Bright and stable luminescent probes for target engagement profiling in live cells.

The pace of progress in biomedical research directly depends on techniques that enable the quantitative interrogation of interactions between proteins and other biopolymers, or with their small-molecule ligands. Time-resolved Förster resonance energy transfer (TR-FRET) assay platforms offer high sensitivity and specificity. However, the paucity of accessible and biocompatible luminescent lanthanide complexes, which are essential reagents for TR-FRET-based approaches, and their poor cellular permeability have limited broader adaptation of TR-FRET beyond homogeneous and extracellular assay applications. Here, we report the development of CoraFluors, a new class of macrotricyclic terbium complexes, which are synthetically readily accessible, stable in biological media and exhibit photophysical and physicochemical properties that are desirable for biological studies. We validate the performance of CoraFluors in cell-free systems, identify cell-permeable analogs and demonstrate their utility in the quantitative domain-selective characterization of Keap1 ligands, as well as in isoform-selective target engagement profiling of HDAC1 inhibitors in live cells.

Job demands, resources and work-related well-being in UK firefighters.

BACKGROUND: There is evidence that firefighters are at risk of work-related stress and mental health problems, but little is known about the organizational hazards they experience. Insight is needed into the work-related factors that are most likely to threaten or protect their work-related well-being. AIMS: To identify levels of job demands and resources (including demands relating to workload, work patterns and the working environment, relationship conflicts, control, support, role clarity and change management) among firefighters, and to use a job demands-resources framework to examine their impacts on work-related well-being. The role played by recovery strategies in predicting work-related well-being was also considered. METHODS: Job demands and resources were assessed by the Health & Safety Executive (HSE) Management Standards Indicator Tool. Validated scales measured recovery strategies (detachment, affective rumination and problem-solving pondering) and work-related well-being (anxiety-contentment and depression-enthusiasm). The impact of job demands, resources and recovery strategies was tested by multiple linear regression. RESULTS: The sample comprised 909 firefighters across seven Fire and Rescue Services in the UK (85% male). Levels of job demands and resources did not meet HSE benchmarks. The main risk factors for poor work-related well-being were relationship conflicts and affective rumination, but resources such as role clarity and job control and the use of problem-solving pondering and detachment were beneficial. CONCLUSIONS: Interventions that aim to reduce relationship conflicts at work and promote problem-solving rather than affective rumination, and detachment from work when off-duty, are likely to improve work-related well-being. Attention to enhancing job resources may also be beneficial.

Promoting and maintaining physical activity in the transition to retirement: a systematic review of interventions for adults around retirement age.

It has been argued that transition points in life, such as the approach towards, and early years of retirement present key opportunities for interventions to improve the health of the population. Research has also highlighted inequalities in health status in the retired population and in response to interventions which should be addressed. We aimed to conduct a systematic review to synthesise international evidence on the types and effectiveness of interventions to increase physical activity among people around the time of retirement. A systematic review of literature was carried out between February 2014 and April 2015. Searches were not limited by language or location, but were restricted by date to studies published from 1990 onwards. Methods for identification of relevant studies included electronic database searching, reference list checking, and citation searching. Systematic search of the literature identified 104 papers which described study populations as being older adults. However, we found only one paper which specifically referred to their participants as being around the time of retirement. The intervention approaches for older adults encompassed: training of health care professionals; counselling and advice giving; group sessions; individual training sessions; in-home exercise programmes; in-home computer-delivered programmes; in-home telephone support; in-home diet and exercise programmes; and community-wide initiatives. The majority of papers reported some intervention effect, with evidence of positive outcomes for all types of programmes. A wide range of different measures were used to evaluate effectiveness, many were self-reported and few studies included evaluation of sedentary time. While the retirement transition is considered a significant point of life change, little research has been conducted to assess whether physical activity interventions at this time may be effective in promoting or maintaining activity, or reducing health inequalities. We were unable to find any evidence that the transition to retirement period was, or was not a significant point for intervention. Studies in older adults more generally indicated that a range of interventions might be effective for people around retirement age.

The return of public health to local government in England: changing the parameters of the public health prioritization debate?

