Cell Reports Methods最新文献_第6页

Characterization of human CMV-specific CD8⁺ T cells using multi-layer single-cell omics. 人cmv特异性CD8+ T细胞的多层单细胞组学研究

IF 4.5

Cell Reports Methods Pub Date : 2025-07-21 Epub Date: 2025-06-23 DOI: 10.1016/j.crmeth.2025.101085

Ioanna Gemünd, Lorenzo Bonaguro, Matthias Becker, Sophie Müller, Clemens Joos, Elena De Domenico, Anna C Aschenbrenner, Joachim L Schultze, Andreas Moosmann, Marc D Beyer

引用次数: 0

Deuterium labeling enables proteome-wide turnover kinetics analysis in cell culture. 氘标记使蛋白质组范围内的周转动力学分析在细胞培养。

IF 4.5

Cell Reports Methods Pub Date : 2025-07-21 Epub Date: 2025-07-10 DOI: 10.1016/j.crmeth.2025.101104

Lorena Alamillo, Dominic C M Ng, Jordan Currie, Alexander Black, Boomathi Pandi, Vyshnavi Manda, Jay Pavelka, Peyton Schaal, Joshua G Travers, Timothy A McKinsey, Maggie P Y Lam, Edward Lau

{"title":"Deuterium labeling enables proteome-wide turnover kinetics analysis in cell culture.","authors":"Lorena Alamillo, Dominic C M Ng, Jordan Currie, Alexander Black, Boomathi Pandi, Vyshnavi Manda, Jay Pavelka, Peyton Schaal, Joshua G Travers, Timothy A McKinsey, Maggie P Y Lam, Edward Lau","doi":"10.1016/j.crmeth.2025.101104","DOIUrl":"10.1016/j.crmeth.2025.101104","url":null,"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.","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":" ","pages":"101104"},"PeriodicalIF":4.5,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12296481/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144620804","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

TransTag enables simple and efficient transgene mapping in zebrafish via tagmentation. TransTag可以通过标记在斑马鱼中进行简单有效的转基因定位。

IF 4.5

Cell Reports Methods Pub Date : 2025-07-21 Epub Date: 2025-07-08 DOI: 10.1016/j.crmeth.2025.101090

Fanju W Meng, Paige Schneider, Xiaolu Wei, Krishan Ariyasiri, Marnie E Halpern, Patrick J Murphy

{"title":"TransTag enables simple and efficient transgene mapping in zebrafish via tagmentation.","authors":"Fanju W Meng, Paige Schneider, Xiaolu Wei, Krishan Ariyasiri, Marnie E Halpern, Patrick J Murphy","doi":"10.1016/j.crmeth.2025.101090","DOIUrl":"10.1016/j.crmeth.2025.101090","url":null,"abstract":"Zebrafish has become a preeminent model for developmental biology research, largely due to the ease of transgenesis. Despite widespread usage of transgenic lines, mapping of transgene insertion sites is rare, which raises complications involving potential local chromatin influences on transgene expression, off-target effects, and issues with allelic variation. To address these shortcomings, we introduce TransTag, a simple and efficient method utilizing Tn5 transposase-mediated tagmentation, for the streamlined identification of Tol2-based transgene insertion sites in zebrafish. TransTag is straightforward to perform and can identify insertion sites without the need for the alignment of raw sequencing data. We also provide a detailed protocol for TransTag, a step-by-step guide for data analysis, and a user-friendly Shiny app, making transgene mapping achievable at a low cost for researchers without programming expertise. Altogether, TransTag emerges as a valuable tool to enhance the precision and utility of transgenesis studies by providing essential chromosome-specific information on transgene locations.","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":" ","pages":"101090"},"PeriodicalIF":4.5,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12296462/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144601757","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Self-supervised learning for generalizable particle picking in cryo-EM micrographs. 低温电镜显微图中可泛化粒子拾取的自监督学习。

IF 4.5

Cell Reports Methods Pub Date : 2025-07-21 Epub Date: 2025-07-07 DOI: 10.1016/j.crmeth.2025.101089

Andreas Zamanos, Panagiotis Koromilas, Giorgos Bouritsas, Panagiotis L Kastritis, Yannis Panagakis

