Nature Protocols最新文献_第6页

Direct and quantitative analysis of tRNA acylation using intact tRNA liquid chromatography-mass spectrometry. 使用完整tRNA液相色谱-质谱法直接定量分析tRNA酰化。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-01 Epub Date: 2025-01-06 DOI: 10.1038/s41596-024-01086-9

Riley Fricke, Isaac Knudson, Cameron Verdayne Swenson, Sarah Smaga, Alanna Schepartz

{"title":"Direct and quantitative analysis of tRNA acylation using intact tRNA liquid chromatography-mass spectrometry.","authors":"Riley Fricke, Isaac Knudson, Cameron Verdayne Swenson, Sarah Smaga, Alanna Schepartz","doi":"10.1038/s41596-024-01086-9","DOIUrl":"https://doi.org/10.1038/s41596-024-01086-9","url":null,"abstract":"Aminoacyl-tRNA synthetases (aaRSs) provide an essential functional link between an mRNA sequence and the protein it encodes. aaRS enzymes catalyze a two-step chemical reaction that acylates specific tRNAs with a cognate α-amino acid. In addition to their role in translation, acylated tRNAs contribute to non-ribosomal natural product biosynthesis and are implicated in multiple human diseases. In synthetic biology, the acylation of tRNAs with a non-canonical α-amino acid or, more recently, a non-α-amino acid monomer is a critical first step in the incorporation of these monomers into proteins, where they can be used for fundamental and applied science. These endeavors all demand an understanding of aaRS activity and specificity. Here, we describe a liquid chromatography-mass spectrometry assay that directly monitors aaRS activity by detecting the intact acyl-tRNA product. After a simple tRNA acylation reaction workup, acyl- and non-acyl-tRNA molecules are resolved by using ion-pairing reverse-phase chromatography, and their exact masses are determined by using high-resolution time-of-flight mass spectrometry. Our assay is fast and simple, quantifies reaction yields as low as 0.23% and can also be used on tRNAs acylated with flexizyme to detect products that are undetectable by using standard techniques. The protocol requires basic expertise in molecular biology, liquid chromatography-mass spectrometry and RNase-free techniques. This protocol takes ≥5 h to complete, depending on the number of samples.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"20 5","pages":"1246-1274"},"PeriodicalIF":13.1,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143993949","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

RNA sample optimization for cryo-EM analysis. 优化用于冷冻电镜分析的 RNA 样品。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-01 Epub Date: 2024-11-15 DOI: 10.1038/s41596-024-01072-1

Xingyu Chen, Liu Wang, Jiahao Xie, Jakub S Nowak, Bingnan Luo, Chong Zhang, Guowen Jia, Jian Zou, Dingming Huang, Sebastian Glatt, Yang Yang, Zhaoming Su

{"title":"RNA sample optimization for cryo-EM analysis.","authors":"Xingyu Chen, Liu Wang, Jiahao Xie, Jakub S Nowak, Bingnan Luo, Chong Zhang, Guowen Jia, Jian Zou, Dingming Huang, Sebastian Glatt, Yang Yang, Zhaoming Su","doi":"10.1038/s41596-024-01072-1","DOIUrl":"10.1038/s41596-024-01072-1","url":null,"abstract":"RNAs play critical roles in most biological processes. Although the three-dimensional (3D) structures of RNAs primarily determine their functions, it remains challenging to experimentally determine these 3D structures due to their conformational heterogeneity and intrinsic dynamics. Cryogenic electron microscopy (cryo-EM) has recently played an emerging role in resolving dynamic conformational changes and understanding structure-function relationships of RNAs including ribozymes, riboswitches and bacterial and viral noncoding RNAs. A variety of methods and pipelines have been developed to facilitate cryo-EM structure determination of challenging RNA targets with small molecular weights at subnanometer to near-atomic resolutions. While a wide range of conditions have been used to prepare RNAs for cryo-EM analysis, correlations between the variables in these conditions and cryo-EM visualizations and reconstructions remain underexplored, which continue to hinder optimizations of RNA samples for high-resolution cryo-EM structure determination. Here we present a protocol that describes rigorous screenings and iterative optimizations of RNA preparation conditions that facilitate cryo-EM structure determination, supplemented by cryo-EM data processing pipelines that resolve RNA dynamics and conformational changes and RNA modeling algorithms that generate atomic coordinates based on moderate- to high-resolution cryo-EM density maps. The current protocol is designed for users with basic skills and experience in RNA biochemistry, cryo-EM and RNA modeling. The expected time to carry out this protocol may range from 3 days to more than 3 weeks, depending on the many variables described in the protocol. For particularly challenging RNA targets, this protocol could also serve as a starting point for further optimizations.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1114-1157"},"PeriodicalIF":13.1,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142639364","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Independent SAGE as an example of effective public dialogue on scientific research. 将独立的 SAGE 作为就科学研究开展有效公众对话的范例。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-01 Epub Date: 2024-12-12 DOI: 10.1038/s41596-024-01089-6

