Experimental and theoretical bulk phase diagram and interfacial tension of ouzo.

Ouzo is a well-known drink in Mediterranean countries, with ingredients water, alcohol and trans-anethole oil. The oil is insoluble in water, but completely soluble in alcohol, so when water is added to the spirit, the available alcohol is depleted and the mixture exhibits spontaneous emulsification. This process is commonly known as the louche or ouzo effect. Although the phase boundaries of this archetypal ternary mixture are well known, the properties of coexisting phases have not previously been studied. Here, we present a detailed experimental investigation into the phase behaviour, including tie-lines connecting coexisting phases, determination of the critical point (also called the plait point in ternary systems) and measurements of the surface tension and density for varying alcohol concentrations. Additionally, we present a theory for the thermodynamics and phase diagram of the system. With suitable selection of the interaction parameters, the theory captures nearly all features of the experimental work. This simple model can be used to determine both bulk and non-uniform (e.g. interfacial) properties, paving the way for a wide range of future applications of the model to ternary mixtures in general. We show how our accurate equilibrium phase diagram can be used to provide improved understanding of non-equilibrium phenomena.

Target deconvolution studies of (2R,6R)-hydroxynorketamine: an elusive search.

The off-label use of racemic ketamine and the FDA approval of (S)-ketamine are promising developments for the treatment of depression. Nevertheless, racemic ketamine and (S)-ketamine are controlled substances with known abuse potential and their use is associated with undesirable side effects. For these reasons, research efforts have focused on identifying alternatives. One candidate is (2R,6R)-hydroxynorketamine ((2R,6R)-HNK), a ketamine metabolite that in preclinical models lacks the dissociative and abuse properties of ketamine while retaining its antidepressant-like behavioral efficacy. (2R,6R)-HNK's mechanism of action however is unclear. The main goals of this study were to perform an in-depth pharmacological characterization of (2R,6R)-HNK at known ketamine targets, to use target deconvolution approaches to discover novel proteins that bind to (2R,6R)-HNK, and to characterize the biodistribution and behavioral effects of (2R,6R)-HNK across several procedures related to substance use disorder liability. We found that unlike (S)- or (R)-ketamine, (2R,6R)-HNK did not directly bind to any known or proposed ketamine targets. Extensive screening and target deconvolution experiments at thousands of human proteins did not identify any other direct (2R,6R)-HNK-protein interactions. Biodistribution studies using radiolabeled (2R,6R)-HNK revealed non-selective brain regional enrichment, and no specific binding in any organ other than the liver. (2R,6R)-HNK was inactive in conditioned place preference, open-field locomotor activity, and intravenous self-administration procedures. Despite these negative findings, (2R,6R)-HNK produced a reduction in immobility time in the forced swim test and a small but significant increase in metabolic activity across a network of brain regions, and this metabolic signature differed from the brain metabolic profile induced by ketamine enantiomers. In sum, our results indicate that (2R,6R)-HNK does not share pharmacological or behavioral profile similarities with ketamine or its enantiomers. However, it could still be possible that both ketamine and (2R,6R)-HNK exert antidepressant-like efficacy through a common and previously unidentified mechanism. Given its pharmacological profile, we predict that (2R,6R)-HNK will exhibit a favorable safety profile in clinical trials, and we must wait for clinical studies to determine its antidepressant efficacy.

Delineation of G Protein-Coupled Receptor Kinase Phosphorylation Sites within the D1 Dopamine Receptor and Their Roles in Modulating ß-Arrestin Binding and Activation.

The D1 dopamine receptor (D1R) is a G protein-coupled receptor that signals through activating adenylyl cyclase and raising intracellular cAMP levels. When activated, the D1R also recruits the scaffolding protein ß-arrestin, which promotes receptor desensitization and internalization, as well as additional downstream signaling pathways. These processes are triggered through receptor phosphorylation by G protein-coupled receptor kinases (GRKs), although the precise phosphorylation sites and their role in recruiting ß-arrestin to the D1R remains incompletely described. In this study, we have used detailed mutational and in situ phosphorylation analyses to completely identify the GRK-mediated phosphorylation sites on the D1R. Our results indicate that GRKs can phosphorylate 14 serine and threonine residues within the C-terminus and the third intracellular loop (ICL3) of the receptor, and that this occurs in a hierarchical fashion, where phosphorylation of the C-terminus precedes that of the ICL3. Using ß-arrestin recruitment assays, we identified a cluster of phosphorylation sites in the proximal region of the C-terminus that drive ß-arrestin binding to the D1R. We further provide evidence that phosphorylation sites in the ICL3 are responsible for ß-arrestin activation, leading to receptor internalization. Our results suggest that distinct D1R GRK phosphorylation sites are involved in ß-arrestin binding and activation.

