Canadian pediatric eating disorder programs and virtual care during the COVID-19 pandemic: a mixed-methods approach to understanding clinicians' perspectives.

BACKGROUND: As a result of the public health measures put in place during the COVID-19 pandemic in Canada, many health services, including those for the treatment of eating disorders, were provided at a distance. This study aims to describe the adaptations made in specialized pediatric eating disorder programs in Canada and the impact of these adaptations on health professionals' experience of providing care. METHODS: A mixed-methods design was used to survey healthcare professionals working in specialized pediatric eating disorder programs about adaptations to treatment made during the pandemic and the impact of these adaptations on their experience of providing care. Data were collected between October 2021 and March 2022 using a cross-sectional survey comprising 25 questions and via semi-structured interviews. Quantitative data were summarized using descriptive statistics and qualitative data were interpreted using qualitative content analysis. RESULTS: Eighteen healthcare professionals in Canada completed the online survey, of whom six also participated in the semi-structured interviews. The cross-sectional survey confirmed that, unlike in pre-pandemic times, the majority of participants provided medical care (15/18) and mental health care (17/18) at a distance during the pandemic, with most participants using telephone (17/18) and videoconferencing (17/18). Most (16/18) health professionals indicated that virtual care would continue to be used as a tool in pediatric ED treatment after the pandemic. Participants used a combination of virtual and in-person care, with most reporting weighing patients both in clinic (16/18) and virtually (15/18). Qualitative content analysis generated five themes: (1) responding to increased demand with insufficient resources; (2) adapting to changes in care due to the COVID-19 pandemic; (3) dealing with uncertainty and apprehension; (4) virtual care as an acceptable and useful clinical tool, and (5) optimal conditions and future expectations. Most interview participants (5/6) had globally positive views of virtual care. CONCLUSIONS: Providing virtual multidisciplinary treatment for children and adolescents with eating disorders seemed feasible and acceptable to professionals during the pandemic. Moving forward, focusing on health professionals' perspectives and providing appropriate training in virtual interventions is essential given their central role in successful implementation and continued use of virtual and hybrid care models.

Mapping the conformational landscape of the stimulatory heterotrimeric G protein.

Heterotrimeric G proteins serve as membrane-associated signaling hubs, in concert with their cognate G-protein-coupled receptors. Fluorine nuclear magnetic resonance spectroscopy was employed to monitor the conformational equilibria of the human stimulatory G-protein α subunit (Gsα) alone, in the intact Gsαß1γ2 heterotrimer or in complex with membrane-embedded human adenosine A2A receptor (A2AR). The results reveal a concerted equilibrium that is strongly affected by nucleotide and interactions with the ßγ subunit, the lipid bilayer and A2AR. The α1 helix of Gsα exhibits significant intermediate timescale dynamics. The α4ß6 loop and α5 helix undergo membrane/receptor interactions and order-disorder transitions respectively, associated with G-protein activation. The αN helix adopts a key functional state that serves as an allosteric conduit between the ßγ subunit and receptor, while a significant fraction of the ensemble remains tethered to the membrane and receptor upon activation.

An Intensive Ambulatory Care Program for Adolescents With Eating Disorders Combining In-Person and Web-Based Care: Protocol for a Single-Site Naturalistic Trial.

BACKGROUND: The incidence of eating disorders (EDs) among adolescents has significantly increased since the beginning of the COVID-19 pandemic. Hybrid care, which combines web-based and in-person modalities, is a promising approach for adolescents with EDs but remains understudied in this population. OBJECTIVE: We aimed to implement a novel hybrid (web-based and in-person) intensive ambulatory care program for youth and evaluate its feasibility, acceptability, and preliminary effectiveness. METHODS: We will use a naturalistic pretest-posttest design to evaluate our proposed pilot Intensive Ambulatory Care Program (IACP). This novel type of day hospital care follows evidence-based principles and uses a family-centered, educational, and motivational approach. It will be tailored to the psychological needs of each participant and will be delivered in a hybrid format. A total of 100 participants meeting the DSM-5 (Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition) criteria for EDs, aged 12-18 years, will be recruited over the 2-year trial period. We will examine recruitment, retention, and adhesion-to-protocol rates; participant and family satisfaction; and preliminary effectiveness using quantitative self-report questionnaires. RESULTS: Rolling recruitment will take place from winter 2022 to fall 2023, during which time we expect to recruit approximately 80% (100/120) of eligible participants, retain at least 75% (75/100) of enrolled participants and have at least 70% (70/100) of enrolled participants complete at least one therapeutic session per week and all pre- and postintervention questionnaires. Data collection will occur concurrently. We base our recruitment and retention estimates on previous literature and consider that the highly flexible design of the IACP and the fact that no extra work will be required of individuals in the program to participate in the study, will lead to high levels of feasibility. We anticipate that participants and their families will be satisfied with both the program and hybrid delivery format. We expect that participation in the IACP will be associated with a medium effect size reduction in ED psychopathology from baseline to end of treatment. The data analysis and manuscript writing are expected to be completed by the summer of 2024. CONCLUSIONS: Given the high clinical burden associated with EDs, this study has the potential to fill an important research gap by testing the implementation of a novel hybrid mode of intervention. If feasible, acceptable, and effective, the IACP could lead to important improvements in health care services for adolescents with EDs. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/37420.

