Low-Affinity Binding Sites and the Transcription Factor Specificity Paradox in Eukaryotes.

Eukaryotic transcription factors (TFs) from the same structural family tend to bind similar DNA sequences, despite the ability of these TFs to execute distinct functions in vivo. The cell partly resolves this specificity paradox through combinatorial strategies and the use of low-affinity binding sites, which are better able to distinguish between similar TFs. However, because these sites have low affinity, it is challenging to understand how TFs recognize them in vivo. Here, we summarize recent findings and technological advancements that allow for the quantification and mechanistic interpretation of TF recognition across a wide range of affinities. We propose a model that integrates insights from the fields of genetics and cell biology to provide further conceptual understanding of TF binding specificity. We argue that in eukaryotes, target specificity is driven by an inhomogeneous 3D nuclear distribution of TFs and by variation in DNA binding affinity such that locally elevated TF concentration allows low-affinity binding sites to be functional.

Context-Dependent Gene Regulation by Homeodomain Transcription Factor Complexes Revealed by Shape-Readout Deficient Proteins.

Eukaryotic transcription factors (TFs) form complexes with various partner proteins to recognize their genomic target sites. Yet, how the DNA sequence determines which TF complex forms at any given site is poorly understood. Here, we demonstrate that high-throughput in vitro DNA binding assays coupled with unbiased computational analysis provide unprecedented insight into how different DNA sequences select distinct compositions and configurations of homeodomain TF complexes. Using inferred knowledge about minor groove width readout, we design targeted protein mutations that destabilize homeodomain binding both in vitro and in vivo in a complex-specific manner. By performing parallel systematic evolution of ligands by exponential enrichment sequencing (SELEX-seq), chromatin immunoprecipitation sequencing (ChIP-seq), RNA sequencing (RNA-seq), and Hi-C assays, we not only classify the majority of in vivo binding events in terms of complex composition but also infer complex-specific functions by perturbing the gene regulatory network controlled by a single complex.

Chromatin modules and their implication in genomic organization and gene regulation.

Regulation of gene expression is a complex but highly guided process. While genomic technologies and computational approaches have allowed high-throughput mapping of cis-regulatory elements (CREs) and their interactions in 3D, their precise role in regulating gene expression remains obscure. Recent complementary observations revealed that interactions between CREs frequently result in the formation of small-scale functional modules within topologically associating domains. Such chromatin modules likely emerge from a complex interplay between regulatory machineries assembled at CREs, including site-specific binding of transcription factors. Here, we review the methods that allow identifying chromatin modules, summarize possible mechanisms that steer CRE interactions within these modules, and discuss outstanding challenges to uncover how chromatin modules fit in our current understanding of the functional 3D genome.

ZAP70, too little, too much can lead to autoimmunity.

Establishing both central and peripheral tolerance requires the appropriate TCR signaling strength to discriminate self- from agonist-peptide bound to self MHC molecules. ZAP70, a cytoplasmic tyrosine kinase, directly interacts with the TCR complex and plays a central and requisite role in TCR signaling in both thymocytes and peripheral T cells. By studying ZAP70 hypomorphic mutations in mice and humans with a spectrum of hypoactive or hyperactive activities, we have gained insights into mechanisms of central and peripheral tolerance. Interestingly, both hypoactive and hyperactive ZAP70 can lead to the development of autoimmune diseases, albeit through distinct mechanisms. Immature thymocytes and mature T cells rely on normal ZAP70 function to complete their development in the thymus and to modulate T cell responses in the periphery. Hypoactive ZAP70 function compromises key developmental checkpoints required to establish central tolerance, allowing thymocytes with potentially self-reactive TCRs a greater chance to escape negative selection. Such 'forbidden clones' may escape into the periphery and may pose a greater risk for autoimmune disease development since they may not engage negative regulatory mechanisms as effectively. Hyperactive ZAP70 enhances thymic negative selection but some thymocytes will, nonetheless, escape negative selection and have greater sensitivity to weak and self-ligands. Such cells must be controlled by mechanisms involved in anergy, expansion of Tregs, and upregulation of inhibitory receptors or signaling molecules. However, such potentially autoreactive cells may still be able to escape control by peripheral negative regulatory constraints. Consistent with findings in Zap70 mutants, the signaling defects in at least one ZAP70 substrate, LAT, can also lead to autoimmune disease. By dissecting the similarities and differences among mouse models of patient disease or mutations in ZAP70 that affect TCR signaling strength, we have gained insights into how perturbed ZAP70 function can lead to autoimmunity. Because of our work and that of others on ZAP70, it is likely that perturbations in other molecules affecting TCR signaling strength will be identified that also overcome tolerance mechanisms and cause autoimmunity. Delineating these molecular pathways could lead to the development of much needed new therapeutic targets in these complex diseases.

