Structural and functional diversity among agonist-bound states of the GLP-1 receptor.

Recent advances in G-protein-coupled receptor (GPCR) structural elucidation have strengthened previous hypotheses that multidimensional signal propagation mediated by these receptors depends, in part, on their conformational mobility; however, the relationship between receptor function and static structures is inherently uncertain. Here, we examine the contribution of peptide agonist conformational plasticity to activation of the glucagon-like peptide 1 receptor (GLP-1R), an important clinical target. We use variants of the peptides GLP-1 and exendin-4 (Ex4) to explore the interplay between helical propensity near the agonist N terminus and the ability to bind to and activate the receptor. Cryo-EM analysis of a complex involving an Ex4 analog, the GLP-1R and Gs heterotrimer revealed two receptor conformers with distinct modes of peptide-receptor engagement. Our functional and structural data, along with molecular dynamics (MD) simulations, suggest that receptor conformational dynamics associated with flexibility of the peptide N-terminal activation domain may be a key determinant of agonist efficacy.

Structures of the human cholecystokinin 1 (CCK1) receptor bound to Gs and Gq mimetic proteins provide insight into mechanisms of G protein selectivity.

G protein-coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here, we used cryo-electron microscopy (cryo-EM) to determine structures of the cholecystokinin (CCK) type 1 receptor (CCK1R) bound to the CCK peptide agonist, CCK-8 and 2 distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11-GPCR complexes, the Gq-α5 helix (αH5) bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket were essentially equivalent when Gs was bound, with the Gs αH5 displaying a conformation that arises from "unwinding" of the far carboxyl-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein are likely to play critical roles in the promiscuous coupling of individual GPCRs.

Membranes under the Magnetic Lens: A Dive into the Diverse World of Membrane Protein Structures Using Cryo-EM.

Membrane proteins are highly diverse in both structure and function and can, therefore, present different challenges for structure determination. They are biologically important for cells and organisms as gatekeepers for information and molecule transfer across membranes, but each class of membrane proteins can present unique obstacles to structure determination. Historically, many membrane protein structures have been investigated using highly engineered constructs or using larger fusion proteins to improve solubility and/or increase particle size. Other strategies included the deconstruction of the full-length protein to target smaller soluble domains. These manipulations were often required for crystal formation to support X-ray crystallography or to circumvent lower resolution due to high noise and dynamic motions of protein subdomains. However, recent revolutions in membrane protein biochemistry and cryo-electron microscopy now provide an opportunity to solve high resolution structures of both large, >1 megadalton (MDa), and small, <100 kDa (kDa), drug targets in near-native conditions, routinely reaching resolutions around or below 3 Å. This review provides insights into how the recent advances in membrane biology and biochemistry, as well as technical advances in cryo-electron microscopy, help us to solve structures of a large variety of membrane protein groups, from small receptors to large transporters and more complex machineries.

A multidimensional approach for differentiating the clinical needs of young people presenting for primary mental health care.

OBJECTIVES: There is an ongoing necessity to match clinical interventions with the multidimensional needs of young people. A key step toward better service planning and the design of optimal models of care is to use multidimensional assessment to understand the clinical needs of those presenting to primary mental health care. METHODS: 1284 people aged 12-25 years presenting to primary youth mental health services completed an online assessment at service entry. Latent class analysis was conducted for seven scales assessing anxiety, depression, psychosis, mania, functioning (indexed by Work and Social Adjustment Scale), and suicidality. RESULTS: A three-class solution was identified as the optimal solution. Class 1 (n = 305, 23.75%), an early illness stage group, had low and mixed symptomatology with limited functional impairment, class 2 (n = 353, 27.49%) was made up of older persons with established depression and functional impairment, and class 3 (n = 626, 48.75%) had very high and complex needs, with functional impairment, suicidality, and at-risk mental states (psychosis or mania). Additional differentiating characteristics included psychological distress, circadian disturbances, social support, mental health history, eating disorder behaviours, and symptoms of post-traumatic stress disorder. CONCLUSIONS: A large proportion of help-seeking young people present with symptoms and functional impairment that may exceed the levels of care available from basic primary care or brief intervention services. These subgroups highlight the importance of multidimensional assessments to determine appropriate service pathways and care options.

