Biology subjects

Scott, J.

Publications and source records attributed to Scott, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Dose-additive and dose-dependent fetal testis toxicity of a nine-phthalate mixture in the rat

Phthalates (ortho-phthalic acid diesters) are used in a variety of manufacturing processes, particularly as non-covalent plasticizers in flexible polyvinyl chloride. Phthalates are male reproductive toxicants that disrupt the development of the fetal testis, resulting in malformed seminiferous cords, multinucleated germ cells (MNGs), and decreased testosterone production. We previously reported that MNGs were a sensitive, dose-dependent marker of dipentyl phthalate (DPEP) germ cell toxicity. To test whether germ cell effects of phthalates are dose-additive, we measured the induction of MNGs by a phthalate mixture. We treated timed pregnant Sprague Dawley rats with an equipotent mixture of nine phthalates, spanning a two-log dose range, from GD 17-21. We found that the phthalate mixture effects on morphogenesis of the testis, assessed through histological analysis of MNGs and seminiferous cord diameter were dose-additive. Consistent with prior results, we also found that the testosterone-reducing effect of the phthalate mixture was dose-additive and dose-dependent. Induction of MNGs had the lowest effective dose of any endpoint that we measured, with a benchmark dose (BMD10) of 9.402 (6.093, 13.13) mg ({sum} all phthalates)/kg/d, which is equivalent to a BMD10 of 3.517 (2.275, 4.919) mg DPEP/kg/d, based on relative potency factors. Quantitative analysis of phthalate metabolites in dam serum showed that the metabolites were present in approximately the same relative concentrations as the parent compounds in the dosing mixture, but with lower levels of monocyclohexyl phthalate and monobenzyl phthalate. Proteomic and spatial transcriptomic analyses showed significant decreases in expression of Leydig cell genes and proteins involved in steroid hormone biosynthesis, as well as enrichment of proteins related to metabolism of lipids and amino acids. Proteomic BMD analysis produced a lowest BMDmedian of 2.156 (0.204, 27.830) mg ({sum} all phthalates)/kg/d, which was within a 4.36-fold margin of the lowest apical endpoint BMD.

pharmacology and toxicology↗

bioRxiv2026

Elucidating pathogen interactions in Tanacetum cinerariifolium (pyrethrum) using fluorescently labelled Didymella tanaceti and Stagonosporopsis tanaceti

Australia is the largest producer of Pyrethrum (Tanacetum cinerariifolium) globally. Amongst the constraints on production are the fungal pathogens Didymella tanaceti and Stagonosporopsis tanaceti, which pose a significant threat to the industry, causing substantial yield losses. While the infection biology of S. tanaceti is well characterised, knowledge of D. tanaceti and its potential interaction with S. tanaceti on plants remains limited, hindering disease management. We developed fluorescently labelled strains of both pathogens via Agrobacterium tumefaciens-mediated transformation (ATMT). Binary vectors carrying the mNeonGreen or tdTomato fluorescent protein genes were introduced into D. tanaceti and S. tanaceti, respectively, and expression of the fluorescent proteins was confirmed by microscopy. Genome sequencing revealed single-copy T-DNA insertions in all transformants, with minor genomic rearrangements at insertion sites. Detached leaf assays demonstrated that transformed strains retained pathogenicity, producing disease symptoms indistinguishable from those of the wild type. These fluorescently labelled variants enabled detailed visualisation of D. tanaceti infection biology and its interactions with S. tanaceti, including co-infection dynamics. Co-infection assays using fluorescent strains further facilitated simultaneous visualisation and differentiation of both pathogens within host tissues. Importantly, these tools also allowed the first description of the early stages of infection by D. tanaceti in pyrethrum leaves. This study represents the first successful transformation of D. tanaceti and S. tanaceti, providing valuable resources to investigate their infection processes.

pathology↗

bioRxiv2026

Estrogen receptor-positive cell line xenograft models recapitulate metastatic dissemination and endocrine response of invasive lobular breast carcinoma

