Biology subjects

Robinson, C.

Publications and source records attributed to Robinson, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Lipid-associated architecture and divergent ammonium transport distinguish a bacterial Rhesus protein

Nitrification is a major process in the global nitrogen cycle, initiated by the oxidation of ammonia to nitrite. The ammonia-oxidising bacterium Nitrosomonas europaea catalyses this first and rate-limiting step and depends on ammonium both as an energy substrate and as a nitrogen source, making ammonium acquisition central to its physiology. Cellular ammonium transport is mediated by the Amt/Mep/Rh superfamily, whose members share a conserved fold despite divergent physiological roles. Remarkably, N. europaea lacks a canonical prokaryotic Amt transporter and instead expresses NeRh50, a Rh-family protein proposed to have been acquired from a eukaryotic lineage. Here, we determine a cryo-EM structure of NeRh50 that reveals tightly associated lipids at the monomer-monomer interfaces, including density consistent with an unusual inverted lipid orientation. Native mass spectrometry and molecular dynamics simulations further support persistent lipid association at these interfaces. NeRh50 also differs functionally from canonical AmtB, selectively mediating electrogenic ammonium transport with ~70-fold lower apparent affinity. Transport is enhanced under alkaline conditions but not by an imposed proton gradient, suggesting membrane-potential dependence. Together, these findings reveal divergence from canonical Amt proteins in both membrane-associated architecture and transport properties, providing insight into the functional and evolutionary diversification of the Amt/Mep/Rh superfamily.

biochemistry↗

bioRxiv2026

Biosensor-guided evolution of chalcone synthase enhances biosynthesis of natural and non-natural flavanones

Flavonoids constitute a large class of natural products widely investigated for their bioactive properties, with microbial production offering a potentially scalable alternative to plant extraction. However, achieving structural diversification of these compounds in microbial systems remains challenging, as modification of the flavonoid B-ring typically relies on downstream tailoring enzymes. An alternative strategy is to exploit the intrinsic promiscuity of the canonical flavanone biosynthesis pathway to introduce structural variation at an early stage. Here, we sought to improve microbial production of diverse flavanones by systematically leveraging pathway promiscuity. By constructing a combinatorial library of pathways comprising 4-coumarate-CoA ligase, chalcone synthase, and chalcone isomerase, we enabled the conversion of a panel of ring-substituted cinnamic acid precursors into ten natural and non-natural flavanones. In parallel, we established a genetically encoded biosensor based on the transcriptional regulator FdeR and demonstrated its responsiveness across all ten flavanones. Leveraging this biosensor for high-throughput screening, we performed directed evolution of chalcone synthases from Hordeum vulgare and Arabidopsis thaliana, identifying enzyme variants that led to improved production of O-methylated flavanones, including isosakuranetin, hesperetin, and homoeriodictyol, as well as fluoro-substituted flavanones. In addition, we demonstrated that specific variants of H. vulgare chalcone synthase promoted the formation of isoferuloyl-derived derailment products. Collectively, this work establishes the FdeR-based biosensor as a versatile platform for pathway and enzyme engineering, enabling efficient early-stage diversification of flavanones in microbial systems and providing insight into the mutational landscape of chalcone synthases.

Synthetic Biology↗

bioRxiv2026

First Usutu virus detections in wild birds in Scotland, 2025

In summer 2025, several Eurasian Blackbird (Turdus merula) deaths were reported on the Isle of Arran in Scotland. Initial investigation included post-mortem examination, where no diagnosis was achieved. Following Orthoflavivirus and avian paramyxovirus testing, Usutu virus RNA was detected in two Blackbirds by reverse transcription-PCR. Phylogenetic analysis identified Usutu virus Africa 3.2 lineage which clustered closely with existing UK detections, indicating geographic expansion rather than a new incursion. Subsequent surveillance confirmed the presence of several potential mosquito vector species.

microbiology↗

bioRxiv2026

Physical continuity at biomaterial-ECM interfaces regulates fibroblast activation via NF-κB

