Biology subjects

Vuylsteke, M.

Publications and source records attributed to Vuylsteke, M..

7 recordsLinked to original sources

bioRxiv2024

Warm temperature modifies cell fates to reduce stomata production in Arabidopsis

O_LIStomatal abundance decrease in Arabidopsis triggered by warm-temperature is attributed to PIF4-mediated repression of SPEECHLESS (SPCH) expression. We identified the unknown developmental and transcriptional basis of this adaptive response. C_LIO_LIWe traced stomatal lineages in vivo using cell-identity marker lines and mutants, quantified epidermal traits and conducted RNA sequencing under oscillating temperatures. C_LIO_LIProlonged warm-temperature or PIF4-overexpression altered cell-fates, inducing diverted stomatal precursors (DPs) that lacked stomatal fate, accounting for stomata reduction. DPs originated from meristemoids that lost SPCH expression, lacked MUTE expression and exited the cell cycle. Short warm-temperature pulses allowed later recovery of SPCH expression, and did not induce DPs or stomata reduction. Comparison of transcriptomes obtained during warm-temperature pulses with stomatal lineage cell-specific profiles identified gene expression changes and contrasted their reversibility. Though warm-temperature silenced key stomatal drivers, most lineages formed stomata through partly modified transcriptional landscapes that promoted uncommitted cell identities and included alternative pathways. C_LIO_LIExpression changes in stomatal regulators and cell-fate changes explain lineage progression under fluctuating temperatures. Since short-term temperature oscillations prevail in natural conditions, the requirement of long warm-temperature exposure to trigger DPs would prevent stomata reduction by occasional temperature rises. Promoting uncommitted lineage stages provides flexibility to stomatal development under environmental changes. C_LI

plant biology↗

bioRxiv2023

Continual improvement of multiplex mutagenesis in Arabidopsis

CRISPR/Cas9 is currently the most powerful tool to generate mutations in plant genomes and more efficient tools are needed as the scale of experiments increases. In the model plant Arabidopsis, the choice of promoter driving Cas9 expression is critical to generate germline mutations. Several optimal promoters have been reported. However, it is unclear which promoter is ideal as they have not been thoroughly tested side-by-side. Furthermore, most plant vectors still use one of the two Cas9 nuclear localization sequence (NLS) configurations initially reported and can still be optimized. We genotyped more than 6,000 Arabidopsis T2 plants to test seven promoters and eleven NLS architectures across 14 targets to systematically improve the generation of single and multiplex inheritable mutations. We find that the RPS5A promoter and double-BP NLS architecture were individually the most efficient components. When combined, 99% of T2 plant contained at least one knockout mutation and 84% contained 4-7-plex knock-outs. These optimizations will be useful to generate higher-order knockouts in the germline of Arabidopsis and likely be applicable to other CRISPR systems as well.

synthetic biology↗

bioRxiv2023

GDF15 mediates inflammation-associated bone loss through a brain-bone axis

Metabolic mediators play an important role in regulating chronic inflammation in the body. Here we report an unexpected role for GDF15 (Growth Differentiation Factor 15), a central mediator of food intake, in inflammation-associated bone loss. GDF15 serum levels were found to be elevated in arthritis patients and inversely correlated with bone density. Despite being associated with inflammation, we found that GDF15 itself does not cause, nor contribute to, clinical or histopathological arthritis. Rather, under inflammatory conditions, GDF15 mediates trabecular bone loss through its receptor GFRAL, which is exclusively expressed in the hindbrain. GDF15-GFRAL binding results in {beta}-adrenergic activation of MALPs (Marrow Adipocytic Lineage Precursors) in the bone marrow, which stimulate osteoclasts and trigger bone loss. These data suggest a metabolic mediator-controlled brain-bone axis in inflammation, through which bone loss is induced in a contextual rather than general manner. These findings may lead to more specific therapeutic interventions to protect bone.

immunology↗

bioRxiv2023

The antigenic landscape of human influenza N2 neuraminidases from 2009 until 2017

Human H3N2 influenza viruses are subject to rapid antigenic evolution which translates into frequent updates of the composition of seasonal influenza vaccines. Despite these updates, the effectiveness of influenza vaccines against H3N2-associated disease is suboptimal. Seasonal influenza vaccines primarily induce hemagglutinin-specific antibody responses. However, antibodies directed against influenza neuraminidase (NA) also contribute to protection. Here, we analyzed the antigenic diversity of a panel of N2 NAs derived from human H3N2 viruses that circulated between 2009 and 2017. The antigenic breadth of these NAs was determined based on the NA inhibition (NAI) of a broad panel of ferret and mouse immune sera that were raised by infection and recombinant N2 NA immunization. This assessment allowed us to distinguish at least 4 antigenic groups in the N2 NAs derived from human H3N2 viruses that circulated between 2009 and 2017. Computational analysis further revealed that the amino acid residues in N2 NA that have a major impact on susceptibility to NAI by immune sera are in proximity of the catalytic site. Finally, a machine learning method was developed that allowed to accurately predict the impact of mutations that are present in our N2 NA panel on NAI. These findings have important implications for the renewed interest to develop improved influenza vaccines based on the inclusion of a protective NA antigen formulation.

