{"title":"Minimally invasive, wide-field two-photon imaging of the brainstem at cellular resolution.","authors":"Masakazu Agetsuma, Azumi Hatakeyama, Daisuke Yamada, Hiroshi Kuniishi, Chihiro Ito, Eri Takeuchi, Shinji Tsuji, Motosuke Tsutsumi, Takako Ichiki, Kohei Otomo, Toshinori Yoshioka, Tomoko Kobayashi, Atsushi Noritake, Yoshitsugu Aoki, Tomomi Nemoto, Hiroshi Yukawa, Akiyoshi Saitoh, Junichi Nabekura, Masayuki Sekiguchi","doi":"10.1016/j.crmeth.2025.101010","DOIUrl":"https://doi.org/10.1016/j.crmeth.2025.101010","url":null,"abstract":"<p><p>Brain-viscera communication is crucial for regulating mental health, with the vagus nerve being a key structure mediating this interaction. Clinically, artificial vagus nerve stimulation (VNS) is used to treat various neuropsychiatric disorders, highlighting the importance of vagal afferent fibers in emotion regulation. The nucleus tractus solitarii (NTS) is a brainstem structure proposed to receive signals from vagal afferents and relay them to brain networks for emotion regulation. However, due to the anatomical complexity and difficulty in accessing the deep-brain NTS region in vivo, its underlying mechanisms remain unclear. Here, we developed a wide-field and deep-brain two-photon imaging method using a double-prism optical interface. This approach enables cellular-resolution imaging to specifically detect NTS neural activity while largely preserving the overlying cerebellum, a region also implicated in emotion regulation. We evaluated NTS neuronal responses to VNS and a gastrointestinal hormone, demonstrating the method's utility for investigating the vagus-NTS pathway in vivo.</p>","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":" ","pages":"101010"},"PeriodicalIF":4.3,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143789245","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Cell Reports MethodsPub Date : 2025-03-24Epub Date: 2025-03-12DOI: 10.1016/j.crmeth.2025.100991
Wolu Chukwu, Siyun Lee, Alexander Crane, Shu Zhang, Sophie Webster, Oumayma Dakhama, Ipsa Mittra, Carlos Rauert, Marcin Imielinski, Rameen Beroukhim, Frank Dubois, Simona Dalin
{"title":"A sequence context-based approach for classifying tumor structural variants without paired normal samples.","authors":"Wolu Chukwu, Siyun Lee, Alexander Crane, Shu Zhang, Sophie Webster, Oumayma Dakhama, Ipsa Mittra, Carlos Rauert, Marcin Imielinski, Rameen Beroukhim, Frank Dubois, Simona Dalin","doi":"10.1016/j.crmeth.2025.100991","DOIUrl":"10.1016/j.crmeth.2025.100991","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":" ","pages":"100991"},"PeriodicalIF":4.3,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143626361","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Xueying Lyu, Russell Wing-Yeung Mok, Hoi-Ying Chan, Tina Suoangbaji, Qian Li, Fanhong Zeng, Renwen Long, Irene Oi-Lin Ng, Loey Lung-Yi Mak, Daniel Wai-Hung Ho
{"title":"AVID enables sensitive and accurate viral integration detection across human cancers.","authors":"Xueying Lyu, Russell Wing-Yeung Mok, Hoi-Ying Chan, Tina Suoangbaji, Qian Li, Fanhong Zeng, Renwen Long, Irene Oi-Lin Ng, Loey Lung-Yi Mak, Daniel Wai-Hung Ho","doi":"10.1016/j.crmeth.2025.101007","DOIUrl":"https://doi.org/10.1016/j.crmeth.2025.101007","url":null,"abstract":"<p><p>Oncovirus infection is a key etiological risk factor of human cancers, which triggers virus integration in the host genome. Viral integration can lead to structural variation, gene dysfunction, and genome instability, promoting tumorigenesis. To support the investigation of virus-associated cancer and improve the detection of virus infection, we developed an algorithm called AVID (accurate viral integration detector) for viral integration detection. AVID was built by overcoming the existing detection limitations, enhancing sensitivity and accuracy, and expanding additional functions of viral integration detection. The performance of AVID was estimated in simulated datasets and experimentally validated datasets compared with other tools. To demonstrate its wide applicability, we also tested AVID on viral integration detection in multiple oncovirus-associated human cancers, including hepatocellular carcinoma (HCC), cervical cancer, and nasopharyngeal carcinoma. Taken together, our study developed an improved and applicable tool for viral integration detection and visualization to facilitate further exploration of virus-infected diseases.</p>","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":"5 3","pages":"101007"},"PeriodicalIF":4.3,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143711473","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sean Keeley, Miriam Fernández-Lajarín, David Bergemann, Nicolette John, Lily Parrott, Brittany E Andrea, Juan Manuel González-Rosa