{"title":"Self-supervised learning for generalizable particle picking in cryo-EM micrographs.","authors":"Andreas Zamanos, Panagiotis Koromilas, Giorgos Bouritsas, Panagiotis L Kastritis, Yannis Panagakis","doi":"10.1016/j.crmeth.2025.101089","DOIUrl":"10.1016/j.crmeth.2025.101089","url":null,"abstract":"We present cryoelectron microscopy masked autoencoder (cryo-EMMAE), a self-supervised method designed to overcome the need for manually annotated cryo-EM data. cryo-EMMAE leverages the representation space of a masked autoencoder to pick particle pixels through clustering of the MAE latent representation. Evaluation across different EMPIAR datasets demonstrates that cryo-EMMAE outperforms state-of-the-art supervised methods in terms of generalization capabilities. Importantly, our method showcases consistent performance, independent of the dataset used for training. Additionally, cryo-EMMAE is data efficient, as we experimentally observe that it converges with as few as five micrographs. Further, 3D reconstruction results indicate that our method has superior performance in reconstructing the volumes in both single-particle datasets and multi-particle micrographs derived from cell extracts. Our results underscore the potential of self-supervised learning in advancing cryo-EM image analysis, offering an alternative for more efficient and cost-effective structural biology research. Code is available at https://github.com/azamanos/Cryo-EMMAE.","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":" ","pages":"101089"},"PeriodicalIF":4.5,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12296464/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144592461","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A fluorescent STING ligand sensor for high-throughput screening of compounds that can enhance tumor immunotherapy. 用于高通量筛选可增强肿瘤免疫治疗的化合物的荧光STING配体传感器。

IF 4.5

Cell Reports Methods Pub Date : 2025-07-21 Epub Date: 2025-07-15 DOI: 10.1016/j.crmeth.2025.101106

Pengkai Sun, Bin Wang, Caiyun Liu, Zixiong Wang, Yang Liu, Yuan-Biao Qiao, Xinjian Li

引用次数: 0

Combination of in vivo and in vitro phosphoproteomics determines the PP2A target repertoire on proteome scale. 体内和体外磷酸化蛋白质组学的结合决定了蛋白质组学尺度上PP2A的靶库。

IF 4.5

Cell Reports Methods Pub Date : 2025-07-21 Epub Date: 2025-06-19 DOI: 10.1016/j.crmeth.2025.101084

Melanie Brunner, Zehan Hu, Heidy Elkhaligy, Gloria Lampo, Carole Roubaty, Christine Vionnet, Devanarayanan Siva Sankar, Sean J McIlwain, Stéphanie Kaeser-Pebernard, Yongna Xing, Jörn Dengjel

{"title":"Combination of in vivo and in vitro phosphoproteomics determines the PP2A target repertoire on proteome scale.","authors":"Melanie Brunner, Zehan Hu, Heidy Elkhaligy, Gloria Lampo, Carole Roubaty, Christine Vionnet, Devanarayanan Siva Sankar, Sean J McIlwain, Stéphanie Kaeser-Pebernard, Yongna Xing, Jörn Dengjel","doi":"10.1016/j.crmeth.2025.101084","DOIUrl":"10.1016/j.crmeth.2025.101084","url":null,"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.","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":" ","pages":"101084"},"PeriodicalIF":4.5,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12296510/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144337124","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Dynamic stimulation promotes functional tissue-like organization of a 3D human lymphoid microenvironment model in vitro. 动态刺激促进体外三维人淋巴微环境模型的功能组织样组织。

IF 4.5

Cell Reports Methods Pub Date : 2025-07-21 Epub Date: 2025-07-11 DOI: 10.1016/j.crmeth.2025.101105

Dafne Barozzi, Fiorella Scagnoli, Federica Barbaglio, Daniela Belloni, Davide Ribezzi, Silvia Farè, Valeria Berno, Riccardo Pinos, Marta Sampietro, Margherita Pauri, Barbara Vergani, Francesco Mantegazza, Paolo Ghia, Cristina Scielzo

{"title":"Dynamic stimulation promotes functional tissue-like organization of a 3D human lymphoid microenvironment model in vitro.","authors":"Dafne Barozzi, Fiorella Scagnoli, Federica Barbaglio, Daniela Belloni, Davide Ribezzi, Silvia Farè, Valeria Berno, Riccardo Pinos, Marta Sampietro, Margherita Pauri, Barbara Vergani, Francesco Mantegazza, Paolo Ghia, Cristina Scielzo","doi":"10.1016/j.crmeth.2025.101105","DOIUrl":"10.1016/j.crmeth.2025.101105","url":null,"abstract":"This work focused on generating a three-dimensional (3D) in vitro dynamic model to study chronic lymphocytic leukemia (CLL) cell dissemination, homing, and mechanisms of therapy resistance. We used a gelatin-based, hard porous biomaterial as a support matrix to develop 3D tissue-like models of the human lymph node and bone marrow, which were matured inside bioreactors under dynamic perfusion of medium. Comparing static and dynamic cultures of these 3D constructs revealed that perfusion promoted a tissue-like internal organization of cells, characterized by the expression of specific functional markers and deposition of an intricate extracellular matrix protein network. Recirculation of CLL cells within the dynamic system led to changes in leukemic cell behavior and in the expression of key markers involved in tumor progression. These findings suggest that the model is well suited for investigating the pathophysiological mechanisms of CLL and potentially other hematological malignancies.","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":" ","pages":"101105"},"PeriodicalIF":4.5,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12296514/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144620805","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A generalizable and targeted molecular biopsy approach for in situ cryogenic electron tomography of vitreous brain tissue. 玻璃体脑组织原位低温电子断层扫描的一种可推广的靶向分子活检方法。