Pharmacological and behavioral divergence of ketamine enantiomers: implications for abuse liability.

Ketamine, a racemic mixture of (S)-ketamine and (R)-ketamine enantiomers, has been used as an anesthetic, analgesic and more recently, as an antidepressant. However, ketamine has known abuse liability (the tendency of a drug to be used in non-medical situations due to its psychoactive effects), which raises concerns for its therapeutic use. (S)-ketamine was recently approved by the United States' FDA for treatment-resistant depression. Recent studies showed that (R)-ketamine has greater efficacy than (S)-ketamine in preclinical models of depression, but its clinical antidepressant efficacy has not been established. The behavioral effects of racemic ketamine have been studied extensively in preclinical models predictive of abuse liability in humans (self-administration and conditioned place preference [CPP]). In contrast, the behavioral effects of each enantiomer in these models are unknown. We show here that in the intravenous drug self-administration model, the gold standard procedure to assess potential abuse liability of drugs in humans, rats self-administered (S)-ketamine but not (R)-ketamine. Subanesthetic, antidepressant-like doses of (S)-ketamine, but not of (R)-ketamine, induced locomotor activity (in an opioid receptor-dependent manner), induced psychomotor sensitization, induced CPP in mice, and selectively increased metabolic activity and dopamine tone in medial prefrontal cortex (mPFC) of rats. Pharmacological screening across thousands of human proteins and at biological targets known to interact with ketamine yielded divergent binding and functional enantiomer profiles, including selective mu and kappa opioid receptor activation by (S)-ketamine in mPFC. Our results demonstrate divergence in the pharmacological, functional, and behavioral effects of ketamine enantiomers, and suggest that racemic ketamine's abuse liability in humans is primarily due to the pharmacological effects of its (S)-enantiomer.

Coronavirus 2019 (COVID-19) venovenous extracorporeal oxygenation: Single community hospital results and insights.

BACKGROUND: The role of extracorporeal membrane oxygenation (ECMO) for patients with refractory respiratory failure due to coronavirus 2019 (COVID-19) is still unclear even now over a year into the pandemic. ECMO is becoming more commonplace even at smaller community hospitals. While the advantages of venovenous (VV) ECMO in acute respiratory distress syndrome (ARDS) from COVID-19 have not been fully determined, we believe the benefits outweighed the risks in our patient population. Here we describe all patients who underwent VV ECMO at our center. METHODS: All patients placed on ECMO at our center since the beginning of the pandemic, May 5, 2020, until February 20, 2021 were included in our study. All patients placed on ECMO during the time period described above were followed until discharge or death. The primary endpoint was in-hospital death. Secondary outcomes included discharge disposition, that is, whether patients were sent to a long-term acute care center (LTAC), inpatient rehabilitation, or went directly home. RESULTS: A total of 41 patients were placed on VV ECMO for refractory acute respiratory failure. Survival to discharge, the primary end point, was 63.4% (26/41). Inpatient mortality was 36.6% (15/41). CONCLUSIONS: We show here that a successful high-volume VV ECMO program for ARDS is achievable at even a medium-size community hospital. We think our success can be replicated by most small- and medium-size community hospitals with cardiothoracic surgery programs and intensivist teams.

Pharmacological Characterization of the Imipridone Anticancer Drug ONC201 Reveals a Negative Allosteric Mechanism of Action at the D2 Dopamine Receptor.

