Biology subjects

Tomlinson, S.

Publications and source records attributed to Tomlinson, S..

13 recordsLinked to original sources

bioRxiv2025

Direct recruitment of RNA Polymerase II by NANOG to activated target genes

Cell identity relies upon transcription factors (TFs). The concentration of the TF NANOG determines the efficiency of maintenance of mouse embryonic stem cell (ESC) identity. However, the mechanisms by which NANOG acts are not fully understood. Models of mammalian transcription generally propose that TFs bind DNA and connect to the transcriptional machinery indirectly via intermediary proteins. Accordingly, examples of direct contact between cell-type specific TFs and RNA synthesis enzymes in mammalian cells remain elusive. Here we show that NANOG directly contacts RNA Polymerase II (RNAPII) via aromatic residues within the low complexity domains of both proteins. NANOG can localize RNAPII to a specific DNA site, with RNAPII enhancing NANOG DNA affinity. The NANOG-RNAPII complex is dissociated by the transcriptional pause-release enzyme, CDK9. Inhibition of CDK9 enhances NANOG chromatin binding, while induction of NANOG localizes RNAPII to specific NANOG chromatin sites. Induced RNAPII localization is selective for targets activated by NANOG. A NANOG mutant that retains DNA, but not RNAPII binding, cannot stimulate RNAPII localization to chromatin, shows an impaired transcriptional response and does not drive LIF-independent ESC self-renewal. These results identify a novel, regulatable interaction between a cell-type specific TF and RNAPII that can change transcription and alter cell fate.

cell biology↗

bioRxiv2025

Age Associated Increase in Microglia Inflammation and Phagocytosis in the Adult Neural Stem Cell Niche.

Adult SVZ neurogenesis declines with age, but the niche mechanisms remain incompletely defined. Here, we show that SVZ microglia acquire activation-associated morphology with aging in male and female C57BL/6 mice. Reanalysis of published SVZ single-cell RNA-Seq data revealed that aged microglia upregulate inflammatory and lysosome/phagosome-related gene programs, including multiple phagocytosis-associated receptors and effectors. Functionally, young SVZ-derived microglia exhibit reduced basal phagocytic activity compared with non-SVZ microglia in vitro, whereas aged SVZ microglia show increased phagocytosis. In vivo SVZ wholemounts reveal increased progenitor-derived material within lysosomal compartments of microglia with age, without an accompanying increase in apoptosis. Inhibiting complement C3 activation partially restores SVZ proliferation in aged mice and coincides with a reduction of progenitor-derived material within lysosomal compartments of aged microglia. These data support a model in which microglial inflammatory signaling and engagement with neural progenitors contributes to age-related suppression of SVZ proliferation. Significance StatementAdult neural stem cells support plasticity and may aid repair after brain injury, but this capacity declines with age. We show that microglia in the aging subventricular zone acquire inflammatory and phagocytic programs, increase their lysosomal association with progenitor-derived material, and are linked to reduced progenitor proliferation. Complement inhibition partially restores proliferative output in aged mice, connecting inflammatory signaling to this niche defect. These findings identify age-dependent microglia-progenitor interactions as a mechanism that may limit neural stem cell activity in the adult brain and suggest immune pathways as possible targets for preserving or restoring neurogenic potential during aging.

cell biology↗

bioRxiv2025

Luminal Vascular Dysfunction Drives Rapid Blood Brain Barrier Injury in Hyperglycemic Stroke: Key Roles for Luminal Glycocalyx and Complement

