Pub Date : 2024-03-20DOI: 10.1038/s41596-024-00958-4
Degong Ruan, Yiyi Xuan, Timothy Theodore Ka Ki Tam, ZhuoXuan Li, Xiao Wang, Shao Xu, Doris Herrmann, Heiner Niemann, Liangxue Lai, Xuefei Gao, Monika Nowak-Imialek, Pentao Liu
Pigs share anatomical and physiological traits with humans and can serve as a large-animal model for translational medicine. Bona fide porcine pluripotent stem cells (PSCs) could facilitate testing cell and drug therapies. Agriculture and biotechnology may benefit from the ability to produce immune cells for studying animal infectious diseases and to readily edit the porcine genome in stem cells. Isolating porcine PSCs from preimplantation embryos has been intensively attempted over the past decades. We previously reported the derivation of expanded potential stem cells (EPSCs) from preimplantation embryos and by reprogramming somatic cells of multiple mammalian species, including pigs. Porcine EPSCs (pEPSCs) self-renew indefinitely, differentiate into embryonic and extra-embryonic lineages, and permit precision genome editing. Here we present a highly reproducible experimental procedure and data of an optimized and robust porcine EPSC culture system and its use in deriving new pEPSC lines from preimplantation embryos and reprogrammed somatic cells. No particular expertise is required for the protocols, which take ~4–6 weeks to complete. Importantly, we successfully established pEPSC lines from both in vitro fertilized and somatic cell nuclear transfer-derived embryos. These new pEPSC lines proliferated robustly over long-term passaging and were amenable to both simple indels and precision genome editing, with up to 100% targeting efficiency. The pEPSCs differentiated into embryonic cell lineages in vitro and teratomas in vivo, and into porcine trophoblast stem cells in human trophoblast stem cell medium. We show here that pEPSCs have unique epigenetic features, particularly H3K27me3 levels substantially lower than fibroblasts. The protocol presents for an optimized culture system for deriving porcine expanded potential stem cells from preimplantation embryos and reprogrammed somatic cells, and for validation and characterization.
{"title":"An optimized culture system for efficient derivation of porcine expanded potential stem cells from preimplantation embryos and by reprogramming somatic cells","authors":"Degong Ruan, Yiyi Xuan, Timothy Theodore Ka Ki Tam, ZhuoXuan Li, Xiao Wang, Shao Xu, Doris Herrmann, Heiner Niemann, Liangxue Lai, Xuefei Gao, Monika Nowak-Imialek, Pentao Liu","doi":"10.1038/s41596-024-00958-4","DOIUrl":"10.1038/s41596-024-00958-4","url":null,"abstract":"Pigs share anatomical and physiological traits with humans and can serve as a large-animal model for translational medicine. Bona fide porcine pluripotent stem cells (PSCs) could facilitate testing cell and drug therapies. Agriculture and biotechnology may benefit from the ability to produce immune cells for studying animal infectious diseases and to readily edit the porcine genome in stem cells. Isolating porcine PSCs from preimplantation embryos has been intensively attempted over the past decades. We previously reported the derivation of expanded potential stem cells (EPSCs) from preimplantation embryos and by reprogramming somatic cells of multiple mammalian species, including pigs. Porcine EPSCs (pEPSCs) self-renew indefinitely, differentiate into embryonic and extra-embryonic lineages, and permit precision genome editing. Here we present a highly reproducible experimental procedure and data of an optimized and robust porcine EPSC culture system and its use in deriving new pEPSC lines from preimplantation embryos and reprogrammed somatic cells. No particular expertise is required for the protocols, which take ~4–6 weeks to complete. Importantly, we successfully established pEPSC lines from both in vitro fertilized and somatic cell nuclear transfer-derived embryos. These new pEPSC lines proliferated robustly over long-term passaging and were amenable to both simple indels and precision genome editing, with up to 100% targeting efficiency. The pEPSCs differentiated into embryonic cell lineages in vitro and teratomas in vivo, and into porcine trophoblast stem cells in human trophoblast stem cell medium. We show here that pEPSCs have unique epigenetic features, particularly H3K27me3 levels substantially lower than fibroblasts. The protocol presents for an optimized culture system for deriving porcine expanded potential stem cells from preimplantation embryos and reprogrammed somatic cells, and for validation and characterization.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140171858","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 : 2024-03-19DOI: 10.1038/s41596-024-00969-1
