The dynamic process of ß(2)-adrenergic receptor activation.

G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the ß(2)-adrenergic receptor (ß(2)AR), a prototypical GPCR. We labeled ß(2)AR with (13)CH(3)ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for ß(2)AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for ß(2)AR's ability to engage multiple signaling and regulatory proteins.

Association between peripheral perfusion index and postoperative acute kidney injury in major noncardiac surgery patients receiving continuous vasopressors: a post hoc exploratory analysis of the VEGA-1 trial.

BACKGROUND: The peripheral perfusion index is the ratio of pulsatile to nonpulsatile static blood flow obtained by photoplethysmography and reflects peripheral tissue perfusion. We investigated the association between intraoperative perfusion index and postoperative acute kidney injury in patients undergoing major noncardiac surgery and receiving continuous vasopressor infusions. METHODS: In this exploratory post hoc analysis of a pragmatic, cluster-randomised, multicentre trial, we obtained areas and cumulative times under various thresholds of perfusion index and investigated their association with acute kidney injury in multivariable logistic regression analyses. In secondary analyses, we investigated the association of time-weighted average perfusion index with acute kidney injury. The 30-day mortality was a secondary outcome. RESULTS: Of 2534 cases included, 8.9% developed postoperative acute kidney injury. Areas and cumulative times under a perfusion index of 3% and 2% were associated with an increased risk of acute kidney injury; the strongest association was observed for area under a perfusion index of 1% (adjusted odds ratio [aOR] 1.32, 95% confidence interval [CI] 1.00-1.74, P=0.050, per 100%∗min increase). Additionally, time-weighted average perfusion index was associated with acute kidney injury (aOR 0.82, 95% CI 0.74-0.91, P<0.001) and 30-day mortality (aOR 0.68, 95% CI 0.49-0.95, P=0.024). CONCLUSIONS: Larger areas and longer cumulative times under thresholds of perfusion index and lower time-weighted average perfusion index were associated with postoperative acute kidney injury in patients undergoing major noncardiac surgery and receiving continuous vasopressor infusions. CLINICAL TRIAL REGISTRATION: NCT04789330.

Intraoperative Use of Albumin in Major Noncardiac Surgery: Incidence, Variability, and Association With Outcomes.

BACKGROUND: The impact of albumin use during major surgery is unknown, and a dearth of evidence governing its use in major noncardiac surgery has long precluded its standardization in clinical guidelines. OBJECTIVE: In this study, we investigate institutional variation in albumin use among medical centers in the United States during major noncardiac surgery and explore the association of intraoperative albumin administration with important postoperative outcomes. METHODS: The study is an observational retrospective cohort analysis performed among 54 U.S. hospitals in the Multicenter Perioperative Outcomes Group and includes adult patients who underwent major noncardiac surgery under general anesthesia between January 2014 and June 2020. The primary endpoint was the incidence of albumin administration. Secondary endpoints are acute kidney injury (AKI), net-positive fluid balance, pulmonary complications, and 30-day mortality. Albumin-exposed and albumin-unexposed cases were compared within a propensity score-matched cohort to evaluate associations of albumin use with outcomes. RESULTS: Among 614,215 major surgeries, predominantly iso-oncotic albumin was administered in 15.3% of cases and featured significant inter-institutional variability in use patterns. Cases receiving intraoperative albumin involved patients of higher American Society of Anesthesiologists physical status and featured larger infused crystalloid volumes, greater blood loss, and vasopressor use. Overall, albumin was most often administered at high-volume surgery centers with academic affiliation, and within a propensity score-matched cohort (n=153,218), the use of albumin was associated with AKI (aOR 1.24, 95% CI 1.20-1.28, P <0.001), severe AKI (aOR 1.45, 95% CI 1.34-1.56, P <0.001), net-positive fluid balance (aOR 1.18, 95% CI 1.16-1.20, P <0.001), pulmonary complications (aOR 1.56, 95% CI 1.30-1.86, P <0.001), and 30-day all-cause mortality (aOR 1.37, 95% CI 1.26-1.49, P <0.001). CONCLUSIONS: Intravenous albumin is commonly administered among noncardiac surgeries with significant inter-institutional variability in use in the United States. Albumin administration was associated with an increased risk of postoperative complications.

Lactate concentration at the end of liver transplant: Early predictor of graft function or just one piece of the puzzle?

