Biology subjects

Zilbauer, M.

Publications and source records attributed to Zilbauer, M..

12 recordsLinked to original sources

bioRxiv2025

Flexibility and modulation of translation initiation in enterovirus genomes

Enteroviruses comprise a large group of mammalian pathogens that often utilize two open reading frames (ORFs) to encode their proteins: the upstream protein (UP) and the main polyprotein. In some enteroviruses, in addition to the canonical upstream AUG (uAUG), there is another AUG that may represent an alternative upstream initiation site. An analysis of enterovirus sequences containing additional upstream AUGs identified several clusters, including strains of pathogenic Enterovirus alphacoxsackie and E. coxsackiepol. Using ribosome profiling on coxsackievirus CVA-13 (E. coxsackiepol), we demonstrate that both upstream AUG codons can be used for translation initiation in infected cells. Moreover, we confirm translation from both upstream AUGs using a reporter system. Mutating the additional upstream AUG in the context of CVA-13 did not result in phenotypic changes in immortalized cell lines. However, the wild-type virus outcompeted this mutant in human intestinal organoids and differentiated neuronal systems, representing an advantage in physiologically relevant infection sites. Mutation of the stop codon of the shorter upstream ORF led to dysregulated translation of the other ORFs in the reporter system, suggesting a potential role for the additional uORF in modulating the expression level of the other ORFs. These findings demonstrate the remarkable plasticity of enterovirus IRES-mediated initiation and the competitive advantage of double-upstream-AUG-containing viruses in terminally differentiated intestinal organoids and neuronal systems.

microbiology↗

bioRxiv2025

Human Milk Oligosaccharides Modulate Inflammatory Responses and Lipid Metabolism in a Human Intestinal Organoid Model

Human milk oligosaccharides (HMOs) are a major component of human breast milk and have significant protective effects on infant gut health. HMOs are also present in the amniotic fluid surrounding the developing fetus in the womb. Beginning at 10 weeks of gestational age, HMOs enter the fetal gut through the swallowing of amniotic fluid. The effects of this earliest exposure to HMOs on the gut epithelium, which pre-term infants partially miss, remains unknown. In this study, we investigate the effect of a blend of HMOs, including 2-fucosyllactose (2FL), 3-sialyllactose (3SL), and difucosyllactose (DFL), on intestinal epithelial cells, examining their role in steady-state conditions and during inflammation. By utilizing advanced intestinal epithelial organoid models derived from fetal and pediatric donors, we reveal developmental-stage-specific responses to this HMO blend, as well as its potential to mitigate inflammatory damage. These findings offer important insights into the role of HMOs in early-life nutrition and gut health, lending compelling evidence to support their inclusion in infant nutrition strategies.

cell biology↗

bioRxiv2024

Reconstructing bat antiviral immunity using epithelial organoids

Bats are remarkably resilient to viruses with pandemic potential. To resolve largely unknown molecular mechanisms governing their exceptional antiviral immunity, we established an organoid platform to model the entire respiratory airway and intestinal epithelium of the important viral reservoir species Rousettus aegyptiacus (Egyptian fruit bat). These bat organoids exhibit an unexpected diversity of cell types and support replication of highly pathogenic zoonotic viruses including Marburg virus (MARV) and MERS-Coronavirus. Following virus infection, bat organoids unleash a strong interferon response, uniquely regulated through virus-dependent and virus-independent mechanisms. By contrast, MARV infected human organoids fail to induce an antiviral gene response and express pro-inflammatory cytokines after interferon stimulation, revealing important molecular differences between bats and humans with implications for lethal Marburg virus infections in primates. These data provide the most comprehensive organoid platform in bats to decode species-specific differences and uncover fundamental principles of bat disease resilience to emerging viruses with pandemic potential.

immunology↗

bioRxiv2024

PTEN and ARID1A haploinsufficiency equip colonic epithelium for oncogenic transformation

