Biology subjects

Hawkins, E.

Publications and source records attributed to Hawkins, E..

14 recordsLinked to original sources

bioRxiv2026

Anti-TGFβ peptide P144 augments CAR-T cell efficacy through neutrophil recruitment and TME remodeling in malignant mesothelioma

Malignant mesothelioma is a rare, aggressive and highly desmoplastic cancer characterised by a profoundly immunosuppressive tumour microenvironment (TME). Chimeric antigen receptor (CAR)-T cell therapy has transformed the treatment of hematological malignancies, but its activity against solid tumours such as mesothelioma remains limited, in part through transforming growth factor-beta (TGF{beta})-mediated immunosuppression. To address this barrier, we engineered murine CAR-T cells that simultaneously target tumour antigen and deliver TGF{beta} blockade to the tumour. We first compared two anti-TGF{beta} peptides, the beta glycan-derived P144 and the phage-display-derived P17, in vitro; both attenuated TGF{beta}-induced epithelial-to-mesenchymal transition and reporter activity, but P144 suppressed canonical Smad3 phosphorylation more effectively and was selected for downstream work. Murine CAR constructs targeting either mesothelin (M11) or fibroblast activation protein (FAP) were modified to secrete P144, and the resulting products were assessed in co-culture assays and in the syngeneic AE17m model of mesothelioma. P144 secretion did not alter transduction efficiency, expansion or antigen-specific cytotoxicity, but markedly increased antigen-driven IFN{gamma} production, most notably at low effector-to-target ratios. In vivo, both M11-P144 and FAP-P144 CAR-T cells-controlled tumour growth better than their parental, peptide-free counterparts, although complete tumour resolution was not achieved. Flow cytometric profiling of the tumour immune infiltrate showed that P144 delivery increased intratumoral neutrophil frequency approximately three-fold, and neutrophils recovered from these tumours were substantially more cytotoxic ex vivo than those from control tumours. Antibody-mediated neutrophil depletion attenuated the additional benefit conferred by P144 while leaving parental CAR-T activity largely intact, indicating that neutrophils contribute functionally to the effect. Local, CAR-T-delivered TGF{beta} blockade therefore remodels the mesothelioma TME and engages a tumoricidal neutrophil response, offering a modular strategy for enhancing adoptive cell therapy in desmoplastic solid tumours.

immunology↗

bioRxiv2026

Long-term dysregulation of histone acetylation, BDNF expression, and the hippocampal transcriptome in adult rats exhibiting a depressive-like phenotype following repeated chlorpyrifos exposure

Epidemiological evidence indicates that occupational exposures to the organophosphate pesticide chlorpyrifos (CPF) in adults are associated with an increased risk for depression. In this study, we use a rat model of occupational-like CPF exposure associated with a chronic depressive phenotype to investigate underlying molecular mechanisms. Adult rats received CPF (5 mg/kg/day) for 21 days. When assessed three months later, blood and brain cholinesterase activity had returned to control levels despite the presence of depressive signs. Hippocampal tissue was collected at this endpoint for molecular analyses. Western blotting analysis showed significant upregulation of histone deacetylase protein levels of HDAC3 and HDAC5, whereas HDAC1 and HDAC2 were unchanged. HDAC5 regulates histone acetylation at the brain-derived neurotrophic factor (Bdnf) gene. We used chromatin immunoprecipitation to assay histone 3 lysine 9 acetylation (H3K9ac) occupancy at the Bdnf promoter IV. H3K9ac levels at the Bdnf promoter IV were significantly reduced in CPF rats, accompanied by a concomitant reduction in BDNF protein expression. Exploratory RNA-sequencing identified 100 significantly differentially expressed genes in CPF-exposed rats at a false discovery rate < 5%, including significant downregulation in genes such as insulin-like growth factor 2 (Igf2), fibroblast growth factor 14 (Fgf14), and slit guidance ligand 2 (Slit2) involved in neuronal growth, synaptic function, and axon guidance. Gene Ontology analysis revealed enrichment in pathways related to neurogenesis, neuronal projections, and myelination. Overall, this study indicates persistent epigenetic and transcriptional dysregulation associated with reduced hippocampal BDNF and alterations in neuronal plasticity-related pathways linked to a long-term depressive-like phenotype following CPF exposure.

pharmacology and toxicology↗

bioRxiv2026

Barcoded-Plasmid DNA library construction for recording cell lineage trees enabled by a Scalable and modular Biofoundry-based Automated Robotic Pipeline