HighlightsO_LIER+, CDH1-deficient ILC xenografts recapitulate single-file histology in vivo C_LIO_LISpontaneous metastases faithfully mirror unique ILC clinical dissemination patterns C_LIO_LIER+ leptomeningeal metastases confirmed histologically in ILC xenograft models C_LIO_LIFulvestrant inhibits ILC tumor growth and metastatic burden, improving survival C_LIO_LIER signaling is enriched in ILC brain metastases, with upregulation of RET C_LI Invasive lobular breast carcinoma (ILC), the most common special histological subtype of breast cancer, is characterized by nearly universal expression of estrogen receptor alpha (ER) and unique sites of metastases, neither of which is fully recapitulated by genetically engineered mouse models. Using reporter-labeled ILC mouse xenografts, herein we used mammary fat pad, tail vein and intracardiac orthotopic growth to analyze spontaneous and experimental metastasis and gene expression. We observed ER-positive primary tumors with single-file histology and collagen deposition, and spontaneous metastasis from the mammary fat pad to bones, ovaries, and brain including the leptomeninges, thereby closely mirroring the growth and metastatic spread of human ILC. Brain metastases showed strong ER staining, confirmed by sequencing analyses which identified estrogen signaling as top activated pathway, and the lesions exhibited robust response to endocrine therapy. In summary, we report endocrine responsive mammary fat pad, tail vein and intracardiac xenografts that faithfully demonstrate unique ILC features and can serve as invaluable pre-clinical translational platforms for validating candidate ILC genetic drivers and testing novel therapeutics.

cancer biology↗

bioRxiv2026

BEEhaviourLab: A high-throughput platform for sublethal stressor screening in insects

Behavioural responses often provide the earliest detectable signs of physiological stress yet quantifying them at scale remains challenging. Acoustic behaviours such as buzzing are central to insect locomotion and communication but are rarely measured systematically, and their potential as sensitive indicators of environmental stress remains largely unexplored. Here, we test whether changes in insect acoustic behaviour can reveal sublethal effects of pesticide exposure. Using a newly developed automated behavioural platform (BEEhaviourLab), we recorded synchronised video and audio from insects, enabling simultaneous, high-throughput quantification of locomotor activity and buzzing behaviour across multiple individuals and species without the need for tagging. We applied this system to characterise the effects of the veterinary antiparasitic moxidectin on the bumble bee Bombus terrestris. Acute contact exposure produced dose-dependent reductions in locomotion and buzzing behaviour at concentrations below lethal thresholds. Acoustic measures detected behavioural disruption with sensitivity comparable to video-based activity metrics, demonstrating that buzzing behaviour provides a sensitive and previously unused indicator of neurotoxic stress. More broadly, scalable multimodal behavioural phenotyping enables subtle behavioural disruption to be detected across large experiments, opening new opportunities to incorporate acoustic endpoints into studies of animal behaviour, environmental stress, and ecological risk assessment.

animal behavior and cognition↗

bioRxiv2026

Predicting how perturbations reshape cellular trajectories with PerturbGen

A major challenge in biology is predicting how cells transition between states over time and how perturbations disrupt these transitions. Understanding such dynamics is critical for identifying interventions that reverse pathological programs or reprogram cells toward desired states. Although recent computational approaches can predict single-cell perturbation responses in silico, they cannot predict responses across dynamic cell trajectories, for example how early perturbations reconfigure later cell states. To address this gap, we introduce PerturbGen, a generative foundation model trained on over 100 million single-cell transcriptomes that predicts perturbation responses along cellular trajectories. PerturbGen predicts how genetic perturbation at source state shapes downstream states, alters gene programs and trajectories across time, for example in differentiation or disease progression. We apply PerturbGen to three newly generated multi-condition human single-cell datasets spanning immune responses, hematopoiesis and skin development. In an in vivo immune challenge, PerturbGen predicts that knocking out an IL1B signal in myeloid cells attenuates later cytokine-interferon programs, with downstream changes consistent with a reversal of IL-1{beta} stimulation signature. In hematopoiesis, anchoring perturbation-induced programs to human genetics enables simulation of monogenic blood disorders, recapitulates established disease-associated biology whilst systematically revealing lineage-specific programs, including in lineages where this was not previously possible. In skin organoids, PerturbGen predicted that Wnt activation enhances stromal differentiation recapitulating the trajectory observed in human prenatal skin, findings that were functionally validated by experimentally activating Wnt signaling. Together, PerturbGen extends modeling of gene perturbations from static to dynamic cellular systems. We envision PerturbGen enabling the creation of in silico, trajectory-aware perturbation atlases and virtual cells across diverse biological scenarios, supporting optimization of disease models and prioritization of candidate molecular interventions for therapeutic discovery.