Trisha Greenhalgh, Anthony Costello, Sheena Cruickshank, Stephen Griffin, Aris Katzourakis, Lennard Lee, Martin McKee, Susan Michie, Christina Pagel, Stephen Reicher, Alice Roberts, Duncan Robertson, Helen Salisbury, Kit Yates

{"title":"Independent SAGE as an example of effective public dialogue on scientific research.","authors":"Trisha Greenhalgh, Anthony Costello, Sheena Cruickshank, Stephen Griffin, Aris Katzourakis, Lennard Lee, Martin McKee, Susan Michie, Christina Pagel, Stephen Reicher, Alice Roberts, Duncan Robertson, Helen Salisbury, Kit Yates","doi":"10.1038/s41596-024-01089-6","DOIUrl":"10.1038/s41596-024-01089-6","url":null,"abstract":"The World Health Organization declared COVID-19 to be a public health emergency of international concern on 30 January 2020 and then a pandemic on 11 March 2020. In early 2020, a group of UK scientists volunteered to provide the public with up-to-date and transparent scientific information. The group formed the Independent Scientific Advisory Group for Emergencies (Independent SAGE) and provided live weekly briefings to the public via YouTube. In this Perspective, we describe how and why this group came together and the challenges it faced. We reflect on 4 years of scientific information broadcasting and discuss the guiding principles followed by Independent SAGE, which may be broadly transferable for strengthening the scientist-public dialogue during public health emergencies in future settings. We discuss the provision of clarity and transparency, engagement with the science-policy interface, the practice of interdisciplinarity, the centrality of addressing inequity, the need for dialogue and partnership with the public, the importance of support for advocacy groups, the diversification of communication channels and modalities, the adoption of regular and organized internal communications, the resourcing and support of the group's communications and the active opposition of misinformation and disinformation campaigns. We reflect on what we might do differently next time and propose research aimed at building the evidence base for optimizing informal scientific advisory groups in crisis situations.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1103-1113"},"PeriodicalIF":13.1,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142818696","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Versatile MRI acquisition and processing protocol for population-based neuroimaging. 基于人群的神经成像的多功能MRI采集和处理协议。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-01 Epub Date: 2024-12-13 DOI: 10.1038/s41596-024-01085-w

Alexandra Koch, Rüdiger Stirnberg, Santiago Estrada, Weiyi Zeng, Valerie Lohner, Mohammad Shahid, Philipp Ehses, Eberhard D Pracht, Martin Reuter, Tony Stöcker, Monique M B Breteler

{"title":"Versatile MRI acquisition and processing protocol for population-based neuroimaging.","authors":"Alexandra Koch, Rüdiger Stirnberg, Santiago Estrada, Weiyi Zeng, Valerie Lohner, Mohammad Shahid, Philipp Ehses, Eberhard D Pracht, Martin Reuter, Tony Stöcker, Monique M B Breteler","doi":"10.1038/s41596-024-01085-w","DOIUrl":"10.1038/s41596-024-01085-w","url":null,"abstract":"Neuroimaging has an essential role in studies of brain health and of cerebrovascular and neurodegenerative diseases, requiring the availability of versatile magnetic resonance imaging (MRI) acquisition and processing protocols. We designed and developed a multipurpose high-resolution MRI protocol for large-scale and long-term population neuroimaging studies that includes structural, diffusion-weighted and functional MRI modalities. This modular protocol takes almost 1 h of scan time and is, apart from a concluding abdominal scan, entirely dedicated to the brain. The protocol links the acquisition of an extensive set of MRI contrasts directly to the corresponding fully automated data processing pipelines and to the required quality assurance of the MRI data and of the image-derived phenotypes. Since its successful implementation in the population-based Rhineland Study (ongoing, currently more than 11,000 participants, target participant number of 20,000), the proposed MRI protocol has proved suitable for epidemiological and clinical cross-sectional and longitudinal studies, including multisite studies. The approach requires expertise in magnetic resonance image acquisition, in computer science for the data management and the execution of processing pipelines, and in brain anatomy for the quality assessment of the MRI data. The protocol takes ~1 h of MRI acquisition and ~20 h of data processing to complete for a single dataset, but parallelization over multiple datasets using high-performance computing resources reduces the processing time. By making the protocol, MRI sequences and pipelines available, we aim to contribute to better comparability, interoperability and reusability of large-scale neuroimaging data.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1223-1245"},"PeriodicalIF":13.1,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142822226","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Design, performance, processing, and validation of a pooled CRISPR perturbation screen for bacterial toxins. 针对细菌毒素的集合 CRISPR 扰乱筛选的设计、性能、处理和验证。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-01 Epub Date: 2024-11-01 DOI: 10.1038/s41596-024-01075-y

