Correlated diffusion in lipid bilayers.

Lipid membranes are complex quasi-two-dimensional fluids, whose importance in biology and unique physical/materials properties have made them a major target for biophysical research. Recent single-molecule tracking experiments in membranes have caused some controversy, calling the venerable Saffman-Delbrück model into question and suggesting that, perhaps, current understanding of membrane hydrodynamics is imperfect. However, single-molecule tracking is not well suited to resolving the details of hydrodynamic flows; observations involving correlations between multiple molecules are superior for this purpose. Here dual-color molecular tracking with submillisecond time resolution and submicron spatial resolution is employed to reveal correlations in the Brownian motion of pairs of fluorescently labeled lipids in membranes. These correlations extend hundreds of nanometers in freely floating bilayers (black lipid membranes) but are severely suppressed in supported lipid bilayers. The measurements are consistent with hydrodynamic predictions based on an extended Saffman-Delbrück theory that explicitly accounts for the two-leaflet bilayer structure of lipid membranes.

Dynamic correlations in lipid bilayer membranes over finite time intervals.

Recent single-molecule measurements [Schoch et al., Proc. Natl. Acad. Sci. U. S. A. 118, e2113202118 (2021)] have observed dynamic lipid-lipid correlations in membranes with submicrometer spatial resolution and submillisecond temporal resolution. While short from an instrumentation standpoint, these length and time scales remain long compared to microscopic molecular motions. Theoretical expressions are derived to infer experimentally measurable correlations from the two-body diffusion matrix appropriate for membrane-bound bodies coupled by hydrodynamic interactions. The temporal (and associated spatial) averaging resulting from finite acquisition times has the effect of washing out correlations as compared to naive predictions (i.e., the bare elements of the diffusion matrix), which would be expected to hold for instantaneous measurements. The theoretical predictions are shown to be in excellent agreement with Brownian dynamics simulations of experimental measurements. Numerical results suggest that the experimental measurement of membrane protein diffusion, in complement to lipid diffusion measurements, might help to resolve the experimental ambiguities encountered for certain black lipid membranes.

Preoperative Assessment of Anxiety and Depression Using Computerized Adaptive Screening Tools: A Pilot Prospective Cohort Study.

BACKGROUND: Serious mental health conditions (eg, anxiety and depression) are common in surgical patients, yet likely underassessed due to the time-consuming and cumbersome traditional screening process. A recently developed computerized adaptive mental health assessment tool (computerized adaptive test-mental health [CAT-MH]) allows rapid, precise, and accurate assessment of numerous mental health disorders, including anxiety and depression, without the need for a trained interviewer. The goal of this investigation was to determine the feasibility of administering CAT-MH for anxiety and depression in the preoperative setting and to obtain preliminary evidence of the prevalence of anxiety and depression in preoperative patients. METHODS: In this prospective cohort study, 100 adult patients scheduled for elective surgery were enrolled and asked to complete the CAT-MH in the preoperative clinic. Urgent and emergency surgeries were excluded as were pregnant patients. Primary feasibility outcomes were completion rate and time to completion. Secondary outcomes were prevalence estimates of anxiety and depression. RESULTS: All 100 enrolled patients completed the study. All patients were able to complete the mental health assessment (mean time: 3.6 ± 1.8 minutes standard deviation). Sixteen patients (16%) screened positive for anxiety (severity: mild, n = 7 [7%]; moderate, n = 7 [7%]); severe, n = 2 [2%]); 12 of 16 (75%) did not have a previous diagnosis of anxiety disorder. Twenty-eight (28%) patients screened positive for depression (severity: mild, n = 26 [26%]; moderate and severe, n = 1 each [1%]); 23 of 28 (82%) had no previous diagnosis of depressive disorder. Nineteen patients (19%) met the screening criteria for major depressive disorder; 14 of 19 (74%) of which had no previous diagnosis of major depressive disorder. CONCLUSIONS: The results of this pilot study support the feasibility of using CAT-MH in a preoperative evaluation and indicate that there is a substantial prevalence of undiagnosed anxiety and depression in surgical patients.

Predictors at Admission of Mechanical Ventilation and Death in an Observational Cohort of Adults Hospitalized With Coronavirus Disease 2019.

