Elucidation of a dynamic interplay between a beta-2 adrenergic receptor, its agonist, and stimulatory G protein.

G protein-coupled receptors (GPCRs) represent the largest group of membrane receptors for transmembrane signal transduction. Ligand-induced activation of GPCRs triggers G protein activation followed by various signaling cascades. Understanding the structural and energetic determinants of ligand binding to GPCRs and GPCRs to G proteins is crucial to the design of pharmacological treatments targeting specific conformations of these proteins to precisely control their signaling properties. In this study, we focused on interactions of a prototypical GPCR, beta-2 adrenergic receptor (ß2AR), with its endogenous agonist, norepinephrine (NE), and the stimulatory G protein (Gs). Using molecular dynamics (MD) simulations, we demonstrated the stabilization of cationic NE, NE(+), binding to ß2AR by Gs protein recruitment, in line with experimental observations. We also captured the partial dissociation of the ligand from ß2AR and the conformational interconversions of Gs between closed and open conformations in the NE(+)-ß2AR-Gs ternary complex while it is still bound to the receptor. The variation of NE(+) binding poses was found to alter Gs α subunit (Gsα) conformational transitions. Our simulations showed that the interdomain movement and the stacking of Gsα α1 and α5 helices are significant for increasing the distance between the Gsα and ß2AR, which may indicate a partial dissociation of Gsα The distance increase commences when Gsα is predominantly in an open state and can be triggered by the intracellular loop 3 (ICL3) of ß2AR interacting with Gsα, causing conformational changes of the α5 helix. Our results help explain molecular mechanisms of ligand and GPCR-mediated modulation of G protein activation.

MISpheroID: a knowledgebase and transparency tool for minimum information in spheroid identity.

Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.

Phenonaut: multiomics data integration for phenotypic space exploration.

SUMMARY: Data integration workflows for multiomics data take many forms across academia and industry. Efforts with limited resources often encountered in academia can easily fall short of data integration best practices for processing and combining high-content imaging, proteomics, metabolomics, and other omics data. We present Phenonaut, a Python software package designed to address the data workflow needs of migration, control, integration, and auditability in the application of literature and proprietary techniques for data source and structure agnostic workflow creation. AVAILABILITY AND IMPLEMENTATION: Source code: https://github.com/CarragherLab/phenonaut, Documentation: https://carragherlab.github.io/phenonaut, PyPI package: https://pypi.org/project/phenonaut/.

High throughput screening identifies dasatinib as synergistic with trametinib in low grade serous ovarian carcinoma.

BACKGROUND: Low grade serous ovarian carcinoma (LGSOC) is a distinct histotype of ovarian cancer characterised high levels of intrinsic chemoresistance, highlighting the urgent need for new treatments. High throughput screening in clinically-informative cell-based models represents an attractive strategy for identifying candidate treatment options for prioritisation in clinical studies. METHODS: We performed a high throughput drug screen of 1610 agents across a panel of 6 LGSOC cell lines (3 RAS/RAF-mutant, 3 RAS/RAF-wildtype) to identify novel candidate therapeutic approaches. Validation comprised dose-response analysis across 9 LGSOC models and 5 high grade serous comparator lines. RESULTS: 16 hits of 1610 screened compounds were prioritised for validation based on >50% reduction in nuclei counts in over half of screened cell lines at 1000 nM concentration. 11 compounds passed validation, and the four agents of greatest interest (dasatinib, tyrosine kinase inhibitor; disulfiram, aldehyde dehydrogenase inhibitor; carfilzomib, proteasome inhibitor; romidepsin, histone deacetylase inhibitor) underwent synergy profiling with the recently approved MEK inhibitor trametinib. Disulfiram demonstrated excellent selectivity for LGSOC versus high grade serous ovarian carcinoma comparator lines (P = 0.003 for IC50 comparison), while the tyrosine kinase inhibitor dasatinib demonstrated favourable synergy with trametinib across multiple LGSOC models (maximum zero interaction potency synergy score 46.9). The novel, highly selective Src family kinase (SFK) inhibitor NXP900 demonstrated a similar trametinib synergy profile to dasatinib, suggesting that SFK inhibition is the likely driver of synergy. CONCLUSION: Dasatinib and other SFK inhibitors represent novel candidate treatments for LGSOC and demonstrate synergy with trametinib. Disulfiram represents an additional treatment strategy worthy of investigation.