Songhai Tian, Yuhang Qin, Yuxuan Wu, Min Dong

{"title":"Design, performance, processing, and validation of a pooled CRISPR perturbation screen for bacterial toxins.","authors":"Songhai Tian, Yuhang Qin, Yuxuan Wu, Min Dong","doi":"10.1038/s41596-024-01075-y","DOIUrl":"10.1038/s41596-024-01075-y","url":null,"abstract":"Unbiased forward genetic screens have been extensively employed in biological research to elucidate functional genomics. In pooled clustered regularly interspaced short palindromic repeats (CRISPR) perturbation screens, various genetically encoded gain-of-function or loss-of-function mutations are introduced into a heterogeneous population of cells. Subsequently, these cells are screened for phenotypes, perturbation-associated genotypes are analyzed and a connection between genotype and phenotype is determined. CRISPR screening techniques enable the investigation of important biological questions, such as how bacterial toxins kill cells and cause disease. However, the broad spectrum of effects caused by diverse toxins presents a challenge when selecting appropriate screening strategies. Here, we provide a step-by-step protocol for a genome-wide pooled CRISPR perturbation screen to study bacterial toxins. We describe technical considerations, pilot experiments, library construction, screen execution, result analysis and validation of the top enriched hits. These screens are applicable for many different types of toxins and are anticipated to reveal a repertoire of host factors crucial in the intoxication pathway, such as receptors, trafficking/translocation factors and substrates. The entire protocol takes 21-27 weeks and does not require specialized knowledge beyond basic biology.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1158-1195"},"PeriodicalIF":13.1,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564733","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Comprehensive microcrystal electron diffraction sample preparation for cryo-EM. 低温电镜综合微晶电子衍射样品制备。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-01 Epub Date: 2024-12-20 DOI: 10.1038/s41596-024-01088-7

William J Nicolas, Cody Gillman, Sara J Weaver, Max T B Clabbers, Anna Shiriaeva, Ampon Sae Her, Michael W Martynowycz, Tamir Gonen

{"title":"Comprehensive microcrystal electron diffraction sample preparation for cryo-EM.","authors":"William J Nicolas, Cody Gillman, Sara J Weaver, Max T B Clabbers, Anna Shiriaeva, Ampon Sae Her, Michael W Martynowycz, Tamir Gonen","doi":"10.1038/s41596-024-01088-7","DOIUrl":"10.1038/s41596-024-01088-7","url":null,"abstract":"Microcrystal electron diffraction (MicroED) has advanced structural methods across a range of sample types, from small molecules to proteins. This cryogenic electron microscopy (cryo-EM) technique involves the continuous rotation of small 3D crystals in the electron beam, while a high-speed camera captures diffraction data in the form of a movie. The crystal structure is subsequently determined by using established X-ray crystallographic software. MicroED is a technique still under development, and hands-on expertise in sample preparation, data acquisition and processing is not always readily accessible. This comprehensive guide on MicroED sample preparation addresses commonly used methods for various sample categories, including room temperature solid-state small molecules and soluble and membrane protein crystals. Beyond detailing the steps of sample preparation for new users, and because every crystal requires unique growth and sample-preparation conditions, this resource provides instructions and optimization strategies for MicroED sample preparation. The protocol is suitable for users with expertise in biochemistry, crystallography, general cryo-EM and crystallography data processing. MicroED experiments, from sample vitrification to final structure, can take anywhere from one workday to multiple weeks, especially when cryogenic focused ion beam milling is involved.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1275-1309"},"PeriodicalIF":13.1,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12173078/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142872610","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Generating allogeneic CAR-NKT cells for off-the-shelf cancer immunotherapy with genetically engineered HSP cells and feeder-free differentiation culture. 用基因工程HSP细胞和无饲料分化培养产生用于现成癌症免疫治疗的同种异体CAR-NKT细胞。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-01 Epub Date: 2025-01-17 DOI: 10.1038/s41596-024-01077-w

Yan-Ruide Li, Kuangyi Zhou, Derek Lee, Yichen Zhu, Tyler Halladay, Jiaji Yu, Yang Zhou, Zibai Lyu, Ying Fang, Yuning Chen, Sasha Semaan, Lili Yang

