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The impact of albumin use during major surgery is unknown, and a dearth of evidence governing its use in major non-cardiac surgery has long precluded its standardization in clinical guidelines.In this study we investigate institutional variation in albumin use among medical centers in the United States during major non-cardiac surgery and explore the association of intraoperative albumin administration with important post-operative outcomes.The study is an observational retrospective cohort analysis performed among 54 hospitals in the Multicenter Perioperative Outcomes Group (MPOG) and includes adult patients who underwent major non-cardiac surgery under general anesthesia between January 2014 and June 2020. The primary endpoint was the incidence of albumin administration. Secondary endpoints acute kidney injury (AKI), net-positive fluid balance, pulmonary complications, and 30-day mortality. Albumin-exposed and -unexposed cases were compared within a propensity score-matched cohort to evaluate associations of albumin use with outcomes.Among 614,215 major surgery cases, albumin was used in 15.3% (mostly isooncotic albumin) but with significant inter-institutional variability in use patterns. Cases involving intraoperative albumin administration were of a higher American Society of Anesthesiologists (ASA) physical status and received larger infused crystalloid volumes, had higher 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, P0.001), severe AKI (aOR 1.45, 95% CI 1.34-1.56, P0.001), net-positive fluid balance (aOR 1.18, 95% CI 1.16-1.20, P0.001), pulmonary complications (aOR 1.56, 95% CI 1.30-1.86, P0.001), and 30-day all-cause mortality (aOR 1.37, 95% CI 1.26-1.49, P0.001).Intravenous albumin is commonly administered among non-cardiac surgeries with significant inter-institutional variability in use in the United States. Albumin was associated with an increased risk of postoperative complications.

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.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.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, P0.001). Subpectoral patients also recorded higher maximum pain scores compared with prepectoral reconstructions while admitted (7 of 10 vs 5 of 10, P0.004). Multivariable linear regression suggests that mastectomy type and subpectoral reconstruction were significant contributors to postoperative opioid use (P0.05).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.

BACKGROUND Acute kidney injury (AKI) after liver transplantation is associated with increased morbidity and mortality. It remains controversial whether the choice of vena cava reconstruction technique impacts AKI. METHODS This is a single-center retrospective cohort of 897 liver transplants performed between June 2009 and September 2018 using either the vena cava preserving piggyback technique or caval replacement technique without veno-venous bypass or shunts. The association between vena cava reconstruction technique and stage of postoperative AKI was assessed using multivariable ordinal logistic regression. Causal mediation analysis was used to evaluate warm ischemia time as a potential mediator of this association. RESULTS The incidence of AKI (AKI stage ≥2) within 48 h after transplant was lower in the piggyback group (40.3%) compared to the caval replacement group (51.8%, P < 0.001). Piggyback technique was associated with a reduced risk of developing a higher stage of postoperative AKI (odds ratio, 0.49; 95% confidence interval, 0.37-0.65, P < 0.001). Warm ischemia time was shorter in the piggyback group and identified as potential mediator of this effect. There was no difference in renal function (estimated glomerular filtration rate and the number of patients alive without dialysis) 1 y after transplant. CONCLUSIONS Piggyback technique, compared with caval replacement, was associated with a reduced incidence of AKI after liver transplantation. There was no difference in long-term renal outcomes between the 2 groups.

BACKGROUND: Pulmonary hypertension (PH) is frequently reported in patients with advanced chronic kidney disease (CKD) and is associated with early allograft failure and death. However, the causes of PH are heterogeneous, and patient prognosis may vary by etiologic subtype. METHODS: Data from the UNC Cardiorenal Registry were examined to determine associations between pulmonary hypertension (PH), with or without elevated left atrial pressure (eLAP), and mortality in candidates for kidney transplantation. PH and eLAP were determined by Doppler echocardiography and by tissue Doppler imaging, respectively. RESULTS: From 2006-2013, 778 registry patients were screened preoperatively by echocardiography. Most patients were black (64%) and male (56%); the mean age was 56 years. PH was identified in 97 (12%) patients; of these, eLAP was prevalent in half. During a median follow up of 4.4 years, 179 (23%) received a kidney transplant, and 195 (25%) died. After adjustments for demographics, comorbidities, dialysis vintage and kidney transplantation, PH was associated with twice the 5-year mortality (HR = 2.11; 95% CI: 1.48 – 3.03), with stronger associations in the absence of eLAP (HR = 2.87; 95% CI: 1.83 – 4.49) than with eLAP (HR = 1.11; 95% CI: 0.57 – 2.17); P for interaction = 0.01. CONCLUSION: The mortality risk associated with PH among patients with advanced CKD appears to differ by etiology. Patients with PH in the absence of eLAP are at high risk of death and in need of focused attention. Future research efforts should investigate potential strategies to improve outcomes for these patients.

