Pub Date : 2024-04-11DOI: 10.1038/s41596-024-00983-3
Ying Chen, Yiwei Yang, Fan Zhang
In vivo microscopy of single cells enables following pathological changes in tissues, revealing signaling networks and cell interactions critical to disease progression. However, conventional intravital microscopy at visible and near-infrared wavelengths <900 nm (NIR-I) suffers from attenuation and is typically performed following the surgical creation of an imaging window. Such surgical procedures cause the alteration of the local vasculature and induce inflammation in skin, muscle and skull, inevitably altering the microenvironment in the imaging area. Here, we detail the use of near-infrared fluorescence (NIR-II, 1,000–1,700 nm) for in vivo microscopy to circumvent attenuation in living tissues. This approach enables the noninvasive visualization of cell migration in deep tissues by labeling specific cells with NIR-II lanthanide downshifting nanoparticles exhibiting high physicochemical stability and photostability. We further developed a NIR-II fluorescence microscopy setup for in vivo imaging through the intact skull with high spatiotemporal resolution, which we use for the real-time dynamic visualization of single-neutrophil behavior in the deep brain of a mouse model of ischemic stroke. The labeled downshifting nanoparticle synthesis takes 5–6 d, the imaging system setup takes 1–2 h, the in vivo cell labeling takes 1–3 h, the in vivo NIR-II microscopic imaging takes 3–5 h and the data analysis takes 3–8 h. The procedures can be performed by users with standard laboratory training in nanomaterials research and appropriate animal handling. Activated neutrophils labeled with NIR-II lanthanide downshifting nanoparticles can be sequentially imaged through the intact skull of a mouse model of ischemic stroke during adhesion, crawling and extravasation processes
{"title":"Noninvasive in vivo microscopy of single neutrophils in the mouse brain via NIR-II fluorescent nanomaterials","authors":"Ying Chen, Yiwei Yang, Fan Zhang","doi":"10.1038/s41596-024-00983-3","DOIUrl":"10.1038/s41596-024-00983-3","url":null,"abstract":"In vivo microscopy of single cells enables following pathological changes in tissues, revealing signaling networks and cell interactions critical to disease progression. However, conventional intravital microscopy at visible and near-infrared wavelengths <900 nm (NIR-I) suffers from attenuation and is typically performed following the surgical creation of an imaging window. Such surgical procedures cause the alteration of the local vasculature and induce inflammation in skin, muscle and skull, inevitably altering the microenvironment in the imaging area. Here, we detail the use of near-infrared fluorescence (NIR-II, 1,000–1,700 nm) for in vivo microscopy to circumvent attenuation in living tissues. This approach enables the noninvasive visualization of cell migration in deep tissues by labeling specific cells with NIR-II lanthanide downshifting nanoparticles exhibiting high physicochemical stability and photostability. We further developed a NIR-II fluorescence microscopy setup for in vivo imaging through the intact skull with high spatiotemporal resolution, which we use for the real-time dynamic visualization of single-neutrophil behavior in the deep brain of a mouse model of ischemic stroke. The labeled downshifting nanoparticle synthesis takes 5–6 d, the imaging system setup takes 1–2 h, the in vivo cell labeling takes 1–3 h, the in vivo NIR-II microscopic imaging takes 3–5 h and the data analysis takes 3–8 h. The procedures can be performed by users with standard laboratory training in nanomaterials research and appropriate animal handling. Activated neutrophils labeled with NIR-II lanthanide downshifting nanoparticles can be sequentially imaged through the intact skull of a mouse model of ischemic stroke during adhesion, crawling and extravasation processes","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140574645","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 : 2024-04-09DOI: 10.1038/s41596-024-00977-1
Emma J. Vontalge, Tamar Kavlashvili, Steven N. Dahmen, Matthew T. Cranford, James M. Dewar
A major obstacle to studying DNA replication is that it involves asynchronous and highly delocalized events. A reversible replication barrier overcomes this limitation and allows replication fork movement to be synchronized and localized, facilitating the study of replication fork function and replication coupled repair. Here we provide details on establishing a reversible replication barrier in vitro and using it to monitor different aspects of DNA replication. DNA template containing an array of lac operator (lacO) sequences is first bound to purified lac repressor (LacR). This substrate is then replicated in vitro using a biochemical replication system, which results in replication forks stalled on either side of the LacR array regardless of when or where they arise. Once replication forks are synchronized at the barrier, isopropyl-β-d-thiogalactopyranoside can be added to disrupt LacR binding so that replication forks synchronously resume synthesis. We describe how this approach can be employed to control replication fork elongation, termination, stalling and uncoupling, as well as assays that can be used to monitor these processes. We also explain how this approach can be adapted to control whether replication forks encounter a DNA lesion on the leading or lagging strand template and whether a converging fork is present. The required reagents can be prepared in 1–2 weeks and experiments using this approach are typically performed over 1–3 d. The main requirements for utilizing the LacR replication barrier are basic biochemical expertise and access to an in vitro system to study DNA replication. Investigators should also be trained in working with radioactive materials. This protocol describes the establishment of a reversible replication barrier using plasmid templates containing a lacO array bound by LacR repressor. The method allows fine control of replication fork movement and replication fork encounter with DNA lesions.