{"title":"Generating allogeneic CAR-NKT cells for off-the-shelf cancer immunotherapy with genetically engineered HSP cells and feeder-free differentiation culture.","authors":"Yan-Ruide Li, Kuangyi Zhou, Derek Lee, Yichen Zhu, Tyler Halladay, Jiaji Yu, Yang Zhou, Zibai Lyu, Ying Fang, Yuning Chen, Sasha Semaan, Lili Yang","doi":"10.1038/s41596-024-01077-w","DOIUrl":"10.1038/s41596-024-01077-w","url":null,"abstract":"The clinical potential of current chimeric antigen receptor-engineered T (CAR-T) cell therapy is hampered by its autologous nature that poses considerable challenges in manufacturing, costs and patient selection. This spurs demand for off-the-shelf therapies. Here we introduce an ex vivo feeder-free culture method to differentiate gene-engineered hematopoietic stem and progenitor (HSP) cells into allogeneic invariant natural killer T (AlloNKT) cells and their CAR-armed derivatives (AlloCAR-NKT cells). We include detailed information on lentivirus generation and titration, as well as the five stages of ex vivo culture required to generate AlloCAR-NKT cells, including HSP cell engineering, HSP cell expansion, NKT cell differentiation, NKT cell deep differentiation and NKT cell expansion. In addition, we describe procedures for evaluating the pharmacology, antitumor efficacy and mechanism of action of AlloCAR-NKT cells. It takes ~2 weeks to generate and titrate lentiviruses and ~6 weeks to generate mature AlloCAR-NKT cells. Competence with human stem cell and T cell culture, gene engineering and flow cytometry is required for optimal results.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1352-1388"},"PeriodicalIF":13.1,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008632","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Validation and quantification of peptide antigens presented on MHCs using SureQuant. 使用 SureQuant 验证和量化呈现在 MHC 上的多肽抗原。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-01 Epub Date: 2024-10-22 DOI: 10.1038/s41596-024-01076-x

Owen Leddy, Yufei Cui, Ryuhjin Ahn, Lauren Stopfer, Elizabeth Choe, Do Hun Kim, Malte Roerden, Stefani Spranger, Bryan D Bryson, Forest M White

{"title":"Validation and quantification of peptide antigens presented on MHCs using SureQuant.","authors":"Owen Leddy, Yufei Cui, Ryuhjin Ahn, Lauren Stopfer, Elizabeth Choe, Do Hun Kim, Malte Roerden, Stefani Spranger, Bryan D Bryson, Forest M White","doi":"10.1038/s41596-024-01076-x","DOIUrl":"10.1038/s41596-024-01076-x","url":null,"abstract":"Vaccines and immunotherapies that target peptide-major histocompatibility complexes (peptide-MHCs) have the potential to address multiple unmet medical needs in cancer and infectious disease. Designing vaccines and immunotherapies to target peptide-MHCs requires accurate identification of target peptides in infected or cancerous cells or tissue, and may require absolute or relative quantification to identify abundant targets and measure changes in presentation under different treatment conditions. Internal standard parallel reaction monitoring (also known as 'SureQuant') can be used to validate and/or quantify MHC peptides previously identified by using untargeted methods such as data-dependent acquisition. SureQuant MHC has three main use cases: (i) conclusive confirmation of the identities of putative MHC peptides via comparison with an internal synthetic stable isotope labeled (SIL) peptide standard; (ii) accurate relative quantification by using pre-formed heavy isotope-labeled peptide-MHC complexes (hipMHCs) containing SIL peptides as internal controls for technical variation; and (iii) absolute quantification of each target peptide by using different amounts of hipMHCs loaded with synthetic peptides containing one, two or three SIL amino acids to provide an internal standard curve. Absolute quantification can help determine whether the abundance of a peptide-MHC is sufficient for certain therapeutic modalities. SureQuant MHC therefore provides unique advantages for immunologists seeking to confidently validate antigenic targets and understand the dynamics of the MHC repertoire. After synthetic standards are ordered (3-4 weeks), this protocol can be carried out in 3-4 days and is suitable for individuals with mass spectrometry experience who are comfortable with customizing instrument methods.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1196-1222"},"PeriodicalIF":13.1,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12012165/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504449","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Establishment of human expanded potential stem cell lines via preimplantation embryo cultivation and somatic cell reprogramming. 通过胚胎着床前培养和体细胞重编程建立人类扩展潜能干细胞系。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-04-29 DOI: 10.1038/s41596-025-01168-2

