Biology subjects

Stevenson, K.

Publications and source records attributed to Stevenson, K..

15 recordsLinked to original sources

bioRxiv2025

Radiotherapy drives lung metastasis in PDAC via a RhoGTPasesignaling shift towards MRCK-dependency

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with an extremely poor prognosis, partly owing to its highly metastatic nature. Although radiotherapy is an effective and potentially curative treatment modality, research over the last few decades has indicated that it may induce a more metastatic phenotype in surviving cancer cells. We demonstrate for the first time that clinically relevant doses of image-guided targeted radiotherapy induce metastasis in a genetically engineered KPC mouse model of PDAC. Furthermore, this induction is largely driven by an organotropic switch towards lung metastasis. Using an in vitro RNAi screening approach, we identified a key role for myotonic dystrophy-related Cdc42-binding kinase (MRCK) in driving this response. MRCK activity was spatially upregulated at the plasma membrane in response to radiotherapy in PDAC cell lines as well as at the invasive margins of PDAC tumors. This upregulation of activity was maintained in metastases, suggesting an important role for MRCK in not only triggering local invasion in response to radiotherapy but also promoting distant metastases. Importantly, inhibition of MRCK with a small-molecule inhibitor (BDP9066) specifically opposed radiation-driven MRCK upregulation and pro-metastatic response.

cancer biology↗

bioRxiv2025

Beyond Gatekeeping: Efflux Pumps Remotely Destabilize Cytoplasmic Drug-Target Interactions by Limiting Rebinding.

Bacterial efflux pumps are major contributors to multidrug resistance, classically described as "gatekeepers" that reduce drug entry. Here, we uncover an equally potent post-entry mechanism of efflux pumps, revealing that their function extends deep into intracellular drug-target interactions. Using quantitative live-cell fluorescence imaging, we monitored the activity of major efflux systems in Escherichia coli and Pseudomonas aeruginosa with Hoechst (HCT), a DNA-binding inhibitor. As expected, more HCT entered efflux-deficient cells than efflux-active cells. Unexpectedly, the intracellular HCT in efflux-deficient cells also bound DNA more stably, resulting in higher binding affinity. Statistical-physics-based modeling and experimental testing show that that unlike in dilute in vitro conditions, drug molecules that unbind from their targets in intracellular environments rapidly undergo successive rebinding, prolonging the lifetime of the drug-target complex. However, efflux pumps counteract this effect by suppressing rebinding, thereby kinetically destabilizing drug-target interactions and lowering the binding affinity. This kinetic destabilization acts synergistically with canonical gatekeeping to broaden and amplify drug resistance. Our findings reveal an underappreciated biophysical mechanism of efflux pumps, expanding the classical biochemical view of how efflux pumps confer multidrug resistance.

microbiology↗

bioRxiv2025

On-Body Measure of Reaction Time Correlates With Intoxication Level

Excessive alcohol use has profound effects on individual health and healthcare systems worldwide. Despite this, there is currently no system or device that can detect robustly the physiologic and functional effects of alcohol-based impairment in real-world conditions. A practical, on-body, device capable of rapidly and accurately determining the functional capacity of an individual to drive, before they can start the ignition of the automobile, is required. The goal of this pilot study was to evaluate the effect of acute alcohol intoxication on premotor time (PMT) and reaction time (RT), both highly sensitive of individual cognition, as evaluated by the Pison Technology wrist-worn wearable. Nineteen participants were included in the study, 14 subjects who consumed alcohol sufficient to raise blood alcohol concentration (BAC) to 0.12% within a 30-minute period and 5 controls who did not consume alcohol. Changes in reaction time data were correlated with blood alcohol levels as measured by breathalyzer testing, identifying a statistically significant difference between those participants under the legal limit and those over the legal limit. Both group and individual analyses confirmed that as the BAC increased in subjects, the PMT also increased. The PMT also decreased as the BAC returned to levels under the legal intoxication threshold (0.08%). A significant effect of BAC levels on changes in PMT at the p < 0.05 level for two conditions. This study demonstrated the first use case of an on-body, neuro-physiological sensor capable of detecting sensitive changes of reaction time, in real time, that serves as an easy-to-measure proxy for blood alcohol content and impairment.

