Pub Date : 2024-07-23DOI: 10.1038/s41596-024-01019-6
Zhenping Cao, Jinyao Liu
Bacteria have been extensively utilized as living therapeutics for disease treatment due to their unique characteristics, such as genetic manipulability, rapid proliferation and specificity to target disease sites. Various in vivo insults can, however, decrease the vitality of dosed bacteria, leading to low overall bioavailability. Additionally, the innate antigens on the bacterial surface and the released toxins and metabolites may cause undesired safety issues. These limitations inevitably result in inadequate treatment outcomes, thereby hindering the clinical transformation of living bacterial therapeutics. Recently, we have developed a versatile platform to prepare advanced living bacterial therapeutics by nanocoating bacteria individually via either chemical decoration or physical encapsulation, which can improve bioavailability and reduce side effects for enhanced microbial therapy. Here we use interfacial self-assembly to prepare lipid membrane-coated bacteria (LCB), exhibiting increased resistance against a variety of harsh environmental conditions owing to the nanocoating’s protective capability. Meanwhile, we apply mechanical extrusion to generate cell membrane-coated bacteria (CMCB), displaying improved biocompatibility owing to the nanocoating’s shielding effect. We describe their detailed preparation procedures and demonstrate the expected functions of the coated bacteria. We also show that following oral delivery and intravenous injection in mouse models, LCB and CMCB present appealing potential for treating colitis and tumors, respectively. Compared with bioengineering that lacks versatile molecular tools for heterogeneous expression, the surface nanocoating technique is convenient to introduce functional components without restriction on bacterial strain types. Excluding bacterial culture, the fabrication of LCB takes ~2 h, while the preparation of CMCB takes ~5 h. This protocol adds a surface nanocoating to bacteria to improve bioavailability and reduce side effects for enhanced microbial therapy. Living bacterial therapeutics are produced by nanocoating bacteria individually via either chemical decoration or physical encapsulation.
{"title":"Surface nanocoating of bacteria as a versatile platform to develop living therapeutics","authors":"Zhenping Cao, Jinyao Liu","doi":"10.1038/s41596-024-01019-6","DOIUrl":"10.1038/s41596-024-01019-6","url":null,"abstract":"Bacteria have been extensively utilized as living therapeutics for disease treatment due to their unique characteristics, such as genetic manipulability, rapid proliferation and specificity to target disease sites. Various in vivo insults can, however, decrease the vitality of dosed bacteria, leading to low overall bioavailability. Additionally, the innate antigens on the bacterial surface and the released toxins and metabolites may cause undesired safety issues. These limitations inevitably result in inadequate treatment outcomes, thereby hindering the clinical transformation of living bacterial therapeutics. Recently, we have developed a versatile platform to prepare advanced living bacterial therapeutics by nanocoating bacteria individually via either chemical decoration or physical encapsulation, which can improve bioavailability and reduce side effects for enhanced microbial therapy. Here we use interfacial self-assembly to prepare lipid membrane-coated bacteria (LCB), exhibiting increased resistance against a variety of harsh environmental conditions owing to the nanocoating’s protective capability. Meanwhile, we apply mechanical extrusion to generate cell membrane-coated bacteria (CMCB), displaying improved biocompatibility owing to the nanocoating’s shielding effect. We describe their detailed preparation procedures and demonstrate the expected functions of the coated bacteria. We also show that following oral delivery and intravenous injection in mouse models, LCB and CMCB present appealing potential for treating colitis and tumors, respectively. Compared with bioengineering that lacks versatile molecular tools for heterogeneous expression, the surface nanocoating technique is convenient to introduce functional components without restriction on bacterial strain types. Excluding bacterial culture, the fabrication of LCB takes ~2 h, while the preparation of CMCB takes ~5 h. This protocol adds a surface nanocoating to bacteria to improve bioavailability and reduce side effects for enhanced microbial therapy. Living bacterial therapeutics are produced by nanocoating bacteria individually via either chemical decoration or physical encapsulation.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 11","pages":"3162-3190"},"PeriodicalIF":13.1,"publicationDate":"2024-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141752147","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 : 2024-07-22DOI: 10.1038/s41596-024-01021-y
Seung Won Shin, Prakriti Mudvari, Shravan Thaploo, Michael A. Wheeler, Daniel C. Douek, Francisco J. Quintana, Eli A. Boritz, Adam R. Abate, Iain C. Clark
Rare cells have an important role in development and disease, and methods for isolating and studying cell subsets are therefore an essential part of biology research. Such methods traditionally rely on labeled antibodies targeted to cell surface proteins, but large public databases and sophisticated computational approaches increasingly define cell subsets on the basis of genomic, epigenomic and transcriptomic sequencing data. Methods for isolating cells on the basis of nucleic acid sequences powerfully complement these approaches by providing experimental access to cell subsets discovered in cell atlases, as well as those that cannot be otherwise isolated, including cells infected with pathogens, with specific DNA mutations or with unique transcriptional or splicing signatures. We recently developed a nucleic acid cytometry platform called ‘focused interrogation of cells by nucleic acid detection and sequencing’ (FIND-seq), capable of isolating rare cells on the basis of RNA or DNA markers, followed by bulk or single-cell transcriptomic analysis. This platform has previously been used to characterize the splicing-dependent activation of the transcription factor XBP1 in astrocytes and HIV persistence in memory CD4 T cells from people on long-term antiretroviral therapy. Here, we outline the molecular and microfluidic steps involved in performing FIND-seq, including protocol updates that allow detection and whole transcriptome sequencing of rare HIV-infected cells that harbor genetically intact virus genomes. FIND-seq requires knowledge of microfluidics, optics and molecular biology. We expect that FIND-seq, and this comprehensive protocol, will enable mechanistic studies of rare HIV+ cells, as well as other cell subsets that were previously difficult to recover and sequence. FIND-seq is a nucleic acid cytometry platform capable of isolating rare cells on the basis of RNA or DNA markers. This protocol outlines the molecular and microfluidic steps to perform FIND-seq, followed by bulk or single-cell transcriptomic analysis.
