Pub Date : 2024-09-17DOI: 10.1038/s41596-024-01049-0
Ziqing Pan, J. P. Martin Trusler, Zhijun Jin, Kaiqiang Zhang
The properties of the interface between materials have practical implications in various fields, encompassing capillary action, foam and emulsion stability, adhesion properties of materials and mass and heat transfer processes. Studying the dynamics of interfaces is also fundamental for understanding intermolecular interactions, change of molecular conformations and molecular aggregations. Pendant-drop tensiometry and its extension, the oscillating drop method, are simple, versatile methods used to measure surface tension, interfacial tension and interfacial rheological properties. These methods can, however, generate unreliable results because of inadequate material preparation, an incorrect calibration method, inappropriate selection of data for analysis, neglect of optical influences or operating the system outside the linear viscoelastic regime. In addition, many studies fail to report accurate uncertainties. This protocol addresses all these critical points and provides detailed descriptions of some operation tips relating to purifying methods for different kinds of material, the time frame for analyzing measurement data, the correction method for optical effects, implementation of the oscillating method with a common programmable pump and remedies for some common problems encountered during the measurement. Examples of interfacial tension measurements for two- and three-phase systems, as well as interfacial dilational modulus measurements for N2 and surfactant solutions, are provided to illustrate procedural details and results. A single measurement takes minutes to hours to complete, while the entire protocol, including the leak test, cleaning, repeated measurements and data analysis, may take several days.
{"title":"Interfacial property determination from dynamic pendant-drop characterizations","authors":"Ziqing Pan, J. P. Martin Trusler, Zhijun Jin, Kaiqiang Zhang","doi":"10.1038/s41596-024-01049-0","DOIUrl":"https://doi.org/10.1038/s41596-024-01049-0","url":null,"abstract":"<p>The properties of the interface between materials have practical implications in various fields, encompassing capillary action, foam and emulsion stability, adhesion properties of materials and mass and heat transfer processes. Studying the dynamics of interfaces is also fundamental for understanding intermolecular interactions, change of molecular conformations and molecular aggregations. Pendant-drop tensiometry and its extension, the oscillating drop method, are simple, versatile methods used to measure surface tension, interfacial tension and interfacial rheological properties. These methods can, however, generate unreliable results because of inadequate material preparation, an incorrect calibration method, inappropriate selection of data for analysis, neglect of optical influences or operating the system outside the linear viscoelastic regime. In addition, many studies fail to report accurate uncertainties. This protocol addresses all these critical points and provides detailed descriptions of some operation tips relating to purifying methods for different kinds of material, the time frame for analyzing measurement data, the correction method for optical effects, implementation of the oscillating method with a common programmable pump and remedies for some common problems encountered during the measurement. Examples of interfacial tension measurements for two- and three-phase systems, as well as interfacial dilational modulus measurements for N<sub>2</sub> and surfactant solutions, are provided to illustrate procedural details and results. A single measurement takes minutes to hours to complete, while the entire protocol, including the leak test, cleaning, repeated measurements and data analysis, may take several days.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142269497","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}
Pub Date : 2024-09-16DOI: 10.1038/s41596-024-01047-2
Omar S. M. El Nahhas, Marko van Treeck, Georg Wölflein, Michaela Unger, Marta Ligero, Tim Lenz, Sophia J. Wagner, Katherine J. Hewitt, Firas Khader, Sebastian Foersch, Daniel Truhn, Jakob Nikolas Kather
Hematoxylin- and eosin-stained whole-slide images (WSIs) are the foundation of diagnosis of cancer. In recent years, development of deep learning-based methods in computational pathology has enabled the prediction of biomarkers directly from WSIs. However, accurately linking tissue phenotype to biomarkers at scale remains a crucial challenge for democratizing complex biomarkers in precision oncology. This protocol describes a practical workflow for solid tumor associative modeling in pathology (STAMP), enabling prediction of biomarkers directly from WSIs by using deep learning. The STAMP workflow is biomarker agnostic and allows for genetic and clinicopathologic tabular data to be included as an additional input, together with histopathology images. The protocol consists of five main stages that have been successfully applied to various research problems: formal problem definition, data preprocessing, modeling, evaluation and clinical translation. The STAMP workflow differentiates itself through its focus on serving as a collaborative framework that can be used by clinicians and engineers alike for setting up research projects in the field of computational pathology. As an example task, we applied STAMP to the prediction of microsatellite instability (MSI) status in colorectal cancer, showing accurate performance for the identification of tumors high in MSI. Moreover, we provide an open-source code base, which has been deployed at several hospitals across the globe to set up computational pathology workflows. The STAMP workflow requires one workday of hands-on computational execution and basic command line knowledge.