Fibrotic responses at biomaterial-tissue interfaces limit implant integration and regenerative healing, yet how the interaction between biomaterials and the extracellular matrix (ECM) regulates fibroblast activation remains poorly understood. Granular hydrogels including microporous annealed particle scaffolds (MAP) reduce fibrosis, while chemically and mechanically matched hydrogels do not, suggesting a dominant role for scaffold architecture. In this model, MAP scaffolds allow collagen infiltration and form physically continuous composites, whereas hydrogels exclude collagen and generate interfacial slip planes. To isolate how biomaterial architecture influences extracellular matrix (ECM) integration and fibroblast activation, we developed a reductionist in vitro model that integrates collagen type I with either microporous annealed particle (MAP) scaffolds or chemically and mechanically matched bulk hydrogels. This physical integration stabilizes collagen architecture, limits fibroblast-mediated matrix compaction, suppresses contractility, and attenuates myofibroblast transition. Fibroblasts in mechanically integrated environments exhibit reduced expression and nuclear localization of NF-{kappa}B and are enriched for quiescent phenotypes. Together, these findings identify biomaterial-ECM physical continuity as a design principle for limiting fibrotic signaling.

bioengineering↗

bioRxiv2026

Glucocorticoid signaling regulates expression of the EBI3 subunit of IL-27 in neonatal macrophages: Implications for antenatal corticosteroid therapy

BackgroundHumans and mice display elevated levels of IL-27, an immunosuppressive cytokine shown to increase during neonatal bacterial sepsis and compromise survival. This study explores two hypotheses for regulation of IL-27 expression: 1) decreased DNA methylation in newborns that contributes to increased expression of IL-27 genes; 2) neonatal hormones regulate IL-27 expression through upstream hormone response elements (HREs). MethodsWhole genome methyl-seq analysis of neonatal and adult blood-derived macrophages identified differentially methylated regions (DMRs) at steady-state. Quantitative PCR (qPCR) measured expression of IL-27 genes (IL27p28 and EBI3) in human and murine neonatal macrophages stimulated in vitro with synthetic glucocorticoid or progesterone. Confocal microscopy and chromatin immunoprecipitation (ChIP) of glucocorticoid receptor (GR) assessed translocation into the nucleus and binding to the EBI3 promoter. ResultsThe IL-27p28 promoter contained DMRs that were increased in the neonatal cohort. The analysis did not identify DMRs within the EBI3 promoter. Dexamethasone stimulation increased EBI3 gene expression in human and murine neonatal macrophages. GR localized to the nucleus in response to dexamethasone and was enriched at the EBI3 upstream regulatory region. ConclusionThese data suggest glucocorticoid (GC) signaling increases EBI3 expression. This has importance in the context of antenatal GC administration that may increase IL-27 levels. Impact Statement{blacksquare} Elevated expression of IL-27 in early life impairs the host response to invasive bacterial infection in neonates. {blacksquare}Understanding the regulatory mechanisms contributing to increased IL-27 during the neonatal period is necessary to reduce susceptibility to infection in this vulnerable population. {blacksquare}The methylation status of the IL-27 genes in macrophages from neonatal and adult blood donors does not suggest regulation of differential expression with age. {blacksquare}Glucocorticoids are a signal that can induce EBI3 gene expression in a GR-dependent manner. {blacksquare}Glucocorticoid therapy for premature infants may increase IL-27 expression and promote enhanced susceptibility to infection.

immunology↗

bioRxiv2026

The impact of intermittent palatable food consumption on microbiota structure in male and female rats