The application of the "inverse problem" method for constructing confining potentials that make N-soliton waveforms exact solutions in the Gross-Pitaevskii equation.

In this work, we discuss an application of the "inverse problem" method to find the external trapping potential, which has particular N trapped soliton-like solutions of the Gross-Pitaevskii equation (GPE) also known as the cubic nonlinear Schrödinger equation (NLSE). This inverse method assumes particular forms for the trapped soliton wave function, which then determines the (unique) external (confining) potential. The latter renders these assumed waveforms exact solutions of the GPE (NLSE) for both attractive (g<0) and repulsive (g>0) self-interactions. For both signs of g, we discuss the stability with respect to self-similar deformations and translations. For g<0, a critical mass Mc or equivalently the number of particles for instabilities to arise can often be found analytically. On the other hand, for the case with g>0 corresponding to repulsive self-interactions which is often discussed in the atomic physics realm of Bose-Einstein condensates, the bound solutions are found to be always stable. For g<0, we also determine the critical mass numerically by using linear stability or Bogoliubov-de Gennes analysis, and compare these results with our analytic estimates. Various analytic forms for the trapped N-soliton solutions in one, two, and three spatial dimensions are discussed, including sums of Gaussians or higher-order eigenfunctions of the harmonic oscillator Hamiltonian.

Anti-obesity and metabolic benefits of metformin: Comparison of different delivery routes.

Obesity is a severe public health problem. Healthy lifestyle interventions are commonly recommended for fighting obesity. But they are hard to follow and have low efficacy. Pharmacotherapy and surgery are of high efficacy but are beset with side effects. Browning subcutaneous white adipose tissue (WAT) is a practical and efficient approach for combating obesity. Metformin, a commonly used FDA-approved antidiabetic drug, is potent to induce browning of WAT through phosphorylation and activation of AMP-activated protein kinase. However, oral administration of metformin has low oral bioavailability, fast renal clearance, and low target specificity that limit metformin's application in browning WAT. Local and transdermal delivery of metformin directly to subcutaneous WAT using injection or microneedle (MN) in combination with iontophoresis (INT) may solve these problems. In this paper, we administered metformin to C57BL/6J obese mice using the following three routes: transdermal delivery (MN and INT), local injection into inguinal WAT (IgWAT, a type of subcutaneous WAT in mice), and oral gavage. The anti-obesity and metabolic effects of metformin via these delivery routes were determined and compared. As compared to local IgWAT injection and oral gavage delivery, transdermal delivery of metformin using MN and INT resulted in 9% lower body weight and 7% decrease in body fat% accompanied by improved energy metabolism and decreased inflammation through browning IgWAT in obese C57BL/6J mice. Transdermal delivery of metformin using MN and INT is an effective approach in browning subcutaneous WAT for combating obesity and improving metabolic health.

A multiscale predictive digital twin for neurocardiac modulation.

