Estimation of the direct health and indirect societal costs of diabetes in the UK using a cost of illness model.

AIMS: The direct cost of diabetes to the UK health system was estimated at around £10 billion in 2012. This analysis updates that estimate using more recent and accurate data sources. METHODS: A pragmatic review of relevant data sources for UK nations was conducted, including population-level data sets and published literature, to generate estimates of costs separately for Type 1, Type 2 and gestational diabetes. A comprehensive cost framework, developed in collaboration with experts, was used to create a population-based cost of illness model. The key driver of the analysis was prevalence of diabetes and its complications. Estimates were made of the excess costs of diagnosis, treatment and diabetes-related complications compared with the general UK population. Estimates of the indirect costs of diabetes focused on productivity losses due to absenteeism and premature mortality. RESULTS: The direct costs of diabetes in 2021/22 for the UK were estimated at £10.7 billion, of which just over 40% related to diagnosis and treatment, with the rest relating to the excess costs of complications. Indirect costs were estimated at £3.3 billion. CONCLUSIONS: Diabetes remains a considerable cost burden in the UK, and the majority of those costs are still spent on potentially preventable complications. Although rates of some complications are reducing, prevalence continues to increase and effective approaches to primary and secondary prevention continue to be needed. Improvements in data capture, data quality and reporting, and further research on the human and financial implications of increasing incidence of Type 2 diabetes in younger people are recommended.

Community engagement strategies for population health research with culturally diverse adults.

The purpose of this report is to describe the community engagement research (CEnR) strategies used to implement the Florida Statewide Registry for Aging Studies (FSRAS), a tri-institutional research project conducted during the height of the COVID-19 pandemic. We describe the CEnR strategies used to enroll adults aged ≥ 25 years old self-identifying as African American (AA), Caribbean (CN), or Hispanic/Latinx (H/L) into FSRAS health research studies. The second goal is to report the number of AA, CN, and H/L adults involved in FSRAS and discuss the implications of CEnR strategies used throughout this research. More than 1600 adults aged 25 years or older participated in FSRAS health-related research activities or studies. Specifically, 25 community leaders from throughout Florida served on the FL-SAGE Council, 587 AA, CN, and H/L adults aged ≥ 25 years old participated in listening sessions and completed surveys exploring intergenerational influence, 292 AA, CN, and H/L adults participated in marketing research, and at least 702 adults have enrolled in AgeWell, FSRAS's health registry for persons interested in healthy aging research. Implications are researchers should continue using several CEnR strategies including technology and social media. Examining how the foundational principles of trust and authenticity are maintained when using CEnR strategies in virtual settings is warranted. Research implications are that simultaneously using CEnR strategies to recruit and enroll underrepresented populations into research is most effective although further research is needed to identify which CEnR strategy is most effective for enrolling AA, CN, and H/L older adults in aging research.

Using Community Engagement to Move Upstream to Address Social Determinants of Health.

ABSTRACT: Given its role as a safety net institution, the University of Florida Health (UF Health) Jacksonville has responded to the community's needs through partnerships with the community for decades. Such academic-community partnerships have a broad emphasis on population health and primary care that expands the model of care to include community engagement, which allows such partnerships to promote health and well-being and reduce health inequalities by addressing social determinants of health (SDOH).This report describes the UF Health Jacksonville and University of Florida College of Medicine - Jacksonville's creation of the Urban Health Alliance (UHA) in June 2019 due to continued poor health outcomes and inequities within the community. The mission of the UHA is to improve community health using community-focused, self-sustainable strategies and solutions to impact SDOH (i.e., more upstream interventions). Using the tenets of the collective impact model, the UHA acts as a backbone organization to achieve these objectives by empowering community partners to affect changes in policy, systems, and other structures necessary for the optimal health of the community. The UHA's work is divided across 4 pillars: services, research, education, and policy. These pillars reflect the traditional missions of academic medical centers-clinical care, research, and education-and the need to address structural changes to improve community health-namely, policy. By addressing the issues that most impact the patients and community of UF Health Jacksonville, the UHA can serve as an example of how an academic medical center can use the traditional missions to improve the community's health and move toward health equity.