The gut microbiome plays a vital role in metabolism, behavior, and overall health, with diet being a key factor shaping its composition. This study examines the impact of intermittent palatable food (PF) consumption on microbiota structure in male and female rats, focusing on feeding preferences and sex differences. Rats were characterized as high preferring (HP) or low preferring (LP) based on PF intake, and microbial analyses were conducted across different gastrointestinal regions, including the colon, feces, cecum, and cecal contents. However, microbiota composition varied with significant differences in Firmicutes, Bacteroidetes, and Actinobacteria abundances. Sex-based differences were evident particularly in fecal and cecal samples, where Proteobacteria and Actinobacteria populations varied between males and females within the same feeding groups. Our findings support the notion that dietary habits and microbiota composition may form a feedback loop, reinforcing food preferences through gut-brain axis signaling. While alpha diversity remained unchanged, beta diversity analysis indicated subtle, but significant differences in microbial community structures based on sex and feeding behavior. This research provides novel insights into the interplay between diet, gut microbiota, and behavior, while emphasizing the importance of considering sex as a variable in microbiome studies. Understanding these relationships may inform dietary interventions aimed at optimizing microbiota composition to improve metabolic and mental health outcomes linked to diet-induced microbiota shifts. ImportanceThe gut microbiome plays a critical role in various bodily functions, from the brains protective mechanisms to dietary behaviors and food choices. In this study, we sought to broaden the understanding of how various levels of high fat and high carbohydrate diet consumption alter gut microbiota, and ultimately, shape food preference behaviors. We assessed preference behaviors by categorizing rats into high-preference and low-preference groups based on their consumption of high calorie, palatable food, then analyzed their gut bacterial composition, comparing diet preference groups and examining whether sex differences were reflected in the results. The findings from this research hold significant implications for understanding the complex interplay between diet, microbiota, and behavior. Understanding the bidirectional relationship between feeding behavior and microbiota may provide novel insights into the mechanisms underlying eating disorders and metabolic dysregulation.

microbiology↗

bioRxiv2026

Dual regulation of the receptor-like kinase BIR1 involves site-directed transcript cleavage and 5-leader-mediated translational control

In Arabidopsis, receptor-like kinase BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE 1 (BAK1)-INTERACTING RECEPTOR-LIKE KINASE 1 (BIR1) is a negative regulator of plant immunity and cell death. BIR1 was earlier described as a target of epigenetic and post-transcriptional silencing. Degradome analysis mapped predominant mRNA cleavage sites at the 5-untranslated leader region (site A) and the protein-coding sequence (sites B and C). Here, we identified another cleavage site (D) within the BIR1 coding region and investigated the contribution of site-directed mRNA cleavage to BIR1 regulation. Mutations at B, C, and D sites enhanced mRNA stability by impairing transcript cleavage, resulting in increased BIR1 mRNA and protein accumulation. This regulation is disrupted in RNA silencing mutants, supporting a model of cis-directed small interfering RNA (siRNA)-mediated degradation. Cleavage events are highly localized, occurring at a limited number of sites that involve only a few siRNAs. Furthermore, our data reveal a repressive role for the 5-leader in regulating BIR1 translation, potentially mediated by upstream open reading frames (uORFs) and a long non-coding RNA (lncRNA) derived from the natural antisense At4g39838 locus. This lncRNA is complementary to a TCA-rich repeat region encompassing cleavage site A, which may serve as a putative binding site. Together, these findings reveal a multilayered regulatory mechanism that integrates sRNA-mediated cleavage with translational control, with broader implications for the fine-tuning of stress-responsive gene expression during infection.

plant biology↗

bioRxiv2025

Experience from an HTS campaign: Investigation of susceptibility and rescue of SARS-CoV-2 nsp3 protease assay from metal contamination

Different biochemical assays yield different rates of false positives than others either due to the nature of the enzyme, the technology associated with the assay, or properties of the compounds being screened. Ensuring that the right counter-screens are in place to identify false positives without wasting time and resources on them is of great importance. Herein we describe the results of a high throughput screen (HTS) against non-structural protein 3 (nsp3) protease PLPro, which resulted exclusively in false-positive hits. By triaging hit compounds through purification of metal chelating resin, we identified contamination by either copper or palladium as the most likely source of false positives from the library screening campaign. We then performed a systematic assessment of the vulnerability of nsp3 protease screening to metal contamination and evaluated common additives to combat the inhibitory effects of different metal salts. We further conducted a thorough survey of the literature reports of nsp3 HTS campaigns with a focus on the presence of additives and what metal susceptibility was likely, given the results of our work. We conclude that the majority of reported nsp3 screens are susceptible to copper contamination with a smaller proportion also potentially susceptible to palladium contamination.

