Overdestabilization vs Overstabilization in the Theoretical Analysis of f-Orbital Covalency.

The complex nature of the f-orbital electronic structures and their interaction with the chemical environment pose significant computational challenges. Advanced computational techniques that variationally include scalar relativities and spin-orbit coupling directly at the molecular orbital level have been developed to address this complexity. Among these, variational relativistic multiconfigurational multireference methods stand out for their high accuracy and systematic improvement in studies of f-block complexes. Additionally, these advanced methods offer the potential for calibrating low-scaling electronic structure methods such as density functional theory. However, studies on the Cl K-edge X-ray absorption spectra of the [Ce(III)Cl6]3- and [Ce(IV)Cl6]2- complexes show that time-dependent density functional theory with approximate exchange-correlation kernels can lead to inaccuracies, resulting in an overstabilization of 4f orbitals and incorrect assessments of covalency. In contrast, approaches utilizing small active space wave function methods may understate the stability of these orbitals. The results herein demonstrate the need for large active space, multireference, and variational relativistic methods in studying f-block complexes.

Validated Clinical Score to Predict Gastroesophageal Reflux in Patients With Chronic Laryngeal Symptoms: COuGH RefluX.

BACKGROUND & AIMS: Discerning whether laryngeal symptoms result from gastroesophageal reflux is clinically challenging and a reliable tool to stratify patients is needed. We aimed to develop and validate a model to predict the likelihood of gastroesophageal reflux disease (GERD) among patients with chronic laryngeal symptoms. METHODS: This multicenter international study collected data from adults with chronic laryngeal symptoms who underwent objective testing (upper gastrointestinal endoscopy and/or ambulatory reflux monitoring) between March 2018 and May 2023. The training phase identified a model with optimal receiver operating characteristic curves, and ß coefficients informed a weighted model. The validation phase assessed performance characteristics of the weighted model. RESULTS: A total of 856 adults, 304 in the training cohort and 552 in the validation cohort, were included. In the training phase, the optimal predictive model (area under the curve, 0.68; 95% CI, 0.62-0.74), was the Cough, Overweight/obesity, Globus, Hiatal Hernia, Regurgitation, and male seX (COuGH RefluX) score, with a lower threshold of 2.5 and an upper threshold of 5.0 to predict proven GERD. In the validation phase, the COuGH RefluX score had an area under the curve of 0.67 (95% CI, 0.62-0.71), with 79% sensitivity and 81% specificity for proven GERD. CONCLUSIONS: The externally validated COuGH RefluX score is a clinically practical model to predict the likelihood of proven GERD. The score classifies most patients with chronic laryngeal symptoms as low/high likelihood of proven GERD, with only 38% remaining as indeterminate. Thus, the COuGH RefluX score can guide diagnostic strategies and reduce inappropriate proton pump inhibitor use or testing for patients referred for evaluation of chronic laryngeal symptoms.

Quantified Metrics of Gastric Emptying Delay by Glucagon-Like Peptide-1 Agonists: A Systematic Review and Meta-Analysis With Insights for Periprocedural Management.

INTRODUCTION: Divergent recommendations for periprocedural management of glucagon-like peptide-1 (GLP-1) receptor agonist (GLP-1 RA) medications rely on limited evidence. We performed a systematic review and meta-analysis to provide quantitative measures of gastric emptying relevant to mechanisms of weight loss and to periprocedural management of GLP-1 RA. We hypothesized that the magnitude of gastric emptying delay would be low and of limited clinical significance to procedural sedation risks. METHODS: A protocolized search identified studies on GLP-1 RA that quantified gastric emptying measures. Pooled estimates using random effects were presented as a weighted mean difference with 95% confidence intervals (CIs). Univariate meta-regression was performed to assess the influence of GLP-1 RA type, short-acting vs long-acting mechanism of action, and duration of treatment on gastric emptying. RESULTS: Fifteen studies met the inclusion criteria. Five studies (n = 247) utilized gastric emptying scintigraphy. Mean T 1/2 was 138.4 minutes (95% CI 74.5-202.3) for GLP-1 RA vs 95.0 minutes (95% CI 54.9-135.0) for placebo, with a pooled mean difference of 36.0 minutes (95% CI 17.0-55.0, P < 0.01, I2 = 79.4%). Ten studies (n = 411) utilized the acetaminophen absorption test, with no significant delay in gastric emptying measured by T max , area under the curve (AUC) 4hr , and AUC 5hr with GLP-1 RA ( P > 0.05). On meta-regression, the type of GLP-1 RA, mechanism of action, and treatment duration did not impact gastric emptying ( P > 0.05). DISCUSSION: While a gastric emptying delay of ∼36 minutes is quantifiable on GLP-1 RA medications, it is of limited magnitude relative to standard periprocedural fasting periods. There were no substantial differences in gastric emptying on modalities reflective of liquid emptying (acetaminophen absorption test), particularly at time points relevant to periprocedural care.