{"title":"From whole-slide image to biomarker prediction: end-to-end weakly supervised deep learning in computational pathology","authors":"Omar S. M. El Nahhas, Marko van Treeck, Georg Wölflein, Michaela Unger, Marta Ligero, Tim Lenz, Sophia J. Wagner, Katherine J. Hewitt, Firas Khader, Sebastian Foersch, Daniel Truhn, Jakob Nikolas Kather","doi":"10.1038/s41596-024-01047-2","DOIUrl":"https://doi.org/10.1038/s41596-024-01047-2","url":null,"abstract":"<p>Hematoxylin- and eosin-stained whole-slide images (WSIs) are the foundation of diagnosis of cancer. In recent years, development of deep learning-based methods in computational pathology has enabled the prediction of biomarkers directly from WSIs. However, accurately linking tissue phenotype to biomarkers at scale remains a crucial challenge for democratizing complex biomarkers in precision oncology. This protocol describes a practical workflow for solid tumor associative modeling in pathology (STAMP), enabling prediction of biomarkers directly from WSIs by using deep learning. The STAMP workflow is biomarker agnostic and allows for genetic and clinicopathologic tabular data to be included as an additional input, together with histopathology images. The protocol consists of five main stages that have been successfully applied to various research problems: formal problem definition, data preprocessing, modeling, evaluation and clinical translation. The STAMP workflow differentiates itself through its focus on serving as a collaborative framework that can be used by clinicians and engineers alike for setting up research projects in the field of computational pathology. As an example task, we applied STAMP to the prediction of microsatellite instability (MSI) status in colorectal cancer, showing accurate performance for the identification of tumors high in MSI. Moreover, we provide an open-source code base, which has been deployed at several hospitals across the globe to set up computational pathology workflows. The STAMP workflow requires one workday of hands-on computational execution and basic command line knowledge.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142263727","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}
Pub Date : 2024-09-16DOI: 10.1038/s41596-024-01045-4
Suoqin Jin, Maksim V. Plikus, Qing Nie
Recent advances in single-cell sequencing technologies offer an opportunity to explore cell–cell communication in tissues systematically and with reduced bias. A key challenge is integrating known molecular interactions and measurements into a framework to identify and analyze complex cell–cell communication networks. Previously, we developed a computational tool, named CellChat, that infers and analyzes cell–cell communication networks from single-cell transcriptomic data within an easily interpretable framework. CellChat quantifies the signaling communication probability between two cell groups using a simplified mass-action-based model, which incorporates the core interaction between ligands and receptors with multisubunit structure along with modulation by cofactors. Importantly, CellChat performs a systematic and comparative analysis of cell–cell communication using a variety of quantitative metrics and machine-learning approaches. CellChat v2 is an updated version that includes additional comparison functionalities, an expanded database of ligand–receptor pairs along with rich functional annotations, and an Interactive CellChat Explorer. Here we provide a step-by-step protocol for using CellChat v2 on single-cell transcriptomic data, including inference and analysis of cell–cell communication from one dataset and identification of altered intercellular communication, signals and cell populations from different datasets across biological conditions. The R implementation of CellChat v2 toolkit and its tutorials together with the graphic outputs are available at https://github.com/jinworks/CellChat. This protocol typically takes ~5 min depending on dataset size and requires a basic understanding of R and single-cell data analysis but no specialized bioinformatics training for its implementation.