The use of technology in the treatment of youth with eating disorders: A scoping review.

BACKGROUND: Adolescence and young adulthood is a high-risk period for the development of eating disorders. In recent years, there has been an increase in use of technology-based interventions (TBIs) for the treatment of eating disorders. The objective of this study was to determine the types of technology used for eating disorder treatment in youth and their effectiveness. METHODS: A scoping review was conducted according to PRISMA-ScR guidelines. Four databases were searched. Eligible articles included: (1) a TBI (2) participants with a mean age between 10- and 25-years and meeting DSM-IV or DSM-5 criteria for any eating disorder and (3) qualitative or quantitative designs. Quantitative and qualitative studies were assessed for quality. RESULTS: The search identified 1621 articles. After screening of titles and abstracts, 130 articles were read in full and assessed for eligibility by two raters. Forty-nine (29 quantitative and 20 qualitative, observational, or mixed methods studies) met inclusion criteria. Quality ratings indicated that 78% of quantitative studies had a low risk of bias and 22% had a moderate risk. Technologies reviewed in our study included videoconference therapy, mobile applications, and online self-help. We considered interventions used both within sessions with clinicians as well as those used in between sessions by patients alone. Fifteen of 18 (83%) quantitative studies found that TBIs reduce eating disorder symptomatology, with nine of those reporting medium-to-large effect sizes. Qualitative data was of high quality and suggested that virtual interventions are acceptable in this population. CONCLUSIONS: Although identified studies are of high quality, they are limited in number. More research is needed, particularly regarding videoconferencing and mobile applications. Nonetheless, TBIs show promise for the treatment of eating disorders in youth. TRIAL REGISTRATION: Not applicable.

The use of virtual care to treat adolescents and young adults with eating disorders has increased in recent years. To better understand what types of technologies are being used in virtual care and these technologies' effectiveness, we systematically reviewed research studies that discussed the use of any technology to treat youth (which was defined as young people aged 25 years or under) with an eating disorder. We found 49 relevant studies. They revealed that videoconference therapy, mobile applications, and online self-help are most frequently used for virtual treatment. Of the 18 studies evaluating the effectiveness of these technologies, 15 found virtual care to be effective for reducing eating disorder symptoms. Studies describing patient experiences with these technologies, of which there were nine, suggested that patients overall were satisfied with receiving virtual care. Although these results are promising, research on virtual treatment is still lacking, especially on videoconferencing therapy and mobile applications.

Allosteric modulation of the adenosine A2A receptor by cholesterol.

Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A2A receptor (A2AR) in reconstituted phospholipid nanodiscs. This model system conveniently excludes possible effects arising from cholesterol-induced phase separation or receptor oligomerization and focuses on the question of allostery. GTP hydrolysis assays show that cholesterol weakly enhances the basal signaling of A2AR while decreasing the agonist EC50. Fluorine nuclear magnetic resonance (19F NMR) spectroscopy shows that this enhancement arises from an increase in the receptor's active state population and a G-protein-bound precoupled state. 19F NMR of fluorinated cholesterol analogs reveals transient interactions with A2AR, indicating a lack of high-affinity binding or direct allosteric modulation. The combined results suggest that the observed allosteric effects are largely indirect and originate from cholesterol-mediated changes in membrane properties, as shown by membrane fluidity measurements and high-pressure NMR.

Structures and Dynamics of Native-State Transmembrane Protein Targets and Bound Lipids.

Membrane proteins work within asymmetric bilayers of lipid molecules that are critical for their biological structures, dynamics and interactions. These properties are lost when detergents dislodge lipids, ligands and subunits, but are maintained in native nanodiscs formed using styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA) copolymers. These amphipathic polymers allow extraction of multicomponent complexes of post-translationally modified membrane-bound proteins directly from organ homogenates or membranes from diverse types of cells and organelles. Here, we review the structures and mechanisms of transmembrane targets and their interactions with lipids including phosphoinositides (PIs), as resolved using nanodisc systems and methods including cryo-electron microscopy (cryo-EM) and X-ray diffraction (XRD). We focus on therapeutic targets including several G protein-coupled receptors (GPCRs), as well as ion channels and transporters that are driving the development of next-generation native nanodiscs. The design of new synthetic polymers and complementary biophysical tools bodes well for the future of drug discovery and structural biology of native membrane:protein assemblies (memteins).