BACKGROUND: Coronavirus disease (COVID-19) can cause severe illness and death. Predictors of poor outcome collected on hospital admission may inform clinical and public health decisions. METHODS: We conducted a retrospective observational cohort investigation of 297 adults admitted to 8 academic and community hospitals in Georgia, United States, during March 2020. Using standardized medical record abstraction, we collected data on predictors including admission demographics, underlying medical conditions, outpatient antihypertensive medications, recorded symptoms, vital signs, radiographic findings, and laboratory values. We used random forest models to calculate adjusted odds ratios (aORs) and 95% confidence intervals (CIs) for predictors of invasive mechanical ventilation (IMV) and death. RESULTS: Compared with age <45 years, ages 65-74 years and ≥75 years were predictors of IMV (aORs, 3.12 [95% CI, 1.47-6.60] and 2.79 [95% CI, 1.23-6.33], respectively) and the strongest predictors for death (aORs, 12.92 [95% CI, 3.26-51.25] and 18.06 [95% CI, 4.43-73.63], respectively). Comorbidities associated with death (aORs, 2.4-3.8; P < .05) included end-stage renal disease, coronary artery disease, and neurologic disorders, but not pulmonary disease, immunocompromise, or hypertension. Prehospital use vs nonuse of angiotensin receptor blockers (aOR, 2.02 [95% CI, 1.03-3.96]) and dihydropyridine calcium channel blockers (aOR, 1.91 [95% CI, 1.03-3.55]) were associated with death. CONCLUSIONS: After adjustment for patient and clinical characteristics, older age was the strongest predictor of death, exceeding comorbidities, abnormal vital signs, and laboratory test abnormalities. That coronary artery disease, but not chronic lung disease, was associated with death among hospitalized patients warrants further investigation, as do associations between certain antihypertensive medications and death.

Characteristics and Risk Factors of Hospitalized and Nonhospitalized COVID-19 Patients, Atlanta, Georgia, USA, March-April 2020.

We compared the characteristics of hospitalized and nonhospitalized patients who had coronavirus disease in Atlanta, Georgia, USA. We found that risk for hospitalization increased with a patient's age and number of concurrent conditions. We also found a potential association between hospitalization and high hemoglobin A1c levels in persons with diabetes.

Characteristics Associated with Hospitalization Among Patients with COVID-19 - Metropolitan Atlanta, Georgia, March-April 2020.

The first reported U.S. case of coronavirus disease 2019 (COVID-19) was detected in January 2020 (1). As of June 15, 2020, approximately 2 million cases and 115,000 COVID-19-associated deaths have been reported in the United States.* Reports of U.S. patients hospitalized with SARS-CoV-2 infection (the virus that causes COVID-19) describe high proportions of older, male, and black persons (2-4). Similarly, when comparing hospitalized patients with catchment area populations or nonhospitalized COVID-19 patients, high proportions have underlying conditions, including diabetes mellitus, hypertension, obesity, cardiovascular disease, chronic kidney disease, or chronic respiratory disease (3,4). For this report, data were abstracted from the medical records of 220 hospitalized and 311 nonhospitalized patients aged ≥18 years with laboratory-confirmed COVID-19 from six acute care hospitals and associated outpatient clinics in metropolitan Atlanta, Georgia. Multivariable analyses were performed to identify patient characteristics associated with hospitalization. The following characteristics were independently associated with hospitalization: age ≥65 years (adjusted odds ratio [aOR] = 3.4), black race (aOR = 3.2), having diabetes mellitus (aOR = 3.1), lack of insurance (aOR = 2.8), male sex (aOR = 2.4), smoking (aOR = 2.3), and obesity (aOR = 1.9). Infection with SARS-CoV-2 can lead to severe outcomes, including death, and measures to protect persons from infection, such as staying at home, social distancing (5), and awareness and management of underlying conditions should be emphasized for those at highest risk for hospitalization with COVID-19. Measures that prevent the spread of infection to others, such as wearing cloth face coverings (6), should be used whenever possible to protect groups at high risk. Potential barriers to the ability to adhere to these measures need to be addressed.

Characteristics and Clinical Outcomes of Adult Patients Hospitalized with COVID-19 - Georgia, March 2020.

