Biology subjects

Walker, A. J.

Publications and source records attributed to Walker, A. J..

11 recordsLinked to original sources

bioRxiv2025

The circadian clock regulates scavenging of fluid-borne substrates by brain border-associated macrophages

Circadian disruptions perturb the brain and immune system and increase the risk of developing Alzheimers Disease (AD), yet whether this involves dysregulation of brain immunity remains less clear. Here, we perform single-cell RNA sequencing of the brain immune compartment around the day-night cycle and identify brain border-associated macrophages (BAMs) as highly rhythmic cells. During the rest phase, we find that BAMs exhibit coordinated upregulation of endocytic genes and enhanced uptake of extracellular fluid-borne material including amyloid-beta (A{beta}). Rhythmicity in BAM scavenging is regulated by the clock gene Bmal1, mediated by the endocytic receptor CD206, and perturbed with age. In a mouse model of AD, we show that deletion of Bmal1 in BAMs worsens perivascular and leptomeningeal A{beta} plaque burden. Our results identify endocytosis as a specialized and rhythmic BAM function and identify perturbed timing of brain border immune functions as a potential mechanism by which circadian disruptions precipitate amyloidosis.

neuroscience↗

bioRxiv2025

Early-life stromal niches orchestrate B lymphopoiesis at the brain's borders

The dura mater serves as a critical immunological niche for the central nervous system, yet the mechanisms governing the emergence of this niche in early life remain understudied. Here, we chart the trajectory of dural immune development, uncovering a distinctive function for the murine dura as a transient niche for B lymphopoiesis in the early-postnatal window. Shared embryonic progenitors initiate dural B cell development in concert with a multi-organ wave of extramedullary lymphopoiesis that contributes distinctively to the peripheral B cell pool. In the dura, B cells develop locally in discrete sinus-proximal foci, occupying an anatomically defined and developmentally restricted fibroblast niche. Sinus-proximal fibroblasts express the pro-hematopoietic chemokine CXCL12, and local deletion of this crucial factor severely impairs dural B lymphopoiesis. These data reveal a critical function for dural fibroblasts in shaping the early-life B cell compartment and provide a model for how extramedullary niches may support early-life leukocyte production.

immunology↗

bioRxiv2025

A Proteolytic Switch: USP5 controls SDE2 function via UBL-directed cleavage

Proteolytic processing is a critical regulatory mechanism in eukaryotic cells, yet the molecular identities and mechanisms underlying these events often remain elusive. Silencing Defective 2 (SDE2) is an essential human protein involved in multiple aspects of genome regulation, including DNA repair, ribosome biogenesis, and mRNA splicing. SDE2 is expressed with an N-terminal ubiquitin-like domain (SDE2UBL) which must be proteolytically cleaved to release the functional C-terminal domain (SDE2CT). This cleavage not only activates SDE2CT but also marks it for subsequent degradation, highlighting the importance of this tightly regulated processing. Despite the crucial role of this cleavage event, the human protease responsible has remained unknown. Here, we identify that the deubiquitinating enzyme, ubiquitin-specific protease 5 (USP5), catalyses the cleavage of SDE2. Using an integrated workflow combining biochemical assays, proteomic profiling, and mass spectrometry, we demonstrate that USP5 selectively processes SDE2 in vitro and in cell. To validate the specificity of this interaction, we engineered SDE2UBL into an activity-based probe, and developed a cellular reporter assay, both of which confirmed USP5 as the primary effector. Biophysical analysis further revealed that SDE2UBL binds to USP5 with similar characteristics to ubiquitin, albeit with reduced affinity, supporting a mechanism of substrate mimicry. Together, these findings uncover a novel regulatory axis for SDE2 function, highlighting an underappreciated role for DUBs in regulating protein maturation events. They also establish a versatile approach for identifying and validating substrate-protease interactions with broader implications for the study of post-translational regulation.

biochemistry↗

bioRxiv2024

The meninges host a unique compartment of regulatory T cells that bulwarks adult hippocampal neurogenesis

