Ethanol exposure increases mutation rate through error-prone polymerases

International audienceEthanol is a ubiquitous environmental stressor that is toxic to all lifeforms. Here, we use the model eukaryote Saccharomyces cerevisiae to show that exposure to sublethal ethanol concentrations causes DNA replication stress and an increased mutation rate. Specifically, we find that ethanol slows down replication and affects localization of Mrc1, a conserved protein that helps stabilize the replisome. In addition, ethanol exposure also results in the recruitment of error-prone DNA polymerases to the replication fork. Interestingly, preventing this recruitment through mutagenesis of the PCNA/Pol30 polymerase clamp or deleting specific error-prone polymerases abolishes the mutagenic effect of ethanol. Taken together, this suggests that the mutagenic effect depends on a complex mechanism, where dysfunctional replication forks lead to recruitment of error-prone polymerases. Apart from providing a general mechanistic framework for the mutagenic effect of ethanol, our findings may also provide a route to better understand and prevent ethanol-associated carcinogenesis in higher eukaryotes

Application of in vivo µCT to identify novel imaging derived biomarkers in a mouse model of silica-induced fibrosis

status: publishe

Using in vivo µCT for longitudinal evaluation of silica-induced pulmonary fibrosis in mouse lungs

status: publishe

Novel µCT-derived biomarkers for longitudinal evaluation of silica-induced pulmonary fibrosis in a mouse model

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From Mouse to Man and Back: Closing the Correlation Gap between Imaging and Histopathology for Lung Diseases

Lung diseases such as fibrosis, asthma, cystic fibrosis, infection and cancer are life-threatening conditions that slowly deteriorate quality of life and for which our diagnostic power is high, but our knowledge on etiology and/or effective treatment options still contains important gaps. In the context of day-to-day practice, clinical and preclinical studies, clinicians and basic researchers team up and continuously strive to increase insights into lung disease progression, diagnostic and treatment options. To unravel disease processes and to test novel therapeutic approaches, investigators typically rely on end-stage procedures such as serum analysis, cyto-/chemokine profiles and selective tissue histology from animal models. These techniques are useful but provide only a snapshot of disease processes that are essentially dynamic in time and space. Technology allowing evaluation of live animals repeatedly is indispensable to gain a better insight into the dynamics of lung disease progression and treatment effects. Computed tomography (CT) is a clinical diagnostic imaging technique that can have enormous benefits in a research context too. Yet, the implementation of imaging techniques in laboratories lags behind. In this review we want to showcase the integrated approaches and novel developments in imaging, lung functional testing and pathological techniques that are used to assess, diagnose, quantify and treat lung disease and that may be employed in research on patients and animals. Imaging approaches result in often novel anatomical and functional biomarkers, resulting in many advantages, such as better insight in disease progression and a reduction in the numbers of animals necessary. We here showcase integrated assessment of lung disease with imaging and histopathological technologies, applied to the example of lung fibrosis. Better integration of clinical and preclinical imaging technologies with pathology will ultimately result in improved clinical translation of (therapy) study results.status: publishe

From Mouse to Man and Back: Closing the Correlation Gap between Imaging and Histopathology for Lung Diseases

Lung diseases such as fibrosis, asthma, cystic fibrosis, infection and cancer are life-threatening conditions that slowly deteriorate quality of life and for which our diagnostic power is high, but our knowledge on etiology and/or effective treatment options still contains important gaps. In the context of day-to-day practice, clinical and preclinical studies, clinicians and basic researchers team up and continuously strive to increase insights into lung disease progression, diagnostic and treatment options. To unravel disease processes and to test novel therapeutic approaches, investigators typically rely on end-stage procedures such as serum analysis, cyto-/chemokine profiles and selective tissue histology from animal models. These techniques are useful but provide only a snapshot of disease processes that are essentially dynamic in time and space. Technology allowing evaluation of live animals repeatedly is indispensable to gain a better insight into the dynamics of lung disease progression and treatment effects. Computed tomography (CT) is a clinical diagnostic imaging technique that can have enormous benefits in a research context too. Yet, the implementation of imaging techniques in laboratories lags behind. In this review we want to showcase the integrated approaches and novel developments in imaging, lung functional testing and pathological techniques that are used to assess, diagnose, quantify and treat lung disease and that may be employed in research on patients and animals. Imaging approaches result in often novel anatomical and functional biomarkers, resulting in many advantages, such as better insight in disease progression and a reduction in the numbers of animals necessary. We here showcase integrated assessment of lung disease with imaging and histopathological technologies, applied to the example of lung fibrosis. Better integration of clinical and preclinical imaging technologies with pathology will ultimately result in improved clinical translation of (therapy) study results

Radiosafe micro-computed tomography for longitudinal evaluation of murine disease models