{"title":"CellChat for systematic analysis of cell–cell communication from single-cell transcriptomics","authors":"Suoqin Jin, Maksim V. Plikus, Qing Nie","doi":"10.1038/s41596-024-01045-4","DOIUrl":"https://doi.org/10.1038/s41596-024-01045-4","url":null,"abstract":"<p>Recent advances in single-cell sequencing technologies offer an opportunity to explore cell–cell communication in tissues systematically and with reduced bias. A key challenge is integrating known molecular interactions and measurements into a framework to identify and analyze complex cell–cell communication networks. Previously, we developed a computational tool, named CellChat, that infers and analyzes cell–cell communication networks from single-cell transcriptomic data within an easily interpretable framework. CellChat quantifies the signaling communication probability between two cell groups using a simplified mass-action-based model, which incorporates the core interaction between ligands and receptors with multisubunit structure along with modulation by cofactors. Importantly, CellChat performs a systematic and comparative analysis of cell–cell communication using a variety of quantitative metrics and machine-learning approaches. CellChat v2 is an updated version that includes additional comparison functionalities, an expanded database of ligand–receptor pairs along with rich functional annotations, and an Interactive CellChat Explorer. Here we provide a step-by-step protocol for using CellChat v2 on single-cell transcriptomic data, including inference and analysis of cell–cell communication from one dataset and identification of altered intercellular communication, signals and cell populations from different datasets across biological conditions. The R implementation of CellChat v2 toolkit and its tutorials together with the graphic outputs are available at https://github.com/jinworks/CellChat. This protocol typically takes ~5 min depending on dataset size and requires a basic understanding of R and single-cell data analysis but no specialized bioinformatics training for its implementation.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142269498","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}
Pub Date : 2024-09-12DOI: 10.1038/s41596-024-01041-8
Ana Mora-Boza, Adriana Mulero-Russe, Nikolas Di Caprio, Jason A. Burdick, Eric O’Neill, Ankur Singh, Andrés J. García
Perfusable hydrogels have garnered substantial attention in recent years for the fabrication of microphysiological systems. However, current methodologies to fabricate microchannels in hydrogel platforms involve sophisticated equipment and techniques, which hinder progress of the field. In this protocol, we present a cost-effective, simple, versatile and ultrafast method to create perfusable microchannels of complex shapes in photopolymerizable hydrogels. Our method uses one-step UV photocross-linking and a photomask printed on inexpensive transparent films, to photopattern both synthetic (PEG-norbornene) and natural (hyaluronic acid-norbornene) hydrogels in just 0.8 s. Moreover, these perfusable hydrogels are fully integrated into a custom-made microfluidic device that allows continuous fluid perfusion when connected to an external pump system. This methodology can be easily reproduced by professionals with basic laboratory skills and a fundamental knowledge of polymers and materials science. In this protocol, we demonstrate the functionality of our photopatterned hydrogels by seeding human endothelial cells into the microchannels, culturing them under dynamic conditions for 7 d, and exposing them to inflammatory stimuli to elicit cellular responses. This highlights the versatility of our platform in fabricating microphysiological systems and different microenvironments. The fabrication of perfusable channels within the hydrogels, including the fabrication of the microfluidic devices, requires ~3 d. The development of the cell-seeded microphysiological system, including the stimulation of cells, takes ~7 d. In conclusion, our approach provides a straightforward and widely applicable solution to simplify and reduce the cost of biofabrication techniques for developing functional in vitro models using perfusable three-dimensional hydrogels.