Cardiac function is tightly regulated by the autonomic nervous system (ANS). Activation of the sympathetic nervous system increases cardiac output by increasing heart rate and stroke volume, while parasympathetic nerve stimulation instantly slows heart rate. Importantly, imbalance in autonomic control of the heart has been implicated in the development of arrhythmias and heart failure. Understanding of the mechanisms and effects of autonomic stimulation is a major challenge because synapses in different regions of the heart result in multiple changes to heart function. For example, nerve synapses on the sinoatrial node (SAN) impact pacemaking, while synapses on contractile cells alter contraction and arrhythmia vulnerability. Here, we present a multiscale neurocardiac modelling and simulator tool that predicts the effect of efferent stimulation of the sympathetic and parasympathetic branches of the ANS on the cardiac SAN and ventricular myocardium. The model includes a layered representation of the ANS and reproduces firing properties measured experimentally. Model parameters are derived from experiments and atomistic simulations. The model is a first prototype of a digital twin that is applied to make predictions across all system scales, from subcellular signalling to pacemaker frequency to tissue level responses. We predict conditions under which autonomic imbalance induces proarrhythmia and can be modified to prevent or inhibit arrhythmia. In summary, the multiscale model constitutes a predictive digital twin framework to test and guide high-throughput prediction of novel neuromodulatory therapy. KEY POINTS: A multi-layered model representation of the autonomic nervous system that includes sympathetic and parasympathetic branches, each with sparse random intralayer connectivity, synaptic dynamics and conductance based integrate-and-fire neurons generates firing patterns in close agreement with experiment. A key feature of the neurocardiac computational model is the connection between the autonomic nervous system and both pacemaker and contractile cells, where modification to pacemaker frequency drives initiation of electrical signals in the contractile cells. We utilized atomic-scale molecular dynamics simulations to predict the association and dissociation rates of noradrenaline with the ß-adrenergic receptor. Multiscale predictions demonstrate how autonomic imbalance may increase proclivity to arrhythmias or be used to terminate arrhythmias. The model serves as a first step towards a digital twin for predicting neuromodulation to prevent or reduce disease.

Increasing the calcium sensitivity of muscle using trifluoperazine-induced manipulations in silico, in vitro and in vivo systems.

Many therapeutics for cardiomyopathy treat the symptoms of the disease rather than the underlying mechanism. The mechanism of cardiomyopathy onset is believed to include two means: calcium sensitivity changes and myosin activity alteration. Trifluoperazine is a compound that binds troponin, and other components of the calcium pathway, which impacts calcium regulation of contraction. Here, the ability of TFP to shift calcium sensitivity was examined in vitro with purified proteins and the impact of TFP on heart function was assessed in vivo using embryonic zebrafish. The binding of TFP to troponin was modeled in silico and a model of zebrafish troponin was generated. TFP increased regulated cardiac actomyosin activity in vitro and elevated embryonic zebrafish heart rates at effective drug concentrations. Troponin structural changes predicted in silico suggest altered protein interactions within thin filaments that would affect the regulation of heart function.

Use of advance directives to promote supported decision-making in mental health care: Implications of international trends for reform in New Zealand.

Advance directives are advocated, in many jurisdictions, as a way to promote supported decision-making for people who use mental health services and to promote countries' compliance with their obligations under the United Nations Convention on the Rights of Persons with Disabilities. The United Nations Convention on the Rights of Persons with Disabilities promotes the use of tools to further personal autonomy which would include integrating the use of advance directives into mental health law, to clarify the effect (or force) an advance directive carries when its maker comes under the relevant mental health legislation. In addition, securing the active use of advance directives requires adoption of certain supportive practices and policies within health services. Here, we discuss a number of approaches taken to advance directives in revised mental health legislation, and the associated practices we think are required.

A Novel High-Content Phenotypic Screen To Identify Inhibitors of Mitochondrial DNA Maintenance in Trypanosomes.

Kinetoplastid parasites cause diverse neglected diseases in humans and livestock, with an urgent need for new treatments. The survival of kinetoplastids depends on their uniquely structured mitochondrial genome (kDNA), the eponymous kinetoplast. Here, we report the development of a high-content screen for pharmacologically induced kDNA loss, based on specific staining of parasites and automated image analysis. As proof of concept, we screened a diverse set of ∼14,000 small molecules and exemplify a validated hit as a novel kDNA-targeting compound.

A Computational Pipeline to Predict Cardiotoxicity: From the Atom to the Rhythm.