SARS-CoV-2, the novel coronavirus that causes coronavirus disease 2019 (COVID-19), was first detected in the United States during January 2020 (1). Since then, >980,000 cases have been reported in the United States, including >55,000 associated deaths as of April 28, 2020 (2). Detailed data on demographic characteristics, underlying medical conditions, and clinical outcomes for persons hospitalized with COVID-19 are needed to inform prevention strategies and community-specific intervention messages. For this report, CDC, the Georgia Department of Public Health, and eight Georgia hospitals (seven in metropolitan Atlanta and one in southern Georgia) summarized medical record-abstracted data for hospitalized adult patients with laboratory-confirmed* COVID-19 who were admitted during March 2020. Among 305 hospitalized patients with COVID-19, 61.6% were aged <65 years, 50.5% were female, and 83.2% with known race/ethnicity were non-Hispanic black (black). Over a quarter of patients (26.2%) did not have conditions thought to put them at higher risk for severe disease, including being aged ≥65 years. The proportion of hospitalized patients who were black was higher than expected based on overall hospital admissions. In an adjusted time-to-event analysis, black patients were not more likely than were nonblack patients to receive invasive mechanical ventilation† (IMV) or to die during hospitalization (hazard ratio [HR] = 0.63; 95% confidence interval [CI] = 0.35-1.13). Given the overrepresentation of black patients within this hospitalized cohort, it is important for public health officials to ensure that prevention activities prioritize communities and racial/ethnic groups most affected by COVID-19. Clinicians and public officials should be aware that all adults, regardless of underlying conditions or age, are at risk for serious illness from COVID-19.

Complete permanent mandibular dentition of early Homo from the upper Burgi Member of the Koobi Fora Formation, Ileret, Kenya.

The KNM-ER 64060 dentition derives from a horizon that most likely dates to between 2.02 and 2.03 Ma. A proximate series of postcranial bones (designated KNM-ER 64061) derives from the same siltstone unit and may be associated with the dentition, but their separation on the surface of the site leaves some room for doubt. KNM-ER 64060 is one of fewer than ten hominin specimens from the Early Pleistocene of East Africa that comprises a full or nearly complete mandibular dentition. Its taxonomic attribution is potentially significant, especially if the postcranial elements are related. At least three, and probably four hominin species, including Paranthropus boisei and Homo erectus (= H. ergaster), are known at about this time in East Africa. Other penecontemporaneous fossils have been referred to a single, highly variable species, H. habilis, or two taxa, namely H. habilis and H. rudolfensis. Although the weight of evidence supports the attribution of these specimens to two species, there is notable lack of agreement over the assignation of individual fossils. We take a conservative approach and group all such specimens under the designation "early Homo sp." for comparative purposes. KNM-ER 64060 is clearly attributable to Homo rather than Paranthropus. The preponderance of the evidence suggests that the affinities of KNM-ER 64060 are with fossils assigned to the early Homo sp. category rather than with H. erectus. This is indicated by the overall sizes of the KNM-ER 64060 canine, premolar and molar crowns, the size relationships of the P3 to P4, the relative narrowness of its premolar crowns, the cusp proportions of the M1 and especially those of the M2 and M3, and seemingly the possession of a two-rooted P4. Some of these comparisons suggest further that among the fossils comprising the early Homo sp. sample, the KNM-ER 64060 dentition exhibits greater overall similarity to specimens such as OH 7 and OH 16 that represent Homo habilis sensu stricto.

Motion of objects embedded in lipid bilayer membranes: Advection and effective viscosity.

An interfacial regularized Stokeslet scheme is presented to predict the motion of solid bodies (e.g., proteins or gel-phase domains) embedded within flowing lipid bilayer membranes. The approach provides a numerical route to calculate velocities and angular velocities in complex flow fields that are not amenable to simple Faxén-like approximations. Additionally, when applied to shearing motions, the calculations yield predictions for the effective surface viscosity of dilute rigid-body-laden membranes. In the case of cylindrical proteins, effective viscosity calculations are compared to two prior analytical predictions from the literature. Effective viscosity predictions for a dilute suspension of rod-shaped objects in the membrane are also presented.

Integrated rate laws for processive and distributive enzymatic turnover.