BackgroundAcute hyperglycemia affects approximately 40% of stroke patients and is associated with worse outcomes. The underlying mechanisms linking this metabolic stress to stroke-induced brain injury remains unclear, and effective therapies are lacking. MethodsIn a mouse model of acute hyperglycemic stroke, luminal disruption, blood-brain barrier (BBB) leakage, neurological deficit, motor function, and mortality were evaluated. Vascular luminal glycocalyx and complement activation were assessed by immunostaining, with glycocalyx loss confirmed by electron microscopy. Complement C3s causal role was tested using C3 knockout mice and site-targeted inhibition with CR2-Crry. To enhance translational relevance, post-mortem human stroke and control brains were immunostained to assess the association between endothelial glycocalyx loss and vascular complement activation. In a separate stroke patient cohort, soluble complement activation products were measured in pre-thrombectomy plasma, and their predictive value for modified Rankin Scale (mRS) outcomes evaluated using elastic net regression. ResultsHyperglycemic stroke mice exhibited accelerated and more severe BBB breakdown, greater functional deficits, and higher mortality than normoglycemic controls, mirroring clinical observations. Acute hyperglycemia triggered rapid vascular luminal injury characterized by loss of endothelial luminal glycocalyx, luminal IgM/IgG deposition, and vascular complement C3 activation, leading to BBB disruption. This vascular luminal injury was corroborated in human stroke brain tissue. These luminal changes persisted despite glucose normalization and were exacerbated by reperfusion, driving injury into the brain parenchyma. Genetic and pharmacological approaches confirmed vascular complement activation as a causal driver of severe BBB disruption and poor outcomes. Importantly, site-targeted pharmacological inhibition of complement after reperfusion preserved BBB integrity and improved outcomes, defining a time-specific, luminal-directed strategy as a promising adjunct to thrombectomy. Notably, soluble complement activation markers in pre-thrombectomy stroke plasma predicted clinical outcomes, highlighting their potential as pre-intervention markers for patient stratification and tailored therapy. ConclusionThis study reframes acute hyperglycemic stroke as a vascular luminal disorder, establishing a novel Metabolic-Complement-Vascular (MCV) axis linking metabolic stress to endothelial luminal glycocalyx loss, vascular complement activation, and BBB breakdown in both mice and humans. This new mechanistic understanding transforms the therapeutic landscape of hyperglycemic stroke, offering a potential time-defined, luminal-focused adjunct therapy alongside thrombectomy. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABS- This study reframes hyperglycemic stroke as an acute vascular luminal problem, marked by rapid loss of endothelial luminal glycocalyx and complement C3 activation at the vascular luminal surface. - The rapid luminal changes, identified in both rodent and human stroke brain tissues, establish a novel Metabolic-Complement-Vascular (MCV) axis linking metabolic stress to luminal damage, blood-brain barrier (BBB) breakdown, and injury progression into the brain parenchyma. - The first clinical evidence that pre-thrombectomy plasma complement activation markers independently predict stroke outcomes -- laying the foundation for risk stratification before reperfusion and precision adjunct therapies. What Are the Clinical Implications?- The rapidity and persistence of the MCV axis activation--even after glucose normalization--help explain the limited efficacy of insulin therapy, shifting the therapeutic focus from glycemic control to luminal-targeted interventions. - Identification of a narrow but actionable window following reperfusion where complement C3 inhibition preserves the BBB and limits injury progression to the parenchyma, offers a promising adjunct to thrombectomy in metabolically vulnerable stroke patients. - Targeting the vascular luminal surface reshapes the therapeutic landscape for hyperglycemic stroke by enabling systemic interventions--bypassing the challenge of BBB penetration, supporting rapid clinical translation using existing FDA-approved C3 inhibitors, and framing the endothelial glycocalyx as a promising area for therapeutic and diagnostic exploration in hyperglycemic stroke and broader cerebrovascular disease.

neuroscience↗

bioRxiv2024

Return of intracranial beta oscillations and traveling waves with recovery from traumatic brain injury

Traumatic brain injury (TBI) remains a pervasive clinical problem associated with significant morbidity and mortality. However, TBI remains clinically and biophysically ill-defined, and prognosis remains difficult even with the standardization of clinical guidelines and advent of multimodality monitoring. Here we leverage a unique data set from TBI patients implanted with either intracranial strip electrodes during craniotomy or quad-lumen intracranial bolts with depth electrodes as part of routine clinical practice. By extracting spectral profiles of this data, we found that the presence of narrow-band oscillatory activity in the beta band (12-30 Hz) closely corresponds with the neurological exam as quantified with the standard Glasgow Coma Scale (GCS). Further, beta oscillations were distributed over the cortical surface as traveling waves, and the evolution of these waves corresponded to recovery from coma, consistent with the putative role of waves in perception and cognitive activity. We consequently propose that beta oscillations and traveling waves are potential biomarkers of recovery from TBI. In a broader sense, our findings suggest that emergence from coma results from recovery of thalamo-cortical interactions that coordinate cortical beta rhythms.