Marley Downes, Christopher E. Shuck, Bernard McBride, Jeffrey Busa, Yury Gogotsi
MXenes are a large family of two-dimensional materials that have attracted attention across many fields due to their desirable optoelectronic, biological, mechanical and chemical properties. There currently exist many synthesis procedures that lead to differences in flake size, defects and surface chemistry, which in turn affect their properties. Herein, we describe the steps to synthesize Ti3C2Tx—the most important and widely used MXene, from a Ti3AlC2 MAX phase precursor. The procedure contains three main sections: synthesis of Ti3AlC2 MAX, wet chemical etching of the MAX in hydrofluoric acid/HCl solution to yield multilayer Ti3C2Tx and its delamination into single-layer flakes. Three delamination options are described; these use LiCl, tertiary amines (tetramethyl ammonium hydroxide/ tetrabutyl ammonium hydroxide) and dimethylsulfoxide respectively. These procedures can be adapted for the synthesis of MXenes beyond Ti3C2Tx. The MAX phase synthesis takes about 1 week, with the etching and delamination each requiring 2 d. This protocol requires users to have experience working with hydrofluoric acid, and it is recommended that users have experience with wet chemistry and centrifugation; characterization techniques such as X-ray diffraction and particle size analysis are also essential for the success of the protocol. While alternative synthesis methods, such as minimally intensive layer delamination, are desirable for certain MXenes (such as Ti2CTx) or specific applications, this protocol aims to standardize the more commonly used hydrofluoric acid/HCl etching method, which produces Ti3C2Tx with minimal concentration of defects and the highest conductivity and serves as a guideline for those working with MXenes for the first time. MXenes are two-dimensional materials with diverse optoelectronic, biological, mechanical and chemical properties. This protocol describes how to prepare single-layer flakes of Ti3C2Tx, the most important and widely used MXene, from a Ti3AlC2 MAX phase precursor.
{"title":"Comprehensive synthesis of Ti3C2Tx from MAX phase to MXene","authors":"Marley Downes, Christopher E. Shuck, Bernard McBride, Jeffrey Busa, Yury Gogotsi","doi":"10.1038/s41596-024-00969-1","DOIUrl":"10.1038/s41596-024-00969-1","url":null,"abstract":"MXenes are a large family of two-dimensional materials that have attracted attention across many fields due to their desirable optoelectronic, biological, mechanical and chemical properties. There currently exist many synthesis procedures that lead to differences in flake size, defects and surface chemistry, which in turn affect their properties. Herein, we describe the steps to synthesize Ti3C2Tx—the most important and widely used MXene, from a Ti3AlC2 MAX phase precursor. The procedure contains three main sections: synthesis of Ti3AlC2 MAX, wet chemical etching of the MAX in hydrofluoric acid/HCl solution to yield multilayer Ti3C2Tx and its delamination into single-layer flakes. Three delamination options are described; these use LiCl, tertiary amines (tetramethyl ammonium hydroxide/ tetrabutyl ammonium hydroxide) and dimethylsulfoxide respectively. These procedures can be adapted for the synthesis of MXenes beyond Ti3C2Tx. The MAX phase synthesis takes about 1 week, with the etching and delamination each requiring 2 d. This protocol requires users to have experience working with hydrofluoric acid, and it is recommended that users have experience with wet chemistry and centrifugation; characterization techniques such as X-ray diffraction and particle size analysis are also essential for the success of the