Diagnostic Yield of Ambulatory Reflux Monitoring Systems for Evaluation of Chronic Laryngeal Symptoms.

INTRODUCTION: Among patients with chronic laryngeal symptoms, ambulatory reflux monitoring off acid suppression is recommended to evaluate for laryngopharyngeal reflux (LPR). However, reflux monitoring systems are diverse in configuration and monitoring capabilities, which present a challenge in creating a diagnostic reference standard in these patients. This study aimed to compare diagnostic yield and performance between reflux monitoring systems in patients with chronic laryngeal symptoms. METHODS: This multicenter, international study of adult patients referred for evaluation of LPR over a 5-year period (March 2018-May 2023) assessed and compared diagnostic yield of pathologic gastroesophageal reflux (GER+) on ambulatory reflux monitoring off acid suppression. RESULTS: Of 813 patients, 296 (36%) underwent prolonged wireless pH, 532 (65%) underwent 24-hour pH-impedance monitoring, and 15 (2%) underwent both tests. Overall diagnostic yield for GER+ was 36% and greater for prolonged wireless pH compared with that for 24-hour pH-impedance monitoring (50% vs 27%; P < 0.01). Among 15 patients who underwent both prolonged wireless pH and 24-h pH-impedance monitoring, concordance between systems for GER+ was 40%. The most common source of discordance was strong evidence of GER+ across multiple days on prolonged wireless pH compared with no evidence of GER+ on pH-impedance. DISCUSSION: In this multicenter international study of patients with chronic laryngeal symptoms referred for LPR evaluation, diagnostic yield of ambulatory reflux monitoring off acid suppression was 36% and rose to 50% when using wireless pH monitoring. In patients referred for chronic laryngeal symptoms, 24-hour pH-impedance monitoring may risk a low negative predictive value in patients with unproven GER+ disease.

Allergic phenotype identified on allergen testing is associated with proton pump inhibitor nonresponse in eosinophilic esophagitis.

BACKGROUND AND AIM: Food/environmental allergens have been associated with eosinophilic esophagitis (EoE); however, the correlation between allergy profiles and disease responsiveness to proton pump inhibitor (PPI) therapy remains unclear. We aimed to assess the association between food/environmental allergies identified on allergen testing and histologic response to PPI in patients with treatment-naive EoE. METHODS: Adults with newly diagnosed EoE who underwent formal testing for food/environmental allergies at a tertiary center were included. All patients underwent twice-daily PPI for 8 weeks with subsequent repeat endoscopy and biopsy to assess histologic response. Patients with <15 eosinophils/hpf on post-PPI mucosal biopsies were classified as responders (PPI-r-EoE), while those with ≥15 eosinophils/hpf were nonresponders (PPI-nr-EoE). RESULTS: Sixty-one patients met inclusion criteria (21 PPI-r-EoE vs 40 PPI-nr-EoE). Demographic, clinical, and endoscopic finding variables were similar between groups. Positive food allergen test was more prevalent among PPI-nr-EoE patients (82.5% vs 42.9%, P = 0.003). On multivariable analysis, positive food allergen testing remained an independent predictor for PPI nonresponse (aOR 0.15, CI: 0.04-0.58, P = 0.0006). Positive environmental allergen testing was highly prevalent, with no significant differences between groups (77.5% vs 95.2%, P = 0.14). However, higher number of positive environmental allergens (23.3% [≥5 allergens] vs 73.3% [<5 allergens], P = 0.003) and specific aeroallergens correlated with PPI-nr-EoE. CONCLUSION: Positive food allergy testing and increased environmental allergens predicted lower likelihood of histologic response to PPI in EoE. Our findings support an allergic phenotype of EoE that may less likely respond to PPI therapy. Formal allergen testing may play a role in therapy selection and tailored management in EoE.

