Pub Date : 2024-05-09DOI: 10.1038/s41596-024-00988-y
Jacob Bobonis, Alessio Ling Jie Yang, Carlos Geert Pieter Voogdt, Athanasios Typas
Toxin–antitoxin systems (TAs) are abundant in bacterial chromosomes and can arrest growth under stress, but usually remain inactive. TAs have been increasingly implicated in halting the growth of infected bacteria from bacteriophages or foreign genetic elements1,2 to protect the population (abortive infection, Abi). The vast diversity and abundance of TAs and other Abi systems3 suggest they play an important immunity role, yet what allows them to sense attack remains largely enigmatic. Here, we describe a method called toxin activation–inhibition conjugation (TAC–TIC), which we used to identify gene products that trigger or block the toxicity of phage-defending tripartite retron-TAs4. TAC–TIC employs high-density arrayed mobilizable gene-overexpression libraries, which are transferred into cells carrying the full TA system or only its toxic component, on inducible vectors. The double-plasmid transconjugants are then pinned on inducer-containing agar plates and their colony fitness is quantified to identify gene products that trigger a TA to inhibit growth (TAC), or that block it from acting (TIC). TAC–TIC is optimized for the Singer ROTOR pinning robot, but can also be used with other robots or manual pinners, and allows screening tens of thousands of genes against any TA or Abi (with toxicity) within a week. Finally, we present a dual conjugation donor/cloning strain (Escherichia coli DATC), which accelerates the construction of TAC–TIC gene-donor libraries from phages, enabling the use of TAC–TIC for identifying TA triggers and antidefense mechanisms in phage genomes. This protocol describes toxin activation–inhibition conjugation (TAC–TIC), a reverse genetics screening approach that can be used to identify triggers or blockers of bacterial toxin–antitoxin or phage immunity systems.
毒素-抗毒素系统（TAs）在细菌染色体中含量丰富，可在压力下阻止生长，但通常保持非活性。越来越多的人认为，TAs 可使受噬菌体或外来遗传因子1,2 感染的细菌停止生长，从而保护种群（中止感染，Abi）。TAs和其他Abi系统3的多样性和丰富性表明它们发挥着重要的免疫作用，然而是什么让它们能够感知攻击，这在很大程度上仍然是个谜。在这里，我们介绍了一种名为毒素激活-抑制共轭（TAC-TIC）的方法，用来鉴定触发或阻断噬菌体防御三方再创-TAs毒性的基因产物4。TAC-TIC 采用高密度阵列可移动基因表达库，通过可诱导载体将其转移到携带完整 TA 系统或仅携带其毒性成分的细胞中。然后将双质粒转染体固定在含诱导剂的琼脂平板上，并对其菌落适应性进行量化，以确定触发 TA 抑制生长（TAC）或阻止 TA 起作用（TIC）的基因产物。TAC-TIC针对辛格ROTOR钉螺机器人进行了优化，但也可用于其他机器人或人工钉螺，可在一周内筛选出数万个针对任何TA或Abi（具有毒性）的基因。最后，我们介绍了一种双共轭供体/克隆菌株（大肠杆菌 DATC），它能加速从噬菌体构建 TAC-TIC 基因供体文库，从而利用 TAC-TIC 鉴定噬菌体基因组中的 TA 触发器和抗防御机制。
{"title":"TAC–TIC, a high-throughput genetics method to identify triggers or blockers of bacterial toxin–antitoxin systems","authors":"Jacob Bobonis, Alessio Ling Jie Yang, Carlos Geert Pieter Voogdt, Athanasios Typas","doi":"10.1038/s41596-024-00988-y","DOIUrl":"10.1038/s41596-024-00988-y","url":null,"abstract":"Toxin–antitoxin systems (TAs) are abundant in bacterial chromosomes and can arrest growth under stress, but usually remain inactive. TAs have been increasingly implicated in halting the growth of infected bacteria from bacteriophages or foreign genetic elements1,2 to protect the population (abortive infection, Abi). The vast diversity and abundance of TAs and other Abi systems3 suggest they play an important immunity role, yet what allows them to sense attack remains largely enigmatic. Here, we describe a method called toxin activation–inhibition conjugation (TAC–TIC), which we used to identify gene products that trigger or block the toxicity of phage-defending tripartite retron-TAs4. TAC–TIC employs high-density arrayed mobilizable gene-overexpression libraries, which are transferred into cells carrying the full TA system or only its toxic component, on inducible vectors. The double-plasmid transconjugants are then pinned on inducer-containing agar plates and their colony fitness is quantified to identify gene products that trigger a TA to inhibit growth (TAC), or that block it from acting (TIC). TAC–TIC is optimized for the Singer ROTOR pinning robot, but can also be used with other robots or manual pinners, and allows screening tens of thousands of genes against any TA or Abi (with toxicity) within a week. Finally, we present a dual conjugation donor/cloning strain (Escherichia coli DATC), which accelerates the construction of TAC–TIC gene-donor libraries from phages, enabling the use of TAC–TIC for identifying TA triggers and antidefense mechanisms in phage genomes. This protocol describes toxin activation–inhibition conjugation (TAC–TIC), a reverse genetics screening approach that can be used to identify triggers or blockers of bacterial toxin–antitoxin or phage immunity systems.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140897947","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 : 2024-05-03DOI: 10.1038/s41596-024-00994-0
Michael Q. Fitzgerald, Tiffany Chu, Francesca Puppo, Rebeca Blanch, Miguel Chillón, Shankar Subramaniam, Alysson R. Muotri
Temporal development of neural electrophysiology follows genetic programming, similar to cellular maturation and organization during development. The emergent properties of this electrophysiological development, namely neural oscillations, can be used to characterize brain development. Recently, we utilized the innate programming encoded in the human genome to generate functionally mature cortical organoids. In brief, stem cells are suspended in culture via continuous shaking and naturally aggregate into embryoid bodies before being exposed to media formulations for neural induction, differentiation and maturation. The specific culture format, media composition and duration of exposure to these media distinguish organoid protocols and determine whether a protocol is guided or unguided toward specific neural fate. The ‘semi-guided’ protocol presented here has shorter induction and differentiation steps