研究 DNA 复制的一个主要障碍是它涉及非同步和高度分散的事件。可逆复制屏障克服了这一限制，使复制叉运动同步化和定位化，促进了对复制叉功能和复制耦合修复的研究。在此，我们将详细介绍如何在体外建立可逆复制屏障，并用它来监测 DNA 复制的不同方面。含有 lac 操作者（lacO）序列阵列的 DNA 模板首先与纯化的 lac 抑制剂（LacR）结合。然后利用生化复制系统在体外复制这种底物，结果是复制叉停滞在 LacR 阵列的两侧，而不管它们何时何地出现。一旦复制叉在屏障处同步化，就可以加入异丙基-β-d-硫代半乳糖苷来破坏 LacR 的结合，从而使复制叉同步恢复合成。我们介绍了如何利用这种方法来控制复制叉的伸长、终止、停滞和解偶联，以及可用于监测这些过程的检测方法。我们还解释了如何调整这种方法，以控制复制叉是否遇到前向链或滞后链模板上的 DNA 病变，以及是否存在会聚叉。利用 LacR 复制屏障的主要要求是具备基本的生化专业知识和使用体外系统研究 DNA 复制的能力。研究人员还应接受过使用放射性材料的培训。
{"title":"Control of DNA replication in vitro using a reversible replication barrier","authors":"Emma J. Vontalge, Tamar Kavlashvili, Steven N. Dahmen, Matthew T. Cranford, James M. Dewar","doi":"10.1038/s41596-024-00977-1","DOIUrl":"10.1038/s41596-024-00977-1","url":null,"abstract":"A major obstacle to studying DNA replication is that it involves asynchronous and highly delocalized events. A reversible replication barrier overcomes this limitation and allows replication fork movement to be synchronized and localized, facilitating the study of replication fork function and replication coupled repair. Here we provide details on establishing a reversible replication barrier in vitro and using it to monitor different aspects of DNA replication. DNA template containing an array of lac operator (lacO) sequences is first bound to purified lac repressor (LacR). This substrate is then replicated in vitro using a biochemical replication system, which results in replication forks stalled on either side of the LacR array regardless of when or where they arise. Once replication forks are synchronized at the barrier, isopropyl-β-d-thiogalactopyranoside can be added to disrupt LacR binding so that replication forks synchronously resume synthesis. We describe how this approach can be employed to control replication fork elongation, termination, stalling and uncoupling, as well as assays that can be used to monitor these processes. We also explain how this approach can be adapted to control whether replication forks encounter a DNA lesion on the leading or lagging strand template and whether a converging fork is present. The required reagents can be prepared in 1–2 weeks and experiments using this approach are typically performed over 1–3 d. The main requirements for utilizing the LacR replication barrier are basic biochemical expertise and access to an in vitro system to study DNA replication. Investigators should also be trained in working with radioactive materials. This protocol describes the establishment of a reversible replication barrier using plasmid templates containing a lacO array bound by LacR repressor. The method allows fine control of replication fork movement and replication fork encounter with DNA lesions.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140574230","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 : 2024-04-04DOI: 10.1038/s41596-024-00973-5
Rebeccah A. Warmack, Belinda B. Wenke, Thomas Spatzal, Douglas C. Rees
Single-particle cryo-electron microscopy (cryoEM) provides an attractive avenue for advancing our atomic resolution understanding of materials, molecules and living systems. However, the vast majority of published cryoEM methodologies focus on the characterization of aerobically purified samples. Air-sensitive enzymes and microorganisms represent important yet understudied systems in structural biology. We have recently demonstrated the success of an anaerobic single-particle cryoEM workflow applied to the air-sensitive nitrogenase enzymes. In this protocol, we detail the use of Schlenk lines and anaerobic chambers to prepare samples, including a protein tag for monitoring sample exposure to oxygen in air. We describe how to use a plunge freezing apparatus inside of a soft-sided vinyl chamber of the type we routinely use for anaerobic biochemistry and crystallography of oxygen-sensitive proteins. Manual control of the airlock allows for introduction of liquid cryogens into the tent. A custom vacuum port provides slow, continuous evacuation of the tent atmosphere to avoid accumulation of flammable vapors within the enclosed chamber. These methods allowed us to obtain high-resolution structures of both nitrogenase proteins using single-particle cryoEM. The procedures involved can be generally subdivided into a 4 d anaerobic sample generation procedure, and a 1 d anaerobic cryoEM sample preparation step, followed by conventional cryoEM imaging and processing steps. As nitrogen is a substrate for nitrogenase, the Schlenk lines and anaerobic chambers described in this procedure are operated under an argon atmosphere; however, the system and these procedures are compatible with other controlled gas environments. It can be challenging to obtain meaningful and accurate structural information for air-sensitive proteins. This protocol describes the application of customized vacuum manifold and anaerobic chamber setups for the purification and cryo-electron microscopy analysis of air-sensitive nitrogenase enzymes.