High-quality plasmid DNA purification at high throughput remains a significant bottleneck in molecular biology and bioengineering. Current methods frequently fail to deliver sufficient yields of pure, transfection-grade DNA required for genetic engineering applications in mammalian cells. Here, we present a Biofoundry-based automated pipeline using the CyBio FeliX robotic liquid handling platform to rapidly purify plasmid DNA with minimal manual intervention. The protocol leverages Solid Phase Reversible Immobilisation (SPRI)-based magnetic bead technology to ensure consistency, scalability, and DNA purity suitable for downstream viral particle production and mammalian cell transfection. The pipeline supports flexible processing of between 8 and 96 samples per run, making it adaptable across a wide range of experimental scales. The protocol is openly available via Earlham Institute GitHub repository, enabling broad adoption across the bioscientific community and contributing to the growing toolkit of reproducible, scalable engineering biology workflows. In this work, we employed an integrated robotic pipeline to process 528 pooled DNA plasmids and built a Lentiviral DNA plasmid library for lineage tracing, validated the library by sequencing, and demonstrated efficacy in downstream mammalian cell transfection experiments.

synthetic biology↗

bioRxiv2026

Menin regulates oncogenic cell identity transcriptional networks in multiple myeloma

Multiple myeloma (MM) is a heterogenous cancer that remains mostly incurable. A unifying feature of MM cells is a highly interconnected network of transcription factors and co-factors that coordinate the activity of super-enhancers to enforce myeloma cell identity. Targeting this network offers a promising avenue for therapeutic intervention across genetically diverse myeloma sub-types. Using integrated molecular and functional genomic approaches we identified Menin as a key driver of oncogenic gene expression in MM cells. Menin and its co-factor KMT2A bind at super-enhancers and maintain expression of essential myeloma genes, including IRF4. We demonstrate that Menin inhibitors, which have recently been approved for treatment of acute myeloid leukaemia, are highly active in myeloma cell lines and in vivo models. Combining Menin inhibitors with other super-enhancer targeting therapies such EP300/CREBBP inhibitors or immunomodulatory drugs (IMiDs) overcomes epigenetic plasticity in cells resistant to single agent treatment.

Cancer Biology↗

bioRxiv2025

Assessment of pharmacokinetics, safety, and neuroprotective efficacy of an adjunct intramuscular verapamil therapy in a rat model of organophosphate DFP-induced status epilepticus

Lethal organophosphate (OP) exposure leads to status epilepticus (SE), which, despite standard-of-care therapy, is associated with acute mortality and long-term morbidities. Neuronal injury and inflammation are reported following OP-SE, and drugs targeted at these processes have produced beneficial outcomes. Verapamil (VPM) is a calcium-channel blocker used as an antihypertensive drug. It exhibits neuroprotective and anti-inflammatory actions in experimental models of CNS injuries. Here, we investigated the feasibility of an adjunctive intramuscular (i.m.) VPM therapy in OP Diisopropyl Fluorophosphate (DFP)-induced SE. We also investigated the safety and toxicity of i.m. VPM and compared its pharmacokinetic (PK) profile to oral (p.o.) administration. Rats were injected with DFP (4 mg/kg, s.c.). One minute later, atropine (0.5 mg/kg, i.m.) and 2-PAM (25 mg/kg, i.m.) were injected, and at 1-hour post-SE, midazolam (1.78 mg/kg, i.m.) was administered. Rats were then treated with VPM (10 mg/kg, i.m, 3 days). Histological analysis was conducted to assess neuronal injury and injection-site pathology. In a separate group of rats, PK studies were conducted on blood and brain homogenates treated once with saline or VPM (10 mg/kg, p.o. or i.m.). Our data demonstrated that following DFP-SE, i.m. VPM achieved higher blood and brain levels and exhibited a favorable PK profile compared to p.o. route. VPM therapy did not cause significant muscle pathology and produced a robust neuroprotective response. Our studies provide evidence that the i.m. route is an effective method for delivering VPM following SE producing significant neuroprotective outcomes compared to treatment with the standard-of-care alone in OP-SE.