{"title":"Facile photopatterning of perfusable microchannels in hydrogels for microphysiological systems","authors":"Ana Mora-Boza, Adriana Mulero-Russe, Nikolas Di Caprio, Jason A. Burdick, Eric O’Neill, Ankur Singh, Andrés J. García","doi":"10.1038/s41596-024-01041-8","DOIUrl":"https://doi.org/10.1038/s41596-024-01041-8","url":null,"abstract":"<p>Perfusable hydrogels have garnered substantial attention in recent years for the fabrication of microphysiological systems. However, current methodologies to fabricate microchannels in hydrogel platforms involve sophisticated equipment and techniques, which hinder progress of the field. In this protocol, we present a cost-effective, simple, versatile and ultrafast method to create perfusable microchannels of complex shapes in photopolymerizable hydrogels. Our method uses one-step UV photocross-linking and a photomask printed on inexpensive transparent films, to photopattern both synthetic (PEG-norbornene) and natural (hyaluronic acid-norbornene) hydrogels in just 0.8 s. Moreover, these perfusable hydrogels are fully integrated into a custom-made microfluidic device that allows continuous fluid perfusion when connected to an external pump system. This methodology can be easily reproduced by professionals with basic laboratory skills and a fundamental knowledge of polymers and materials science. In this protocol, we demonstrate the functionality of our photopatterned hydrogels by seeding human endothelial cells into the microchannels, culturing them under dynamic conditions for 7 d, and exposing them to inflammatory stimuli to elicit cellular responses. This highlights the versatility of our platform in fabricating microphysiological systems and different microenvironments. The fabrication of perfusable channels within the hydrogels, including the fabrication of the microfluidic devices, requires ~3 d. The development of the cell-seeded microphysiological system, including the stimulation of cells, takes ~7 d. In conclusion, our approach provides a straightforward and widely applicable solution to simplify and reduce the cost of biofabrication techniques for developing functional in vitro models using perfusable three-dimensional hydrogels.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176229","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}
Pub Date : 2024-09-11DOI: 10.1038/s41596-024-01042-7
Carlos W. Gantner, Bailey A. T. Weatherbee, Yuntao Wang, Magdalena Zernicka-Goetz
The embryonic and extraembryonic tissue interactions underlying human embryogenesis at implantation stages are not currently understood. We have generated a pluripotent stem cell-derived model that mimics aspects of peri-implantation development, allowing tractable experimentation otherwise impossible in the human embryo. Activation of the extraembryonic lineage-specific transcription factors GATA6 and SOX17 (hypoblast factors) or GATA3 and TFAP2C (encoding AP2γ; trophoblast factors) in human embryonic stem (ES) cells drive conversion to extraembryonic-like cells. When combined with wild-type ES cells, self-organized embryo-like structures form in the absence of exogenous factors, termed human inducible embryoids (hiEmbryoids). The epiblast-like domain of hiEmbryoids polarizes and differentiates in response to extraembryonic-secreted extracellular matrix and morphogen cues. Extraembryonic mesenchyme, amnion and primordial germ cells are specified in hiEmbryoids in a stepwise fashion. After establishing stable inducible ES lines and converting ES cells to RSeT culture media, the protocol takes 7–10 d to generate hiEmbryoids. Generation of hiEmbryoids can be performed by researchers with basic expertise in stem cell culture.