with less-specific patterning molecules than most guided protocols but maintains the use of neurotrophic factors such as brain-derived growth factor and neurotrophin-3, unlike unguided approaches. This approach yields the cell type diversity of unguided approaches while maintaining reproducibility for disease modeling. Importantly, we characterized the electrophysiology of these organoids and found that they recapitulate the maturation of neural oscillations observed in the developing human brain, a feature not shown with other approaches. This protocol represents the potential first steps toward bridging molecular and cellular biology to human cognition, and it has already been used to discover underlying features of human brain development, evolution and neurological conditions. Experienced cell culture technicians can expect the protocol to take 1 month, with extended maturation, electrophysiology recording, and adeno-associated virus transduction procedure options. This protocol details the generation of cortical organoids with complex neural oscillations through a ‘semi-guided’ protocol, and their functional characterization using microelectrode array measurements, calcium imaging and adeno-associated virus transduction.
{"title":"Generation of ‘semi-guided’ cortical organoids with complex neural oscillations","authors":"Michael Q. Fitzgerald, Tiffany Chu, Francesca Puppo, Rebeca Blanch, Miguel Chillón, Shankar Subramaniam, Alysson R. Muotri","doi":"10.1038/s41596-024-00994-0","DOIUrl":"10.1038/s41596-024-00994-0","url":null,"abstract":"Temporal development of neural electrophysiology follows genetic programming, similar to cellular maturation and organization during development. The emergent properties of this electrophysiological development, namely neural oscillations, can be used to characterize brain development. Recently, we utilized the innate programming encoded in the human genome to generate functionally mature cortical organoids. In brief, stem cells are suspended in culture via continuous shaking and naturally aggregate into embryoid bodies before being exposed to media formulations for neural induction, differentiation and maturation. The specific culture format, media composition and duration of exposure to these media distinguish organoid protocols and determine whether a protocol is guided or unguided toward specific neural fate. The ‘semi-guided’ protocol presented here has shorter induction and differentiation steps with less-specific patterning molecules than most guided protocols but maintains the use of neurotrophic factors such as brain-derived growth factor and neurotrophin-3, unlike unguided approaches. This approach yields the cell type diversity of unguided approaches while maintaining reproducibility for disease modeling. Importantly, we characterized the electrophysiology of these organoids and found that they recapitulate the maturation of neural oscillations observed in the developing human brain, a feature not shown with other approaches. This protocol represents the potential first steps toward bridging molecular and cellular biology to human cognition, and it has already been used to discover underlying features of human brain development, evolution and neurological conditions. Experienced cell culture technicians can expect the protocol to take 1 month, with extended maturation, electrophysiology recording, and adeno-associated virus transduction procedure options. This protocol details the generation of cortical organoids with complex neural oscillations through a ‘semi-guided’ protocol, and their functional characterization using microelectrode array measurements, calcium imaging and adeno-associated virus transduction.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140835576","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 : 2024-05-03DOI: 10.1038/s41596-024-00989-x
Rory M. Power, Aline Tschanz, Timo Zimmermann, Jonas Ries
Single-molecule localization microscopy (SMLM) enables imaging scientists to visualize biological structures with unprecedented resolution. Particularly powerful implementations of SMLM are capable of three-dimensional, multicolor and high-throughput imaging and can yield key biological insights. However, widespread access to these technologies is limited, primarily by the cost of commercial options and complexity of de novo development of custom systems. Here we provide a comprehensive guide for interested researchers who wish to establish a high-end, custom-built SMLM setup in their laboratories. We detail the initial configuration and subsequent assembly of the SMLM, including the instructions for the alignment of all the optical pathways, the software and hardware integration, and the operation of the instrument. We describe the validation steps, including the preparation and imaging of test and biological samples with structures of well-defined geometries, and assist the user in troubleshooting and benchmarking the system’s performance. Additionally, we provide a walkthrough of the reconstruction of a super-resolved dataset from acquired raw images using the Super-resolution Microscopy Analysis Platform. Depending on the instrument configuration, the cost of the components is in the range US$95,000–180,000, similar to other open-source advanced SMLMs, and substantially lower than the cost of a commercial instrument. A builder with some experience of optical systems is expected to require 4–8 months from the start of the system construction to attain high-quality three-dimensional and multicolor biological images. The buildup and operation of a custom single-molecule localization microscope with state-of-the-art performance and advanced features bridges the gap between entry-level open-source projects and costly commercial systems.