稀有细胞在发育和疾病中起着重要作用，因此分离和研究细胞亚群的方法是生物学研究的重要组成部分。这些方法传统上依赖于针对细胞表面蛋白的标记抗体，但大型公共数据库和复杂的计算方法越来越多地根据基因组、表观基因组和转录组测序数据来定义细胞亚群。根据核酸序列分离细胞的方法是对这些方法的有力补充，可通过实验获得细胞图谱中发现的细胞亚群，以及那些无法通过其他方法分离的细胞亚群，包括感染病原体的细胞、具有特定DNA突变或具有独特转录或剪接特征的细胞。我们最近开发了一种名为 "通过核酸检测和测序对细胞进行重点检测"（FIND-seq）的核酸细胞测量平台，能够根据 RNA 或 DNA 标记分离稀有细胞，然后进行大量或单细胞转录组分析。该平台曾被用于描述星形胶质细胞中转录因子 XBP1 的剪接依赖性激活，以及长期接受抗逆转录病毒治疗者记忆 CD4 T 细胞中 HIV 的持续存在。在这里，我们概述了执行 FIND-seq 所涉及的分子和微流控步骤，包括允许对携带基因完整病毒基因组的罕见 HIV 感染细胞进行检测和全转录组测序的方案更新。FIND-seq 需要微流控、光学和分子生物学方面的知识。我们希望 FIND-seq 和这一综合方案能对罕见的 HIV+ 细胞以及以前难以恢复和测序的其他细胞亚群进行机理研究。
{"title":"FIND-seq: high-throughput nucleic acid cytometry for rare single-cell transcriptomics","authors":"Seung Won Shin, Prakriti Mudvari, Shravan Thaploo, Michael A. Wheeler, Daniel C. Douek, Francisco J. Quintana, Eli A. Boritz, Adam R. Abate, Iain C. Clark","doi":"10.1038/s41596-024-01021-y","DOIUrl":"10.1038/s41596-024-01021-y","url":null,"abstract":"Rare cells have an important role in development and disease, and methods for isolating and studying cell subsets are therefore an essential part of biology research. Such methods traditionally rely on labeled antibodies targeted to cell surface proteins, but large public databases and sophisticated computational approaches increasingly define cell subsets on the basis of genomic, epigenomic and transcriptomic sequencing data. Methods for isolating cells on the basis of nucleic acid sequences powerfully complement these approaches by providing experimental access to cell subsets discovered in cell atlases, as well as those that cannot be otherwise isolated, including cells infected with pathogens, with specific DNA mutations or with unique transcriptional or splicing signatures. We recently developed a nucleic acid cytometry platform called ‘focused interrogation of cells by nucleic acid detection and sequencing’ (FIND-seq), capable of isolating rare cells on the basis of RNA or DNA markers, followed by bulk or single-cell transcriptomic analysis. This platform has previously been used to characterize the splicing-dependent activation of the transcription factor XBP1 in astrocytes and HIV persistence in memory CD4 T cells from people on long-term antiretroviral therapy. Here, we outline the molecular and microfluidic steps involved in performing FIND-seq, including protocol updates that allow detection and whole transcriptome sequencing of rare HIV-infected cells that harbor genetically intact virus genomes. FIND-seq requires knowledge of microfluidics, optics and molecular biology. We expect that FIND-seq, and this comprehensive protocol, will enable mechanistic studies of rare HIV+ cells, as well as other cell subsets that were previously difficult to recover and sequence. FIND-seq is a nucleic acid cytometry platform capable of isolating rare cells on the basis of RNA or DNA markers. This protocol outlines the molecular and microfluidic steps to perform FIND-seq, followed by bulk or single-cell transcriptomic analysis.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 11","pages":"3191-3218"},"PeriodicalIF":13.1,"publicationDate":"2024-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141741617","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 : 2024-07-18DOI: 10.1038/s41596-024-01022-x
Ting Zhang, Yuxi Wang, Xucong Teng, Ruijie Deng, Jinghong Li
The evolution and mutation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgent concerns as they pose the risk of vaccine failure and increased viral transmission. However, affordable and scalable tools allowing rapid identification of SARS-CoV-2 variants are not readily available, which impedes diagnosis and epidemiological surveillance. Here we present a colorimetric nucleic acid assay named MARVE (multiplexed, preamplification-free, single-nucleotide-resolved viral evolution) that is convenient to perform and yields single-nucleotide resolution. The assay integrates nucleic acid strand displacement reactions with enzymatic amplification to colorimetrically sense viral RNA using a metal ion-incorporated DNA probe (TEprobe). We provide detailed guidelines to design TEprobes for discriminating single-nucleotide variations in viral RNAs, and to fabricate a test paper for the detection of SARS-CoV-2 variants of concern. Compared with other nucleic acid assays, our assay is preamplification-free, single-nucleotide-resolvable and results are visible via a color change. Besides, it is smartphone readable, multiplexed, quick and cheap ($0.30 per test). The protocol takes ~2 h to complete, from the design and preparation of the DNA probes and test papers (~1 h) to the detection of SARS-CoV-2 or its variants (30–45 min). The design of the TEprobes requires basic knowledge of molecular biology and familiarity with NUPACK or the Python programming language. The fabrication of the origami papers requires access to a wax printer using the CAD and PDF files provided or requires users to be familiar with AutoCAD to design new origami papers. The protocol is also applicable for designing assays to detect other pathogens and their variants. The authors present MARVE, a paper-based, preamplification-free diagnostic tool for fast but accurate colorimetric detection of viral variants at single-nucleotide resolution.