Comparison of Variational and Perturbative Spin-Orbit Coupling within Two-Component CASSCF.

The modeling of spin-orbit coupling (SOC) remains a challenge in computational chemistry due to the high computational cost. With the rising popularity of spin-driven processes and f-block metals in chemistry and materials science, it is incumbent on the community to develop accurate multiconfigurational SOC methods that scale to large systems and understand the limits of different treatments of SOC. Herein, we introduce an implementation of perturbative SOC in scalar-relativistic two-component CASSCF (srX2C-CASSCF-SO). Perspectives on the limitations and accuracy of srX2C-CASSCF-SO are presented via benchmark calculations.

De novo CLPTM1 variants with reduced GABAA R current response in patients with epilepsy.

OBJECTIVE: To investigate the clinical features and potential pathogenesis mechanism of de novo CLPTM1 variants associated with epilepsy. METHODS: Identify de novo genetic variants associated with epilepsy by reanalyzing trio-based whole-exome sequencing data. We analyzed the clinical characteristics of patients with these variants and performed functional in vitro studies in cells expressing mutant complementary DNA for these variants using whole-cell voltage-clamp current recordings and outside-out patch-clamp recordings from transiently transfected human embryonic kidney (HEK) cells. RESULTS: Two de novo missense variants related to epilepsy were identified in the CLPTM1 gene. Functional studies indicated that CLPTM1-p.R454H and CLPTM1-p.R568Q variants reduced the γ-aminobutyric acid A receptor (GABAA R) current response amplitude recorded under voltage clamp compared to the wild-type receptors. These variants also reduced the charge transfer and altered the time course of desensitization and deactivation following rapid removal of GABA. The surface expression of the GABAA R γ2 subunit from the CLPTM1-p.R568Q group was significantly reduced compared to CLPTM1-WT. SIGNIFICANCE: This is the first report of functionally relevant variants within the CLPTM1 gene. Patch-clamp recordings showed that these de novo CLPTM1 variants reduce GABAA R currents and charge transfer, which should promote excitation and hypersynchronous activity. This study may provide insights into the molecular mechanisms of the CLPTM1 variants underlying the patients' phenotypes, as well as for exploring potential therapeutic targets for epilepsy.

Resilience and diabetes self-management among African-American men receiving primary care at an urban safety-net hospital: a cross-sectional survey.

BACKGROUND: Resilience is the ability to adapt to adverse life events. Studies that explore diabetes self-management interventions integrating resilience in African-Americans with diabetes include few African-American men, who have higher diabetes-related mortality and complication rates compared to African-American women. DESIGN: We conducted a cross-sectional study of African-American men with uncontrolled diabetes living in diabetes hotspots. We measured resilience levels using the General Self Efficacy Scale (GSES), adherence to diabetes self-management behaviors using the Diabetes Self-Management Questionnaire (DSMQ), and incarceration history by phone survey. We categorized participants as higher or lower resilience level and higher or lower adherence to diabetes self-management behaviors. Using multivariable logistic regression, we examined the relationship between resilience and adherence to diabetes self-management behaviors. Our model accounted for potential confounders, including age, incarceration history, and socioeconomic factors. RESULTS: Of 234 patients contacted by mail and phone, 94 (40.2%) completed the survey. Mean age was 60.6 years, 59.5% reported an annual household income of less than $20,000, and 29.8% reported a history of incarceration. The mean unadjusted GSES score was 25.0 (sd 5.2; range: 0-30, higher scores indicate greater resilience), and the mean DSMQ score was 7.34 (sd 1.78; range: 0-10, higher scores indicate greater adherence to diabetes self-management behaviors). In multivariable analyses, higher levels of resilience were associated with higher adherence to diabetes self-management behaviors (aOR = 9.68, 95% CI 3.01, 31.12). History of incarceration was negatively associated with higher adherence to diabetes self-management behaviors (aOR = 0.23, 95% CI 0.06, 0.81). CONCLUSIONS: Resilience and personal history of incarceration are associated with adherence to diabetes self-management behaviors among African-American men residing in diabetes hotspots. Future interventions should incorporate resilience training to improve diabetes self-management behaviors. At a societal level, social determinants of health that adversely affect African-American men, such as structural racism and mass incarceration, need to be eliminated.