neuroscience↗

bioRxiv2023

Reconstructing colonization dynamics to establish how human activities transformed island biodiversity

AimThe drivers and dynamics of initial human migrations across individual islands and archipelagos are poorly understood, affecting assessments of human-modification of island biodiversity. Here, we describe and test a process-explicit approach for reconstructing human arrival and expansion on islands, which combines archaeological and climate records with high-resolution spatial population models. Using Polynesian colonisation of New Zealand as an example, we show that our new method can generate information crucial for assessing how humans affected biodiversity on islands. InnovationThe transition of islands from prehuman to human dominated ecosystems has typically been assessed by comparing biodiversity before and after time of first arrival, without considering the potential importance of the spatiotemporal dynamics of the human expansion event. Our new approach, which uses pattern-oriented modelling methods to combine inferences of human colonisation dynamics from dated archaeological material with spatially explicit population models, produces validated reconstructions of the pattern and pace of human migration across islands at high spatiotemporal resolutions. From these reconstructions, demographic and environmental drivers of human colonization can be identified, and the role that people had on biodiversity established. Using this technique, we show that closely reconciling inferences of Polynesian colonisation of New Zealand requires there to have been a single founding population of approximately 500 people, arriving between 1233 and 1257 AD, settling multiple areas, and expanding quickly over both North and South islands. The resultant maps of M[a]ori colonisation dynamics provide new opportunities to better determine how human activities transformed biodiversity of New Zealand in space and time. Main conclusionsProcess-explicit models can reconstruct human migration across large islands, producing validated, high resolution spatiotemporal projections of human occupancy and abundance that account for dispersal and population dynamics. This modelling framework should prove effective across any islands and archipelagos where climate and archaeological records are available.

ecology↗

bioRxiv2022

Fifty years of reduction in sulphur deposition drives recovery in soil pH and plant communities

O_LISulphur deposition through rainfall has led to species loss and ecosystem degradation globally, and across Europe huge reductions in sulphur emissions since the 1970s were expected to promote the recovery of acidified ecosystem. However, the rate and ecological impact of recovery from acidification in terrestrial ecosystems is still unclear as is the influence of management and climate, as to date there has been no long-term spatially extensive evaluation of these changes. C_LIO_LIHere we present data from thousands of sites across Great Britain surveyed repeatedly from 1978-2019 and assess change in soil pH and plant acidity preference (Ellenberg R) in response to atmospheric deposition of sulphur and nitrogen. We analyse change in grasslands managed for pasture, referred to as high-intensity habitats, and compare to semi-natural habitats comprising rough grassland, broadleaved woodland, bog and heathland, referred to as low-intensity habitats. C_LIO_LISoil pH increased from 1978 to 2007 but then decreased between 2007 and 2019, resulting in a net increase of ~0.2 pH units in low-intensity habitats but no change in high-intensity habitats. The community average Ellenberg R increased in semi-natural habitats by ~0.2 units but remained stable in intensive grasslands. C_LIO_LIIn semi-natural habitats, but not intensive grasslands, these changes in plant community composition were associated with the soil pH changes which were in turn linked to decreasing sulphur deposition and differences in rainfall. C_LIO_LINitrogen deposition, which was relatively stable over the survey period, showed no additional effect upon soil acidity once sulphur deposition was accounted for. C_LIO_LISynthesis: Our results provide conclusive evidence that reductions in acid emissions are stimulating the gradual recovery of chronically acidified terrestrial ecosystems at a whole-country scale, while also suggesting this recovery is being compromised by changing climate and land management. C_LI

ecology↗

bioRxiv2022

B1 SINE-binding ZFP266 impedes reprogramming through suppression of chromatin opening by pioneering factors