{"title":"Rapid and robust generation of cardiomyocyte-specific crispants in zebrafish using the cardiodeleter system.","authors":"Sean Keeley, Miriam Fernández-Lajarín, David Bergemann, Nicolette John, Lily Parrott, Brittany E Andrea, Juan Manuel González-Rosa","doi":"10.1016/j.crmeth.2025.101003","DOIUrl":"https://doi.org/10.1016/j.crmeth.2025.101003","url":null,"abstract":"<p><p>CRISPR-Cas9 has accelerated loss-of-function studies in zebrafish, but creating tissue-specific mutant lines is still labor intensive. While some tissue-specific Cas9 zebrafish lines exist, standardized methods for gene targeting, including guide RNA (gRNA) delivery, are lacking, limiting broader use in the community. To tackle these limitations, we develop a cardiomyocyte-specific Cas9 line, the cardiodeleter, that efficiently generates biallelic mutations in combination with gene-specific gRNAs. We create transposon-based guide shuttles that deliver gRNAs targeting a gene of interest while permanently labeling cells susceptible to becoming mutant. We validate this modular approach by deleting five genes (ect2, tnnt2a, cmlc2, amhc, and erbb2), resulting in the loss of the corresponding protein or phenocopy of established mutants. We provide detailed protocols for generating guide shuttles, facilitating the adoption of these techniques in the zebrafish community. Our approach enables rapid generation of tissue-specific crispants and analysis of mosaic phenotypes, making it a valuable tool for cell-autonomous studies and genetic screening.</p>","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":"5 3","pages":"101003"},"PeriodicalIF":4.3,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143711481","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ting An Lee, Jan Morlock, John Allan, Harrison Steel
{"title":"Directing microbial co-culture composition using cybernetic control.","authors":"Ting An Lee, Jan Morlock, John Allan, Harrison Steel","doi":"10.1016/j.crmeth.2025.101009","DOIUrl":"https://doi.org/10.1016/j.crmeth.2025.101009","url":null,"abstract":"<p><p>We demonstrate a cybernetic approach to control the composition of a P. putida and E. coli co-culture that does not rely on genetic engineering to interface cells with computers. We first show how composition information can be extracted from different bioreactor measurements and then combined with a system model using an extended Kalman filter to generate accurate estimates of a noisy system. We then demonstrate that adjusting the culture temperature can drive the composition due to the species' different optimal temperatures. Using a proportional-integral control algorithm, we are able to track dynamic references with real-time noise rejection and independence from starting conditions such as inoculation ratio. We stabilize the co-culture for 7 days (∼250 generations) with the experiment ending before the cells could adapt out of the control. This cybernetic framework is broadly applicable, with different microbes' unique characteristics enabling robust control over diverse co-cultures.</p>","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":"5 3","pages":"101009"},"PeriodicalIF":4.3,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143711476","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Daan B Boltje, Radim Skoupý, Clémence Taisne, Wiel H Evers, Arjen J Jakobi, Jacob P Hoogenboom