BACKGROUND: The post-operative course after Liver Transplantation (LT) can be complicated by early allograft dysfunction (EAD), primary nonfunction (PNF) and death. A lactate concentration at the end of transplant of ≥5 mmol/L was recently proposed as a predictive marker of PNF, EAD, and mortality; this study aimed to validate these previous reports in a large single center cohort. METHODS: This retrospective cohort study included adult liver transplant recipients who received grafts from deceased donors at our center between June 2012 and May 2021. Receiver operating characteristic (ROC) curves for the lactate concentration at the end of transplantation were computed to determine the AUC for PNF, EAD and mortality at 90 days. RESULTS: In our cohort of 1137 cases, the AUCs for lactate to predict EAD, PNF and mortality were respectively .56 (95% confidence interval [CI]: .53-.60), .69 (95% CI: .52-.85), and .74 (95% CI: .63-.84). CONCLUSION: The clinical value of lactate concentration at the end of transplantation to predict PNF, EAD and mortality at 90 days was, at best, modest, as shown by the relatively low AUCs. Our findings cannot validate previous reports that the lactate level alone is a good predictor of poor outcomes after liver transplantation.

Norepinephrine versus phenylephrine for treating hypotension during general anaesthesia in adult patients undergoing major noncardiac surgery: a multicentre, open-label, cluster-randomised, crossover, feasibility, and pilot trial.

BACKGROUND: Intraoperative hypotension is associated with postoperative complications. The use of vasopressors is often required to correct hypotension but the best vasopressor is unknown. METHODS: A multicentre, cluster-randomised, crossover, feasibility and pilot trial was conducted across five hospitals in California. Phenylephrine (PE) vs norepinephrine (NE) infusion as the first-line vasopressor in patients under general anaesthesia alternated monthly at each hospital for 6 months. The primary endpoint was first-line vasopressor administration compliance of 80% or higher. Secondary endpoints were acute kidney injury (AKI), 30-day mortality, myocardial injury after noncardiac surgery (MINS), hospital length of stay, and rehospitalisation within 30 days. RESULTS: A total of 3626 patients were enrolled over 6 months; 1809 patients were randomised in the NE group, 1817 in the PE group. Overall, 88.2% received the assigned first-line vasopressor. No drug infiltrations requiring treatment were reported in either group. Patients were median 63 yr old, 50% female, and 58% white. Randomisation in the NE group vs PE group did not reduce readmission within 30 days (adjusted odds ratio=0.92; 95% confidence interval, 0.6-1.39), 30-day mortality (1.01; 0.48-2.09), AKI (1.1; 0.92-1.31), or MINS (1.63; 0.84-3.16). CONCLUSIONS: A large and diverse population undergoing major surgery under general anaesthesia was successfully enrolled and randomised to receive NE or PE infusion. This pilot and feasibility trial was not powered for adverse postoperative outcomes and a follow-up multicentre effectiveness trial is planned. CLINICAL TRIAL REGISTRATION: NCT04789330 (ClinicalTrials.gov).

Optimizing the Design and Analysis of Future AKI Trials.

AKI is a complex clinical syndrome associated with an increased risk of morbidity and mortality, particularly in critically ill and perioperative patient populations. Most AKI clinical trials have been inconclusive, failing to detect clinically important treatment effects at predetermined statistical thresholds. Heterogeneity in the pathobiology, etiology, presentation, and clinical course of AKI remains a key challenge in successfully testing new approaches for AKI prevention and treatment. This article, derived from the "AKI" session of the "Kidney Disease Clinical Trialists" virtual workshop held in October 2021, reviews barriers to and strategies for improving the design and implementation of clinical trials in patients with, or at risk of, developing AKI. The novel approaches to trial design included in this review span adaptive trial designs that increase the knowledge gained from each trial participant; pragmatic trial designs that allow for the efficient enrollment of sufficiently large numbers of patients to detect small, but clinically significant, treatment effects; and platform trial designs that use one trial infrastructure to answer multiple clinical questions simultaneously. This review also covers novel approaches to clinical trial analysis, such as Bayesian analysis and assessing heterogeneity in the response to therapies among trial participants. We also propose a road map and actionable recommendations to facilitate the adoption of the reviewed approaches. We hope that the resulting road map will help guide future clinical trial planning, maximize learning from AKI trials, and reduce the risk of missing important signals of benefit (or harm) from trial interventions.

Prepectoral Breast Reconstruction Reduces Opioid Consumption and Pain After Mastectomy: A Head-to-Head Comparison With Submuscular Reconstruction.