Acute kidney injury (AKI) after liver transplantation is associated with increased morbidity and mortality. It remains controversial whether the choice of vena cava reconstruction technique impacts AKI.This is a single-center retrospective cohort of 897 liver transplants performed between June 2009 and September 2018 using either the vena cava preserving piggyback technique or caval replacement technique without veno-venous bypass or shunts. The association between vena cava reconstruction technique and stage of postoperative AKI was assessed using multivariable ordinal logistic regression. Causal mediation analysis was used to evaluate warm ischemia time as a potential mediator of this association.The incidence of AKI (AKI stage ≥2) within 48 h after transplant was lower in the piggyback group (40.3%) compared to the caval replacement group (51.8%, P0.001). Piggyback technique was associated with a reduced risk of developing a higher stage of postoperative AKI (odds ratio, 0.49; 95% confidence interval, 0.37-0.65, P0.001). Warm ischemia time was shorter in the piggyback group and identified as potential mediator of this effect. There was no difference in renal function (estimated glomerular filtration rate and the number of patients alive without dialysis) 1 y after transplant.Piggyback technique, compared with caval replacement, was associated with a reduced incidence of AKI after liver transplantation. There was no difference in long-term renal outcomes between the 2 groups.

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.

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 asso

Patients with end-stage liver disease are often hyponatremic due to multiple physiological processes associated with hepatic failure. For severely hyponatremic patients undergoing liver transplantation, intraoperative management of serum sodium concentration ([Na]s) is challenging. [Na]s tends to increase during transplantation by the administration of fluids with higher sodium concentration than the patient's [Na]s. An overly rapid increase in [Na]s (>1 mEq·L·hour) is difficult to avoid and increases the risk of serious perioperative complications. We report the successful use of intravenous desmopressin to reverse an overly rapid rise in [Na]s during living donor liver transplantation.

The proper diagnosis of hyponatremia is a vital component of the perioperative care of liver transplant patients, as hyponatremia is common among patients with end-stage liver disease. The general recommendation for the treatment of chronic hyponatremia is to limit the speed of correction. Unfortunately, the peri-transplantation period is often punctuated by large fluctuations in serum sodium concentration, which increases the risk of neurologic complications and osmotic demyelination syndrome. Consequently, the therapeutic goals and management strategies for hyponatremia vary greatly between the pre-transplantation, intraoperative, and post-transplantation periods. In this chapter, the definition, prognosis, pathophysiology, and treatment of hyponatremia in patients with cirrhosis are reviewed.

Hypertension is the major risk factor for premature cardiovascular disease, morbidity and mortality. It is the leading cause of death and disability globally, contributing to cardiovascular and renal diseases, including stroke, heart failure, coronary heart disease, and chronic kidney disease. We sought to determine the transcriptional changes that occur in cardiac endothelial cells secondary to hypertension, using ribosomal profiling of mice expressing the ribosomal protein Rpl22 tagged with the hemagglutinin (HA) epitope under control of Tie2-Cre (Tie2-Cre:RiboTag mice). Immunoprecipitation of HA-Rpl22 from heart tissue isolated from Tie2-Cre:RiboTag mice resulted in an approximate 10-fold enrichment of endothelial genes (Pecam-1, VE-cadherin) relative to genes highly expressed in cardiomyocytes (Tnnt2, Fabp3) as assessed by RT-PCR. Subsequently, Tie2-Cre:RiboTag mice were implanted with osmotic pumps delivering saline or angiotensin II (1000ng/kg/min) for 4 weeks. Angiotensin II infusion significantly increased blood pressure (p1.5, p

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 β(2)-adrenergic receptor (β(2)AR), a prototypical GPCR. We labeled β(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 β(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 β(2)AR's ability to engage multiple signaling and regulatory proteins.

G-protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters, and these membrane proteins are the largest group of therapeutic targets for a broad range of diseases. It is very difficult to obtain high-resolution X-ray crystal structures of GPCRs; little is known about the functional role(s) of the extracellular surface in receptor activation or about the conformational coupling of the extracellular surface to the native ligand-binding pocket. In this study, Bokoch et al. used NMR spectroscopy to investigate ligand-specific conformational changes around a salt bridge linking extracellular loops 2 and 3 of the β2 adrenergic receptor. They found that drugs that bind within the transmembrane core (and exhibit different efficacies towards G-protein activation) stabilize distinct conformations of the extracellular surface. New therapeutic agents that target this diverse surface could function as allosteric modulators with high subtype selectivity. G-protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters and are the largest group of therapeutic targets for a range of diseases. The extracellular surface (ECS) of GPCRs is diverse and therefore an ideal target for the discovery of subtype-selective drugs. Here, NMR spectroscopy is used to investigate ligand-specific conformational changes around a central structural feature in the ECS of a GPCR. 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 GPCRs1,2,3,4,5 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 β2 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.