{"title":"Thickness- and quality-controlled fabrication of fluorescence-targeted frozen-hydrated lamellae.","authors":"Daan B Boltje, Radim Skoupý, Clémence Taisne, Wiel H Evers, Arjen J Jakobi, Jacob P Hoogenboom","doi":"10.1016/j.crmeth.2025.101004","DOIUrl":"https://doi.org/10.1016/j.crmeth.2025.101004","url":null,"abstract":"<p><p>Cryogenic focused ion beam (FIB) milling is essential for fabricating thin lamella-shaped samples out of frozen-hydrated cells for high-resolution structure determination. Structural information can only be resolved at high resolution if the lamella thickness is between 100 and 200 nm. While the lamella fabrication workflow has improved significantly since its conception, quantitative, live feedback on lamella thickness, quality, and biological target inclusion remains lacking. Using coincident light microscopy integrated into the FIB scanning electron microscope (FIB-SEM), we present three strategies that enable accurate, live control during lamella fabrication. First, we combine four-dimensional (4D) STEM with fluorescence microscopy (FM) targeting to determine lamella thickness. Second, with reflected light microscopy (RLM), we screen target sites for ice contamination and monitor lamella thickness and protective Pt coating integrity during FIB milling. Third, we exploit thin-film interference for fine-grained feedback on thickness uniformity below 500 nm. Finally, we present a fluorescence-targeted, quality-controlled workflow for frozen-hydrated lamellae, benchmarked with excellent agreement with energy-filtered transmission electron microscopy (EFTEM) measurements and tomograms from electron cryotomography.</p>","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":"5 3","pages":"101004"},"PeriodicalIF":4.3,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143711493","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Małgorzata Lisowska, Erin G Worrall, Filip Zavadil-Kokas, Keith Charlton, Euan Murray, M Aiman Mohtar, Radovan Krejcir, Vaclav Hrabal, Jack Brydon, Ainhoa Gonzalez Urionabarrenetxea, David G Saliba, Mariana Grima, Umesh Kalathiya, Petr Muller, Adam Krejci, Borivoj Vojtesek, Kathryn L Ball, Robin Fahraeus, David J Argyle, Maciej Parys, Ted R Hupp
{"title":"The development of a canine single-chain phage antibody library to isolate recombinant antibodies for use in translational cancer research.","authors":"Małgorzata Lisowska, Erin G Worrall, Filip Zavadil-Kokas, Keith Charlton, Euan Murray, M Aiman Mohtar, Radovan Krejcir, Vaclav Hrabal, Jack Brydon, Ainhoa Gonzalez Urionabarrenetxea, David G Saliba, Mariana Grima, Umesh Kalathiya, Petr Muller, Adam Krejci, Borivoj Vojtesek, Kathryn L Ball, Robin Fahraeus, David J Argyle, Maciej Parys, Ted R Hupp","doi":"10.1016/j.crmeth.2025.101008","DOIUrl":"https://doi.org/10.1016/j.crmeth.2025.101008","url":null,"abstract":"<p><p>The development of canine immunotolerant monoclonal antibodies can accelerate the invention of new medicines for both canine and human diseases. We develop a methodology to clone the naive, somatically mutated variable domain repertoire from canine B cell mRNA using 5'RACE PCR. A set of degenerate primers were then designed and used to clone variable domain genes into archival \"holding\" plasmid libraries. These archived variable domain genes were then combinatorially ligated to produce a scFv M13 phage library. Next-generation long-read and short-read DNA sequencing methodologies were developed to annotate features of the cloned library including CDR diversity and IGHV/IGKV/IGLV subfamily distribution. A synthetic immunoglobulin G was developed from this scFv library to the canine immune checkpoint receptor PD-1. This synthetic platform can be used to clone and annotate archived antibody variable domain genes for use in perpetuity in order to develop improved preclinical models for the treatment of complex human diseases.</p>","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":"5 3","pages":"101008"},"PeriodicalIF":4.3,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143711486","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Shengyuan He, Shangqin Sun, Kun Liu, Bo Pang, Yun Xiao