{"title":"Build and operation of a custom 3D, multicolor, single-molecule localization microscope","authors":"Rory M. Power, Aline Tschanz, Timo Zimmermann, Jonas Ries","doi":"10.1038/s41596-024-00989-x","DOIUrl":"10.1038/s41596-024-00989-x","url":null,"abstract":"Single-molecule localization microscopy (SMLM) enables imaging scientists to visualize biological structures with unprecedented resolution. Particularly powerful implementations of SMLM are capable of three-dimensional, multicolor and high-throughput imaging and can yield key biological insights. However, widespread access to these technologies is limited, primarily by the cost of commercial options and complexity of de novo development of custom systems. Here we provide a comprehensive guide for interested researchers who wish to establish a high-end, custom-built SMLM setup in their laboratories. We detail the initial configuration and subsequent assembly of the SMLM, including the instructions for the alignment of all the optical pathways, the software and hardware integration, and the operation of the instrument. We describe the validation steps, including the preparation and imaging of test and biological samples with structures of well-defined geometries, and assist the user in troubleshooting and benchmarking the system’s performance. Additionally, we provide a walkthrough of the reconstruction of a super-resolved dataset from acquired raw images using the Super-resolution Microscopy Analysis Platform. Depending on the instrument configuration, the cost of the components is in the range US$95,000–180,000, similar to other open-source advanced SMLMs, and substantially lower than the cost of a commercial instrument. A builder with some experience of optical systems is expected to require 4–8 months from the start of the system construction to attain high-quality three-dimensional and multicolor biological images. The buildup and operation of a custom single-molecule localization microscope with state-of-the-art performance and advanced features bridges the gap between entry-level open-source projects and costly commercial systems.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140835632","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 : 2024-04-26DOI: 10.1038/s41596-024-00975-3
Alberto J. Taurozzi, Patrick L. Rüther, Ioannis Patramanis, Claire Koenig, Ryan Sinclair Paterson, Palesa P. Madupe, Florian Simon Harking, Frido Welker, Meaghan Mackie, Jazmín Ramos-Madrigal, Jesper V. Olsen, Enrico Cappellini
In temperate and subtropical regions, ancient proteins are reported to survive up to about 2 million years, far beyond the known limits of ancient DNA preservation in the same areas. Accordingly, their amino acid sequences currently represent the only source of genetic information available to pursue phylogenetic inference involving species that went extinct too long ago to be amenable for ancient DNA analysis. Here we present a complete workflow, including sample preparation, mass spectrometric data acquisition and computational analysis, to recover and interpret million-year-old dental enamel protein sequences. During sample preparation, the proteolytic digestion step, usually an integral part of conventional bottom-up proteomics, is omitted to increase the recovery of the randomly degraded peptides spontaneously generated by extensive diagenetic hydrolysis of ancient proteins over geological time. Similarly, we describe other solutions we have adopted to (1) authenticate the endogenous origin of the protein traces we identify, (2) detect and validate amino acid variation in the ancient protein sequences and (3) attempt phylogenetic inference. Sample preparation and data acquisition can be completed in 3–4 working days, while subsequent data analysis usually takes 2–5 days. The workflow described requires basic expertise in ancient biomolecules analysis, mass spectrometry-based proteomics and molecular phylogeny. Finally, we describe the limits of this approach and its potential for the reconstruction of evolutionary relationships in paleontology and paleoanthropology. Ancient proteins carry genetic information from fossils that are too old or degraded for ancient DNA recovery. This protocol describes the extraction and tandem mass spectrometry sequencing of million-year-old dental enamel proteins for phylogenetic inference.
