Biology subjects

Ahluwalia, A.

Publications and source records attributed to Ahluwalia, A..

14 recordsLinked to original sources

bioRxiv2026

Immune determinants of leuprolide-associated cardiovascular risk in prostate cancer

Leuprolide, a widely used androgen-deprivation therapy (ADT) for prostate cancer (PC), has been associated with increased cardiovascular events (CVE), although the underlying mechanisms remain unclear. Through integrated spatial transcriptomic analyses of murine heart and immune-stromal profiling in patient blood, we identified immune programs linked to leuprolide-associated CVE risk. Spatial analyses revealed the distinct organization of lymphoid (T+B cells) aggregates and inflammatory macrophages in the plaque following leuprolide exposure that accompanied by ventricular hypertrophy, relative to normal vasculature. Parallel profiling of circulatory soluble and cellular components in patients demonstrated CD8+T cell enriched pattern in ~44% of patients, including memory and type 1 cytotoxic subsets that linked to treatment-induced CVE, such as ventricular dysfunctions, but no contribution of adipocytokines. These translational findings highlight the stratification of leuprolide-associated CVE risk in PC patients based on circulating T cell profiling and could guide the risk mitigation with alternative ADT agents.

immunology↗

bioRxiv2026

Hepatic Xanthine Oxidoreductase Sustains Antithrombotic Nitric Oxide Signalling Through the Nitrate-Nitrite-Nitric Oxide Pathway

Background: Tonic endothelium-derived nitric oxide (NO) suppresses platelet activation physiologically, and its loss underlies the thrombotic risk of endothelial dysfunction. Inorganic nitrate and nitrite provide an alternative NO source, and xanthine oxidoreductase (XOR) is a candidate nitrite reductase, but whether endogenous XOR sustains platelet NO signalling in vivo, and its source is unknown. Methods: Dietary nitrate (15 mmol/L KNO3) was given to eNOS-/- and ApoE-/- mice. XOR was interrogated pharmacologically (allopurinol) and genetically (global Xdh+/- and hepatocyte-specific XOR knockout, HXOR KO). Haemostasis and thrombosis were assessed by tail bleeding and intravital microscopy imaging of FeCl3-induced mesenteric arterial thrombosis, alongside aggregometry, flow cytometry, platelet VASPSer239 phosphorylation, cGMP and ozone chemiluminescence-based analysis of nitrate and nitrite. Results: Dietary nitrate raised plasma nitrate and nitrite levels in all genotypes. In eNOS-/- and ApoE-/- mice, it prolonged bleeding time and normalised thrombus burden. In ApoE-/- mice nitrate treatment improved vasorelaxation, without altering XOR or eNOS expression. Allopurinol suppressed nitrite reductase activity in liver and plasma but not aorta, elevated plasma nitrite, shortened bleeding time and reduced platelet P-VASPSer239 expression. Xdh+/- mice were spontaneously prothrombotic, showed reduced P-VASPSer239 and exaggerated calcium mobilisation, and were refractory to dietary nitrate treatment despite equivalent nitrite elevation. XOR was undetectable in platelets. HXOR KO mice phenocopied global deficiency, with blunted nitrite-induced vasorelaxation but preserved acetylcholine and spermine-NO responses, reduced P-VASPSer239, enhanced aggregation and shortened bleeding time. Conclusions: Hepatic XOR sustains platelet NO-cGMP signalling and thromboresistance through an inter-organ, endocrine-like axis. Dietary nitrate restores antithrombotic protection when endothelial NO generation fails, whereas XOR inhibition removes physiological tonic antithrombotic signal.

pharmacology and toxicology↗

bioRxiv2026

Mapping the thymus in the viscoelastic landscape of biological tissues

The thymus plays a pivotal role in the generation of immunocompetent T cells. Although its function is dependent on its complex extracellular matrix, its 3D architecture and mechanical properties remain poorly characterised This knowledge gap limits efforts to model and engineer the organ, which is a critical step towards the development of strategies for the treatment of many haematological and autoimmune diseases. Here, we provide the first comprehensive multiscale dataset of bovine thymic extracellular matrix architecture and viscoelastic behaviour, including quantitiative descriptors such as relaxation times, instantaneous and equilibrium elastic moduli, storage and loss moduli, and spatial mechanical heterogeneity. Taken together, our data define the thymus as a compliant, highly dissipative viscoelastic organ with a fibrillar architecture. They also represent a unique database, which, for the first time, paves the way for quantitative thymus tissue engineering.

