A Computational and Conceptual DFT Study of the Reactivity of Anionic Compounds: Implications for Enzymatic Catalysis

We present a DFT study on the reactivity of an alkoxide and a thiolate toward the neutral and all ionic forms of arsenate and phosphate. The isolated neutral, mono-, di-, and trianionic arsenate/phosphate (electrophiles) and thiolate/methanolate (nucleophiles) species are used as model systems of reacting partners in the enzymatic reactions catalyzed by arsenate reductase and phosphatase. The onset of the displacement reaction at the electrophilic center by the attacking nucleophile is described by the DFT descriptor local softness, applied in a HSAB context. The instability of multiply charged anions in gas phase necessitates the use of a solvent model with an appropriate dielectric constant mimicking the enzymatic environment. Gas-phase and solvent-reactivity studies of isolated compounds indicate that the nucleophilic attack of a thiolate during the first catalysis step of arsenate reductase (ArsC) and low molecular weight phosphatase (LMWPTPase) preferably occurs via the dianionic arsenate and phosphate. This computational approach confirms and supplements earlier experimental data

Reproducibility of telomere length assessment: an international collaborative study

BACKGROUND: Telomere length is a putative biomarker of ageing, morbidity and mortality. Its application is hampered by lack of widely applicable reference ranges and uncertainty regarding the present limits of measurement reproducibility within and between laboratories. METHODS: We instigated an international collaborative study of telomere length assessment: 10 different laboratories, employing 3 different techniques [Southern blotting, single telomere length analysis (STELA) and real-time quantitative PCR (qPCR)] performed two rounds of fully blinded measurements on 10 human DNA samples per round to enable unbiased assessment of intra- and inter-batch variation between laboratories and techniques. RESULTS: Absolute results from different laboratories differed widely and could thus not be compared directly, but rankings of relative telomere lengths were highly correlated (correlation coefficients of 0.63-0.99). Intra-technique correlations were similar for Southern blotting and qPCR and were stronger than inter-technique ones. However, inter-laboratory coefficients of variation (CVs) averaged about 10% for Southern blotting and STELA and more than 20% for qPCR. This difference was compensated for by a higher dynamic range for the qPCR method as shown by equal variance after z-scoring. Technical variation per laboratory, measured as median of intra- and inter-batch CVs, ranged from 1.4% to 9.5%, with differences between laboratories only marginally significant (P = 0.06). Gel-based and PCR-based techniques were not different in accuracy. CONCLUSIONS: Intra- and inter-laboratory technical variation severely limits the usefulness of data pooling and excludes sharing of reference ranges between laboratories. We propose to establish a common set of physical telomere length standards to improve comparability of telomere length estimates between laboratories