Our knowledge about the meningeal immune system has recently burgeoned, particularly our understanding of how innate and adaptive effector cells are mobilized to meet brain challenges. However, information on how meningeal immunocytes guard brain homeostasis in healthy individuals remains sparse. This study highlights the heterogeneous and polyfunctional regulatory-T (Treg) cell compartment in the meninges. A Treg subtype specialized in controlling Th1-cell responses and another known to control responses in B-cell follicles were substantial components of this compartment, foretelling that punctual Treg-cell ablation rapidly unleashed interferon-gamma production by meningeal lymphocytes, unlocked their access to the brain parenchyma, and altered meningeal B-cell profiles. Distally, the hippocampus assumed a reactive state, with morphological and transcriptional changes in multiple glial-cell types; within the dentate gyrus, neural stem cells showed exacerbated death and desisted from further differentiation, associated with inhibition of spatial-reference memory. Thus, meningeal Treg cells are a multifaceted bulwark to brain homeostasis at steady-state. One sentence summaryA distinct population of regulatory T cells in the murine meninges safeguards homeostasis by keeping local interferon-{gamma}-producing lymphocytes in check, thereby preventing their invasion of the parenchyma, activation of hippocampal glial cells, death of neural stem cells, and memory decay.

immunology↗

bioRxiv2020

Single Cell Sequencing Reveals Glial Specific Responses to Tissue Processing & Enzymatic Dissociation in Mice and Humans

A key aspect of nearly all single cell experiments is the necessity to dissociate intact tissues into single cell suspensions for processing. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression changes during this process. Microglia, the brains resident macrophages, are a highly dynamic population that are extremely sensitive to their microenvironment and have been shown to dramatically alter their transcriptome upon stimulation. We demonstrate that use of enzymatic dissociation methods on mouse central nervous system (CNS) tissue induces an aberrant gene expression signature in microglia that can significantly confound downstream analysis. To minimize this issue, we developed a flexible protocol, that can be used with existing enzymatic protocols for fresh tissue, to eliminate artifactual gene expression while allowing for increased cell type diversity and yield. We demonstrate efficacy of this protocol in analysis of diverse CNS cell types and sorted myeloid populations while using enzymatic dissociation. Generation of new and reanalysis of previously published human brain single nucleus RNAseq (snRNA-seq) datasets reveal that a similar signature is also present in post-mortem tissue. Through novel snRNA-seq analysis of acutely-resected neurosurgical tissue we demonstrate that this signature can be induced in human tissue due to technical differences in sample processing. These results provide key insight into the potential confounds of enzymatic digestion and provide a solution to allow for enzymatic digestion for scRNA-seq while avoiding ex vivo transcriptional artifacts. Analysis of human tissue reveals potential for artifacts in current and future snRNA-seq datasets that will require deeper analysis and careful consideration to separate true biology from artifacts related to post-mortem processes.

neuroscience↗

bioRxiv2019

An 11-bit High Dynamic Range (HDR) Luminance Display and Its Use to Discover Contextual Mechanisms of Real-World Luminance Normalization for Visual Acuity and Target Discrimination

Luminance can vary widely when scanning across a scene, by up to 10^9 to 1, requiring multiple normalizing mechanisms spanning from the retina to cortex to support visual acuity and recognition. Vision models based on standard dynamic range luminance contrast ratios below 100 to 1 have limited ability to generalize to real-world scenes with contrast ratios over 10,000 to 1 (high dynamic range [HDR]). Understanding and modeling brain mechanisms of HDR luminance normalization is thus important for military applications, including automatic target recognition, display tone mapping, and camouflage. Yet, computer display of HDR stimuli was until recently unavailable or impractical for research. Here we describe procedures for setup, calibration, and precision check of an HDR display system with over 100,000 to 1 luminance dynamic range (650-0.0065 cd/m^2), pseudo 11-bit grayscale precision, and 3-ms temporal precision in the MATLAB/Psychtoolbox software environment. The setup is synchronized with electroencephalography and IR eye-tracking measurements. We report measures of HDR visual acuity and the discovery of a novel phenomenon--that abrupt darkening (from 400 to 4 cd/m^2) engages contextual facilitation, distorting the perceived orientation of a high-contrast central target. Surprisingly, the facilitation effect depended on luminance similarity, contradicting both classic divisive and subtractive models of contextual normalization.