OBJECTIVES: To explore the influence of values and context in public health priority-setting in local government in England. STUDY DESIGN: Qualitative interview study. METHODS: Decision-makers' views were identified through semi-structured interviews and prioritization tools relevant for public health were reviewed. Interviews (29) were carried out with Health and Wellbeing Board members and other key stakeholders across three local authorities in England, following an introductory workshop. RESULTS: There were four main influences on priorities for public health investment in our case study sites: an organizational context where health was less likely to be associated with health care and where accountability was to a local electorate; a commissioning and priority-setting context (plan, do, study, act) located within broader local authority priority-setting processes; different views of what counts as evidence and, in particular, the role of local knowledge; and debates over what constitutes a public health intervention, triggered by the transfer of a public health budget from the NHS to local authorities in England. CONCLUSIONS: The relocation of public health into local authorities exposes questions over prioritizing public health investment, including the balance across lifestyle interventions and broader action on social determinants of health and the extent to which the public health evidence base influences local democratic decision-making. Action on wider social determinants reinforces not only the art and science but also the values and politics of public health.

Protocol for the comprehensive biochemical and cellular profiling of small-molecule degraders using CoraFluor TR-FRET technology.

Comprehensive characterization of small-molecule degraders, including binary and ternary complex formation and degradation efficiency, is critical for bifunctional ligand development and understanding structure-activity relationships. Here, we present a protocol for the biochemical and cellular profiling of small-molecule degraders based on CoraFluor time-resolved fluorescence resonance energy transfer (TR-FRET) technology. We describe steps for labeling antibodies and proteins, tracer saturation binding, binary target engagement, ternary complex profiling, and off-rate determination. We then detail procedures for the quantification of endogenous and GFP fusion proteins in cell lysates. For complete details on the use and execution of this protocol, please refer to Ichikawa et al.1.

The cyclimids: Degron-inspired cereblon binders for targeted protein degradation.

Cereblon (CRBN) is an E3 ligase substrate adapter widely exploited for targeted protein degradation (TPD) strategies. However, achieving efficient and selective target degradation is a preeminent challenge with ligands that engage CRBN. Here, we report that the cyclimids, ligands derived from the C-terminal cyclic imide degrons of CRBN, exhibit distinct modes of interaction with CRBN and offer a facile approach for developing potent and selective bifunctional degraders. Quantitative TR-FRET-based characterization of 60 cyclimid degraders in binary and ternary complexes across different substrates revealed that ternary complex binding affinities correlated strongly with cellular degradation efficiency. Our studies establish the unique properties of the cyclimids as versatile warheads in TPD and a systematic biochemical approach for quantifying ternary complex formation to predict their cellular degradation activity, which together will accelerate the development of ligands that engage CRBN.

Variation in mortality following hip fracture across the Asia Pacific region: Systematic review and proportional meta-analysis.

OBJECTIVE: To determine country/region-specific mortality (in-hospital, 30-day and 1-year) following hip fracture across the Asia Pacific region. METHODS: Five databases MEDLINE, PUBMED, EMBASE, Web of Science and the Cochrane Library were searched to identify studies that reported mortality following hospitalisation for low-trauma hip fracture in adults aged ≥50 years with data from 2010 to 30 September 2021. There were no restrictions on study design or language. Pooled mortality estimates for countries/regions with ≥2 studies were calculated using random-effects models. RESULTS: In total 244 studies were included in the meta-analysis. 123 studies (1,382,810 patients, 13 countries/regions) reported in-hospital mortality which ranged from 1.4 % in Japan [95 %CI 1.2-1.7], Singapore [95 %CI 1.0-1.6], China [95 %CI 0.8-2.3] and Hong Kong SAR [95 %CI 0.8-2.6] to 5.5 % [95 %CI 4.1-7.2] in New Zealand. 92 studies (628,450 patients, 13 countries/regions) reported 30-day mortality which ranged from 1.2 % in Japan [95 %CI 0.9-1.5] and Thailand [95 %CI 0.7-2.0] to 7.4 % [95 %CI 7.0-7.8] in Australia. 142 studies (1,139,752 patients, 14 countries/regions) reported 1-year mortality which ranged from 10.8 % [95 %CI 9.6-12.1] in Singapore to 23.3 % [95 %CI 22.3-24.5] in Australia and 23.8 % in New Zealand. CONCLUSION: There is substantial variation in mortality across the Asia Pacific region. Short-term mortality rates in Asian countries, notably Japan and Singapore, are up to four-fold lower than for Australia and New Zealand. This difference, although less marked, is sustained at 1-year with a two-fold lower mortality rate in Asia. This meta-analysis is the first to delineate these differences, further studies are required to understand the reasons for this variation.