{"title":"Anaerobic cryoEM protocols for air-sensitive nitrogenase proteins","authors":"Rebeccah A. Warmack, Belinda B. Wenke, Thomas Spatzal, Douglas C. Rees","doi":"10.1038/s41596-024-00973-5","DOIUrl":"10.1038/s41596-024-00973-5","url":null,"abstract":"Single-particle cryo-electron microscopy (cryoEM) provides an attractive avenue for advancing our atomic resolution understanding of materials, molecules and living systems. However, the vast majority of published cryoEM methodologies focus on the characterization of aerobically purified samples. Air-sensitive enzymes and microorganisms represent important yet understudied systems in structural biology. We have recently demonstrated the success of an anaerobic single-particle cryoEM workflow applied to the air-sensitive nitrogenase enzymes. In this protocol, we detail the use of Schlenk lines and anaerobic chambers to prepare samples, including a protein tag for monitoring sample exposure to oxygen in air. We describe how to use a plunge freezing apparatus inside of a soft-sided vinyl chamber of the type we routinely use for anaerobic biochemistry and crystallography of oxygen-sensitive proteins. Manual control of the airlock allows for introduction of liquid cryogens into the tent. A custom vacuum port provides slow, continuous evacuation of the tent atmosphere to avoid accumulation of flammable vapors within the enclosed chamber. These methods allowed us to obtain high-resolution structures of both nitrogenase proteins using single-particle cryoEM. The procedures involved can be generally subdivided into a 4 d anaerobic sample generation procedure, and a 1 d anaerobic cryoEM sample preparation step, followed by conventional cryoEM imaging and processing steps. As nitrogen is a substrate for nitrogenase, the Schlenk lines and anaerobic chambers described in this procedure are operated under an argon atmosphere; however, the system and these procedures are compatible with other controlled gas environments. It can be challenging to obtain meaningful and accurate structural information for air-sensitive proteins. This protocol describes the application of customized vacuum manifold and anaerobic chamber setups for the purification and cryo-electron microscopy analysis of air-sensitive nitrogenase enzymes.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140574641","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 : 2024-04-02DOI: 10.1038/s41596-024-00986-0
Mohammed Alser, Brendan Lawlor, Richard J. Abdill, Sharon Waymost, Ram Ayyala, Neha Rajkumar, Nathan LaPierre, Jaqueline Brito, André M. Ribeiro-dos-Santos, Nour Almadhoun, Varuni Sarwal, Can Firtina, Tomasz Osinski, Eleazar Eskin, Qiyang Hu, Derek Strong, Byoung-Do (B.D) Kim, Malak S. Abedalthagafi, Onur Mutlu, Serghei Mangul
Methods for analyzing the full complement of a biomolecule type, e.g., proteomics or metabolomics, generate large amounts of complex data. The software tools used to analyze omics data have reshaped the landscape of modern biology and become an essential component of biomedical research. These tools are themselves quite complex and often require the installation of other supporting software, libraries and/or databases. A researcher may also be using multiple different tools that require different versions of the same supporting materials. The increasing dependence of biomedical scientists on these powerful tools creates a need for easier installation and greater usability. Packaging and containerization are different approaches to satisfy this need by delivering omics tools already wrapped in additional software that makes the tools easier to install and use. In this systematic review, we describe and compare the features of prominent packaging and containerization platforms. We outline the challenges, advantages and limitations of each approach and some of the most widely used platforms from the perspectives of users, software developers and system administrators. We also propose principles to make the distribution of omics software more sustainable and robust to increase the reproducibility of biomedical and life science research. Attempts to reproduce the computational steps described in published omics research often fail. This review provides guidelines for the packaging and containerization of software so that readers can use the exact programs used in published work.