neuroscience↗

bioRxiv2023

Interplay between ATRX and IDH1 mutations governs innate immune responses in diffuse gliomas

Stimulating the innate immune system has been explored as a therapeutic option for the treatment of gliomas. Inactivating mutations in ATRX, defining molecular alterations in IDH-mutant astrocytomas, have been implicated in dysfunctional immune signaling. However, little is known about the interplay between ATRX loss and IDH mutation on innate immunity. To explore this, we generated ATRX knockout glioma models in the presence and absence of the IDH1R132H mutation. ATRX-deficient glioma cells were sensitive to dsRNA-based innate immune agonism and exhibited impaired lethality and increased T-cell infiltration in vivo. However, the presence of IDH1R132Hdampened baseline expression of key innate immune genes and cytokines in a manner restored by genetic and pharmacological IDH1R132H inhibition. IDH1R132H co-expression did not interfere with the ATRX KO-mediated sensitivity to dsRNA. Thus, ATRX loss primes cells for recognition of dsRNA, while IDH1R132H reversibly masks this priming. This work reveals innate immunity as a therapeutic vulnerability of astrocytoma.

cancer biology↗

bioRxiv2022

Slice'N'Dice: Maximising the value of predicted models for structural biologists

With the advent of next generation modelling methods, such as AlphaFold2, structural biologists are increasingly using predicted structures as search models for Molecular Replacement (MR) when experimental structures of homologues are unavailable. Inaccuracy in domain-domain orientations is often a key limitation when using predicted models for MR. SliceNDice is a software package designed to address this issue by first slicing models into distinct structural units and then automatically placing the slices using Phaser. The slicing step can use AlphaFold2s predicted aligned error (PAE), or can operate via a variety of C atom clustering algorithms, extending applicability to structures of any origin. The number of splits can be selected by the user. SliceNDice is available in CCP4 8.0 and is currently being adapted for cryo-EM use cases.

bioinformatics↗

bioRxiv2022

Polio Virotherapy of Malignant Glioma Engages the Tumor Myeloid Infiltrate and Induces Diffuse Microglia Activation

Malignant gliomas commandeer abundant inflammatory infiltrates with glioma-associated macrophages and microglia (GAMM) actively promoting tumor progression. Like all cells in the mononuclear phagocytic system, macrophages and microglia constitutively express the poliovirus receptor, CD155. Besides myeloid cells, CD155 is widely upregulated ectopically in the neoplastic compartment of malignant gliomas (and solid cancers in general). Intratumor treatment with the highly attenuated rhino:poliovirus chimera, PVSRIPO, yielded long-term survival with durable radiographic responses in patients with recurrent glioblastoma (Desjardins et al. New England Journal of Medicine, 2018). Here, we studied mechanisms of PVSRIPO immunotherapy in mouse brain tumor models to decipher contributions of myeloid vs. malignant cells to antitumor efficacy. PVSRIPO treatment caused intense engagement of the GAMM infiltrate associated with substantial, but transient tumor regression. This was accompanied by diffuse microglia activation and proliferation in the normal central nervous system (CNS) surrounding the tumor, extending to the ipsilateral and even the contralateral hemispheres. PVSRIPO-instigated microglia activation occurred against a backdrop of sustained innate antiviral inflammation, associated with induction of the PD-L1 immune checkpoint on GAMM. Combining PVSRIPO with PD1/PD-L1 blockade led to durable remissions. Our work implicates GAMM as active drivers of PVSRIPO-induced antitumor inflammation and reveals profound and widespread neuroinflammatory activation of the CNS-resident myeloid compartment by PVSRIPO.

cancer biology↗

bioRxiv2022

Glioblastoma exosomes influence glial cell hyaluronic acid deposition to promote invasiveness