据报道，在温带和亚热带地区，古蛋白质的存活时间长达约 200 万年，远远超出了已知的古 DNA 在同一地区的保存极限。因此，它们的氨基酸序列是目前唯一可用来进行系统发育推断的遗传信息来源，涉及的物种在很久以前就已经灭绝，不适合进行古 DNA 分析。在这里，我们介绍了一个完整的工作流程，包括样品制备、质谱数据采集和计算分析，以恢复和解释百万年前的牙釉质蛋白质序列。在样品制备过程中，我们省略了通常是传统自下而上蛋白质组学不可或缺的一部分的蛋白质分解步骤，以提高对古蛋白质在地质年代中大量成因水解自发产生的随机降解肽的回收率。同样，我们还介绍了我们采用的其他解决方案，以便：(1) 鉴定蛋白质踪迹的内源来源；(2) 检测和验证古蛋白质序列中的氨基酸变异；(3) 尝试进行系统发育推断。样品制备和数据采集可在 3-4 个工作日内完成，而后续的数据分析通常需要 2-5 天。所述工作流程要求具备古代生物分子分析、基于质谱的蛋白质组学和分子系统学方面的基本专业知识。最后，我们将介绍这种方法的局限性及其在重建古生物学和古人类学进化关系方面的潜力。
{"title":"Deep-time phylogenetic inference by paleoproteomic analysis of dental enamel","authors":"Alberto J. Taurozzi, Patrick L. Rüther, Ioannis Patramanis, Claire Koenig, Ryan Sinclair Paterson, Palesa P. Madupe, Florian Simon Harking, Frido Welker, Meaghan Mackie, Jazmín Ramos-Madrigal, Jesper V. Olsen, Enrico Cappellini","doi":"10.1038/s41596-024-00975-3","DOIUrl":"10.1038/s41596-024-00975-3","url":null,"abstract":"In temperate and subtropical regions, ancient proteins are reported to survive up to about 2 million years, far beyond the known limits of ancient DNA preservation in the same areas. Accordingly, their amino acid sequences currently represent the only source of genetic information available to pursue phylogenetic inference involving species that went extinct too long ago to be amenable for ancient DNA analysis. Here we present a complete workflow, including sample preparation, mass spectrometric data acquisition and computational analysis, to recover and interpret million-year-old dental enamel protein sequences. During sample preparation, the proteolytic digestion step, usually an integral part of conventional bottom-up proteomics, is omitted to increase the recovery of the randomly degraded peptides spontaneously generated by extensive diagenetic hydrolysis of ancient proteins over geological time. Similarly, we describe other solutions we have adopted to (1) authenticate the endogenous origin of the protein traces we identify, (2) detect and validate amino acid variation in the ancient protein sequences and (3) attempt phylogenetic inference. Sample preparation and data acquisition can be completed in 3–4 working days, while subsequent data analysis usually takes 2–5 days. The workflow described requires basic expertise in ancient biomolecules analysis, mass spectrometry-based proteomics and molecular phylogeny. Finally, we describe the limits of this approach and its potential for the reconstruction of evolutionary relationships in paleontology and paleoanthropology. Ancient proteins carry genetic information from fossils that are too old or degraded for ancient DNA recovery. This protocol describes the extraction and tandem mass spectrometry sequencing of million-year-old dental enamel proteins for phylogenetic inference.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41596-024-00975-3.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140800429","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 : 2024-04-25DOI: 10.1038/s41596-024-00978-0
Bhaskar Paul, Sabuj Kundu
Methanol is a key building block in the chemical industry. In recent years, it has been used as a C1 source in various organic transformations in the presence of a transition-metal catalyst. This protocol describes the ruthenium- and cobalt-catalyzed utilization of methanol in different types of methylation reactions and heterocycle synthesis. Initially, we describe the synthesis of tridentate ligands (L1–L3) and their corresponding Ru(II) complexes (Ru-1, -2 and -3) and then detail how to apply these Ru(II) complexes and Co/PP3 (PP3 = P(CH2CH2PPh2)3) in various methanol dehydrogenative coupling reactions. We discuss six types of transformations by using methanol or a methanol/water mixture. The experimental setup for all the catalytic reactions is similar and involves adding all the respective reagents and solvents to an argon-filled pressure tube, which is sealed (by screw cap) and refluxed at the indicated temperature before the desired products are isolated and characterized. The catalytic systems described in this protocol work well for both small-scale and preparative-scale synthesis of various N-methylated amines/amides, C-methylated products and quinazolinones. These catalytic reactions are greener and more sustainable than conventional synthesis methods, with only H2 and/or H2O as by-products, and we evaluate the ‘green chemistry metrics’ for a typical substrate. The total time required for the catalytic experiments described in this protocol is 16–28 h, and the operation time is 4 h. An average level of expertise in organic synthesis is required to carry out these protocols. This protocol details methods for using methanol in methylation reactions, including the synthesis of suitable transition metal-containing catalysts, and in the synthesis of heterocycles. The methods described produce only H2 and H2O as by-products.