Repeated immunization with ATRA-containing liposomal adjuvant transdifferentiates Th17 cells to a Tr1-like phenotype.

In many autoimmune diseases, autoantigen-specific Th17 cells play a pivotal role in disease pathogenesis. Th17 cells can transdifferentiate into other T cell subsets in inflammatory conditions, however, there have been no attempts to target Th17 cell plasticity using vaccines. We investigated if autoantigen-specific Th17 cells could be specifically targeted using a therapeutic vaccine approach, where antigen was formulated in all-trans retinoic acid (ATRA)-containing liposomes, permitting co-delivery of antigen and ATRA to the same target cell. Whilst ATRA was previously found to broadly reduce Th17 responses, we found that antigen formulated in ATRA-containing cationic liposomes only inhibited Th17 cells in an antigen-specific manner and not when combined with an irrelevant antigen. Furthermore, this approach shifted existing Th17 cells away from IL-17A expression and transcriptomic analysis of sorted Th17 lineage cells from IL-17 fate reporter mice revealed a shift of antigen-specific Th17 cells to exTh17 cells, expressing functional markers associated with T cell regulation and tolerance. In the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, vaccination with myelin-specific (MOG) antigen in ATRA-containing liposomes reduced Th17 responses and alleviated disease. This highlights the potential of therapeutic vaccination for changing the phenotype of existing Th17 cells in the context of immune mediated diseases.

Patient-Reported Outcome Measures: How to Maximize Their Utility and Mitigate Health Care Disparities.

Patient-reported outcome measures (PROMs) provide a standardized assessment from the patient about their own health status. Although originally developed as research tools, PROMs can be used in clinical orthopaedic care to complement objective functional measures (eg, range of motion). When PROMs are used during clinical care, they can improve patient outcomes, engagement, well-being, and patient-physician communication. Therefore, PROMs are increasingly integrated into shared decision-making approaches to guide treatment decisions, enhance treatment plans, and predict outcomes. They are also being used in quality measurement and value-based health care arrangements. However, methods to communicate PROMs with patients have not been fully developed, and there continue to be barriers to implementing their collection and communication at scale. In addition, measuring care quality and communicating PROMs with patients may have unintended consequences, such as when used in measurement without accounting for confounding factors (eg, psychological and social health), or in perpetuating health care disparities when used imprecisely (eg, lack of linguistic or cultural validation). It is important to describe the current state of PROM use in orthopaedic surgery, highlight opportunities and challenges of PROM use in clinical care, and provide a roadmap for how to incorporate PROMs to equitably improve patient health and build orthopaedic surgery practices.

Inflammasome inhibition protects dopaminergic neurons from α-synuclein pathology in a model of progressive Parkinson's disease.

Neuroinflammation has been suggested as a pathogenetic mechanism contributing to Parkinson's disease (PD). However, anti-inflammatory treatment strategies have not yet been established as a therapeutic option for PD patients. We have used a human α-synuclein mouse model of progressive PD to examine the anti-inflammatory and neuroprotective effects of inflammasome inhibition on dopaminergic (DA) neurons in the substantia nigra (SN). As the NLRP3 (NOD-, LRR- and pyrin domain-containing 3)-inflammasome is a core interface for both adaptive and innate inflammation and is also highly druggable, we investigated the implications of its inhibition. Repeat administration of MCC950, an inhibitor of NLRP3, in a PD model with ongoing pathology reduced CD4+ and CD8+ T cell infiltration into the SN. Furthermore, the anti-inflammasome treatment mitigated microglial activation and modified the aggregation of α-synuclein protein in DA neurons. MCC950-treated mice showed significantly less neurodegeneration of DA neurons and a reduction in PD-related motor behavior. In summary, early inflammasome inhibition can reduce neuroinflammation and prevent DA cell death in an α-synuclein mouse model for progressive PD.

Practice Variations in Managing Infantile Hemangiomas.

