We report on a prototype protocol for the automatic and fast construction of congruous sets of QM/MM models of rhodopsin-like photoreceptors and of their mutants. In the present implementation the information required for the construction of each model is essentially a crystallographic structure or a comparative model complemented with information on the protonation state of ionizable side chains and distributions of external counterions. Starting with such information, a model formed by a fixed environment system, a flexible cavity system, and a chromophore system is automatically generated. The results of the predicted vertical excitation energy for 27 different rhodopsins including vertebrate, invertebrate, and microbial pigments indicate that such basic models could be employed for predicting trends in spectral changes and/or correlate the spectral changes with structural variations in large sets of proteins.

The engineering of microbial rhodopsins with enhanced fluorescence is of great importance in the expanding field of optogenetics. Here we report the discovery of two mutants (W76S/Y179F and L83Q) of a sensory rhodopsin from the cyanobacterium Anabaena PCC7120 with opposite fluorescence behavior. In fact, while W76S/Y179F displays, with respect to the wild-type protein, a nearly tenfold increase in red-light emission, the second is not emissive. Thus, the W76S/ Y179F, L83Q pair offers an unprecedented opportunity for the investigation of fluorescence enhancement in microbial rhodopsins, which is pursued by combining transient absorption spectroscopy and multi-configurational quantum chemistry. The results of such an investigation point to an isomerization-blocking electronic effect as the direct cause of instantaneous (subpicosecond) fluorescence enhancement.

Microbial rhodopsins are photoreceptive membrane proteins that transport various ions using light energy. While they are widely used in optogenetics to optically control neuronal activity, rhodopsins that function with longer-wavelength light are highly demanded because of their low phototoxicity and high tissue penetration. Here, we achieve a 40-nm red-shift in the absorption wavelength of a sodium-pump rhodopsin (KR2) by altering dipole moment of residues around the retinal chromophore (KR2 P219T/S254A) without impairing its ion-transport activity. Structural differences in the chromophore of the red-shifted protein from that of the wildtype are observed by Fourier transform infrared spectroscopy. QM/MM models generated with an automated protocol show that the changes in the electrostatic interaction between protein and chromophore induced by the amino-acid replacements, lowered the energy gap between the ground and the first electronically excited state. Based on these insights, a natural sodium pump with red-shifted absorption is identified from Jannaschia seosinensis .

The presence of DAD appears to define a specific subphenotype in patients with ARDS. Targeting patients with DAD within the population of patients with the clinical diagnosis of ARDS might be appropriate to find effective therapies for this condition.

A positive cuff-leak test (absence of leak) should alert the clinician of a high risk of upper airway obstruction.

A novel atomistic methodology to perform free energy geometry optimization of a retinal chromophore covalently bound to any rhodopsin-like protein cavity is presented and benchmarked by computing the absorption maxima wavelengths (λ) of distant rhodopsin systems. The optimization is achieved by computing the Nagaoka's Free Energy Gradient (FEG) within an Average Solvent Electrostatic Configuration (ASEC) atomistic representation of the thermodynamic equilibrium and minimizing such quantity via an iterative procedure based on sequential classical MD and constrained QM/MM geometry optimization steps. The performance of such an ASEC-FEG protocol is assessed at the CASPT2//CASSCF/Amber level by reproducing the λ values observed for 12 mutants of redesigned human cellular retinol binding protein II (hCRBPII) systems; a set of 10 distant wild-type rhodopsins from vertebrates, invertebrates, eubacteria, and archaea organisms; and finally a set of 10 rhodopsin mutants from an eubacterial rhodopsin. The results clearly show that the proposed protocol, which can be easily extended to any protein incorporating a covalently bound ligand, yields correct λ trends with limited absolute errors.

Purpose: Variations in clinical characteristics and management and in the mortality of mechanically ventilated patients have not been sufficiently evaluated. We hypothesized that mortality shows a variability associated with country after adjustment for clinical characteristics and management. Methods: Analysis of four studies carried out at 6-year intervals over an 18-year period. The studies included 26,024 patients (5183 in 1998, 4968 in 2004, 8108 in 2010, and 7765 in 2016) admitted to 1253 units from 38 countries. The primary outcome was 28-day mortality. We performed analyses using multilevel logistic modeling with mixed-random effects, including country as a random variable. To evaluate the effect of management strategies on mortality, a mediation analysis was performed. Results: Adjusted 28-day mortality decreased significantly over time (first study as reference): 2004: odds ratio 0.82 (95% confidence interval [CI] 0.72-0.93); 2010: 0.63 (95% CI 0.53-0.75); 2016: 0.49 (95% CI 0.39-0.61). A protective ventilatory strategy and the use of continuous sedation mediated a moderate fraction of the effect of time on mortality in patients with moderate hypoxemia and without hypoxemia, respectively. Logistic multilevel modeling showed a significant effect of country on mortality:

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