BackgroundInfiltration of glioblastoma (GBM) throughout the brain leads to its inevitable recurrence following standard-of-care treatments, such as surgical resection, chemo- and radio-therapy. A deeper understanding of the mechanisms invoked by GMB to infiltrate the brain is needed to develop approaches to contain the disease and reduce recurrence. The aim of this study was to discover mechanisms through which extracellular vesicles (EVs) released by GBM influence the brain microenvironment to facilitate infiltration, and to determine how altered extracellular matrix (ECM) deposition by glial cells might contribute to this. MethodsCRISPR was used to delete genes, previously established to drive carcinoma invasiveness and EV production, from patient-derived primary and GBM cell lines. We purified and characterised EVs released by these cells, assessed their capacity to foster pro-migratory microenvironments in mouse brain slices, and evaluated the contribution made by astrocyte-derived extracellular matrix (ECM) to this. Finally, we determined how CRISPR-mediated deletion of genes, which we had found to control EV-mediated communication between GBM cells and astrocytes, influenced GBM infiltration when orthotopically injected into CD1-nude mice. ResultsGBM cells expressing a p53 mutant (p53273H) with established pro-invasive gain-of-function release EVs containing a sialomucin, podocalyxin (PODXL), which encourages astrocytes to deposit ECM with increased levels of hyaluronic acid (HA). This HA-rich ECM, in turn, promotes migration of GBM cells. Consistently, CRISPR-mediated deletion of PODXL opposes infiltration of GBM in vivo. ConclusionsThis work describes several key components of an EV-mediated mechanism though which GBM cells educate astrocytes to support infiltration of the surrounding healthy brain tissue. KEY POINTSThe p53R273H oncogene encourages GBM cells to release EVs containing podocalyxin. Podocalyxin-containing EVs from GBM increase hyaluronic acid production by astrocytes. Hyaluronic acid production by astrocytes drives GBM migration. IMPORTANCE OF THE STUDYThe infiltrative behaviour of glioblastoma (GBM) leads to widespread dissemination of cancer cells throughout the brain. Thus, even following successful resection of the primary tumour these disseminated cells inevitably contribute to post-surgical relapse. In this study, we have discovered a new mechanism through which GBM can release small extracellular vesicles (EVs) to reprogramme extracellular matrix (ECM) production by astrocytes in a way that supports increased invasive behaviour of the GBM cells. Moreover, we have discovered several key components of the pathway which contribute to this EV-mediated GBM-glial cell communication. Principal amongst these, we show that a particular mutant of the p53 tumour suppressor, p53273H drives the release of EVs which foster the deposition of pro-invasive ECM by astrocytes. This study provides mechanistic insight into why brain tumours expressing p53273H are associated with particularly poor patient survival and highlights the possibility of deploying agents which target astrocyte ECM deposition to reduce the morbidity of p53273H- expressing GBM.

cancer biology↗

bioRxiv2021

The development of ovine gastric and intestinal organoids for studying ruminant host-pathogen interactions

Gastrointestinal (GI) infections in sheep have significant implications for animal health, welfare and productivity, as well as being a source of zoonotic pathogens. Interactions between pathogens and epithelial cells at the mucosal surface play a key role in determining the outcome of GI infections; however, the inaccessibility of the GI tract in vivo significantly limits the ability to study such interactions in detail. We therefore developed ovine epithelial organoids representing physiologically important gastric and intestinal sites of infection, specifically the abomasum (analogous to the stomach in monogastrics) and ileum. We show that both abomasal and ileal organoids form self-organising three-dimensional structures with a single epithelial layer and a central lumen that are stable in culture over serial passage. We performed RNA-seq analysis on abomasal and ileal tissue from multiple animals and on organoids across multiple passages and show the transcript profile of both abomasal and ileal organoids cultured under identical conditions are reflective of the tissue from which they were derived and that the transcript profile in organoids is stable over at least five serial passages. In addition, we demonstrate that the organoids can be successfully cryopreserved and resuscitated, allowing long-term storage of organoid lines, thereby reducing the number of animals required as a source of tissue. We also report the first published observations of a helminth infecting gastric and intestinal organoids by challenge with the sheep parasitic nematode Teladorsagia circumcincta, demonstrating the utility of these organoids for pathogen co-culture experiments. Finally, the polarity in the abomasal and ileal organoids can be inverted to make the apical surface directly accessible to pathogens or their products, here shown by infection of apical-out organoids with the zoonotic enteric bacterial pathogen Salmonella enterica serovar Typhimurium. In summary, we report a simple and reliable in vitro culture system for generation and maintenance of small ruminant intestinal and gastric organoids. In line with 3Rs principals, use of such organoids will reduce and replace animals in host-pathogen research.

