@article{MTMT:36186439, title = {Quiescent horizontal basal stem cells act as a niche for olfactory neurogenesis in a mouse 3D organoid model}, url = {https://m2.mtmt.hu/api/publication/36186439}, author = {Gameiro, J.G. and Hintschich, C.A. and Dekeyser, A. and Hox, V. and Schwob, J.E. and Holbrook, E.H. and Fornazieri, M.A. and Lin, B.}, doi = {10.1016/j.crmeth.2025.101055}, journal-iso = {CELL REP METH}, journal = {CELL REPORTS METHODS}, unique-id = {36186439}, issn = {2667-2375}, abstract = {The olfactory epithelium contains two basal stem cell populations that facilitate the usually life-long ability for neuronal regeneration that is required for maintaining our sense of smell. Horizontal basal cells (HBCs) are generally quiescent and only become active after direct injury to the epithelium that eliminates more than just the olfactory sensory neurons (OSNs). Globose basal cells (GBCs) lie apical to HBCs and are solely responsible for the generation of olfactory neurons in the undamaged epithelium. Understanding how these two neurogenic stem cell populations are regulated as OSNs are replenished is hampered by a lack of robust culture models. Here, we report the development of a 3D mouse organoid model that recapitulates the neurogenic cascade, forming immature OSNs while maintaining both HBCs and GBCs in culture. We use this model to demonstrate that, despite their relative quiescence, HBCs form a critical niche for the emergence and composition of the organoid. © 2025 The Author(s)}, keywords = {stem cell; NEUROGENESIS; olfaction; Olfactory epithelium; Organoid; olfactory; 3D culture; CP: Neuroscience; CP: Stem cell; globose basal cell; horizontal basal cell}, year = {2025}, eissn = {2667-2375} } @article{MTMT:36255952, title = {Combination of in vivo and in vitro phosphoproteomics determines the PP2A target repertoire on proteome scale}, url = {https://m2.mtmt.hu/api/publication/36255952}, author = {Brunner, M. and Hu, Z. and Elkhaligy, H. and Lampo, G. and Roubaty, C. and Vionnet, C. and Sankar, D.S. and McIlwain, S.J. and Kaeser-Pebernard, S. and Xing, Y. and Dengjel, J.}, doi = {10.1016/j.crmeth.2025.101084}, journal-iso = {CELL REP METH}, journal = {CELL REPORTS METHODS}, unique-id = {36255952}, issn = {2667-2375}, abstract = {Dynamics of protein phosphorylation are regulated by the interplay of kinases and phosphatases. Current mass spectrometry-based phosphoproteomic approaches are extremely powerful in identifying and quantifying tens of thousands of phosphosites in single biological samples. However, whereas the mapping of phosphosites is successfully automated supporting high sample throughput, the characterization of responsible kinases and phosphatases still largely depends on laborious protein biochemical assays. To show direct (de)phosphorylation events, in vitro kinase or phosphatase assays using single substrates or peptide arrays are often used. Here, we describe the development of an in vitro phosphatase assay using whole proteome under native conditions as input. We employ this approach to study the PP1 and PP2A target repertoire, characterizing thousands of potential target sites. Focusing on PPP2R5E/B56ε-containing complexes, we combine in vitro with in vivo phosphoproteomics to characterize bona fide target sites, which highlight the role of PP2A in regulating stress granule assembly. © 2025 The Author(s)}, keywords = {PHOSPHATASE; KINASE; Mass spectrometry; proteomics; signaling; PP2A; stress granule; PP1; CAPRIN1; CP: Biotechnology; PPP2R5E}, year = {2025}, eissn = {2667-2375} } @article{MTMT:36291651, title = {Deuterium labeling enables proteome-wide turnover kinetics analysis in cell culture}, url = {https://m2.mtmt.hu/api/publication/36291651}, author = {Alamillo, L. and Ng, D.C.M. and Currie, J. and Black, A. and Pandi, B. and Manda, V. and Pavelka, J. and Schaal, P. and Travers, J.G. and McKinsey, T.A. and Lam, M.P.Y. and Lau, E.