严重急性呼吸系统综合症冠状病毒 2（SARS-CoV-2）的进化和变异是亟待解决的问题，因为它们会带来疫苗失效和病毒传播增加的风险。然而，目前还没有可快速识别 SARS-CoV-2 变异体的经济实惠且可扩展的工具，这阻碍了诊断和流行病学监测。在这里，我们提出了一种名为 MARVE（多路复用、无前置扩增、单核苷酸分辨病毒进化）的比色核酸测定法，它操作方便，并能获得单核苷酸分辨率。该检测方法将核酸链置换反应与酶扩增相结合，利用金属离子掺入 DNA 探针（TEprobe）对病毒 RNA 进行比色检测。我们提供了设计 TE 探针以分辨病毒 RNA 中单核苷酸变异的详细指南，并制作了检测 SARS-CoV-2 变异的试纸。与其他核酸检测方法相比，我们的检测方法无需扩增前处理，可区分单核苷酸，检测结果通过颜色变化可见。此外，它还具有智能手机可读性、多重性、快速性和廉价性（每次检测 0.30 美元）。从设计和制备 DNA 探针和试纸（约 1 小时）到检测 SARS-CoV-2 或其变种（30-45 分钟），整个过程约需 2 小时。TE探针的设计需要分子生物学的基础知识和对NUPACK或Python编程语言的熟悉。制作折纸需要使用提供的 CAD 和 PDF 文件或熟悉 AutoCAD 的用户使用蜡打印机设计新的折纸。该方案也适用于设计检测其他病原体及其变体的方法。
{"title":"Preamplification-free viral RNA diagnostics with single-nucleotide resolution using MARVE, an origami paper-based colorimetric nucleic acid test","authors":"Ting Zhang, Yuxi Wang, Xucong Teng, Ruijie Deng, Jinghong Li","doi":"10.1038/s41596-024-01022-x","DOIUrl":"10.1038/s41596-024-01022-x","url":null,"abstract":"The evolution and mutation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgent concerns as they pose the risk of vaccine failure and increased viral transmission. However, affordable and scalable tools allowing rapid identification of SARS-CoV-2 variants are not readily available, which impedes diagnosis and epidemiological surveillance. Here we present a colorimetric nucleic acid assay named MARVE (multiplexed, preamplification-free, single-nucleotide-resolved viral evolution) that is convenient to perform and yields single-nucleotide resolution. The assay integrates nucleic acid strand displacement reactions with enzymatic amplification to colorimetrically sense viral RNA using a metal ion-incorporated DNA probe (TEprobe). We provide detailed guidelines to design TEprobes for discriminating single-nucleotide variations in viral RNAs, and to fabricate a test paper for the detection of SARS-CoV-2 variants of concern. Compared with other nucleic acid assays, our assay is preamplification-free, single-nucleotide-resolvable and results are visible via a color change. Besides, it is smartphone readable, multiplexed, quick and cheap ($0.30 per test). The protocol takes ~2 h to complete, from the design and preparation of the DNA probes and test papers (~1 h) to the detection of SARS-CoV-2 or its variants (30–45 min). The design of the TEprobes requires basic knowledge of molecular biology and familiarity with NUPACK or the Python programming language. The fabrication of the origami papers requires access to a wax printer using the CAD and PDF files provided or requires users to be familiar with AutoCAD to design new origami papers. The protocol is also applicable for designing assays to detect other pathogens and their variants. The authors present MARVE, a paper-based, preamplification-free diagnostic tool for fast but accurate colorimetric detection of viral variants at single-nucleotide resolution.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 11","pages":"3426-3455"},"PeriodicalIF":13.1,"publicationDate":"2024-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141724008","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 : 2024-07-18DOI: 10.1038/s41596-024-01017-8
Garret Couture, Shawn Ehlers Cheang, Christopher Suarez, Ye Chen, Nikita P. Bacalzo Jr, Jiani Jiang, Cheng-Yu Charlie Weng, Aaron Stacy, Juan J. Castillo, Omar Delannoy-Bruno, Daniel M. Webber, Michael J. Barratt, Jeffrey I. Gordon, David A. Mills, J. Bruce German, Naomi K. Fukagawa, Carlito B. Lebrilla
Carbohydrates comprise the largest fraction of most diets and exert a profound impact on health. Components such as simple sugars and starch supply energy, while indigestible components, deemed dietary fiber, reach the colon to provide food for the tens of trillions of microbes that make up the gut microbiota. The interactions between dietary carbohydrates, our gastrointestinal tracts, the gut microbiome and host health are dictated by their structures. However, current methods for analysis of food glycans lack the sensitivity, specificity and throughput needed to quantify and elucidate these myriad structures. This protocol describes a multi-glycomic approach to food carbohydrate analysis in which the analyte might be any food item or biological material such as fecal and cecal samples. The carbohydrates are extracted by ethanol precipitation, and the resulting samples are subjected to rapid-throughput liquid chromatography (LC)-tandem mass spectrometry (LC-MS/MS) methods. Quantitative analyses of monosaccharides, glycosidic linkages, polysaccharides and alcohol-soluble carbohydrates are performed in 96-well plates at the milligram scale to reduce the biomass of sample required and enhance throughput. Detailed stepwise processes for sample preparation, LC-MS/MS and data analysis are provided. We illustrate the application of the protocol to a diverse set of foods as well as different apple cultivars and various fermented foods. Furthermore, we show the utility of these methods in elucidating glycan–microbe interactions in germ-free and colonized mice. These methods provide a framework for elucidating relationships between dietary fiber, the gut microbiome and human physiology. These structures will further guide nutritional and clinical feeding studies that enhance our understanding of the role of diet in nutrition and health. Complex carbohydrates that are not broken down by human enzymes are food sources for gut microbiota. Toward understanding this process, this protocol describes the quantitative analysis of carbohydrates in food and fecal samples by using LC-MS/MS.