bioinformatics↗

bioRxiv2026

Therapeutic Targeting of Microglial Hexokinase-2 Recalibrates Inflammasome Activation and Improves Functional Recovery After Traumatic Brain Injury

Traumatic brain injury (TBI) initiates a secondary inflammatory cascade in which sustained microglial activation contributes to long-term neurological dysfunction. Microglial inflammatory states depend on glycolytic reprogramming, suggesting that targeted modulation of metabolic regulators may attenuate post-traumatic inflammation while preserving essential immune functions. Hexokinase-2 (HK2), a rate-limiting glycolytic enzyme, regulates inflammatory signaling and inflammasome activation in microglia in neurodegenerative contexts; however, its role in TBI remains undefined. We therefore examined whether partial suppression of microglial HK2 modulates inflammatory responses following severe TBI. HK2 was robustly induced in microglia during the sub-acute phase after injury. Pharmacological inhibition of HK2 improved motor coordination without impairing locomotion or cognitive performance and selectively reduced inflammasome-related gene expression and ASC accumulation, particularly within the hippocampal hilus. Importantly, HK2 antagonism slowed microglial proliferation while preserving efferocytic capacity. Partial genetic reduction of microglial HK2 phenocopied these molecular and behavioral effects, supporting an HK2-dependent mechanism. Together, these findings identify microglial HK2 as a therapeutically targetable regulator of inflammatory amplification after TBI. Partial modulation of this pathway attenuates secondary neuroinflammation while maintaining critical microglial functions, highlighting HK2 as a promising strategy to improve functional recovery after traumatic brain injury.

neuroscience↗

bioRxiv2026

Selection for targeted therapy resistance leads to an indirect selection for higher phenotypic plasticity and enhanced evolvability to orthogonal stressors

Acquired resistance to targeted therapies is a primary barrier to durable cancer control. Resistance frequently coincides with stem-like features and EMT-associated cell-state changes (partial EMT), yet whether these programs directly cause resistance or instead enable escape by increasing phenotypic plasticity remains debated. Integrating computational modeling, functional experimental assays, and lineage tracing, we investigated how cell-state plasticity shapes acquired resistance to ALK inhibition in ALK+ lung cancer models. Our results support a model in which drug exposure selects for cells with higher phenotypic plasticity, progressively increasing their representation as resistance emerges. Consequently, resistant populations show enhanced capacity to adapt to orthogonal therapeutic and environmental stressors and exhibit heightened metastatic potential. Bulk ATAC-seq showed that highly plastic cells have increased chromatin accessibility at regulators of EMT and stemness. Consistent with this, boosting plasticity via Yamanaka-factor induction or EMT-factor expression reduced ALKi sensitivity over time. In contrast, constraining plasticity (SOX2 knockdown or epigenetic inhibition) reduced long-term resistance outgrowth and prolonged ALKi response. Together, our results indicate that targeted therapy indirectly selects for cells with increased phenotypic plasticity, providing the substrate from which multifactorial resistance and metastatic competence evolve. Further, it suggests that constraining plasticity could delay resistance and extend response durability.

cancer biology↗

bioRxiv2025

UGT35B1 is the principal enzyme mediating nicotine glycosylation in Drosophila melanogaster