Advances in the study of GPCRs by 19F NMR.

Crystallography and cryo-electron microscopy have advanced atomic resolution perspectives of inactive and active states of G protein-coupled receptors (GPCRs), alone and in complex with G proteins or arrestin. 19F NMR can play a role in ascertaining activation mechanisms and understanding the complete energy landscape associated with signal transduction. Fluorinated reporters are introduced biosynthetically via fluorinated amino acid analogs or chemically, via thiol-specific fluorinated reporters. The chemical shift sensitivity of these reporters makes it possible to discern details of conformational ensembles. In addition to spectroscopic details, paramagnetic species can be incorporated through orthogonal techniques to obtain distance information on fluorinated reporters, while T2-and T1-based relaxation experiments provide details on exchange kinetics in addition to fluctuations within a given state.

Hybridization of ß-Adrenergic Agonists and Antagonists Confers G Protein Bias.

Starting from the ß-adrenoceptor agonist isoprenaline and beta-blocker carvedilol, we designed and synthesized three different chemotypes of agonist/antagonist hybrids. Investigations of ligand-mediated receptor activation using bioluminescence resonance energy transfer biosensors revealed a predominant effect of the aromatic head group on the intrinsic activity of our ligands, as ligands with a carvedilol head group were devoid of agonistic activity. Ligands composed of a catechol head group and an antagonist-like oxypropylene spacer possess significant intrinsic activity for the activation of Gαs, while they only show weak or even no ß-arrestin-2 recruitment at both ß1- and ß2-AR. Molecular dynamics simulations suggest that the difference in G protein efficacy and ß-arrestin recruitment of the hybrid ( S)-22, the full agonist epinephrine, and the ß2-selective, G protein-biased partial agonist salmeterol depends on specific hydrogen bonding between Ser5.46 and Asn6.55, and the aromatic head group of the ligands.

Bioluminescence resonance energy transfer-based imaging of protein-protein interactions in living cells.

Bioluminescence resonance energy transfer (BRET) is a transfer of energy between a luminescence donor and a fluorescence acceptor. Because BRET occurs when the distance between the donor and acceptor is <10 nm, and its efficiency is inversely proportional to the sixth power of distance, it has gained popularity as a proximity-based assay to monitor protein-protein interactions and conformational rearrangements in live cells. In such assays, one protein of interest is fused to a bioluminescent energy donor (luciferases from Renilla reniformis or Oplophorus gracilirostris), and the other protein is fused to a fluorescent energy acceptor (such as GFP or YFP). Because the BRET donor does not require an external light source, it does not lead to phototoxicity or autofluorescence. It therefore represents an interesting alternative to fluorescence-based imaging such as FRET. However, the low signal output of BRET energy donors has limited the spatiotemporal resolution of BRET imaging. Here, we describe how recent improvements in detection devices and BRET probes can be used to markedly improve the resolution of BRET imaging, thus widening the field of BRET imaging applications. The protocol described herein involves three main stages. First, cell preparation and transfection require 3 d, including cell culture time. Second, image acquisition takes 10-120 min per sample, after an initial 60 min for microscope setup. Finally, image analysis typically takes 1-2 h. The choices of energy donor, acceptor, luminescent substrates, cameras and microscope setup, as well as acquisition modes to be used for different applications, are also discussed.

Structural Insight into G Protein-Coupled Receptor Signaling Efficacy and Bias between Gs and ß-Arrestin.

G protein-coupled receptors (GPCRs) form the largest family of membrane proteins involved in signal transduction. Because of their ability to regulate a wide range of cellular responses and their dysregulation being associated with many diseases, GPCRs remain a key therapeutic target for several clinical indications. In recent years, it has been demonstrated that ligands for a given receptor can engage distinct pathways with different relative efficacies, a concept known as biased signaling or functional selectivity. However, the structural determinants of this phenomenon remain poorly understood. Using the ß2-adrenergic receptor as a model, we identified a linker residue (L1243.43) between the known PIF and NPxxY structural motifs, that plays a central role in the differential efficacy of biased ligands toward the Gs and ß-arrestin pathways. Given the high level of conservation of this linker residue, the study provides structural explanations for biased signaling that can be extrapolated to other GPCRs.

Publisher Correction: Structural insights into binding specificity, efficacy and bias of a ß2AR partial agonist.

