Nature Protocols最新文献_第5页

Visualizing epigenetic modifications and their spatial proximities in single cells using three DNA-encoded amplifying FISH imaging strategies: BEA-FISH, PPDA-FISH and Cell-TALKING. 利用三种 DNA 编码放大 FISH 成像策略观察单细胞中的表观遗传修饰及其空间邻近性：BEA-FISH、PPDA-FISH 和 Cell-TALKING。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-09-04 DOI: 10.1038/s41596-024-01036-5

Feng Chen, Xinyin Li, Min Bai, Yongxi Zhao

{"title":"Visualizing epigenetic modifications and their spatial proximities in single cells using three DNA-encoded amplifying FISH imaging strategies: BEA-FISH, PPDA-FISH and Cell-TALKING.","authors":"Feng Chen, Xinyin Li, Min Bai, Yongxi Zhao","doi":"10.1038/s41596-024-01036-5","DOIUrl":"https://doi.org/10.1038/s41596-024-01036-5","url":null,"abstract":"Epigenetic modifications and spatial proximities of nucleic acids and proteins play important roles in regulating physiological processes and disease progression. Currently available cell imaging methods, such as fluorescence in situ hybridization (FISH) and immunofluorescence, struggle to detect low-abundance modifications and their spatial proximities. Here we describe a step-by-step protocol for three DNA-encoded amplifying FISH-based imaging strategies to overcome these challenges for varying applications: base-encoded amplifying FISH (BEA-FISH), pairwise proximity-differentiated amplifying FISH (PPDA-FISH) and cellular macromolecules-tethered DNA walking indexing (Cell-TALKING). They all use the similar core principle of DNA-encoded amplification, which transforms different nonsequence molecular features into unique DNA barcodes for in situ rolling circle amplification and FISH analysis. This involves three key reactions in fixed cell samples: target labeling, DNA encoding and rolling circle amplification imaging. Using this protocol, these three imaging strategies achieve in situ counting of low-abundance modifications alone, the pairwise proximity-differentiated visualization of two modifications and the exploration of multiple modifications around one protein (one-to-many proximity), respectively. Low-abundance modifications, including 5-hydroxymethylcytosine, 5-formylcytosine, 5-hydroxymethyluracil and 5-formyluracil, are clearly visualized in single cells. Various combinatorial patterns of nucleic acid modifications and/or histone modifications are found. The whole protocol takes ~2-4 d to complete, depending on different imaging applications.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142133271","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

Tutorial: a guide to diffusion MRI and structural connectomics. 教程：弥散核磁共振成像和结构连接组学指南。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-09-04 DOI: 10.1038/s41596-024-01052-5

Ittai Shamir, Yaniv Assaf

{"title":"Tutorial: a guide to diffusion MRI and structural connectomics.","authors":"Ittai Shamir, Yaniv Assaf","doi":"10.1038/s41596-024-01052-5","DOIUrl":"https://doi.org/10.1038/s41596-024-01052-5","url":null,"abstract":"Diffusion magnetic resonance imaging (dMRI) is a versatile imaging technique that has gained popularity thanks to its sensitive ability to measure displacement of water molecules within a living tissue on a micrometer scale. Although dMRI has been around since the early 1990s, its applications are constantly evolving, primarily regarding the inference of structural connectomics from nerve fiber trajectories. However, these applications require expertise in image processing and statistics, and it can be difficult for a newcomer to choose an appropriate pipeline to fit their research needs, not least because dMRI is such a flexible methodology that dozens of acquisition and analysis pipelines have been developed over the years. This introductory guide is designed for graduate students and researchers in the neuroscience community who are interested in integrating this new methodology regardless of their background in neuroimaging and computational tools. The guide provides a brief overview of the basic dMRI methodologies but focuses on its applications in neuroplasticity and connectomics. The guide starts with dMRI experimental designs and a complete step-by-step pipeline for structural connectomics. The following section covers the basics of dMRI, including parameters and clinical applications (apparent diffusion coefficient, mean diffusivity, fractional anisotropy and microscopic fractional anisotropy), as well as different approaches and models. The final section focuses on structural connectomics, covering subjects from fiber tracking (techniques, evaluation and limitations) to structural networks (constructing, analyzing and visualizing a network).","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142133270","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

A bioswitchable delivery system for microRNA therapeutics based on a tetrahedral DNA nanostructure. 基于四面体 DNA 纳米结构的微 RNA 治疗生物开关输送系统。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-08-30 DOI: 10.1038/s41596-024-01050-7