Nicotine is a plant-derived pyridine alkaloid with potent neurotoxic properties. A major pathway for detoxification of nicotine in mammals is via glucuronidation to produce nicotine N-glucuronide, but this process in insects remains poorly understood. Using mass spectrometry, we demonstrate that Drosophila melanogaster detoxifies nicotine through glycosylation, producing nicotine N-glycoside. Given that many new agrochemicals contain pyridine rings, we also investigated the metabolism of flonicamid and imidacloprid. We detected glycosylation of flonicamid, but not imidacloprid. A targeted RNAi screen across 21 UDP-glycosyltransferases (Ugts) identified Ugt35B1 as important for survival of nicotine exposure. CRISPR-based knockout of Ugt35B1 increases sensitivity to nicotine and flonicamid, but not to imidacloprid, nor to a structurally distinct neonicotinoid (thiamethoxam). Mass spectrometry of knockout and control flies confirms that Ugt35B1 glycosylates nicotine, its metabolite cotinine, and flonicamid. Together these findings establish Ugt35B1 as the principal UGT mediating nicotine detoxification in D. melanogaster, revealing a previously uncharacterized insect glycosylation pathway with potential implications for herbivory, insecticide detoxification and toxicology. Highlights- Drosophila detoxifies nicotine by glycosylation into nicotine N-glycoside. - A targeted RNAi screen identifies Ugt35B1 as critical for nicotine survival. - Ugt35B1 knockout sensitizes flies to nicotine and flonicamid, but not to imidacloprid or thiamethoxam. - First demonstration of an insect UGT mediating in vivo glycosylation of nicotine and cotinine.

pharmacology and toxicology↗

bioRxiv2025

Nutrition of honeybees is constrained by the ratios of essential amino acids in pollen protein

Bees pollinate most of the worlds flowering plants and collect and eat floral pollen as their sole source of dietary protein, yet we know relatively little about nutritional constraints imposed on them by pollinivory. Pollen protein provides bees with essential amino acid (EAA) profiles that vary as a function of plant species, but the extent of this variation and its impact on bee feeding behaviour and performance are unknown. Here, we measured the EAA profiles of pollen, bee bread, honeybees, and royal jelly to understand natural variation in protein quality and tested how mismatches in EAAs relative to bee tissues impacted protein-to-carbohydrate regulation in adult workers. Bees fed diets with an EAA profile that matched their own tissues consumed more food, gained more weight, and ate proportionally more protein relative to carbohydrate (1:72 EAA:C) while those fed with pollen sources including bee bread ate proportionally less protein and less food overall. Deficiencies found in pollen led us to discover that nutrient balancing for protein and carbohydrate in bees was driven by the inverse relationship between quantities of the branched-chain amino acids relative to histidine in dietary protein. We predict that the creation of bee bread, a mixture of bee-collected pollen, is a general adaptation to pollen feeding that reduces the impact of imbalances in the EAA profile of pollen protein.

ecology↗

bioRxiv2025

On the imbalance between production and exploitation of marine fish assemblages: a case study from the Celtic Seas

Sustainable management of marine ecosystems has to take into account the conservation of many species that are not themselves targets of fishing, in addition to those that are commercially exploited. Dynamic size-spectrum models suggest that fishing which leads species to have similar ratios of yield to production (i.e. similar exploitation ratios) is not sufficient to protect those that are rare: these species need to experience lower exploitation ratios. Here, the status of the demersal fish assemblage in the Celtic Seas is examined from the perspective of yield and production, using survey data collected over the period 2012 to 2016, and incorporating both common and rare species. The results give no evidence that rarer species have lower exploitation ratios than common ones. This suggests that current management methods are not not operating in a way that conserves the fish assemblage as a whole.

ecology↗

bioRxiv2025

In Vivo Multiplexed Modeling Reveals Diverse Roles of the TBX2 Subfamily and Egr1 in Ras-Driven Lung Adenocarcinoma

The TBX2 subfamily of T-box transcription factors (including Tbx2, Tbx3, Tbx4, Tbx5) plays an essential role in lung development. Downregulation of these genes in human Lung adenocarcinoma (LUAD) suggests that these genes may be tumor suppressive, however because downregulation appears to occur primarily via epigenetic change, it remains unclear if these changes causally drive tumor progression or are merely the consequence of upstream events. Herein, we developed the first multiplexed mouse model to study the impact of TBX2 subfamily loss, alongside associated signaling genes Egr1, Chd2, Tnfaip3a, and Atf3, in Ras-driven lung cancer. Using TuBa-seq, a high-throughput tumor-barcoding system, we quantified the growth effects of these knockouts during early and late tumorigenesis. Chd2 loss consistently suppressed tumor progression, while Tbx2 loss exhibited stage-dependent effects. Notably, Egr1 emerged as a potent tumor suppressor, with its knockout increasing tumor size ([~]5x) at 20 weeks, surpassing Rb1 loss. Transcriptomic analyses of Egr1-deficient tumors suggested immune dysregulation, including heightened inflammation and potential markers of T cell exhaustion in the tumor microenvironment. These findings indicate that Egr1 may play a role in suppressing tumor growth through modulating immune dynamics, offering new insights into the interplay between tumor progression and immune regulation in LUAD.