bioengineering↗

bioRxiv2026

An in vitro model of breast cancer metastatic niche priming

Metastatic breast cancer is responsible for around 11,500 deaths a year in the UK. The primary tumour likely plays a major role in priming the distant site for metastasis and crosstalk between primary and metastatic sites may be essential for secondary tumour growth. We have developed a novel in vitro model in which we can further study these interactions; evaluating niche priming and cancer cell conditioning as well as assessing their influence on cell homing and colonisation. In this paper we describe a model that we believe adds to the array of in vitro tools available to study various stages of the metastatic cascade, offering a unique opportunity to assess bidirectional, primary to niche interactions in vitro. We show that proliferation, migration and chemotaxis, and stem cell activity are altered in both cancer cell lines and in lung epithelial cells following linked, fluidic culture. Changes in cell homing and colonisation can be modelled in cell lines and within viable lung tissue explants taken from mice, with breast cancer cells settling and growing within the lung epithelial cells and tissue explants over 7 days. The colonisation/growth of cells injected into the system closely represents that seen following tail vein injection and cancer cells can be seen to settle and grow within the lung epithelial cells.

cancer biology↗

bioRxiv2025

Addressing cultural and knowledge barriers to enable preclinical sex inclusive research

For over thirty years, research has highlighted a sex bias in early research, risking the validity of biological knowledge. The first step towards change is effectively challenging misconceptions allowing researchers to perceive sex inclusive research as do-able. Utilising the theory of planned behaviour, we quantified researchers intention as a proxy measure for conducting sex inclusive research and explored attitude (value of the behaviour), subjective norm (perceived social pressure) and behavioural control (ability to conduct the behaviour). Additionally, we quantified the knowledge gap, prevalence of misconceptions, and assessed perceived benefits and barriers. We tested a workshop intervention that directly challenges the cultural embedded barriers. The data shows researchers intentions were high, but they had weak statistical knowledge and misunderstandings leading to a perception that inclusive research is prohibitive due to cost and animal use. We demonstrate that participation in the training intervention improved knowledge, altered the perceived barriers and cultural expectations.

scientific communication and education↗

bioRxiv2024

The nitrite reductase activity of xanthine oxidoreductase sustains cardiovascular health

BackgroundXanthine oxidoreductase (XOR) is a multi-functional enzyme that metabolises purines generating uric acid and is a generator of reactive oxygen species. Both functions have been implicated in the pathogenesis of cardiovascular disease. More recently, a third function of XOR as a nitrite reductase has been identified and been shown to play a key role in the benefits of targeting the non-canonical pathway for nitric oxide (NO) generation in the cardiovascular disease setting. This effect has been specifically attributed to XOR dependent recovery of NO levels. However, whether XOR derived NO plays any role in maintaining cardiovascular homeostasis in health is unknown. To explore this, we used global and hepatocyte-specific Xdh-deleted mice to assess cardiovascular homeostasis. MethodsXdh+/+ and Xdh+/-, Xdhfl/fl and Xdhfl/flAlbCre+/-(HXOR KO) mice littermates, matched for sex and age, were used for in vivo cardiovascular phenotyping; blood pressure, cardiac function, endothelial reactivity, and leukocyte trafficking. Tissues from these mice were used for biochemical measurements of nitrate, nitrite, and markers of NO downstream signalling. ResultsXdh+/- and HXOR KO mice expressed significantly attenuated liver and plasma nitrite reductase activity and platelet cGMP levels versus littermate controls. These effects were associated with increased systolic blood pressure, left ventricular remodelling, and increased leukocyte activation. These effects were associated with and likely driven by endothelial dysfunction evident in both mouse models. This dysfunction was reflected by increased endothelial adhesion molecule expression (P-selectin), increased ischaemia-induced vasoconstriction, during vessel occlusion, and an impaired flow-mediated dilation response of the iliac artery in vivo. ConclusionsIn summary, XOR derived NO is critical for maintaining vascular homeostasis under physiological conditions and is key in mediating the benefits of dietary nitrate regimes in cardiovascular pathology

pharmacology and toxicology↗

bioRxiv2024

Evolvoid: A genetic algorithm for shaping optimal cellular constructs

We describe an in-silico pipeline, Evolvoid, based on Genetic Algorithms (GAs) for identifying the optimal morphologies of cell-laden constructs. Driven by an ad hoc selection rule (i.e., the so-called fitness function (FF)), Evolvoid iteratively identifies the characteristics (i.e., the genome) of the survival of the fittest individual of a given population throughout generations. The FF is based on universally observed biophysical laws, representing the optimal trade-off between i) high cell viability and robustness to changes in environmental oxygen and ii) a low surface energy. The Shannon entropy is used to evaluate genome complexity, with the most complex fittest individuals showing quantitative and qualitative biological resemblance to in vitro constructs. Evolvoid paves the way for the development of "lab on a laptop": high-fidelity and cost-effective digital twins of cellular constructs which could augment or even substitute costly in vitro models.

bioengineering↗

bioRxiv2023

Digitoids: a novel computational platform for mimicking oxygen-dependent firing dynamics in in vitro neuronal networks