Patterns of Sustainability Capacity Among Organizations That Deliver the National Diabetes Prevention Program: A Latent Profile Analysis.

INTRODUCTION: Since the launch of the National Diabetes Prevention Program (DPP) in 2010, more than 3,000 organizations have registered with the Centers for Disease and Control and Prevention to deliver the program; today, however, only approximately 2,000 organizations are registered, indicating challenges with sustainability. We used the Program Sustainability Assessment Tool (PSAT) to explore patterns of sustainability capacity among National DPP delivery organizations. METHODS: We used data from a cross-sectional online survey conducted in August and September 2021 of staff members (N = 440) at National DPP delivery organizations. We conducted a latent profile analysis to identify latent subpopulations on the basis of respondent PSAT domain scores. Regression analyses were used to estimate associations between derived latent classes, PSAT scores, and respondent characteristics. RESULTS: The 4-class model included 4 groups of capacity for program sustainability, ranging from low to high: low (class 1) with 8.0% of the sample, medium-low (class 2) with 22.0%, medium-high (class 3) with 41.6%, and high (class 4) with 28.4%. Program evaluation (mean score = 5.1 [SD = 1.4]) and adaptation (mean score = 5.3 [SD = 1.3]) were the domains with the highest scores, while funding stability (mean score = 4.0 [SD = 1.6]) and Partnerships (mean score = 4.0 [SD = 1.7]) had the lowest scores. In our sample of National DPP delivery organizations, most reported relatively high capacity for program sustainability, and key indicators associated with sustainability capacity were virtual delivery, location of delivery, funding sources, and organization type. DISCUSSION: Similar to sustainability capacity findings from other PSAT studies, our study found that funding stability and partnerships are areas to strengthen. This insight is useful in sustainability planning at organizational and national levels across multiple programs.

Secretin Amino-Terminal Structure-Activity Relationships and Complementary Mutagenesis at the Site of Docking to the Secretin Receptor.

Class B1 G protein-coupled receptors are activated by peptides, with amino-terminal regions critical for biologic activity. Although high resolution structures exist, understanding of key features of the peptide activation domain that drive signaling is limited. In the secretin receptor (SecR) structure, interactions are observed between peptide residues His1 and Ser2 and seventh transmembrane segment (TM7) receptor residue E373. We interrogated these interactions using systematic structure-activity analysis of peptide and receptor. His1 was critical for binding and cAMP responses, but its orientation was not critical, and substitution could independently modify affinity and efficacy. Ser2 was also critical, with all substitutions reducing peptide affinity and functional responses proportionally. Mutation of E373 to conserved acidic Asp (E373D), uncharged polar Gln (E373Q), or charge-reversed basic Arg (E373R) did not alter receptor expression, with all exhibiting secretin-dependent cAMP accumulation. All position 373 mutants displayed reduced binding affinities and cAMP potencies for many peptide analogs, although relative effects of position 1 peptides were similar whereas position 2 peptides exhibited substantial differences. The peptide including basic Lys in position 2 was active at SecR having acidic Glu in position 373 and at E373D while exhibiting minimal activity at those receptors in which an acidic residue is absent in this position (E373Q and E373R). In contrast, the peptide including acidic Glu in position 2 was equipotent with secretin at E373R while being much less potent than secretin at wild-type SecR and E373D. These data support functional importance of a charge-charge interaction between the amino-terminal region of secretin and the top of TM7. SIGNIFICANCE STATEMENT: This work refines our molecular understanding of the activation mechanisms of class B1 G protein-coupled receptors. The amino-terminal region of secretin interacts with the seventh transmembrane segment of its receptor with structural specificity and with a charge-charge interaction helping to drive functional activation.

Targeting VIP and PACAP Receptor Signaling: New Insights into Designing Drugs for the PACAP Subfamily of Receptors.

Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and Vasoactive Intestinal Peptide (VIP) are neuropeptides involved in a diverse array of physiological and pathological processes through activating the PACAP subfamily of class B1 G protein-coupled receptors (GPCRs): VIP receptor 1 (VPAC1R), VIP receptor 2 (VPAC2R), and PACAP type I receptor (PAC1R). VIP and PACAP share nearly 70% amino acid sequence identity, while their receptors PAC1R, VPAC1R, and VPAC2R share 60% homology in the transmembrane regions of the receptor. PACAP binds with high affinity to all three receptors, while VIP binds with high affinity to VPAC1R and VPAC2R, and has a thousand-fold lower affinity for PAC1R compared to PACAP. Due to the wide distribution of VIP and PACAP receptors in the body, potential therapeutic applications of drugs targeting these receptors, as well as expected undesired side effects, are numerous. Designing selective therapeutics targeting these receptors remains challenging due to their structural similarities. This review discusses recent discoveries on the molecular mechanisms involved in the selectivity and signaling of the PACAP subfamily of receptors, and future considerations for therapeutic targeting.

Cryo-EM structure of the dual incretin receptor agonist, peptide-19, in complex with the glucagon-like peptide-1 receptor.

Dual agonists that can activate both the glucagon-like peptide-1 receptor (GLP-1R) and the gastric inhibitory polypeptide receptor (GIPR) have demonstrated high efficacy for the treatment of metabolic disease. Peptide-19 is a prototypical dual agonist that has high potency at both GLP-1R and GIPR but has a distinct signalling profile relative to the native peptides at the cognate receptors. In this study, we solved the structure of peptide-19 bound to the GLP-1R in complex with Gs protein, and compared the structure and dynamics of this complex to that of published structures of GLP-1R:Gs in complex with other receptor agonists. Unlike other peptide-bound receptor complexes, peptide-19:GLP-1R:Gs demonstrated a more open binding pocket where transmembrane domain (TM) 6, TM7 and the interconnecting extracellular loop 3 (ECL3) were located away from the peptide, with no interactions between peptide-19 and TM6/ECL3. Analysis of conformational variance of the complex revealed that peptide-19 was highly dynamic and underwent binding and unbinding motions facilitated by the more open TM binding pocket. Both the consensus structure of the GLP-1R complex with peptide-19 and the dynamics of this complex were distinct from previously described GLP-1R structures providing unique insights into the mode of GLP-1R activation by this dual agonist.

Implementing a digital health model of care in Australian youth mental health services: protocol for impact evaluation.

BACKGROUND: The World Economic Forum has recently highlighted substantial problems in mental health service provision and called for the rapid deployment of smarter, digitally-enhanced health services as a means to facilitate effective care coordination and address issues of demand. In mental health, the biggest enabler of digital solutions is the implementation of an effective model of care that is facilitated by integrated health information technologies (HITs); the latter ensuring the solution is easily accessible, scalable and sustainable. The University of Sydney's Brain and Mind Centre (BMC) has developed an innovative digital health solution - delivered through the Youth Mental Health and Technology Program - which incorporates two components: 1) a highly personalised and measurement-based (data-driven) model of youth mental health care; and 2) an industrial grade HIT registered on the Australian Register of Therapeutic Goods. This paper describes a research protocol to evaluate the impact of implementing the BMC's digital health solution into youth mental health services (i.e. headspace - a highly accessible, youth-friendly integrated service that responds to the mental health, physical health, alcohol or other substance use, and vocational concerns of young people aged 12 to 25 years) within urban and regional areas of Australia. METHODS: The digital health solution will be implemented into participating headspace centres using a naturalistic research design. Quantitative and qualitative data will be collected from headspace health professionals, service managers and administrators, as well as from lead agency and local Primary Health Network (PHN) staff, via service audits, Implementation Officer logs, online surveys, and semi-structured interviews, at baseline and then three-monthly intervals over the course of 12 months. DISCUSSION: At the time of publication, six headspace centres had been recruited to this study and had commenced implementation and impact evaluation. The first results are expected to be submitted for publication in 2021. This study will focus on the impact of implementing a digital health solution at both a service and staff level, and will evaluate digital readiness of service and staff adoption; quality, usability and acceptability of the solution by staff; staff self-reported clinical competency; overall impact on headspace centres as well as their lead agencies and local PHNs; and social return on investment.

Recommendations for Designing Health Information Technologies for Mental Health Drawn From Self-Determination Theory and Co-design With Culturally Diverse Populations: Template Analysis.