Induced pluripotent stem cell reprogramming is inherently inefficient and understanding the molecular mechanisms underlying this inefficiency holds the key to successfully control cellular identity. Here, we report 16 novel reprogramming roadblock genes identified by CRISPR/Cas9-mediated genome-wide knockout (KO) screening. Of these, depletion of the predicted KRAB zinc finger protein (KRAB-ZFP) Zfp266 strongly and consistently enhanced iPSC generation in several iPSC reprogramming settings, emerging as the most robust roadblock. Further analyses revealed that ZFP266 binds Short Interspersed Nuclear Elements (SINEs) adjacent to binding sites of pioneering factors, OCT4 (POU5F1), SOX2 and KLF4, and impedes chromatin opening. Replacing the KRAB co-suppressor with a co-activator domain converted ZFP266 from a reprogramming inhibitor to a potent reprogramming facilitator. This work proposes SINE-KRAB-ZFP interaction to be a critical regulator of chromatin accessibility at enhancers for efficient cellular identity changes and also serves as a resource to further illuminate molecular mechanisms hindering reprogramming.

cell biology↗

bioRxiv2021

p57Kip2 regulates embryonic haematopoietic stem cell numbers by controlling the size of the sympathoadrenal progenitor pool

Haematopoietic stem cells (HSCs) are of major clinical importance, and finding methods for their in vitro generation is a prime research focus. We demonstrate that the cell cycle inhibitor p57Kip2/Cdkn1c limits HSC numbers by restricting the size of the sympathetic nervous system (SNS) and the amount of HSC-supportive catecholamines secreted by these cells, specifically in the aorta-gonads-mesonephros (AGM) region via {beta}2-adrenergic receptor signalling. This regulation occurs at the SNS progenitor level and is in contrast to the cell-intrinsic function of p57Kip2 in maintaining adult HSCs. Using single-cell RNA-Seq we dissect the differentiation pathway of neural crest cells into SNS cells in the AGM and reveal that they are able to take an alternative differentiation pathway, giving rise to a subset of mesenchymal cells expressing HSC-supportive factors. Neural crest cells thus appear to contribute to the AGM HSC niche via two different mechanisms: SNS-mediated catecholamine secretion and HSC-supportive mesenchymal cell production.

developmental biology↗

bioRxiv2021

Identification of a rapidly-spreading triple mutant for high-level metabolic insecticide resistance in Anopheles gambiae provides a real-time molecular diagnostic for anti-malarial intervention deployment.

Insecticide resistance provides both an increasingly pressing threat to the control of vector-borne diseases and insights into the remarkable capacity of natural populations to show rapid evolutionary responses to contemporary selection. Malaria control remains heavily dependent on deployment of pyrethroid insecticides, primarily in long lasting insecticidal nets (LLINs), but resistance in the major malaria vectors has increased over the last 15 years in concert with dramatic expansion of LLIN distributions. Identifying genetic mechanisms underlying high-level resistance in mosquitoes, which may almost entirely overcome pyrethroid efficacy, is crucial for the development and deployment of potentially resistance-breaking tools. Using the Anopheles gambiae 1000 genomes (Ag1000g) data we identified a very recent selective sweep in mosquitoes from Uganda which localized to a cluster of cytochrome P450 genes, including some commonly implicated in resistance. Further interrogation revealed a haplotype involving a trio of mutations, a nonsynonymous point mutation in Cyp6p4 (I236M), an upstream insertion of a partial Zanzibar-like transposable element (TE) and a duplication of the Cyp6aa1 gene. The mutations appear to have originated recently in An. gambiae from the Kenya-Uganda border region around Lake Victoria, with stepwise replacement of the double-mutant (Zanzibar-like TE and Cyp6p4-236M) with the triple-mutant haplotype (including Cyp6aa1 duplication), which has spread into the Democratic Republic of Congo and Tanzania. The triple-mutant haplotype is strongly associated with increased expression of genes able to metabolise pyrethroids and is strongly predictive of resistance to pyrethroids most notably deltamethrin, a commonly-used LLIN insecticide. Importantly, there was increased mortality in mosquitoes carrying the triple-mutation when exposed to nets co-treated with the synergist piperonyl butoxide (PBO). Frequencies of the triple-mutant haplotype remain spatially variable within countries, suggesting an effective marker system to guide deployment decisions for limited supplies of PBO-pyrethroid co-treated LLINs across African countries. Duplications of the Cyp6aa1 gene are common in An. gambiae across Africa and, given the enzymes metabolic activity, are likely to be a useful diagnostic for high levels of pyrethroid resistance.

