Development of fluorescent peptide G protein-coupled receptor activation biosensors for NanoBRET characterization of intracellular allosteric modulators

G protein-coupled receptors (GPCRs) are widely therapeutically targeted, and recent advances in allosteric modulator development at these receptors offer further potential for exploitation. Intracellular allosteric modulators (IAM) represent a class of ligands that bind to the receptor–effector interface (e.g., G protein) and inhibit agonist responses noncompetitively. This potentially offers greater selectivity between receptor subtypes compared to classical orthosteric ligands. However, while examples of IAM ligands are well described, a more general methodology for assessing compound interactions at the IAM site is lacking. Here, fluorescent labeled peptides based on the Gα peptide C terminus are developed as novel binding and activation biosensors for the GPCR-IAM site. In TR-FRET binding studies, unlabeled peptides derived from the Gαs subunit were first characterized for their ability to positively modulate agonist affinity at the β2-adrenoceptor. On this basis, a tetramethylrhodamine (TMR) labeled tracer was synthesized based on the 19 amino acid Gαs peptide (TMR-Gαs19cha18, where cha = cyclohexylalanine). Using NanoBRET technology to detect binding, TMR-Gαs19cha18 was recruited to Gs coupled β2-adrenoceptor and EP2 receptors in an agonist-dependent manner, but not the Gi-coupled CXCR2 receptor. Moreover, NanoBRET competition binding assays using TMR-Gαs19cha18 enabled direct assessment of the affinity of unlabeled ligands for β2-adrenoceptor IAM site. Thus, the NanoBRET platform using fluorescent-labeled G protein peptide mimetics offers novel potential for medium-throughput screens to identify IAMs, applicable across GPCRs coupled to a G protein class. Using the same platform, Gs peptide biosensors also represent useful tools to probe orthosteric agonist efficacy and the dynamics of receptor activation

The importance of spawning behavior in understanding the vulnerability of exploited marine fishes in the U.S. Gulf of Mexico

The vulnerability of a fish stock to becoming overfished is dependent upon biological traits that influence productivity and external factors that determine susceptibility or exposure to fishing effort. While a suite of life history traits are traditionally incorporated into management efforts due to their direct association with vulnerability to overfishing, spawning behavioral traits are seldom considered. We synthesized the existing biological and fisheries information of 28 fish stocks in the U.S. Gulf of Mexico to investigate relationships between life history traits, spawning behavioral traits, management regulations, and vulnerability to fishing during the spawning season. Our results showed that spawning behavioral traits were not correlated with life history traits but improved identification of species that have been historically overfished. Species varied widely in their intrinsic vulnerability to fishing during spawning in association with a broad range of behavioral strategies. Extrinsic vulnerability was high for nearly all species due to exposure to fishing during the spawning season and few management measures in place to protect spawning fish. Similarly, several species with the highest vulnerability scores were historically overfished in association with spawning aggregations. The most vulnerable species included several stocks that have not been assessed and should be prioritized for further research and monitoring. Collectively, the results of this study illustrate that spawning behavior is a distinct aspect of fish ecology that is important to consider for predictions of vulnerability and resilience to fisheries exploitation

A protective role for BRCA2 at stalled replication forks

The hereditary breast and ovarian cancer predisposition genes BRCA1 and BRCA2 account for the lion's share of heritable breast cancer risk in the human population. Loss of function of either gene results in defective homologous recombination (HR) and triggers genomic instability, accelerating breast tumorigenesis. A long-standing hypothesis proposes that BRCA1 and BRCA2 mediate HR following attempted replication across damaged DNA, ensuring error-free processing of the stalled replication fork. A recent paper describes a new replication fork protective function of BRCA2, which appears to collaborate with its HR function to suppress genomic instability

Update to the study protocol for an implementation-effectiveness trial comparing two education strategies for improving the uptake of noninvasive ventilation in patients with severe COPD exacerbation

