Fetal RHD detection from circulating cell-free fetal DNA in maternal plasma: validation of a diagnostic kit using automatic extraction and frozen DNA.

OBJECTIVE: This study aimed to validate non-invasive RHD genotyping of cell-free fetal DNA (cff-DNA) using different DNA extraction methods and of fresh and frozen extracted cff-DNA. BACKGROUND: Non-invasive RHD genotyping of cff-DNA predicts fetal RhD phenotype, allowing for the rational implementation of antenatal immunoprophylaxis and representing a big step forward in the management of RhD-immunised women. Validation of a diagnostic method is mandatory before its clinical application. METHODS: RhD-negative pregnant women were recruited at different gestational ages. The cff-DNA extraction was carried out using manual and automatic methods in order to improve cff-DNA yield and optimise the extraction. Fetal RHD genotyping was performed using a commercial real-time polymerase chain reaction (PCR) kit, and the results were compared with postnatal serological RhD determination on cord blood. RESULTS: Overall, 133 plasma samples were examined for the validation process, and a total of 423 tests were performed. No differences have been observed between the two extraction methods or between fresh or frozen cff-DNA regarding cff-DNA stability and quality parameters. There was 100% concordance between fetal RHD genotyping of cff-DNA and RhD phenotype on cord blood for both extraction methods on both fresh and frozen cff-DNA. CONCLUSION: Our study shows the reliability of automatic and manual cff-DNA extraction methods and the possibility of freezing extracted cff-DNA when performing RHD genotyping. This result might be relevant for improving laboratory work and organisation through the development of a standardised procedure for fetal RHD genotyping on cff-DNA, laying the foundations for evidence-based use of anti-D Ig prophylaxis in RhD pregnant women.

In vitro and in silico evaluation of fucosterol from Sargassum horridum as potential human acetylcholinesterase inhibitor.

The fucosterol has been reported numerous biological activities. In this study, the activity in vitro of the fucosterol from Sargassum horridum as potential human acetylcholinesterase inhibitor was evaluated. The structural identification was obtained by nuclear magnetic resonance (NMR) spectroscopy and based on experimental data, we combined docking and molecular dynamics simulations coupled to the molecular-mechanics-generalized-born-surface-area approach to evaluating the structural and energetic basis for the molecular recognition of fucosterol and neostigmine at the binding site of acetylcholinesterase (AChE). In addition, the Lineweaver-Burk plot showed the nature of a non-competitive inhibition. The maximum velocity (Vmax) and the constant of Michaelis-Menten (Km) estimated for fucosterol (0.006 µM) were 0.015 1/Vo (ΔA/h and 6.399 1/[ACh] mM-1, respectively. While, for neostigmine (0.14 µM), the Vmax was 0.022 1/Vo (ΔA/h) and Km of 6.726 1/[ACh] mM-1, these results showed a more effective inhibition by fucosterol respect to neostigmine. Structural analysis revealed that neostigmine reaches the AChE binding site reported elsewhere, whereas fucosterol can act as a no-competitive and competitive acetylcholinesterase inhibitor, in agree with kinetic enzymatic experiments. Binding free energy calculations revealed that fucosterol reaches the acetylcholinesterase binding site with higher affinity than neostigmine, which is according to experimental results. Whereas the per-residue decomposition free energy analysis let us identify crucial residues involved in the molecular recognition of ligands by AChE. Results corroborate the ability of theoretical methods to provide crucial information at the atomic level about energetic and structural differences in the binding interaction and affinity from fucosterol with AChE. Communicated by Ramaswamy H. Sarma.