Providing telemedicine services to persons living with HIV in an urban community: a demonstration project.

Although HIV incidence and mortality rates have declined over the past 20 years, HIV health disparities continue to persist among patients living in urban communities. Barriers to proficient health outcomes for persons with HIV (PWH) in urban communities include lack of access to care, resulting from limited transportation or clinic availability. While healthcare systems in rural communities provide telemedicine (TM) services to PWH to eliminate transportation and accessibility barriers, few examples exist regarding TM use for PWH in urban communities. This project's goal was to increase the provision of healthcare services in an urban setting to PWH, using TM. As guided by "Integration of Healthcare Delivery Service" theories and key principles, we created an integration framework comprised of several simultaneous, overlapping activities to include: (1) capacity building (2) clinical standardization (3) community and patient engagement and (4) evaluation performance and measurements. This paper describes major activities involved with developing, implementing and evaluating a TM program for PWH. We discuss results, challenges, and lessons learned from integrating this program into our existing healthcare system.

Smart Meds: Using Pharmacists to Address Health Literacy Disparities Among Medically- and Socially-Vulnerable Populations.

Patients in historically underserved communities are most vulnerable to uncontrolled chronic conditions and report a lack of health knowledge to manage them. This report aims to describe the development of SMART MEDS, a pharmacy-led program implemented to address health literacy disparities among medically and socially vulnerable patients.

Synthetic genetic circuits to uncover the OCT4 trajectories of successful reprogramming of human fibroblasts.

Reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs) is inefficient, with heterogeneity among transcription factor (TF) trajectories driving divergent cell states. Nevertheless, the impact of TF dynamics on reprogramming efficiency remains uncharted. We develop a system that accurately reports OCT4 protein levels in live cells and use it to reveal the trajectories of OCT4 in successful reprogramming. Our system comprises a synthetic genetic circuit that leverages noise to generate a wide range of OCT4 trajectories and a microRNA targeting endogenous OCT4 to set total cellular OCT4 protein levels. By fusing OCT4 to a fluorescent protein, we are able to track OCT4 trajectories with clonal resolution via live-cell imaging. We discover that a supraphysiological, stable OCT4 level is required, but not sufficient, for efficient iPSC colony formation. Our synthetic genetic circuit design and high-throughput live-imaging pipeline are generalizable for investigating TF dynamics for other cell fate programming applications.

Long-term muscle-specific overexpression of DOK7 in mice using AAV9-tMCK-DOK7.

Neuromuscular junction (NMJ) dysfunction underlies several diseases, including congenital myasthenic syndromes (CMSs) and motor neuron disease (MND). Molecular pathways governing NMJ stability are therefore of interest from both biological and therapeutic perspectives. Muscle-specific kinase (MuSK) is necessary for the formation and maintenance of post-synaptic elements of the NMJ, and downstream of tyrosine kinases 7 (DOK7) is crucial for activation of the MuSK pathway. Overexpression of DOK7 using AAV9 has been shown to ameliorate neuromuscular pathology in pre-clinical disease models of CMS and MND. However, long-term consequences of DOK7 expression have been sparsely investigated and targeted overexpression of DOK7 in skeletal muscle yet to be established. Here, we developed and characterized a novel AAV9-DOK7 facilitating forced expression of DOK7 under a skeletal muscle-specific promoter. AAV9-tMCK-DOK7 was systemically delivered to newborn mice that were monitored over 6 months. DOK7 overexpression was restricted to skeletal muscles. Body weight, blood biochemistry, and histopathological assessments were unaffected by AAV9-tMCK-DOK7 treatment. In contrast, forced expression of DOK7 resulted in enlargement of both the pre- and post-synaptic components of the NMJ, without causing denervation. We conclude that muscle-specific DOK7 overexpression can be achieved in a safe manner, with the capacity to target NMJs in vivo.