{"title":"A multi-glycomic platform for the analysis of food carbohydrates","authors":"Garret Couture, Shawn Ehlers Cheang, Christopher Suarez, Ye Chen, Nikita P. Bacalzo Jr, Jiani Jiang, Cheng-Yu Charlie Weng, Aaron Stacy, Juan J. Castillo, Omar Delannoy-Bruno, Daniel M. Webber, Michael J. Barratt, Jeffrey I. Gordon, David A. Mills, J. Bruce German, Naomi K. Fukagawa, Carlito B. Lebrilla","doi":"10.1038/s41596-024-01017-8","DOIUrl":"10.1038/s41596-024-01017-8","url":null,"abstract":"Carbohydrates comprise the largest fraction of most diets and exert a profound impact on health. Components such as simple sugars and starch supply energy, while indigestible components, deemed dietary fiber, reach the colon to provide food for the tens of trillions of microbes that make up the gut microbiota. The interactions between dietary carbohydrates, our gastrointestinal tracts, the gut microbiome and host health are dictated by their structures. However, current methods for analysis of food glycans lack the sensitivity, specificity and throughput needed to quantify and elucidate these myriad structures. This protocol describes a multi-glycomic approach to food carbohydrate analysis in which the analyte might be any food item or biological material such as fecal and cecal samples. The carbohydrates are extracted by ethanol precipitation, and the resulting samples are subjected to rapid-throughput liquid chromatography (LC)-tandem mass spectrometry (LC-MS/MS) methods. Quantitative analyses of monosaccharides, glycosidic linkages, polysaccharides and alcohol-soluble carbohydrates are performed in 96-well plates at the milligram scale to reduce the biomass of sample required and enhance throughput. Detailed stepwise processes for sample preparation, LC-MS/MS and data analysis are provided. We illustrate the application of the protocol to a diverse set of foods as well as different apple cultivars and various fermented foods. Furthermore, we show the utility of these methods in elucidating glycan–microbe interactions in germ-free and colonized mice. These methods provide a framework for elucidating relationships between dietary fiber, the gut microbiome and human physiology. These structures will further guide nutritional and clinical feeding studies that enhance our understanding of the role of diet in nutrition and health. Complex carbohydrates that are not broken down by human enzymes are food sources for gut microbiota. Toward understanding this process, this protocol describes the quantitative analysis of carbohydrates in food and fecal samples by using LC-MS/MS.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 11","pages":"3321-3359"},"PeriodicalIF":13.1,"publicationDate":"2024-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141724007","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}
With the advent of multiomics, software capable of multidimensional enrichment analysis has become increasingly crucial for uncovering gene set variations in biological processes and disease pathways. This is essential for elucidating disease mechanisms and identifying potential therapeutic targets. clusterProfiler stands out for its comprehensive utilization of databases and advanced visualization features. Importantly, clusterProfiler supports various biological knowledge, including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, through performing over-representation and gene set enrichment analyses. A key feature is that clusterProfiler allows users to choose from various graphical outputs to visualize results, enhancing interpretability. This protocol describes innovative ways in which clusterProfiler has been used for integrating metabolomics and metagenomics analyses, identifying and characterizing transcription factors under stress conditions, and annotating cells in single-cell studies. In all cases, the computational steps can be completed within ~2 min. clusterProfiler is released through the Bioconductor project and can be accessed via https://bioconductor.org/packages/clusterProfiler/ . clusterProfiler is a tool for characterizing and visualizing omics data. The example procedures show integration of metabolomics and metagenomics analyses, characterization of transcription factors and annotation of cells in single-cell studies.