In vitro models of neural tissues are crucial to gain new insights on the pathophysiology of the brain. However, cell cultures are associated with many drawbacks and difficulties, e.g., technical complexities, ethical problems and high cost. Computational model-based solutions could represent an important tool to support the study of neuron function. In this work we present a novel computational platform where digital neuronal networks, i.e., Digitoids, can be developed with different size and layouts. The Digitoids rely on a novel firing model where the dependence on oxygen concentration is introduced, since it is a crucial limiting factor in cell cultures. To validate the performance of the platform, Digitoids were developed with the same morphological arrangement as observed in neuron monolayers in vitro. The comparison between the functional output of the Digitoids and the experimental data are not statistically different. The platform delivers a flexible digital tool that can easily be adapted for mimicking in vitro models with increasing complexity and can be exploited to optimize the laboratory experiments involving neuron cultures. Author SummaryThe use of cell models within laboratories is crucial to gain new understandings of the functioning of neuronal assemblies. However, culturing cells requires highly skilled personnel, a huge quantity of disposable materials and is thus associated with elevated costs. To overcome these limitations, the use of computer-based systems that reproduce the electrical behaviour of neurons can be employed. Since in vitro models are vessel-free, we present a novel computational model of neuron electrophysiology, where we introduce the dependence on local oxygen concentration Indeed, oxygen affects the metabolism and the function of cultured neurons. Thanks to our model and platform, we are able to reproduce the morphology of the cultured networks of neurons and build the so-called Digitoids. We then simulate the Digitoids and obtain their electrophysiological output. We compared the Digitoids output with experimental data from neurons cultured on microelectrode arrays. We did not find any differences between the electrophysiological output of the Digitoids and the experimental data, therefore we conclude that our novel oxygen-dependent model can be used to develop more physiologically relevant tools for simulating the activity of cultured neurons.

neuroscience↗

bioRxiv2023

Natural mutations of human XDH promote the nitrite (NO2-)-reductase capacity of xanthine oxidoreductase: a novel mechanism to promote redox health?

Several rare genetic variations of human XDH have been shown to alter xanthine oxidoreductase (XOR) activity leading to impaired purine catabolism. However, XOR is a multi-functional enzyme that depending upon the environmental conditions also expresses oxidase activity leading to both O {middle dot}- and H O and nitrite ({middle dot}NO -) reductase activity leading to NO. Since these products express important, and often diametrically opposite, biological activity consideration of the impact of XOR mutations in the context of each aspect of the biochemical activity of the enzyme is needed to determine the potential full impact of these variants. Herein, we show that known naturally occurring hXDH mutations do not have a uniform impact upon the biochemical activity of the enzyme in terms of uric acid (UA), reactive oxygen species (ROS) and nitric oxide ({middle dot}NO) formation. We show that the His1221Arg mutant, in the presence of xanthine, increases UA, O2{middle dot}- and NO generation compared to the WT, whilst the Ile703Val increases UA and {middle dot}NO formation, but not O2{middle dot}-. We speculate that this change in the balance of activity of the enzyme is likely to endow those carrying these mutations with a harmful or protective influence over health that may explain the current equipoise underlying the perceived importance of XDH mutations. We also suggest that targeting enzyme activity to enhance the NO2--reductase profile in those carrying such mutations may provide novel therapeutic options, particularly in cardiovascular disease. HighlightsO_LIMutations of xanthine oxidoreductase modulate both its expression and activity C_LIO_LIThe His1221Arg natural mutation increases xanthine oxidoreductase activity C_LIO_LIRaised xanthine oxidoreductase activity coupled with increased availability of nitrite substrate leads to increased NO provision C_LI

biochemistry↗

bioRxiv2019

The ARRIVE guidelines 2019: updated guidelines for reporting animal research

Reproducible science requires transparent reporting. The ARRIVE guidelines were originally developed in 2010 to improve the reporting of animal research. They consist of a checklist of information to include in publications describing in vivo experiments to enable others to scrutinise the work adequately, evaluate its methodological rigour, and reproduce the methods and results. Despite considerable levels of endorsement by funders and journals over the years, adherence to the guidelines has been inconsistent, and the anticipated improvements in the quality of reporting in animal research publications have not been achieved.\n\nHere we introduce ARRIVE 2019. The guidelines have been updated and information reorganised to facilitate their use in practice. We used a Delphi exercise to prioritise the items and split the guidelines into two sets, the ARRIVE Essential 10, which constitute the minimum requirement, and the Recommended Set, which describes the research context. This division facilitates improved reporting of animal research by supporting a stepwise approach to implementation. This helps journal editors and reviewers to verify that the most important items are being reported in manuscripts. We have also developed the accompanying Explanation and Elaboration document that serves 1) to explain the rationale behind each item in the guidelines, 2) to clarify key concepts and 3) to provide illustrative examples. We aim through these changes to help ensure that researchers, reviewers and journal editors are better equipped to improve the rigour and transparency of the scientific process and thus reproducibility.