Neuromuscular junction denervation and terminal Schwann cell loss in the hTDP-43 overexpression mouse model of amyotrophic lateral sclerosis.

AIMS: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with complex aetiology. Despite evidence of neuromuscular junction (NMJ) denervation and 'dying-back' pathology in models of SOD1-dependent ALS, evidence in other genetic forms of ALS is limited by a lack of suitable animal models. TDP-43, a key mediator protein in ALS, is overexpressed in neurons in Thy1-hTDP-43WT mice. We therefore aimed to comprehensively analyse NMJ pathology in this model of ALS. METHODS: Expression of TDP-43 was assessed via western blotting. Immunohistochemistry techniques, alongside NMJ-morph quantification, were used to analyse motor neuron number, NMJ denervation status and terminal Schwann cell morphology. RESULTS: We present a time course of progressive, region-specific motor neuron pathology in Thy1-hTDP-43WT mice. Thy1-driven hTDP-43 expression increased steadily, correlating with developing hindlimb motor weakness and associated motor neuron loss in the spinal cord with a median survival of 21 days. Pronounced NMJ denervation was observed in hindlimb muscles, mild denervation in cranial muscles but no evidence of denervation in either forelimb or trunk muscles. NMJ pathology was restricted to motor nerve terminals, with denervation following the same time course as motor neuron loss. Terminal Schwann cells were lost from NMJs in hindlimb muscles, directly correlating with denervation status. CONCLUSIONS: Thy1-hTDP-43WT mice represent a severe model of ALS, with NMJ pathology/denervation of distal muscles and motor neuron loss, as observed in ALS patients. This model therefore provides an ideal platform to investigate mechanisms of dying-back pathology, as well as NMJ-targeting disease-modifying therapies in ALS.

Sex biased human thymic architecture guides T cell development through spatially defined niches.

Within the thymus, regulation of the cellular cross-talk directing T cell development is dependent on spatial interactions within specialized niches. To create a holistic, spatially defined map of tissue niches guiding postnatal T cell development we employed the multidimensional imaging platform CO-detection by indEXing (CODEX), as well as CITE-seq and ATAC-seq. We generated age-matched 4-5-month-old postnatal thymus datasets for male and female donors, and identify significant sex differences in both T cell and thymus biology. We demonstrate a crucial role for JAG ligands in directing thymic-like dendritic cell development, reveal important functions of a novel population of ECM- fibroblasts, and characterize the medullary niches surrounding Hassall's corpuscles. Together, these data represent a unique age-matched spatial multiomic resource to investigate how sex-based differences in thymus regulation and T cell development arise, and provide an essential resource to understand the mechanisms underlying immune function and dysfunction in males and females.

Rapid Development of Cell State Identification Circuits with Poly-Transfection.

Mammalian genetic circuits have demonstrated the potential to sense and treat a wide range of disease states, but optimization of the levels of circuit components remains challenging and labor-intensive. To accelerate this process, our lab developed poly-transfection, a high-throughput extension of traditional mammalian transfection. In poly-transfection, each cell in the transfected population essentially performs a different experiment, testing the behavior of the circuit at different DNA copy numbers and allowing users to analyze a large number of stoichiometries in a single-pot reaction. So far, poly-transfections that optimize ratios of three-component circuits in a single well of cells have been demonstrated; in principle, the same method can be used for the development of even larger circuits. Poly-transfection results can be easily applied to find optimal ratios of DNA to co-transfect for transient circuits or to choose expression levels for circuit components for the generation of stable cell lines. Here, we demonstrate the use of poly-transfection to optimize a three-component circuit. The protocol begins with experimental design principles and explains how poly-transfection builds upon traditional co-transfection methods. Next, poly-transfection of cells is carried out and followed by flow cytometry a few days later. Finally, the data is analyzed by examining slices of the single-cell flow cytometry data that correspond to subsets of cells with certain component ratios. In the lab, poly-transfection has been used to optimize cell classifiers, feedback and feedforward controllers, bistable motifs, and many more. This simple but powerful method speeds up design cycles for complex genetic circuits in mammalian cells.