microbiology↗

bioRxiv2021

Increased apoptotic priming of glioblastoma enables therapeutic targeting by BH3-mimetics

IDH wild-type glioblastoma (GBM) is the most prevalent malignant primary brain tumour in adults. GBM typically has a poor prognosis, mainly due to a lack of effective treatment options leading to tumour persistence or recurrence. Tackling this, we investigated the therapeutic potential of targeting anti-apoptotic BCL-2 proteins in GBM. Levels of anti- apoptotic BCL-xL and MCL-1 were consistently increased in GBM compared with non- malignant cells and tissue. Moreover, we found that relative to their differentiated counterparts, patient-derived GBM stem-like cells also displayed higher expression of anti- apoptotic BCL-2 family members. Surprisingly, high anti-apoptotic BCL-xL and MCL-1 expression correlated with heightened susceptibility of GBM to BCL-2 family protein- targeting BH3-mimetics. This is indicative of increased apoptotic priming. Indeed, GBM displayed an obligate requirement for MCL-1 expression in both tumour development and maintenance. Investigating this apoptotic sensitivity, we found that sequential inhibition of BCL-xL and MCL-1 led to robust anti-tumour responses in vivo, in the absence of overt toxicity. These data demonstrate that BCL-xL and MCL-1 pro-survival function is a fundamental prerequisite for GBM survival that can be therapeutically exploited by BH3- mimetics.

cancer biology↗

bioRxiv2021

Low-dose lung radiotherapy for COVID-19 lung disease: a pre-clinical efficacy study in a bleomycin model of pneumonitis.

PurposeLow-dose whole lung radiotherapy (LDLR) has been proposed as a treatment for patients with acute respiratory distress syndrome associated with SARS-CoV-2 infection and clinical trials are underway. There is an urgent need for preclinical evidence to justify this approach and inform dose, scheduling and mechanisms of action. Materials and methodsFemale C57BL/6 mice were treated with intranasal bleomycin sulphate (7.5 or 11.25 units/kg, day 0), then exposed to whole lung radiation therapy (0.5, 1.0, 1.5 Gy or sham, day 3). Bodyweight was measured daily and lung tissue harvested for histology and flow cytometry on day 10. Computed tomography (CT) lung imaging was performed pre-radiation (day 3) and pre-endpoint (day 10). ResultsBleomycin caused pneumonitis of variable severity which correlated with weight loss. LDLR at 1.0 Gy was associated with a significant increase in the proportion of mice recovering to 98% of initial bodyweight and a proportion of these mice exhibited less severe histopathological lung changes. Mice experiencing moderate initial weight loss were more likely to respond to LDLR than those experiencing severe initial weight loss. Additionally, LDLR (1.0 Gy) significantly reduced bleomycin-induced increases in interstitial macrophages, CD103+ dendritic cells and neutrophil-DC hybrids. Overall,bleomycin-treated mice exhibited significantly higher percentages of non-aerated lung in left than right lungs and LDLR (1.0 Gy) prevented further reductions in aerated lung volume in right but not left lungs. LDLR at 0.5 and 1.5 Gy did not modulate bodyweight or flow cytometric readouts of bleomycin-induced pneumonitis. ConclusionsOur data support the concept that LDLR can ameliorate acute inflammatory lung injury, identify 1.0 Gy as the most effective dose and provide preliminary evidence that it is more effective in the context of moderate than severe pneumonitis. Mechanistically, LDLR at 1.0 Gy significantly suppressed bleomycin-induced accumulation of pulmonary interstitial macrophages, CD103+ dendritic cells and neutrophil-DC hybrids.