BACKGROUND: Acute pain after mastectomy is increased with concurrent breast reconstruction. One postulated advantage of prepectoral breast reconstruction is less postoperative pain; however, no comparisons to partial submuscular reconstruction have been made to date. Here, we examined the postoperative pain experienced between patients with prepectoral and subpectoral breast reconstruction after mastectomy. METHODS: We performed a retrospective chart review of all patients undergoing immediate breast reconstruction with tissue expanders from 2012 to 2019 by a single plastic surgeon. Patient demographics, surgical details, and anesthetic techniques were evaluated, and our primary outcome compared postoperative opioid usage between prepectoral and subpectoral reconstructions. Our secondary outcome compared pain scores between techniques. RESULTS: A total of 211 subpectoral and 117 prepectoral reconstruction patients were included for analysis. Patients with subpectoral reconstructions had higher postoperative opioid usage (80.0 vs 45.0 oral morphine equivalents, P < 0.001). Subpectoral patients also recorded higher maximum pain scores compared with prepectoral reconstructions while admitted (7 of 10 vs 5 of 10, P < 0.004). Multivariable linear regression suggests that mastectomy type and subpectoral reconstruction were significant contributors to postoperative opioid use (P < 0.05). CONCLUSIONS: Prepectoral breast reconstruction was associated with less postoperative opioid consumption and lower postoperative pain scores as compared with subpectoral reconstruction, when controlling for other surgical and anesthesia factors. Future randomized controlled trials are warranted to study how postoperative pain and chronic pain are influenced by the location of prosthesis placement in implant-based postmastectomy breast reconstruction.

Suppression of pupillary unrest by general anesthesia and propofol sedation.

The pupil undergoes irregular oscillations when exposed to light. These oscillations, known as pupillary unrest in ambient light, originate from oscillatory activity within the Edinger-Westphal nucleus in the midbrain. The midbrain and upper pons also contain nuclei known to be very sensitive to the effects of anesthetics that play a central role in maintaining wakefulness. We hypothesized that anesthetics may display similar effects on wakefulness and pupillary unrest. Repeat measurements of pupillary unrest using infrared pupillometry were performed in 16 patients undergoing general anesthesia and 8 patients undergoing propofol sedation. Pupil scans were analyzed using fast Fourier transformation to quantify the effects of the anesthetics on pupillary unrest. During general anesthesia and deep sedation, observed pupillary unrest values below 0.1 (AU) indicate complete suppression of pupillary oscillations. Pupillary unrest decreased more during general anesthesia [to 24% of baseline (95% CI 17-30%)] than pupil size [51% of baseline (95% CI 45-57%)]. Sedation with propofol was associated with a reduction in pupillary unrest that was correlated to the depth of sedation as assessed by the Richmond Agitation-Sedation Scale and the processed electroencephalogram. Pupillary unrest is caused by oscillatory activity within the midbrain that is affected by the state of wakefulness or by hypnotics directly. Increased sedation and general anesthesia reduce and then abolish pupillary unrest as wakefulness decreases. We speculate that midbrain nuclei responsible for wakefulness and pupillary unrest are either communicating or share a similar sensitivity to the effects of commonly used anesthetics.

Entry from the Lipid Bilayer: A Possible Pathway for Inhibition of a Peptide G Protein-Coupled Receptor by a Lipophilic Small Molecule.

The pathways that G protein-coupled receptor (GPCR) ligands follow as they bind to or dissociate from their receptors are largely unknown. Protease-activated receptor-1 (PAR1) is a GPCR activated by intramolecular binding of a tethered agonist peptide that is exposed by thrombin cleavage. By contrast, the PAR1 antagonist vorapaxar is a lipophilic drug that binds in a pocket almost entirely occluded from the extracellular solvent. The binding and dissociation pathway of vorapaxar is unknown. Starting with the crystal structure of vorapaxar bound to PAR1, we performed temperature-accelerated molecular dynamics simulations of ligand dissociation. In the majority of simulations, vorapaxar exited the receptor laterally into the lipid bilayer through openings in the transmembrane helix (TM) bundle. Prior to full dissociation, vorapaxar paused in metastable intermediates stabilized by interactions with the receptor and lipid headgroups. Derivatives of vorapaxar with alkyl chains predicted to extend between TM6 and TM7 into the lipid bilayer inhibited PAR1 with apparent on rates similar to that of the parent compound in cell signaling assays. These data are consistent with vorapaxar binding to PAR1 via a pathway that passes between TM6 and TM7 from the lipid bilayer, in agreement with the most consistent pathway observed by molecular dynamics. While there is some evidence of entry of the ligand into rhodopsin and lipid-activated GPCRs from the cell membrane, our study provides the first such evidence for a peptide-activated GPCR and suggests that metastable intermediates along drug binding and dissociation pathways can be stabilized by specific interactions between lipids and the ligand.