Degong Ruan, Andy Chun Hang Chen, Timothy Theodore Ka Ki Tam, Wen Huang, Jilong Guo, Shao Xu, Hanzhang Ruan, Sze Wan Fong, Xueyan Liu, Xuefei Gao, William Shu Biu Yeung, Yin Lau Lee, Pentao Liu

{"title":"Establishment of human expanded potential stem cell lines via preimplantation embryo cultivation and somatic cell reprogramming.","authors":"Degong Ruan, Andy Chun Hang Chen, Timothy Theodore Ka Ki Tam, Wen Huang, Jilong Guo, Shao Xu, Hanzhang Ruan, Sze Wan Fong, Xueyan Liu, Xuefei Gao, William Shu Biu Yeung, Yin Lau Lee, Pentao Liu","doi":"10.1038/s41596-025-01168-2","DOIUrl":"https://doi.org/10.1038/s41596-025-01168-2","url":null,"abstract":"We previously reported the derivation of expanded potential stem cells (EPSCs) by modulating signaling pathways involved in preimplantation embryogenesis. These cells exhibit expanded developmental potential into embryonic and extraembryonic lineages, and we have shown that human EPSCs (hEPSCs) possess trophoblast differentiation potency for generating human trophoblast stem cells. Here we report protocols for deriving stable hEPSC lines directly from morula or early blastocyst stages of human preimplantation embryos (hEPSC-em) and by reprogramming human dermal fibroblasts (human induced EPSCs) using six exogenous factors, as an extension to our previous protocols on deriving porcine EPSCs from preimplantation embryos and by reprogramming somatic cells. These hEPSC lines proliferate robustly over long-term passaging and are amenable to both simple indels and precision genome editing. We provide guidance for characterizing these newly established hEPSCs, including cell-cycle analysis, pluripotency validation and karyotyping. The hEPSCs form teratomas with embryonic and extraembryonic cell lineages and readily differentiate into human trophoblast stem cells in vitro. At the molecular level, hEPSCs have unique features such as high expression of core histone genes and low H3K27me3 levels resembling eight-cell/morula stage embryos. These properties make hEPSCs a valuable tool not only for studying early human development but also for potential applications in regenerative medicine. The protocols presented in this manuscript can be readily performed by postgraduate students or postdoctoral fellows and completed within around 2 months.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144044911","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Thioether-mediated protein ubiquitination in constructing affinity- and activity-based ubiquitinated protein probes. 构建基于亲和和活性的泛素化蛋白探针的硫醚介导的蛋白质泛素化。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-04-25 DOI: 10.1038/s41596-025-01162-8

Gregory A Davidson, Zeinab Moafian, Amanda R Sensi, Zhihao Zhuang

{"title":"Thioether-mediated protein ubiquitination in constructing affinity- and activity-based ubiquitinated protein probes.","authors":"Gregory A Davidson, Zeinab Moafian, Amanda R Sensi, Zhihao Zhuang","doi":"10.1038/s41596-025-01162-8","DOIUrl":"https://doi.org/10.1038/s41596-025-01162-8","url":null,"abstract":"Protein ubiquitination, a critical regulatory mechanism and post-translational modification in eukaryotic cells, involves the formation of an isopeptide bond between ubiquitin (Ub) and targeted proteins. Despite extensive investigation into the roles played by protein ubiquitination in various cellular processes, many questions remain to be answered. A major challenge in the biochemical and biophysical characterization of protein ubiquitination, along with its associated pathways and protein players, lies in the generation of ubiquitinated proteins, either in mono- or poly-ubiquitinated forms. Enzymatic and chemical strategies have been reported to address this challenge; however, there are still unmet needs for the facile generation of ubiquitinated proteins in the quantity and homogeneity required to precisely decipher the role of various protein-specific ubiquitination events. In this protocol, we provide the ubiquitin research community with a chemical ubiquitination method enabled by an α-bromoketone-mediated ligation strategy. This method can be readily adapted to generate mono- and poly-ubiquitinated proteins of interest through a cysteine introduced to replace the target lysine, with the native cysteines mutated to serine. Using proliferating cell nuclear antigen (PCNA) as an example, we present herein a detailed protocol for generating di- and tri-Ub PCNA that contains a photo-activatable cross-linker for capturing potential reader proteins. The thioether-mediated protein ligation and purification typically takes 2-3 weeks. An important feature of our ubiquitination strategy is the ability to introduce a Michael-acceptor warhead to the linkage, allowing the generation of activity-based probes for deubiquitinases and ubiquitin-carrying enzymes such as HECT and RBR E3 ubiquitin ligases and E2 enzymes. As such, our method is highly versatile and can be readily adapted to investigate the readers and erasers of many proteins that undergo reversible ubiquitination.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144020115","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0