}, doi = {10.1016/j.crmeth.2025.101104}, journal-iso = {CELL REP METH}, journal = {CELL REPORTS METHODS}, volume = {5}, unique-id = {36291651}, issn = {2667-2375}, abstract = {Protein turnover is a critical component of gene expression regulation and cellular homeostasis, yet methods for measuring turnover rates that are scalable and applicable to different models are still needed. We introduce an improved D2O (heavy water) labeling strategy to investigate the landscape of protein turnover in cell culture, with accurate calibration of per-residue deuterium incorporation in multiple cell types. Applying this method, we mapped the proteome-wide turnover landscape of pluripotent and differentiating human induced pluripotent stem cells (hiPSCs). Our analysis highlights the role of APC/C (anaphase-promoting complex/cyclosome) and SPOP (speckle-type POZ protein) degrons in the fast turnover of cell-cycle-related and DNA-binding hiPSC proteins. Upon pluripotency exit, many short-lived hiPSC proteins are depleted, while RNA-binding and -splicing proteins become hyperdynamic. The ability to identify fast-turnover proteins also facilitates secretome profiling, as exemplified in hiPSC-cardiomyocyte and primary human cardiac fibroblast analysis. This method is broadly applicable to protein turnover studies in primary, pluripotent, and transformed cells. © 2025 The Author(s)}, keywords = {Adult; Humans; metabolism; ARTICLE; FIBROBLASTS; MOUSE; KINETICS; KINETICS; human; Chemistry; Cell Differentiation; Cell Differentiation; protein analysis; controlled study; Cytology; nonhuman; animal experiment; animal cell; Cells, Cultured; DEUTERIUM; DEUTERIUM; Mass spectrometry; Mass spectrometry; FIBROBLAST; human cell; cell culture; cell culture; human tissue; protein synthesis; Cell Culture Techniques; proteomics; proteomics; Protein turnover; protein metabolism; Myocytes, Cardiac; proteome; proteome; amino acid analysis; cell kinetics; atom; deuterium oxide; induced pluripotent stem cell; Induced pluripotent stem cells; secretome; Heavy water; procedures; cell culture technique; cardiac muscle cell; Secretomics; degron; hiPSC; CP: Molecular biology; CP: Systems biology; turnover kinetics}, year = {2025}, eissn = {2667-2375} } @article{MTMT:36303083, title = {Automated denoising of CITE-seq data with ThresholdR}, url = {https://m2.mtmt.hu/api/publication/36303083}, author = {Oliaeimotlagh, M. and Kumar, S. and Taraskin, A. and Armstrong, Suthahar S.S. and Suryawanshi, V. and Chiang, A.W.T. and Ley, K.}, doi = {10.1016/j.crmeth.2025.101088}, journal-iso = {CELL REP METH}, journal = {CELL REPORTS METHODS}, volume = {5}, unique-id = {36303083}, issn = {2667-2375}, year = {2025}, eissn = {2667-2375} } @article{MTMT:36396039, title = {Report Combination of in vivo and in vitro phosphoproteomics determines the PP2A target repertoire on proteome scale}, url = {https://m2.mtmt.hu/api/publication/36396039}, author = {Brunner, Melanie and Hu, Zehan and Elkhaligy, Heidy and Lampo, Gloria and Roubaty, Carole and Vionnet, Christine and Sankar, Devanarayanan Siva and McIlwain, Sean J. and Kaeser-Pebernard, Stephanie and Xing, Yongna and Dengjel, Jorn}, doi = {10.1016/j.crmeth.2025.101084}, journal-iso = {CELL REP METH}, journal = {CELL REPORTS METHODS}, volume = {5}, unique-id = {36396039}, issn = {2667-2375}, abstract = {Dynamics of protein phosphorylation are regulated by the interplay of kinases and phosphatases. Current mass spectrometry-based phosphoproteomic approaches are extremely powerful in identifying and quantifying tens of thousands of phosphosites in single biological samples. However, whereas the mapping of phosphosites is successfully automated supporting high sample throughput, the characterization of responsible kinases and phosphatases still largely depends on laborious protein biochemical assays. To show direct (de)phosphorylation events, in vitro kinase or phosphatase assays using single substrates or peptide arrays are often used. Here, we describe the development of an in vitro phosphatase assay using whole proteome under native conditions as input. We employ this approach to study the PP1 and PP2A target repertoire, characterizing thousands of potential target sites. Focusing on PPP2R5E/B56 epsilon-containing complexes, we combine in vitro with in vivo phosphoproteomics to characterize bona fide target sites, which highlight the role of PP2A in regulating stress granule assembly.