{"title":"Using clusterProfiler to characterize multiomics data","authors":"Shuangbin Xu, Erqiang Hu, Yantong Cai, Zijing Xie, Xiao Luo, Li Zhan, Wenli Tang, Qianwen Wang, Bingdong Liu, Rui Wang, Wenqin Xie, Tianzhi Wu, Liwei Xie, Guangchuang Yu","doi":"10.1038/s41596-024-01020-z","DOIUrl":"10.1038/s41596-024-01020-z","url":null,"abstract":"With the advent of multiomics, software capable of multidimensional enrichment analysis has become increasingly crucial for uncovering gene set variations in biological processes and disease pathways. This is essential for elucidating disease mechanisms and identifying potential therapeutic targets. clusterProfiler stands out for its comprehensive utilization of databases and advanced visualization features. Importantly, clusterProfiler supports various biological knowledge, including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, through performing over-representation and gene set enrichment analyses. A key feature is that clusterProfiler allows users to choose from various graphical outputs to visualize results, enhancing interpretability. This protocol describes innovative ways in which clusterProfiler has been used for integrating metabolomics and metagenomics analyses, identifying and characterizing transcription factors under stress conditions, and annotating cells in single-cell studies. In all cases, the computational steps can be completed within ~2 min. clusterProfiler is released through the Bioconductor project and can be accessed via https://bioconductor.org/packages/clusterProfiler/ . clusterProfiler is a tool for characterizing and visualizing omics data. The example procedures show integration of metabolomics and metagenomics analyses, characterization of transcription factors and annotation of cells in single-cell studies.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 11","pages":"3292-3320"},"PeriodicalIF":13.1,"publicationDate":"2024-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141633981","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 : 2024-07-15DOI: 10.1038/s41596-024-01016-9
Ji Luan, Chaoyi Song, Yan Liu, Ruoting He, Ruofei Guo, Qingwen Cui, Chanjuan Jiang, Xiaochen Li, Kexin Hao, A. Francis Stewart, Jun Fu, Youming Zhang, Hailong Wang
Seamless site-directed mutagenesis is an important technique for studying protein functions, tuning enzyme catalytic activities and modifying genetic elements in multiple rounds because it can insert, delete or substitute nucleotides, DNA segments or even entire genes at the target site without introducing any unwanted change. To facilitate seamless site-directed mutagenesis in large plasmids and bacterial artificial chromosomes (BACs) with repetitive sequences, we recently developed the RedEx strategy. Compared with previous methods, our approach achieves the recovery of correct recombinants with high accuracy by circumventing unwanted recombination between repetitive sequences. RedEx readily yields more than 80% accuracy in seamless DNA insertion and deletion in large multimodular polyketide synthase gene clusters, which are among the most difficult targets due to the large number of repetitive DNA sequences in modules encoding almost identical enzymes. Here we present the RedEx method by describing in detail the seamless site-directed mutagenesis in a BAC vector. Overall, the process includes three parts: (1) insertion of the RedEx cassette containing the desired mutation together with selection–counterselection markers flanked by unique restriction sites and 20-bp overlapping sequences into the target site by recombineering, (2) removal of the selection–counterselection markers in the BAC by restriction digestion and (3) circularization of the linear BAC by exonuclease-mediated in vitro DNA annealing. This protocol can be performed within 3 weeks and will enable researchers with DNA cloning experience to master seamless site-directed mutagenesis to accelerate their research. RedEx achieves seamless genetic modification of large, highly repetitive, DNA targets by combining Redαβ-mediated linear–circular homologous recombination, ccdB counterselection and exonuclease-mediated in vitro annealing.
无缝定点诱变是研究蛋白质功能、调整酶催化活性和多轮修饰遗传元件的重要技术，因为它可以在目标位点插入、删除或替换核苷酸、DNA片段甚至整个基因，而不会引入任何不必要的变化。为了便于在具有重复序列的大型质粒和细菌人工染色体（BAC）上进行无缝定点诱变，我们最近开发了 RedEx 策略。与以前的方法相比，我们的方法通过规避重复序列之间不必要的重组，实现了正确重组子的高准确率回收。由于在编码几乎相同酶的模块中存在大量重复 DNA 序列，因此这些基因簇是最难处理的目标之一。在这里，我们详细介绍了在 BAC 载体中进行无缝定点诱变的 RedEx 方法。总的来说，该过程包括三个部分：（1）通过重组将含有所需突变的 RedEx 盒以及两侧有独特限制性位点和 20-bp 重叠序列的选择-反选标记插入目标位点；（2）通过限制性消化去除 BAC 中的选择-反选标记；（3）通过外切酶介导的体外 DNA 退火将线性 BAC 环化。该方案可在 3 周内完成，可使具有 DNA 克隆经验的研究人员掌握无缝定点诱变技术，加快研究进度。
{"title":"Seamless site-directed mutagenesis in complex cloned DNA sequences using the RedEx method","authors":"Ji Luan, Chaoyi Song, Yan Liu, Ruoting He, Ruofei Guo, Qingwen Cui, Chanjuan Jiang, Xiaochen Li, Kexin Hao, A. Francis Stewart, Jun Fu, Youming Zhang, Hailong Wang","doi":"10.1038/s41596-024-01016-9","DOIUrl":"10.1038/s41596-024-01016-9","url":null,"abstract":"Seamless site-directed mutagenesis is an important technique for studying protein functions, tuning enzyme catalytic activities and modifying genetic elements in multiple rounds because it can insert, delete or substitute nucleotides, DNA segments or even entire genes at the target site without introducing any unwanted change. To facilitate seamless site-directed mutagenesis in large plasmids and bacterial artificial chromosomes (BACs) with repetitive sequences, we recently developed the RedEx strategy. Compared with previous methods, our approach achieves the recovery of correct recombinants with high accuracy by circumventing unwanted recombination between repetitive sequences. RedEx readily yields more than 80% accuracy in seamless DNA insertion and deletion in large multimodular polyketide synthase gene clusters, which are among the most difficult targets due to the large number of repetitive DNA sequences in modules encoding almost identical enzymes. Here we present the RedEx method by describing in detail the seamless site-directed mutagenesis in a BAC vector. Overall, the process includes three parts: (1) insertion of the RedEx cassette containing the desired mutation together with selection–counterselection markers flanked by unique restriction sites and 20-bp overlapping sequences into the target site by recombineering, (2) removal of the selection–counterselection markers in the BAC by restriction digestion and (3) circularization of the linear BAC by exonuclease-mediated in vitro DNA annealing. This protocol can be performed within 3 weeks and will enable researchers with DNA cloning experience to master seamless site-directed mutagenesis to accelerate their research. RedEx achieves seamless genetic modification of large, highly repetitive, DNA targets by combining Redαβ-mediated linear–circular homologous recombination, ccdB counterselection and exonuclease-mediated in vitro annealing.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 11","pages":"3360-3388"},"PeriodicalIF":13.1,"publicationDate":"2024-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141620411","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 : 2024-07-11DOI: 10.1038/s41596-024-00998-w