Infantile hemangioma (IH) is the most common benign tumor of infancy. For children with IH who require treatment, propranolol and other beta blockers have been shown to be safe and effective. Although consensus guidelines for managing IH have been published, anecdotal experience suggests that there remain variations in management. This study was performed to document these variations amongst providers and to identify areas for future research. We conducted an Internet-based survey of clinicians who treat patients with IH. Hypothetical cases and management scenarios were presented. Twenty-nine respondents participated in the survey. Most respondents use generic propranolol in infants with growing IH of the head and neck, with a goal dose of 2 mg/kg/d, until ~1 year of age. A variety of management strategies were documented including which patients should be treated, optimal dose and duration of therapy, how patients should be monitored, which patients should get additional workup, how propranolol should best be discontinued, and how often to see patients in follow-up. This study demonstrates wide practice variations in managing patients with IH. Further research is indicated to address these variations and develop additional/updated evidence-based guidelines.

The Role of Autopsy in Quality Assurance: Pilot Study of a Method for Prospective Reporting of Diagnostic Errors Discovered at Autopsy.

ABSTRACT: Hospital autopsies frequently reveal errors in diagnosis that could have affected the patient's clinical outcome. The aims of this study were (1) to investigate the ability of autopsy at our institution to elucidate unrecognized antemortem diagnoses and (2) to pilot a method for tabulating diagnostic discrepancies on a prospective basis. The study sample consisted of 296 cases from our hybrid hospital/forensic autopsy service during the period 2016 to 2018. Discrepancies in autopsy and clinical diagnosis were reported by pathologists at the time of autopsy report generation using a standard form. The rates of major discrepancies between autopsy and clinical diagnoses were 37.5% for in-hospital cases and 25% for patients who died outside our hospital (P < 0.05). The most common discrepant category was infection. The overall rates of discrepant causes of death were 14% (in hospital) and 8% (out of hospital) (ns). Overall percentages of cases with major diagnostic discrepancies were higher in our study than have been previously reported. It is possible that the nature of our patient population plays a role in this result. This study describes an important prospective reporting tool that will allow us to track rates of medical errors and improve diagnosis and treatment of the critically ill.

How we approach the diagnosis of a vascular anomaly.

Vascular anomalies represent a diverse group of complex disorders that can cause significant complications, including coagulopathies, pain, and decreased function. The diagnosis of vascular anomalies is often challenging due to heterogeneity of presenting phenotypes and overlapping clinical features with other pediatric conditions. Pediatric hematologists/oncologists (PHO) are uniquely positioned for an essential role in diagnosing, managing, and coordinating the multidisciplinary care required to maximize the quality of life of these patients. Here, we review the diagnostic approach involved in patients with vascular anomalies and utilize cases to highlight the challenges involved, and how PHOs can play a vital part in the care of these patients.

Sirolimus efficacy in the treatment of critically ill infants with congenital primary chylous effusions.

BACKGROUND: Chylothorax can be a presenting symptom of complex lymphatic anomaly in children and is associated with significant respiratory morbidity. Historically, the traditional pharmacological treatment has been octreotide. There are several treatments that have been utilized in the past few years including sirolimus; however, data regarding their efficacy and outcomes is limited. Furthermore, sirolimus has proven efficacy in complex vascular malformations, and hence, its utility/efficacy in infantile primary chylous effusions warrants further investigation. METHODS: In this retrospective study at Texas Children's Hospital, data were extracted for all infants with chylothorax who were treated with sirolimus between 2009 and 2020. Details regarding underlying diagnosis, comorbidities, and number of days from sirolimus initiation to resolution of effusion were collected. RESULTS: Initially a total of 12 infants were identified. Among them, seven patients had complete data and were included in the study. Reasons for chylous effusions include presumed complex lymphatic anomaly, generalized lymphatic anomaly, and complex congenital lymphatic anomaly. The mean duration of sirolimus treatment needed for chest tube removal was 16 days, with a median of 19 days and range of 7-22 days. No patients had progression of effusions while on sirolimus. CONCLUSION: With close monitoring, sirolimus appears to be an effective therapy for pediatric lymphatic effusions even in critically ill infants. The study also demonstrates shorter duration of chest tube requirement after initiation of sirolimus compared to previous studies. Larger multi-institutional studies are needed to further support our findings.