BACKGROUND: There is strong evidence that noninvasive ventilation (NIV) improves the outcomes of patients hospitalized with severe COPD exacerbation, and NIV is recommended as the first-line therapy for these patients. Yet, several studies have demonstrated substantial variation in NIV use across hospitals, leading to preventable morbidity and mortality. In addition, prior studies suggested that efforts to increase NIV use in COPD need to account for the complex and interdisciplinary nature of NIV delivery and the need for team coordination. Therefore, our initial project aimed to compare two educational strategies: online education (OLE) and interprofessional education (IPE), which targets complex team-based care in NIV delivery. Due to the impact of the COVID-19 pandemic on recruitment and planned intervention, we had made several changes in the study design, statistical analysis, and implementation strategies delivery as outlined in the methods. METHODS: We originally proposed a two-arm, pragmatic, cluster, randomized hybrid implementation-effectiveness trial comparing two education strategies to improve NIV uptake in patients with severe COPD exacerbation in 20 hospitals with a low baseline rate of NIV use. Due to logistical constrains and slow recruitment, we changed the study design to an opened cohort stepped-wedge design with three steps which will allow the institutions to enroll when they are ready to participate. Only the IPE strategy will be implemented, and the education will be provided in an online virtual format. Our primary outcome will be the hospital-level risk-standardized NIV proportion for the period post-IPE training, along with the change in rate from the period prior to training. Aim 1 will compare the change over time of NIV use among patients with COPD in the step-wedged design. Aim 2 will explore the mediators\u27 role (respiratory therapist autonomy and team functionality) on the relationship between the implementation strategies and effectiveness. Finally, in Aim 3, through interviews with providers, we will assess the acceptability and feasibility of the educational training. CONCLUSION: The changes in study design will result in several limitation. Most importantly, the hospitals in the three cohorts are not randomized as they enroll based on their readiness. Second, the delivery of the IPE is virtual, and it is not known if remote education is conducive to team building. However, this study will be among the first to test the impact of IPE in the inpatient setting carefully and may generalize to other interventions directed to seriously ill patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT04206735 . Registered on December 20, 2019

APOΕ4 Lowers Energy Expenditure in Females and Impairs Glucose Oxidation by Increasing Flux through Aerobic Glycolysis

BACKGROUND: Cerebral glucose hypometabolism is consistently observed in individuals with Alzheimer\u27s disease (AD), as well as in young cognitively normal carriers of the Ε4 allele of Apolipoprotein E (APOE), the strongest genetic predictor of late-onset AD. While this clinical feature has been described for over two decades, the mechanism underlying these changes in cerebral glucose metabolism remains a critical knowledge gap in the field. METHODS: Here, we undertook a multi-omic approach by combining single-cell RNA sequencing (scRNAseq) and stable isotope resolved metabolomics (SIRM) to define a metabolic rewiring across astrocytes, brain tissue, mice, and human subjects expressing APOE4. RESULTS: Single-cell analysis of brain tissue from mice expressing human APOE revealed E4-associated decreases in genes related to oxidative phosphorylation, particularly in astrocytes. This shift was confirmed on a metabolic level with isotopic tracing of 13C-glucose in E4 mice and astrocytes, which showed decreased pyruvate entry into the TCA cycle and increased lactate synthesis. Metabolic phenotyping of E4 astrocytes showed elevated glycolytic activity, decreased oxygen consumption, blunted oxidative flexibility, and a lower rate of glucose oxidation in the presence of lactate. Together, these cellular findings suggest an E4-associated increase in aerobic glycolysis (i.e. the Warburg effect). To test whether this phenomenon translated to APOE4 humans, we analyzed the plasma metabolome of young and middle-aged human participants with and without the Ε4 allele, and used indirect calorimetry to measure whole body oxygen consumption and energy expenditure. In line with data from E4-expressing female mice, a subgroup analysis revealed that young female E4 carriers showed a striking decrease in energy expenditure compared to non-carriers. This decrease in energy expenditure was primarily driven by a lower rate of oxygen consumption, and was exaggerated following a dietary glucose challenge. Further, the stunted oxygen consumption was accompanied by markedly increased lactate in the plasma of E4 carriers, and a pathway analysis of the plasma metabolome suggested an increase in aerobic glycolysis. CONCLUSIONS: Together, these results suggest astrocyte, brain and system-level metabolic reprogramming in the presence of APOE4, a \u27Warburg like\u27 endophenotype that is observable in young females decades prior to clinically manifest AD