pharmacology and toxicology↗

bioRxiv2023

An immunohistochemical atlas of necroptotic pathway expression

Necroptosis is a lytic form of regulated cell death reported to contribute to inflammatory diseases of the gut, skin and lung, as well as ischemic-reperfusion injuries of the kidney, heart and brain. However, precise identification of the cells and tissues that undergo necroptotic cell death in vivo has proven challenging in the absence of robust protocols for immunohistochemical detection. Here, we provide automated immunohistochemistry protocols to detect core necroptosis regulators - Caspase-8, RIPK1, RIPK3 and MLKL - in formalin-fixed mouse and human tissues. We observed surprising heterogeneity in protein expression within tissues, whereby short-lived immune barrier cells were replete with necroptotic effectors, whereas long-lived cells lacked RIPK3 or MLKL expression. Local changes in the expression of necroptotic effectors occurred in response to insults such as inflammation, dysbiosis or immune challenge, consistent with necroptosis being dysregulated in disease contexts. These methods will facilitate the precise localisation and evaluation of necroptotic signaling in vivo. HighlightsO_LI13 automated immunohistochemistry protocols for detecting the necroptotic pathway C_LIO_LINecroptotic pathway expression is confined to fast-cycling immune barriers C_LIO_LINecroptotic pathway expression changes at sites of immunoinflammatory challenge C_LIO_LIImmunodetection of necrosomes in IBD patients is a putative new diagnostic tool C_LI

cell biology↗

bioRxiv2023

Comparison of spontaneous recurrent seizures in rats following status epilepticus induced by organophosphate paraoxon, DFP, and sarin

Organophosphate (OP) compounds are highly toxic and include household, industrial, agricultural, and chemical warfare nerve agents (CWNA). OP exposure inhibits acetylcholinesterase enzyme, causing cholinergic overstimulation that can evolve into status epilepticus (SE) and produce lethality. Furthermore, OP-SE survival is associated with mood and memory dysfunction and spontaneous recurrent seizures (SRS). Here we assessed hippocampal pathology and chronic SRS following SE induced by OP agents in rats. Male Sprague-Dawley rats were injected with 1.5x LD50 of various OP agents, followed by atropine and 2-PAM. At 1-h post-OP-SE onset, midazolam was administered to control SE. Approximately 6 months following OP-SE, SRS were evaluated using continuous video-EEG monitoring. Histopathology was conducted using Hematoxylin and Eosin (H&E), while silver sulfide (Timm) staining was utilized to assess Mossy Fiber Sprouting (MFS). Over 60% of OP-SE surviving rats developed SRS with varying seizure frequencies, durations, and Racine severity scores. H&E staining revealed a significant hippocampal neuronal loss, while Timm staining revealed extensive MFS within the inner molecular region of the dentate gyrus of SRS-expressing OP-SE rats. This study demonstrates that OP-SE is associated with hippocampal neuronal loss, extensive MFS, and SRS, all hallmarks of chronic epilepsy.

pharmacology and toxicology↗

bioRxiv2022

SELECTIVE INHIBITION OF SOLUBLE TNF ATTENUATES HIPPOCAMPAL NEUROINFLAMMATION AND PSD-95 EXPRESSION TO IMPROVE NEUROLOGICAL FUNCTIONS IN A RAT MODEL OF GULF WAR ILLNESS

BACKGROUNDSystemic inflammation is a major contributor to poor brain pathology across many disease conditions. Specifically, the upregulation of the pro-inflammatory cytokine TNF in the hippocampus activates its receptor TNFR1, reducing AMPA receptor trafficking to impair LTP and associated behavioral outcomes. Studies using animal models of GWI have shown both a chronic upregulation of TNF and impaired neurological function. Therefore, this study aimed to investigate whether selectively inhibiting only the soluble form of TNF (solTNF) that preferentially activates TNFR1 can reverse neuroinflammation to improve neuroplasticity and neurological function. METHODSGWI was induced in rats by treating with DFP (or vehicle) for 5 consecutive days. Six months later, the rats were treated with XPro1595 (or vehicle) for 2 weeks to selectively inhibit solTNF, after which they were subjected to a battery of behavioral tests (cognition, anxiety-related, depressive-like behavior, and neuropathic pain). MRI brain scans were performed, and the animals were euthanized for brain pathological analysis. RESULTSThe hippocampus of the GWI rats had significantly increased neuroinflammatory levels, resulting in edema and reduced AMPA receptor trafficking to the post-synaptic membrane that collectively promoted impairments in memory, anxiety, depressive-like behavior, and neuropathic pain. However, treating the rats with XPro1595 in the chronic environment attenuated the neuroinflammatory response, that reduced edema and impaired AMPA receptor trafficking, allowing for improvements in all areas of neurological function. CONCLUSIONOverall findings suggest that selectively inhibiting solTNF using XPro1595 reduces neuroinflammation, synaptic plasticity, and overall function when administered in the chronic setting of a rat model of GWI. This data supports the use of XPro1595 in Veterans with GWI.