Implementation of in vivo high-resolution micro-computed tomography (µCT), a powerful tool for longitudinal analysis of murine lung disease models, is hampered by the lack of data on cumulative low-dose radiation effects on the investigated disease models. We aimed to measure radiation doses and effects of repeated µCT scans, to establish cumulative radiation levels and scan protocols without relevant toxicity. Lung metastasis, inflammation and fibrosis models and healthy mice were weekly scanned over one-month with µCT using high-resolution respiratory-gated 4D and expiration-weighted 3D protocols, comparing 5-times weekly scanned animals with controls. Radiation dose was measured by ionization chamber, optical fiberradioluminescence probe and thermoluminescent detectors in a mouse phantom. Dose effects were evaluated by in vivo µCT and bioluminescence imaging read-outs, gold standard endpoint evaluation and blood cell counts. Weekly exposure to 4D µCT, dose of 540-699 mGy/scan, did not alter lung metastatic load nor affected healthy mice. We found a disease-independent decrease in circulating blood platelets and lymphocytes after repeated 4D µCT. This effect was eliminated by optimizing a 3D protocol, reducing dose to 180-233 mGy/scan while maintaining equally high-quality images. We established µCT safety limits and protocols for weekly repeated whole-body acquisitions with proven safety for the overall health status, lung, disease process and host responses under investigation, including the radiosensitive blood cell compartment.status: accepte

Ethanol exposure increases mutation rate through error-prone polymerases

Ethanol is a ubiquitous environmental stressor that is toxic to all lifeforms. Here, we use the model eukaryote Saccharomyces cerevisiae to show that exposure to sublethal ethanol concentrations causes DNA replication stress and an increased mutation rate. Specifically, we find that ethanol slows down replication and affects localization of Mrc1, a conserved protein that helps stabilize the replisome. In addition, ethanol exposure also results in the recruitment of error-prone DNA polymerases to the replication fork. Interestingly, preventing this recruitment through mutagenesis of the PCNA/Pol30 polymerase clamp or deleting specific error-prone polymerases abolishes the mutagenic effect of ethanol. Taken together, this suggests that the mutagenic effect depends on a complex mechanism, where dysfunctional replication forks lead to recruitment of error-prone polymerases. Apart from providing a general mechanistic framework for the mutagenic effect of ethanol, our findings may also provide a route to better understand and prevent ethanol-associated carcinogenesis in higher eukaryotes.Voordeckers K, Colding C, Grasso L, Pardo B, Hoes L, Kominek J, Gielens K, Dekoster K, Gordon J, Van der Zande E, Bircham P, Swings T, Michiels J, Van Loo P, Nuyts S, Pasero P, Lisby M, Verstrepen KJ.Voordeckers K, et al. Among authors: nuyts s. Nat Commun. 2020 Jul 21;11(1):3664. doi: 10.1038/s41467-020-17447-3.Nat Commun. 2020. PMID: 32694532status: publishe

DataSheet_1_Innate Lymphoid Cells Are Required to Induce Airway Hyperreactivity in a Murine Neutrophilic Asthma Model.docx

RationaleNon-allergic asthma is driven by multiple endotypes of which neutrophilic and pauci-granulocytic asthma have been best established. However, it is still puzzling what drives inflammation and airway hyperreactivity (AHR) in these patients and how it can be treated effectively. Recently, a potential role of the innate immune system and especially the innate lymphoid cells (ILC) has been proposed.ObjectiveIn this study, we investigated the effects of LPS inhalation on airway inflammation and AHR as a potential model for elucidating the pathogenesis of non-allergic asthma.MethodsWild-type (BALB/c), SCID, IL-17A-/-, and Rag2-/- γC-/- mice were endonasally exposed to lipopolysaccharide (LPS, 2 µg) on four consecutive days. Twenty-four hours after the last exposure, AHR to methacholine was assessed. Cytokine levels and ILC subpopulations were determined in lung tissue. Cellular differential analysis was performed in BAL fluid.Main ResultsIn this study, we developed a murine model for non-allergic neutrophilic asthma. We found that repeated endonasal applications of low-dose LPS in BALB/c mice led to AHR, BAL neutrophilia, and a significant increase in lung ILC3 as well as a significant increase in lung chemokines KC and MIP-2 and cytokines IL-1β, IL-17A, IL-22, and TNF. The adoptive transfer of ILC in Rag2-/- γC-/- mice showed that ILC played a causal role in the induction of AHR in this model. Antagonising IL-1β, but not IL-17A or neutrophils, resulted in a partial reduction in LPS-induced AHR.ConclusionIn conclusion, we report here a murine model for neutrophilic asthma where ILC are required to induce airway hyperreactivity.</p