人类胚胎发育在植入阶段所依赖的胚胎和胚外组织相互作用目前尚不清楚。我们已经生成了一个多能干细胞衍生模型,该模型可模仿着床前发育的各个方面,从而可进行人类胚胎不可能进行的实验。在人类胚胎干细胞(ES)中激活胚外系特异性转录因子GATA6和SOX17(胚下细胞因子)或GATA3和TFAP2C(编码AP2γ;滋养层细胞因子),可推动向胚外样细胞转化。当与野生型 ES 细胞结合时,在没有外源因子的情况下会形成自组织胚胎样结构,称为人类可诱导胚胎(hiEmbryoids)。hiEmbryoids的上胚层样域会根据胚外分泌的细胞外基质和形态发生因子进行极化和分化。胚外间充质、羊膜和原始生殖细胞在 hiEmbryoids 中逐步分化。在建立稳定的可诱导 ES 系并将 ES 细胞转化为 RSeT 培养基后,该方案需要 7-10 天才能生成高胚胎性细胞。具有干细胞培养基础知识的研究人员都可以进行干胚胎的生成。
{"title":"Assembly of a stem cell-derived human postimplantation embryo model","authors":"Carlos W. Gantner, Bailey A. T. Weatherbee, Yuntao Wang, Magdalena Zernicka-Goetz","doi":"10.1038/s41596-024-01042-7","DOIUrl":"https://doi.org/10.1038/s41596-024-01042-7","url":null,"abstract":"<p>The embryonic and extraembryonic tissue interactions underlying human embryogenesis at implantation stages are not currently understood. We have generated a pluripotent stem cell-derived model that mimics aspects of peri-implantation development, allowing tractable experimentation otherwise impossible in the human embryo. Activation of the extraembryonic lineage-specific transcription factors <i>GATA6</i> and <i>SOX17</i> (hypoblast factors) or <i>GATA3</i> and <i>TFAP2C</i> (encoding AP2γ; trophoblast factors) in human embryonic stem (ES) cells drive conversion to extraembryonic-like cells. When combined with wild-type ES cells, self-organized embryo-like structures form in the absence of exogenous factors, termed human inducible embryoids (hiEmbryoids). The epiblast-like domain of hiEmbryoids polarizes and differentiates in response to extraembryonic-secreted extracellular matrix and morphogen cues. Extraembryonic mesenchyme, amnion and primordial germ cells are specified in hiEmbryoids in a stepwise fashion. After establishing stable inducible ES lines and converting ES cells to RSeT culture media, the protocol takes 7–10 d to generate hiEmbryoids. Generation of hiEmbryoids can be performed by researchers with basic expertise in stem cell culture.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176228","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}
One of the foremost challenges in nanobiotechnology is obtaining direct evidence of nanoparticles' absorption and internalization in plants. Although confocal laser scanning microscopy (CLSM) or transmission electron microscopy (TEM) are currently the most commonly used tools to characterize nanoparticles in plants, subjectivity of researchers, incorrect sample handling, inevitable fluorescence leakage and limitations of imaging instruments lead to false positives and non-reproducibility of experimental results. This protocol provides an easy-to-operate dual-step method, combining CLSM for macroscopic tissue examination and TEM for cellular-level analysis, to effectively trace single particles in plant roots with accuracy and precision. In addition, we also provide detailed methods for processing plant materials before imaging, including cleaning, and staining, to maximize the accuracy and reliability of imaging. This protocol involves currently commonly used nanomaterial types, such as metal-based and doped carbon-based materials, and enables accurate localization of nanoparticles with different sizes at the cell level in Arabidopsis thaliana root samples either through contrast or element mapping analysis. It serves as a valuable reference and benchmark for scholars in plant science, chemistry and environmental studies to understand the interaction between plant roots and nanomaterials and to detect the distribution of nanomaterials in plants. Excluding plant culture time, the protocol can be completed in 4-5 d.
纳米生物技术的首要挑战之一是获得纳米粒子在植物体内吸收和内化的直接证据。尽管共焦激光扫描显微镜(CLSM)或透射电子显微镜(TEM)是目前表征植物中纳米粒子的最常用工具,但研究人员的主观性、不正确的样品处理、不可避免的荧光泄漏以及成像仪器的局限性导致了假阳性和实验结果的不可再现性。本方案提供了一种易于操作的双步骤方法,将用于宏观组织检查的 CLSM 与用于细胞级分析的 TEM 相结合,可有效、准确地追踪植物根中的单个颗粒。此外,我们还提供了成像前处理植物材料的详细方法,包括清洁和染色,以最大限度地提高成像的准确性和可靠性。该方案涉及目前常用的纳米材料类型,如金属基和掺杂碳基材料,通过对比或元素图谱分析,可在拟南芥根部样本的细胞水平准确定位不同大小的纳米颗粒。它为植物科学、化学和环境研究领域的学者了解植物根系与纳米材料之间的相互作用以及检测纳米材料在植物中的分布提供了宝贵的参考和基准。不包括植物培养时间,该方案可在 4-5 d 内完成。