Prediction of Opioid Analgesic Efficacy by Measurement of Pupillary Unrest.

BACKGROUND: Pupillary unrest under ambient light (PUAL) is the fluctuation in pupil diameter in time around a mean value. PUAL is augmented by light and diminished by administration of opioids. We hypothesized that, because pupillary unrest is a marker of opioid effect, low levels of PUAL may be associated with reduced opioid efficacy, as measured by changes in the numerical rating scale (NRS) pain scores of patients in the postanesthesia care unit (PACU). METHODS: We used an infrared pupillometer to measure PUAL in patients recovering from ambulatory surgery at 2 different institutions. At both sites, PUAL was quantified using spectral analysis of the Fourier transform of pupil diameter versus time. We measured PUAL and pain scores before and after opioid administration. Protocols for total capture time and lighting conditions varied between the 2 sites. Correlations between PUAL and change in NRS scores were examined using significance testing of Pearson correlation coefficients. Correlations between change in PUAL and change in NRS scores were also examined. Patients were divided into high and low PUAL groups, and high and low response to opioid. A Fisher exact test was used to determine whether there was a significant association between PUAL and opioid response. RESULTS: For patients with pain in the PACU, low levels of pupillary unrest before opioid therapy were associated with minimal or no reduction in pain scores after opioid administration. We noted a significant correlation at both sites between PUAL and pain score reduction with opioids (r = 0.59, P = .0053, and r = 0.57, P = .022.) The Fisher exact test confirmed that patients with PUAL levels above the mean had a more beneficial analgesic effect from opioids than those with low PUAL levels (P = .018). We also noted that change in PUAL was significantly correlated with change in pain score at both sites (r = 0.56, P = .03 and r = 0.55, P = .01). CONCLUSIONS: We observe that the pretreatment magnitude of PUAL is correlated with the analgesic response to opioid therapy, and that patients who exhibit higher levels of PUAL change after opioid administration have a more beneficial analgesic effect from opioids. Larger studies with uniform measurement protocols are required to confirm these preliminary results.

Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor.

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the beta(2) adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

From the journal archives: cyclopropane: induction and recovery with a bang!

PURPOSE: To review the history of the early development of cyclopropane PRINCIPAL FINDINGS: Cyclopropane was initially investigated because it was thought to be the toxic element in ethylene. Instead, it turned out to be an excellent anesthetic with very rapid onset and recovery while maintaining stable hemodynamics. Its use was ultimately limited because it was highly explosive. Development required collaboration among laboratory scientists and clinicians in Toronto, Canada, clinicians in Madison, USA, and industry in both countries. CONCLUSIONS: The phenomenal success of cyclopropane in over 40 years of clinical use resulted from a lucky, but incorrect, hypothesis that it was a toxic contaminant.

Mismatched Postsurgical Opioid Prescription to Liver Transplant Patients: A Retrospective Cohort Study From a Single High-volume Transplant Center.

BACKGROUND: Improper opioid prescription after surgery is a well-documented iatrogenic contributor to the current opioid epidemic in North America. In fact, opioids are known to be overprescribed to liver transplant patients, and liver transplant patients with high doses or prolonged postsurgical opioid use have higher risks of graft failure and death. METHODS: This is a retrospective cohort study of 552 opioid-naive patients undergoing liver transplant at an academic center between 2012 and 2019. The primary outcome was the discrepancy between the prescribed discharge opioid daily dose and each patient's own inpatient opioid consumption 24 h before discharge. Variables were analyzed with Wilcoxon and chi-square tests and logistic regression. RESULTS: Opioids were overprescribed in 65.9% of patients, and 54.3% of patients who required no opioids the day before discharge were discharged with opioid prescriptions. In contrast, opioids were underprescribed in 13.4% of patients, among whom 27.0% consumed inpatient opioids but received no discharge opioid prescription. The median prescribed opioid daily dose was 333.3% and 56.3% of the median inpatient opioid daily dose in opioid overprescribed and underprescribed patients, respectively. Importantly, opioid underprescribed patients had higher rates of opioid refill 1 to 30 and 31 to 90 d after discharge, and the rate of opioid underprescription more than doubled from 2016 to 2019. CONCLUSIONS: Opioids are both over- and underprescribed to liver transplant patients, and opioid underprescribed patients had higher rates of opioid refill. Therefore, we proposed to prescribe discharge opioid prescriptions based on liver transplant patients' inpatient opioid consumption to provide patient-centered opioid prescriptions.

