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- Carreras-Puigvert, Jordi, et al.
(author)
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A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family
- 2017
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In: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 8:1
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Journal article (peer-reviewed)abstract
- The NUDIX enzymes are involved in cellular metabolism and homeostasis, as well as mRNA processing. Although highly conserved throughout all organisms, their biological roles and biochemical redundancies remain largely unclear. To address this, we globally resolve their individual properties and inter-relationships. We purify 18 of the human NUDIX proteins and screen 52 substrates, providing a substrate redundancy map. Using crystal structures, we generate sequence alignment analyses revealing four major structural classes. To a certain extent, their substrate preference redundancies correlate with structural classes, thus linking structure and activity relationships. To elucidate interdependence among the NUDIX hydrolases, we pairwise deplete them generating an epistatic interaction map, evaluate cell cycle perturbations upon knockdown in normal and cancer cells, and analyse their protein and mRNA expression in normal and cancer tissues. Using a novel FUSION algorithm, we integrate all data creating a comprehensive NUDIX enzyme profile map, which will prove fundamental to understanding their biological functionality.
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| 4. |
- Espinoza, JA, et al.
(author)
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The antimalarial drug amodiaquine stabilizes p53 through ribosome biogenesis stress, independently of its autophagy-inhibitory activity
- 2020
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In: Cell death and differentiation. - : Springer Science and Business Media LLC. - 1476-5403 .- 1350-9047. ; 27:2, s. 773-789
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Journal article (peer-reviewed)abstract
- Pharmacological inhibition of ribosome biogenesis is a promising avenue for cancer therapy. Herein, we report a novel activity of the FDA-approved antimalarial drug amodiaquine which inhibits rRNA transcription, a rate-limiting step for ribosome biogenesis, in a dose-dependent manner. Amodiaquine triggers degradation of the catalytic subunit of RNA polymerase I (Pol I), with ensuing RPL5/RPL11-dependent stabilization of p53. Pol I shutdown occurs in the absence of DNA damage and without the subsequent ATM-dependent inhibition of rRNA transcription. RNAseq analysis revealed mechanistic similarities of amodiaquine with BMH-21, the first-in-class Pol I inhibitor, and with chloroquine, the antimalarial analog of amodiaquine, with well-established autophagy-inhibitory activity. Interestingly, autophagy inhibition caused by amodiaquine is not involved in the inhibition of rRNA transcription, suggesting two independent anticancer mechanisms. In vitro, amodiaquine is more efficient than chloroquine in restraining the proliferation of human cell lines derived from colorectal carcinomas, a cancer type with predicted susceptibility to ribosome biogenesis stress. Taken together, our data reveal an unsuspected activity of a drug approved and used in the clinics for over 30 years, and provide rationale for repurposing amodiaquine in cancer therapy.
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| 5. |
- Gad, Helge, et al.
(author)
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MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool
- 2014
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In: Nature. - : Nature Publishing Group. - 0028-0836 .- 1476-4687. ; 508:7495, s. 215-221
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Journal article (peer-reviewed)abstract
- Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bindin the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.
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| 7. |
- Gupta, Ankit, et al.
(author)
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Is brightfield all you need for MoA prediction?
- 2022
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Conference paper (peer-reviewed)abstract
- Fluorescence staining techniques, such as Cell Painting, together with fluorescence microscopy have proven invaluable for visualizing and quantifying the effects that drugs and other perturbations have on cultured cells. However, fluorescence microscopy is expensive, time-consuming, and labor-intensive, and the stains applied can be cytotoxic, interfering with the activity under study. The simplest form of microscopy, brightfield microscopy, lacks these downsides, but the images produced have low contrast and the cellular compartments are difficult to discern. Nevertheless, by harnessing deep learning, these brightfield images may still be sufficient for various predictive purposes. In this study, we compared the predictive performance of models trained on fluorescence images to those trained on brightfield images for predicting the mechanism of action (MoA) of different drugs. We also extracted CellProfiler features from the fluorescence images and used them to benchmark the performance. Overall, we found comparable and correlated predictive performance for the two imaging modalities. This is promising for future studies of MoAs in time-lapse experiments.
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| 8. |
- Harrison, Philip J., et al.
(author)
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Exploring the evolution of cellular morphological changes after drug administration based on brightfield image data
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Other publication (other academic/artistic)abstract
- Most image based studies of the morphological effects of compound treatments on cells, such as those for elucidating a compound's mechanism of action, use fixed-cell based approaches whereby the cells are fixated, stained, and imaged with fluorescence microscopy some time after compound administration. This snapshot data, however, cannot uncover any information on the temporal dynamics of the induced morphological changes. For instance regarding the rate at which these changes occur following compound perturbation. Live-cell compatible dyes can be used although are limited by technical difficulties, cytotoxicity and photobleaching. A simpler, cheaper and less harmful option is to use brightfield microscopy. Although brightfield images have less contrast than fluorescence images and cannot separate out the different cellular compartments, we here show that for compounds inducing morphological changes on cells, brightfield data, together with convolutional neural networks and feature projection techniques, can be used to extract such temporal information from time-lapse experiments.