biochemistry↗

bioRxiv2025

Crystallographic characterisation and development of bi-substrate inhibitors of coronavirus nsp14 methyltransferase

SARS-CoV-2 non-structural protein 14 (nsp14) is essential for viral mRNA cap guanine-N7 methylation and represents a promising but underexplored antiviral target. Herein we describe a structure-guided campaign based on a hit from a focussed SAM mimetic library. Systematic SAR exploration guided by six X-ray co-crystal structures in complex with SARS-CoV-2 led to compound 26, a bi-substrate inhibitor that bridges the SAM and RNA cap binding sites. Compound 26 achieved nanomolar potency against nsp14 from SARS-CoV-2 (IC50 = 53 nM), SARS-CoV-1, and two alphacoronaviruses, with excellent selectivity over human RNMT and flaviviral MTase. In general, the compounds demonstrated favourable metabolic stability, passive permeability, and no HepG2 cytotoxicity. However, cellular antiviral activity was limited, revealing disconnects between enzyme inhibition and phenotypic response. These findings provide a structural framework for optimizing bi-substrate methyltransferase inhibitors against coronaviruses with a view for pan-coronaviral activity.

biochemistry↗

bioRxiv2025

Rewilding beyond the wilderness: Beavers can restore stream biodiversity from urban to agricultural to natural landscapes

1 Beavers have been promoted as a cost-effective nature-based solution to restore degraded stream ecosystems and enhance overall biodiversity. However, the extent to which beaver-engineering boosts biodiversity in human-impacted landscapes remains poorly understood. 2 We assessed the responses of aquatic and terrestrial taxa (amphibians, dragonflies, fish, macrophytes, plankton, terrestrial vegetation, bats, aquatic, flying and terrestrial invertebrates) to beaver-engineering along a gradient of human land-use intensity in Switzerland. In 16 streams, we measured species richness, abundance, unique species richness, evenness, beta and gamma diversity in a beaver-engineered site with beaver dams and a control site without beaver activity. 3 Especially aquatic and semi-aquatic species profited from beaver-engineering. In response to beaver-engineering, amphibians, dragonflies, macrophytes, and plankton increased in species richness, abundance and unique species richness, while fish and terrestrial plants increased in species richness and unique species richness. Bats only increased in unique species richness, while for the other terrestrial taxa, we found no difference between beaver-engineered and control sites. Community evenness remained largely unaffected across all groups. 4 Overall, beaver-engineering increased biodiversity across a land-use gradient. For macrophytes and amphibians (species richness, unique species richness and abundance), as well for dragonflies (species richness and unique species richness), increases were smaller under higher land-use intensity. For the remaining taxa and metrics, we found no relationship between land-use intensity and responses to beaver engineering. 5 However, for beta-diversity, we found that sites with a high land-use intensity had a lower component of nestedness and stronger community turnover in comparison to more natural sites, with a significant effect for macrophytes. Gamma species richness was generally unaffected by land- use intensity, except for amphibians, which were often only found in beaver-engineered sites in high- land-use intensity areas. 6 Synthesis and applications. Our findings demonstrate that beaver-engineering significantly enhances local biodiversity across aquatic and terrestrial ecosystems, even at sites with high land-use intensity. Hence, beavers can effectively restore stream biodiversity from urban to agricultural to natural landscapes. Integrating beaver-engineering into river restoration strategies can substantially advance biodiversity goals under frameworks such as the EU Water Framework Directive and national conservation policies.

ecology↗

bioRxiv2025

Improving Recombinant Antibody Production Using FcBAR: An In Situ Approach to Detect and Amplify Protein-Protein Interactions