{"title":"Precise tracking of nanoparticles in plant roots.","authors":"Xiao-Dong Sun, Jing-Ya Ma, Li-Juan Feng, Jian-Lu Duan, Xian-Zheng Yuan","doi":"10.1038/s41596-024-01044-5","DOIUrl":"https://doi.org/10.1038/s41596-024-01044-5","url":null,"abstract":"<p><p>One of the foremost challenges in nanobiotechnology is obtaining direct evidence of nanoparticles' absorption and internalization in plants. Although confocal laser scanning microscopy (CLSM) or transmission electron microscopy (TEM) are currently the most commonly used tools to characterize nanoparticles in plants, subjectivity of researchers, incorrect sample handling, inevitable fluorescence leakage and limitations of imaging instruments lead to false positives and non-reproducibility of experimental results. This protocol provides an easy-to-operate dual-step method, combining CLSM for macroscopic tissue examination and TEM for cellular-level analysis, to effectively trace single particles in plant roots with accuracy and precision. In addition, we also provide detailed methods for processing plant materials before imaging, including cleaning, and staining, to maximize the accuracy and reliability of imaging. This protocol involves currently commonly used nanomaterial types, such as metal-based and doped carbon-based materials, and enables accurate localization of nanoparticles with different sizes at the cell level in Arabidopsis thaliana root samples either through contrast or element mapping analysis. It serves as a valuable reference and benchmark for scholars in plant science, chemistry and environmental studies to understand the interaction between plant roots and nanomaterials and to detect the distribution of nanomaterials in plants. Excluding plant culture time, the protocol can be completed in 4-5 d.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142140587","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}
Pub Date : 2024-09-05DOI: 10.1038/s41596-024-01032-9
Annie Maslan, Nicolas Altemose, Jeremy Marcus, Reet Mishra, Lucy D Brennan, Kousik Sundararajan, Gary Karpen, Aaron F Straight, Aaron Streets
We recently developed directed methylation with long-read sequencing (DiMeLo-seq) to map protein-DNA interactions genome wide. DiMeLo-seq is capable of mapping multiple interaction sites on single DNA molecules, profiling protein binding in the context of endogenous DNA methylation, identifying haplotype-specific protein-DNA interactions and mapping protein-DNA interactions in repetitive regions of the genome that are difficult to study with short-read methods. With DiMeLo-seq, adenines in the vicinity of a protein of interest are methylated in situ by tethering the Hia5 methyltransferase to an antibody using protein A. Protein-DNA interactions are then detected by direct readout of adenine methylation with long-read, single-molecule DNA sequencing platforms such as Nanopore sequencing. Here we present a detailed protocol and practical guidance for performing DiMeLo-seq. This protocol can be run on nuclei from fresh, lightly fixed or frozen cells. The protocol requires 1-2 d for performing in situ targeted methylation, 1-5 d for library preparation depending on desired fragment length and 1-3 d for Nanopore sequencing depending on desired sequencing depth. The protocol requires basic molecular biology skills and equipment, as well as access to a Nanopore sequencer. We also provide a Python package, dimelo, for analysis of DiMeLo-seq data.
我们最近开发了定向甲基化长读程测序(DiMeLo-seq)技术,用于绘制全基因组蛋白质-DNA相互作用图谱。DiMeLo-seq 能够绘制单个 DNA 分子上多个相互作用位点的图谱,分析内源性 DNA 甲基化背景下的蛋白质结合情况,确定单倍型特异性蛋白质-DNA 相互作用,并绘制基因组重复区域中的蛋白质-DNA 相互作用图谱,而这些区域是短线程方法难以研究的。通过 DiMeLo-seq 技术,利用蛋白 A 将 Hia5 甲基转移酶系在抗体上,使感兴趣的蛋白质附近的腺嘌呤发生原位甲基化,然后利用长读程单分子 DNA 测序平台(如 Nanopore 测序平台)直接读出腺嘌呤甲基化,检测蛋白质与 DNA 的相互作用。在此,我们介绍了执行 DiMeLo-seq 的详细方案和实用指南。该方案可在新鲜、轻度固定或冷冻的细胞核上运行。原位靶向甲基化需要 1-2 天,文库制备需要 1-5 天,具体取决于所需的片段长度,Nanopore 测序需要 1-3 天,具体取决于所需的测序深度。该方案需要基本的分子生物学技能和设备,以及一台 Nanopore 测序仪。我们还提供了一个 Python 软件包 dimelo,用于分析 DiMeLo-seq 数据。