In the version of this paper originally published, the structure for epinephrine shown in Figure 1a was redrawn with an extra carbon. The structure has been replaced in the HTML and PDF versions of the article. The original and corrected versions of the structure are shown below.

Structural insights into binding specificity, efficacy and bias of a ß2AR partial agonist.

Salmeterol is a partial agonist for the ß2 adrenergic receptor (ß2AR) and the first long-acting ß2AR agonist to be widely used clinically for the treatment of asthma and chronic obstructive pulmonary disease. Salmeterol's safety and mechanism of action have both been controversial. To understand its unusual pharmacological action and partial agonism, we obtained the crystal structure of salmeterol-bound ß2AR in complex with an active-state-stabilizing nanobody. The structure reveals the location of the salmeterol exosite, where sequence differences between ß1AR and ß2AR explain the high receptor-subtype selectivity. A structural comparison with the ß2AR bound to the full agonist epinephrine reveals differences in the hydrogen-bond network involving residues Ser2045.43 and Asn2936.55. Mutagenesis and biophysical studies suggested that these interactions lead to a distinct active-state conformation that is responsible for the partial efficacy of G-protein activation and the limited ß-arrestin recruitment for salmeterol.

Bioluminescence resonance energy transfer-based biosensors allow monitoring of ligand- and transducer-mediated GPCR conformational changes.

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate a variety of cellular response which make them a target of choice for drug development in many indications. It is now well established that GPCRs can adopt several distinct conformations that can be differentially stabilized by various ligands resulting in different biological outcomes, a concept known as functional selectivity. However, due to the highly hydrophobic nature of GPCRs, tools to monitor these conformational ensembles are limited and addressing their conformation dynamics remains a challenge with current structural biology approaches. Here we describe new bioluminescent resonance energy transfer-based biosensors that can probe the conformational rearrangement promoted by ligands with different signaling efficacies as well as the impact of transducers such as G proteins and ß-arrestin on these conformational transitions. The design of such sensors for other receptors should be useful to further explore the structural determinants of GPCR functional selectivity.

Evolutionary action and structural basis of the allosteric switch controlling ß2AR functional selectivity.

Functional selectivity of G-protein-coupled receptors is believed to originate from ligand-specific conformations that activate only subsets of signaling effectors. In this study, to identify molecular motifs playing important roles in transducing ligand binding into distinct signaling responses, we combined in silico evolutionary lineage analysis and structure-guided site-directed mutagenesis with large-scale functional signaling characterization and non-negative matrix factorization clustering of signaling profiles. Clustering based on the signaling profiles of 28 variants of the ß2-adrenergic receptor reveals three clearly distinct phenotypical clusters, showing selective impairments of either the Gi or ßarrestin/endocytosis pathways with no effect on Gs activation. Robustness of the results is confirmed using simulation-based error propagation. The structural changes resulting from functionally biasing mutations centered around the DRY, NPxxY, and PIF motifs, selectively linking these micro-switches to unique signaling profiles. Our data identify different receptor regions that are important for the stabilization of distinct conformations underlying functional selectivity.

Multimodal and Polymorphic Interactions between Anillin and Actin: Their Implications for Cytokinesis.

Cytokinesis of animal cells requires the assembly of a contractile ring, which promotes daughter cell splitting. Anillin is a conserved scaffold protein involved in organizing the structural components of the contractile ring including filamentous actin (F-actin), myosin, and septins and in forming the subsequent midbody ring. Like other metazoan homologs, Drosophila anillin contains a conserved domain that can bind and bundle F-actin, but the importance and molecular details of its interaction with F-actin remain unclear. Here, we show that in a depletion-and-rescue assay in Drosophila S2 cells, anillin lacking the entire actin-binding domain (ActBD) exhibits defective cortical localization during mitosis and a greatly diminished ability to support cytokinesis. Using in vitro binding assays and electron microscopy on recombinant fragments, we determine that the anillin ActBD harbors three distinct actin-binding sites (ABS 1-3). We show that each ABS binds to a distinct place on F-actin. Importantly, ABS1 and ABS3 partially overlap on the surface of actin and, therefore, interact with F-actin in a mutually exclusive fashion. Although ABS2 and ABS3 are sufficient for bundling, ABS1 contributes to the overall F-actin bundling activity of anillin and enables anillin to switch between two actin-bundling morphologies and promote the formation of three-dimensional F-actin bundles. Finally, we show that in live S2 cells, ABS2 and ABS3 are each required and together sufficient for the robust cortical localization of the ActBD during cytokinesis. Collectively, our structural, biochemical, and cell biological data suggest that multiple anillin-actin interaction modes promote the faithful progression of cytokinesis.