G protein-coupled receptors (GPCRs) respond to a diverse array of ligands, mediating cellular responses to hormones and neurotransmitters, as well as the senses of smell and taste. The structures of the GPCR rhodopsin and several G proteins have been determined by x-ray crystallography, yet the organization of the signaling complex between GPCRs and G proteins is poorly understood. The observations that some GPCRs are obligate heterodimers, and that many GPCRs form both homo- and heterodimers, has led to speculation that GPCR dimers may be required for efficient activation of G proteins. However, technical limitations have precluded a definitive analysis of G protein coupling to monomeric GPCRs in a biochemically defined and membrane-bound system. Here we demonstrate that a prototypical GPCR, the β 2 -adrenergic receptor (β 2 AR), can be incorporated into a reconstituted high-density lipoprotein (rHDL) phospholipid bilayer particle together with the stimulatory heterotrimeric G protein, Gs. Single-molecule fluorescence imaging and FRET analysis demonstrate that a single β 2 AR is incorporated per rHDL particle. The monomeric β 2 AR efficiently activates Gs and displays GTP-sensitive allosteric ligand-binding properties. These data suggest that a monomeric receptor in a lipid bilayer is the minimal functional unit necessary for signaling, and that the cooperativity of agonist binding is due to G protein association with a receptor monomer and not receptor oligomerization.

Cholesterol oxidase is immobilized in electrode-supported lipid bilayer membranes. Platinum electrodes are initially modified with a self-assembled monolayer of thiolipid. A vesicle fusion method is used to deposit an outer leaflet of phospholipids onto the thiolipid monolayer forming a thiolipid/lipid bilayer membrane on the electrode surface. Cholesterol oxidase spontaneously inserts into the electrode-supported lipid bilayer membrane from solution and is consequently immobilized to the electrode surface. Cholesterol partitions into the membrane from buffer solutions containing cyclodextrin. Cholesterol oxidase catalyzes the oxidation of cholesterol by molecular oxygen, forming hydrogen peroxide as a product. Amperometric detection of hydrogen peroxide for continuous solution flow experiments are presented, where flow was alternated between cholesterol solution and buffer containing no cholesterol. Steady-state anodic currents were observed during exposures of cholesterol solutions ranging in concentration from 10 to 1000 μM. These data are consistent with the Michaelis–Menten kinetic model for oxidation of cholesterol as catalyzed by cholesterol oxidase immobilized in the lipid bilayer membrane. The cholesterol detection limit is below 1 μM for cholesterol solution prepared in buffered cyclodextrin. The response of the electrodes to low density lipoprotein solutions is increased upon addition of cyclodextrin. Evidence for adsorption of low density lipoprotein to the electrode surface is presented.

Lucas G.H.W. Can Anaesth Soc J 1960; 7: 237-56. To review the history of the early development of cyclopropane 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. 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.

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, e-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.

Recent crystal structures reveal the inactive states of non-rhodopsin G-protein coupled receptors (GPCRs) in beautiful detail. Solution NMR spectroscopy is ideally suited to contribute dynamic information regarding GPCR activation. However, these eukaryotically-expressed membrane proteins remain challenging NMR targets. We apply selective labeling with [13C]methyl probes and two-dimensional NMR to analyze ligand-induced conformational changes in beta2-adrenergic receptor (b2AR).Lysine side chains were labeled with [13C]dimethyl probes to explore conformational changes in the b2AR extracellular surface. Lys305 forms a salt bridge connecting the extracellular end of transmembrane (TM) helix 7 with extracellular loop 2. The Lys305 NMR resonances are sensitive to conformational changes in the receptor extracellular surface. Using NMR, we observe disruption of the Lys305 salt bridge upon receptor activation by agonist. Computational modeling suggests that a lateral displacement of TM7 occurs in concert with an inward motion at the extracellular end of TM6 (thus extending the “global toggle switch” model of Schwartz (2006) Annu. Rev. Pharmacol. Toxicol.) Different conformational changes occur upon inverse agonist binding. Molecular dynamics simulations suggest that a conserved phenylalanine (Phe193) in the orthosteric ligand binding site is key for inverse agonism. Taken as a whole, these results demonstrate conformational coupling between the GPCR extracellular surface and orthosteric ligand binding site within the transmembrane domains (Ahuja (2009) Nat. Struct. Mol. Biol.) This provides rationale for developing allosteric pharmaceuticals targeting the GPCR extracellular surface.Conformational changes within the b2AR transmembrane core are also observed by NMR using selective epsilon-[13CH3] labeling of methionines. While assignments are pending, clear conformational changes are seen with activation or inverse agonist binding. [13C]methyl NMR spectroscopy, in combination with crystal structures and molecular dynamics simulation, provides a dynamic view of the conformational changes intrinsic to GPCR function.