evolutionary biology↗

bioRxiv2019

Resistance to dieldrin evolution in African malaria vectors is driven by interspecific and interkaryotypic introgression

The evolution of insecticide resistance mechanisms in natural populations of Anopheles malaria vectors is a major public health concern across Africa. Using genome sequence data, we study the evolution of resistance mutations in the resistance to dieldrin locus (Rdl), a GABA receptor targeted by several insecticides, but most notably by the long-discontinued cyclodiene, dieldrin. The two Rdl resistance mutations (296G and 296S) spread across West and Central African Anopheles via two independent hard selective sweeps that included likely compensatory nearby mutations, and were followed by a rare combination of introgression across species (from A. gambiae and A. arabiensis to A. coluzzii) and across non-concordant karyotypes of the 2La chromosomal inversion. Rdl resistance evolved in the 1950s as the first known adaptation to a large-scale insecticide-based intervention, but the evolutionary lessons from this system highlight contemporary and future dangers for management strategies designed to combat development of resistance in malaria vectors.

evolutionary biology↗

bioRxiv2019

Open source 3D printable replacement parts for the WHO insecticide susceptibility bioassay system

BackgroundMalaria vector control and research rely heavily on monitoring mosquito populations for the development of resistance to public health insecticides. One standard method for determining susceptibility in adult mosquito populations is the World Health Organization test (WHO bioassay). The WHO bioassay kit consists of several acrylic pieces that are assembled into a unit. Parts of the kit commonly break, reducing the capacity of insectaries to carry out resistance profiling. Since there is at present only a single supplier for the test kits, replacement parts can be hard to procure in a timely fashion. Here, we present 3D printable versions for all pieces of the WHO bioassay kit.\n\nResultsUsing widely available polylactic acid (PLA) filament as a printing material, we were able to design and print functional replacements for each piece of the WHO bioassay kit. We note no significant difference in mortality results obtained from PLA printed tubes and WHO acrylic tubes. Additionally, we observed no degradation of PLA in response to prolonged exposure times of commonly used cleaning solutions.\n\nConclusionsOur designs can be used to produce replacement parts for the WHO bioassay kit in any facility with a 3D printer, which are becoming increasingly widespread. 3D printing technologies can affordably and rapidly address equipment shortages and be used to develop bespoke equipment in laboratories.

evolutionary biology↗

bioRxiv2018

Complement C3-targeted gene therapy restricts onset and progression of neurodegeneration in chronic mouse glaucoma

Dysregulation of the complement system is implicated in neurodegeneration, including human and animal glaucoma. Optic nerve and retinal damage in glaucoma is preceded by local complement upregulation and activation, but whether targeting this early innate immune response could have therapeutic benefit remains undefined. Because complement signals through three pathways that intersect at complement C3 activation, here we targeted this step to restore complement balance in the glaucomatous retina, and to determine its contribution to degeneration onset and/or progression. To achieve this, we combined adeno-associated viral retinal gene therapy with the targeted C3 inhibitor CR2-Crry. We show that intravitreal injection of AAV2.CR2-Crry produced sustained Crry overexpression in the retina, and reduced deposition of the activation product complement C3d on retinal ganglion cells and the inner retina of DBA/2J mice. This resulted in neuroprotection of retinal ganglion cell axons and somata despite continued intraocular pressure elevation, suggesting a direct restriction of neurodegeneration onset and progression, and significant delay to terminal disease stages. Our study uncovers a damaging effect of complement C3 or downstream complement activation in glaucoma and establishes AAV2.CR2-Crry as a viable therapeutic strategy to target pathogenic C3-mediated complement activation in the glaucomatous retina.

neuroscience↗

bioRxiv2018

An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein

CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5-30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain tumour-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of sixty different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells.

cell biology↗