Nearest-neighbour transition-state analysis for nucleic acid kinetics.

We used stopped-flow to monitor hypochromicity for 43 oligonucleotide duplexes to study nucleic acid kinetics and extract transition-state parameters for association and dissociation. Reactions were performed in 1.0 M NaCl (for literature comparisons) and 2.2 mM MgCl2 (PCR conditions). Dissociation kinetics depended on sequence, increased exponentially with temperature, and transition-state parameters inversely correlated to thermodynamic parameters (r = -0.99). Association had no consistent enthalpic component, varied little with temperature or sequence, and poorly correlated to thermodynamic parameters (r = 0.28). Average association rates decreased 78% in MgCl2 compared to NaCl while dissociation was relatively insensitive to ionic conditions. A nearest-neighbour kinetic model for dissociation predicted rate constants within 3-fold of literature values (n = 11). However, a nearest-neighbour model for association appeared overparameterized and inadequate for predictions. Kinetic predictions were used to simulate published high-speed (<1 min) melting analysis and extreme (<2 min) PCR experiments. Melting simulations predicted apparent melting temperatures increase on average 2.4°C when temperature ramp rates increased from 0.1 to 32°C/s, compared to 2.8°C reported in the literature. PCR simulations revealed that denaturation kinetics are dependent on the thermocycling profile. Simulations overestimated annealing efficiencies at shorter annealing times and suggested that polymerase interactions contribute to primer-template complex stability at extension temperatures.

Recurrent miscarriages and the association with regulatory T cells; A systematic review.

Regulatory T cells (Tregs) are essential in tolerizing the maternal immune system toward the semi-allogeneic embryo. In this systematic review, we evaluated the association of levels and function of Tregs in peripheral blood and decidua with recurrent miscarriage (RM), defined as two unexplained miscarriages. We included 18 studies. Ten studies showed a significantly decreased level of Tregs in peripheral blood of non-pregnant women with RM, compared to controls (p < 0.05). In pregnant women with RM, levels of Tregs in the peripheral blood were significantly lower compared to control groups (p = 0.0004), as shown in nine studies. Moreover, seven studies described a decrease of Treg levels in the placenta of pregnant women with RM (p < 0.0001) compared to controls. Accordingly, the median of the relative changes (MRC) between cases and controls in the non-pregnant group (peripheral blood), and the two pregnant groups (peripheral blood and decidua) were -0.18 (-0.27-0), -0.26 (-0.35 to -0.17), and -0.52 (0.63--0.31), respectively. In addition to the assessment of Tregs by phenotype, six out of the 18 included studies investigated the functionality of these cells. These studies showed a lower inhibitory effect of Tregs cells on the proliferation of effector T cells of women with RM compared to fertile women. Also, the expression of IL-10 and TGF-beta was diminished. This systematic review shows that Treg levels and their function are significantly decreased in peripheral blood and decidua of pregnant and non-pregnant women with RM. This underlines the hypothesis that Tregs play a role in the pathogenesis of RM.