BACKGROUND: Culturally diverse populations (including Aboriginal and Torres Strait Islander people, people of diverse genders and sexualities, and culturally and linguistically diverse people) in nonurban areas face compounded barriers to accessing mental health care. Health information technologies (HITs) show promising potential to overcome these barriers. OBJECTIVE: This study aims to identify how best to improve a mental health and well-being HIT for culturally diverse Australians in nonurban areas. METHODS: We conducted 10 co-design workshops (N=105 participants) in primary youth mental health services across predominantly nonurban areas of Australia and conducted template analysis on the workshop outputs. Owing to local (including service) demographics, the workshop participants naturalistically reflected culturally diverse groups. RESULTS: We identified 4 main themes: control, usability, affirmation, and health service delivery factors. The first 3 themes overlap with the 3 basic needs postulated by self-determination theory (autonomy, competence, and relatedness) and describe participant recommendations on how to design an HIT. The final theme includes barriers to adopting HITs for mental health care and how HITs can be used to support care coordination and delivery. Hence, it describes participant recommendations on how to use an HIT. CONCLUSIONS: Although culturally diverse groups have specific concerns, their expressed needs fall broadly within the relatively universal design principles identified in this study. The findings of this study provide further support for applying self-determination theory to the design of HITs and reflect the tension in designing technologies for complex problems that overlap multiple medical, regulatory, and social domains, such as mental health care. Finally, we synthesize the identified themes into general recommendations for designing HITs for mental health and provide concrete examples of design features recommended by participants.

Project Synergy: co-designing technology-enabled solutions for Australian mental health services reform.

Project Synergy aims to test the potential of new and emerging technologies to enhance the quality of mental health care provided by traditional face-to-face services. Specifically, it seeks to ensure that consumers get the right care, first time (delivery of effective mental health care early in the course of illness). Using co-design with affected individuals, Project Synergy has built, implemented and evaluated an online platform to assist the assessment, feedback, management and monitoring of people with mental disorders. It also promotes the maintenance of wellbeing by collating health and social information from consumers, their supportive others and health professionals. This information is reported back openly to consumers and their service providers to promote genuine collaborative care. The online platform does not provide stand-alone medical or health advice, risk assessment, clinical diagnosis or treatment; instead, it supports users to decide what may be suitable care options. Using an iterative cycle of research and development, the first four studies of Project Synergy (2014-2016) involved the development of different types of online prototypes for young people (i) attending university; (ii) in three disadvantaged communities in New South Wales; (iii) at risk of suicide; and (iv) attending five headspace centres. These contributed valuable information concerning the co-design, build, user testing and evaluation of prototypes, as well as staff experiences during development and service quality improvements following implementation. Through ongoing research and development (2017-2020), these prototypes underpin one online platform that aims to support better multidimensional mental health outcomes for consumers; more efficient, effective and appropriate use of health professional knowledge and clinical skills; and quality improvements in mental health service delivery.

Impact of secretin receptor homo-dimerization on natural ligand binding.

Class B G protein-coupled receptors can form dimeric complexes important for high potency biological effects. Here, we apply pharmacological, biochemical, and biophysical techniques to cells and membranes expressing the prototypic secretin receptor (SecR) to gain insights into secretin binding to homo-dimeric and monomeric SecR. Spatial proximity between peptide and receptor residues, probed by disulfide bond formation, demonstrates that the secretin N-terminus moves from adjacent to extracellular loop 3 (ECL3) at wild type SecR toward ECL2 in non-dimerizing mutants. Analysis of fluorescent secretin analogs demonstrates stable engagement of the secretin C-terminal region within the receptor extracellular domain (ECD) for both dimeric and monomeric receptors, while the mid-region exhibits lower mobility while docked at the monomer. Moreover, decoupling of G protein interaction reduces mobility of the peptide mid-region at wild type receptor to levels similar to the mutant, whereas it has no further impact on the monomer. These data support a model of peptide engagement whereby the ability of SecR to dimerize promotes higher conformational dynamics of the peptide-bound receptor ECD and ECLs that likely facilitates more efficient G protein recruitment and activation, consistent with the higher observed functional potency of secretin at wild type SecR relative to the monomeric mutant receptor.

The Polarization of Clinician and Service Staff Perspectives After the Use of Health Information Technology in Youth Mental Health Services: Implementation and Evaluation Study.