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| 10. |
- Li, Xuexin, et al.
(author)
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The anti-leprosy drug clofazimine reduces polyQ toxicity through activation of PPARγ
- 2024
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In: EBioMedicine. - : Elsevier. - 2352-3964. ; 103
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Journal article (peer-reviewed)abstract
- Background: PolyQ diseases are autosomal dominant neurodegenerative disorders caused by the expansion of CAG repeats. While of slow progression, these diseases are ultimately fatal and lack effective therapies.Methods: A high-throughput chemical screen was conducted to identify drugs that lower the toxicity of a protein containing the fi rst exon of Huntington's disease (HD) protein huntingtin (HTT) harbouring 94 glutamines (Htt-Q94). 94 ). Candidate drugs were tested in a wide range of in vitro and in vivo models of polyQ toxicity.Findings: The chemical screen identified fi ed the anti-leprosy drug clofazimine as a hit, which was subsequently validated in several in vitro models. Computational analyses of transcriptional signatures revealed that the effect of clofazimine was due to the stimulation of mitochondrial biogenesis by peroxisome proliferator-activated receptor gamma (PPARγ). In agreement with this, clofazimine rescued mitochondrial dysfunction triggered by Htt-Q94 expression. Importantly, clofazimine also limited polyQ toxicity in developing zebrafish fish and neuron-specific worm models of polyQ disease.Interpretation: Our results support the potential of repurposing the antimicrobial drug clofazimine for the treatment of polyQ diseases.
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| 11. |
- Matuszewski, Damian J., et al.
(author)
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Comparison of Flow Cytometry and Image-Based Screening for Cell Cycle Analysis
- 2016
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In: Image Analysis And Recognition (ICIAR 2016). - Cham : Springer. ; , s. 623-630
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Conference paper (peer-reviewed)abstract
- Quantitative cell state measurements can provide a wealth of information about mechanism of action of chemical compounds and gene functionality. Here we present a comparison of cell cycle disruption measurements from commonly used flow cytometry (generating onedimensional signal data) and bioimaging (producing two-dimensional image data). Our results show high correlation between the two approaches indicating that image-based screening can be used as an alternative to flow cytometry. Furthermore, we discuss the benefits of image informatics over conventional single-signal flow cytometry.
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| 12. |
- Matuszewski, Damian J., et al.
(author)
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PopulationProfiler : A Tool for Population Analysis and Visualization of Image-Based Cell Screening Data
- 2016
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:3
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Journal article (peer-reviewed)abstract
- Image-based screening typically produces quantitative measurements of cell appearance. Large-scale screens involving tens of thousands of images, each containing hundreds of cells described by hundreds of measurements, result in overwhelming amounts of data. Reducing per-cell measurements to the averages across the image(s) for each treatment leads to loss of potentially valuable information on population variability. We present PopulationProfiler-a new software tool that reduces per-cell measurements to population statistics. The software imports measurements from a simple text file, visualizes population distributions in a compact and comprehensive way, and can create gates for subpopulation classes based on control samples. We validate the tool by showing how PopulationProfiler can be used to analyze the effect of drugs that disturb the cell cycle, and compare the results to those obtained with flow cytometry.
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| 13. |
- Ouyang, Wei, et al.
(author)
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An Open-Source Modular Framework for Automated Pipetting and Imaging Applications
- 2022
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In: Advanced Biology. - : Wiley. - 2701-0198. ; 6:4, s. 2101063-
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Journal article (peer-reviewed)abstract
- The number of samples in biological experiments is continuously increasing, but complex protocols and human error in many cases lead to suboptimal data quality and hence difficulties in reproducing scientific findings. Laboratory automation can alleviate many of these problems by precisely reproducing machine-readable protocols. These instruments generally require high up-front investments, and due to the lack of open application programming interfaces (APIs), they are notoriously difficult for scientists to customize and control outside of the vendor-supplied software. Here, automated, high-throughput experiments are demonstrated for interdisciplinary research in life science that can be replicated on a modest budget, using open tools to ensure reproducibility by combining the tools OpenFlexure, Opentrons, ImJoy, and UC2. This automated sample preparation and imaging pipeline can easily be replicated and established in many laboratories as well as in educational contexts through easy-to-understand algorithms and easy-to-build microscopes. Additionally, the creation of feedback loops, with later pipetting or imaging steps depending on the analysis of previously acquired images, enables the realization of fully autonomous “smart” microscopy experiments. All documents and source files are publicly available to prove the concept of smart lab automation using inexpensive, open tools. It is believed this democratizes access to the power and repeatability of automated experiments.
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