Synthetic genetic circuits to uncover and enforce the OCT4 trajectories of successful reprogramming of human fibroblasts.

Reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs) is inefficient, with heterogeneity among transcription factor (TF) trajectories driving divergent cell states. Nevertheless, the impact of TF dynamics on reprogramming efficiency remains uncharted. Here, we identify the successful reprogramming trajectories of the core pluripotency TF, OCT4, and design a genetic controller that enforces such trajectories with high precision. By combining a genetic circuit that generates a wide range of OCT4 trajectories with live-cell imaging, we track OCT4 trajectories with clonal resolution and find that a distinct constant OCT4 trajectory is required for colony formation. We then develop a synthetic genetic circuit that yields a tight OCT4 distribution around the identified trajectory and outperforms in terms of reprogramming efficiency other circuits that less accurately regulate OCT4. Our synthetic biology approach is generalizable for identifying and enforcing TF dynamics for cell fate programming applications.

Virtual cells in a virtual microenvironment recapitulate early development-like patterns in human pluripotent stem cell colonies.

The mechanism by which morphogenetic signals engage the regulatory networks responsible for early embryonic tissue patterning is incompletely understood. Here, we developed a minimal gene regulatory network (GRN) model of human pluripotent stem cell (hPSC) lineage commitment and embedded it into "cellular" agents that respond to a dynamic morphogenetic signaling microenvironment. Simulations demonstrated that GRN wiring had significant non-intuitive effects on tissue pattern order, composition, and dynamics. Experimental perturbation of GRN connectivities supported model predictions and demonstrated the role of OCT4 as a master regulator of peri-gastrulation fates. Our so-called GARMEN strategy provides a multiscale computational platform to understand how single-cell-based regulatory interactions scale to tissue domains. This foundation provides new opportunities to simulate the impact of network motifs on normal and aberrant tissue development.

Synthetic gene circuits for cell state detection and protein tuning in human pluripotent stem cells.

During development, cell state transitions are coordinated through changes in the identity of molecular regulators in a cell type- and dose-specific manner. The ability to rationally engineer such transitions in human pluripotent stem cells (hPSC) will enable numerous applications in regenerative medicine. Herein, we report the generation of synthetic gene circuits that can detect a desired cell state using AND-like logic integration of endogenous miRNAs (classifiers) and, upon detection, produce fine-tuned levels of output proteins using an miRNA-mediated output fine-tuning technology (miSFITs). Specifically, we created an "hPSC ON" circuit using a model-guided miRNA selection and circuit optimization approach. The circuit demonstrates robust PSC-specific detection and graded output protein production. Next, we used an empirical approach to create an "hPSC-Off" circuit. This circuit was applied to regulate the secretion of endogenous BMP4 in a state-specific and fine-tuned manner to control the composition of differentiating hPSCs. Our work provides a platform for customized cell state-specific control of desired physiological factors in hPSC, laying the foundation for programming cell compositions in hPSC-derived tissues and beyond.

Metformin protects against vascular calcification through the selective degradation of Runx2 by the p62 autophagy receptor.

