Cation binding to 15-TBA quadruplex DNA is a multiple-pathway cation-dependent process

A combination of explicit solvent molecular dynamics simulation (30 simulations reaching 4 µs in total), hybrid quantum mechanics/molecular mechanics approach and isothermal titration calorimetry was used to investigate the atomistic picture of ion binding to 15-mer thrombin-binding quadruplex DNA (G-DNA) aptamer. Binding of ions to G-DNA is complex multiple pathway process, which is strongly affected by the type of the cation. The individual ion-binding events are substantially modulated by the connecting loops of the aptamer, which play several roles. They stabilize the molecule during time periods when the bound ions are not present, they modulate the route of the ion into the stem and they also stabilize the internal ions by closing the gates through which the ions enter the quadruplex. Using our extensive simulations, we for the first time observed full spontaneous exchange of internal cation between quadruplex molecule and bulk solvent at atomistic resolution. The simulation suggests that expulsion of the internally bound ion is correlated with initial binding of the incoming ion. The incoming ion then readily replaces the bound ion while minimizing any destabilization of the solute molecule during the exchange

Characterization of a K+-induced conformational switch in a human telomeric DNA oligonucleotide using 2-aminopurine fluorescence

Human telomeric DNA consists of tandem repeats of the DNA sequence d(GGGTTA). Oligodeoxynucletotide telomere models such as d[A(GGGTTA)(3)GGG] (Tel22) fold in a cation-dependent manner into quadruplex structures consisting of stacked G-quartets linked by d(TTA) loops. NMR has shown that in Na(+) solutions Tel22 forms a ‘basket’ topology of four antiparallel strands; in contrast, Tel22 in K(+) solutions consists of a mixture of unknown topologies. Our previous studies on the mechanism of folding of Tel22 and similar telomere analogs utilized changes in UV absorption between 270 and 325 nm that report primarily on G-quartet formation and stacking showed that quadruplex formation occurs within milliseconds upon mixing with an appropriate cation. In the current study, we assessed the dynamics and equilibria of folding of specific loops by using Tel22 derivatives in which the dA residues were serially substituted with the fluorescent reporter base, 2-aminopurine (2-AP). Tel22 folding induced by Na(+) or K(+) assessed by changes in 2-AP fluorescence consists of at least three kinetic steps with time constants spanning a range of ms to several hundred seconds. Na(+)-dependent equilibrium titrations of Tel22 folding could be approximated as a cooperative two-state process. In contrast, K(+)-dependent folding curves were biphasic, revealing that different conformational ensembles are present in 1 mM and 30 mM K(+). This conclusion was confirmed by (1)H NMR. Molecular dynamics simulations revealed a K(+) binding pocket in Tel22 located near dA1 that is specific for the so-called hybrid-1 conformation in which strand 1 is in a parallel arrangement. The possible presence of this topologically specific binding site suggests that K(+) may play an allosteric role in regulating telomere conformation and function by modulating quadruplex tertiary structure

Temporal changes in the epidemiology, management, and outcome from acute respiratory distress syndrome in European intensive care units: a comparison of two large cohorts

Background: Mortality rates for patients with ARDS remain high. We assessed temporal changes in the epidemiology and management of ARDS patients requiring invasive mechanical ventilation in European ICUs. We also investigated the association between ventilatory settings and outcome in these patients. Methods: This was a post hoc analysis of two cohorts of adult ICU patients admitted between May 1–15, 2002 (SOAP study, n = 3147), and May 8–18, 2012 (ICON audit, n = 4601 admitted to ICUs in the same 24 countries as the SOAP study). ARDS was defined retrospectively using the Berlin definitions. Values of tidal volume, PEEP, plateau pressure, and FiO2 corresponding to the most abnormal value of arterial PO2 were recorded prospectively every 24 h. In both studies, patients were followed for outcome until death, hospital discharge or for 60 days. Results: The frequency of ARDS requiring mechanical ventilation during the ICU stay was similar in SOAP and ICON (327[10.4%] vs. 494[10.7%], p = 0.793). The diagnosis of ARDS was established at a median of 3 (IQ: 1–7) days after admission in SOAP and 2 (1–6) days in ICON. Within 24 h of diagnosis, ARDS was mild in 244 (29.7%), moderate in 388 (47.3%), and severe in 189 (23.0%) patients. In patients with ARDS, tidal volumes were lower in the later (ICON) than in the earlier (SOAP) cohort. Plateau and driving pressures were also lower in ICON than in SOAP. ICU (134[41.1%] vs 179[36.9%]) and hospital (151[46.2%] vs 212[44.4%]) mortality rates in patients with ARDS were similar in SOAP and ICON. High plateau pressure (> 29 cmH2O) and driving pressure (> 14 cmH2O) on the first day of mechanical ventilation but not tidal volume (> 8 ml/kg predicted body weight [PBW]) were independently associated with a higher risk of in-hospital death. Conclusion: The frequency of and outcome from ARDS remained relatively stable between 2002 and 2012. Plateau pressure > 29 cmH2O and driving pressure > 14 cmH2O on the first day of mechanical ventilation but not tidal volume > 8 ml/kg PBW were independently associated with a higher risk of death. These data highlight the continued burden of ARDS and provide hypothesis-generating data for the design of future studies