Automated Intracellular Pharmacological Electrophysiology for Ligand-Gated Ionotropic Receptor and Pharmacology Screening.

Communication between neuronal cells, which is central to brain function, is performed by several classes of ligand-gated ionotropic receptors. The gold-standard technique for measuring rapid receptor response to agonist is manual patch-clamp electrophysiology, capable of the highest temporal resolution of any current electrophysiology technique. We report an automated high-precision patch-clamp system that substantially improves the throughput of these time-consuming pharmacological experiments. The patcherBotPharma enables recording from cells expressing receptors of interest and manipulation of them to enable millisecond solution exchange to activate ligand-gated ionotropic receptors. The solution-handling control allows for autonomous pharmacological concentration-response experimentation on adherent cells, lifted cells, or excised outside-out patches. The system can perform typical ligand-gated ionotropic receptor experimentation protocols autonomously, possessing a high success rate in completing experiments and up to a 10-fold reduction in research effort over the duration of the experiment. Using it, we could rapidly replicate previous data sets, reducing the time it took to produce an eight-point concentration-response curve of the effect of propofol on GABA type A receptor deactivation from likely weeks of recording to ∼13 hours of recording. On average, the rate of data collection of the patcherBotPharma was a data point every 2.1 minutes that the operator spent interacting with the patcherBotPharma The patcherBotPharma provides the ability to conduct complex and comprehensive experimentation that yields data sets not normally within reach of conventional systems that rely on constant human control. This technical advance can contribute to accelerating the examination of the complex function of ion channels and the pharmacological agents that act on them. SIGNIFICANCE STATEMENT: This work presents an automated intracellular pharmacological electrophysiology robot, patcherBotPharma, that substantially improves throughput and reduces human time requirement in pharmacological patch-clamp experiments. The robotic system includes millisecond fluid exchange handling and can perform highly efficient ligand-gated ionotropic receptor experiments. The patcherBotPharma is built using a conventional patch-clamp rig, and the technical advances shown in this work greatly accelerate the ability to conduct high-fidelity pharmacological electrophysiology.

Evaluating the Cost of Prevention Programming and Universal Screening with Discrete Event Simulation.

Discrete Event Simulation (DES) is a novel system modeling technique that allows for the evaluation of the potential costs and personnel needed for mental health services in school. A case study is presented to illustrate how DES could be used by a school's decision makers to help plan for implementation of an integrated mental health service model. Discrete Event Simulation was used to model the personnel, time, and costs of an integrated mental health service model within a school setting. In addition, costs are calculated and then compared to a business as usual model. Data from the present investigation indicate substantial cost savings of implementing a prevention oriented mental health intervention model within a school setting. In a school of 1000 students, the prevention model could result in an annual cost savings of approximately $30,000 as well as a 50% reduction in disciplinary referrals and 22% reduction in suspensions. Results from the present investigation indicate substantial savings in financial resources and overall numbers of disciplinary infractions when implementing a prevention model. The DES allows for customization of personnel and time to modify the model and resulting output to local conditions. These data may allow school administrators to modify resources to meet student needs. In addition, cost data can help address some of the common implementation barriers associated with adoption of universal screening and preventative mental health services.

Correction to: Detection of Candidatus Neoehrlichia mikurensis in Norway up to the northern limit of Ixodes ricinus distribution using a novel real time PCR test targeting the groEL gene.

After publication of our article [1] it came to our notice that the source of the sequence for the control plasmid, pNeo (Materials and methods: Controls) was incorrectly stated as AB094461. The correct accession number is AB074461. The authors apologize for any confusion this may have caused.

The quantum-Ehrenfest method with the inclusion of an IR pulse: Application to electron dynamics of the allene radical cation.