scientific communication and education↗

bioRxiv2019

Reporting animal research: Explanation and Elaboration for the ARRIVE guidelines 2019

Improving the reproducibility of biomedical research is a major challenge. Transparent and accurate reporting are vital to this process; it allows readers to assess the reliability of the findings, and repeat or build upon the work of other researchers. The NC3Rs developed the ARRIVE guidelines in 2010 to help authors and journals identify the minimum information necessary to report in publications describing in vivo experiments.\n\nDespite widespread endorsement by the scientific community, the impact of the ARRIVE guidelines on the transparency of reporting in animal research publications has been limited. We have revised the ARRIVE guidelines to update them and facilitate their use in practice. The revised guidelines are published alongside this paper. This Explanation and Elaboration document was developed as part of the revision. It provides further information about each of the 21 items in ARRIVE 2019, including the rationale and supporting evidence for their inclusion in the guidelines, elaboration of details to report, and examples of good reporting from the published literature.

scientific communication and education↗

bioRxiv2019

Allometric Scaling of physiologically-relevant organoids

The functional and structural resemblance of organoids to mammalian organs suggests that they might follow the same allometric scaling rules. However, despite their remarkable likeness to downscaled organs, non-luminal organoids are often reported to possess necrotic cores due to oxygen diffusion limits. To assess their potential as physiologically relevant in vitro models, we determined the range of organoid masses in which quarter power scaling as well as a minimum threshold oxygen concentration is maintained. Using data on brain organoids as a reference, computational models were developed to estimate oxygen consumption and diffusion at different stages of growth. The results show that mature brain (or other non-luminal) organoids generated using current protocols must lie within a narrow range of masses to maintain both quarter power scaling and viable cores. However, micro-fluidic oxygen delivery methods could be designed to widen this range, ensuring a minimum viable oxygen threshold throughout the constructs and mass dependent metabolic scaling. The results provide new insights into the significance of the allometric exponent in systems without a resource-supplying network and may be used to guide the design of more predictive and physiologically relevant in vitro models, providing an effective alternative to animals in research.

bioengineering↗

bioRxiv2018

Smart Region-Growing: a novel algorithm for the segmentation of 3D clarified confocal image stacks

MotivationAccurately mapping the brain at the micro-scale is still a challenge in cellular neuroscience. While notable success has been reached in the field of tissue clarification and confocal imaging to obtain high-fidelity maps of three-dimensional neuron organization, neuron segmentation is still far away of ground-truth and manual segmentation performed by experts may be needed. The need of an expert is in part related to the limited success of the algorithms and tools performing single-neuron segmentation from 3D microscopic image data available in the State of Art, in part to the non-complete information given by these methods, which typically perform neuron tracing and thus limit the interpret-ability of results.\n\nResultsIn this paper, a novel algorithm for segmenting single neurons in their own arrangement within the brain is presented. The algorithm performs a region growing procedure with local thresholds based on the pixel intensity statistics typical of confocal acquisitions of biological samples and described by a mixture model. The algorithm is developed and tested on 3D confocal datasets obtained from clarified tissues. We compare the result of our algorithm with those obtained by manual segmentation performed by 6 different experts in terms of neuron surface area, volume and Sholl profiles. Statistical analysis performed on morphologic features extracted from the segmented structures confirms the feasibility of our approach.\n\nAvailabilityThe Smart Region Growing (SmRG) algorithm used in the analysis along with test confocal image stacks is available on request to the authors.\n\nContactalejandrocallara@gmail.com\n\nSupplementary informationSupplementary data are available on request to the authors.

bioinformatics↗

bioRxiv2017

Systemic and vascular inflammation in an in-vitro model of central obesity

Metabolic disorders due to over-nutrition are a major global health problem, often associated with obesity and related morbidities. Obesity is peculiar to humans, as it is associated with lifestyle and diet, and so difficult to reproduce in animal models.\n\nHere we describe a model of human central adiposity based on a 3-tissue system consisting of a series of interconnected fluidic modules. Given the causal link between obesity and systemic inflammation, we focused primarily on pro-inflammatory markers, examining the similarities and differences between the 3-tissue model and evidence from human studies in the literature. When challenged with high levels of adiposity, the in-vitro system manifests cardiovascular stress through expression of E-selectin and von Willebrand factor as well as systemic inflammation (expressing IL-6 and MCP-1) as observed in humans. Interestingly, most of the responses are dependent on the synergic interaction between adiposity and the presence of multiple tissue types. The set-up has the potential to reduce animal experiments in obesity research and may help unravel specific cellular mechanisms which underlie tissue response to nutritional overload.

bioengineering↗