{"title":"The use of methanol as a C1 building block","authors":"Bhaskar Paul, Sabuj Kundu","doi":"10.1038/s41596-024-00978-0","DOIUrl":"10.1038/s41596-024-00978-0","url":null,"abstract":"Methanol is a key building block in the chemical industry. In recent years, it has been used as a C1 source in various organic transformations in the presence of a transition-metal catalyst. This protocol describes the ruthenium- and cobalt-catalyzed utilization of methanol in different types of methylation reactions and heterocycle synthesis. Initially, we describe the synthesis of tridentate ligands (L1–L3) and their corresponding Ru(II) complexes (Ru-1, -2 and -3) and then detail how to apply these Ru(II) complexes and Co/PP3 (PP3 = P(CH2CH2PPh2)3) in various methanol dehydrogenative coupling reactions. We discuss six types of transformations by using methanol or a methanol/water mixture. The experimental setup for all the catalytic reactions is similar and involves adding all the respective reagents and solvents to an argon-filled pressure tube, which is sealed (by screw cap) and refluxed at the indicated temperature before the desired products are isolated and characterized. The catalytic systems described in this protocol work well for both small-scale and preparative-scale synthesis of various N-methylated amines/amides, C-methylated products and quinazolinones. These catalytic reactions are greener and more sustainable than conventional synthesis methods, with only H2 and/or H2O as by-products, and we evaluate the ‘green chemistry metrics’ for a typical substrate. The total time required for the catalytic experiments described in this protocol is 16–28 h, and the operation time is 4 h. An average level of expertise in organic synthesis is required to carry out these protocols. This protocol details methods for using methanol in methylation reactions, including the synthesis of suitable transition metal-containing catalysts, and in the synthesis of heterocycles. The methods described produce only H2 and H2O as by-products.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140653880","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 : 2024-04-23DOI: 10.1038/s41596-024-00981-5
Kristýna Kantnerová, Nils Kuhlbusch, Dieter Juchelka, Andreas Hilkert, Sebastian Kopf, Cajetan Neubauer
Stable isotopes of carbon, hydrogen, nitrogen, oxygen and sulfur are widespread in nature. Nevertheless, their relative abundance is not the same everywhere. This is due to kinetic isotope effects in enzymes and other physical principles such as equilibrium thermodynamics. Variations in isotope ratios offer unique insights into environmental pollution, trophic relationships in ecology, metabolic disorders and Earth history including climate history. Although classical isotope ratio mass spectrometry (IRMS) techniques still struggle to access intramolecular information like site-specific isotope abundance, electrospray ionization–Orbitrap mass spectrometry can be used to achieve precise and accurate intramolecular quantification of isotopically substituted molecules (‘isotopocules’). This protocol describes two procedures. In the first one, we provide a step-by-step beginner’s guide for performing multi-elemental, intramolecular and site-specific stable isotope analysis in unlabeled polar solutes by direct infusion. Using a widely available calibration solution, isotopocules of trifluoroacetic acid and immonium ions from the model peptide MRFA are quantified. In the second approach, nitrate is used as a simple model for a flow injection routine that enables access to a diverse range of naturally occurring isotopic signatures in inorganic oxyanions. Each procedure takes 2–3 h to complete and requires expertise only in general mass spectrometry. The workflows use optimized Orbitrap IRMS data-extraction and -processing software and are transferable to various analytes amenable to soft ionization, including metabolites, peptides, drugs and environmental pollutants. Optimized mass spectrometry systems will enable intramolecular isotope research in many areas of biology. Isotope ratio mass spectrometry as described in this protocol can be used to determine natural variation in the abundance of stable isotopes in individual compounds to provide information relevant to metabolism, ecology or climate change.
{"title":"A guide to precise measurements of isotope abundance by ESI-Orbitrap MS","authors":"Kristýna Kantnerová, Nils Kuhlbusch, Dieter Juchelka, Andreas Hilkert, Sebastian Kopf, Cajetan Neubauer","doi":"10.1038/s41596-024-00981-5","DOIUrl":"10.1038/s41596-024-00981-5","url":null,"abstract":"Stable isotopes of carbon, hydrogen, nitrogen, oxygen and sulfur are widespread in nature. Nevertheless, their relative abundance is not the same everywhere. This is due to kinetic isotope effects in enzymes and other physical principles such as equilibrium thermodynamics. Variations in isotope ratios offer unique insights into environmental pollution, trophic relationships in ecology, metabolic disorders and Earth history including climate history. Although classical isotope ratio mass spectrometry (IRMS) techniques still struggle to access intramolecular information like site-specific isotope abundance, electrospray ionization–Orbitrap mass spectrometry can be used to achieve precise and accurate intramolecular quantification of isotopically substituted molecules (‘isotopocules’). This protocol describes two procedures. In the first one, we provide a step-by-step beginner’s guide for performing multi-elemental, intramolecular and site-specific stable isotope analysis in unlabeled polar solutes by direct infusion. Using a widely available calibration solution, isotopocules of trifluoroacetic acid and immonium ions from the model peptide MRFA are quantified. In the second approach, nitrate is used as a simple model for a flow injection routine that enables access to a diverse range of naturally occurring isotopic signatures in inorganic oxyanions. Each procedure takes 2–3 h to complete and requires expertise only in general mass spectrometry. The workflows use optimized Orbitrap IRMS data-extraction and -processing software and are transferable to various analytes amenable to soft ionization, including metabolites, peptides, drugs and environmental pollutants. Optimized mass spectrometry systems will enable intramolecular isotope research in many areas of biology. Isotope ratio mass spectrometry as described in this protocol can be used to determine natural variation in the abundance of stable isotopes in individual compounds to provide information relevant to metabolism, ecology or climate change.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140666146","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 : 2024-04-23DOI: 10.1038/s41596-024-00992-2