ONC201 is a first-in-class imipridone compound that is in clinical trials for the treatment of high-grade gliomas and other advanced cancers. Recent studies identified that ONC201 antagonizes D2-like dopamine receptors at therapeutically relevant concentrations. In the current study, characterization of ONC201 using radioligand binding and multiple functional assays revealed that it was a full antagonist of the D2 and D3 receptors (D2R and D3R) with low micromolar potencies, similar to its potency for antiproliferative effects. Curve-shift experiments using D2R-mediated ß-arrestin recruitment and cAMP assays revealed that ONC201 exhibited a mixed form of antagonism. An operational model of allostery was used to analyze these data, which suggested that the predominant modulatory effect of ONC201 was on dopamine efficacy with little to no effect on dopamine affinity. To investigate how ONC201 binds to the D2R, we employed scanning mutagenesis coupled with a D2R-mediated calcium efflux assay. Eight residues were identified as being important for ONC201's functional antagonism of the D2R. Mutation of these residues followed by assessing ONC201 antagonism in multiple signaling assays highlighted specific residues involved in ONC201 binding. Together with computational modeling and simulation studies, our results suggest that ONC201 interacts with the D2R in a bitopic manner where the imipridone core of the molecule protrudes into the orthosteric binding site, but does not compete with dopamine, whereas a secondary phenyl ring engages an allosteric binding pocket that may be associated with negative modulation of receptor activity. SIGNIFICANCE STATEMENT: ONC201 is a novel antagonist of the D2 dopamine receptor with demonstrated efficacy in the treatment of various cancers, especially high-grade glioma. This study demonstrates that ONC201 antagonizes the D2 receptor with novel bitopic and negative allosteric mechanisms of action, which may explain its high selectivity and some of its clinical anticancer properties that are distinct from other D2 receptor antagonists widely used for the treatment of schizophrenia and other neuropsychiatric disorders.

New roles for dopamine D2 and D3 receptors in pancreatic beta cell insulin secretion.

Although long-studied in the central nervous system, there is increasing evidence that dopamine (DA) has important roles in the periphery including in metabolic regulation. Insulin-secreting pancreatic ß-cells express the machinery for DA synthesis and catabolism, as well as all five DA receptors. In these cells, DA functions as a negative regulator of glucose-stimulated insulin secretion (GSIS), which is mediated by DA D2-like receptors including D2 (D2R) and D3 (D3R) receptors. However, the fundamental mechanisms of DA synthesis, storage, release, and signaling in pancreatic ß-cells and their functional relevance in vivo remain poorly understood. Here, we assessed the roles of the DA precursor L-DOPA in ß-cell DA synthesis and release in conjunction with the signaling mechanisms underlying DA's inhibition of GSIS. Our results show that the uptake of L-DOPA is essential for establishing intracellular DA stores in ß-cells. Glucose stimulation significantly enhances L-DOPA uptake, leading to increased DA release and GSIS reduction in an autocrine/paracrine manner. Furthermore, D2R and D3R act in combination to mediate dopaminergic inhibition of GSIS. Transgenic knockout mice in which ß-cell D2R or D3R expression is eliminated exhibit diminished DA secretion during glucose stimulation, suggesting a new mechanism where D2-like receptors modify DA release to modulate GSIS. Lastly, ß-cell-selective D2R knockout mice exhibit marked postprandial hyperinsulinemia in vivo. These results reveal that peripheral D2R and D3R receptors play important roles in metabolism through their inhibitory effects on GSIS. This opens the possibility that blockade of peripheral D2-like receptors by drugs including antipsychotic medications may significantly contribute to the metabolic disturbances observed clinically.

Development of pyrimidone D1 dopamine receptor positive allosteric modulators.

MLS1082 is a structurally novel pyrimidone-based D1-like dopamine receptor positive allosteric modulator. Potentiation of D1 dopamine receptor (D1R) signaling is a therapeutic strategy for treating neurocognitive disorders. Here, we investigate the relationship between D1R potentiation and two prominent structural features of MLS1082, namely the pendant N-aryl and C-alkyl groups on the pyrimidone ring. To this end, we synthesized 24 new analogues and characterized their ability to potentiate dopamine signaling at the D1R and the closely related D5R. We identified structure-activity relationship trends for both aryl and alkyl modifications and our efforts afforded several analogues with improvements in activity. The most effective analogues demonstrated an approximately 8-fold amplification of dopamine-mediated D1R signaling. These findings advance the understanding of structural moieties underlying the activity of pyrimidone-based D1R positive allosteric modulators.