Pulmonary Injury Causing a Massive Air Leak During Liver Transplantation: A Case Report and Discussion of Decision-Making.

Pulmonary injury can occur during liver transplantation in patients with prior liver surgery, infection, or hepatocellular carcinoma treatments. Compromise of gas exchange during liver transplantation mandates rapid, multidisciplinary decision-making. We present a case of lung parenchymal injury causing a massive air leak during the dissection phase of a liver transplant. An endobronchial blocker was used for emergency lung isolation. Since oxygenation and pH were stable, we proceeded with liver transplantation to minimize graft ischemic time, followed by thoracic repair. The postoperative course was notable for adequate early liver function and discharge after prolonged postoperative ventilation and tube thoracostomy drainage.

Angiotensin II in liver transplantation (AngLT-1): protocol of a randomised, double-blind, placebo-controlled trial.

INTRODUCTION: Catecholamine vasopressors such as norepinephrine are the standard drugs used to maintain mean arterial pressure during liver transplantation. At high doses, catecholamines may impair organ perfusion. Angiotensin II is a peptide vasoconstrictor that may improve renal perfusion pressure and glomerular filtration rate, a haemodynamic profile that could reduce acute kidney injury. Angiotensin II is approved for vasodilatory shock but has not been rigorously evaluated for treatment of hypotension during liver transplantation. The objective is to assess the efficacy of angiotensin II as a second-line vasopressor infusion during liver transplantation. This trial will establish the efficacy of angiotensin II in decreasing the dose of norepinephrine to maintain adequate blood pressure. Completion of this study will allow design of a follow-up, multicentre trial powered to detect a reduction of organ injury in liver transplantation. METHODS AND ANALYSIS: This is a double-blind, randomised clinical trial. Eligible subjects are adults with a Model for End-Stage Liver Disease Sodium Score ≥25 undergoing deceased donor liver transplantation. Subjects are randomised 1:1 to receive angiotensin II or saline placebo as the second-line vasopressor infusion. The study drug infusion is initiated on reaching a norepinephrine dose of 0.05 µg kg-1 min-1 and titrated per protocol. The primary outcome is the dose of norepinephrine required to maintain a mean arterial pressure ≥65 mm Hg. Secondary outcomes include vasopressin or epinephrine requirement and duration of hypotension. Safety outcomes include incidence of thromboembolism within 48 hours of the end of surgery and severe hypertension. An intention-to-treat analysis will be performed for all randomised subjects receiving the study drug. The total dose of norepinephrine will be compared between the two arms by a one-tailed Mann-Whitney U test. ETHICS AND DISSEMINATION: The trial protocol was approved by the local Institutional Review Board (#20-30948). Results will be posted on ClinicalTrials.gov and published in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: ClinicalTrials.govNCT04901169.

Lysine methylation strategies for characterizing protein conformations by NMR.

In the presence of formaldehyde and a mild reducing agent, reductive methylation is known to achieve near complete dimethylation of protein amino groups under non-denaturing conditions, in aqueous media. Amino methylation of proteins is employed in mass spectrometric, crystallographic, and NMR studies. Where biosynthetic labeling is prohibitive, amino (13)C-methylation provides an attractive option for monitoring folding, kinetics, protein-protein and protein-DNA interactions by NMR. Here, we demonstrate two improvements over traditional (13)C-reductive methylation schemes: (1) By judicious choice of stoichiometry and pH, ε-aminos can be preferentially monomethylated. Monomethyl tags are less perturbing and generally exhibit improved resolution over dimethyllysines, and (2) By use of deuterated reducing agents and (13)C-formaldehyde, amino groups can be labeled with (13)CH(2)D tags. Use of deutero-(13)C-formaldehyde affords either (13)CHD(2), or (13)CD(3) probes depending on choice of reducing agent. Making use of (13)C-(2)H scalar couplings, we demonstrate a filtering scheme that eliminates natural abundance (13)C signal.