Recombinant proteins, in particular monoclonal antibodies and related molecules, have become dominant therapeutics. As they are produced in mammalian cells, they require the concerted function of hundreds of host cell proteins in the protein secretion pathway. However, the comprehensive set of host cell machinery involved remains unclear. Thus, it is often unknown why some recombinant proteins fail to express well. Here we present and deploy an approach called Fc-targeting Biotinylation by Antibody Recognition (FcBAR), which allows for the in situ detection of protein-protein interactions for any recombinant protein with Fc domain. Briefly, cells are permeabilized and incubated with an anti-Fc antibody, conjugated with horseradish peroxidase. All proteins interacting with Fc-bearing proteins are then biotinylated, pulled down and identified via mass spectrometry. We applied this method on a panel of rituximab-producing CHO-S clones with a range of productivity levels. Through analysis of FcBAR protein-protein interactions and RNA-Seq, we identified protein interactions positively correlated with rituximab secretion, and tested 7 of these targets. We found overexpression of AGPAT4, EPHX1, and NSDHL significantly increased rituximab production. Thus, FcBAR provides an unbiased approach to measure PPIs supporting recombinant antibody production in situ, and can guide efforts to boost production of biotherapeutics and biosimilars by addressing production bottlenecks.

systems biology↗

bioRxiv2025

Enhanced Production of HCV E1E2 Subunit Vaccine Candidates via Protein-Protein Interaction Identification in Glycoengineered CHO cells

Hepatitis C Virus (HCV) is a bloodborne virus that affects 57 million people globally with infections that can often go unnoticed, and it is the leading cause of chronic liver disease and cancer. Thus, development of an HCV vaccine is a major medical and public health concern. While prior work has developed secreted E1E2 (sE1E2) protein vaccine candidates, efforts to express it recombinantly in Chinese hamster ovary (CHO) cells have resulted in very low titers. To address this challenge, here we employed a multi-omics approach to identify protein interactors that enhance the secretion of sE1E2. By detecting Protein-Protein Interactions (PPIs) using Biotinylation by Antibody Recognition (BAR) and integrating the data with RNA-Seq, we identified proteins within the secretory pathway that interact with sE1E2 and validated their impact by overexpressing the interacting proteins. Among these, CUL4A and YWHAH enhanced sE1E2 secretion in glycoengineered CHO (geCHO) cells. The integration of omics techniques and genetic engineering in this study provides valuable insights into improving protein secretion in CHO cells, paving the way for the development of more affordable and accessible biotherapeutics.

systems biology↗

bioRxiv2025

Heat stress disrupts early development and photosymbiosis in Cassiopea jellyfish

Photosymbioses between Cnidarians and algae are widespread in marine ecosystems. The jellyfish Cassiopea-Symbiodinium symbiosis serves as a valuable model for studying host-symbiont interactions in photosymbiotic organisms. Despite its ecological similarity to coral symbiosis, the effects of rising sea surface temperatures on Cassiopea symbiosis, particularly during early developmental stages, remain unexplored. By exposing Symbiodinium cultures to heat stress and subsequently using these symbionts to colonize jellyfish polyps under ambient and elevated temperature conditions, we study the impact of heat on microbe-stimulating metamorphosis. We observed a significant reduction in chlorophyll concentration in heat-stressed Symbiodinium algae. Polyps colonized with these symbionts exhibited delayed metamorphosis under ambient conditions and failed to undergo metamorphosis under continued heat stress. Additionally, we found abnormal ephyra morphology and increased rates of asexual reproduction under heat stress. Our findings suggest that ocean warming may disrupt critical stages of Cassiopea metamorphosis and development by impairing symbiosis, ultimately threatening their population stability under warming marine environments.

physiology↗

bioRxiv2025

The receptor-like kinases BIR1 and BIR3 modulates antiviral resistance by different mechanisms