Reporters of TCR signaling identify arthritogenic T cells in murine and human autoimmune arthritis.

How pathogenic cluster of differentiation 4 (CD4) T cells in rheumatoid arthritis (RA) develop remains poorly understood. We used Nur77-a marker of T cell antigen receptor (TCR) signaling-to identify antigen-activated CD4 T cells in the SKG mouse model of autoimmune arthritis and in patients with RA. Using a fluorescent reporter of Nur77 expression in SKG mice, we found that higher levels of Nur77-eGFP in SKG CD4 T cells marked their autoreactivity, arthritogenic potential, and ability to more readily differentiate into interleukin-17 (IL-17)-producing cells. The T cells with increased autoreactivity, nonetheless had diminished ex vivo inducible TCR signaling, perhaps reflective of adaptive inhibitory mechanisms induced by chronic autoantigen exposure in vivo. The enhanced autoreactivity was associated with up-regulation of IL-6 cytokine signaling machinery, which might be attributable, in part, to a reduced amount of expression of suppressor of cytokine signaling 3 (SOCS3)-a key negative regulator of IL-6 signaling. As a result, the more autoreactive GFPhi CD4 T cells from SKGNur mice were hyperresponsive to IL-6 receptor signaling. Consistent with findings from SKGNur mice, SOCS3 expression was similarly down-regulated in RA synovium. This suggests that despite impaired TCR signaling, autoreactive T cells exposed to chronic antigen stimulation exhibit heightened sensitivity to IL-6, which contributes to the arthritogenicity in SKG mice, and perhaps in patients with RA.

Nurse Leader Preparation for Disasters: A Cross-sectional Survey.

OBJECTIVE: The aim of this study was to evaluate nurse leader confidence in emergency management and disaster preparedness. BACKGROUND: There is a dearth of evidence in the literature regarding the preparation of nurse leaders to manage emergencies and disasters. Research suggests significant gaps in nurse leader confidence across roles and in structured education that prepares nurse leaders across the spectrum of experience to manage in a crisis. METHODS: An exploratory, cross-sectional survey included a sample of 432 RNs who are members of the Northwest Organization for Nurse Leaders. RESULTS: Results indicate significant variance in nurse leader confidence across roles, experience, and previous disaster-related education. Positive associations regarding nurse leaders' confidence in managing disasters exist with more advanced positions, advanced education, and structured training. CONCLUSIONS: Nursing leaders lack consistent education that prepares them for emergency and disaster management. Nurse leaders across all levels would benefit from formal education in these areas.

Teams Model of Clinical Nursing Education: An Innovative Approach.

The need for registered nurses has never been greater. Only through thoughtful academic-practice partnerships will it be possible to align the current and future demands of the workplace with the knowledge, attitudes, and skills that are required. Outdated nursing education models that treat practice partners as "real estate" and try to expose all students, in the same way, to the full array of patient populations, most often in acute care settings, do not serve the profession well. Attending to the heightened expectations for nurses to lead complex, interprofessional teams across an array of settings led to the development of the Teams Model of clinical nursing education. In this model, students match to practice partner facilities where they complete the majority of their clinical hours. They do not rotate through various settings, they do not spend precious practice time reorienting to new facilities, and they are not relegated to the periphery of the interprofessional team when in the student role. They become expected and valuable members of the care delivery team. They focus on developing depth as a nurse rather than breath across patient populations. The Teams Model is predicated on advancing academic-practice partnerships that provide a pipeline for workforce development and prepares the new graduate as a work-ready hire. Ongoing evaluation of the students' development and their knowledge acquisition is linked to practice partner assessments and provides support for this model as an innovative alternative to preparing the next generation of nurses. This partnership program builds confidence through immersion experiences in a consistent, welcoming, and prepared clinical environment, with opportunities to advance the priorities of the practice setting in addition to promoting positive patient outcomes. Benefits include student exposure to nontraditional fields of nursing and areas of critical shortage as well as the ability to pivot quickly as workforce needs shift.

SelexGLM differentiates androgen and glucocorticoid receptor DNA-binding preference over an extended binding site.