Ruotian Chen, Chenwei Ni, Jian Zhu, Fengtao Fan, Can Li
Solar-driven photocatalytic reactions offer a promising route to clean and sustainable energy, and the spatial separation of photogenerated charges on the photocatalyst surface is the key to determining photocatalytic efficiency. However, probing the charge-separation properties of photocatalysts is a formidable challenge because of the spatially heterogeneous microstructures, complicated charge-separation mechanisms and lack of sensitivity for detecting the low density of separated photogenerated charges. Recently, we developed surface photovoltage microscopy (SPVM) with high spatial and energy resolution that enables the direct mapping of surface-charge distributions and quantitative assessment of the charge-separation properties of photocatalysts at the nanoscale, potentially providing unprecedented insights into photocatalytic charge-separation processes. Here, this protocol presents detailed procedures that enable researchers to construct the SPVM instruments by integrating Kelvin probe force microscopy with an illumination system and the modulated surface photovoltage (SPV) approach. It then describes in detail how to perform SPVM measurements on actual photocatalyst particles, including sample preparation, tuning of the microscope, adjustment of the illuminated light path, acquisition of SPVM images and measurements of spatially resolved modulated SPV signals. Moreover, the protocol also includes sophisticated data analysis that can guide non-experts in understanding the microscopic charge-separation mechanisms. The measurements are ordinarily performed on photocatalysts with a conducting substrate in gases or vacuum and can be completed in 15 h. Surface photovoltage microscopy as described in this protocol allows high spatial and energy resolution mapping of surface-charge distributions on photocatalyst particles, enabling rational design of improved materials.
{"title":"Surface photovoltage microscopy for mapping charge separation on photocatalyst particles","authors":"Ruotian Chen, Chenwei Ni, Jian Zhu, Fengtao Fan, Can Li","doi":"10.1038/s41596-024-00992-2","DOIUrl":"10.1038/s41596-024-00992-2","url":null,"abstract":"Solar-driven photocatalytic reactions offer a promising route to clean and sustainable energy, and the spatial separation of photogenerated charges on the photocatalyst surface is the key to determining photocatalytic efficiency. However, probing the charge-separation properties of photocatalysts is a formidable challenge because of the spatially heterogeneous microstructures, complicated charge-separation mechanisms and lack of sensitivity for detecting the low density of separated photogenerated charges. Recently, we developed surface photovoltage microscopy (SPVM) with high spatial and energy resolution that enables the direct mapping of surface-charge distributions and quantitative assessment of the charge-separation properties of photocatalysts at the nanoscale, potentially providing unprecedented insights into photocatalytic charge-separation processes. Here, this protocol presents detailed procedures that enable researchers to construct the SPVM instruments by integrating Kelvin probe force microscopy with an illumination system and the modulated surface photovoltage (SPV) approach. It then describes in detail how to perform SPVM measurements on actual photocatalyst particles, including sample preparation, tuning of the microscope, adjustment of the illuminated light path, acquisition of SPVM images and measurements of spatially resolved modulated SPV signals. Moreover, the protocol also includes sophisticated data analysis that can guide non-experts in understanding the microscopic charge-separation mechanisms. The measurements are ordinarily performed on photocatalysts with a conducting substrate in gases or vacuum and can be completed in 15 h. Surface photovoltage microscopy as described in this protocol allows high spatial and energy resolution mapping of surface-charge distributions on photocatalyst particles, enabling rational design of improved materials.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140667191","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 : 2024-04-18DOI: 10.1038/s41596-024-00979-z
Martin P. Schwalm, Krishna Saxena, Susanne Müller, Stefan Knapp
The rational development of small-molecule degraders (e.g., proteolysis targeting chimeras) remains a challenge as the rate-limiting steps that determine degrader efficiency are largely unknown. Standard methods in the field of targeted protein degradation mostly rely on classical, low-throughput endpoint assays such as western blots or quantitative proteomics. Here we applied NanoLuciferase- and HaloTag-based screening technologies to determine the kinetics and stability of small-molecule-induced ternary complex formation between a protein of interest and a selected E3 ligase. A collection of live-cell assays were designed to probe the most critical steps of the degradation process while minimizing the number of required expression constructs, making the proposed assay pipeline flexible and adaptable to the requirements of the users. This approach evaluates the underlying mechanism of selective target degraders and reveals the exact characteristics of the developed degrader molecules in living cells. The protocol allows scientists trained in basic cell culture and molecular biology to carry out small-molecule proximity-inducer screening via tracking of the ternary complex formation within 2 weeks of establishment, while degrader screening using the HiBiT system requires a CRISPR–Cas9 engineered cell line whose generation can take up to 3 months. After cell-line generation, degrader screening and validation can be carried out in high-throughput manner within days. NanoLuciferase- and HaloTag-based screening technologies are versatile tools suitable for the live-cell analysis of the entire small-molecule-induced degradation cascade to uncover the mode of action of proximity-inducing compounds such as PROTACs.
