RESUMO
The authors describe a turn-off fluorometric method for the determination of the activity of the T4 polynucleotide kinase (T4 PNK). It is based on the use of DNA-templated silver nanoclusters (AgNCs). DNA probes with terminal 5' hydroxy groups are used as substrates for DNA phosphatases. If subsequently treated with T4 PNK and Lambda exonuclease (λ exo), the AgNC DNA probes with a modified C-rich sequence and the G-rich sequence is separated. Upon their separation, the strong fluorescence (with excitation/emission maxima at 580/650 nm) that is caused by the proximity of the G-rich region and the C-rich region in the AgNCs decreases sharply. This enabled the fluorometric kinetic determination of the activity of T4 PNK. The assay is characterized by a wide linear range (from 0.01 to 12.5 U·mL-1), a low detection limit (0.01 U·mL-1) and short assay time (typically 60 min). This makes it a promising tool for use in studying processes related to DNA phosphorylation, in drug discovery and in diagnostics. Graphical abstract A turn-off fluorometric method for the determination of the activity of the T4 polynucleotide kinase (T4 PNK) has been developed. It is based on the use of DNA-templated silver nanoclusters (AgNCs). This makes it a promising tool for use in studying processes related to DNA phosphorylation, in drug discovery and in diagnostics.
Assuntos
Bacteriófago T4/enzimologia , DNA/química , Fluorometria/métodos , Nanopartículas Metálicas/química , Sondas Moleculares/química , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , DNA/metabolismo , Cinética , Fosforilação , Polinucleotídeo 5'-Hidroxiquinase/análise , PrataRESUMO
Avian influenza is a serious zoonotic infectious disease with huge negative impacts on local poultry farming, human health and social stability. Therefore, the design of new compounds against avian influenza has been the focus in this field. In this study, computational methods were applied to investigate the compounds with neuraminidase inhibitory activity. First, 2D-SAR model was built to recognize neuraminidase inhibitors (NAIs). As a result, the accuracy of 10 cross-validation and independent tests is 96.84% and 98.97%, respectively. Then, the Topomer CoMFA model was constructed to predict the inhibitory activity and analyses molecular fields. Two models were obtained by changing the cutting methods. The second model is employed to predict the activity (q2â¯=â¯0.784 and r2â¯=â¯0.982). Molecular docking was also used to further analyze the binding sites between NAIs and neuraminidase from human and avian virus. As a result, it is found that same binding Total Score has some differences, but the binding sites are basically the same. At last, some potential NAIs were screened and some optimal opinions were taken. It is expected that our study can assist to study and develop new types of NAIs.
RESUMO
A reusable biosensor has been fabricated in this work for the assay of α-glucosidase activity and the inhibitor screening. In this design, the aptamer of ATP is split as split aptamer 1 (Apt 1) and split aptamer 2 (Apt 2), and Apt 2 can link gold nanoparticles (AuNPs) modified with Apt 1 and 4-aminophenyl-α-d-glucopyranoside (pAPG). Consequently, the functional AuNPs can be immobilized onto the surface of gold electrode, allowing for salt-induced regeneration. In the presence of α-glucosidase, the glycosyl of pAPG is cut off, and the electroactive phenolic hydroxyls appear to give a strong current signal. Furthermore, the biosensor can be recovered very easily by incubating it in water to dissociate the AuNPs modified with Apt 1 and pAPG. So, a new biosensor for α-glucosidase activity detection and inhibitor screening is developed based on enzyme-activated signal generation and recovery. The biosensor may also exhibit good sensitivity for α-glucosidase determination with the detection limit 0.005 U/mL and can be reused by water-washing regeneration with good repeatability. Meanwhile this biosensor can also be utilized for inhibitor screening, which may have potential for clinical applications.
Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , alfa-Glucosidases/análise , alfa-Glucosidases/metabolismo , Eletrodos , Inibidores de Glicosídeo Hidrolases/farmacologia , Ouro/química , Humanos , Nanopartículas Metálicas/químicaRESUMO
DNA methylation catalyzed by DNA methyltransferase plays an important role in many biological processes including gene transcription, genomic imprinting and cellular differentiation. Herein, a novel and effective electrochemical method for the assay of DNA methyltransferase 1(DNMT1) activity has been successfully developed by using uracil-specific excision reagent (USER) induced G-quadruplex formation. Briefly, double stranded DNA containing the recognition sequence of DNMT1 is immobilized on the electrode. Among them, one strand (DNA S1) contains G-rich sequence and a cytosine base, while the supplement strand (DNA S2) cotains C-rich sequence and a methylated cytosine. Through the activity of DNMT1, the hemimethylated CG recognition sequence of the double stranded DNA are methylated and DNA S2 strand is cleaved and removed after the subsequently treatment with EpiTect fast bisulfite conversion kits and USER, leaving the DNA S1 to form the G-quadruplex-hemin DNAzyme for signal amplification. Under optimal-conditions, the method shows wide linear range of 0.1-40 U mL-1 with a detection limit of 0.06 U mL-1. Furthermore, the inhibition assay study demonstrates that SGI-1027 can inhibit the DNMT 1 activity with the IC50 values of 6 µM in the presence of 160 µM S-adenosylmethionine. Since this method can detect human DNMT1 activity effectively and has successfully been applied in complex biological samples, it may have great potential in the applications in DNA methylation related clinical practices and biochemical researches.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Quadruplex G , Uracila , DNA , DNA Catalítico , Hemina , HumanosRESUMO
Pd-Pt bimetallic nanoparticles anchored on functionalized reduced graphene oxide (RGO) nanomaterials were synthesized via a one-step in situ reduction process, in which Pt and Pd ions were first attached to poly(diallyldimethylammonium chloride) (PDDA) functionalized graphene oxide (GO) sheets, and then the encased metal ions and GO were subjected to simultaneous reduction by ethylene glycol. The as-prepared Pd3Pt1/PDDA-RGO nanocomposites were characterized by transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), Raman spectroscopy and electrochemical methods. In addition, an electrochemical sensor based on the graphene nanocomposites was fabricated for the simultaneous detection of ascorbic acid (AA), dopamine (DA) and uric acid (UA) in their ternary mixture. Three well-separated voltammetric peaks along with remarkable increasing electro-oxidation currents were obtained in differential pulse voltammetry (DPV) measurements. Under the optimized conditions, there were linear relationships between the peak currents and the concentrations in the range of 40-1200 µM for AA, 4-200 µM for DA and 4-400 µM for UA, with the limit of detection (LOD) (based on S/N=3) of 0.61, 0.04 and 0.10 µM for AA, DA and UA, respectively. This improved electrochemical performance can be attributed to the synergistic effect of metallic nanoparticles and RGO and the combination of the bimetallic nanoparticles. Furthermore, the practical electroanalytical utility of the sensor was demonstrated by the determination of AA, DA and together with UA in human urine and blood serum samples with satisfactory results.
Assuntos
Ácido Ascórbico/análise , Dopamina/análise , Técnicas Eletroquímicas/métodos , Grafite/química , Nanopartículas/química , Paládio/química , Platina/química , Ácido Úrico/análise , Ácido Ascórbico/sangue , Ácido Ascórbico/urina , Dopamina/sangue , Dopamina/urina , Eletrodos , Vidro/química , Humanos , Concentração de Íons de Hidrogênio , Nanopartículas/ultraestrutura , Oxirredução , Espectroscopia Fotoeletrônica , Polietilenos/química , Compostos de Amônio Quaternário/química , Soluções , Análise Espectral Raman , Ácido Úrico/sangue , Ácido Úrico/urinaRESUMO
OBJECTIVE: To study the efficacy of diffusion tensor tracking (DTT) in study of the normal and abnormal cerebral white matter fiber. METHODS: Ten normal adult volunteers, 5 males and 5 females, aged 40.9 (24-65), and 28 patients with cranial tumors, 9 males and 19 females, aged 43.0 (11-77), underwent MR diffusion tensor. The data thus obtained were transferred to a personal computer and processed with dTV. RESULTS: The main cerebral white matter fiber pathways were successfully observed. Association fibers, including arcuate fibers, cingulum, superior and inferior longitudinal fasciculus, and inferior fronto-occipital fasciculus, commissural fibers (corpus callosum), and projection fibers (corticospinal tract) were revealed. The arcuate fibers, cingulum, superior and inferior longitudinal fasciculus, corticospinal tract, external capsule, optic radiation, and corpus callosum were all involved by intracranial tumors. The abnormality of cerebral white matter could be classified into 4 groups: disruption + infiltration (7 cases), disruption + displacement (10 cases), infiltration + displacement (3 cases), and displacement (8 cases). CONCLUSION: DTT is useful for showing the main normal and abnormal cerebral white mater fiber tracts, thus opening a new field foe research of cerebral white matter fiber in vitro.