Rounded Layering Transitions on the Surface of Ice.

Understanding the wetting properties of premelting films requires knowledge of the film's equation of state, which is not usually available. Here we calculate the disjoining pressure curve of premelting films and perform a detailed thermodynamic characterization of premelting behavior on ice. Analysis of the density profiles reveals the signature of weak layering phenomena, from one to two and from two to three water molecular layers. However, disjoining pressure curves, which closely follow expectations from a renormalized mean field liquid state theory, show that there are no layering phase transitions in the thermodynamic sense along the sublimation line. Instead, we find that transitions at mean field level are rounded due to capillary wave fluctuations. We see signatures that true first order layering transitions could arise at low temperatures, for pressures between the metastable line of water-vapor coexistence and the sublimation line. The extrapolation of the disjoining pressure curve above water-vapor saturation displays a true first order phase transition from a thin to a thick film consistent with experimental observations.

Prophylactic Internal Fixation to Avoid Mandible Fracture With Third Molar Removal: Use of Computer-Assisted Surgery to Improve Clinical Outcomes.

Although uncommon, iatrogenic fractures of the mandible may occur during the perioperative time period of a lower third molar removal. The case described used digital technology to apply prophylactic internal fixation before a lower third molar removal thought to be at high risk for a mandible fracture. A medical model of the patient's mandible was 3-dimensionally printed and used as a reference to prebend a titanium plate. Dynamic navigation, a form of computer-assisted surgery, was used during the surgery to accurately place the prebent titanium plate according to the corresponding position on the medical model.

The E2.65A mutation disrupts dynamic binding poses of SB269652 at the dopamine D2 and D3 receptors.

The dopamine D2 and D3 receptors (D2R and D3R) are important targets for antipsychotics and for the treatment of drug abuse. SB269652, a bitopic ligand that simultaneously binds both the orthosteric binding site (OBS) and a secondary binding pocket (SBP) in both D2R and D3R, was found to be a negative allosteric modulator. Previous studies identified Glu2.65 in the SBP to be a key determinant of both the affinity of SB269652 and the magnitude of its cooperativity with orthosteric ligands, as the E2.65A mutation decreased both of these parameters. However, the proposed hydrogen bond (H-bond) between Glu2.65 and the indole moiety of SB269652 is not a strong interaction, and a structure activity relationship study of SB269652 indicates that this H-bond may not be the only element that determines its allosteric properties. To understand the structural basis of the observed phenotype of E2.65A, we carried out molecular dynamics simulations with a cumulative length of ~77 µs of D2R and D3R wild-type and their E2.65A mutants bound to SB269652. In combination with Markov state model analysis and by characterizing the equilibria of ligand binding modes in different conditions, we found that in both D2R and D3R, whereas the tetrahydroisoquinoline moiety of SB269652 is stably bound in the OBS, the indole-2-carboxamide moiety is dynamic and only intermittently forms H-bonds with Glu2.65. Our results also indicate that the E2.65A mutation significantly affects the overall shape and size of the SBP, as well as the conformation of the N terminus. Thus, our findings suggest that the key role of Glu2.65 in mediating the allosteric properties of SB269652 extends beyond a direct interaction with SB269652, and provide structural insights for rational design of SB269652 derivatives that may retain its allosteric properties.

Remote Scene Size-up Using an Unmanned Aerial Vehicle in a Simulated Mass Casualty Incident.