Recently derived steady-state differential rate laws for the catalytic turnover of molecules containing two substrate sites are reformulated as integrated rate laws. The analysis applies to a broad class of Markovian dynamic models, motivated by the varied and often complex mechanisms associated with DNA modifying enzymes. Analysis of experimental data for the methylation kinetics of DNA by Dam (DNA adenine methyltransferase) is drastically improved through the use of integrated rate laws. Data that are too noisy for fitting to differential predictions are reliably interpreted through the integrated rate laws.

Lipid diffusion in the distal and proximal leaflets of supported lipid bilayer membranes studied by single particle tracking.

Supported lipid bilayers (SLBs) have been studied extensively as simple but powerful models for cellular membranes. Yet, potential differences in the dynamics of the two leaflets of a SLB remain poorly understood. Here, using single particle tracking, we obtain a detailed picture of bilayer dynamics. We observe two clearly separate diffusing populations, fast and slow, that we associate with motion in the distal and proximal leaflets of the SLB, respectively, based on fluorescence quenching experiments. We estimate diffusion coefficients using standard techniques as well as a new method based on the blur of images due to motion. Fitting the observed diffusion coefficients to a two-leaflet membrane hydrodynamic model allows for the simultaneous determination of the intermonolayer friction coefficient and the substrate-membrane friction coefficient, without any prior assumptions on the strengths of the relevant interactions. Remarkably, our calculations suggest that the viscosity of the interfacial water confined between the membrane and the substrate is elevated by ∼104 as compared to bulk water. Using hidden Markov model analysis, we then obtain insight into the transbilayer movement of lipids. We find that lipid flip-flop dynamics are very fast, with half times in the range of seconds. Importantly, we find little evidence for membrane defect mediated lipid flip-flop for SLBs at temperatures well above the solid-to-liquid transition, though defects seem to be involved when the SLBs are cooled down. Our work thus shows that the combination of single particle tracking and advanced hydrodynamic modeling provides a powerful means to obtain insight into membrane dynamics.

High-sensitivity Cardiac Troponin Elevation after Electroconvulsive Therapy: A Prospective, Observational Cohort Study.

BACKGROUND: While electroconvulsive therapy is widely regarded as a lifesaving and safe procedure, evidence regarding its effects on myocardial cell injury is sparse. The objective of this investigation was to determine the incidence and magnitude of new cardiac troponin elevation after electroconvulsive therapy using a novel high-sensitivity cardiac troponin I assay. METHODS: This was a prospective cohort study in adult patients undergoing electroconvulsive therapy in a single academic center (up to three electroconvulsive therapy treatments per patient). The primary outcome was new high-sensitivity cardiac troponin I elevation after electroconvulsive therapy, defined as an increase of high-sensitivity cardiac troponin I greater than 100% after electroconvulsive therapy compared to baseline with at least one value above the limit of quantification (10 ng/l). Twelve-lead electrocardiogram and high-sensitivity cardiac troponin I values were obtained before and 15 to 30 min after electroconvulsive therapy; in a subset of patients, an additional 2-h high-sensitivity cardiac troponin I value was obtained. RESULTS: The final study population was 100 patients and a total of 245 electroconvulsive therapy treatment sessions. Eight patients (8 of 100; 8%) experienced new high-sensitivity cardiac troponin I elevation after electroconvulsive therapy with a cumulative incidence of 3.7% (9 of 245 treatments; one patient had two high-sensitivity cardiac troponin I elevations), two of whom had a non-ST-elevation myocardial infarction (incidence 2 of 245; 0.8%). Median high-sensitivity cardiac troponin I concentrations did not increase significantly after electroconvulsive therapy. Tachycardia and/or elevated systolic blood pressure developed after approximately two thirds of electroconvulsive therapy treatments. CONCLUSIONS: Electroconvulsive therapy appears safe from a cardiac standpoint in a large majority of patients. A small subset of patients with preexisting cardiovascular risk factors, however, may develop new cardiac troponin elevation after electroconvulsive therapy, the clinical relevance of which is unclear in the absence of signs of myocardial ischemia.

The evolution of drug design at Merck Research Laboratories.