Stéphanie I. De Vleeschauwer, Marieke van de Ven, Anaïs Oudin, Karlijn Debusschere, Kate Connor, Annette T. Byrne, Doreen Ram, Anne Marie Rhebergen, Yannick D. Raeves, Maik Dahlhoff, Virginie Dangles-Marie, Els R. Hermans
Existing guidelines on the preparation (Planning Research and Experimental Procedures on Animals: Recommendations for Excellence (PREPARE)) and reporting (Animal Research: Reporting of In Vivo Experiments (ARRIVE)) of animal experiments do not provide a clear and standardized approach for refinement during in vivo cancer studies, resulting in the publication of generic methodological sections that poorly reflect the attempts made at accurately monitoring different pathologies. Compliance with the 3Rs guidelines has mainly focused on reduction and replacement; however, refinement has been harder to implement. The Oncology Best-practices: Signs, Endpoints and Refinements for in Vivo Experiments (OBSERVE) guidelines are the result of a European initiative supported by EurOPDX and INFRAFRONTIER, and aim to facilitate the refinement of studies using in vivo cancer models by offering robust and practical recommendations on approaches to research scientists and animal care staff. We listed cancer-specific clinical signs as a reference point and from there developed sets of guidelines for a wide variety of rodent models, including genetically engineered models and patient derived xenografts. In this Consensus Statement, we systematically and comprehensively address refinement and monitoring approaches during the design and execution of murine cancer studies. We elaborate on the appropriate preparation of tumor-initiating biologicals and the refinement of tumor-implantation methods. We describe the clinical signs to monitor associated with tumor growth, the appropriate follow-up of animals tailored to varying clinical signs and humane endpoints, and an overview of severity assessment in relation to clinical signs, implantation method and tumor characteristics. The guidelines provide oncology researchers clear and robust guidance for the refinement of in vivo cancer models. This Consensus Statement, which presents the best practices for implantation, monitoring and assessment of tumors in rodents, aims to improve the refinement of cancer models in vivo.
{"title":"OBSERVE: guidelines for the refinement of rodent cancer models","authors":"Stéphanie I. De Vleeschauwer, Marieke van de Ven, Anaïs Oudin, Karlijn Debusschere, Kate Connor, Annette T. Byrne, Doreen Ram, Anne Marie Rhebergen, Yannick D. Raeves, Maik Dahlhoff, Virginie Dangles-Marie, Els R. Hermans","doi":"10.1038/s41596-024-00998-w","DOIUrl":"10.1038/s41596-024-00998-w","url":null,"abstract":"Existing guidelines on the preparation (Planning Research and Experimental Procedures on Animals: Recommendations for Excellence (PREPARE)) and reporting (Animal Research: Reporting of In Vivo Experiments (ARRIVE)) of animal experiments do not provide a clear and standardized approach for refinement during in vivo cancer studies, resulting in the publication of generic methodological sections that poorly reflect the attempts made at accurately monitoring different pathologies. Compliance with the 3Rs guidelines has mainly focused on reduction and replacement; however, refinement has been harder to implement. The Oncology Best-practices: Signs, Endpoints and Refinements for in Vivo Experiments (OBSERVE) guidelines are the result of a European initiative supported by EurOPDX and INFRAFRONTIER, and aim to facilitate the refinement of studies using in vivo cancer models by offering robust and practical recommendations on approaches to research scientists and animal care staff. We listed cancer-specific clinical signs as a reference point and from there developed sets of guidelines for a wide variety of rodent models, including genetically engineered models and patient derived xenografts. In this Consensus Statement, we systematically and comprehensively address refinement and monitoring approaches during the design and execution of murine cancer studies. We elaborate on the appropriate preparation of tumor-initiating biologicals and the refinement of tumor-implantation methods. We describe the clinical signs to monitor associated with tumor growth, the appropriate follow-up of animals tailored to varying clinical signs and humane endpoints, and an overview of severity assessment in relation to clinical signs, implantation method and tumor characteristics. The guidelines provide oncology researchers clear and robust guidance for the refinement of in vivo cancer models. This Consensus Statement, which presents the best practices for implantation, monitoring and assessment of tumors in rodents, aims to improve the refinement of cancer models in vivo.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 9","pages":"2571-2596"},"PeriodicalIF":13.1,"publicationDate":"2024-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41596-024-00998-w.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141590828","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}
Pub Date : 2024-07-09DOI: 10.1038/s41596-024-01008-9
Amanda C. Ritter, Christopher Cheleuitte-Nieves, Neil S. Lipman
A consensus statement outlines the best practices to refine animal welfare in rodent models of cancer. Their adoption will allow the standardization of experimental interventions, considering the characteristics of each cancer model, thereby improving the reproducibility and translational value of data obtained in rodents.