protocol. While alternative synthesis methods, such as minimally intensive layer delamination, are desirable for certain MXenes (such as Ti2CTx) or specific applications, this protocol aims to standardize the more commonly used hydrofluoric acid/HCl etching method, which produces Ti3C2Tx with minimal concentration of defects and the highest conductivity and serves as a guideline for those working with MXenes for the first time. MXenes are two-dimensional materials with diverse optoelectronic, biological, mechanical and chemical properties. This protocol describes how to prepare single-layer flakes of Ti3C2Tx, the most important and widely used MXene, from a Ti3AlC2 MAX phase precursor.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140175590","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 : 2024-03-19DOI: 10.1038/s41596-024-00972-6
Maria Alieva, Mario Barrera Román, Sam de Blank, Diana Petcu, Amber L. Zeeman, Noël M. M. Dautzenberg, Annelisa M. Cornel, Cesca van de Ven, Rob Pieters, Monique L. den Boer, Stefan Nierkens, Friso G. J. Calkoen, Hans Clevers, Jürgen Kuball, Zsolt Sebestyén, Ellen J. Wehrens, Johanna F. Dekkers, Anne C. Rios
Modeling immuno-oncology by using patient-derived material and immune cell co-cultures can advance our understanding of immune cell tumor targeting in a patient-specific manner, offering leads to improve cellular immunotherapy. However, fully exploiting these living cultures requires analysis of the dynamic cellular features modeled, for which protocols are currently limited. Here, we describe the application of BEHAV3D, a platform that implements multi-color live 3D imaging and computational tools for: (i) analyzing tumor death dynamics at both single-organoid or cell and population levels, (ii) classifying T cell behavior and (iii) producing data-informed 3D images and videos for visual inspection and further insight into obtained results. Together, this enables a refined assessment of how solid and liquid tumors respond to cellular immunotherapy, critically capturing both inter- and intratumoral heterogeneity in treatment response. In addition, BEHAV3D uncovers T cell behavior involved in tumor targeting, offering insight into their mode of action. Our pipeline thereby has strong implications for comparing, prioritizing and improving immunotherapy products by highlighting the behavioral differences between individual tumor donors, distinct T cell therapy concepts or subpopulations. The protocol describes critical wet lab steps, including co-culture preparations and fast 3D imaging with live cell dyes, a segmentation-based image processing tool to track individual organoids, tumor and immune cells and an analytical pipeline for behavioral profiling. This 1-week protocol, accessible to users with basic cell culture, imaging and programming expertise, can easily be adapted to any type of co-culture to visualize and exploit cell behavior, having far-reaching implications for the immuno-oncology field and beyond. BEHAV3D is a 3D live imaging platform for analyzing engineered T cell behavior and tumor response. This provides insights into the mode of action of cellular immunotherapy, capturing heterogeneity within and between tumors during treatment response.
通过使用源自患者的材料和免疫细胞共培养物建立免疫肿瘤学模型，可以推进我们对免疫细胞以患者特异性方式靶向肿瘤的理解，为改进细胞免疫疗法提供线索。然而，充分利用这些活体培养物需要分析建模的动态细胞特征，而目前这方面的方案还很有限。在这里，我们介绍了 BEHAV3D 的应用，这是一个实现多色实时三维成像和计算工具的平台，可用于(i)在单器官或细胞和群体水平上分析肿瘤死亡动态，(ii)对 T 细胞行为进行分类，(iii)生成数据信息三维图像和视频，用于视觉检查和进一步深入了解所获得的结果。这样就能对实体瘤和液体瘤如何对细胞免疫疗法做出反应进行精细评估，并准确捕捉治疗反应中瘤间和瘤内的异质性。此外，BEHAV3D 还能发现参与肿瘤靶向的 T 细胞行为，从而深入了解它们的作用模式。因此，我们的研究方法通过突出单个肿瘤供体、不同 T 细胞疗法概念或亚群之间的行为差异，对比较、优先考虑和改进免疫疗法产品具有重要意义。该方案描述了关键的湿实验室步骤，包括共培养制备和活细胞染料快速三维成像，基于分割的图像处理工具跟踪单个器官组织、肿瘤和免疫细胞，以及行为分析管道。这个为期一周的方案可供具备基本细胞培养、成像和编程专业知识的用户使用，可轻松适用于任何类型的共培养，以可视化和利用细胞行为，对免疫肿瘤学领域及其他领域具有深远影响。
