Biology subjects

Wolvetang, E.

Publications and source records attributed to Wolvetang, E..

6 recordsLinked to original sources

bioRxiv2026

Single-cell analysis of West Nile virus infection in brain organoids identified a serine synthesis enzyme as a determinant of viral neural tropism and a druggable antiviral target

The cellular and molecular determinants of human neuroinvasive West Nile virus (WNV) disease are currently poorly defined. Previous studies of WNV neural infection relied on rodent models and immortalised cell lines that diverge from human brain tissue. Herein, we established a reproducible human cerebral organoid model of WNV infection and profiled it by virus-inclusive single-cell RNA sequencing (scRNA-Seq) to obtain an understanding of WNV-host interactions in the context of human neural tissue. We found that WNV showed preferential tropism to progenitor cells, immature neurons, and astrocytes, while the mature neurons were largely resistant to infection. Astrocytes were identified as the principal source of interferon (IFN) and the main hub of IFN-signalling, whereas mature neurons expressed little viral RNA-sensing machinery or STAT1 and responded weakly to infection and to IFN. The differential gene expression analysis between WNV-permissive and nonpermissive cells demonstrated that virus-susceptible cells had high baseline expression of the serine synthesis and one-carbon metabolism genes PHGDH, SHMT2, and MTHFD2, which were not induced by infection. RNA-interference and pharmacological inhibition of phosphoglycerate dehydrogenase (PHGDH) restricted WNV replication, confirming the requirement of this gene for WNV infection. In addition, the FDA-approved drug disulfiram, known to inhibit PHGDH, also reduced viral replication in neural progenitors and cerebral organoids. Overall, these results provide the first comprehensive single-cell-level characterisation of WNV neural infection in human neural tissue. They identify de novo serine synthesis as a metabolic dependency of WNV in human neural cells that can be targeted as a host-directed antiviral therapy.

microbiology↗

bioRxiv2026

Liver organoid-mediated cyclophosphamide neurotoxicity in CNS organoids in a multi-organ microphysiological system

Cyclophosphamide (CP) is a widely used alkylating agent whose cytotoxic activity depends on hepatic CYP450-mediated bioactivation. While CP-associated neurotoxicity and cognitive impairment are recognized clinically, the mechanisms of secondary organ damage through metabolic cross-talk remain poorly understood due to limitations of conventional monoculture models. Here we employ a multi-organ microphysiological system (MPS) connecting stem cell derived liver and CNS organoids via microfluidic channels to model inter-organ drug metabolism and secondary toxicity. Liver organoids were treated with CP (0-200 {micro}M) for 48 hours, and connected CNS organoids were assessed for secondary damage by confocal Z-stack imaging of DNA damage ({gamma}H2AX), neuronal identity (NeuN), and nuclear content (DAPI). We observe dose-dependent reduction in NeuN expression and {gamma}H2AX signal in connected CNS organoids, consistent with neurotoxic metabolite transfer from liver. Critically, CNS-to-CNS control connections show no comparable damage at equivalent CP concentrations, confirming that hepatic metabolism is required for CNS toxicity. These findings validate the MPS platform for modelling multi-organ drug toxicity and provide direct evidence that liver-derived CP metabolites drive secondary neurotoxicity through inter-organ metabolic communication.

bioengineering↗

bioRxiv2025

Computational reconstruction of Tyrannosaurus rex collagen for biomanufacturing of paleo-inspired biomaterials with tuneable stiffness

Ancient protein fragments preserved in fossilized tissues offer evolutionary sequence information inaccessible through conventional genomics. Here we demonstrate computational reconstruction of full-length Tyrannosaurus rex type I collagen (COL1A1) by integrating paleo-proteomics data with protein language models. Using TOC large protein model with pattern-constrained generation to maintain collagens Gly-X-Y triplet architecture, we generated five computationally designed sequences incorporating validated T. rex peptide fragments. These generated variants maintain triple helix structural features (23.0% Gly-X-Y content) and exhibit enhanced lysine content (up to 84 residues vs. 57 in wild-type chicken) to increase enzymatic crosslinking capacity. To enable spatial and temporal control of material properties such as stiffness, we engineered a doxycycline-inducible lysyl oxidase-4 (LOX4) expression system in TOC t-rex cell line that achieved 9-fold enzyme induction. This biomimetic approach, inspired by functionally graded fish scale architecture, decouples constitutive collagen production from inducible crosslinking, enabling biomanufacturing of materials with tuneable mechanical gradients. We validate stable transgene integration, confirm tight transcriptional control, and demonstrate system compatibility with scalable cell culture. This work establishes a platform combining paleo-proteomics, protein language modelling, and bioinspired engineering to produce next-generation biomaterials from computationally reconstructed extinct proteins.

bioengineering↗

bioRxiv2024

SARS-CoV-2 infection in hiPSC-derived neurons is cathepsin-dependent and causes accumulation of HIF1alpha and phosphorylated tau