{"title":"Luciferase- and HaloTag-based reporter assays to measure small-molecule-induced degradation pathway in living cells","authors":"Martin P. Schwalm, Krishna Saxena, Susanne Müller, Stefan Knapp","doi":"10.1038/s41596-024-00979-z","DOIUrl":"10.1038/s41596-024-00979-z","url":null,"abstract":"The rational development of small-molecule degraders (e.g., proteolysis targeting chimeras) remains a challenge as the rate-limiting steps that determine degrader efficiency are largely unknown. Standard methods in the field of targeted protein degradation mostly rely on classical, low-throughput endpoint assays such as western blots or quantitative proteomics. Here we applied NanoLuciferase- and HaloTag-based screening technologies to determine the kinetics and stability of small-molecule-induced ternary complex formation between a protein of interest and a selected E3 ligase. A collection of live-cell assays were designed to probe the most critical steps of the degradation process while minimizing the number of required expression constructs, making the proposed assay pipeline flexible and adaptable to the requirements of the users. This approach evaluates the underlying mechanism of selective target degraders and reveals the exact characteristics of the developed degrader molecules in living cells. The protocol allows scientists trained in basic cell culture and molecular biology to carry out small-molecule proximity-inducer screening via tracking of the ternary complex formation within 2 weeks of establishment, while degrader screening using the HiBiT system requires a CRISPR–Cas9 engineered cell line whose generation can take up to 3 months. After cell-line generation, degrader screening and validation can be carried out in high-throughput manner within days. NanoLuciferase- and HaloTag-based screening technologies are versatile tools suitable for the live-cell analysis of the entire small-molecule-induced degradation cascade to uncover the mode of action of proximity-inducing compounds such as PROTACs.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140627346","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 : 2024-04-18DOI: 10.1038/s41596-024-00990-4
Chao Chen, Xiaoyan Zhang, Zhiyuan Gao, Guangxue Feng, Dan Ding
Fluorescence imaging represents a vital tool in modern biology, oncology and biomedical applications. Afterglow luminescence (AGL), which circumvents the light scattering and tissue autofluorescence interference associated with real-time excitation source, shows remarkably increased imaging sensitivity and depth. Here we present a protocol for the design and synthesis of AGL nanoprobes with an aggregation-induced emission (AIE) effect to simultaneously red shift and amplify the afterglow signal for tumor imaging and image-guided tumor resection. The nanoprobe (AGL AIE dot) is composed of an enol ether format of Schaap’s agent and a near-infrared AIE fluorogen (AIEgen) (tetraphenylethylene-phenyl-dicyanomethylene-4H-chromene, TPE-Ph-DCM) to suppress the nonradiative dissipation pathway. Pre-irradiating AGL AIE dots with white light could generate singlet oxygen to convert Schaap’s agent to its 1,2-dioxetane format, thus initializing the AGL process. With the aid of AIEgen, the AGL shows simultaneously red shifted emission maximum (from ~540 nm to ~625 nm) and enhanced intensity (by 3.2-fold), facilitating better signal-to-background ratio, imaging sensitivity and depth. Intriguingly, the activated AGL can last for over 10 days. Compared with conventional approaches, our method provides a new solution to concurrently red shift and amplify afterglow signals for better in vivo imaging outcomes. The preparation of AGL AIE dots takes ~2 days, the in vitro characterization takes ~10 days (less than 1 day if not involving afterglow kinetic profile study) and the tumor imaging and image-guided tumor resection takes ~7 days. These procedures can be easily reproduced and amended after standard laboratory training in chemical synthesis and animal handling. This protocol describes the preparation of long-lasting aggregation-induced emission-based, near-infrared afterglow luminescence nanoprobes. Their enhanced afterglow intensity results in improved imaging sensitivity and depth in vivo.