{"title":"Packaging and containerization of computational methods","authors":"Mohammed Alser, Brendan Lawlor, Richard J. Abdill, Sharon Waymost, Ram Ayyala, Neha Rajkumar, Nathan LaPierre, Jaqueline Brito, André M. Ribeiro-dos-Santos, Nour Almadhoun, Varuni Sarwal, Can Firtina, Tomasz Osinski, Eleazar Eskin, Qiyang Hu, Derek Strong, Byoung-Do (B.D) Kim, Malak S. Abedalthagafi, Onur Mutlu, Serghei Mangul","doi":"10.1038/s41596-024-00986-0","DOIUrl":"10.1038/s41596-024-00986-0","url":null,"abstract":"Methods for analyzing the full complement of a biomolecule type, e.g., proteomics or metabolomics, generate large amounts of complex data. The software tools used to analyze omics data have reshaped the landscape of modern biology and become an essential component of biomedical research. These tools are themselves quite complex and often require the installation of other supporting software, libraries and/or databases. A researcher may also be using multiple different tools that require different versions of the same supporting materials. The increasing dependence of biomedical scientists on these powerful tools creates a need for easier installation and greater usability. Packaging and containerization are different approaches to satisfy this need by delivering omics tools already wrapped in additional software that makes the tools easier to install and use. In this systematic review, we describe and compare the features of prominent packaging and containerization platforms. We outline the challenges, advantages and limitations of each approach and some of the most widely used platforms from the perspectives of users, software developers and system administrators. We also propose principles to make the distribution of omics software more sustainable and robust to increase the reproducibility of biomedical and life science research. Attempts to reproduce the computational steps described in published omics research often fail. This review provides guidelines for the packaging and containerization of software so that readers can use the exact programs used in published work.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140574342","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 : 2024-03-28DOI: 10.1038/s41596-024-00976-2
Nils Kriedemann, Wiebke Triebert, Jana Teske, Mira Mertens, Annika Franke, Kevin Ullmann, Felix Manstein, Lika Drakhlis, Alexandra Haase, Caroline Halloin, Ulrich Martin, Robert Zweigerdt
A promising cell-therapy approach for heart failure aims at differentiating human pluripotent stem cells (hPSCs) into functional cardiomyocytes (CMs) in vitro to replace the disease-induced loss of patients’ heart muscle cells in vivo. But many challenges remain for the routine clinical application of hPSC-derived CMs (hPSC-CMs), including good manufacturing practice (GMP)-compliant production strategies. This protocol describes the efficient generation of hPSC-CM aggregates in suspension culture, emphasizing process simplicity, robustness and GMP compliance. The strategy promotes clinical translation and other applications that require large numbers of CMs. Using a simple spinner-flask platform, this protocol is applicable to a broad range of users with general experience in handling hPSCs without extensive know-how in biotechnology. hPSCs are expanded in monolayer to generate the required cell numbers for process inoculation in suspension culture, followed by stirring-controlled formation of cell-only aggregates at a 300-ml scale. After 48 h at checkpoint (CP) 0, chemically defined cardiac differentiation is induced by WNT-pathway modulation through use of the glycogen-synthase kinase-3 inhibitor CHIR99021 (WNT agonist), which is replaced 24 h later by the chemical WNT-pathway inhibitor IWP-2. The exact application of the described process parameters is important to ensure process efficiency and robustness. After 10 d of differentiation (CP I), the production of ≥100 × 106 CMs is expected. Moreover, to ‘uncouple’ cell production from downstream applications, continuous maintenance of CM aggregates for up to 35 d in culture (CP II) is demonstrated without a reduction in CM content, supporting downstream logistics while potentially overcoming the requirement for cryopreservation. We present a protocol for achieving efficient generation of hPSC-CM aggregates in suspension culture, emphasizing process simplicity, robustness and GMP compliance. The strategy promotes clinical translation and other applications that require large numbers of CMs.
{"title":"Standardized production of hPSC-derived cardiomyocyte aggregates in stirred spinner flasks","authors":"Nils Kriedemann, Wiebke Triebert, Jana Teske, Mira Mertens, Annika Franke, Kevin Ullmann, Felix Manstein, Lika Drakhlis, Alexandra Haase, Caroline Halloin, Ulrich Martin, Robert Zweigerdt","doi":"10.1038/s41596-024-00976-2","DOIUrl":"10.1038/s41596-024-00976-2","url":null,"abstract":"A promising cell-therapy approach for heart failure aims at differentiating human pluripotent stem cells (hPSCs) into functional cardiomyocytes (CMs) in vitro to replace the disease-induced loss of patients’ heart muscle cells in vivo. But many challenges remain for the routine clinical application of hPSC-derived CMs (hPSC-CMs), including good manufacturing practice (GMP)-compliant production strategies. This protocol describes the efficient generation of hPSC-CM aggregates in suspension culture, emphasizing process simplicity, robustness and GMP compliance. The strategy promotes clinical translation and other applications that require large numbers of CMs. Using a simple spinner-flask platform, this protocol is applicable to a broad range of users with general experience in handling hPSCs without extensive know-how in biotechnology. hPSCs are expanded in monolayer to generate the required cell numbers for process inoculation in suspension culture, followed by stirring-controlled formation of cell-only aggregates at a 300-ml scale. After 48 h at checkpoint (CP) 0, chemically defined cardiac differentiation is induced by WNT-pathway modulation through use of the glycogen-synthase kinase-3 inhibitor CHIR99021 (WNT agonist), which is replaced 24 h later by the chemical WNT-pathway inhibitor IWP-2. The exact application of the described process parameters is important to ensure process efficiency and robustness. After 10 d of differentiation (CP I), the production of ≥100 × 106 CMs is expected. Moreover, to ‘uncouple’ cell production from downstream applications, continuous maintenance of CM aggregates for up to 35 d in culture (CP II) is demonstrated without a reduction in CM content, supporting downstream logistics while potentially overcoming the requirement for cryopreservation. We present a protocol for achieving efficient generation of hPSC-CM aggregates in suspension culture, emphasizing process simplicity, robustness and GMP compliance. The strategy promotes clinical translation and other applications that require large numbers of CMs.