BACKGROUND: Highly personalized care is substantially improved by technology platforms that assess and track patient outcomes. However, evidence regarding how to successfully implement technology in real-world mental health settings is limited. OBJECTIVE: This study aimed to naturalistically monitor how a health information technology (HIT) platform was used within 2 real-world mental health service settings to gain practical insights into how HIT can be implemented and sustained to improve mental health service delivery. METHODS: An HIT (The Innowell Platform) was naturally implemented in 2 youth mental health services in Sydney, Australia. Web-based surveys (n=19) and implementation logs were used to investigate staff attitudes toward technology before and after implementation. Descriptive statistics were used to track staff attitudes over time, whereas qualitative thematic analysis was used to explore implementation log data to gain practical insights into useful implementation strategies in real-world settings. RESULTS: After the implementation, the staff were nearly 3 times more likely to agree that the HIT would improve care for their clients (3/12, 25% agreed before the implementation compared with 7/10, 70% after the implementation). Despite this, there was also an increase in the number of staff who disagreed that the HIT would improve care (from 1/12, 8% to 2/10, 20%). There was also decreased uncertainty (from 6/12, 50% to 3/10, 30%) about the willingness of the service to implement the technology for its intended purpose, with similar increases in the number of staff who agreed and disagreed with this statement. Staff were more likely to be uncertain about whether colleagues in my service are receptive to changes in clinical processes (not sure rose from 5/12, 42% to 7/10, 70%). They were also more likely to report that their service already provides the best mental health care (agreement rose from 7/12, 58% to 8/10, 80%). After the implementation, a greater proportion of participants reported that the HIT enabled shared or collaborative decision-making with young people (2/10, 20%, compared with 1/12, 8%), enabled clients to proactively work on their mental health care through digital technologies (3/10, 30%, compared with 2/12, 16%), and improved their response to suicidal risk (4/10, 40% compared with 3/12, 25%). CONCLUSIONS: This study raises important questions about why clinicians, who have the same training and support in using technology, develop more polarized opinions on its usefulness after implementation. It seems that the uptake of HIT is heavily influenced by a clinician's underlying beliefs and attitudes toward clinical practice in general as well as the role of technology, rather than their knowledge or the ease of use of the HIT in question.

New Insights into the Structure and Function of Class B1 GPCRs.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors. Class B1 GPCRs constitute a subfamily of 15 receptors that characteristically contain large extracellular domains (ECDs) and respond to long polypeptide hormones. Class B1 GPCRs are critical regulators of homeostasis, and, as such, many are important drug targets. While most transmembrane proteins, including GPCRs, are recalcitrant to crystallization, recent advances in cryo-electron microscopy (cryo-EM) have facilitated a rapid expansion of the structural understanding of membrane proteins. As a testament to this success, structures for all the class B1 receptors bound to G proteins have been determined by cryo-EM in the past 5 years. Further advances in cryo-EM have uncovered dynamics of these receptors, ligands, and signaling partners. Here, we examine the recent structural underpinnings of the class B1 GPCRs with an emphasis on structure-function relationships.

A structural equation model of CFIR inner and outer setting constructs, organization characteristics, and national DPP enrollment.

BACKGROUND: The National Diabetes Prevention Program (DPP) has made great strides in increasing accessibility to its year-long, evidence-based lifestyle change program, with around 3000 organizations having delivered the program. This large dissemination effort offers a unique opportunity to identify organization-level factors associated with program implementation and reach (enrollment) across diverse settings. The purpose of this study was to quantitatively examine the relationships among Consolidated Framework for Implementation Research (CFIR) Inner Setting and Outer Setting constructs and the implementation outcome of reach. METHODS: This study analyzed data from a 2021 cross-sectional online survey with 586 National DPP Staff (lifestyle coaches, master trainers, program coordinators) with information about their organization, implementation outcomes, and responses to quantitative CFIR Inner Setting and Outer Setting construct items. Structural equation modeling was used to test a hypothesized path model with Inner and Outer Setting variables to explore direct and indirect pathways to enrollment. RESULTS: The CFIR items had good internal consistency and indicated areas of implementation strength and weakness. Eight variables included as part of the CFIR structural characteristics and one organization characteristic variable had significant direct relationships with enrollment. The length of delivery, number of lifestyle coaches, number of full-time staff, large organization size, and organizations delivering in rural, suburban, and/or urban settings all had positive significant direct relationships with enrollment, while academic organizations and organizations with only non-White participants enrolled in their National DPP lifestyle change programs had a negative association with enrollment. CONCLUSIONS: Participant reach is an important implementation outcome for the National DPP and vital to making population-level decreases in diabetes incidence in the USA. Our findings suggest that to facilitate enrollment, program implementers should focus on organizational structural characteristics such as staffing. Strengths of this study include the use of adapted and newly developed quantitative CFIR measures and structural equation modeling. Health prevention programs can use the methods and findings from this study to further understand and inform the impact of organization factors on implementation outcomes.