INTRODUCTION: The scene-size-up is a crucial first step in the response to a mass casualty incident (MCI). Unmanned aerial vehicles (UAV) may potentially enhance the scene-size-up with real-time visual feedback during chaotic, evolving or inaccessible events. We performed this study to test the feasibility of paramedics using UAV video from a simulated MCI to identify scene hazards, initiate patient triage, and designate key operational locations. METHODS: We simulated an MCI, including 15 patients plus 4 hazards, on a college campus. A UAV surveyed the scene, capturing video of all patients, hazards, surrounding buildings and streets. We invited attendees of a provincial paramedic meeting to participate. Participants received a lecture on Sort-Assess-Lifesaving Interventions-Treatment/Transport (SALT) Triage and MCI scene management principles. Next, they watched the UAV video footage. We directed participants to sort patients according to SALT Triage Step One, identify injuries, and to localize the patients within the campus. Additionally, we asked them to select a start point for SALT Triage Step Two, identify and locate hazards, and designate locations for an Incident Command Post, Treatment Area, Transport Area and Access/Egress routes. The primary outcome was the number of correctly allocated triage scores. RESULTS: Ninety-six individuals participated. Mean age was 35 years (SD 11); 46% (44) were female and 49% (47) were Primary Care Paramedics. Most participants (79; 82%) correctly sorted at least 12 of 15 patients. Increased age was associated with decreased triage accuracy [-0.04(-0.07, -0.01); p = 0.031]. Fifty-two (54%) correctly localized 12 or more patients to a 27 × 20m grid area. Advanced paramedic certification, and local residency were associated with improved patient localization [2.47(0.23,4.72); p = 0.031], [3.36(1.10,5.61); p = 0.004]. The majority of participants (70; 81%) chose an acceptable location to start SALT Triage Step Two and 75 (78%) identified at least 3 of 4 hazards. Approximately half (53; 56%) of participants appropriately designated 4 or more of 5 key operational areas. CONCLUSION: This study demonstrates the ability of UAV technology to remotely facilitate the scene size-up in an MCI. Additional research is required to further investigate optimal strategies to deploy UAVs in this context.

Identification of Positive Allosteric Modulators of the D1 Dopamine Receptor That Act at Diverse Binding Sites.

The D1 dopamine receptor is linked to a variety of neuropsychiatric disorders and represents an attractive drug target for the enhancement of cognition in schizophrenia, Alzheimer disease, and other disorders. Positive allosteric modulators (PAMs), with their potential for greater selectivity and larger therapeutic windows, may represent a viable drug development strategy, as orthosteric D1 receptor agonists possess known clinical liabilities. We discovered two structurally distinct D1 receptor PAMs, MLS6585 and MLS1082, via a high-throughput screen of the NIH Molecular Libraries program small-molecule library. Both compounds potentiate dopamine-stimulated G protein- and ß-arrestin-mediated signaling and increase the affinity of dopamine for the D1 receptor with low micromolar potencies. Neither compound displayed any intrinsic agonist activity. Both compounds were also found to potentiate the efficacy of partial agonists. We tested maximally effective concentrations of each PAM in combination to determine if the compounds might act at separate or similar sites. In combination, MLS1082 + MLS6585 produced an additive potentiation of dopamine potency beyond that caused by either PAM alone for both ß-arrestin recruitment and cAMP accumulation, suggesting diverse sites of action. In addition, MLS6585, but not MLS1082, had additive activity with the previously described D1 receptor PAM "Compound B," suggesting that MLS1082 and Compound B may share a common binding site. A point mutation (R130Q) in the D1 receptor was found to abrogate MLS1082 activity without affecting that of MLS6585, suggesting this residue may be involved in the binding/activity of MLS1082 but not that of MLS6585. Together, MLS1082 and MLS6585 may serve as important tool compounds for the characterization of diverse allosteric sites on the D1 receptor as well as the development of optimized lead compounds for therapeutic use.

The pressure tensor across a liquid-vapour interface.

Inhomogeneous fluids exhibit physical properties that are neither uniform nor isotropic. The pressure tensor is a case in point, key to the mechanical description of the interfacial region. Kirkwood and Buff and, later, Irving and Kirkwood, obtained a formal treatment based on the analysis of the pressure across a planar surface [J. G. Kirkwood and F. P. Buff, J. Chem. Phys. 17(3), 338 (1949); J. H. Irving and J. G. Kirkwood, J. Chem. Phys. 18, 817 (1950)]. We propose a generalisation of Irving and Kirkwood's argument to fluctuating, non-planar surfaces and obtain an expression for the pressure tensor that is not smeared by thermal fluctuations at the molecular scale and corresponding capillary waves [F. P. Buff et al., Phys. Rev. Lett. 15, 621-623 (1965)]. We observe the emergence of surface tension, defined as an excess tangential stress, acting exactly across the dividing surface at the sharpest molecular resolution. The new statistical mechanical expressions extend current treatments to fluctuating inhomogeneous systems far from equilibrium.