{"title":"Comprehensive assessment of computational methods for cancer immunoediting.","authors":"Shengyuan He, Shangqin Sun, Kun Liu, Bo Pang, Yun Xiao","doi":"10.1016/j.crmeth.2025.101006","DOIUrl":"https://doi.org/10.1016/j.crmeth.2025.101006","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":"5 3","pages":"101006"},"PeriodicalIF":4.3,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143711474","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Cell Reports MethodsPub Date : 2025-03-24Epub Date: 2025-03-17DOI: 10.1016/j.crmeth.2025.101011
Vinicius Daguano Gastaldi, Martin Hindermann, Justus B H Wilke, Anja Ronnenberg, Sahab Arinrad, Sabine Kraus, Anne-Fleur Wildenburg, Antonios Ntolkeras, Micah J Provost, Liu Ye, Yasmina Curto, Jonathan-Alexis Cortés-Silva, Umer Javed Butt, Klaus-Armin Nave, Kamilla Woznica Miskowiak, Hannelore Ehrenreich
{"title":"A comprehensive and standardized pipeline for automated profiling of higher cognition in mice.","authors":"Vinicius Daguano Gastaldi, Martin Hindermann, Justus B H Wilke, Anja Ronnenberg, Sahab Arinrad, Sabine Kraus, Anne-Fleur Wildenburg, Antonios Ntolkeras, Micah J Provost, Liu Ye, Yasmina Curto, Jonathan-Alexis Cortés-Silva, Umer Javed Butt, Klaus-Armin Nave, Kamilla Woznica Miskowiak, Hannelore Ehrenreich","doi":"10.1016/j.crmeth.2025.101011","DOIUrl":"10.1016/j.crmeth.2025.101011","url":null,"abstract":"<p><p>In rodent behavior research, observer-independent methods, such as the IntelliCage, enhance data collection in a social, and thus stress-reduced, environment. The IntelliCage system allows experimenters to create cognitive challenges for mice motivated by rewards. Given the extensive and diverse data from IntelliCage, there is a high demand for automated analysis. Here, we introduce IntelliR, a free and standardized pipeline for analyzing IntelliCage data, including a cognition index for performance comparison across challenges. IntelliR supports the automatic analysis of three challenges that cover spatial, episodic-like, and working memory with their reversal tests and can also be adapted for other designs. Results from three cohorts of adult female C57B6 mice showed improved task proficiency over time. To validate cognitive impairment detection, we used adult female NexCreERT2xRosa26-eGFP-DTA mice after neuron ablation in cortex and hippocampus, in which we observed reduced learning capabilities. IntelliR integrates easily into research, improving time management and reproducibility.</p>","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":" ","pages":"101011"},"PeriodicalIF":4.3,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143658871","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Cell Reports MethodsPub Date : 2025-03-24Epub Date: 2025-03-17DOI: 10.1016/j.crmeth.2025.101005
Hannah M Lynn, Jeffrey I Gordon
{"title":"Sequential co-assembly reduces computational resources and errors in metagenome-assembled genomes.","authors":"Hannah M Lynn, Jeffrey I Gordon","doi":"10.1016/j.crmeth.2025.101005","DOIUrl":"10.1016/j.crmeth.2025.101005","url":null,"abstract":"<p><p>Generating metagenome-assembled genomes from DNA shotgun sequencing datasets can demand considerable computational resources. Here, we describe a sequential co-assembly method that reduces the assembly of duplicate reads through successive application of single-node computing tools for read assembly and mapping. Using a simulated mouse microbiome DNA shotgun sequencing dataset, we demonstrated that this approach shortens assembly time, uses less memory than traditional co-assembly, and produces significantly fewer assembly errors. Applying sequential co-assembly to shotgun sequencing reads from (1) a longitudinal study of gut microbiomes from undernourished Bangladeshi children and (2) a 2.3-terabyte dataset generated from gnotobiotic mice colonized with pooled microbiomes from these children that was too large to be handled by a traditional co-assembly approach also demonstrated significant reductions in assembly time and memory requirements. These results suggest that this approach should be useful in resource-constrained settings, including in low- and middle-income countries.</p>","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":" ","pages":"101005"},"PeriodicalIF":4.3,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143658873","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}