immunology↗

bioRxiv2023

N-terminal proteoforms may engage in different protein complexes

Alternative translation initiation and alternative splicing may give rise to N-terminal proteoforms, proteins that differ at their N-terminus compared to their canonical counterparts. Such proteoforms can have altered localizations, stabilities and functions. While proteoforms generated from splice variants can be engaged in different protein complexes, it remained to be studied to what extent this applies to N-terminal proteoforms. To address this, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. First, we generated a catalogue of N-terminal proteoforms found in the HEK293T cellular cytosol from which 22 pairs were selected for interactome profiling. Additionally, we provide evidence for the expression of several N-terminal proteoforms, identified in our catalogue, across different human tissues as well as tissue-specific expression, highlighting their biological relevance. Protein-protein interaction profiling revealed that the overlap of the interactomes for both proteoforms is generally high, showing their functional relation. We also showed that N-terminal proteoforms can be engaged in new interactions and/or lose several interactions compared to their canonical counterpart, thus further expanding the functional diversity of proteomes.

systems biology↗

bioRxiv2021

Exploration of synergistic action of cell wall-degrading enzymes against Mycobacterium tuberculosis

SynopsisO_ST_ABSBackgroundC_ST_ABSThe major global health threat tuberculosis is caused by Mycobacterium tuberculosis (Mtb). Mtb has a complex cell envelope - a partially covalently linked composite of polysaccharides, peptidoglycan and lipids, including a mycolic acid layer - which conveys pathogenicity but also protects against antibiotics. Given previous successes in treating gram-positive and -negative infections with cell wall degrading enzymes, we investigated such approach for Mtb. Objectives(i) Development of an Mtb microtiter growth inhibition assay that allows undisturbed cell envelope formation, to overcome the invalidation of results by typical clumped Mtb-growth in surfactant-free assays. (ii) Exploring anti-Mtb potency of cell wall layer-degrading enzymes. (iii) Investigation of the concerted action of several such enzymes. MethodsWe inserted a bacterial luciferase-operon in an auxotrophic Mtb strain to develop a microtiter assay that allows proper evaluation of cell wall degrading anti-Mtb enzymes. We assessed growth-inhibition by enzymes (recombinant mycobacteriophage mycolic acid esterase (LysB), fungal -amylase and human and chicken egg white lysozymes) and combinations thereof, in presence or absence of biopharmaceutically acceptable surfactant. ResultsOur biosafety level-2 assay identified both LysB and lysozymes as potent Mtb-inhibitors, but only in presence of surfactant. Moreover, most potent disruption of the mycolic acid hydrophobic barrier was obtained by the highly synergistic combination of LysB, -amylase and polysorbate 80. ConclusionsSynergistically acting cell wall degrading enzymes are potently inhibiting Mtb - which sets the scene for the design of specifically tailored antimycobacterial (fusion) enzymes. Airway delivery of protein therapeutics has already been established and should be studied in animal models for active TB.

microbiology↗

bioRxiv2019

CRISPR-NSID: an in vivo CRISPR/Cas9 negative selection screen reveals EZH2 as a druggable dependency factor in a genetic desmoid tumor model

Cancer precision medicine implies identification of tumor-specific vulnerabilities associated with defined oncogenic pathways. Desmoid tumors are soft-tissue neoplasms strictly driven by Wnt signaling network hyperactivation. Despite this clearly defined genetic etiology and the strict and unique implication of the Wnt/{beta}-catenin pathway, no specific molecular targets for these tumors have been identified. To address this caveat, we developed fast and semi-high throughput genetic Xenopus tropicalis desmoid tumor models to identify and characterize novel drug targets. We used multiplexed CRISPR/Cas9 genome editing in these models to simultaneously target a tumor suppressor gene (apc) and candidate dependency genes. Our methodology CRISPR/Cas9 Selection mediated Identification of Dependencies (CRISPR-SID) uses calculated deviations between experimentally observed gene editing outcomes and deep-learning-predicted double strand break repair patterns, to identify genes under negative selection during tumorigenesis. This revealed EZH2 and SUZ12, both encoding polycomb repressive complex 2 components, and the transcription factor CREB3L1, as genetic dependencies for desmoid tumors. In vivo EZH2 inhibition by Tazemetostat induced partial regression of established autochthonous tumors. In vitro models of patient desmoid tumor cells revealed a direct effect of Tazemetostat on Wnt pathway activity. CRISPR-SID represents a potent novel approach for in vivo mapping of tumor vulnerabilities and drug target identification. Significance StatementCRISPR-SID was established in the diploid frog Xenopus tropicalis for in vivo elucidation of cancer cell vulnerabilities. CRISPR-SID uses deep learning predictions and binomial theory to identify genes under positive or negative selection during autochthonous tumor development. Using CRISPR-SID in a genetic model for desmoid tumors, treatment-recalcitrant mesenchymal tumors driven by hyper-activation of the Wnt signaling pathway, we identified EZH2 and SUZ12, both encoding critical components of the polycomb repressive complex 2, as dependency genes for desmoid. Finally, we demonstrate the promise of EZH2 inhibition as a novel therapeutic strategy for desmoid tumors. With the simplicity of CRISPR sgRNA multiplexing in Xenopus embryos the CRISPR-SID method may be applicable to reveal vulnerabilities in other tumor types.

cancer biology↗