cancer biology↗

bioRxiv2025

The Plankton Imager (Pi-10): An imaging instrument for automated and continuous zooplankton data collection

The Pi-10 is the latest iteration of the Plankton Imager: a high-speed colour line-scan camera that images particles in a flow-through system. The Pi-10 is a cost-effective, easy to install and low maintenance automated instrument that can be used on any platform with access to water and power supply. We tested the Pi-10 on the research vessel Cefas Endeavour, connected to a continuous water supply pumping water at 34 L min-1. The instrument collected images of particles, within the size range of 180 {micro}m - 3.5 cm, automatically and continuously, alongside other vessel operations, in all weathers over a period of 18 days. The Pi-10 successfully captured and saved up 5000 images per minute, translating into a 46 GB of digital storage per day. When particle density exceeded 147 per litre, the instrument stopped saving all images, while still recording the number of particles that passed through the system. This is akin to subsampling, with more sub-sampling required in areas or times of high particle density (e.g at the time of spring plankton bloom or in turbid waters). The Pi-10 collects high volumes of data in a continuous manner, thus providing unprecedented fine spatial data. The high frequency nature of the instrument opens the door to new areas of research. These include, in particular, the observation of fine scale processes, the move towards real-time sampling, and the increased capability to build a digital twin of the oceans. As technologies continue evolving the Pi-10 performance will increase, being able to collect and save more and more images.

ecology↗

bioRxiv2024

CalliCog: an open-source cognitive neuroscience toolkit for freely behaving nonhuman primates

Nonhuman primates (NHPs) are pivotal for unlocking the complexities of human cognition, yet traditional cognitive studies remain constrained to specialized laboratories. To revolutionize this paradigm, we present CalliCog: an open-source, scalable in-cage platform tailored for freely behaving experiments in small primate species such as the common marmoset (Callithrix jacchus). CalliCog includes modular operant chambers that operate autonomously and integrate seamlessly with home cages, eliminating human intervention. Our results showcase the power of CalliCog to train experimentally naive marmosets in touchscreen-based cognitive tasks. Remarkably, across two independent facilities, marmosets achieved touchscreen proficiency within two weeks and successfully completed tasks probing behavioral flexibility and working memory. Moreover, CalliCog enabled precise synchronization of behavioral data with electrocorticography (ECoG) recordings from freely moving animals, opening new frontiers for neurobehavioral research. By making CalliCog openly accessible, we aim to democratize cognitive experimentation with small NHPs, narrowing the translational gap between preclinical models and human cognition. MotivationCognitive neuroscience research involving nonhuman primates (NHPs) has traditionally been confined to a few highly specialized laboratories equipped with advanced infrastructure, expert knowledge, and specialized resources for housing and testing these animals. The common marmoset (Callithrix jacchus), a small NHP species, has gained popularity in cognitive research due to its ability to address some of these challenges. However, behavioral studies in marmosets remain labor-intensive and restricted mainly to experts in the field, making them less accessible to the broader scientific community. To address these barriers, we introduce an open and accessible platform designed for automated cognitive experiments in home cage settings with marmosets. This system supports the integration of cognitive behavioral analysis with wireless neural recordings, is cost-effective, and requires minimal technical expertise to build and operate.