IF 4.5

Cell Reports Methods Pub Date : 2025-07-21 Epub Date: 2025-06-16 DOI: 10.1016/j.crmeth.2025.101080

Calina Glynn, Jake L R Smith, Matthew Case, Rebecca Csöndör, Ana Katsini, Maria E Sanita, Thomas S Glen, Avery Pennington, Michael Grange

{"title":"A generalizable and targeted molecular biopsy approach for in situ cryogenic electron tomography of vitreous brain tissue.","authors":"Calina Glynn, Jake L R Smith, Matthew Case, Rebecca Csöndör, Ana Katsini, Maria E Sanita, Thomas S Glen, Avery Pennington, Michael Grange","doi":"10.1016/j.crmeth.2025.101080","DOIUrl":"10.1016/j.crmeth.2025.101080","url":null,"abstract":"Cellular cryogenic electron tomography (cryo-ET) enables the capture of detailed structural information within a biologically relevant environment. However, information in more complex samples, such as multicellular specimens and tissues, is lacking. Importantly, these observations need to be set in the context of populations. Currently, imaging on the molecular scale is limited to a few observations in situ that struggle to be generalized. This is due to limitations in throughput and versatility employed by current instrumentation. Here, we utilize plasma focused ion beam milling to examine the molecular landscape of mouse hippocampus by cryo-ET. We reveal the complex organization of macromolecules in targeted regions across CA1 stratum pyramidale (sp) to radiatum (sr), representing a molecular atlas of hippocampal architecture in adult mice. The combination of instrumentation and application of technical advancements provides a framework to explore specific structural questions within other tissues in a targeted manner.","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":" ","pages":"101080"},"PeriodicalIF":4.5,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12296509/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144318149","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

NanoFLUID is a flexible bioelectronic delivery patch. 纳米流体是一种柔性的生物电子输送贴片。

IF 4.5

Cell Reports Methods Pub Date : 2025-07-21 DOI: 10.1016/j.crmeth.2025.101112

Rui Ye, Yupei Zhang, Shugang Qin

引用次数: 0

A single-cell transposable element atlas of human cell identity. 人类细胞身份的单细胞转座因子图谱。

IF 4.5

Cell Reports Methods Pub Date : 2025-07-21 Epub Date: 2025-06-20 DOI: 10.1016/j.crmeth.2025.101086

Helena Reyes-Gopar, Jez L Marston, Bhavya Singh, Matthew Greenig, Jonah Lin, Mario A Ostrowski, Kipchoge N Randall, Santiago Sandoval-Motta, Nicholas Dopkins, Elsa Lawrence, Morgan M O'Mara, Tongyi Fei, Rodrigo R R Duarte, Timothy R Powell, Enrique Hernández-Lemus, Luis P Iñiguez, Douglas F Nixon, Matthew L Bendall

{"title":"A single-cell transposable element atlas of human cell identity.","authors":"Helena Reyes-Gopar, Jez L Marston, Bhavya Singh, Matthew Greenig, Jonah Lin, Mario A Ostrowski, Kipchoge N Randall, Santiago Sandoval-Motta, Nicholas Dopkins, Elsa Lawrence, Morgan M O'Mara, Tongyi Fei, Rodrigo R R Duarte, Timothy R Powell, Enrique Hernández-Lemus, Luis P Iñiguez, Douglas F Nixon, Matthew L Bendall","doi":"10.1016/j.crmeth.2025.101086","DOIUrl":"10.1016/j.crmeth.2025.101086","url":null,"abstract":"Single-cell RNA sequencing (scRNA-seq) is revolutionizing the study of complex biological systems. However, most sequencing studies overlook the contribution of transposable element (TE) expression to the transcriptome. The quantification of locus-specific TE expression in scRNA-seq experiments is challenging due to their repetitive sequence content and poorly characterized annotations. Here, we developed a computational tool for single-cell transposable element locus-level analysis of scRNA sequencing (Stellarscope) that reassigns multimapped reads to specific genomic loci using an expectation maximization algorithm. Using Stellarscope, we built an atlas of TE expression in human PBMCs. We found that locus-specific TEs delineate cell types and define cell subsets not identified by standard mRNA expression profiles. Altogether, this study provides comprehensive insights into the influence of TEs in human biology at the single-cell level.","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":" ","pages":"101086"},"PeriodicalIF":4.5,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12296447/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144340482","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0