Molecular insights into peptide agonist engagement with the PTH receptor.

The parathyroid hormone (PTH) 1 receptor (PTH1R) is a G protein-coupled receptor (GPCR) that regulates skeletal development and calcium homeostasis. Here, we describe cryo-EM structures of the PTH1R in complex with fragments of the two hormones, PTH and PTH-related protein, the drug abaloparatide, as well as the engineered tool compounds, long-acting PTH (LA-PTH) and the truncated peptide, M-PTH(1-14). We found that the critical N terminus of each agonist engages the transmembrane bundle in a topologically similar fashion, reflecting similarities in measures of Gαs activation. The full-length peptides induce subtly different extracellular domain (ECD) orientations relative to the transmembrane domain. In the structure bound to M-PTH, the ECD is unresolved, demonstrating that the ECD is highly dynamic when unconstrained by a peptide. High resolutions enabled identification of water molecules near peptide and G protein binding sites. Our results illuminate the action of orthosteric agonists of the PTH1R.

Inner and outer setting factors that influence the implementation of the National Diabetes Prevention Program (National DPP) using the Consolidated Framework for Implementation Research (CFIR): a qualitative study.

BACKGROUND: Scaling evidence-based interventions are key to impacting population health. The National DPP lifestyle change program is one such intervention that has been scaled across the USA over the past 20 years; however, enrollment is an ongoing challenge. Furthermore, little is known about which organizations are most successful with program delivery, enrollment, and scaling. This study aims to understand more about the internal and external organization factors that impact program implementation and reach. METHODS: Between August 2020 and January 2021, data were collected through semi-structured key informant interviews with 30 National DPP delivery organization implementers. This study uses a qualitative cross-case construct rating methodology to assess which Consolidated Framework for Implementation Research (CFIR) inner and outer setting constructs contributed (both in valence and magnitude) to the organization's current level of implementation reach (measured by average participant enrollment per year). A construct by case matrix was created with ratings for each CFIR construct by interviewee and grouped by implementation reach level. RESULTS: Across the 16 inner and outer setting constructs and subconstructs, the interviewees with greater enrollment per year provided stronger and more positive examples related to implementation and enrollment of the program, while the lower reach groups reported stronger and more negative examples across rated constructs. Four inner setting constructs/subconstructs (structural characteristics, compatibility, goals and feedback, and leadership engagement) were identified as "distinguishing" between enrollment reach levels based on the difference between groups by average rating, the examination of the number of extreme ratings within levels, and the thematic analysis of the content discussed. Within these constructs, factors such as organization size and administrative processes; program fit with existing organization services and programs; the presence of enrollment goals; and active leadership involvement in implementation were identified as influencing program reach. CONCLUSIONS: Our study identified a number of influential CFIR constructs and their impact on National DPP implementation reach. These findings can be leveraged to improve efforts in recruiting and assisting delivery organizations to increase the reach and scale of the National DPP as well as other evidence-based interventions.

A Structurally Characterized Staphylococcus aureus Evolutionary Escape Route from Treatment with the Antibiotic Linezolid.

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen that presents great health concerns. Treatment requires the use of last-line antibiotics, such as members of the oxazolidinone family, of which linezolid is the first member to see regular use in the clinic. Here, we report a short time scale selection experiment in which strains of MRSA were subjected to linezolid treatment. Clonal isolates which had evolved a linezolid-resistant phenotype were characterized by whole-genome sequencing. Linezolid-resistant mutants were identified which had accumulated mutations in the ribosomal protein uL3. Multiple clones which had two mutations in uL3 exhibited resistance to linezolid, 2-fold higher than the clinical breakpoint. Ribosomes from this strain were isolated and subjected to single-particle cryo-electron microscopic analysis and compared to the ribosomes from the parent strain. We found that the mutations in uL3 lead to a rearrangement of a loop that makes contact with Helix 90, propagating a structural change over 15 Å away. This distal change swings nucleotide U2504 into the binding site of the antibiotic, causing linezolid resistance. IMPORTANCE Antibiotic resistance poses a critical problem to human health and decreases the utility of these lifesaving drugs. Of particular concern is the "superbug" methicillin-resistant Staphylococcus aureus (MRSA), for which treatment of infection requires the use of last-line antibiotics, including linezolid. In this paper, we characterize the atomic rearrangements which the ribosome, the target of linezolid, undergoes during its evolutionary journey toward becoming drug resistant. Using cryo-electron microscopy, we describe a particular molecular mechanism which MRSA uses to become resistant to linezolid.