Key questions in marine mammal bioenergetics

This work was funded by the Marine Mammal Commission (MMC19-173). The Office of Naval Research funded the bioenergetic workshop (N000142012392) that provided support for this work.Bioenergetic approaches are increasingly used to understand how marine mammal populations could be affected by a changing and disturbed aquatic environment. There remain considerable gaps in our knowledge of marine mammal bioenergetics, which hinder the application of bioenergetic studies to inform policy decisions. We conducted a priority-setting exercise to identify high-priority unanswered questions in marine mammal bioenergetics, with an emphasis on questions relevant to conservation and management. Electronic communication and a virtual workshop were used to solicit and collate potential research questions from the marine mammal bioenergetic community. From a final list of 39 questions, 11 were identified as ‘key’ questions because they received votes from at least 50% of survey participants. Key questions included those related to energy intake (prey landscapes, exposure to human activities) and expenditure (field metabolic rate, exposure to human activities, lactation, time-activity budgets), energy allocation priorities, metrics of body condition and relationships with survival and reproductive success and extrapolation of data from one species to another. Existing tools to address key questions include labelled water, animal-borne sensors, mark-resight data from long-term research programs, environmental DNA and unmanned vehicles. Further validation of existing approaches and development of new methodologies are needed to comprehensively address some key questions, particularly for cetaceans. The identification of these key questions can provide a guiding framework to set research priorities, which ultimately may yield more accurate information to inform policies and better conserve marine mammal populations.Publisher PDFPeer reviewe

The attentional blink is related to phonemic decoding, but not sight-word recognition, in typically reading adults

This research investigated the relationship between the attentional blink (AB) and reading in typical adults. The AB is a deficit in the processing of the second of two rapidly presented targets when it occurs in close temporal proximity to the first target. Specifically, this experiment examined whether the AB was related to both phonological and sight-word reading abilities, and whether the relationship was mediated by accuracy on a single-target rapid serial visual processing task (single-target accuracy). Undergraduate university students completed a battery of tests measuring reading ability, non-verbal intelligence, and rapid automatised naming, in addition to rapid serial visual presentation tasks in which they were required to identify either two (AB task) or one (single target task) target/s (outlined shapes: circle, square, diamond, cross, and triangle) in a stream of random-dot distractors. The duration of the AB was related to phonological reading (n=41, ß=-0.43): participants who exhibited longer ABs had poorer phonemic decoding skills. The AB was not related to sight-word reading. Single-target accuracy did not mediate the relationship between the AB and reading, but was significantly related to AB depth (non-linear fit, R2 = .50): depth reflects the maximal cost in T2 reporting accuracy in the AB. The differential relationship between the AB and phonological versus sight-word reading implicates common resources used for phonemic decoding and target consolidation, which may be involved in cognitive control. The relationship between single-target accuracy and the AB is discussed in terms of cognitive preparation

Improving Prevention of Mother-to-Child Transmission of HIV Care and Related Services in Eastern Rwanda

Younsook Lim and colleagues describe the Rwanda Learning Collaborative on Child Health, which aimed to improve and extend the impact of prevention of mother-to-child transmission of HIV/AIDS

Challenges of maintaining research protocol fidelity in a clinical care setting: A qualitative study of the experiences and views of patients and staff participating in a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Trial research has predominantly focused on patient and staff understandings of trial concepts and/or motivations for taking part, rather than why treatment recommendations may or may not be followed during trial delivery. This study sought to understand why there was limited attainment of the glycaemic target (HbA_1c≤6.5%) among patients who participated in the Treating to Target in Type 2 Diabetes Trial (4-T). The objective was to inform interpretation of trial outcomes and provide recommendations for future trial delivery.</p> <p>Methods</p> <p>In-depth interviews were conducted with 45 patients and 21 health professionals recruited from 11 of 58 trial centres in the UK. Patients were broadly representative of those in the main trial in terms of treatment allocation, demographics and glycaemic control. Both physicians and research nurses were interviewed.</p> <p>Results</p> <p>Most patients were committed to taking insulin as recommended by 4-T staff. To avoid hypoglycaemia, patients occasionally altered or skipped insulin doses, normally in consultation with staff. Patients were usually unaware of the trial's glycaemic target. Positive staff feedback could lead patients to believe they had been 'successful' trial participants even when their HbA_1cexceeded 6.5%. While some staff felt that the 4-T automated insulin dose adjustment algorithm had increased their confidence to prescribe larger insulin doses than in routine clinical practice, all described situations where they had not followed its recommendations. Staff regarded the application of a 'one size fits all' glycaemic target during the trial as contradicting routine clinical practice where they would tailor treatments to individuals. Staff also expressed concerns that 'tight' glycaemic control might impose an unacceptably high risk of hypoglycaemia, thus compromising trust and safety, especially amongst older patients. To address these concerns, staff tended to adapt the trial protocol to align it with their clinical practices and experiences.</p> <p>Conclusions</p> <p>To understand trial findings, foster attainment of endpoints, and promote protocol fidelity, it may be necessary to look beyond individual patient characteristics and experiences. Specifically, the context of trial delivery, the impact of staff involvement, and the difficulties staff may encounter in balancing competing 'clinical' and 'research' roles and responsibilities may need to be considered and addressed.</p