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140318690","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 : 2024-03-28DOI: 10.1038/s41596-024-00974-4
Michael G. J. Doyle, Braeden A. Mair, Anna Sib, Odey Bsharat, Maxime Munch, Volker Derdau, Benjamin H. Rotstein, Rylan J. Lundgren
Isotopically carbon-labeled α-amino acids are valuable synthetic targets that are increasingly needed in pharmacology and medical imaging. Existing preparations rely on early stage introduction of the isotopic label, which leads to prohibitive synthetic costs and time-intensive preparations. Here we describe a protocol for the preparation of C1-labeled α-amino acids using simple aldehyde catalysts in conjunction with [*C]CO2 (* = 14, 13, 11). This late-stage labeling strategy is enabled by the one-pot carboxylate exchange of unprotected α-amino acids with [*C]CO2. The protocol consists of three separate procedures, describing the syntheses of (±)-[1-13C]phenylalanine, (±)-[1-11C]phenylalanine and (±)-[1-14C]phenylalanine from unlabeled phenylalanine. Although the delivery of [*C]CO2 is operationally distinct for each experiment, each procedure relies on the same fundamental chemistry and can be executed by heating the reaction components at 50–90 °C under basic conditions in dimethylsulfoxide. Performed on scales of up to 0.5 mmol, this methodology is amenable to C1-labeling of many proteinogenic α-amino acids and nonnatural derivatives, which is a breakthrough from existing methods. The synthesis of (±)-[1-13C]phenylalanine requires ~2 d, with product typically obtained in a 60–80% isolated yield (n = 3, μ = 71, σ = 8.3) with an isotopic incorporation of 70–88% (n = 18, μ = 72, σ = 9.0). Starting from the preformed imino acid (~3 h preparation time), rapid synthesis of (±)-[1-11C]phenylalanine can be completed in ~1 h with an isolated radiochemical yield of 13%. Finally, (±)-[1-14C]phenylalanine can be accessed in ~2 d with a 51% isolated yield and 11% radiochemical yield. Isotopically labeled amino acids are useful in pharmacology and for medical imaging. In this protocol, C1-labeled α-amino acids are prepared via late-stage carboxylate exchange of unprotected α-amino acids with [*C]CO2 where *C is 13C, 11C or 14C.
{"title":"A practical guide for the preparation of C1-labeled α-amino acids using aldehyde catalysis with isotopically labeled CO2","authors":"Michael G. J. Doyle, Braeden A. Mair, Anna Sib, Odey Bsharat, Maxime Munch, Volker Derdau, Benjamin H. Rotstein, Rylan J. Lundgren","doi":"10.1038/s41596-024-00974-4","DOIUrl":"10.1038/s41596-024-00974-4","url":null,"abstract":"Isotopically carbon-labeled α-amino acids are valuable synthetic targets that are increasingly needed in pharmacology and medical imaging. Existing preparations rely on early stage introduction of the isotopic label, which leads to prohibitive synthetic costs and time-intensive preparations. Here we describe a protocol for the preparation of C1-labeled α-amino acids using simple aldehyde catalysts in conjunction with [*C]CO2 (* = 14, 13, 11). This late-stage labeling strategy is enabled by the one-pot carboxylate exchange of unprotected α-amino acids with [*C]CO2. The protocol consists of three separate procedures, describing the syntheses of (±)-[1-13C]phenylalanine, (±)-[1-11C]phenylalanine and (±)-[1-14C]phenylalanine from unlabeled phenylalanine. Although the delivery of [*C]CO2 is operationally distinct for each experiment, each procedure relies on the same fundamental chemistry and can be executed by heating the reaction components at 50–90 °C under basic conditions in dimethylsulfoxide. Performed on scales of up to 0.5 mmol, this methodology is amenable to C1-labeling of many proteinogenic α-amino acids and nonnatural derivatives, which is a breakthrough from existing methods. The synthesis of (±)-[1-13C]phenylalanine requires ~2 d, with product typically obtained in a 60–80% isolated yield (n = 3, μ = 71, σ = 8.3) with an isotopic incorporation of 70–88% (n = 18, μ = 72, σ = 9.0). Starting from the preformed imino acid (~3 h preparation time), rapid synthesis of (±)-[1-11C]phenylalanine can be completed in ~1 h with an isolated radiochemical yield of 13%. Finally, (±)-[1-14C]phenylalanine can be accessed in ~2 d with a 51% isolated yield and 11% radiochemical yield. Isotopically labeled amino acids are useful in pharmacology and for medical imaging. In this protocol, C1-labeled α-amino acids are prepared via late-stage carboxylate exchange of unprotected α-amino acids with [*C]CO2 where *C is 13C, 11C or 14C.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140318728","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 : 2024-03-26DOI: 10.1038/s41596-024-00971-7
Aude-Anais Olijnik, Antonio Rodriguez-Romera, Zoë C. Wong, Yuqi Shen, Jasmeet S. Reyat, Natalie J. Jooss, Julie Rayes, Bethan Psaila, Abdullah O. Khan