We describe the implementation of a laser control pulse in the quantum-Ehrenfest method, a molecular quantum dynamics method that solves the time-dependent Schrödinger equation for both electrons and nuclei. The oscillating electric field-dipole interaction is incorporated directly in the one-electron Hamiltonian of the electronic structure part of the algorithm. We then use the coupled electron-nuclear dynamics of the π-system in the allene radical cation (•CH2=C=CH2)+ as a simple model of a pump-control experiment. We start (pump) with a two-state superposition of two cationic states. The resulting electron dynamics corresponds to the rapid oscillation of the unpaired electron between the two terminal methylenes. This electron dynamics is, in turn, coupled to the torsional motion of the terminal methylenes. There is a conical intersection at 90° twist, where the electron dynamics collapses because the adiabatic states become degenerate. After passing the conical intersection, the electron dynamics revives. The IR pulse (control) in our simulations is timed to have its maximum at the conical intersection. Our simulations show that the effect of the (control) pulse is to change the electron dynamics at the conical intersection and, as a consequence, the concomitant nuclear dynamics, which is dominated by the change in the torsional angle.

Perspective on Kramers symmetry breaking and restoration in relativistic electronic structure methods for open-shell systems.

Without rigorous symmetry constraints, solutions to approximate electronic structure methods may artificially break symmetry. In the case of the relativistic electronic structure, if time-reversal symmetry is not enforced in calculations of molecules not subject to a magnetic field, it is possible to artificially break Kramers degeneracy in open shell systems. This leads to a description of excited states that may be qualitatively incorrect. Despite this, different electronic structure methods to incorporate correlation and excited states can partially restore Kramers degeneracy from a broken symmetry solution. For single-reference techniques, the inclusion of double and possibly triple excitations in the ground state provides much of the needed correction. Formally, however, this imbalanced treatment of the Kramers-paired spaces is a multi-reference problem, and so methods such as complete-active-space methods perform much better at recovering much of the correct symmetry by state averaging. Using multi-reference configuration interaction, any additional corrections can be obtained as the solution approaches the full configuration interaction limit. A recently proposed "Kramers contamination" value is also used to assess the magnitude of symmetry breaking.

Detection of Candidatus Neoehrlichia mikurensis in Norway up to the northern limit of Ixodes ricinus distribution using a novel real time PCR test targeting the groEL gene.

BACKGROUND: Candidatus Neoehrlichia mikurensis is an emerging tick-borne pathogen. It is widely distributed in Ixodes ricinus ticks in Europe, but knowledge of its distribution in Norway, where I. ricinus reaches its northern limit, is limited. In this study we have developed a real time PCR test for Ca. N. mikurensis and used it to investigate the distribution of Ca. N. mikurensis in Norway. RESULTS: Real time PCR targeting the groEL gene was developed and shown to be highly sensitive. It was used to detect Ca. N. mikurensis in 1651 I. ricinus nymphs and adults collected from twelve locations in Norway, from the eastern Oslo Fjord in the south to near the Arctic Circle in the north. The overall prevalence was 6.5% and varied locally between 0 and 16%. Prevalence in adults and nymphs was similar, suggesting that ticks acquire Ca. N. mikurensis predominantly during their first blood meal. In addition, 123 larvae were investigated; Ca. N. mikurensis was not found in larvae, suggesting that transovarial transmission is rare or absent. Sequence analysis suggests that a single variant dominates in Norway. CONCLUSIONS: Ca. N. mikurensis is widespread and common in ticks in Norway and reaches up to their northern limit near the Arctic Circle. Ticks appear to acquire Ca. N. mikurensis during their first blood meal. No evidence for transovarial transmission was found.

Modulating native GABAA receptors in medulloblastoma with positive allosteric benzodiazepine-derivatives induces cell death.

PURPOSE: Pediatric brain cancer medulloblastoma (MB) standard-of-care results in numerous comorbidities. MB is comprised of distinct molecular subgroups. Group 3 molecular subgroup patients have the highest relapse rates and after standard-of-care have a 20% survival. Group 3 tumors have high expression of GABRA5, which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABAAR). We are advancing a therapeutic approach for group 3 based on GABAAR modulation using benzodiazepine-derivatives. METHODS: We performed analysis of GABR and MYC expression in MB tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional 'druggable' GABAAR in group 3 cells. RESULTS: Analysis of expression of 763 MB tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of GABR genes, which code for GABAAR subunits α5, ß3 and γ2 and 3. There are ~ 1000 functional α5-GABAARs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2 × 109 ions/s. Benzodiazepines, designed to prefer α5-GABAAR, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine ('KRM-II-08') binds to the α5-GABAAR (0.8 µM EC50) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response, TP53 upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter protein localization. CONCLUSION: GABRA5 expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABAAR is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis.