{"title":"BEHAV3D: a 3D live imaging platform for comprehensive analysis of engineered T cell behavior and tumor response","authors":"Maria Alieva, Mario Barrera Román, Sam de Blank, Diana Petcu, Amber L. Zeeman, Noël M. M. Dautzenberg, Annelisa M. Cornel, Cesca van de Ven, Rob Pieters, Monique L. den Boer, Stefan Nierkens, Friso G. J. Calkoen, Hans Clevers, Jürgen Kuball, Zsolt Sebestyén, Ellen J. Wehrens, Johanna F. Dekkers, Anne C. Rios","doi":"10.1038/s41596-024-00972-6","DOIUrl":"10.1038/s41596-024-00972-6","url":null,"abstract":"Modeling immuno-oncology by using patient-derived material and immune cell co-cultures can advance our understanding of immune cell tumor targeting in a patient-specific manner, offering leads to improve cellular immunotherapy. However, fully exploiting these living cultures requires analysis of the dynamic cellular features modeled, for which protocols are currently limited. Here, we describe the application of BEHAV3D, a platform that implements multi-color live 3D imaging and computational tools for: (i) analyzing tumor death dynamics at both single-organoid or cell and population levels, (ii) classifying T cell behavior and (iii) producing data-informed 3D images and videos for visual inspection and further insight into obtained results. Together, this enables a refined assessment of how solid and liquid tumors respond to cellular immunotherapy, critically capturing both inter- and intratumoral heterogeneity in treatment response. In addition, BEHAV3D uncovers T cell behavior involved in tumor targeting, offering insight into their mode of action. Our pipeline thereby has strong implications for comparing, prioritizing and improving immunotherapy products by highlighting the behavioral differences between individual tumor donors, distinct T cell therapy concepts or subpopulations. The protocol describes critical wet lab steps, including co-culture preparations and fast 3D imaging with live cell dyes, a segmentation-based image processing tool to track individual organoids, tumor and immune cells and an analytical pipeline for behavioral profiling. This 1-week protocol, accessible to users with basic cell culture, imaging and programming expertise, can easily be adapted to any type of co-culture to visualize and exploit cell behavior, having far-reaching implications for the immuno-oncology field and beyond. BEHAV3D is a 3D live imaging platform for analyzing engineered T cell behavior and tumor response. This provides insights into the mode of action of cellular immunotherapy, capturing heterogeneity within and between tumors during treatment response.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140171928","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 : 2024-03-19DOI: 10.1038/s41596-024-00967-3
Costanza Borrelli, Alessandra Gurtner, Isabelle C. Arnold, Andreas E. Moor
Eosinophils are a class of granulocytes with pleiotropic functions in homeostasis and various human diseases. Nevertheless, they are absent from conventional single-cell RNA sequencing atlases owing to technical difficulties preventing their transcriptomic interrogation. Consequently, eosinophil heterogeneity and the gene regulatory networks underpinning their diverse functions remain poorly understood. We have developed a stress-free protocol for single-cell RNA capture from murine tissue-resident eosinophils, which revealed distinct intestinal subsets and their roles in colitis. Here we describe in detail how to enrich eosinophils from multiple tissues of residence and how to capture high-quality single-cell transcriptomes by preventing transcript degradation. By combining magnetic eosinophil enrichment with microwell-based single-cell RNA capture (BD Rhapsody), our approach minimizes shear stress and processing time. Moreover, we report how to perform genome-wide CRISPR pooled genetic screening in ex vivo-conditioned bone marrow-derived eosinophils to functionally probe pathways required for their differentiation and intestinal maturation. These protocols can be performed by any researcher with basic skills in molecular biology and flow cytometry, and can be adapted to investigate other granulocytes, such as neutrophils and mast cells, thereby offering potential insights into their roles in both homeostasis and disease pathogenesis. Single-cell transcriptomics of eosinophils can be performed in 2–3 d, while functional genomics assays may require up to 1 month. This protocol presents a method for single-cell RNA sequencing of tissue-resident murine eosinophils, with a complementary method for CRISPR screening of bone marrow-derived eosinophils.