Songhang Li, Taoran Tian, Tao Zhang, Yunfeng Lin, Xiaoxiao Cai

{"title":"A bioswitchable delivery system for microRNA therapeutics based on a tetrahedral DNA nanostructure.","authors":"Songhang Li, Taoran Tian, Tao Zhang, Yunfeng Lin, Xiaoxiao Cai","doi":"10.1038/s41596-024-01050-7","DOIUrl":"10.1038/s41596-024-01050-7","url":null,"abstract":"As microRNAs (miRNA) regulate almost all physiopathological activities in the human body, miRNA therapeutics that deliver miRNA regulators have attracted considerable attention in the field of nucleic acid drug development. The use of tetrahedral DNA nanostructures to deliver miRNA regulators is promising because of their simple fabrication, enhanced cell entry, effective tissue penetration, biocompatibility and functional editability. This protocol extension builds on our previous protocol for the use of tetrahedral DNA nanostructures and was designed to establish an updated bioswitchable delivery system (BDS) for achieving controlled cargo loading and release. A ribonuclease H-sensitive sequence is designed as a bioswitchable apparatus for the targeted release of the miRNA regulator. The functional sequence of the miRNA regulator and minimal secondary structure formation tendency during annealing are two key points in cargo design. We provide two BDS design strategies; BDS-A comprises an intact DNA tetrahedron with the RNA cargo hanging outside, offering the merits of lower cost, simplicity, and more direct structural design. In the BDS-B design, the RNA regulators are embedded into the DNA tetrahedron, which is beneficial for dermal tissue permeation applications. Following sequence design in Oligo 7 and Tiamat, the BDS assembly is completed and then ribonuclease H achieves controlled release of the miRNA regulator by triggering the bioswitchable apparatus. This is verified via polyacrylamide and agarose gel electrophoresis or fluorophore modifications. Both BDSs show promising cellular membrane permeability, tissue permeability and target inhibition in vitro and in vivo. The assembly and characterization of the BDS can be completed in 4 d, and the validation time for biostability and biological applications will depend on the specific use.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142109658","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

Author Correction: Metagenome analysis using the Kraken software suite. 作者更正：使用 Kraken 软件套件进行元基因组分析。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-08-29 DOI: 10.1038/s41596-024-01064-1

Jennifer Lu, Natalia Rincon, Derrick E Wood, Florian P Breitwieser, Christopher Pockrandt, Ben Langmead, Steven L Salzberg, Martin Steinegger

引用次数: 0

Tutorial: fluorescence lifetime microscopy of membrane mechanosensitive Flipper probes. 教程：膜机械敏感 Flipper 探针的荧光寿命显微镜。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-08-29 DOI: 10.1038/s41596-024-01027-6

Chloé Roffay, Juan Manuel García-Arcos, Pierrik Chapuis, Javier López-Andarias, Falk Schneider, Adai Colom, Caterina Tomba, Ilaria Di Meglio, Katia Barrett, Valentin Dunsing, Stefan Matile, Aurélien Roux, Vincent Mercier

引用次数: 0

Genome-wide analysis of the biophysical properties of chromatin and nuclear proteins in living cells with Hi-D. 利用 Hi-D 对活细胞中染色质和核蛋白的生物物理特性进行全基因组分析。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-08-28 DOI: 10.1038/s41596-024-01038-3

Cesar Augusto Valades-Cruz, Roman Barth, Marwan Abdellah, Haitham A Shaban

{"title":"Genome-wide analysis of the biophysical properties of chromatin and nuclear proteins in living cells with Hi-D.","authors":"Cesar Augusto Valades-Cruz, Roman Barth, Marwan Abdellah, Haitham A Shaban","doi":"10.1038/s41596-024-01038-3","DOIUrl":"https://doi.org/10.1038/s41596-024-01038-3","url":null,"abstract":"To understand the dynamic nature of the genome, the localization and rearrangement of DNA and DNA-binding proteins must be analyzed across the entire nucleus of single living cells. Recently, we developed a computational light microscopy technique, called high-resolution diffusion (Hi-D) mapping, which can accurately detect, classify and map diffusion dynamics and biophysical parameters such as the diffusion constant, the anomalous exponent, drift velocity and model physical diffusion from the data at a high spatial resolution across the genome in living cells. Hi-D combines dense optical flow to detect and track local chromatin and nuclear protein motion genome-wide and Bayesian inference to characterize this local movement at nanoscale resolution. Here we present the Python implementation of Hi-D, with an option for parallelizing the calculations to run on multicore central processing units (CPUs). The functionality of Hi-D is presented to the users via user-friendly documented Python notebooks. Hi-D reduces the analysis time to less than 1 h using a multicore CPU with a single compute node. We also present different applications of Hi-D for live-imaging of DNA, histone H2B and RNA polymerase II sequences acquired with spinning disk confocal and super-resolution structured illumination microscopy.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2024-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142093563","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

