reactors (SBRs) operated at temperatures ranging from 5 to 30 °C. The results showed that phosphorus removal efficiency increased with temperature increasing from 5 to 20 °C but slightly decreased when temperature continually increased to 30 °C. The highest phosphorus removal rate of 97.1 % was obtained at 20 °C. The biomass cultured at 20 °C contained more polyphosphate accumulating organisms (PAO) and less glycogen accumulating organisms (GAO) than that cultured at any other temperatures investigated. The mechanism studies revealed that temperature affected the transformations of glycogen and polyhydroxyalkanoates, and the activities of exopolyphosphatase and polyphosphate kinase activities. In addition, phosphorus removal performances of the A/EI and traditional anaerobic/oxic (A/O) SBRs were compared at 5 and 20 °C, respectively. The results showed the A/EI regime drove better phosphorus removal than the A/O regime at both 5 and 20 °C, and more PAO and less GAO abundances in the biomass might be the principal reason for the higher BPR in the A/EI SBRs as compared with the A/O SBRs.