RATIONALE: Drug-induced proarrhythmia is so tightly associated with prolongation of the QT interval that QT prolongation is an accepted surrogate marker for arrhythmia. But QT interval is too sensitive a marker and not selective, resulting in many useful drugs eliminated in drug discovery. OBJECTIVE: To predict the impact of a drug from the drug chemistry on the cardiac rhythm. METHODS AND RESULTS: In a new linkage, we connected atomistic scale information to protein, cell, and tissue scales by predicting drug-binding affinities and rates from simulation of ion channel and drug structure interactions and then used these values to model drug effects on the hERG channel. Model components were integrated into predictive models at the cell and tissue scales to expose fundamental arrhythmia vulnerability mechanisms and complex interactions underlying emergent behaviors. Human clinical data were used for model framework validation and showed excellent agreement, demonstrating feasibility of a new approach for cardiotoxicity prediction. CONCLUSIONS: We present a multiscale model framework to predict electrotoxicity in the heart from the atom to the rhythm. Novel mechanistic insights emerged at all scales of the system, from the specific nature of proarrhythmic drug interaction with the hERG channel, to the fundamental cellular and tissue-level arrhythmia mechanisms. Applications of machine learning indicate necessary and sufficient parameters that predict arrhythmia vulnerability. We expect that the model framework may be expanded to make an impact in drug discovery, drug safety screening for a variety of compounds and targets, and in a variety of regulatory processes.

Trichoplein binds PCM1 and controls endothelial cell function by regulating autophagy.

Autophagy is an essential cellular quality control process that has emerged as a critical one for vascular homeostasis. Here, we show that trichoplein (TCHP) links autophagy with endothelial cell (EC) function. TCHP localizes to centriolar satellites, where it binds and stabilizes PCM1. Loss of TCHP leads to delocalization and proteasome-dependent degradation of PCM1, further resulting in degradation of PCM1's binding partner GABARAP. Autophagic flux under basal conditions is impaired in THCP-depleted ECs, and SQSTM1/p62 (p62) accumulates. We further show that TCHP promotes autophagosome maturation and efficient clearance of p62 within lysosomes, without affecting their degradative capacity. Reduced TCHP and high p62 levels are detected in primary ECs from patients with coronary artery disease. This phenotype correlates with impaired EC function and can be ameliorated by NF-κB inhibition. Moreover, Tchp knock-out mice accumulate of p62 in the heart and cardiac vessels correlating with reduced cardiac vascularization. Taken together, our data reveal that TCHP regulates endothelial cell function via an autophagy-mediated mechanism.

Does pelvic incidence tell us the risk of proximal junctional kyphosis in adult spinal deformity surgery?

PURPOSE: To investigate the relationship between pelvic incidence (PI) and proximal junctional kyphosis (PJK) in patients treated surgically for adult spinal deformity (ASD) with fusion from thoracolumbar junction to sacrum. METHODS: A consecutive series of ASD patients who underwent fusion from the thoracolumbar junction to the sacrum with a minimum of 2-year follow-up was studied. Patients were divided into low PI (≤ 50°) and high PI (> 50°) groups. We compared radiographic parameters and the rates of PJK, between the two groups. A sub-analysis was performed on patients with a postoperative PI minus lumbar lordosis mismatch between - 10° and 10° (i.e., ideally corrected). RESULTS: Sixty-three patients were included: 19 low PI and 44 high PI. Median follow-up was 34 months (range 24-103). Overall PJK rate was 38%. PJK was observed in 16% of low PI and 48% of high PI patients (p = 0.02). The odds ratio for developing PJK with a high PI compared to a low PI was 4.9 (p = 0.03). There were 32 ideally corrected patients. Eleven of these were in the low PI group, and 21 patients were in the high PI group. The incidence of PJK was 25% for ideally corrected patients. PJK occurred in none of these patients in the low PI group and 38% of patients in the high PI group (p = 0.03). CONCLUSION: When the upper-instrumented vertebra includes the thoracolumbar junction, patients with a PI > 50° are at a significantly higher risk of developing PJK compared to patients with a PI ≤ 50°.

Greater protein quality of an egg breakfast may be inadequate to induce satiety during weight loss, compared with a cereal breakfast of equal protein quantity.