neuroscience↗

bioRxiv2022

OPS-γδ: allogeneic opsonin-secreting γδT cell immunotherapy for solid tumours mediates direct and bystander immunity

T cell-based cancer immunotherapy has typically relied on membrane-bound cytotoxicity enhancers such as chimeric antigen receptors expressed in autologous {beta}T cells. These approaches are limited by tonic signalling of synthetic constructs and costs associated with manufacture of bespoke patient products. {gamma}{delta}T cells are an emerging alternative chassis for cellular therapy, possessing innate anti-tumour activity, potent antibody-dependent cytotoxicity (ADCC) and minimal alloreactivity. We present an immunotherapeutic platform technology built around the V{gamma}9V{delta}2 {gamma}{delta}T cell chassis, harnessing specific characteristics of this cell type and offering an allo-compatible means of delivering cellular therapy that recruits bystander immunity. We engineered {gamma}{delta}T cells to secrete synthetic opsonins and stabilized IL15 (stIL15). Using GD2 as a model antigen we show how opsonin-secreting V{gamma}9V{delta}2 (OPS-{gamma}{delta}) have enhanced cytotoxicity and also confer this benefit on lymphoid and myeloid bystander cells. Reflecting the secreted nature of the engineered efficacy modules, the entire product rather than just the gene-modified fraction exhibited enhanced activation and cytotoxic profiles, superior persistence and proliferative capacity even upon repeated tumour challenge. Secretion of stIL15 abrogated the need for exogenous cytokine supplementation during expansion and further mediated functional licensing of bystander NK cells. Compared to unmodified {gamma}{delta}T cells, stIL15-OPS-{gamma}{delta} cells exhibited superior in-vivo control of subcutaneous tumour and persistence in the blood. stIL15-OPS-{gamma}{delta} cells were further efficacious in 3D patient-derived osteosarcoma models, where efficacy could be boosted with the addition of immunomodulatory aminobisphosphonate drug, zoledronic acid. Together the data identify stIL15-OPS-{gamma}{delta} cells as a novel allogeneic platform combining direct cytolysis with bystander activation to effect solid tumour control. One Sentence SummaryArmoured, opsonin-secreting OPS-{gamma}{delta} cell immunotherapy is built on the innate strengths of the V{gamma}9V{delta}2 cell chassis for allogeneic solid tumour targeting.

immunology↗

bioRxiv2022

Contrasting roles of the wheat MRC in promoting starch granule initiation in leaves and repressing the onset of B-type granule initiation in the endosperm

The spatial and temporal patterns by which starch granules initiate vary greatly between species and organs, but molecular factors that contribute to these diverse patterns are poorly understood. We reveal distinct organ-specific roles of the MYOSIN-RESEMBLING CHLOROPLAST PROTEIN (MRC) in regulating granule initiation in the endosperm and leaves of wheat. We isolated three independent TILLING mutants of tetraploid wheat (Triticum turgidum cv. Kronos) with premature stop or missense mutations in the A-genome homeolog, which we showed to be the only active homeolog in tetraploid wheat due to a disruption of the B-genome homeolog. Wheat endosperm contains both large A-type granules initiated during early grain development, and small B-type granules that initiate about 10 - 15 days later. The mrc mutants had significantly smaller A-type granules and a higher relative volume of B-type granules in the endosperm than the wild type. Whereas B-type granules initiated 15 - 20 days post anthesis (dpa) in the wild-type, they appeared as early as 10 dpa in the mrc-1 mutant, suggesting a role for MRC in suppressing B-type granule initiation during early grain development. By contrast, MRC promotes granule initiation in leaves: mutants carrying premature stop mutations in MRC had fewer granules per chloroplast than the wild type. These contrasting roles of MRC among wheat organs provide new insight into functional diversification of granule initiation proteins, and suggest that they may facilitate the diverse patterns of granule initiation observed across species and organs.

plant biology↗

bioRxiv2021

STARCH SYNTHASE 4 is required for normal starch granule initiation in amyloplasts of wheat endosperm