The DNA-binding interfaces of the androgen (AR) and glucocorticoid (GR) receptors are virtually identical, yet these transcription factors share only about a third of their genomic binding sites and regulate similarly distinct sets of target genes. To address this paradox, we determined the intrinsic specificities of the AR and GR DNA-binding domains using a refined version of SELEX-seq. We developed an algorithm, SelexGLM, that quantifies binding specificity over a large (31-bp) binding site by iteratively fitting a feature-based generalized linear model to SELEX probe counts. This analysis revealed that the DNA-binding preferences of AR and GR homodimers differ significantly, both within and outside the 15-bp core binding site. The relative preference between the two factors can be tuned over a wide range by changing the DNA sequence, with AR more sensitive to sequence changes than GR. The specificity of AR extends to the regions flanking the core 15-bp site, where isothermal calorimetry measurements reveal that affinity is augmented by enthalpy-driven readout of poly(A) sequences associated with narrowed minor groove width. We conclude that the increased specificity of AR is correlated with more enthalpy-driven binding than GR. The binding models help explain differences in AR and GR genomic binding and provide a biophysical rationale for how promiscuous binding by GR allows functional substitution for AR in some castration-resistant prostate cancers.

Exploring influences on evaluation practice: a case study of a national physical activity programme.

BACKGROUND: Interventions to improve physical activity behaviour are a core part of public health policy and practice. It is essential that we evaluate these interventions and use the evidence to inform decisions to improve population health. Evaluation of 'real-world' interventions provide an opportunity to generate practice-relevant evidence, however these interventions are difficult to evaluate. Various guidelines have been developed to facilitate evaluation, but evidence about their effectiveness in practice is limited. To explore influences on evaluation practice in an applied context, we conducted a case study of Sport England's 'Get Healthy Get Active' (GHGA) programme. This was a national programme that funded 33 projects that were delivered and evaluated across England. The programme was chosen as it was designed to generate evidence on the role of sport in increasing physical activity and improving health. The study aimed to explore and appraise whether strategies intended to facilitate project evaluation, including funder requirements to use a standardised evaluation framework and specific data collection methods, were effective in generating evidence that enabled the programme to meet its aims. METHODS: We applied a collective case study design involving 35 semi-structured interviews, and documentary analysis of multiple sources of evidence from 23 physical activity projects funded by GHGA. We applied thematic and framework analysis. We developed a logic model and mapped actual outcomes against intended outcomes. A narrative synthesis is provided. We discuss implications for the effective commissioning and evaluation of public health interventions. RESULTS: We identified five main themes of influences on evaluation practices that can act as barriers and facilitators to good practice: programme and project design; evaluation design; partnerships; resources; and organisational structures and systems. These influences are context-specific and operate through a complex set of interactions. CONCLUSION: Developing a better understanding of how influences on evaluation practice can act as facilitators or barriers is vital to help close current gaps in the evidence-based practice cycle. Critically, organisational structures and systems are needed to facilitate collaborative decision making; integration of projects and evaluation across partners organisations; transfer of knowldege and insights between stakeholders; and more rapid feedback and dissemination.

Long-term outcomes of pediatric and young adult patients receiving radiotherapy for nonmalignant vascular anomalies.

BACKGROUND: Nonmalignant vascular anomalies (VA) comprise a heterogeneous spectrum of conditions characterized by aberrant growth or development of blood and/or lymphatic vessels and can cause significant morbidity. Little is known about outcomes after radiotherapy in pediatric and young adult patients with nonmalignant VA. METHODS: Thirty patients who were diagnosed with nonmalignant VA and treated with radiotherapy prior to 2017 and before the age of 30 were identified. Clinical and treatment characteristics and outcomes were recorded. RESULTS: Median age at first radiotherapy was 15 years (range 0.02-27). Median follow-up from completion of first radiotherapy was 9.8 years (range 0.02-67.4). Lymphatic malformations (33%), kaposiform hemangioendothelioma (17%), and venous malformations (17%) were the most common diagnoses. The most common indication for first radiotherapy was progression despite standard therapy and/or urgent palliation for symptoms (57%). After first radiotherapy, 14 patients (47%) had a complete response or partial response, defined as decrease in size of treated lesion or symptomatic improvement. After first radiotherapy, 27 (90%) required additional treatment for progression or recurrence. Long-term complications included telangiectasias, fibrosis, xerophthalmia, radiation pneumonitis, ovarian failure, and central hypothyroidism. No patient developed secondary malignancies. At last follow-up, three patients (10%) were without evidence of disease, 26 (87%) with disease, and one died of complications (3.3%). CONCLUSIONS: A small group of pediatric and young adult patients with nonmalignant, high-risk VA experienced clinical benefit from radiotherapy with expected toxicity; however, most experienced progression. Prospective studies are needed to characterize indications for radiotherapy in VA refractory to medical therapy, including targeted inhibitors.