The C-termini of G protein-coupled receptors (GPCRs) interact with specific kinases and arrestins in an agonist-dependent manner suggesting that conformational changes induced by ligand binding within the transmembrane domains are transmitted to the C-terminus. Förster resonance energy transfer (FRET) can be used to monitor changes in distance between two protein domains if each site can be specifically and efficiently labeled with a donor or acceptor fluorophore. In order to probe GPCR conformational changes, we have developed a FRET technique that uses site-specific donor and acceptor fluorophores introduced by two orthogonal labeling chemistries. Using this strategy, we examined ligand-induced changes in the distance between two labeled sites in the beta(2) adrenoceptor (beta(2)-AR), a well-characterized GPCR model system. The donor fluorophore, LumioGreen, is chelated by a CCPGCC motif [Fluorescein Arsenical Helix or Hairpin binder (FlAsH) site] introduced through mutagenesis. The acceptor fluorophore, Alexa Fluor 568, is attached to a single reactive cysteine (C265). FRET analyses revealed that the average distance between the intracellular end of transmembrane helix (TM) six and the C-terminus of the beta(2)-AR is 62 A. This relatively large distance suggests that the C-terminus is extended and unstructured. Nevertheless, ligand-specific conformational changes were observed (1). The results provide new insight into the structure of the beta(2)-AR C-terminus and ligand-induced conformational changes that may be relevant to arrestin interactions. The FRET labeling technique described herein can be applied to many GPCRs (and other membrane proteins) and is suitable for conformational studies of domains other than the C-terminus.

The Anti-Brownian ELectrokinetic (ABEL) trap uses voltage feedback to electrokinetically cancel the Brownian motion of single particles in solution in microfluidic geometries. This allows trapping of single high density lipoprotein (HDL) particles for extended observation.

The C-termini of G protein-coupled receptors (GPCRs) interact with specific kinases and arrestins in an agonist-dependent manner suggesting that conformational changes induced by ligand binding within the transmembrane domains are transmitted to the C-terminus. Forster resonance energy transfer (FRET) can be used to monitor changes in distance between two protein domains if each site can be specifically and efficiently labeled with a donor or acceptor fluorophore. In order to probe GPCR conformational changes, we have developed a FRET technique that uses site-specific donor and acceptor fluorophores introduced by two orthogonal labeling chemistries. Using this strategy, we examined ligand-induced changes in the distance between two labeled sites in the beta(2) adrenoceptor (beta(2)-AR), a well-characterized GPCR model system. The donor fluorophore, LumioGreen, is chelated by a CCPGCC motif [Fluorescein Arsenical Helix or Hairpin binder (FlAsH) site] introduced through mutagenesis. The acceptor fluorophore, Alexa Fluor 568, is attached to a single reactive cysteine (C265). FRET analyses revealed that the average distance between the intracellular end of transmembrane helix (TM) six and the C-terminus of the beta(2)-AR is 62 A. This relatively large distance suggests that the C-terminus is extended and unstructured. Nevertheless, ligand-specific conformational changes were observed (1). The results provide new insight into the structure of the beta(2)-AR C-terminus and ligand-induced conformational changes that may be relevant to arrestin interactions. The FRET labeling technique described herein can be applied to many GPCRs (and other membrane proteins) and is suitable for conformational studies of domains other than the C-terminus.

As part of the Journal’s 60 anniversary Diamond Jubilee Celebration, a number of seminal articles from the Journal archives are highlighted in the Journal’s 61 printed volume and online at: www.springer.com/12630. The following article was selected on the basis of its novelty at the time of publication, its scientific merit, and its overall importance to clinical practice: Lucas GHW. The discovery and pharmacology of cyclopropane. Can Anaesth Soc J 1960; 7: 237-56. Drs. Michael P. Bokoch and Adrian W. Gelb provide expert commentary on the discovery and use of a remarkable hemodynamically stable, rapid onset of action, anesthetic gas, the widespread use of which ultimately ended due to its proclivity for explosion.