Normal aged tissues are thought to exist as a patchwork of mutant clones. However, the relevance of driver mutations in normal tissue in terms of cancer initiation has not been well described. Here, we sought a quantitative understanding of how different cancer drivers achieve an age-related mutational footprint in the human colonic epithelium and to relate the clonal behaviours they generate to cancer risk. Metanalysis of contemporary multiregional sampling studies of colorectal tumours revealed many of the weak or moderate cancer drivers are trunk mutations present in the last common ancestor from which cancers arise. To study the processes by which such driver mutations could contribute to cancer predisposition, immunohistochemistry was used to detect PTEN, SMAD4 and ARID1A deficient clones in normal colon FFPE surgical resection samples (N=182 patients). Age-related changes in clone size and frequency identified positive biases in clone dynamics that acted to increase the mutational footprint for ARID1A and PTEN but not SMAD4. In vitro engineered monoallelic loss of PTEN and ARID1A implicated specific altered downstream pathways and acquired pro-oncogenic cellular fates corresponding to haploinsufficiency for these genes. In situ analysis confirmed enhanced proliferation in both PTEN and ARID1A deficient clones and creation of an immune exclusive microenvironment associated with ARID1A deficiency. The behaviours resulting from haploinsufficiency of PTEN and ARID1A exemplify how priming of the tissue through somatic mosaicism could contribute alternative combinations of genetic events leading to transformation.

cancer biology↗

bioRxiv2023

Efficient genetic editing of human intestinal organoids using ribonucleoprotein-based CRISPR

Organoids are currently one of the most widely used ex vivo models in epithelial biology. Combined with genetic editing strategies, organoids offer a promise of rapid and efficient investigation of gene function in many models of human disease. However, to date, the editing efficiency of organoids with the use of non-viral electroporation methods has been only up to 30%, with implications for the subsequent need for selection including including turnaround time and exhaustion or adaptation of the organoid population. Here, we describe an efficient method of intestinal organoid editing using a Ribonucleoprotein CRISPR-based approach. Editing efficiencies of up to 98% in target genes were robustly achieved across different anatomical gut locations and developmental timepoints from multiple patient samples with no off-target editing. The method allowed us to study the effect of the loss of the tumour suppressor gene, PTEN, in normal human intestinal cells. Analysis of PTEN deficient organoids defined phenotypes that likely relate to its tumour suppressive function in vivo, such as a proliferative advantage and increased organoid budding. Transcriptional profiling revealed differential expression of genes in pathways commonly known to be associated with PTEN loss including mTORC1 activation.

cell biology↗

bioRxiv2022

Culture Associated DNA Methylation Changes Impact on Cellular Function of Human Intestinal Organoids

Background & AimsHuman intestinal epithelial organoids (IEO) are a powerful tool to model major aspects of intestinal development, health and diseases, as patient derived cultures retain many features found in-vivo. A necessary aspect of the organoid model is the requirement to expand cultures in-vitro through several rounds of passaging. This is of concern, as the passaging of cells has been shown to affect cell morphology, ploidy, and function. In this study, we address concerns around long term passaging of IEO to better characterise and define effects on cell morphology and function. MethodsHere we have analysed 173 human IEO from two sampling sites, terminal ileum and sigmoid colon and examined the effect of culture duration on DNA methylation (DNAm), gene expression and cellular function including their response to proinflammatory cytokines and in-vitro cell differentiation. ResultsOur analyses revealed a major effect of culture duration on DNAm, leading to significant changes at 61,337 loci representing approximately 8% of all CpGs tested. Although global cellular functions such as gut segment-specific gene expression remained stable, a subset of methylation changes correlated with altered gene expression at baseline as well as in response to inflammatory cytokine exposure and in-vitro differentiation. Importantly, epigenetic changes were found to be enriched in genomic regions associated with colonic cancer and distant to the site of replication indicating similarities to malignant transformation. ConclusionsOur study reveals culture-associated epigenetic, transcriptomic and functional changes in human mucosa derived IEO and highlights the importance of considering passage number as a potentially confounding factor. SynopsisThis work describes cell culture induced changes to DNA methylation, gene expression and cellular function in human IEO. Globally organoids lost DNA methylation with time in culture while DNA methylation also became generally more variable. This work suggests a shifted epigenetic profile in organoids cultured long-term.