On October 5, 1981, Fortune magazine published a cover article entitled the "Next Industrial Revolution: Designing Drugs by Computer at Merck". With a 40+ year investment, we have been in the drug design business longer than most. During its history, the Merck drug design group has had several names, but it has always been in the "design" business, with the ultimate goal to provide an actionable hypothesis that could be tested experimentally. Often the result was a small molecule but it could just as easily be a peptide, biologic, predictive model, reaction, process, etc. To this end, the concept of design is now front and center in all aspects of discovery, safety assessment and early clinical development. At present, the Merck design group includes computational chemistry, protein structure determination, and cheminformatics. By bringing these groups together under one umbrella, we were able to align activities and capabilities across multiple research sites and departments. This alignment from 2010 to 2016 resulted in an 80% expansion in the size of the department, reflecting the increase in impact due to a significant emphasis across the organization to "design first" along the entire drug discovery path from lead identification (LID) to first in human (FIH) dosing. One of the major advantages of this alignment has been the ability to access all of the data and create an adaptive approach to the overall LID to FIH pathway for any modality, significantly increasing the quality of candidates and their probability of success. In this perspective, we will discuss how we crafted a new strategy, defined the appropriate phenotype for group members, developed the right skillsets, and identified metrics for success in order to drive continuous improvement. We will not focus on the tactical implementation, only giving specific examples as appropriate.

High-Sensitivity Cardiac Troponin T Improves the Diagnosis of Perioperative MI.

BACKGROUND: The diagnosis of myocardial infarction (MI) after noncardiac surgery has traditionally relied on using relatively insensitive contemporary cardiac troponin (cTn) assays. We hypothesized that using a recently introduced novel high-sensitivity cTnT (hscTnT) assay would increase the detection rate of perioperative MI. METHODS: In this ancillary study of the Vitamins in Nitrous Oxide trial, readjudicated incidence rates of myocardial injury (new isolated cTn elevation) and MI were compared when diagnosed by contemporary cTnI versus hscTnT. We probed various relative (eg, >50%) or absolute (eg, +5 ng/L) hscTnT change metrics. Inclusion criteria for this ancillary study were the presence of a baseline and at least 1 postoperative hscTnT value. RESULTS: Among 605 patients, 70 patients (12%) had electrocardiogram changes consistent with myocardial ischemia; 82 patients (14%) had myocardial injury diagnosed by contemporary cTnI, 31 (5.1%) of which had an adjudicated MI. After readjudication, 67 patients (11%) were diagnosed with MI when using hscTnT, a 2-fold increase. Incidence rates of postoperative myocardial injury ranged from 12% (n = 73) to 65% (n = 393) depending on the hscTnT metric used. Incidence rates of MI using various hscTnT change metrics and the presence of ischemic electrocardiogram changes, but without event adjudication, ranged from 3.6% (n = 22) to 12% (n = 74), a >3-fold difference. New postoperative hscTnT elevation, either by absolute or relative hscTnT change metric, was associated with an up to 5-fold increase in 6-month mortality. CONCLUSIONS: The use of hscTnT compared to contemporary cTnI increases the detection rate of perioperative MI by a factor of 2. Using different absolute or relative hscTnT change metrics may lead to under- or overdiagnosis of perioperative MI.

Improving Prediction of Postoperative Myocardial Infarction With High-Sensitivity Cardiac Troponin T and NT-proBNP.

BACKGROUND: This study sought to determine whether preoperatively measured high-sensitivity cardiac troponin T (hs-cTnT) and N-terminal pro-brain natriuretic peptide (NT-proBNP) improve cardiac risk prediction in patients undergoing major noncardiac surgery compared with the standard risk indices. METHODS: In this ancillary study to the Vitamins in Nitrous Oxide trial, patients were included who had preoperative hs-cTnT and NT-proBNP measured (n = 572). Study outcome was the incidence of postoperative myocardial infarction (MI) within the first 3 postoperative days. hs-cTnT was considered elevated if >14 ng/L and NT-proBNP if >300 ng/L. Additional cutoff values were investigated on the basis of receiver operating characteristic statistics. Biomarker risk prediction was compared with Lee's Revised Cardiac Risk Index (RCRI) with the use of standard methods and net reclassification index. RESULTS: The addition of hs-cTnT (>14 ng/L) and NT-proBNP (>300 ng/L) to RCRI significantly improved the prediction of postoperative MI (event rate 30/572 [5.2%], Area under the receiver operating characteristic curve increased from 0.590 to 0.716 with a 0.66 net reclassification index [95% confidence interval 0.32-0.99], P < .001). The use of 108 ng/L as a cutoff for NT-proBNP improved sensitivity compared with 300 ng/L (0.87 vs 0.53). Sensitivity, specificity, positive, and negative predictive value for hs-cTnT were 0.70, 0.60, 0.09, and 0.97 and for NT-proBNP were 0.53, 0.68, 0.08, and 0.96. CONCLUSIONS: The addition of cardiac biomarkers hs-cTnT and NT-proBNP to RCRI improves the prediction of adverse cardiac events in the immediate postoperative period after major noncardiac surgery. The high negative predictive value of preoperative hs-cTnT and NT-proBNP suggest usefulness as a "rule-out" test to confirm low risk of postoperative MI.