neuroscience↗

bioRxiv2024

Genomic epidemiology of emerging terbinafine-resistant Trichophyton indotineae

Dermatophyte skin infections affect around a quarter of the worlds population and are a growing public health concern due to increasing incidence of novel species causing severe infections that are resistant to antifungal treatments. Trichophyton species cause the greatest burden of dermatophytosis worldwide, with the T. mentagrophytes species complex being particularly associated with the emergence of new aggressive infections. One emerging species, T. indotineae (originally T. mentagrophytes genotype VIII) is notable for the extensive nature of the often inflammatory infection, its clinical resistance to terbinafine antifungal treatment, and its rapid global spread. To better understand the epidemiology of this disease, we sourced isolates from severe cases of dermatophytosis in the United Kingdom, Ireland, France, Canada and India for the period 2018-2023, including the type strain from Japan. We used whole-genome sequencing to confirm 90 isolates were T. indotineae, and antifungal susceptibility testing indicated that over half of these (62%) were resistant to terbinafine (MIC [≥]1 mg/L). Pairwise genetic distances showed very high identity with only 147 (1-414) SNPs separating isolates that were nested within a monophyletic phylogeny, supporting a single evolutionary origin of T. indotineae. That no clear geographic clustering of isolates was observed confirms the rapid transcontinental spread of T. indotineae from its likely centre of diversity in Asia. Genome-wide analyses identified multiple non-synonymous SNPs in SQLE (ERG1), the squalene epoxidase target of terbinafine, that were associated with terbinafine in vitro resistance [≥]1 mg/L. However, five isolates exhibited high MIC values without SQLE mutations, suggesting the presence of alternative resistance mechanisms. Our findings highlight the importance of better genomic surveillance to understand and manage this severe and rapidly emerging terbinafine-resistant dermatophyte.

genomics↗

bioRxiv2024

Human-like APOBEC3 gene expression and anti-viral responses following replacement of mouse Apobec3 with the 7-gene human APOBEC3 locus

The seven human APOBEC3 (hA3) genes encode polynucleotide cytidine deaminases that play vital roles in restricting replication of viruses and retrotransposons. However, off-target A3 deamination of the cellular genome is a major source of somatic mutations in human cancer. The ability to study A3 biology in vivo is hindered by the fact that the solitary murine Apobec3 gene (mA3) encodes a cytoplasmic enzyme, with no apparent mutagenic activity. Transgenic expression of individual hA3 genes in mice has helped to confirm their oncogenic potential but important questions including which hA3 genes are active in different tissue contexts and how they function in concert when under control of their cognate promoters cannot be addressed using these models. Here we describe humanization of the mouse mA3 locus by integration of a modified BAC clone encompassing the entire 7-gene hA3 locus from human chromosome 22 replacing mA3 on mouse chromosome 15. APOBEC3 mice are viable and fertile and hA3 gene expression in cells and tissues correlates strongly with expression in corresponding human cells and tissues, indicating human-like regulation of hA3 gene expression in the mice. Splenocytes from this line display a functional human A3 response to Friend Murine Leukaemia Virus (F-MLV) infection. We propose that the Hs-APOBEC3 mouse will uniquely model the function of the complete hA3 locus in a living organism and that it will serve as a useful background upon which to model human cancer, as well as assisting drug discovery efforts.

cancer biology↗

bioRxiv2024

Peristromal niches protect lung cancers from targeted therapies through a combined effect of multiple molecular mediators

Targeted therapies directed against oncogenic signaling addictions, such as inhibitors of ALK in ALK+ NSCLC often induce strong and durable clinical responses. However, they are not curative in metastatic cancers, as some tumor cells persist through therapy, eventually developing resistance. Therapy sensitivity can reflect not only cell-intrinsic mechanisms but also inputs from stromal microenvironment. Yet, the contribution of tumor stroma to therapeutic responses in vivo remains poorly defined. To address this gap of knowledge, we assessed the contribution of stroma-mediated resistance to therapeutic responses to the frontline ALK inhibitor alectinib in xenograft models of ALK+ NSCLC. We found that stroma-proximal tumor cells are partially protected against cytostatic effects of alectinib. This effect is observed not only in remission, but also during relapse, indicating the strong contribution of stroma-mediated resistance to both persistence and resistance. This therapy-protective effect of the stromal niche reflects a combined action of multiple mechanisms, including growth factors and extracellular matrix components. Consequently, despite improving alectinib responses, suppression of any individual resistance mechanism was insufficient to fully overcome the protective effect of stroma. Focusing on shared collateral sensitivity of persisters offered a superior therapeutic benefit, especially when using an antibody-drug conjugate with bystander effect to limit therapeutic escape. These findings indicate that stroma-mediated resistance might be the major contributor to both residual and progressing disease and highlight the limitation of focusing on suppressing a single resistance mechanism at a time.

cancer biology↗

bioRxiv2024

The sulfur-related metabolic status of Aspergillus fumigatus during infection reveals cytosolic serine hydroxymethyltransferase as a promising antifungal target.