We compared the effects of consuming egg-breakfast of superior protein quality to cereal-breakfast of similar energy density and protein quantity, but lower protein quality. Two, two-week randomised crossover clinical trials included 30 otherwise healthy women with overweight or obesity. Subjects received counselling to follow a reduced-calorie diet. Under supervision, participants consumed either breakfast for one-week then crossed over to the opposite breakfast. Experiment-1 outcome variables included post-breakfast appetite hormones, glucose and insulin, subjective markers of satiety and energy intake at lunch and dinner. In Experiment-2, an appealing food (brownies) was included in lunch. Following the breakfasts, Experiment-1 showed no significant differences in outcome variables. In Experiment-2, the egg-breakfast increased fullness (p = 0.038), but lunch-time energy intake was not different. If these findings apply to other breakfasts, it suggests that in comparing two breakfasts with similar protein quantity, the greater protein quality of a breakfast may not be adequate to induce satiety.

Molecular determinants of pro-arrhythmia proclivity of d- and l-sotalol via a multi-scale modeling pipeline.

Drug isomers may differ in their proarrhythmia risk. An interesting example is the drug sotalol, an antiarrhythmic drug comprising d- and l- enantiomers that both block the hERG cardiac potassium channel and confer differing degrees of proarrhythmic risk. We developed a multi-scale in silico pipeline focusing on hERG channel - drug interactions and used it to probe and predict the mechanisms of pro-arrhythmia risks of the two enantiomers of sotalol. Molecular dynamics (MD) simulations predicted comparable hERG channel binding affinities for d- and l-sotalol, which were validated with electrophysiology experiments. MD derived thermodynamic and kinetic parameters were used to build multi-scale functional computational models of cardiac electrophysiology at the cell and tissue scales. Functional models were used to predict inactivated state binding affinities to recapitulate electrocardiogram (ECG) QT interval prolongation observed in clinical data. Our study demonstrates how modeling and simulation can be applied to predict drug effects from the atom to the rhythm for dl-sotalol and also increased proarrhythmia proclivity of d- vs. l-sotalol when accounting for stereospecific beta-adrenergic receptor blocking.

A short, attribution theory-based video intervention does not reduce weight bias in a nationally representative sample of registered dietitians: a randomized trial.

BACKGROUND/OBJECTIVES: Weight bias among registered dietitians (RDs) is a concern and effective interventions to reduce weight bias are sparse. Our objective was to determine if a short, attribution theory-based online video intervention would reduce weight bias in RDs. SUBJECTS/METHODS: Dietitians from a nationally representative sample were recruited for a randomized, parallel-arm study with online surveys at pre-, post-intervention and 1-month follow-up. One hundred and forty-seven RDs who watched one of three videos embedded in an online survey from June to August 2019 were considered for the analysis. RDs were randomized to watch either the intervention, positive control, or negative control video. The primary outcome was the change in the "blame" component of the Anti-Fat Attitude Test (AFAT) from pre-to immediate post-intervention. Differences in changes in AFAT and Implicit Association Test (IAT) scores across treatment groups were assessed via linear models; multiple imputation were performed for missing data. RESULTS: Baseline demographics, AFAT and IAT scores of the 147 participants who watched a video were not significantly different between the study groups (p > 0.05). The intervention group's AFAT-blame score reduced by an average of 0.05 between pre- and immediate post-intervention but was not statistically significant (p = 0.76, confidence intervals (CI) = -0.40, 0.30). Furthermore, there were no significant changes for AFAT-social, AFAT-physical subscores, and IAT within or between groups between pre- and immediate post-intervention (p > 0.05). Due to high attrition rates, the changes at 1-month follow-up are not reported. CONCLUSIONS: This study was the first to explore the effectiveness of an online video intervention to reduce weight bias in RDs. This study was unable to detect a significant impact of a short, attribution theory-based video intervention on weight bias in practicing RDs and future larger studies are needed to confirm our findings.

Errors in the implementation, analysis, and reporting of randomization within obesity and nutrition research: a guide to their avoidance.