}, keywords = {INHIBITION; APOPTOSIS; SPECIFICITY; SUBSTRATE; MUTATIONS; RECRUITMENT; DISCOVERY; CYTOSCAPE; PHOSPHATASE 2A; Biochemical Research Methods}, year = {2025}, eissn = {2667-2375}, orcid-numbers = {Elkhaligy, Heidy/0000-0002-2767-860X} } @article{MTMT:36343915, title = {CRISPR GENome and epigenome engineering improves loss-of-function genetic-screening approaches}, url = {https://m2.mtmt.hu/api/publication/36343915}, author = {Stadager, Jannis and Bernardini, Chiara and Hartmann, Laura and May, Henrik and Wiepcke, Jessica and Kuban, Monika and Najafova, Zeynab and Johnsen, Steven A. and Legewie, Stefan and Traube, Franziska R. and Jude, Julian and Rathert, Philipp}, doi = {10.1016/j.crmeth.2025.101078}, journal-iso = {CELL REP METH}, journal = {CELL REPORTS METHODS}, volume = {5}, unique-id = {36343915}, issn = {2667-2375}, abstract = {CRISPR-Cas9 technology has revolutionized genotype-to-phenotype assignments through large-scale loss-of-function (LOF) screens. However, limitations like editing inefficiencies and unperturbed genes cause significant noise in data collection. To address this, we introduce CRISPR gene and epigenome engineering (CRISPRgenee), which uses two specific single guide RNAs (sgRNAs) to simultaneously repress and cleave the target gene within the same cell, increasing LOF efficiencies and reproducibility. CRISPRgenee outperforms conventional CRISPR knockout (CRISPRko), CRISPR interference (CRISPRi), and CRISPRoff systems in suppressing challenging targets and regulators of cell proliferation. Additionally, it efficiently suppresses modulators of epithelial-to-mesenchymal transition (EMT) and impairs neuronal differentiation in a human induced pluripotent stem cell (iPSC) model. CRISPRgenee exhibits improved depletion efficiency, reduced sgRNA performance variance, and accelerated gene depletion compared to individual CRISPRi or CRISPRko screens, ensuring consistency in phenotypic effects and identifying more significant gene hits. By combining CRISPRko and CRISPRi, CRISPRgenee increases LOF rates without increasing genotoxic stress, facilitating library size reduction for advanced LOF screens.}, keywords = {BINDING; SPECIFICITY; ACTIVATION; Checkpoint; REVEALS; Biochemical Research Methods; LSD1; CAS9; SEQUENCE DETERMINANTS; Bub1}, year = {2025}, eissn = {2667-2375}, orcid-numbers = {Johnsen, Steven A./0000-0003-1198-5805} } @article{MTMT:36413535, title = {Quiescent horizontal basal stem cells act as a niche for olfactory neurogenesis in a mouse 3D organoid model}, url = {https://m2.mtmt.hu/api/publication/36413535}, author = {Gameiro, Juliana Gutschow and Hintschich, Constantin A. and Dekeyser, Agnes and Hox, Valerie and Schwob, James E. and Holbrook, Eric H. and Fornazieri, Marco Aurelio and Lin, Brian}, doi = {10.1016/j.crmeth.2025.101055}, journal-iso = {CELL REP METH}, journal = {CELL REPORTS METHODS}, volume = {5}, unique-id = {36413535}, issn = {2667-2375}, abstract = {The olfactory epithelium contains two basal stem cell populations that facilitate the usually life-long ability for neuronal regeneration that is required for maintaining our sense of smell. Horizontal basal cells (HBCs) are generally quiescent and only become active after direct injury to the epithelium that eliminates more than just the olfactory sensory neurons (OSNs). Globose basal cells (GBCs) lie apical to HBCs and are solely responsible for the generation of olfactory neurons in the undamaged epithelium. Understanding how these two neurogenic stem cell populations are regulated as OSNs are replenished is hampered by a lack of robust culture models. Here, we report the development of a 3D mouse organoid model that recapitulates the neurogenic cascade, forming immature OSNs while maintaining both HBCs and GBCs in culture. We use this model to demonstrate that, despite their relative quiescence, HBCs form a critical niche for the emergence and composition of the organoid.}, keywords = {EXPRESSION; IN-VITRO; SENSORY NEURONS; MATURATION; epithelium; PROGENITOR CELLS; SELF-RENEWAL; Biochemical Research Methods; C-KIT RECEPTOR; CULTURE MODEL}, year = {2025}, eissn = {2667-2375}, orcid-numbers = {Lin, Brian/0000-0002-8851-3534} } @article{MTMT:36272810, title = {A sequence context-based approach for classifying tumor structural variants without paired normal samples}, url = {https://m2.mtmt.hu/api/publication/36272810}, author = {Chukwu, Wolu and Lee, Siyun and Crane, Alexander and Zhang, Shu and Webster, Sophie and Dakhama, Oumayma and Mittra, Ipsa and Rauert, Carlos and Imielinski, Marcin and Beroukhim, Rameen and Dubois, Frank and Dalin, Simona}, doi = {10.1016/j.crmeth.2025.100991}, journal-iso = {CELL REP METH}, journal = {CELL REPORTS METHODS}, volume = {5}, unique-id = {36272810}, issn = {2667-2375}, abstract = {Although several recent studies have characterized structural variants (SVs) in germline and cancer genomes independently, the genomic contexts of these SVs have not been comprehensively compared. We examined similarities and differences between 2 million germline and 115 thousand tumor SVs from a cohort of 963 patients from The Cancer Genome Atlas. We found significant differences in features related to their genomic sequences and localization that suggest differences between SV-generating processes and selective pressures. For example, our results show that features linked to transposon-mediated processes are associated with germline SVs, while somatic SVs more frequently show features characteristic of chromoanagenesis. These genomic differences enabled us to develop a classifier-the Germline and Tumor Structural Variant or "the great GaTSV"-that accurately distinguishes between germline and cancer SVs in tumor samples that lack a matched normal sample.