Sulfur metabolism is an essential aspect of fungal physiology and is known to be crucial for pathogenicity. Fungal sulfur metabolism comprises anabolic and catabolic routes that are not well- conserved in mammals, and therefore can be considered a promising source of prospective novel antifungal targets. To gain insight into the status of the Aspergillus fumigatus sulfur-related metabolism during infection we used a NanoString custom nCounter TagSet and compared the expression of 68 key metabolic genes in different murine models of invasive pulmonary aspergillosis, at three different time-points, and a variety of in vitro conditions. We identified a set of 15 genes that are consistently expressed at higher levels in vivo than in vitro, suggesting that they may be particularly relevant for intrapulmonary growth and therefore constitute promising drug targets. Indeed, the role of five of the fifteen genes had previously been empirically validated, supporting the likelihood that the remaining candidates are relevant. In addition, the analysis of the dynamics of gene expression at the early (16h), mid (24h-1) and late (72h) time-points uncovered potential disease initiation and progression factors. We further characterised one of the identified genes, encoding the cytosolic serine hydroxymethyltransferase ShmB, and demonstrated that it is an essential gene of A. fumigatus and that it is also required for virulence in a murine model of established pulmonary infection. We further show that the structure of the ligand binding pocket of the fungal enzyme differs significantly from its human counterpart, suggesting that specific inhibitors can be designed. Therefore, in vivo transcriptomics is a powerful tool to identify genes crucial for fungal pathogenicity that might encode promising antifungal target candidates. AUTHOR SUMMARYAspergillus fumigatus is an opportunistic human fungal pathogen that causes devastating chronic and invasive infections in immunocompromised patients. Our arsenal of antifungal drugs to fight this and other fungal pathogens is very limited, partly because of the high similarity between eukaryotic fungal and human cells makes the identification of suitable drug targets a challenging task. Furthermore, targets identified in vitro are often not effective in vivo, as their action is not relevant for fungal virulence. To address this challenge, we compared the expression profiles of a set of genes involved in sulfur metabolism, a promising source of potential drug targets, in numerous in vitro and in vivo conditions to identify favourable antifungal candidates. Subsequently, we validated one of the highlighted genes, demonstrating that it is essential for A. fumigatus viability and virulence, and that it can likely be targeted by specific inhibitors. Hence, we show the potential of using in vivo transcriptomics to identify targets that contribute to virulence, propose various candidates for future studies and present a novel target validated for further antifungal drug development.

microbiology↗

bioRxiv2024

PTEN and the PTEN-like phosphatase CnrN have both distinct and overlapping roles in a Dictyostelium chemorepulsion pathway

The directed movement of eukaryotic cells is crucial for processes such as embryogenesis and immune cell trafficking. The enzyme Phosphatase and tensin homolog (PTEN) dephosphorylates phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Dictyostelium discoideum cells require both PTEN and the PTEN-like phosphatase CnrN to locally inhibit Ras activation to induce biased movement of cells away from the secreted chemorepellent protein AprA. Both PTEN and CnrN decrease basal levels of PI(3,4,5)P3 and increase basal numbers of macropinosomes, and AprA prevents this increase. AprA requires both PTEN and CnrN to increase PI(4,5)P2 levels, decrease PI(3,4,5)P3 levels, inhibit proliferation, decrease myosin II phosphorylation, and increase filopod sizes. AprA causes PTEN, similar to CnrN, to localize to the side of the cell towards AprA in an AprA gradient. However, PTEN and CnrN also have distinct roles in some signaling pathways. PTEN, but not CnrN, decreases basal levels of PI(4,5)P2, AprA requires PTEN, but not CnrN, to induce cell roundness, and CnrN and PTEN have different effects on the number of filopods and pseudopods, and the sizes of filopods. Together, our results suggest that CnrN and PTEN play unique roles in D. discoideum signaling pathways, and possibly dephosphorylate PI(3,4,5)P3 in different membrane domains, to mediate chemorepulsion away from AprA.

cell biology↗