De novo variants in GABRA2 and GABRA5 alter receptor function and contribute to early-onset epilepsy.

GABAA receptors are ligand-gated anion channels that are important regulators of neuronal inhibition. Mutations in several genes encoding receptor subunits have been identified in patients with various types of epilepsy, ranging from mild febrile seizures to severe epileptic encephalopathy. Using whole-genome sequencing, we identified a novel de novo missense variant in GABRA5 (c.880G > C, p.V294L) in a patient with severe early-onset epilepsy and developmental delay. Targeted resequencing of 279 additional epilepsy patients identified 19 rare variants from nine GABAA receptor genes, including a novel de novo missense variant in GABRA2 (c.875C > A, p.T292K) and a recurrent missense variant in GABRB3 (c.902C > T, p.P301L). Patients with the GABRA2 and GABRB3 variants also presented with severe epilepsy and developmental delay. We evaluated the effects of the GABRA5, GABRA2 and GABRB3 missense variants on receptor function using whole-cell patch-clamp recordings from human embryonic kidney 293T cells expressing appropriate α, ß and γ subunits. The GABRA5 p.V294L variant produced receptors that were 10-times more sensitive to GABA but had reduced maximal GABA-evoked current due to increased receptor desensitization. The GABRA2 p.T292K variant reduced channel expression and produced mutant channels that were tonically open, even in the absence of GABA. Receptors containing the GABRB3 p.P301L variant were less sensitive to GABA and produced less GABA-evoked current. These results provide the first functional evidence that de novo variants in the GABRA5 and GABRA2 genes contribute to early-onset epilepsy and developmental delay, and demonstrate that epilepsy can result from reduced neuronal inhibition via a wide range of alterations in GABAA receptor function.

The Ehrenfest method with fully quantum nuclear motion (Qu-Eh): Application to charge migration in radical cations.

An algorithm is described for quantum dynamics where an Ehrenfest potential is combined with fully quantum nuclear motion (Quantum-Ehrenfest, Qu-Eh). The method is related to the single-set variational multi-configuration Gaussian approach (vMCG) but has the advantage that only a single quantum chemistry computation is required at each time step since there is only a single time-dependent potential surface. Also shown is the close relationship to the "exact factorization method." The quantum Ehrenfest method is compared with vMCG for study of electron dynamics in a modified bismethylene-adamantane cation system. Illustrative examples of electron-nuclear dynamics are presented for a distorted allene system and for HCCI+ where one has a degenerate Π system.

Substitutions mimicking deimination and phosphorylation of 18.5-kDa myelin basic protein exert local structural effects that subtly influence its global folding.

Intrinsically-disordered proteins (IDPs) present a complex interplay of conformational variability and multifunctionality, modulated by environment and post-translational modifications. The 18.5-kDa myelin basic protein (MBP) is essential to the formation of the myelin sheath of the central nervous system and is exemplary in this regard. We have recently demonstrated that the unmodified MBP-C1 component undergoes co-operative global conformational changes in increasing concentrations of trifluoroethanol, emulating the decreasing dielectric environment that the protein encounters upon adsorption to the oligodendrocyte membrane [K.A. Vassall et al., Journal of Molecular Biology, 427, 1977-1992, 2015]. Here, we extended this study to the pseudo-deiminated MBP-C8 charge component, one found in greater proportion in developing myelin and in multiple sclerosis. A similar tri-conformational distribution as for MBP-C1 was observed with slight differences in Gibbs free energy. A more dramatic difference was observed by cathepsin D digestion of the protein in both aqueous and membrane environments, which showed significantly greater accessibility of the F42-F43 cut site of MBP-C8, indicative of a global conformational change. In contrast, this modification caused little change in the protein's density of packing on myelin-mimetic membranes as ascertained by double electron-electron resonance spectroscopy [D.R. Kattnig et al., Biochimica et Biophysica Acta (Biomembranes), 1818, 2636-2647, 2012], or in its affinity for Ca(2+)-CaM. Site-specific threonyl pseudo-phosphorylation at residues T92 and/or T95 did not appreciably affect any of the thermodynamic mechanisms of conformational transitions, susceptibility to cathepsin D, or affinity for Ca(2+)-CaM, despite previously having been shown to affect local structure and disposition on the membrane surface.