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been shown to infect the human brain and a subset of human neurons in vitro. We have previously demonstrated that the virus enters the human induced pluripotent stem cell (hiPSC)-derived neurons via an endosomal-lysosomal pathway, which is dependent on low levels of angiotensin-converting enzyme 2 (ACE2) and independent of transmembrane serine protease 2 (TMPRSS2). Here, we use hiPSC-derived neurons overexpressing ACE2 in co-culture with human astrocytes to show that the infection with both SARS-CoV-2 Wuhan and Omicron XBB.1.5 variants is dependent on cathepsins and can be efficiently blocked by an inhibitor of cathepsin B (CA-074-ME). The result was reproducible in non-transgenic hiPSC-derived cortical organoids. The cathepsin L inhibitor SB412515 was less effective against the Wuhan strain but equally effective against the Omicron variant. Using PCR and reinfection assays, we show that SARS-CoV-2 can replicate in neurons in 2D co-cultures. Interestingly, the infectivity of the newly produced virions declined at 24 hours post-infection despite a further increase in released viral RNA at later time points, suggesting the possible activation of an antiviral response in neurons and/or astrocytes, which is supported by a correspondent increase in the levels of secreted cytokines. Furthermore, the number of infected neurons decreased within five days, suggesting that SARS-CoV-2 infection eventually leads to the death of the target neuronal cell in vitro. The infection also caused the accumulation of the hypoxia-inducible stress factor HIF1- in infected neurons under normoxia. Finally, we confirm and expand the previous finding that in SARS-CoV-2 infected neurons, the microtubule-associated protein tau is hyperphosphorylated at multiple loci, including S202/T205, and mislocalized to the soma of the infected neurons. Hyperphosphorylation and mislocalization of tau are hallmarks of Alzheimers disease (AD) and other tauopathies. Our data provides further evidence supporting the neurodegenerative potential of SARS-CoV-2 infection. SummaryThe recent COVID-19 pandemic has raised concerns about the potential for SARS-CoV-2 to infect the brain and worsen brain diseases like Alzheimers disease. Research has shown that SARS-CoV-2 can indeed infect the human brain, including a small number of neurons and other brain cells in laboratory settings. In our previous studies, we identified the endosomal pathway as the route the virus uses to enter neuronal cells. In this study, we build on that work by demonstrating that inhibitors of endo-lysosomal cathepsin proteases can block this neuronal infection. We also found that infectious progeny virions are released from the infected neuronal cells. Importantly, the infection proves harmful to the host cells, as evidenced by a decrease in the number of infected cells in experimental cultures over a five-day period. Additionally, we confirm and expand on earlier findings that SARS-CoV-2 infection leads to the phosphorylation and altered localization of the tau protein, a process associated with brain diseases like Alzheimers. Finally, we observed an increase in the production of inflammatory cytokines following neuronal infection with SARS-CoV-2, along with an accumulation of the stress marker protein HIF-1 in neurons. This protein has been linked to other viral infections and Alzheimers disease. Overall, our data suggest that SARS-CoV-2 exhibits neurodegenerative characteristics.

microbiology↗

bioRxiv2021

Zika virus noncoding RNA cooperates with the viral protein NS5 to inhibit STAT1 phosphorylation and facilitate viral pathogenesis

Zika virus (ZIKV) is a re-emerging pathogenic flavivirus, which causes microcephaly in infants and poses a continuing threat to public health. ZIKV, like all other flaviviruses, produces highly abundant noncoding RNA known as subgenomic flaviviral RNA (sfRNA). Herein we utilized wild-type and mutant ZIKV defective in production of sfRNA to elucidate for the first time how production of sfRNA affects all aspects of ZIKV pathogenesis. We found that in mouse pregnancy model of infection sfRNA is required for trans-placental dissemination of ZIKV and subsequent infection of fetal brain. Using human brain organoids, we showed that sfRNA promotes apoptosis of neural progenitor cells leading to profound cytopathicity and disintegration of organoids. We also found by transcriptome profiling and gene network analysis that in infected human placental cells sfRNA inhibits multiple antiviral pathways and promotes apoptosis with STAT1 identified as a key shared factor linking these two interconnected sfRNA activities. We further showed for the first time that sfRNA inhibits phosphorylation and nuclear translocation of STAT1 by a novel mechanism which involves binding to and stabilizing viral protein NS5. This allows accumulation of NS5 at the levels required for efficient inhibition of STAT1 phosphorylation. Thus, we elucidated the molecular mechanism by which ZIKV sfRNA exerts its functions in vertebrate hosts and discovered a co-operation between viral noncoding RNA and a viral protein as a novel strategy employed by viruses to counteract antiviral responses.

microbiology↗

bioRxiv2017

Transcriptional evaluation of the developmental accuracy, reproducibility and robustness of kidney organoids derived from human pluripotent stem cells

We have previously reported a protocol for the directed differentiation of human induced pluripotent stem cells to kidney organoids comprised of nephrons, proximal and distal epithelium, vasculature and surrounding interstitial elements. The utility of this protocol for applications such as disease modelling will rely implicitly on the developmental accuracy of the model, technical robustness of the protocol and transferability between iPSC lines. Here we report extensive transcriptional analyses of the sources of variation across the timecourse of differentiation from pluripotency to complete kidney organoid, focussing on repeated differentiations to day 18 organoid. Individual organoids generated within the same differentiation experiment show Spearmans correlation coefficients of >0.99. The greatest source of variation was seen between experimental batch, with the enrichment for genes that also varied temporally between day 10 and day 25 organoids implicating nephron maturation as contributing to transcriptional variance between individual differentiation experiments. A morphological analysis revealed a transition from renal vesicle to capillary loop stage nephrons across the same time period. Distinct iPSC clones were also shown to display congruent transcriptional programs with inter-experimental and inter-clonal variation most strongly associated with nephron patterning. Even epithelial cells isolated from organoids showed transcriptional alignment with total organoids of the same day of differentiation. This data provides a framework for managing experimental variation, thereby increasing the utility of this approach for personalised medicine and functional genomics.

bioinformatics↗