Understanding VPAC receptor family peptide binding and selectivity.

The vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) receptors are key regulators of neurological processes. Despite recent structural data, a comprehensive understanding of peptide binding and selectivity among different subfamily receptors is lacking. Here, we determine structures of active, Gs-coupled, VIP-VPAC1R, PACAP27-VPAC1R, and PACAP27-PAC1R complexes. Cryo-EM structural analyses and molecular dynamics simulations (MDSs) reveal fewer stable interactions between VPAC1R and VIP than for PACAP27, more extensive dynamics of VIP interaction with extracellular loop 3, and receptor-dependent differences in interactions of conserved N-terminal peptide residues with the receptor core. MD of VIP modelled into PAC1R predicts more transient VIP-PAC1R interactions in the receptor core, compared to VIP-VPAC1R, which may underlie the selectivity of VIP for VPAC1R over PAC1R. Collectively, our work improves molecular understanding of peptide engagement with the PAC1R and VPAC1R that may benefit the development of novel selective agonists.

Informing the Future of Integrated Digital and Clinical Mental Health Care: Synthesis of the Outcomes From Project Synergy.

BACKGROUND: Globally, there are fundamental shortcomings in mental health care systems, including restricted access, siloed services, interventions that are poorly matched to service users' needs, underuse of personal outcome monitoring to track progress, exclusion of family and carers, and suboptimal experiences of care. Health information technologies (HITs) hold great potential to improve these aspects that underpin the enhanced quality of mental health care. OBJECTIVE: Project Synergy aimed to co-design, implement, and evaluate novel HITs, as exemplified by the InnoWell Platform, to work with standard health care organizations. The goals were to deliver improved outcomes for specific populations under focus and support organizations to enact significant system-level reforms. METHODS: Participating health care organizations included the following: Open Arms-Veterans & Families Counselling (in Sydney and Lismore, New South Wales [NSW]); NSW North Coast headspace centers for youth (Port Macquarie, Coffs Harbour, Grafton, Lismore, and Tweed Heads); the Butterfly Foundation's National Helpline for eating disorders; Kildare Road Medical Centre for enhanced primary care; and Connect to Wellbeing North Coast NSW (administered by Neami National), for population-based intake and assessment. Service users, families and carers, health professionals, and administrators of services across Australia were actively engaged in the configuration of the InnoWell Platform to meet service needs, identify barriers to and facilitators of quality mental health care, and highlight potentially the best points in the service pathway to integrate the InnoWell Platform. The locally configured InnoWell Platform was then implemented within the respective services. A mixed methods approach, including surveys, semistructured interviews, and workshops, was used to evaluate the impact of the InnoWell Platform. A participatory systems modeling approach involving co-design with local stakeholders was also undertaken to simulate the likely impact of the platform in combination with other services being considered for implementation within the North Coast Primary Health Network to explore resulting impacts on mental health outcomes, including suicide prevention. RESULTS: Despite overwhelming support for integrating digital health solutions into mental health service settings and promising impacts of the platform simulated under idealized implementation conditions, our results emphasized that successful implementation is dependent on health professional and service readiness for change, leadership at the local service level, the appropriateness and responsiveness of the technology for the target end users, and, critically, funding models being available to support implementation. The key places of interoperability of digital solutions and a willingness to use technology to coordinate health care system use were also highlighted. CONCLUSIONS: Although the COVID-19 pandemic has resulted in the widespread acceptance of very basic digital health solutions, Project Synergy highlights the critical need to support equity of access to HITs, provide funding for digital infrastructure and digital mental health care, and actively promote the use of technology-enabled, coordinated systems of care.