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As one of the key initiators of the base excision repair process, uracil-DNA glycosylase (UDG) plays an important role in maintaining genomic integrity. It has been found that aberrant expression of UDG is associated with a variety of diseases. Thus, accurate and sensitive detection of UDG activity is of critical significance for biomedical research and early clinical diagnosis. Here, we developed a novel fluorescent sensing platform for UDG activity detection based on a terminal deoxynucleotidyl transferase (TdT) and T7 exonuclease (T7 Exo)-aided recycling amplification strategy. In this strategy, only two DNA oligonucleotides (DNA substrate containing one uracil base and Poly dT probe labeled with a fluorophore/quencher pair) are used. UDG catalyzes the removal of uracil base from the enclosed dumbbell-shape DNA substrate to give an apyrimidinic site, at which the substrate oligonucleotide is cleaved by endonuclease IV. The released 3'-end can be elongated by TdT to form a long deoxyadenine-rich (Poly dA) tail, which may be used as a recyclable template to initiate T7 Exo-mediated hybridization-digestion cycles of the Poly dT probe, giving a significantly enhanced fluorescence output. The proposed UDG-sensing strategy showed excellent selectivity and high sensitivity with a detection limit of 1.5 × 10-4 U/mL. The sensing platform was also demonstrated to work well for UDG inhibitor screening and inhibitory activity evaluation, thus holding great potential in UDG-related disease diagnosis and drug discovery. The proposed strategy can be easily used for the detection of other DNA repair-related enzymes by simply changing the recognition site in DNA substrate and might also be extended to the analysis of some DNA/RNA-processing enzymes, including restriction endonuclease, DNA methyltransferase, polynucleotide kinase, and so on.

A novel, simple, one-step and one-tube detection method was developed for ultrasensitive detection of polynucleotide kinase (PNK) activity on the basis of dual enzyme-synergistic signal amplification. This method was also demonstrated to work well for PNK inhibitor screening and endogenous PNK detection in cell lysates at a single-cell level.

Age-related physiological changes are known to alter the pharmacokinetics (PK) and pharmacodynamics (PD) of drugs. Metformin is commonly used as first-line medication for management of diabetes in elderly patients. However, the PK and PD of metformin have not been sufficiently studied in elderly subjects. Here, 12 elderly subjects, aged 65 to 85 years, and 20 younger healthy volunteers were orally administered 750 mg of metformin 2 hours after dinner, followed by administration of a second dose (500 mg) 12 hours later. An oral glucose tolerance test (OGTT) was performed 2 hours after the second dose, with 75 g of glucose administered. Blood samples were collected at specific time points after the second metformin dose for the assessment of PK and the glucose-lowering effect of metformin. Elderly subjects exhibited 1.7 and 2.0 times higher average Cmax and AUC∞ than the younger subjects, respectively (P = .007 and .001, respectively), and t1/2 was comparable between the elderly and younger subjects. However, relative glucose level changes from baseline after metformin administration tended to be lower in elderly subjects. Systemic exposure to metformin was elevated by 50% or more in elderly subjects, whereas the glucose-lowering effect was similar compared to younger subjects after 2 doses of metformin.