O_LIStarch granule initiation is poorly understood at the molecular level. The glucosyltransferase, STARCH SYNTHASE 4 (SS4), plays a central role in granule initiation in Arabidopsis leaves, but its function in cereal endosperms is unknown. We investigated the role of SS4 in wheat, which has a distinct spatiotemporal pattern of granule initiation during grain development. C_LIO_LIWe generated TILLING mutants in tetraploid wheat (Triticum turgidum) that are defective in both SS4 homoeologs. The morphology of endosperm starch was examined in developing and mature grains. C_LIO_LISS4 deficiency led to severe alterations in endosperm starch granule morphology. During early grain development, while the wild type initiated single A-type granules per amyloplast, most amyloplasts in the mutant formed compound granules due to multiple initiations. This phenotype was similar to mutants deficient in B-GRANULE CONTENT 1 (BGC1). SS4 deficiency also reduced starch content in leaves and pollen grains. C_LIO_LIWe propose that SS4 and BGC1 are required for the proper control of granule initiation during early grain development that leads to a single A-type granule per amyloplast. The absence of either protein results in a variable number of initiations per amyloplast and compound granule formation. C_LI

plant biology↗

bioRxiv2020

The metalloproteinase inhibitor Prinomastat reduces AML growth,prevents stem cell loss and improves chemotherapy effectiveness

Acute myeloid leukemia (AML) is a blood cancer of the myeloid lineage. Its prognosis remains poor, highlighting the need for new therapeutic and precision medicine approaches. AML symptoms often include cytopenias, linked to loss of healthy hematopoietic stem and progenitor cells (HSPCs). The mechanism behind HSPC decline is complex and still poorly understood. Here, intravital microscopy (IVM) of a well-established experimental model of AML allows direct observation of the interactions between healthy and malignant cells in the bone marrow (BM), suggesting that physical dislodgment of healthy cells by AML through damaged vasculature may play an important role. Numerous human leukemia types, particularly MLL-AF9 samples, show high expression levels of multiple matrix metalloproteinases (MMPs). Therefore, we evaluate the therapeutic potential of the MMP inhibitor (MMPI) prinomastat. IVM analyses of treated mice reveal reduced vascular permeability and healthy cell clusters in circulation, and lower AML cell speed. Furthermore, treated mice have decreased BM infiltration, increased retention of healthy HSPCs in the BM and increased survival following chemotherapy. Overall, our results suggest that MMPIs could be a promising complementary therapy to reduce AML growth and limit the loss of HSPC and BM vascular damage caused by MLL-AF9 and possibly other AML subtypes.

cancer biology↗

bioRxiv2020

Loss of erythroblasts in acute myeloid leukemia causes iron redistribution with clinical implications

Acute myeloid leukemia (AML) is a heterogeneous disease with poor prognosis and limited treatment strategies. Determining the role of cell-extrinsic regulators of leukemic cells is vital to gain clinical insights into the biology of AML. Iron is a key extrinsic regulator of cancer but its systemic regulation remains poorly explored in AML. To address this question, we studied iron metabolism in AML patients at diagnosis and mechanisms involved using the syngeneic MLL-AF9-induced AML mouse model. We found that AML is a disorder with a unique iron profile not associated with inflammation or transfusion and characterized by high ferritin, low transferrin, high transferrin saturation (TSAT), and high hepcidin. The increased TSAT in particular, contrasts with observations in other cancer types and in anemia of inflammation. Using the MLL-AF9 mouse model of AML, we demonstrated that leukemic blasts take up iron and that the AML-induced loss of erythroblasts is responsible for iron redistribution and an increase in TSAT. We also show that elevated TSAT at diagnosis is independently associated with increased overall survival in AML and suggest that TSAT may be a relevant prognostic marker in AML.

cancer biology↗

bioRxiv2018

Functional network dynamics in a neurodevelopmental disorder of known genetic origin

Cognitive processing depends on the temporal co-ordination of functional brain networks. This fundamental aspect of neurophysiology potentially bridges the genetic regulation of neuronal activity and developmental cognitive impairments. We investigated brain network dynamics in a neurodevelopmental disorder of known genetic origin, by comparing individuals with ZDHHC9-associated intellectual disability to individuals with no known impairment. We used Hidden Markov Modelling on magnetoencephalography (MEG) data, at rest and during auditory oddball stimulation, to characterise transient network dynamics. At rest, network dynamics distinguished the groups, with ZDHHC9 participants showing longer state activation. Crucially, ZDHHC9 gene expression levels predicted the group differences across networks, supporting an association between molecular pathology and neurophysiology. In contrast, network dynamics during auditory oddball stimulation did not show this association. We demonstrate a link between brain network dynamics and regional gene expression, and present a valuable method for understanding the real-time neural mechanisms linking genetic variation to cognitive difficulties.

neuroscience↗