Vascular calcification is associated with aging, type 2 diabetes, and atherosclerosis, and increases the risk of cardiovascular morbidity and mortality. It is an active, highly regulated process that resembles physiological bone formation. It has previously been established that pharmacological doses of metformin alleviate arterial calcification through adenosine monophosphate-activated protein kinase (AMPK)-activated autophagy, however the specific pathway remains elusive. In the present study we hypothesized that metformin protects against arterial calcification through the direct autophagic degradation of runt-related transcription factor 2 (Runx2). Calcification was blunted in vascular smooth muscle cells (VSMCs) by metformin in a dose-dependent manner (0.5-1.5 mM) compared to control cells (p < 0.01). VSMCs cultured under high-phosphate (Pi) conditions in the presence of metformin (1 mM) showed a significant increase in LC3 puncta following bafilomycin-A1 (Baf-A; 5 nM) treatment compared to control cells (p < 0.001). Furthermore, reduced expression of Runx2 was observed in the nuclei of metformin-treated calcifying VSMCs (p < 0.0001). Evaluation of the functional role of autophagy through Atg3 knockdown in VSMCs showed aggravated Pi-induced calcification (p < 0.0001), failure to induce autophagy (punctate LC3) (p < 0.001) and increased nuclear Runx2 expression (p < 0.0001) in VSMCs cultured under high Pi conditions in the presence of metformin (1 mM). Mechanistic studies employing three-way coimmunoprecipitation with Runx2, p62, and LC3 revealed that p62 binds to both LC3 and Runx2 upon metformin treatment in VSMCs. Furthermore, immunoblotting with LC3 revealed that Runx2 specifically binds with p62 and LC3-II in metformin-treated calcified VSMCs. Lastly, we investigated the importance of the autophagy pathway in vascular calcification in a clinical setting. Ex vivo clinical analyses of calcified diabetic lower limb artery tissues highlighted a negative association between Runx2 and LC3 in the vascular calcification process. These studies suggest that exploitation of metformin and its analogues may represent a novel therapeutic strategy for clinical intervention through the induction of AMPK/Autophagy Related 3 (Atg3)-dependent autophagy and the subsequent p62-mediated autophagic degradation of Runx2.

Recognising the potential of large animals for modelling neuromuscular junction physiology and disease.

The aetiology and pathophysiology of many diseases of the motor unit remain poorly understood and the role of the neuromuscular junction (NMJ) in this group of disorders is particularly overlooked, especially in humans, when these diseases are comparatively rare. However, elucidating the development, function and degeneration of the NMJ is essential to uncover its contribution to neuromuscular disorders, and to explore potential therapeutic avenues to treat these devastating diseases. Until now, an understanding of the role of the NMJ in disease pathogenesis has been hindered by inherent differences between rodent and human NMJs: stark contrasts in body size and corresponding differences in associated axon length underpin some of the translational issues in animal models of neuromuscular disease. Comparative studies in large mammalian models, including examination of naturally occurring, highly prevalent animal diseases and evaluation of their treatment, might provide more relevant insights into the pathogenesis and therapy of equivalent human diseases. This review argues that large animal models offer great potential to enhance our understanding of the neuromuscular system in health and disease, and in particular, when dealing with diseases for which nerve length dependency might underly the pathogenesis.

A method to identify, dissect and stain equine neuromuscular junctions for morphological analysis.

Morphological study of the neuromuscular junction (NMJ), a specialised peripheral synapse formed between a lower motor neuron and skeletal muscle fibre, has significantly contributed to the understanding of synaptic biology and neuromuscular disease pathogenesis. Rodent NMJs are readily accessible, and research into conditions such as amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease (CMT), and spinal muscular atrophy (SMA) has relied heavily on experimental work in these small mammals. However, given that nerve length dependency is an important feature of many peripheral neuropathies, these rodent models have clear shortcomings; large animal models might be preferable, but their size presents novel anatomical challenges. Overcoming these constraints to study the NMJ morphology of large mammalian distal limb muscles is of prime importance to increase cross-species translational neuromuscular research potential, particularly in the study of long motor units. In the past, NMJ phenotype analysis of large muscle bodies within the equine distal pelvic limb, such as the tibialis cranialis, or within muscles of high fibrous content, such as the soleus, has posed a distinct experimental hurdle. We optimised a technique for NMJ location and dissection from equine pelvic limb muscles. Using a quantification method validated in smaller species, we demonstrate their morphology and show that equine NMJs can be reliably dissected, stained and analysed. We reveal that the NMJs within the equine soleus have distinctly different morphologies when compared to the extensor digitorum longus and tibialis cranialis muscles. Overall, we demonstrate that equine distal pelvic limb muscles can be regionally dissected, with samples whole-mounted and their innervation patterns visualised. These methods will allow the localisation and analysis of neuromuscular junctions within the muscle bodies of large mammals to identify neuroanatomical and neuropathological features.