{"title":"Mapping protein-DNA interactions with DiMeLo-seq.","authors":"Annie Maslan, Nicolas Altemose, Jeremy Marcus, Reet Mishra, Lucy D Brennan, Kousik Sundararajan, Gary Karpen, Aaron F Straight, Aaron Streets","doi":"10.1038/s41596-024-01032-9","DOIUrl":"https://doi.org/10.1038/s41596-024-01032-9","url":null,"abstract":"<p><p>We recently developed directed methylation with long-read sequencing (DiMeLo-seq) to map protein-DNA interactions genome wide. DiMeLo-seq is capable of mapping multiple interaction sites on single DNA molecules, profiling protein binding in the context of endogenous DNA methylation, identifying haplotype-specific protein-DNA interactions and mapping protein-DNA interactions in repetitive regions of the genome that are difficult to study with short-read methods. With DiMeLo-seq, adenines in the vicinity of a protein of interest are methylated in situ by tethering the Hia5 methyltransferase to an antibody using protein A. Protein-DNA interactions are then detected by direct readout of adenine methylation with long-read, single-molecule DNA sequencing platforms such as Nanopore sequencing. Here we present a detailed protocol and practical guidance for performing DiMeLo-seq. This protocol can be run on nuclei from fresh, lightly fixed or frozen cells. The protocol requires 1-2 d for performing in situ targeted methylation, 1-5 d for library preparation depending on desired fragment length and 1-3 d for Nanopore sequencing depending on desired sequencing depth. The protocol requires basic molecular biology skills and equipment, as well as access to a Nanopore sequencer. We also provide a Python package, dimelo, for analysis of DiMeLo-seq data.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142140586","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}
Pub Date : 2024-09-05DOI: 10.1038/s41596-024-01040-9
Lauren Gay, Keittisak Suwan, Amin Hajitou
Successful delivery of nucleic acid therapeutics to diseased sites would present a pivotal advancement in cancer treatment. However, progress has been hindered by the lack of efficient tumor-selective vectors via clinical systemic routes, the blood-brain barrier for brain tumors and problems with repeated administrations. We present a new generation of M13 phage-based vectors termed transmorphic phage/adeno-associated virus (AAV) (TPA), wherein the phage genome has been excised to facilitate exclusive packaging of human AAV DNA by phage coat proteins. Here we provide a detailed protocol for the molecular cloning of DNA into the TPA construct, display of disease-specific ligands on the helper phage capsid for cell targeting and entry, and packaging of TPA DNA by helper phage coat proteins in a bacterial host. Furthermore, we provide methods for mammalian cell transduction and assessment of transgene expression in vitro as well as in vivo application of TPA particles in tumor-bearing mice. Unlike other similar methods, our protocol enables high-yield production and control of helper phage quantity in TPA preparations. Moreover, compared with existing M13 phage vectors, TPA particles can accommodate large size transgene inserts, despite being considerably more compact, providing superior gene delivery through enhanced diffusion across the extracellular matrix, improved cellular binding and entry and increased vector DNA accumulation in the nucleus. The protocol encompasses a timeline of 4-5 months, including construction and production of TPA particles with transgene and targeted ligand and in vitro/in vivo testing. This protocol can be conducted by researchers trained in basic molecular biology/bacteriology research techniques.