The bone marrow supports and regulates hematopoiesis, responding to physiological requirements for blood cell production over ontogeny and during pathological challenges. Interactions between hematopoietic cells and niche components are challenging to study mechanistically in the human context, but are important to delineate in order to explore the pathobiology of blood and bone marrow disorders. Organoids are proving transformative in many research settings, but an accurate human bone marrow model incorporating multiple hematopoietic and stromal elements has been lacking. This protocol describes a method to generate three-dimensional, multilineage bone marrow organoids from human induced pluripotent stem cells (hiPSCs), detailing the steps for the directed differentiation of hiPSCs using a series of cytokine cocktails and hydrogel embedding. Over 18 days of differentiation, hiPSCs yield the key lineages that are present in central myelopoietic bone marrow, organized in a well-vascularized architecture that resembles native hematopoietic tissues. This presents a robust, in vitro system that can model healthy and perturbed hematopoiesis in a scalable three-dimensional microenvironment. Bone marrow organoids also support the growth of immortalized cell lines and primary cells from healthy donors and patients with myeloid and lymphoid cancers, including cell types that are poorly viable in standard culture systems. Moreover, we discuss assays for the characterization of organoids, including interrogation of pathogenic remodeling using recombinant TGF-ß treatment, and methods for organoid engraftment with exogenous cells. This protocol can be readily adapted to specific experimental requirements, can be easily implemented by users with tissue culture experience and does not require access to specialist equipment. This protocol can be used to generate three-dimensional vascularized bone marrow organoids from human induced pluripotent stem cells. The organoids contain key stromal and hematopoietic cell types and can be engrafted with normal and malignant cells from adult donors to model niche interactions.
{"title":"Generating human bone marrow organoids for disease modeling and drug discovery","authors":"Aude-Anais Olijnik, Antonio Rodriguez-Romera, Zoë C. Wong, Yuqi Shen, Jasmeet S. Reyat, Natalie J. Jooss, Julie Rayes, Bethan Psaila, Abdullah O. Khan","doi":"10.1038/s41596-024-00971-7","DOIUrl":"10.1038/s41596-024-00971-7","url":null,"abstract":"The bone marrow supports and regulates hematopoiesis, responding to physiological requirements for blood cell production over ontogeny and during pathological challenges. Interactions between hematopoietic cells and niche components are challenging to study mechanistically in the human context, but are important to delineate in order to explore the pathobiology of blood and bone marrow disorders. Organoids are proving transformative in many research settings, but an accurate human bone marrow model incorporating multiple hematopoietic and stromal elements has been lacking. This protocol describes a method to generate three-dimensional, multilineage bone marrow organoids from human induced pluripotent stem cells (hiPSCs), detailing the steps for the directed differentiation of hiPSCs using a series of cytokine cocktails and hydrogel embedding. Over 18 days of differentiation, hiPSCs yield the key lineages that are present in central myelopoietic bone marrow, organized in a well-vascularized architecture that resembles native hematopoietic tissues. This presents a robust, in vitro system that can model healthy and perturbed hematopoiesis in a scalable three-dimensional microenvironment. Bone marrow organoids also support the growth of immortalized cell lines and primary cells from healthy donors and patients with myeloid and lymphoid cancers, including cell types that are poorly viable in standard culture systems. Moreover, we discuss assays for the characterization of organoids, including interrogation of pathogenic remodeling using recombinant TGF-ß treatment, and methods for organoid engraftment with exogenous cells. This protocol can be readily adapted to specific experimental requirements, can be easily implemented by users with tissue culture experience and does not require access to specialist equipment. This protocol can be used to generate three-dimensional vascularized bone marrow organoids from human induced pluripotent stem cells. The organoids contain key stromal and hematopoietic cell types and can be engrafted with normal and malignant cells from adult donors to model niche interactions.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140294000","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 : 2024-03-21DOI: 10.1038/s41596-024-00954-8
Mehrad Hajiaghabozorgi, Mathew Fischbach, Michael Albrecht, Wen Wang, Chad L. Myers
Genetic interactions have the potential to modulate phenotypes, including human disease. In principle, genome-wide association studies (GWAS) provide a platform for detecting genetic interactions; however, traditional methods for identifying them, which tend to focus on testing individual variant pairs, lack statistical power. In this protocol, we describe a novel computational approach, called Bridging Gene sets with Epistasis (BridGE), for discovering genetic interactions between biological pathways from GWAS data. We present a Python-based implementation of BridGE along with instructions for its application to a typical human GWAS cohort. The major stages include initial data processing and quality control, construction of a variant-level genetic interaction network, measurement of pathway-level genetic interactions, evaluation of statistical significance using sample permutations and generation of results in a standardized output format. The BridGE software pipeline includes options for running the analysis on multiple cores and multiple nodes for users who have access to computing clusters or a cloud computing environment. In a cluster computing environment with 10 nodes and 100 GB of memory per node, the method can be run in less than 24 h for typical human GWAS cohorts. Using BridGE requires knowledge of running Python programs and basic shell script programming experience. Genetic interactions have been found to influence phenotypes in a variety of systems, yet their specific contribution to complex diseases remains unclear. This protocol describes Bridging Gene sets with Epistasis (BridGE), a computational approach for discovering interactions between biological pathways from genome-wide association studies data.