Discovery and characterization of a G protein-biased agonist that inhibits ß-arrestin recruitment to the D2 dopamine receptor.

A high-throughput screening campaign was conducted to interrogate a 380,000+ small-molecule library for novel D2 dopamine receptor modulators using a calcium mobilization assay. Active agonist compounds from the primary screen were examined for orthogonal D2 dopamine receptor signaling activities including cAMP modulation and ß-arrestin recruitment. Although the majority of the subsequently confirmed hits activated all signaling pathways tested, several compounds showed a diminished ability to stimulate ß-arrestin recruitment. One such compound (MLS1547; 5-chloro-7-[(4-pyridin-2-ylpiperazin-1-yl)methyl]quinolin-8-ol) is a highly efficacious agonist at D2 receptor-mediated G protein-linked signaling, but does not recruit ß-arrestin as demonstrated using two different assays. This compound does, however, antagonize dopamine-stimulated ß-arrestin recruitment to the D2 receptor. In an effort to investigate the chemical scaffold of MLS1547 further, we characterized a set of 24 analogs of MLS1547 with respect to their ability to inhibit cAMP accumulation or stimulate ß-arrestin recruitment. A number of the analogs were similar to MLS1547 in that they displayed agonist activity for inhibiting cAMP accumulation, but did not stimulate ß-arrestin recruitment (i.e., they were highly biased). In contrast, other analogs displayed various degrees of G protein signaling bias. These results provided the basis to use pharmacophore modeling and molecular docking analyses to build a preliminary structure-activity relationship of the functionally selective properties of this series of compounds. In summary, we have identified and characterized a novel G protein-biased agonist of the D2 dopamine receptor and identified structural features that may contribute to its biased signaling properties.

Dopamine D1-like receptors regulate the α1A-adrenergic receptor in human renal proximal tubule cells and D1-like dopamine receptor knockout mice.

The homeostatic control of blood pressure hinges upon the delicate balance between prohypertensinogenic and antihypertensinogenic systems. D1-like dopamine receptors [dopamine D1 and D5 receptors (D1Rs and D5Rs, respectively)] and the α1A-adrenergic receptor (α1A-AR) are expressed in the renal proximal tubule and engender opposing effects on Na(+) transport, i.e., natriuresis (via D1Rs and D5Rs) or antinatriuresis (via α1A-ARs). We tested the hypothesis that the D1R/D5R regulates the α1A-AR. D1-like dopamine receptors coimmunoprecipitated, colocalized, and cofractionated with α1A-ARs in lipid rafts in immortalized human renal proximal tubule cells. Long-term treatment with the D1R/D5R agonist fenoldopam resulted in decreased D1R and D5R expression but increased α1A-AR abundance in the plasma membrane. Short-term fenoldopam treatment stimulated the translocation of Na(+)-K(+)-ATPase from the plasma membrane to the cytosol that was partially reversed by an α1A-AR agonist, which by itself induced Na(+)-K(+)-ATPase translocation from the cytosol to the plasma membrane. The α1A-AR-specific agonist A610603 also minimized the ability of fenoldopam to inhibit Na(+)-K(+)-ATPase activity. To determine the interaction among D1Rs, D5Rs, and α1A-ARs in vivo, we used phenylephrine and A610603 to decrease Na(+) excretion in several D1-like dopamine receptor knockout mouse strains. Phenylephrine and A61603 treatment resulted in a partial reduction of urinary Na(+) excretion in wild-type mice and its abolition in D1R knockout, D5R knockout, and D1R-D5R double-knockout mice. Our results demonstrate the ability of the D1-like dopamine receptors to regulate the expression and activity of α1A-AR. Elucidating the intricacies of the interaction among these receptors is crucial for a better understanding of the crosstalk between anti- and pro-hypertensive systems.