{"title":"The OBSERVE guidelines provide refinement criteria for rodent oncology models","authors":"Amanda C. Ritter, Christopher Cheleuitte-Nieves, Neil S. Lipman","doi":"10.1038/s41596-024-01008-9","DOIUrl":"10.1038/s41596-024-01008-9","url":null,"abstract":"A consensus statement outlines the best practices to refine animal welfare in rodent models of cancer. Their adoption will allow the standardization of experimental interventions, considering the characteristics of each cancer model, thereby improving the reproducibility and translational value of data obtained in rodents.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 10","pages":"2831-2832"},"PeriodicalIF":13.1,"publicationDate":"2024-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141563832","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 : 2024-07-02DOI: 10.1038/s41596-024-01014-x
Jeremy Leitz, Chuchu Wang, Luis Esquivies, John J. Peters, Nisha Gopal, Richard A. Pfuetzner, Austin L. Wang, Axel T. Brunger
Here, we present a protocol for isolating functionally intact glutamatergic synaptic vesicles from whole-mouse brain tissue and using them in a single-vesicle assay to examine their association and fusion with plasma membrane mimic vesicles. This is a Protocol Extension, building on our previous protocol, which used a purely synthetic system comprised of reconstituted proteins in liposomes. We also describe the generation of a peptide based on the vesicular glutamate transporter, which is essential in the isolation process of glutamatergic synaptic vesicles. This method uses easily accessible reagents to generate fusion-competent glutamatergic synaptic vesicles through immunoisolation. The generation of the vGlut peptide can be accomplished in 6 d, while the isolation of the synaptic vesicles by using the peptide can be accomplished in 2 d, with an additional day to fluorescently label the synaptic vesicles for use in a single-vesicle hybrid fusion assay. The single-vesicle fusion assay can be accomplished in 1 d and can unambiguously delineate synaptic vesicle association, dissociation, Ca2+-independent and Ca2+-dependent fusion modalities. This assay grants control of the synaptic vesicle environment while retaining the complexity of the synaptic vesicles themselves. This protocol can be adapted to studies of other types of synaptic vesicles or, more generally, different secretory or transport vesicles. The workflow described here requires expertise in biochemistry techniques, in particular, protein purification and fluorescence imaging. We assume that the laboratory has protein-purification equipment, including chromatography systems. The authors present a protocol for isolating functionally intact glutamatergic synaptic vesicles from whole-mouse brain tissue and using them in a single-vesicle fusion assay to examine their association and fusion with plasma membrane mimic vesicles.