immunology↗

bioRxiv2020

Quantitative in vivo bioluminescence imaging of orthotopic patient-derived glioblastoma xenografts

Despite extensive research, little progress has been made in glioblastoma therapy, owing in part to a lack of adequate preclinical in vivo models to study this disease. To mitigate this, primary patient-derived cell lines, which maintain their specific stem-like phenotypes, have replaced established glioblastoma cell lines. However, due to heterogenous tumour growth inherent in glioblastoma, the use of primary cells for orthotopic in vivo studies often requires large experimental group sizes. Therefore, when using intracranial patient-derived xenograft (PDX) approaches, it is advantageous to deploy imaging techniques to monitor tumour growth and allow stratification of mice. Here we show that stable expression of near-infrared fluorescent protein (iRFP) in patient-derived glioblastoma cells enables rapid direct non-invasive monitoring of tumour development without compromising tumour stemness or tumorigenicity. Moreover, as this approach does not depend on the use of agents like luciferin, which can cause variability due to changing bioavailability, it can be used for quantitative longitudinal monitoring of tumour growth. Notably, we show that this technique also allows quantitative assessment of tumour burden in highly invasive models spreading throughout the brain. Thus, iRFP transduction of primary patient-derived glioblastoma cells is a reliable, cost- and time-effective way to monitor heterogenous orthotopic PDX growth.

cancer biology↗

bioRxiv2020

Ultra-Low-Cost Integrated Silicon-based Transducer for On-Site, Genetic Detection of Pathogens

Rapid screening and low-cost diagnosis play a crucial role in choosing the correct course of intervention e.g., drug therapy, quarantine, no action etc. when dealing with highly infectious pathogens. This is especially important if the disease-causing agent has no effective treatment, such as the novel coronavirus SARS-CoV-2 (the pathogen causing COVID-19), and shows no or similar symptoms to other common infections. We report a silicon-based integrated Point-of-Need (PoN) transducer (TriSilix) that can chemically-amplify and detect pathogen-specific sequences of nucleic acids (NA) quantitatively in real-time. Unlike other silicon-based technologies, TriSilix can be produced at wafer-scale in a standard laboratory; we have developed a series of methodologies based on metal-assisted chemical (wet) etching, electroplating, thermal bonding and laser-cutting to enable a cleanroom-free low-cost fabrication that does not require processing in an advanced semiconductor foundry. TriSilix is, therefore, resilient to disruptions in the global supply chain as the devices can be produced anywhere in the world. To create an ultra-low-cost device, the architecture proposed exploits the intrinsic properties of silicon and integrates three modes of operation in a single chip: i) electrical (Joule) heater, ii) temperature sensor (i.e. thermistor) with a negative temperature coefficient that can provide the precise temperature of the sample solution during reaction and iii) electrochemical sensor for detecting target NA. Using TriSilix, the sample solution can be maintained at a single, specific temperature (needed for isothermal amplification of NA such as Recombinase Polymerase Amplification (RPA) or cycled between different temperatures (with a precision of {+/-}1.3{degrees}C) for Polymerase Chain Reaction (PCR) while the exact concentration of amplicons is measured quantitatively and in real-time electrochemically. A single 4-inch Si wafer yields 37 TriSilix chips of 10x10x0.65 mm in size and can be produced in 7 hours, costing ~US $0.35 per device. The system is operated digitally, portable and low power - capable of running up to 35 tests with a 4000 mAh battery (a typical battery capacity of a modern smartphone). We were able to quantitatively detect a 563-bp fragment (Insertion Sequence IS900) of the genomic DNA of M. avium subsp. paratuberculosis (extracted from cultured field samples) through PCR in real-time with a Limit-of-Detection of 20 fg, equivalent to a single bacterium, at the 30th cycle. Using TriSilix, we also detected the cDNA from SARS-CoV-2 (1 pg), through PCR, with high specificity against SARS-CoV (2003).

bioengineering↗

bioRxiv2019

Healthcare related activity following kidney transplantation: an observational cohort study.