On the generality of Michaelian kinetics.

The reversible Michaelis-Menten equation is shown to follow from a very broad class of steady-state kinetic models involving enzymes that adopt a unique free (i.e., not complexed to substrate/product) state in solution. In the case of enzymes with multiple free states/conformations (e.g., fluctuating, hysteretic, or co-operative monomeric enzymes), Michaelian behavior is still assured if the relative steady-state populations of free enzyme states are independent of substrate and product concentration. Prior models for Michaelian behavior in multiple conformer enzymes are shown to be special cases of this single condition.

Theory of single molecule emission spectroscopy.

A general theory and calculation framework for the prediction of frequency-resolved single molecule photon counting statistics is presented. Expressions for the generating function of photon counts are derived, both for the case of naive "detection" based solely on photon emission from the molecule and also for experimentally realizable detection of emitted photons, and are used to explicitly calculate low-order photon-counting moments. The two cases of naive detection versus physical detection are compared to one another and it is demonstrated that the physical detection scheme resolves certain inconsistencies predicted via the naive detection approach. Applications to two different models for molecular dynamics are considered: a simple two-level system and a two-level absorber subject to spectral diffusion.

Extracting enzyme processivity from kinetic assays.

A steady-state analysis for the catalytic turnover of molecules containing two substrate sites is presented. A broad class of Markovian dynamic models, motivated by the action of DNA modifying enzymes and the rich variety of translocation mechanisms associated with these systems (e.g., sliding, hopping, intersegmental transfer, etc.), is considered. The modeling suggests an elementary and general method of data analysis, which enables the extraction of the enzyme's processivity directly and unambiguously from experimental data. This analysis is not limited to the initial velocity regime. The predictions are validated both against detailed numerical models and by revisiting published experimental data for EcoRI endonuclease acting on DNA.

Strong influence of periodic boundary conditions on lateral diffusion in lipid bilayer membranes.

The Saffman-Delbrück hydrodynamic model for lipid-bilayer membranes is modified to account for the periodic boundary conditions commonly imposed in molecular simulations. Predicted lateral diffusion coefficients for membrane-embedded solid bodies are sensitive to box shape and converge slowly to the limit of infinite box size, raising serious doubts for the prospects of using detailed simulations to accurately predict membrane-protein diffusivities and related transport properties. Estimates for the relative error associated with periodic boundary artifacts are 50% and higher for fully atomistic models in currently feasible simulation boxes. MARTINI simulations of LacY membrane protein diffusion and LacY dimer diffusion in DPPC membranes and lipid diffusion in pure DPPC bilayers support the underlying hydrodynamic model.

Determination of biomembrane bending moduli in fully atomistic simulations.

The bilayer bending modulus (Kc) is one of the most important physical constants characterizing lipid membranes, but precisely measuring it is a challenge, both experimentally and computationally. Experimental measurements on chemically identical bilayers often differ depending upon the techniques employed, and robust simulation results have previously been limited to coarse-grained models (at varying levels of resolution). This Communication demonstrates the extraction of Kc from fully atomistic molecular dynamics simulations for three different single-component lipid bilayers (DPPC, DOPC, and DOPE). The results agree quantitatively with experiments that measure thermal shape fluctuations in giant unilamellar vesicles. Lipid tilt, twist, and compression moduli are also reported.