Epigenetic modulation through BET bromodomain inhibitors as a novel therapeutic strategy for progranulin-deficient frontotemporal dementia.

Frontotemporal dementia (FTD) is a debilitating neurodegenerative disorder with currently no disease-modifying treatment options available. Mutations in GRN are one of the most common genetic causes of FTD, near ubiquitously resulting in progranulin (PGRN) haploinsufficiency. Small molecules that can restore PGRN protein to healthy levels in individuals bearing a heterozygous GRN mutation may thus have therapeutic value. Here, we show that epigenetic modulation through bromodomain and extra-terminal domain (BET) inhibitors (BETi) potently enhance PGRN protein levels, both intracellularly and secreted forms, in human central nervous system (CNS)-relevant cell types, including in microglia-like cells. In terms of potential for disease modification, we show BETi treatment effectively restores PGRN levels in neural cells with a GRN mutation known to cause PGRN haploinsufficiency and FTD. We demonstrate that BETi can rapidly and durably enhance PGRN in neural progenitor cells (NPCs) in a manner dependent upon BET protein expression, suggesting a gain-of-function mechanism. We further describe a CNS-optimized BETi chemotype that potently engages endogenous BRD4 and enhances PGRN expression in neuronal cells. Our results reveal a new epigenetic target for treating PGRN-deficient forms of FTD and provide mechanistic insight to aid in translating this discovery into therapeutics.

Inhibiting the Keap1/Nrf2 Protein-Protein Interaction with Protein-Like Polymers.

Successful and selective inhibition of the cytosolic protein-protein interaction (PPI) between nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associating protein 1 (Keap1) can enhance the antioxidant response, with the potential for a therapeutic effect in a range of settings including in neurodegenerative disease (ND). Small molecule inhibitors have been developed, yet many have off-target effects, or are otherwise limited by poor cellular permeability. Peptide-based strategies have also been attempted to enhance specificity, yet face challenges due to susceptibility to degradation and lack of cellular penetration. Herein, these barriers are overcome utilizing a polymer-based proteomimetics. The protein-like polymer (PLP) consists of a synthetic, lipophilic polymer backbone displaying water soluble Keap1-binding peptides on each monomer unit forming a brush polymer architecture. The PLPs are capable of engaging Keap1 and displacing the cellular protective transcription factor Nrf2, which then translocates to the nucleus, activating the antioxidant response element (ARE). PLPs exhibit increased Keap1 binding affinity by several orders of magnitude compared to free peptides, maintain serum stability, are cell-penetrant, and selectively activate the ARE pathway in cells, including in primary cortical neuronal cultures. Keap1/Nrf2-inhibitory PLPs have the potential to impact the treatment of disease states associated with dysregulation of oxidative stress, such as NDs.

KBTBD4 Cancer Hotspot Mutations Drive Neomorphic Degradation of HDAC1/2 Corepressor Complexes.

Cancer mutations can create neomorphic protein-protein interactions to drive aberrant function 1 . As a substrate receptor of the CULLIN3-RBX1 E3 ubiquitin ligase complex, KBTBD4 is recurrently mutated in medulloblastoma (MB) 2 , the most common embryonal brain tumor in children, and pineoblastoma 3 . These mutations impart gain-of-function to KBTBD4 to induce aberrant degradation of the transcriptional corepressor CoREST 4 . However, their mechanism of action remains unresolved. Here, we elucidate the mechanistic basis by which KBTBD4 mutations promote CoREST degradation through engaging HDAC1/2, the direct neomorphic target of the substrate receptor. Using deep mutational scanning, we systematically map the mutational landscape of the KBTBD4 cancer hotspot, revealing distinct preferences by which insertions and substitutions can promote gain-of-function and the critical residues involved in the hotspot interaction. Cryo-electron microscopy (cryo-EM) analysis of two distinct KBTBD4 cancer mutants bound to LSD1-HDAC1-CoREST reveals that a KBTBD4 homodimer asymmetrically engages HDAC1 with two KELCH-repeat propeller domains. The interface between HDAC1 and one of the KBTBD4 propellers is stabilized by the MB mutations, which directly insert a bulky side chain into the active site pocket of HDAC1. Our structural and mutational analyses inform how this hotspot E3-neo-substrate interface can be chemically modulated. First, our results unveil a converging shape complementarity-based mechanism between gain-of-function E3 mutations and a molecular glue degrader, UM171. Second, we demonstrate that HDAC1/2 inhibitors can block the mutant KBTBD4-HDAC1 interface, the aberrant degradation of CoREST, and the growth of KBTBD4-mutant MB models. Altogether, our work reveals the structural and mechanistic basis of cancer mutation-driven neomorphic protein-protein interactions and pharmacological strategies to modulate their action for therapeutic applications.