{"title":"Preparation of AIEgen-based near-infrared afterglow luminescence nanoprobes for tumor imaging and image-guided tumor resection","authors":"Chao Chen, Xiaoyan Zhang, Zhiyuan Gao, Guangxue Feng, Dan Ding","doi":"10.1038/s41596-024-00990-4","DOIUrl":"10.1038/s41596-024-00990-4","url":null,"abstract":"Fluorescence imaging represents a vital tool in modern biology, oncology and biomedical applications. Afterglow luminescence (AGL), which circumvents the light scattering and tissue autofluorescence interference associated with real-time excitation source, shows remarkably increased imaging sensitivity and depth. Here we present a protocol for the design and synthesis of AGL nanoprobes with an aggregation-induced emission (AIE) effect to simultaneously red shift and amplify the afterglow signal for tumor imaging and image-guided tumor resection. The nanoprobe (AGL AIE dot) is composed of an enol ether format of Schaap’s agent and a near-infrared AIE fluorogen (AIEgen) (tetraphenylethylene-phenyl-dicyanomethylene-4H-chromene, TPE-Ph-DCM) to suppress the nonradiative dissipation pathway. Pre-irradiating AGL AIE dots with white light could generate singlet oxygen to convert Schaap’s agent to its 1,2-dioxetane format, thus initializing the AGL process. With the aid of AIEgen, the AGL shows simultaneously red shifted emission maximum (from ~540 nm to ~625 nm) and enhanced intensity (by 3.2-fold), facilitating better signal-to-background ratio, imaging sensitivity and depth. Intriguingly, the activated AGL can last for over 10 days. Compared with conventional approaches, our method provides a new solution to concurrently red shift and amplify afterglow signals for better in vivo imaging outcomes. The preparation of AGL AIE dots takes ~2 days, the in vitro characterization takes ~10 days (less than 1 day if not involving afterglow kinetic profile study) and the tumor imaging and image-guided tumor resection takes ~7 days. These procedures can be easily reproduced and amended after standard laboratory training in chemical synthesis and animal handling. This protocol describes the preparation of long-lasting aggregation-induced emission-based, near-infrared afterglow luminescence nanoprobes. Their enhanced afterglow intensity results in improved imaging sensitivity and depth in vivo.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140614061","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 : 2024-04-17DOI: 10.1038/s41596-024-00984-2
Jia Ping Tan, Xiaodong Liu, Jose M. Polo
The study of early human embryogenesis has relied on the use of blastocysts donated to research or simple stem cell culture systems such as pluripotent and trophoblast stem cells, which have been seminal in shedding light on many key developmental processes. However, simple culture systems lack the necessary complexity to adequately model the spatiotemporal, cellular and molecular dynamics occurring during the early phases of embryonic development. As such, an in vitro model of the human blastocyst is advantageous in many aspects to decipher human embryogenesis. Here we describe a step-by-step protocol for the generation of induced blastoids (iBlastoids), an in vitro integrated model of the human blastocyst derived via somatic reprogramming. This protocol details the workflow for reprogramming of human dermal fibroblasts and subsequent generation of iBlastoids using the reprogramming intermediates, which together takes ~27 days (21 days for reprogramming and 6 days for iBlastoid generation). We also discuss several characterization/functional assays that can be used on the iBlastoids. We believe that a person trained in cell culture with ~1 year of experience with human somatic cell and reprogramming/cell differentiation assays would be able to perform this protocol. In short, the iBlastoids present an alternative tool as a model to the blastocyst to facilitate the scientific community in the exploration of early human development. A protocol for the generation of induced blastoids, an in vitro integrated model of the human blastocyst derived via somatic reprogramming. This model overcomes restrictions associated with the use of human blastocysts in embryology research.
{"title":"Reprogramming fibroblast into human iBlastoids","authors":"Jia Ping Tan, Xiaodong Liu, Jose M. Polo","doi":"10.1038/s41596-024-00984-2","DOIUrl":"10.1038/s41596-024-00984-2","url":null,"abstract":"The study of early human embryogenesis has relied on the use of blastocysts donated to research or simple stem cell culture systems such as pluripotent and trophoblast stem cells, which have been seminal in shedding light on many key developmental processes. However, simple culture systems lack the necessary complexity to adequately model the spatiotemporal, cellular and molecular dynamics occurring during the early phases of embryonic development. As such, an in vitro model of the human blastocyst is advantageous in many aspects to decipher human embryogenesis. Here we describe a step-by-step protocol for the generation of induced blastoids (iBlastoids), an in vitro integrated model of the human blastocyst derived via somatic reprogramming. This protocol details the workflow for reprogramming of human dermal fibroblasts and subsequent generation of iBlastoids using the reprogramming intermediates, which together takes ~27 days (21 days for reprogramming and 6 days for iBlastoid generation). We also discuss several characterization/functional assays that can be used on the iBlastoids. We believe that a person trained in cell culture with ~1 year of experience with human somatic cell and reprogramming/cell differentiation assays would be able to perform this protocol. In short, the iBlastoids present an alternative tool as a model to the blastocyst to facilitate the scientific community in the exploration of early human development. A protocol for the generation of induced blastoids, an in vitro integrated model of the human blastocyst derived via somatic reprogramming. This model overcomes restrictions associated with the use of human blastocysts in embryology research.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140614052","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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