Clarithromycin in γ-aminobutyric acid-Related hypersomnolence: A randomized, crossover trial.

OBJECTIVE: Some central hypersomnolence syndromes are associated with a positive allosteric modulator of γ-aminobutyric acid (GABA)-A receptors in cerebrospinal fluid. Negative allosteric modulators of GABA-A receptors, including clarithromycin, have been reported to reduce sleepiness in these patients. We sought to systematically assess the effects of clarithromycin on objective vigilance and subjective sleepiness. METHODS: This was a 5-week, randomized, placebo-controlled, double-blind, crossover trial of clarithromycin 500mg with breakfast and lunch, in patients with hypersomnolence syndromes (excluding narcolepsy with cataplexy) and evidence for abnormal cerebrospinal fluid potentiation of GABA-A receptors. The study occurred at a university-affiliated medical center. The primary outcome measure was median reaction time on the psychomotor vigilance task (PVT) at week 2 in each condition. Secondary outcomes included the Epworth Sleepiness Scale, Stanford Sleepiness Scale, Functional Outcomes of Sleep Questionnaire, Pittsburgh Sleep Quality Index, SF-36, and additional PVT measures. RESULTS: Twenty-three patients began treatment. Three patients dropped out, and final analyses were performed on 20 complete cases. Median reaction time was not significantly different between clarithromycin and placebo. Subjective measures of sleepiness were significantly improved on clarithromycin versus placebo. Altered taste perception occurred, but was the only side effect more common on clarithromycin than placebo. No serious adverse events occurred. INTERPRETATION: Subjective sleepiness, but not psychomotor vigilance, improved during a 2-week course of clarithromycin. Although additional studies are needed, this suggests that clarithromycin may be a reasonable treatment option in patients with treatment-refractory hypersomnolence. This trial was registered at ClinicalTrials.gov (NCT01146600) and supported by the American Sleep Medicine Foundation.

Effects of γ-Aminobutyric Acid Type A Receptor Modulation by Flumazenil on Emergence from General Anesthesia.

BACKGROUND: Transitions into conscious states are partially mediated by inactivation of sleep networks and activation of arousal networks. Pharmacologic hastening of emergence from general anesthesia has largely focused on activating subcortical monoaminergic networks, with little attention on antagonizing the γ-aminobutyric acid type A receptor (GABAAR). As the GABAAR mediates the clinical effects of many common general anesthetics, the authors hypothesized that negative GABAAR modulators would hasten emergence, possibly via cortical networks involved in sleep. METHODS: The authors investigated the capacity of the benzodiazepine rescue agent, flumazenil, which had been recently shown to promote wakefulness in hypersomnia patients, to alter emergence. Using an in vivo rodent model and an in vitro GABAAR heterologous expression system, they measured flumazenil's effects on behavioral, neurophysiologic, and electrophysiologic correlates of emergence from isoflurane anesthesia. RESULTS: Animals administered intravenous flumazenil (0.4 mg/kg, n = 8) exhibited hastened emergence compared to saline-treated animals (n = 8) at cessation of isoflurane anesthesia. Wake-like electroencephalographic patterns occurred sooner and exhibited more high-frequency electroencephalography power after flumazenil administration (median latency ± median absolute deviation: 290 ± 34 s) compared to saline administration (473 ± 186 s; P = 0.042). Moreover, in flumazenil-treated animals, there was a decreased impact on postanesthesia sleep. In vitro experiments in human embryonic kidney-293T cells demonstrated that flumazenil inhibited isoflurane-mediated GABA current enhancement (n = 34 cells, 88.7 ± 2.42% potentiation at 3 µM). Moreover, flumazenil exhibited weak agonist activity on the GABAAR (n = 10 cells, 10.3 ± 3.96% peak GABA EC20 current at 1 µM). CONCLUSIONS: Flumazenil can modulate emergence from isoflurane anesthesia. The authors highlight the complex role GABAARs play in mediating consciousness and provide mechanistic links between emergence from anesthesia and arousal.