Can diabetes prevention programmes be translated effectively into real-world settings and still deliver improved outcomes? A synthesis of evidence.

OBJECTIVE: Randomized trials provide evidence that intensive lifestyle interventions leading to dietary and physical activity change can delay or prevent Type 2 diabetes. Translational studies have assessed the impact of interventions based on, but less intensive than, trial protocols delivered in community settings with high-risk populations. The aim of this review was to synthesize evidence from translational studies of any design to assess the impact of interventions delivered outside large randomized trials. METHODS: Medical and scientific databases were searched using specified inclusion and exclusion criteria. Studies were included that used a tested diabetes preventive study protocol with an adult population at risk from Type 2 diabetes. Included papers were quality assessed and data extracted using recommended methods. RESULTS: From an initial 793 papers, 19 papers reporting 17 studies were included. Translational studies from a range of settings utilized a variety of methods. All were based on the US Diabetes Prevention Programme protocol or the Finnish Diabetes Prevention Study, with modifications that increased feasibility and access. The main outcome that was reported in all studies was weight change. Weight loss, which occurred in all but one study, was greater in intervention arms than in control subjects. No consistent differences were found in blood glucose or waist circumference. CONCLUSIONS: Translational studies based on the intensive diabetes prevention programmes showed that there is potential for less intensive interventions both to be feasible and to have an impact on future progression to diabetes in at-risk individuals.

Community-based dietary and physical activity interventions in low socioeconomic groups in the UK: a mixed methods systematic review.

OBJECTIVE: Low socioeconomic status (SES) is a risk factor for type 2 diabetes and changes in diet and physical activity can prevent diabetes. We assessed the effectiveness and acceptability of community-based dietary and physical activity interventions among low-SES groups in the UK. METHOD: We searched relevant databases and web resources from 1990 to November 2009 to identify relevant published and grey literature using an iterative approach, focusing on UK studies. RESULTS: Thirty-five relevant papers (nine quantitative, 23 qualitative and three mixed methods studies) were data extracted, quality assessed and synthesised using narrative synthesis and thematic analysis. The relationship between interventions and barriers and facilitators was also examined. Dietary/nutritional, food retail, physical activity and multi-component interventions demonstrated mixed effectiveness. Qualitative studies indicated a range of barriers and facilitators, which spanned pragmatic, social and psychological issues. The more effective interventions used a range of techniques to address some surface-level psychological and pragmatic concerns, however many deeper-level social, psychological and pragmatic concerns were not addressed. CONCLUSION: Evidence on the effectiveness of community-based dietary and physical activity interventions is inconclusive. A range of barriers and facilitators exist, some of which were addressed by interventions but some of which require consideration in future research.

Overview of trials on artificial intelligence algorithms in breast cancer screening - A roadmap for international evaluation and implementation.

Accumulating evidence from retrospective studies demonstrate at least non-inferior performance when using AI algorithms with different strategies versus double-reading in mammography screening. In addition, AI algorithms for mammography screening can reduce work load by moving to single human reading. Prospective trials are essential to avoid unintended adverse consequences before incorporation of AI algorithms into UK's National Health Service (NHS) Breast Screening Programme (BSP). A stakeholders' meeting was organized in Newnham College, Cambridge, UK to undertake a review of the current evidence to enable consensus discussion on next steps required before implementation into a screening programme. It was concluded that a multicentre multivendor testing platform study with opt-out consent is preferred. AI thresholds from different vendors should be determined while maintaining non-inferior screening performance results, particularly ensuring recall rates are not increased. Automatic recall of cases using an agreed high sensitivity AI score versus automatic rule out with a low AI score set at a high sensitivity could be used. A human reader should still be involved in decision making with AI-only recalls requiring human arbitration. Standalone AI algorithms used without prompting maintain unbiased screening reading performance, but reading with prompts should be tested prospectively and ideally provided for arbitration.