BACKGROUND BACKGROUND METHODS RESULTS DISCUSSION Post-Transplant Activity Applicability Summary Competing interests Patient consent for publication Funding REFERENCES Renal transplantation is regarded as the gold standard of renal replacement therapy (RRT) with evidence of improved life expectancy, quality of life and superior cost-effectiveness when compared to dialysis (1-6). In recent years there has been a steady rise in the incidence of renal transplantation within most developed healthcare economies (7). Such has been the observed benefit that transplantation is now being considered across an increasingly wide range of patient age, BMI and comor ...

epidemiology↗

bioRxiv2018

Synthetic Lethality of Wnt Pathway Activation and Asparaginase in Drug-Resistant Acute Leukemias

Resistance to asparaginase, an antileukemic enzyme that depletes asparagine, is a common clinical problem. Using a genome-wide CRISPR/Cas9 screen, we found a synthetic lethal interaction between Wnt pathway activation and asparaginase in acute leukemias resistant to this enzyme. Wnt pathway activation induced asparaginase sensitivity in distinct treatment-resistant subtypes of acute leukemia, including T-lymphoblastic, hypodiploid B-lymphoblastic, and acute myeloid leukemias, but not in normal hematopoietic progenitors. Sensitization to asparaginase was mediated by Wnt-dependent stabilization of proteins (Wnt/STOP), which inhibits GSK3-dependent protein ubiquitination and degradation. Inhibiting the alpha isoform of GSK3 phenocopied this effect, and pharmacologic GSK3 inhibition profoundly sensitized drug-resistant leukemias to asparaginase. Our findings provide a molecular rationale for activation of Wnt/STOP signaling to improve the therapeutic index of asparaginase.\n\nSIGNIFICANCEThe intensification of asparaginase-based therapy has improved outcomes for several subtypes of acute leukemia, but the development of treatment resistance has a poor prognosis. We hypothesized, from the concept of synthetic lethality, that gain-of-fitness alterations in drug-resistant cells had conferred a survival advantage that could be exploited therapeutically. We found a synthetic lethal interaction between activation of Wnt-dependent stabilization of proteins (Wnt/STOP) and asparaginase in acute leukemias resistant to this enzyme. Inhibition of the alpha isoform of GSK3 was sufficient to phenocopy this effect, and the combination of GSK3-selective inhibitors and asparaginase had marked therapeutic activity against leukemias resistant to monotherapy with either agent. These data indicate that drug-drug synthetic lethal interactions can improve the therapeutic index of cancer therapy.

cancer biology↗

bioRxiv2018

Detection of Mycobacterium leprae DNA in soil; Multiple needles in the haystack?

BackgroundLeprosy is an infectious disease caused by Mycobacterium leprae and Mycobacterium lepromatosis affecting the skin and nerves. Despite decades of availability of adequate treatment, transmission is unabated and routes of transmission are not completely understood. Notwithstanding the general assumption that untreated M. leprae infected humans represent the major source of transmission, scarce reports indicate that environmental specimens could play a role as a reservoir as well.\n\nMethodologyIn order to identify patterns of bacterial transmission, this study investigated whether M. leprae DNA is present in soil of regions where leprosy is endemic or areas with possible animal reservoirs (armadillos and red squirrels). Soil samples were collected in Bangladesh, Suriname and the British Isles. The presence of M. leprae DNA was determined by RLEP PCR and M. leprae SNP types were further identified by Sanger sequencing of loci 1-3.\n\nResultsM. leprae DNA was identified in soil samples from environments inhabited by leprosy patients (Bangladesh), armadillos (Suriname) and the lepromatous Eurasian red squirrels (British Isles). In Bangladesh SNP type 1 was detected, Suriname soil contained SNP type 1 or 2, whilst SNP typing was not feasible for the British samples.\n\nConclusionsIt is conceivable that, besides humans and animals, environmental reservoirs may play a role in transmission. Frequent, intense contact with multibacillary leprosy patients poses the highest risk of transmission, and even though the risk of environmental contamination is low, it may offer a possible explanation for the occurrence of leprosy in individuals in areas without any reported human leprosy.

microbiology↗