The BAK1-INTERACTING RECEPTOR-LIKE KINASE (BIR) proteins, including BIR1, are negative regulators of cell death and defense in Arabidopsis. Although BIR1 is upregulated during viral infections, its role in virus defense remains unclear. Here, we demonstrate a conserved role for the BIR family as modulators of antiviral resistance in Arabidopsis. During tobacco rattle virus (TRV) infection, BIR gene expression is regulated by antagonistic interactions between salicylic acid (SA) and jasmonic acid (JA) signaling pathways. Genetic evidence shows that BIR1, BIR2, and BIR3 negatively regulate TRV resistance via distinct mechanisms. The antiviral phenotype in bir1-1 mutants is unaffected by mutations in the receptor-like kinases (RLK) BAK1, SOBIR1, or in the lipase-like PAD4. While BIR1 induction partially suppresses PTI marker gene expression, it does not affect early PTI responses such as reactive oxygen species (ROS) production or ethylene accumulation. BIR1 also inhibits plasmodesmata (PD) callose deposition and facilitates virus spread. Our data suggest that BIR1 modulates antiviral defense through mechanisms that include PTI gene expression, PD callose deposition alongside yet unidentified pathways independent of ROS or BAK1, SOBIR1, or PAD4 signaling components. In contrast, BIR3 suppresses an antiviral resistance pathway against TRV that requires BAK1 and the lipases PAD4 and EDS1. Our results suggest that BIR3 controls an NLR-dependent ETI-like response that restricts virus proliferation without triggering a hypersensitive response. These findings highlight the distinct roles of BIR family members in regulating antiviral defense in Arabidopsis.

plant biology↗

bioRxiv2025

Identification of a novel false-positive mechanism in RapidFire mass spectrometry and application in drug discovery

False-positives plague High Throughput Screening in general and are costly as they consume resource and time to resolve. Methods that can rapidly identify such compounds at the initial screen are therefore of great value. Advances in mass spectrometry have led to the ability to screen inhibitors in drug discovery applications by direct detection of an enzyme reaction product. The technique is free from some of the artefacts that trouble classical assays such as fluorescence interference. Its direct nature negates the need for coupling enzymes and hence is simpler with fewer opportunities for artefacts. Despite its myriad advantages, we report here a mechanism for false-positive hits which has not been reported in the literature. Further we have developed a pipeline for detecting these false-positive hits and suggest a method to mitigate against them. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=156 HEIGHT=200 SRC="FIGDIR/small/634670v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@a99654org.highwire.dtl.DTLVardef@1cc7ec8org.highwire.dtl.DTLVardef@978abaorg.highwire.dtl.DTLVardef@114c0d1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

iPSC-derived hepatocytes accurately recapitulate population diversity in alpha-1-antitrypsin deficiency and offer a novel in vitro model for large-scale drug efficacy screening studies.

BackgroundAlpha-1 antitrypsin deficiency (A1ATD) is a hereditary recessive disorder caused by mutations in the SERPINA1 gene. It is a clinically under-recognised disease characterised by low circulating A1AT levels and intracellular accumulation of misfolded A1AT in hepatocytes. Deposition of excessive abnormal A1AT in the liver leads to liver failure, yet no specific treatments are available due to the lack of physiologically relevant disease modelling platforms. MethodsWe have hypothesised that human induced pluripotent stem cell (iPSC)-derived hepatocytes can provide an efficient platform to study A1ATD. Using CRISPR/Cas9, we have generated wild-type and A1ATD iPSC-derived hepatocytes (Opti-HEP) from healthy and A1ATD donors and developed a bioassay that mimics the accumulation of misfolded A1AT in the liver. Responses to the reference drug carbamazepine (CBZ), known to reduce intracellular misfolded A1AT levels, and RNA-based therapeutics were subsequently investigated. ResultsAll lines successfully differentiated into hepatocytes as measured by comparable key hepatic and disease markers to those seen in primary human hepatocytes. The diseased lines displayed increased intracellular accumulation of misfolded A1AT compared to isogenic controls. Diseased cell lines showed significant decreases in intracellular accumulation of polymeric A1AT following transfection with RNA-based therapeutics, but a differential response upon treatment with CBZ. ConclusionWe have developed a specific and robust in vitro model of A1ATD that recapitulates disease pathophysiology and responds to small molecule-based treatments and advanced therapeutic strategies. These data demonstrate the suitability of this model for large-scale efficacy screening studies for the treatment of A1ATD and help pave the way towards the development of novel therapies.