}, keywords = {MECHANISMS; CANCER; Elements; GERMLINE; Biochemical Research Methods}, year = {2025}, eissn = {2667-2375}, orcid-numbers = {Crane, Alexander/0000-0003-1930-279X} } @article{MTMT:36343811, title = {Comprehensive assessment of computational methods for cancer immunoediting}, url = {https://m2.mtmt.hu/api/publication/36343811}, author = {He, Shengyuan and Sun, Shangqin and Liu, Kun and Pang, Bo and Xiao, Yun}, doi = {10.1016/j.crmeth.2025.101006}, journal-iso = {CELL REP METH}, journal = {CELL REPORTS METHODS}, volume = {5}, unique-id = {36343811}, issn = {2667-2375}, abstract = {Cancer immunoediting reflects the role of the immune system in eliminating tumor cells and shaping tumor immunogenicity, which leaves marks in the genome. In this study, we systematically evaluate four methods for quantifying immunoediting. In colorectal cancer samples from The Cancer Genome Atlas, we found that these methods identified 78.41%, 46.17%, 36.61%, and 4.92% of immunoedited samples, respectively, covering 92.90% of all colorectal cancer samples. Comparison of 10 patient-derived xenografts (PDXs) with their original tumors showed that different methods identified reduced immune selection in PDXs ranging from 44.44% to 60.0%. The proportion of such PDX-tumor pairs increases to 77.78% when considering the union of results from multiple methods, indicating the complementarity of these methods. We find that observed-to-expected ratios highly rely on neoantigen selection criteria and reference datasets. In contrast, HLA-binding mutation ratio, immune dN/dS, and enrichment score of cancer cell fraction were less affected by these factors. Our findings suggest integration of multiple methods may benefit future immunoediting analyses.}, keywords = {MUTATIONS; MODELS; EVOLUTION; prediction; immunosurveillance; Immune; Biochemical Research Methods; HYPERMUTATED TUMORS}, year = {2025}, eissn = {2667-2375} } @article{MTMT:35780815, title = {Exploring common mechanisms of adverse drug reactions and disease phenotypes through network-based analysis}, url = {https://m2.mtmt.hu/api/publication/35780815}, author = {Firoozbakht, F. and Elkjaer, M.L. and Handy, D.E. and Wang, R.-S. and Chervontseva, Z. and Rarey, M. and Loscalzo, J. and Baumbach, J. and Tsoy, O.}, doi = {10.1016/j.crmeth.2025.100990}, journal-iso = {CELL REP METH}, journal = {CELL REPORTS METHODS}, volume = {5}, unique-id = {35780815}, issn = {2667-2375}, abstract = {The need for a deeper understanding of adverse drug reaction (ADR) mechanisms is vital for improving drug safety and repurposing. This study introduces Drug Adverse Reaction Mechanism Explainer (DREAMER), a network-based framework that uses a comprehensive knowledge graph to uncover molecular mechanisms underlying ADRs and disease phenotypes. By examining shared phenotypes of drugs and diseases and their effects on protein-protein interaction networks, DREAMER identifies proteins linked to ADR mechanisms. Applied to 649 ADRs, DREAMER identified molecular mechanisms for 67 ADRs, including ventricular arrhythmia and metabolic acidosis, and emphasized pathways like GABAergic signaling and coagulation proteins in personality disorders and intracranial hemorrhage. We further demonstrate the application of DREAMER in drug repurposing and propose sotalol, ranolazine, and diltiazem as candidate drugs to be repurposed for cardiac arrest. In summary, DREAMER effectively detects molecular mechanisms underlying phenotypes, emphasizing the importance of network-based analyses with integrative data for enhancing drug safety and accelerating the discovery of novel therapeutic strategies. © 2025 The Author(s)}, keywords = {Disease phenotype; drug safety; adverse drug reaction; drug repurposing; CLINICAL PHENOTYPES; Network-based analysis; Network diffusion; CP: Systems biology}, year = {2025}, eissn = {2667-2375} }