genomics↗

bioRxiv2022

Single-cell atlas of human liver development reveals pathways directing hepatic cell fates

The liver has been studied extensively due to the broad number of diseases affecting its vital functions. However, therapeutic advances, especially in regenerative medicine, are currently hampered by the lack of knowledge concerning human hepatic cell development. Here, we addressed this limitation by describing the developmental trajectories of different cell types comprising the human fetal liver at single-cell resolution. These transcriptomic analyses revealed that sequential cell-to-cell interactions direct functional maturation of hepatocytes, with non-parenchymal cells playing critical, supportive roles during organogenesis. We utilised this information to derive bipotential hepatoblast organoids and then exploited this novel model system to validate the importance of key signalling pathways and developmental cues. Furthermore, these insights into hepatic maturation enabled the identification of stage-specific transcription factors to improve the functionality of hepatocyte-like cells generated from human pluripotent stem cells. Thus, our study establishes a new platform to investigate the basic mechanisms of human liver development and to produce cell types for clinical applications.

developmental biology↗

bioRxiv2021

Cells of the human intestinal tract mapped across space and time

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. To comprehensively map cell lineages in the healthy developing, pediatric and adult human gut from ten distinct anatomical regions, as well as draining lymph nodes, we used singlecell RNA-seq and VDJ analysis of roughly one third of a million cells. This reveals the presence of BEST4+ absorptive cells throughout the human intestinal tract, demonstrating the existence of this cell type beyond the colon for the first time. Furthermore, we implicate IgG sensing as a novel function of intestinal tuft cells, and link these cells to the pathogenesis of inflammatory bowel disease. We define novel glial and neuronal cell populations in the developing enteric nervous system, and predict cell-type specific expression of Hirschsprungs disease-associated genes. Finally, using a systems approach, we identify key cell players across multiple cell lineages driving secondary lymphoid tissue formation in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. These data provide an unprecedented catalogue of intestinal cells, and new insights into cellular programs in development, homeostasis and disease.

cell biology↗

bioRxiv2020

CD8+ T-cell transcription and DNA methylation show age specific differences and lack correlation with clinical outcome in pediatric Inflammatory Bowel Disease

Background & AimsCD8+ T-cell gene expression has been implicated in the pathogenesis of Inflammatory Bowel Diseases (IBD) and has been shown to correlate with disease outcome in adult patients. Moreover, CD8+ T-cell exhaustion was identified as a contributing mechanism that impacts on disease behaviour. We aimed to explore CD8+ T-cell gene expression and DNA methylation in children newly diagnosed with IBD and test their correlation with disease outcome. MethodsWe prospectively recruited 112 children with IBD at the point of diagnosis and 19 non-IBD controls. Follow-up samples were obtained from a subset of patients at 3-month intervals (n=62). CD8+ T-cells were purified from peripheral blood samples using magnetic bead sorting and genome-wide transcriptional (n=192) and DNA methylation (n=66) profiles were generated using Affymetrix and Illumina arrays respectively. Publicly available adult CD8+ T-cell transcriptomes and DNA methylomes were included in data analyses to investigate age dependant differences. ResultsVariation amongst CD8+ T-cell transcriptomes obtained from children showed association with disease, systemic inflammation, age and gender but lacked correlation with disease outcome in pediatric IBD. In contrast to CD8+ T-cell transcriptomes in adult Crohns Disease (CD), samples from pediatric patients did not show variation within genes forming part of the previously reported prognostic expression or T-cell exhaustion signatures. Pediatric CD patient derived DNA methylation profiles also lacked correlation with disease outcome but in comparison to adult CD8+ methylomes showed a higher predicted proportion of CD8+ naive T-cells. ConclusionsOur findings indicate age-related differences in IBD pathogenesis and highlight the importance of validating adult clinical biomarkers in pediatric cohorts.

