Riboflavin Stabilizes Abasic, Oxidized G-Quadruplex Structures.

The G-quadruplex is a noncanonical fold of DNA commonly found at telomeres and within gene promoter regions of the genome. These guanine-rich sequences are highly susceptible to damages such as base oxidation and depurination, leading to abasic sites. In the present work, we address whether a vacancy, such as an abasic site, in a G-quadruplex serves as a specific ligand recognition site. When the G-tetrad is all guanines, the vacant (abasic) site is recognized and bound by free guanine nucleobase. However, we aim to understand whether the preference for a specific ligand recognition changes with the presence of a guanine oxidation product 8-oxo-7,8-dihydroguanine (OG) adjacent to the vacancy in the tetrad. Using molecular dynamics simulation, circular dichroism, and nuclear magnetic resonance, we examined the ability for riboflavin to stabilize abasic site-containing G-quadruplex structures. Through structural and free energy binding analysis, we observe riboflavin's ability to stabilize an abasic site-containing G-quadruplex only in the presence of an adjacent OG-modified base. Further, when compared to simulation with the vacancy filled by free guanine, we observe that the free guanine nucleobase is pushed outside of the tetrad by OG to interact with other parts of the structure, including loop residues. These results support the preference of riboflavin over free guanine to fill an OG-adjacent G-quadruplex abasic vacancy.

Evaluation of Minimum Reporting Limits to Determine In-Competition Use of Stimulants.

The applicability of urinary minimum reporting limits (MRLs) to determine in-competition use of prohibited substances is an evolving topic. Most stimulants are subject to a universal MRL, despite the wide range of commercially available dosages for commonly used stimulants. Further, it is unknown whether the urinary MRL is reflective of a pharmacological dose ingested after the start of the in-competition period. To evaluate whether urinary MRLs can distinguish between in-competition and out-of-competition use, a controlled administration study was performed with three commonly used stimulants-amphetamine, methylphenidate, and modafinil at relatively low but therapeutically relevant dosages. Four to six volunteers were administered a particular drug once per day for five consecutive days. Urine, serum, dried blood spots (DBS), and oral fluid (OF) were collected during the active administration period and for 48 h after cessation of use. For all participants, urinary concentrations for all target analytes exceeded the MRL even 48 h after cessation of use. In serum and DBS, most volunteers showed detectable amounts at 48 h post use. Peak concentrations were variable between target compounds even with similar administered dosages. Further, there was a reproducible difference between serum and DBS concentrations. Interpretation of results from OF measurements was challenging due to the inability to normalize for hydration status and OF viscosity. Analyte concentrations decreased steadily over the washout period but did not correlate across matrices for all target analytes. The study reiterates the challenges associated with determining in-competition use by relying on urinary concentrations.