{"title":"Observing isolated synaptic vesicle association and fusion ex vivo","authors":"Jeremy Leitz, Chuchu Wang, Luis Esquivies, John J. Peters, Nisha Gopal, Richard A. Pfuetzner, Austin L. Wang, Axel T. Brunger","doi":"10.1038/s41596-024-01014-x","DOIUrl":"10.1038/s41596-024-01014-x","url":null,"abstract":"Here, we present a protocol for isolating functionally intact glutamatergic synaptic vesicles from whole-mouse brain tissue and using them in a single-vesicle assay to examine their association and fusion with plasma membrane mimic vesicles. This is a Protocol Extension, building on our previous protocol, which used a purely synthetic system comprised of reconstituted proteins in liposomes. We also describe the generation of a peptide based on the vesicular glutamate transporter, which is essential in the isolation process of glutamatergic synaptic vesicles. This method uses easily accessible reagents to generate fusion-competent glutamatergic synaptic vesicles through immunoisolation. The generation of the vGlut peptide can be accomplished in 6 d, while the isolation of the synaptic vesicles by using the peptide can be accomplished in 2 d, with an additional day to fluorescently label the synaptic vesicles for use in a single-vesicle hybrid fusion assay. The single-vesicle fusion assay can be accomplished in 1 d and can unambiguously delineate synaptic vesicle association, dissociation, Ca2+-independent and Ca2+-dependent fusion modalities. This assay grants control of the synaptic vesicle environment while retaining the complexity of the synaptic vesicles themselves. This protocol can be adapted to studies of other types of synaptic vesicles or, more generally, different secretory or transport vesicles. The workflow described here requires expertise in biochemistry techniques, in particular, protein purification and fluorescence imaging. We assume that the laboratory has protein-purification equipment, including chromatography systems. The authors present a protocol for isolating functionally intact glutamatergic synaptic vesicles from whole-mouse brain tissue and using them in a single-vesicle fusion assay to examine their association and fusion with plasma membrane mimic vesicles.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 11","pages":"3139-3161"},"PeriodicalIF":13.1,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141492702","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 : 2024-06-28DOI: 10.1038/s41596-024-01013-y
Negin Farzad, Archibald Enninful, Shuozhen Bao, Di Zhang, Yanxiang Deng, Rong Fan
Spatial epigenetic mapping of tissues enables the study of gene regulation programs and cellular functions with the dependency on their local tissue environment. Here we outline a complete procedure for two spatial epigenomic profiling methods: spatially resolved genome-wide profiling of histone modifications using in situ cleavage under targets and tagmentation (CUT&Tag) chemistry (spatial-CUT&Tag) and transposase-accessible chromatin sequencing (spatial-ATAC-sequencing) for chromatin accessibility. Both assays utilize in-tissue Tn5 transposition to recognize genomic DNA loci followed by microfluidic deterministic barcoding to incorporate spatial address codes. Furthermore, these two methods do not necessitate prior knowledge of the transcription or epigenetic markers for a given tissue or cell type but permit genome-wide unbiased profiling pixel-by-pixel at the 10 μm pixel size level and single-base resolution. To support the widespread adaptation of these methods, details are provided in five general steps: (1) sample preparation; (2) Tn5 transposition in spatial-ATAC-sequencing or antibody-controlled pA–Tn5 tagmentation in CUT&Tag; (3) library preparation; (4) next-generation sequencing; and (5) data analysis using our customed pipelines available at: https://github.com/dyxmvp/Spatial_ATAC-seq and https://github.com/dyxmvp/spatial-CUT-Tag . The whole procedure can be completed on four samples in 2–3 days. Familiarity with basic molecular biology and bioinformatics skills with access to a high-performance computing environment are required. A rudimentary understanding of pathology and specimen sectioning, as well as deterministic barcoding in tissue-specific skills (e.g., design of a multiparameter barcode panel and creation of microfluidic devices), are also advantageous. In this protocol, we mainly focus on spatial profiling of tissue region-specific epigenetic landscapes in mouse embryos and mouse brains using spatial-ATAC-sequencing and spatial-CUT&Tag, but these methods can be used for other species with no need for species-specific probe design. Deterministic barcoding in tissue allows the mapping of chromatin accessibility and histone modifications with high spatial resolution via next-generation sequencing. The method enables rapid identification of cell types and their spatial distribution.
{"title":"Spatially resolved epigenome sequencing via Tn5 transposition and deterministic DNA barcoding in tissue","authors":"Negin Farzad, Archibald Enninful, Shuozhen Bao, Di Zhang, Yanxiang Deng, Rong Fan","doi":"10.1038/s41596-024-01013-y","DOIUrl":"10.1038/s41596-024-01013-y","url":null,"abstract":"Spatial epigenetic mapping of tissues enables the study of gene regulation programs and cellular functions with the dependency on their local tissue environment. Here we outline a complete procedure for two spatial epigenomic profiling methods: spatially resolved genome-wide profiling of histone modifications using in situ cleavage under targets and tagmentation (CUT&Tag) chemistry (spatial-CUT&Tag) and transposase-accessible chromatin sequencing (spatial-ATAC-sequencing) for chromatin accessibility. Both assays utilize in-tissue Tn5 transposition to recognize genomic DNA loci followed by microfluidic deterministic barcoding to incorporate spatial address codes. Furthermore, these two methods do not necessitate prior knowledge of the transcription or epigenetic markers for a given tissue or cell type but permit genome-wide unbiased profiling pixel-by-pixel at the 10 μm pixel size level and single-base resolution. To support the widespread adaptation of these methods, details are provided in five general steps: (1) sample preparation; (2) Tn5 transposition in spatial-ATAC-sequencing or antibody-controlled pA–Tn5 tagmentation in CUT&Tag; (3) library preparation; (4) next-generation sequencing; and (5) data analysis using our customed pipelines available at: https://github.com/dyxmvp/Spatial_ATAC-seq and https://github.com/dyxmvp/spatial-CUT-Tag . The whole procedure can be completed on four samples in 2–3 days. Familiarity with basic molecular biology and bioinformatics skills with access to a high-performance computing environment are required. A rudimentary understanding of pathology and specimen sectioning, as well as deterministic barcoding in tissue-specific skills (e.g., design of a multiparameter barcode panel and creation of microfluidic devices), are also advantageous. In this protocol, we mainly focus on spatial profiling of tissue region-specific epigenetic landscapes in mouse embryos and mouse brains using spatial-ATAC-sequencing and spatial-CUT&Tag, but these methods can be used for other species with no need for species-specific probe design. Deterministic barcoding in tissue allows the mapping of chromatin accessibility and histone modifications with high spatial resolution via next-generation sequencing. The method enables rapid identification of cell types and their spatial distribution.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"19 11","pages":"3389-3425"},"PeriodicalIF":13.1,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469524","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}
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