neuroscience↗

bioRxiv2018

Complex cell-state changes revealed by single cell RNA sequencing of 76,149 microglia throughout the mouse lifespan and in the injured brain

Microglia, the resident immune cells of the brain, rapidly change states in response to their environment, but we lack molecular and functional signatures of different microglial populations. In this study, we analyzed the RNA expression patterns of more than 76,000 individual microglia during development, old age and after brain injury. Analysis uncovered at least nine transcriptionally distinct microglial states, which expressed unique sets of genes and were localized in the brain using specific markers. The greatest microglial heterogeneity was found at young ages; however, several states - including chemokine-enriched inflammatory microglia - persisted throughout the lifespan or increased in the aged brain. Multiple reactive microglial subtypes were also found following demyelinating injury in mice, at least one of which was also found in human MS lesions. These unique microglia signatures can be used to better understand microglia function and to identify and manipulate specific subpopulations in health and disease.

neuroscience↗

bioRxiv2018

Why did some practices not implement new antibiotic prescribing guidelines on urinary tract infection? A cohort study and survey in NHS England primary care

ObjectivesTo describe prescribing trends and geographic variation for trimethoprim and nitrofurantoin; to describe variation in implementing guideline change; and to compare actions taken to reduce trimethoprim use in high- and low-using Clinical Commissioning Groups (CCGs).\n\nDesignA retrospective cohort study and interrupted time series analysis in English NHS primary care prescribing data; complemented by information obtained through Freedom of Information Act requests to CCGs. The main outcome measures were: variation in practice and CCG prescribing ratios geographically and over time, including an interrupted time-series; and responses to Freedom of Information requests.\n\nResultsThe amount of trimethoprim prescribed, as a proportion of nitrofurantoin and trimethoprim combined, remained stable and high until 2014, then fell gradually to below 50% in 2017; this reduction was more rapid following the introduction of the Quality Premium. There was substantial variation in the speed of change between CCGs. As of April 2017, for the 10 worst CCGs (with the highest trimethoprim ratios): 9 still had trimethoprim as first line treatment for uncomplicated UTI (one CCG had no formulary); none had active work plans to facilitate change in prescribing behaviour away from trimethoprim; and none had implemented an incentive scheme for change in prescribing behaviour. For the 10 best CCGs: 2 still had trimethoprim as first line treatment (all CCGs had a formulary); 5 (out of 7 who answered this question) had active work plans to facilitate change in prescribing behaviour away from trimethoprim; and 5 (out of 10 responding) had implemented an incentive scheme for change in prescribing behaviour. 9 of the best 10 CCGs reported at least one of: formulary change, work plan, or incentive scheme. None of the worst 10 CCGs did so.\n\nConclusionsMany CCGs failed to implement an important change in antibiotic prescribing guidance; and report strong evidence suggesting that CCGs with minimal prescribing change did little to implement the new guidance. We strongly recommend a national programme of training and accreditation for medicines optimisation pharmacists; and remedial action for CCGs that fail to implement guidance; with all materials and data shared publicly for both such activities.

microbiology↗

bioRxiv2018

Measuring the Impact of an Open Online Prescribing Data Analysis Service on Clinical Practice: a Cohort Study in NHS England Data

BackgroundOpenPrescribing is a freely accessible service that enables any user to view and analyse NHS primary care prescribing data at the level of individual practices. This tool is intended to improve the quality, safety, and cost-effectiveness of prescribing.\n\nObjectivesWe set out to measure the impact of OpenPrescribing being viewed on subsequent prescribing.\n\nMethodsHaving pre-registered our protocol and code, we measured three different metrics of prescribing quality (mean percentile across 34 existing OpenPrescribing quality measures, available \"price-per-unit\" savings, and total \"low-priority prescribing\" spend) to see if they changed after CCG and practice pages were viewed. We also measured whether practices whose data were viewed on OpenPrescribing differed in prescribing, prior to viewing, to those who were not. We used fixed effects and between effects linear panel regression, to isolate change over time and differences between practices respectively. We adjusted for month of prescribing in the fixed effects model, to remove underlying trends in outcome measures.\n\nResultsWe found a reduction in available price-per-unit savings for both practices and CCGs after their pages were viewed. The saving was greater at the practice level (-{pound}40.42 per thousand patients per month, 95% confidence interval -54.04 to -26.01) than at CCG level (-{pound}14.70 per thousand patients per month, 95% confidence interval -25.56 to -3.84). We estimate a total saving since launch of {pound}243k at practice level and {pound}1.47m at CCG level between the feature launch and end of follow-up (August to November 2017) among practices viewed. If the observed savings from practices viewed were extrapolated to all practices, this would generate {pound}26.8m in annual savings for the NHS, approximately 20% of the total possible savings from this method. The other two measures were not different after CCGs/practices were viewed. Practices which were viewed had worse prescribing quality scores overall, prior to viewing.\n\nConclusionsWe found a clinically significant positive impact from use of OpenPrescribing, specifically for the class of savings opportunities that can only be identified by using this tool. We also show that it is possible to conduct a robust analysis of the impact of such an online service on clinical practice.