STRAIGHT-IN: a platform for rapidly generating panels of genetically modified human pluripotent stem cell lines. STRAIGHT-IN：快速生成转基因人类多能干细胞系的平台。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-08-23 DOI: 10.1038/s41596-024-01039-2

Albert Blanch-Asensio, Catarina Grandela, Christine L Mummery, Richard P Davis

{"title":"STRAIGHT-IN: a platform for rapidly generating panels of genetically modified human pluripotent stem cell lines.","authors":"Albert Blanch-Asensio, Catarina Grandela, Christine L Mummery, Richard P Davis","doi":"10.1038/s41596-024-01039-2","DOIUrl":"https://doi.org/10.1038/s41596-024-01039-2","url":null,"abstract":"Targeted integration of large DNA cargoes (>10 kb) or genomic replacements in mammalian cells, such as human pluripotent stem cells (hPS cells), remains challenging. Here we describe a platform termed serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation (STRAIGHT-IN) to circumvent this. First, a landing pad cassette is precisely inserted or used to replace specific genomic regions. The site-specific integrase Bxb1 then enables DNA constructs, including those >50 kb, to be integrated into the genome, while Cre recombinase excises auxiliary DNA sequences to prevent postintegrative silencing. Using a strategy whereby the positive selection marker is only expressed if the donor plasmid carrying the payload is correctly targeted, we can obtain 100% enrichment for cells containing the DNA payload. Procedures for expressing Cre efficiently also mean that a clonal isolation step is no longer essential to derive the required genetically modified hPS cells containing the integrated DNA, potentially reducing clonal variability. Furthermore, STRAIGHT-IN facilitates rapid and multiplexed generation of genetically matched hPS cells when multiple donor plasmids are delivered simultaneously. STRAIGHT-IN has various applications, which include integrating complex genetic circuits for synthetic biology, as well as creating panels of hPS cells lines containing, as necessary, hundreds of disease-linked variants for disease modeling and drug discovery. After establishing the hPS cell line containing the landing pad, the entire procedure, including donor plasmid synthesis, takes 1.5-3 months, depending on whether single or multiple DNA payloads are integrated. This protocol only requires the researcher to be skilled in molecular biology and standard cell culture techniques.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2024-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142046915","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

Fabrication of cyborg bacterial cells as living cell-material hybrids using intracellular hydrogelation. 利用细胞内水凝胶技术制造作为活细胞-材料混合物的半机械细菌细胞。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-08-22 DOI: 10.1038/s41596-024-01035-6

Ofelya Baghdasaryan, Luis E Contreras-Llano, Shahid Khan, Aijun Wang, Che-Ming Jack Hu, Cheemeng Tan

{"title":"Fabrication of cyborg bacterial cells as living cell-material hybrids using intracellular hydrogelation.","authors":"Ofelya Baghdasaryan, Luis E Contreras-Llano, Shahid Khan, Aijun Wang, Che-Ming Jack Hu, Cheemeng Tan","doi":"10.1038/s41596-024-01035-6","DOIUrl":"https://doi.org/10.1038/s41596-024-01035-6","url":null,"abstract":"The production of living therapeutics, cell-based delivery of drugs and gene-editing tools and the manufacturing of bio-commodities all share a common concept: they use either a synthetic or a living cell chassis to achieve their primary engineering or therapeutic goal. Live-cell chassis face limitations inherent to their auto-replicative nature and the complexity of the cellular context. This limitation highlights the need for a new chassis combining the engineering simplicity of synthetic materials and the functionalities of natural cells. Here, we describe a protocol to assemble a synthetic polymeric network inside bacterial cells, rendering them incapable of cell division and allowing them to resist environmental stressors such as high pH, hydrogen peroxide and cell-wall-targeting antibiotics that would otherwise kill unmodified bacteria. This cellular bioengineering protocol details how bacteria can be transformed into single-lifespan devices that are resistant to environmental stressors and possess programable functionality. We designate the modified bacteria as cyborg bacterial cells. This protocol expands the synthetic biology toolset, conferring precise control over living cells and creating a versatile cell chassis for biotechnology, biomedical engineering and living therapeutics. The protocol, including the preparation of gelation reagents and chassis strain, can be completed in 4 d. The implementation of the protocol requires expertise in microbiology techniques, hydrogel chemistry, fluorescence microscopy and flow cytometry. Further functionalization of the cyborg bacterial cells and adaptation of the protocol requires skills ranging from synthetic genetic circuit engineering to hydrogel polymerization chemistries.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142036441","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