{"title":"BridGE: a pathway-based analysis tool for detecting genetic interactions from GWAS","authors":"Mehrad Hajiaghabozorgi, Mathew Fischbach, Michael Albrecht, Wen Wang, Chad L. Myers","doi":"10.1038/s41596-024-00954-8","DOIUrl":"10.1038/s41596-024-00954-8","url":null,"abstract":"Genetic interactions have the potential to modulate phenotypes, including human disease. In principle, genome-wide association studies (GWAS) provide a platform for detecting genetic interactions; however, traditional methods for identifying them, which tend to focus on testing individual variant pairs, lack statistical power. In this protocol, we describe a novel computational approach, called Bridging Gene sets with Epistasis (BridGE), for discovering genetic interactions between biological pathways from GWAS data. We present a Python-based implementation of BridGE along with instructions for its application to a typical human GWAS cohort. The major stages include initial data processing and quality control, construction of a variant-level genetic interaction network, measurement of pathway-level genetic interactions, evaluation of statistical significance using sample permutations and generation of results in a standardized output format. The BridGE software pipeline includes options for running the analysis on multiple cores and multiple nodes for users who have access to computing clusters or a cloud computing environment. In a cluster computing environment with 10 nodes and 100 GB of memory per node, the method can be run in less than 24 h for typical human GWAS cohorts. Using BridGE requires knowledge of running Python programs and basic shell script programming experience. Genetic interactions have been found to influence phenotypes in a variety of systems, yet their specific contribution to complex diseases remains unclear. This protocol describes Bridging Gene sets with Epistasis (BridGE), a computational approach for discovering interactions between biological pathways from genome-wide association studies data.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140184984","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}
The forthcoming massive genome data generated by the Earth BioGenome Project will open up a new era of comparative genomics, for which genome synteny analysis provides an important framework. Profiling genome synteny represents an essential step in elucidating genome architecture, regulatory blocks/elements and their evolutionary history. Here we describe PanSyn, ( https://github.com/yhw320/PanSyn ), the most comprehensive and up-to-date genome synteny pipeline, providing step-by-step instructions and application examples to demonstrate its usage. PanSyn inherits both basic and advanced functions from existing popular tools, offering a user-friendly, highly customized approach for genome macrosynteny analysis and integrated pan-evolutionary and regulatory analysis of genome architecture, which are not yet available in public synteny software or tools. The advantages of PanSyn include: (i) advanced microsynteny analysis by functional profiling of microsynteny genes and associated regulatory elements; (ii) comprehensive macrosynteny analysis, including the inference of karyotype evolution from ancestors to extant species; and (iii) functional integration of microsynteny and macrosynteny for pan-evolutionary profiling of genome architecture and regulatory blocks, as well as integration with external functional genomics datasets from three- or four-dimensional genome and ENCODE projects. PanSyn requires basic knowledge of the Linux environment and Perl programming language and the ability to access a computer cluster, especially for large-scale genomic comparisons. Our protocol can be easily implemented by a competent graduate student or postdoc and takes several days to weeks to execute for dozens to hundreds of genomes. PanSyn provides yet the most comprehensive and powerful tool for integrated evolutionary and functional genomics. This protocol is for using PanSyn, the first software package for the identification of micro- and macrosynteny and their functional integration for comprehensive characterization of genome architecture and regulatory evolution.