Structural and biochemical characterization establishes a detailed understanding of KEAP1-CUL3 complex assembly.

KEAP1 promotes the ubiquitin-dependent degradation of NRF2 by assembling into a CUL3-dependent ubiquitin ligase complex. Oxidative and electrophilic stress inhibit KEAP1 allowing NRF2 to accumulate for the transactivation of stress response genes. To date there are no structures of the KEAP1-CUL3 interaction nor binding data to show the contributions of different domains to their binding affinity. We determined a crystal structure of the BTB and 3-box domains of human KEAP1 in complex with the CUL3 N-terminal domain that showed a heterotetrameric assembly with 2:2 stoichiometry. To support the structural data, we developed a versatile TR-FRET-based assay system to profile the binding of BTB-domain-containing proteins to CUL3 and determine the contribution of distinct protein features, revealing the importance of the CUL3 N-terminal extension for high affinity binding. We further provide direct evidence that the investigational drug CDDO does not disrupt the KEAP1-CUL3 interaction, even at high concentrations, but reduces the affinity of KEAP1-CUL3 binding. The TR-FRET-based assay system offers a generalizable platform for profiling this protein class and may form a suitable screening platform for ligands that disrupt these interactions by targeting the BTB or 3-box domains to block E3 ligase function.

Prolyl-tRNA synthetase as a novel therapeutic target in multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy characterised by aberrant production of immunoglobulins requiring survival mechanisms to adapt to proteotoxic stress. We here show that glutamyl-prolyl-tRNA synthetase (GluProRS) inhibition constitutes a novel therapeutic target. Genomic data suggest that GluProRS promotes disease progression and is associated with poor prognosis, while downregulation in MM cells triggers apoptosis. We developed NCP26, a novel ATP-competitive ProRS inhibitor that demonstrates significant anti-tumour activity in multiple in vitro and in vivo systems and overcomes metabolic adaptation observed with other inhibitor chemotypes. We demonstrate a complex phenotypic response involving protein quality control mechanisms that centers around the ribosome as an integrating hub. Using systems approaches, we identified multiple downregulated proline-rich motif-containing proteins as downstream effectors. These include CD138, transcription factors such as MYC, and transcription factor 3 (TCF3), which we establish as a novel determinant in MM pathobiology through functional and genomic validation. Our preclinical data therefore provide evidence that blockade of prolyl-aminoacylation evokes a complex pro-apoptotic response beyond the canonical integrated stress response and establish a framework for its evaluation in a clinical setting.

Diagnostic TR-FRET assays for detection of antibodies in patient samples.

Serological assays are important diagnostic tools for surveying exposure to the pathogen, monitoring immune response post vaccination, and managing spread of the infectious agent among the population. Current serological laboratory assays are often limited because they require the use of specialized laboratory technology and/or work with a limited number of sample types. Here, we evaluate an alternative by developing time-resolved Förster resonance energy transfer (TR-FRET) homogeneous assays that exhibited exceptional versatility, scalability, and sensitivity and outperformed or matched currently used strategies in terms of sensitivity, specificity, and precision. We validated the performance of the assays measuring total immunoglobulin G (IgG) levels; antibodies against severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle Eastern respiratory syndrome (MERS)-CoV spike (S) protein; and SARS-CoV-2 S and nucleocapsid (N) proteins and applied it to several large sample sets and real-world applications. We further established a TR-FRET-based ACE2-S competition assay to assess the neutralization propensity of the antibodies. Overall, these TR-FRET-based serological assays can be rapidly extended to other antigens and are compatible with commonly used plate readers.

Discovery of a series of 2-(1H-pyrazol-1-yl)pyridines as ALK5 inhibitors with potential utility in the prevention of dermal scarring.

A series of 2-(1H-pyrazol-1-yl)pyridines are described as inhibitors of ALK5 (TGFß receptor I kinase). Modeling compounds in the ALK5 kinase domain enabled some optimization of potency via substitutions on the pyrazole core. One of these compounds PF-03671148 gave a dose dependent reduction in TGFß induced fibrotic gene expression in human fibroblasts. A similar reduction in fibrotic gene expression was observed when PF-03671148 was applied topically in a rat wound repair model. Thus these compounds have potential utility for the prevention of dermal scarring.