Grammatical processing in two languages: How individual differences in language experience and cognitive abilities shape comprehension in heritage bilinguals.

Recent studies have demonstrated variation in language processing for monolingual and bilingual speakers alike, suggesting that only by considering individual differences will an accurate picture of the consequences of language experience be adequately understood. This approach can be illustrated in ERP research that has shown that sentence contexts that traditionally elicit a P600 component in response to a syntactic violation, elicit an N400 response for a subset of individuals. That result has been reported for monolingual speakers processing sentences in their L1 and also for bilinguals processing sentences in their L2. To date, no studies have compared variation in L1 and L2 ERP effects in the very same bilingual speakers. In the present paper, we do that by examining sentence processing in heritage bilinguals who acquired both languages from early childhood but for whom the L2 typically becomes the dominant language. Variation in ERPs produced by the non-dominant L1 and dominant L2 of heritage bilinguals was compared to variation found in monolingual L1 processing. The group-averaged results showed the smallest N400 and P600 responses in the native, but no longer dominant, L1 of heritage bilinguals, and largest in the monolinguals. Individual difference analyses linking ERP variation to working memory and language proficiency showed that working memory was the primary factor related to monolingual L1 processing, whereas bilinguals did not show this relationship. In contrast, proficiency was the primary factor related to ERP responses for no longer dominant L1 for bilinguals, but unrelated to monolingual L1 processing, whereas bilinguals' dominant L2 processing showed an intermediate relationship. Finally, the N400 was absent for bilinguals performing the task in the same language in which they initially learned to read, but significantly larger when bilinguals performed the task in the other language. The results support the idea that proficient bilinguals utilize the same underlying mechanisms to process both languages, although the factors that affect processing in each language may differ. More broadly, we find that bilingualism is an experience that opens the language system to perform fluidly under changing circumstances, such as increasing proficiency. In contrast, language processing in monolinguals was primarily related to relatively stable factors (working memory).

Accurate and sensitive quantification of protein-DNA binding affinity.

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

A systems approach to the exploration of research activity and relationships within a local authority.

BACKGROUND: Organizations with responsibilities for public health are increasingly required to use evidence-based practice to inform programme delivery, requiring research to generate relevant evidence, and dissemination and use of evidence to inform decisions and practices. Understanding how relationships between organizational structures, systems and processes influence evidence-based practices is critical to improving practice at both an institutional and system level, yet how these relationships should best operate is not well understood. Understanding how to better support research within local authorities, the elected administrative bodies responsible for services including public health at a regional level in the United Kingdom, is a priority for the National Institute for Health Research (NIHR) Public Health Research. This study is based on Norfolk County Council, a local authority in the east of England. We aimed to apply a systems perspective to develop a better understanding of the structures, systems and processes that support a local authority in becoming research-active, identifying gaps in understanding and recommendations for action to address them. METHODS: Taking a participatory action research approach, we applied qualitative methods to explore research activity and relationships in Norfolk County Council. We surveyed employees and used network analysis to map individuals, departments and external partners involved in research activities and the connections between them. We then applied participatory approaches to conduct a series of focus groups and semi-structured interviews to explore stakeholders' experiences and perceptions of being involved in research at, or with, the authority, and their ideas for recommendations for future actions. RESULTS: A range of research activity is undertaken at the local authority, with an emphasis on applied work to improve service delivery. We identified several examples of effective practice and models of research collaboration in some departments. Challenges such as limitations in resources, capacity and knowledge exchange were evident, yet there was a readiness amongst key stakeholders to develop and implement actions that may better support the authority in becoming more research-active. CONCLUSION: In large complex organizations, a key challenge is how to share learning across teams and implement good practice at an organizational and system level. Our findings highlight the potential for developing improved collaborative partnership models and systems to support sustainable processes and practices for research and knowledge exchange at an institutional and interorganizational level. The insights gained and shared will support other local authorities and similar large, multilevel organizations with responsibilities for evidence-based public health to explore their own setting and implement change where needed, and provide stimulus for further research into system-level change.