{"title":"Pan-evolutionary and regulatory genome architecture delineated by an integrated macro- and microsynteny approach","authors":"Hongwei Yu, Yuli Li, Wentao Han, Lisui Bao, Fuyun Liu, Yuanting Ma, Zhongqi Pu, Qifan Zeng, Lingling Zhang, Zhenmin Bao, Shi Wang","doi":"10.1038/s41596-024-00966-4","DOIUrl":"10.1038/s41596-024-00966-4","url":null,"abstract":"The forthcoming massive genome data generated by the Earth BioGenome Project will open up a new era of comparative genomics, for which genome synteny analysis provides an important framework. Profiling genome synteny represents an essential step in elucidating genome architecture, regulatory blocks/elements and their evolutionary history. Here we describe PanSyn, ( https://github.com/yhw320/PanSyn ), the most comprehensive and up-to-date genome synteny pipeline, providing step-by-step instructions and application examples to demonstrate its usage. PanSyn inherits both basic and advanced functions from existing popular tools, offering a user-friendly, highly customized approach for genome macrosynteny analysis and integrated pan-evolutionary and regulatory analysis of genome architecture, which are not yet available in public synteny software or tools. The advantages of PanSyn include: (i) advanced microsynteny analysis by functional profiling of microsynteny genes and associated regulatory elements; (ii) comprehensive macrosynteny analysis, including the inference of karyotype evolution from ancestors to extant species; and (iii) functional integration of microsynteny and macrosynteny for pan-evolutionary profiling of genome architecture and regulatory blocks, as well as integration with external functional genomics datasets from three- or four-dimensional genome and ENCODE projects. PanSyn requires basic knowledge of the Linux environment and Perl programming language and the ability to access a computer cluster, especially for large-scale genomic comparisons. Our protocol can be easily implemented by a competent graduate student or postdoc and takes several days to weeks to execute for dozens to hundreds of genomes. PanSyn provides yet the most comprehensive and powerful tool for integrated evolutionary and functional genomics. This protocol is for using PanSyn, the first software package for the identification of micro- and macrosynteny and their functional integration for comprehensive characterization of genome architecture and regulatory evolution.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140184986","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 : 2024-03-21DOI: 10.1038/s41596-024-00970-8
Mohamed Abdelrahim, Quan Gao, Yichuan Zhang, Weishuo Li, Qi Xing, Mark Bradley, Jin Geng
The synthesis of synthetic intracellular polymers offers groundbreaking possibilities in cellular biology and medical research, allowing for novel experiments in drug delivery, bioimaging and targeted cancer therapies. These macromolecules, composed of biocompatible monomers, are pivotal in manipulating cellular functions and pathways due to their bioavailability, cytocompatibility and distinct chemical properties. This protocol details two innovative methods for intracellular polymerization. The first one uses 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) as a photoinitiator for free radical polymerization under UV light (365 nm, 5 mW/cm2). The second method employs photoinduced electron transfer-reversible addition–fragmentation chain-transfer polymerization with visible light (470 nm, 100 mW/cm2). We further elaborate on isolating these intracellular polymers by streptavidin/biotin interaction or immobilized metal ion affinity chromatography for polymers tagged with biotin or histidine. The entire process, from polymerization to isolation, takes ~48 h. Moreover, the intracellular polymers thus generated demonstrate significant potential in enhancing actin polymerization, in bioimaging applications and as a novel avenue in cancer treatment strategies. The protocol extends to animal models, providing a comprehensive approach from cellular to systemic applications. Users are advised to have a basic understanding of organic synthesis and cell biology techniques. Cell engineering using polymeric material is an area that remains largely unexplored. This protocol describes two light-driven approaches for synthesizing bioactive polymers within intricate intracellular settings.
{"title":"Light-mediated intracellular polymerization","authors":"Mohamed Abdelrahim, Quan Gao, Yichuan Zhang, Weishuo Li, Qi Xing, Mark Bradley, Jin Geng","doi":"10.1038/s41596-024-00970-8","DOIUrl":"10.1038/s41596-024-00970-8","url":null,"abstract":"The synthesis of synthetic intracellular polymers offers groundbreaking possibilities in cellular biology and medical research, allowing for novel experiments in drug delivery, bioimaging and targeted cancer therapies. These macromolecules, composed of biocompatible monomers, are pivotal in manipulating cellular functions and pathways due to their bioavailability, cytocompatibility and distinct chemical properties. This protocol details two innovative methods for intracellular polymerization. The first one uses 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) as a photoinitiator for free radical polymerization under UV light (365 nm, 5 mW/cm2). The second method employs photoinduced electron transfer-reversible addition–fragmentation chain-transfer polymerization with visible light (470 nm, 100 mW/cm2). We further elaborate on isolating these intracellular polymers by streptavidin/biotin interaction or immobilized metal ion affinity chromatography for polymers tagged with biotin or histidine. The entire process, from polymerization to isolation, takes ~48 h. Moreover, the intracellular polymers thus generated demonstrate significant potential in enhancing actin polymerization, in bioimaging applications and as a novel avenue in cancer treatment strategies. The protocol extends to animal models, providing a comprehensive approach from cellular to systemic applications. Users are advised to have a basic understanding of organic synthesis and cell biology techniques. Cell engineering using polymeric material is an area that remains largely unexplored. This protocol describes two light-driven approaches for synthesizing bioactive polymers within intricate intracellular settings.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140184985","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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