epidemiology↗

bioRxiv2017

Trends and variation in Prescribing of Low-Priority Medicines Identified by NHS England: A Cross-Sectional Study and Interactive Data Tool in English Primary Care

BackgroundRoutine accessible audit of prescribing data presents significant opportunities to identify cost-saving opportunities. NHS England recently announced a consultation seeking to discourage use of medicines it considers to be low-value. We set out to produce an interactive data resource to show savings in each NHS general practice, and to assess the current use of these medicines, their change in use over time, and the extent and reasons for variation in such prescribing.\n\nResultsThe total NHS spend on all low-value medicines identified by NHS England was {pound}153.5m in the last year, across 5.8m prescriptions (mean {pound}26 per prescription). Among individual medications, liothyronine had the highest prescribing cost at {pound}29.6m, followed by trimipramine ({pound}20.2m) and gluten-free foods ({pound}18.7m). Over time, the overall total number of low-value prescriptions decreased, but the cost increased, although this varied greatly between medications. Annual practice level spending varied widely (median, {pound}2,262 per thousand patients, IQR {pound}1,439 to {pound}3,298). The proportion of patients over 65 showed the strongest association with low-value prescribing; CCG was also strongly associated. Our interactive data tool was deployed to OpenPrescribing.net where monthly updated figures and graphs can be viewed.\n\nConclusionsPrescribing of low-value medications is extensive but varies widely by medication, geographic area and individual practice. Despite a fall in prescription numbers, the overall cost of prescribing for low-value items has risen. Prescribing behaviour is clustered by CCG, which may represent variation in medicines optimisation efficiency, or in some cases access inequality.\n\nAbbreviations

epidemiology↗

bioRxiv2017

A New Mechanism for Cost Savings in NHS Prescribing: Minimising "Price-Per-Unit"

BackgroundMinimising prescription costs while maintaining quality is a core element of delivering high value healthcare. There are various strategies to achieve savings, but almost no research to date on determining the most effective approach. We describe a new method of identifying potential savings due to large national variations in drug cost, including variation in generic drug cost; and compare these with potential savings from an established method (generic prescribing).\n\nMethodsWe used English NHS Digital prescribing data, from October 2015 to September 2016. Potential cost savings were calculated by determining the price-per-unit (e.g. pill, ml) for each drug and dose within each general practice. This was compared against the same cost for the practice at the lowest cost decile, to determine achievable savings. We compared these price-per-unit savings to the savings possible from generic switching; and determined the chemicals with the highest savings nationally. A senior pharmacist manually assessed whether a random sample of savings were practically achievable.\n\nResultsWe identified a theoretical maximum of {pound}410M of savings over 12 months. {pound}273M of these savings were for individual prescribing changes worth over {pound}50 per practice per month; this compares favorably with generic switching, where only {pound}35M of achievable savings were identified. The biggest savings nationally were on glucose blood testing reagents ({pound}12M), fluticasone propionate ({pound}9M) and venlafaxine ({pound}8M). Approximately half of all savings were deemed practically achievable.\n\nDiscussionWe have developed a new method to identify and enable large potential cost savings within NHS community prescribing. Given the current pressures on the NHS, it is vital that these potential savings are realised. Our tool enabling doctors to achieve these savings is now launched in pilot form. However savings could potentially be achieved more simply through national policy change.\n\nAbbreviations

epidemiology↗