成功地将核酸治疗药物输送到患病部位将是癌症治疗领域的一大进步。然而,临床系统途径缺乏高效的肿瘤选择性载体、脑肿瘤的血脑屏障以及重复给药等问题阻碍了这一研究的进展。我们提出了新一代基于 M13 噬菌体的载体,称为跨形态噬菌体/腺相关病毒(AAV)(TPA),其中的噬菌体基因组已被切除,以方便噬菌体衣壳蛋白对人类 AAV DNA 进行专属包装。在此,我们提供了一个详细的方案,用于将 DNA 分子克隆到 TPA 构建体中,在辅助噬菌体外壳上显示疾病特异性配体以实现细胞靶向和进入,以及在细菌宿主中通过辅助噬菌体衣壳蛋白包装 TPA DNA。此外,我们还提供了体外转导哺乳动物细胞和评估转基因表达的方法,以及在肿瘤小鼠体内应用 TPA 粒子的方法。与其他类似方法不同,我们的方案能够高产生产并控制 TPA 制剂中辅助噬菌体的数量。此外,与现有的 M13 噬菌体载体相比,尽管 TPA 颗粒的结构要紧凑得多,但仍能容纳大尺寸的转基因插入物,通过增强在细胞外基质中的扩散、改善细胞结合和进入以及增加载体 DNA 在细胞核中的积累,提供更优越的基因递送。该方案需要 4-5 个月的时间,包括构建和生产带有转基因和靶向配体的 TPA 颗粒以及体外/体内测试。受过基础分子生物学/细菌学研究技术培训的研究人员可以完成该方案。
{"title":"Construction and utilization of a new generation of bacteriophage-based particles, or TPA, for guided systemic delivery of nucleic acids to tumors.","authors":"Lauren Gay, Keittisak Suwan, Amin Hajitou","doi":"10.1038/s41596-024-01040-9","DOIUrl":"https://doi.org/10.1038/s41596-024-01040-9","url":null,"abstract":"<p><p>Successful delivery of nucleic acid therapeutics to diseased sites would present a pivotal advancement in cancer treatment. However, progress has been hindered by the lack of efficient tumor-selective vectors via clinical systemic routes, the blood-brain barrier for brain tumors and problems with repeated administrations. We present a new generation of M13 phage-based vectors termed transmorphic phage/adeno-associated virus (AAV) (TPA), wherein the phage genome has been excised to facilitate exclusive packaging of human AAV DNA by phage coat proteins. Here we provide a detailed protocol for the molecular cloning of DNA into the TPA construct, display of disease-specific ligands on the helper phage capsid for cell targeting and entry, and packaging of TPA DNA by helper phage coat proteins in a bacterial host. Furthermore, we provide methods for mammalian cell transduction and assessment of transgene expression in vitro as well as in vivo application of TPA particles in tumor-bearing mice. Unlike other similar methods, our protocol enables high-yield production and control of helper phage quantity in TPA preparations. Moreover, compared with existing M13 phage vectors, TPA particles can accommodate large size transgene inserts, despite being considerably more compact, providing superior gene delivery through enhanced diffusion across the extracellular matrix, improved cellular binding and entry and increased vector DNA accumulation in the nucleus. The protocol encompasses a timeline of 4-5 months, including construction and production of TPA particles with transgene and targeted ligand and in vitro/in vivo testing. This protocol can be conducted by researchers trained in basic molecular biology/bacteriology research techniques.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142140585","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}
Pub Date : 2024-09-04DOI: 10.1038/s41596-024-01036-5
Feng Chen, Xinyin Li, Min Bai, Yongxi Zhao
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.
核酸和蛋白质的表观遗传修饰及其空间邻近性在调节生理过程和疾病进展中发挥着重要作用。目前可用的细胞成像方法,如荧光原位杂交(FISH)和免疫荧光,很难检测到低丰度修饰及其空间邻近性。在此,我们将逐步介绍三种基于 DNA 编码放大 FISH 的成像策略,以克服这些挑战,满足不同的应用需求:碱基编码放大 FISH (BEA-FISH)、成对邻近分化放大 FISH (PPDA-FISH) 和细胞大分子系留 DNA 步行索引 (Cell-TALKING)。它们都使用类似的 DNA 编码扩增核心原理,将不同的非序列分子特征转化为独特的 DNA 条形码,用于原位滚圆扩增和 FISH 分析。这涉及固定细胞样本中的三个关键反应:靶标标记、DNA 编码和滚动圈扩增成像。利用该方案,这三种成像策略可分别实现低丰度修饰的原位计数、两种修饰的成对临近差异可视化以及一种蛋白质周围多种修饰的探索(一对多临近)。低丰度修饰,包括 5-羟甲基胞嘧啶、5-甲酰基胞嘧啶、5-羟甲基尿嘧啶和 5-甲酰基尿嘧啶,在单细胞中清晰可见。可发现核酸修饰和/或组蛋白修饰的各种组合模式。根据不同的成像应用,整个过程大约需要 2-4 天。
{"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":"<p><p>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.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"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}
Pub Date : 2024-09-04DOI: 10.1038/s41596-024-01052-5
Ittai Shamir, Yaniv Assaf
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).
{"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":"<p><p>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).</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"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}