Randomization is an important tool used to establish causal inferences in studies designed to further our understanding of questions related to obesity and nutrition. To take advantage of the inferences afforded by randomization, scientific standards must be upheld during the planning, execution, analysis, and reporting of such studies. We discuss ten errors in randomized experiments from real-world examples from the literature and outline best practices for their avoidance. These ten errors include: representing nonrandom allocation as random, failing to adequately conceal allocation, not accounting for changing allocation ratios, replacing subjects in nonrandom ways, failing to account for non-independence, drawing inferences by comparing statistical significance from within-group comparisons instead of between-groups, pooling data and breaking the randomized design, failing to account for missing data, failing to report sufficient information to understand study methods, and failing to frame the causal question as testing the randomized assignment per se. We hope that these examples will aid researchers, reviewers, journal editors, and other readers to endeavor to a high standard of scientific rigor in randomized experiments within obesity and nutrition research.

The Dark Side of Actin: Cardiac actin variants highlight the role of allostery in disease development.

Mutations in the α-cardiac actin ACTC1 gene cause dilated or hypertrophic cardiomyopathy. These diseases are the result of changes in protein interactions between ACTC protein and force-generating ß-myosin or the calcium-dependent cardiac-tropomyosin (cTm) and cardiac troponin (cTn) regulatory complex, altering the overall contractile force. The T126I and S271F ACTC variants possess amino acid substitutions on the other side of actin relative to the myosin or regulatory protein binding sites on what we call the "dark side" of actin. The T126I change results in hyposensitivity to calcium, in accordance with the calcium sensitivity pathway of cardiomyopathy development while the S271F change alters the maximum in vitro motility sliding speed, reflecting a change in maximum force. These results demonstrate the role of actin allostery in the cardiac disease development.

High content phenotypic screening identifies serotonin receptor modulators with selective activity upon breast cancer cell cycle and cytokine signaling pathways.

Heterogeneity in disease mechanisms between genetically distinct patients contributes to high attrition rates in late stage clinical drug development. New personalized medicine strategies aim to identify predictive biomarkers which stratify patients most likely to respond to a particular therapy. However, for complex multifactorial diseases not characterized by a single genetic driver, empirical approaches to identifying predictive biomarkers and the most promising therapies for personalized medicine are required. In vitro pharmacogenomics seeks to correlate in vitro drug sensitivity testing across panels of genetically distinct cell models with genomic, gene expression or proteomic data to identify predictive biomarkers of drug response. However, the vast majority of in vitro pharmacogenomic studies performed to date are limited to dose-response screening upon a single viability assay endpoint. In this article we describe the application of multiparametric high content phenotypic screening and the theta comparative cell scoring method to quantify and rank compound hits, screened at a single concentration, which induce a broad variety of divergent phenotypic responses between distinct breast cancer cell lines. High content screening followed by transcriptomic pathway analysis identified serotonin receptor modulators which display selective activity upon breast cancer cell cycle and cytokine signaling pathways correlating with inhibition of cell growth and survival. These methods describe a new evidence-led approach to rapidly identify compounds which display distinct response between different cell types. The results presented also warrant further investigation of the selective activity of serotonin receptor modulators upon breast cancer cell growth and survival as a potential drug repurposing opportunity.

Assembly and Maintenance of Sarcomere Thin Filaments and Associated Diseases.

Sarcomere assembly and maintenance are essential physiological processes required for cardiac and skeletal muscle function and organism mobility. Over decades of research, components of the sarcomere and factors involved in the formation and maintenance of this contractile unit have been identified. Although we have a general understanding of sarcomere assembly and maintenance, much less is known about the development of the thin filaments and associated factors within the sarcomere. In the last decade, advancements in medical intervention and genome sequencing have uncovered patients with novel mutations in sarcomere thin filaments. Pairing this sequencing with reverse genetics and the ability to generate patient avatars in model organisms has begun to deepen our understanding of sarcomere thin filament development. In this review, we provide a summary of recent findings regarding sarcomere assembly, maintenance, and disease with respect to thin filaments, building on the previous knowledge in the field. We highlight debated and unknown areas within these processes to clearly define open research questions.