immunology↗

bioRxiv2020

Single-cell sequencing of developing human gut reveals transcriptional links to childhood Crohns disease

Human gut development requires the orchestrated interaction of various differentiating cell types. Here we generate an in-depth single-cell map of the developing human intestine at 6-10 weeks post-conception, a period marked by crypt-villus formation. Our analysis reveals the transcriptional profile of cycling epithelial precursor cells, which are distinct from LGR5-expressing cells. We use computational analyses to show that these cells contribute to differentiated cell subsets directly and indirectly via the generation of LGR5-expressing stem cells and receive signals from the surrounding mesenchymal cells. Furthermore, we draw parallels between the transcriptomes of ex vivo tissues and in vitro fetal organoids, revealing the maturation of organoid cultures in a dish. Lastly, we compare scRNAseq profiles from paediatric Crohns disease epithelium alongside matched healthy controls to reveal disease associated changes in epithelial composition. Contrasting these with the fetal profiles reveals re-activation of fetal transcription factors in Crohns disease epithelium. Our study provides a unique resource, available at www.gutcellatlas.org, and underscores the importance of unravelling fetal development in understanding disease.

cell biology↗

bioRxiv2019

Norovirus replication in human intestinal epithelial cells is restricted by the interferon-induced JAK/STAT signalling pathway and RNA Polymerase II mediated transcriptional responses.

Human noroviruses (HuNoV) are a leading cause of viral gastroenteritis worldwide and a significant cause of morbidity and mortality in all age groups. The recent finding that HuNoV can be propagated in B cells and mucosa derived intestinal epithelial organoids (IEOs), has transformed our capability to dissect the life cycle of noroviruses. Using RNA-Seq of HuNoV infected intestinal epithelial cells (IECs), we have found that replication of HuNoV in IECs results in interferon-induced transcriptional responses and that HuNoV replication in IECs is sensitive to IFN. This contrasts with previous studies that suggest that the innate immune response may play no role in the restriction of HuNoV replication in immortalised cells. We demonstrate that the inhibition of JAK1/JAK2 enhances HuNoV replication in IECs. Surprisingly, targeted inhibition of cellular RNA polymerase II-mediated transcription was not detrimental to HuNoV replication, but enhanced replication to a greater degree compared to blocking of JAK signalling directly. Furthermore, we demonstrate for the first time that IECs generated from genetically modified intestinal organoids, engineered to be deficient in the interferon response, are more permissive to HuNoV infection. Together our work identifies the IFN-induced transcriptional responses restrict HuNoV replication in IECs and demonstrates that the inhibition of these responses by modifications to the culture conditions can greatly enhance the robustness of the norovirus culture system.\n\nImportanceNoroviruses are a major cause of gastroenteritis worldwide yet the challenges associated with their growth culture has greatly hampered the development of therapeutic approaches and has limited our understanding of cellular pathways that control infection. Here we show that human intestinal epithelial cells, the first point of entry of human noroviruses into the host, limit virus replication by the induction of the innate responses. Furthermore we show that modulating the ability of intestinal epithelial cells to induce transcriptional responses to HuNoV infection can significantly enhance human norovirus replication in culture. Collectively our findings provide new insights into the biological pathways that control norovirus infection but also identify mechanisms to enhance the robustness of norovirus culture.

microbiology↗

bioRxiv2018

The landscape of somatic mutation in normal colorectal epithelial cells

The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions leading to cancer. As for most cancer types, however, understanding of the earliest phases of colorectal neoplastic change, which may occur in morphologically normal tissue, is comparatively limited because of the difficulty of detecting somatic mutations in normal cells. Each colorectal crypt is a small clone of cells derived from a single recently-existing stem cell. Here, we whole genome sequenced hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed, some ubiquitous and continuous, others only found in some individuals, in some crypts or during some phases of the cell lineage from zygote to adult cell. Likely driver mutations were present in [~]1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium.

cancer biology↗