The theory of receptor-ligand binding equilibria has long been well-established in biochemistry, and was primarily constructed to describe dilute aqueous solutions. Accordingly, few computational approaches have been developed for making quantitative predictions of binding probabilities in environments other than dilute isotropic solution. Existing techniques, ranging from simple automated docking procedures to sophisticated thermodynamics-based methods, have been developed with soluble proteins in mind. Biologically and pharmacologically relevant protein-ligand interactions often occur in complex environments, including lamellar phases like membranes and crowded, non-dilute solutions. Here we revisit the theoretical bases of ligand binding equilibria, avoiding overly specific assumptions that are nearly always made when describing receptor-ligand binding. Building on this formalism, we extend the asymptotically exact Alchemical Free Energy Perturbation technique to quantifying occupancies of sites on proteins in a complex bulk, including phase-separated, anisotropic, or non-dilute solutions, using a thermodynamically consistent and easily generalized approach that resolves several ambiguities of current frameworks. To incorporate the complex bulk without overcomplicating the overall thermodynamic cycle, we simplify the common approach for ligand restraints by using a single distance-from-bound-configuration (DBC) ligand restraint during AFEP decoupling from protein. DBC restraints should be generalizable to binding modes of most small molecules, even those with strong orientational dependence. We apply this approach to compute the likelihood that membrane cholesterol binds to known crystallographic sites on 3 GPCRs (beta2-adrenergic, 5HT-2B, and mu-opioid) at a range of concentrations. Non-ideality of cholesterol in a binary cholesterol:phosphatidylcholine (POPC) bilayer is characterized and consistently incorporated into the interpretation. We find that the three sites exhibit very different affinities for cholesterol: the site on the adrenergic receptor is predicted to be high affinity, with 50% occupancy for 1:109 CHOL:POPC mixtures. The site on the 5HT-2B and mu-opioid receptor are predicted to be lower affinity, with 50% occupancy for 1 ∶ 103 CHOL:POPC and 1 ∶ 102 CHOL:POPC respectively. These results could not have been predicted from the crystal structures alone.

Background Among asymptomatic patients with severe carotid artery stenosis but no recent stroke or transient cerebral ischaemia, either carotid artery stenting (CAS) or carotid endarterectomy (CEA) can restore patency and reduce long-term stroke risks. However, from recent national registry data, each option causes about 1% procedural risk of disabling stroke or death. Comparison of their long-term protective effects requires large-scale randomised evidence.Methods ACST-2 is an international multicentre randomised trial of CAS versus CEA among asymptomatic patients with severe stenosis thought to require intervention, interpreted with all other relevant trials. Patients were eligible if they had severe unilateral or bilateral carotid artery stenosis and both doctor and patient agreed that a carotid procedure should be undertaken, but they were substantially uncertain which one to choose. Patients were randomly allocated to CAS or CEA and followed up at 1 month and then annually, for a mean 5 years. Procedural events were those within 30 days of the intervention. Intention-to-treat analyses are provided. Analyses including procedural hazards use tabular methods. Analyses and meta-analyses of non-procedural strokes use Kaplan-Meier and log-rank methods. The trial is registered with the ISRCTN registry, ISRCTN21144362.

Background Awake intubation is the standard of care for management of the anticipated difficult airway. The performance of awake intubation may be perceived as complex and time-consuming, potentially leading clinicians to avoid this technique of airway management. This retrospective review of awake intubations at a large academic medical center was performed to determine the average time taken to perform awake intubation, its effects on hemodynamics, and the incidence and characteristics of complications and failure. Methods Anesthetic records from 2007 to 2014 were queried for the performance of an awake intubation. Of the 1,085 awake intubations included for analysis, 1,055 involved the use of a flexible bronchoscope. Each awake intubation case was propensity matched with two controls (1:2 ratio), with similar comorbidities and intubations performed after the induction of anesthesia (n = 2,170). The time from entry into the operating room until intubation was compared between groups. The anesthetic records of all patients undergoing awake intubation were also reviewed for failure and complications. Results The median time to intubation for patients intubated post induction was 16.0 min (interquartile range: 13 to 22) from entrance into the operating room. The median time to intubation for awake patients was 24.0 min (interquartile range: 19 to 31). The complication rate was 1.6% (17 of 1,085 cases). The most frequent complications observed were mucous plug, endotracheal tube cuff leak, and inadvertent extubation. The failure rate for attempted awake intubation was 1% (n = 10). Conclusions Awake intubations have a high rate of success and low rate of serious complications and failure. Awake intubations can be performed safely and rapidly.

Pentameric ligand-gated ion channels (pLGICs) mediate synaptic transmission and are sensitive to their lipid environment. The mechanism of phospholipid modulation of any pLGIC is not well understood. We demonstrate that the model pLGIC, ELIC (Erwinia ligand-gated ion channel), is positively modulated by the anionic phospholipid, phosphatidylglycerol, from the outer leaflet of the membrane. To explore the mechanism of phosphatidylglycerol modulation, we determine a structure of ELIC in an open-channel conformation. The structure shows a bound phospholipid in an outer leaflet site, and structural changes in the phospholipid binding site unique to the open-channel. In combination with streamlined alchemical free energy perturbation calculations and functional measurements in asymmetric liposomes, the data support a mechanism by which an anionic phospholipid stabilizes the activated, open-channel state of a pLGIC by specific, state-dependent binding to this site.