Synthesis of N-heterocycles through alcohol dehydrogenative coupling. 通过醇脱氢偶联合成 N-杂环。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-08-22 DOI: 10.1038/s41596-024-01031-w

Bhaskar Paul, Dibyajyoti Panja, Sabuj Kundu

{"title":"Synthesis of N-heterocycles through alcohol dehydrogenative coupling.","authors":"Bhaskar Paul, Dibyajyoti Panja, Sabuj Kundu","doi":"10.1038/s41596-024-01031-w","DOIUrl":"https://doi.org/10.1038/s41596-024-01031-w","url":null,"abstract":"Nitrogen heterocycles are found in the structures of many biologically important compounds, as well as materials used in the synthesis of fine chemicals. Notably, ~59% of US Food and Drug Administration-approved small-molecule drugs contain nitrogen heterocycles. It is therefore meaningful to explore greener or more sustainable methods for their synthesis. The use of alcohols as reagents is attractive as they can be readily obtained from biomass derived natural resources. In the last two decades, alcohol dehydrogenative coupling reaction to synthesize various heterocycles were extensively explored which furnished hydrogen (H2) and water (H2O) as the two greener byproducts. In this protocol, we describe several efficient catalytic transformations to synthesize quinolines, 1,8-naphthyridines, quinoxalines, quinazolines, pyrimidines, benzimidazoles, pyrroles and pyridines, using alcohol as starting materials. We also describe the synthesis of several homogeneous iridium/ruthenium catalysts and heterogeneous cobalt/copper catalysts that can be used in these transformations. The reaction setup is simple; in a Schlenk/reaction tube with magnetic stir-bar, alcohol, corresponding coupling reagents (nucleophiles), catalyst, base and solvent (water or organic solvent such as toluene, dioxane or p-xylene) are added. The reaction mixture is refluxed at the specified temperature (110-150 °C)-either in air or under argon-to furnish these heterocycles. Synthesis of the catalysts takes 3-5 h and the coupling reactions take 4-5 h depending on the target product. The cobalt- and copper-based heterogeneous catalytic systems displayed an good catalyst recyclability.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142036443","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

Native MS-guided lipidomics to define endogenous lipid microenvironments of eukaryotic receptors and transporters. 原生 MS 引导的脂质组学定义真核受体和转运体的内源性脂质微环境。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-08-22 DOI: 10.1038/s41596-024-01037-4

Di Wu, Haiping Tang, Xingyu Qiu, Siyuan Song, Siyun Chen, Carol V Robinson

{"title":"Native MS-guided lipidomics to define endogenous lipid microenvironments of eukaryotic receptors and transporters.","authors":"Di Wu, Haiping Tang, Xingyu Qiu, Siyuan Song, Siyun Chen, Carol V Robinson","doi":"10.1038/s41596-024-01037-4","DOIUrl":"https://doi.org/10.1038/s41596-024-01037-4","url":null,"abstract":"The mammalian membrane is composed of various eukaryotic lipids interacting with extensively post-translationally modified proteins. Probing interactions between these mammalian membrane proteins and their diverse and heterogeneous lipid cohort remains challenging. Recently, native mass spectrometry (MS) combined with bottom-up 'omics' approaches has provided valuable information to relate structural and functional lipids to membrane protein assemblies in eukaryotic membranes. Here we provide a step-by-step protocol to identify and provide relative quantification for endogenous lipids bound to mammalian membrane proteins and their complexes. Using native MS to guide our lipidomics strategies, we describe the necessary sample preparation steps, followed by native MS data acquisition, tailored lipidomics and data interpretation. We also highlight considerations for the integration of different levels of information from native MS and lipidomics and how to deal with the various challenges that arise during the experiments. This protocol begins with the preparation of membrane proteins from mammalian cells and tissues for native MS. The results enable not only direct assessment of copurified endogenous lipids but also determination of the apparent affinities of specific lipids. Detailed sample preparation for lipidomics analysis is also covered, along with comprehensive settings for liquid chromatography-MS analysis. This protocol is suitable for the identification and quantification of endogenous lipids, including fatty acids, sterols, glycerolipids, phospholipids and glycolipids and can be used to interrogate proteins from recombinant sources to native membranes.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142036442","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