Study protocol for a type III hybrid effectiveness-implementation trial to evaluate scaling interoperable clinical decision support for patient-centered chronic pain management in primary care.

BACKGROUND: The US continues to face public health crises related to both chronic pain and opioid overdoses. Thirty percent of Americans suffer from chronic noncancer pain at an estimated yearly cost of over $600 billion. Most patients with chronic pain turn to primary care clinicians who must choose from myriad treatment options based on relative risks and benefits, patient history, available resources, symptoms, and goals. Recently, with attention to opioid-related risks, prescribing has declined. However, clinical experts have countered with concerns that some patients for whom opioid-related benefits outweigh risks may be inappropriately discontinued from opioids. Unfortunately, primary care clinicians lack usable tools to help them partner with their patients in choosing pain treatment options that best balance risks and benefits in the context of patient history, resources, symptoms, and goals. Thus, primary care clinicians and patients would benefit from patient-centered clinical decision support (CDS) for this shared decision-making process. METHODS: The objective of this 3-year project is to study the adaptation and implementation of an existing interoperable CDS tool for pain treatment shared decision making, with tailored implementation support, in new clinical settings in the OneFlorida Clinical Research Consortium. Our central hypothesis is that tailored implementation support will increase CDS adoption and shared decision making. We further hypothesize that increases in shared decision making will lead to improved patient outcomes, specifically pain and physical function. The CDS implementation will be guided by the Exploration, Preparation, Implementation, Sustainment (EPIS) framework. The evaluation will be organized by the Reach, Effectiveness, Adoption, Implementation, and Maintenance (RE-AIM) framework. We will adapt and tailor PainManager, an open source interoperable CDS tool, for implementation in primary care clinics affiliated with the OneFlorida Clinical Research Consortium. We will evaluate the effect of tailored implementation support on PainManager's adoption for pain treatment shared decision making. This evaluation will establish the feasibility and obtain preliminary data in preparation for a multi-site pragmatic trial targeting the effectiveness of PainManager and tailored implementation support on shared decision making and patient-reported pain and physical function. DISCUSSION: This research will generate evidence on strategies for implementing interoperable CDS in new clinical settings across different types of electronic health records (EHRs). The study will also inform tailored implementation strategies to be further tested in a subsequent hybrid effectiveness-implementation trial. Together, these efforts will lead to important new technology and evidence that patients, clinicians, and health systems can use to improve care for millions of Americans who suffer from pain and other chronic conditions. TRIAL REGISTRATION: ClinicalTrials.gov, NCT05256394 , Registered 25 February 2022.

Robust and tunable signal processing in mammalian cells via engineered covalent modification cycles.

Engineered signaling networks can impart cells with new functionalities useful for directing differentiation and actuating cellular therapies. For such applications, the engineered networks must be tunable, precisely regulate target gene expression, and be robust to perturbations within the complex context of mammalian cells. Here, we use bacterial two-component signaling proteins to develop synthetic phosphoregulation devices that exhibit these properties in mammalian cells. First, we engineer a synthetic covalent modification cycle based on kinase and phosphatase proteins derived from the bifunctional histidine kinase EnvZ, enabling analog tuning of gene expression via its response regulator OmpR. By regulating phosphatase expression with endogenous miRNAs, we demonstrate cell-type specific signaling responses and a new strategy for accurate cell type classification. Finally, we implement a tunable negative feedback controller via a small molecule-stabilized phosphatase, reducing output expression variance and mitigating the context-dependent effects of off-target regulation and resource competition. Our work lays the foundation for establishing tunable, precise, and robust control over cell behavior with synthetic signaling networks.

Anatomical approaches in locally advanced and recurrent pelvic malignancy.

Pelvic exenteration is a complex, technically challenging procedure requiring detailed anatomical knowledge. Understanding the complexity of the pelvis beyond TME is an essential requirement for both operative planning and execution. This paper highlights the key anatomical approaches to extended pelvic resection as relevant to the Colorectal Surgeon.