cell biology↗

bioRxiv2024

Development of optimised human iPSC-derived hepatocytes with improved liver function for in vitro metabolic disease modelling and toxicity studies

Background & AimsLiver disease is a rising cause of mortality worldwide. Primary human hepatocytes (PHH) and hepatocellular cancer cells are currently used in drug development, however, they come with limitations, including limited supply, rapid loss of function, and tumorigenic origin. In addition, current iPSC differentiation protocols lead to the generation of hepatocyte-like cells with compromised liver-related features. We hypothesised that optimisation of iPSC differentiation protocols can lead to the generation of hepatocyte-like cells with improved metabolic functionality for disease modelling and toxicity screening studies. MethodsHealthy human iPSCs were differentiated to hepatocyte-like cells (Opti-HEP) using a novel 3-step differentiation protocol. Hepatocyte functionality was assessed, including liver maturity marker expression, urea synthesis, de novo gluconeogenesis, and CYP450 expression, activity, and induction. Suitability of Opti-HEP to predict drug-induced liver injury (DILI) was evaluated by cell viability assays. CRISPR/Cas9 gene editing was employed to generate in vitro inherited metabolic disease models. ResultsOpti-HEP expressed similar liver maturity marker levels to those seen in primary human hepatocytes (PHH), in addition to functional urea and gluconeogenesis pathways. CYP450 expression and activity were comparable between Opti-HEP and PHH, with both cell types showing similar levels of CYP3A4 induction upon 1,25-hydroxy-vitamin D3 treatment. Opti-HEP accurately predicted DILI, following treatment with 7 drugs of known DILI liability. CRISPR-derived Opti-HEP harbouring mutations for inherited metabolic disorders (Ornithine Transcarbamylase Deficiency, Progressive Familial Intrahepatic Cholestasis Type 2, Citrullinemia Type 1) recapitulated key pathophysiological disease features, including reduced protein expression, impaired urea secretion, and bile acid transport. ConclusionsWe demonstrate the generation of optimised iPSC-derived hepatocytes with enhanced liver functionality that is comparable to PHH. These data alongside the expansion capacity and amenability of these cells highlight the opportunities this model can offer in the space of disease modelling and large-scale drug efficacy and hepatotoxicity screening.

cell biology↗

bioRxiv2024

Metastatic organotropism in small cell lung cancer

Metastasis is the leading cause of cancer-related deaths, yet its regulatory mechanisms are not fully understood. Small-cell lung cancer (SCLC) is the most metastatic form of lung cancer, with most patients presenting with widespread disease, making it an ideal model for studying metastasis. However, the lack of suitable preclinical models has limited such studies. We utilized rapid autopsy-derived tumors to develop xenograft models that mimic key features of SCLC, including histopathology, rapid and widespread development of metastasis to the liver, brain, adrenal, bone marrow, and kidneys within weeks, and response to chemotherapy. By integrating in vivo lineage selection with comprehensive bulk and single cell multiomic profiling of transcriptomes and chromatin accessibility, we identified critical cellular programs driving metastatic organotropism to the liver and brain, the most common sites of SCLC metastasis. Our findings reveal the key role of nuclear-cytoskeletal interactions in SCLC liver metastasis. Specifically, the loss of the nuclear envelope protein lamin A/C, encoded by the LMNA gene, increased nuclear deformability and significantly increased the incidence of liver metastasis. Human liver metastases exhibited reduced LMNA expression compared to other metastatic sites, correlating with poorer patient outcomes and increased mortality. This study introduces novel preclinical models for SCLC metastasis and highlights pathways critical for organ-specific metastasis, offering new avenues for the development of targeted therapies to prevent or treat metastatic disease.

cancer biology↗