{"title":"Stress-free single-cell transcriptomic profiling and functional genomics of murine eosinophils","authors":"Costanza Borrelli, Alessandra Gurtner, Isabelle C. Arnold, Andreas E. Moor","doi":"10.1038/s41596-024-00967-3","DOIUrl":"10.1038/s41596-024-00967-3","url":null,"abstract":"Eosinophils are a class of granulocytes with pleiotropic functions in homeostasis and various human diseases. Nevertheless, they are absent from conventional single-cell RNA sequencing atlases owing to technical difficulties preventing their transcriptomic interrogation. Consequently, eosinophil heterogeneity and the gene regulatory networks underpinning their diverse functions remain poorly understood. We have developed a stress-free protocol for single-cell RNA capture from murine tissue-resident eosinophils, which revealed distinct intestinal subsets and their roles in colitis. Here we describe in detail how to enrich eosinophils from multiple tissues of residence and how to capture high-quality single-cell transcriptomes by preventing transcript degradation. By combining magnetic eosinophil enrichment with microwell-based single-cell RNA capture (BD Rhapsody), our approach minimizes shear stress and processing time. Moreover, we report how to perform genome-wide CRISPR pooled genetic screening in ex vivo-conditioned bone marrow-derived eosinophils to functionally probe pathways required for their differentiation and intestinal maturation. These protocols can be performed by any researcher with basic skills in molecular biology and flow cytometry, and can be adapted to investigate other granulocytes, such as neutrophils and mast cells, thereby offering potential insights into their roles in both homeostasis and disease pathogenesis. Single-cell transcriptomics of eosinophils can be performed in 2–3 d, while functional genomics assays may require up to 1 month. This protocol presents a method for single-cell RNA sequencing of tissue-resident murine eosinophils, with a complementary method for CRISPR screening of bone marrow-derived eosinophils.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140171926","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 : 2024-03-15DOI: 10.1038/s41596-024-00968-2
Yupeng Wang, Haibao Tang, Xiyin Wang, Ying Sun, Paule V. Joseph, Andrew H. Paterson
As different taxa evolve, gene order often changes slowly enough that chromosomal ‘blocks’ with conserved gene orders (synteny) are discernible. The MCScanX toolkit ( https://github.com/wyp1125/MCScanX ) was published in 2012 as freely available software for the detection of such ‘colinear blocks’ and subsequent synteny and evolutionary analyses based on genome-wide gene location and protein sequence information. Owing to its simplicity and high efficiency for colinear block detection, MCScanX provides a powerful tool for conducting diverse synteny and evolutionary analyses. Moreover, the detection of colinear blocks has been embraced as an integral step for pangenome graph construction. Here, new application trends of MCScanX are explored, striving to better connect this increasingly used tool to other tools and accelerate insight generation from exponentially growing sequence data. We provide a detailed protocol that covers how to install MCScanX on diverse platforms, tune parameters, prepare input files from data from the National Center for Biotechnology Information, run MCScanX and its visualization and evolutionary analysis tools, and connect MCScanX with external tools, including MCScanX-transposed, Circos and SynVisio. This protocol is easily implemented by users with minimal computational background and is adaptable to new data of interest to them. The data and utility programs for this protocol can be obtained from http://bdx-consulting.com/mcscanx-protocol . Synteny and colinearity are important parameters that delineate the evolution of genomes and gene families. This protocol describes MCScanX, a user-friendly toolkit that facilitates rapid evolutionary analysis of chromosomal structural changes.
{"title":"Detection of colinear blocks and synteny and evolutionary analyses based on utilization of MCScanX","authors":"Yupeng Wang, Haibao Tang, Xiyin Wang, Ying Sun, Paule V. Joseph, Andrew H. Paterson","doi":"10.1038/s41596-024-00968-2","DOIUrl":"10.1038/s41596-024-00968-2","url":null,"abstract":"As different taxa evolve, gene order often changes slowly enough that chromosomal ‘blocks’ with conserved gene orders (synteny) are discernible. The MCScanX toolkit ( https://github.com/wyp1125/MCScanX ) was published in 2012 as freely available software for the detection of such ‘colinear blocks’ and subsequent synteny and evolutionary analyses based on genome-wide gene location and protein sequence information. Owing to its simplicity and high efficiency for colinear block detection, MCScanX provides a powerful tool for conducting diverse synteny and evolutionary analyses. Moreover, the detection of colinear blocks has been embraced as an integral step for pangenome graph construction. Here, new application trends of MCScanX are explored, striving to better connect this increasingly used tool to other tools and accelerate insight generation from exponentially growing sequence data. We provide a detailed protocol that covers how to install MCScanX on diverse platforms, tune parameters, prepare input files from data from the National Center for Biotechnology Information, run MCScanX and its visualization and evolutionary analysis tools, and connect MCScanX with external tools, including MCScanX-transposed, Circos and SynVisio. This protocol is easily implemented by users with minimal computational background and is adaptable to new data of interest to them. The data and utility programs for this protocol can be obtained from http://bdx-consulting.com/mcscanx-protocol . Synteny and colinearity are important parameters that delineate the evolution of genomes and gene families. This protocol describes MCScanX, a user-friendly toolkit that facilitates rapid evolutionary analysis of chromosomal structural changes.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":13.1,"publicationDate":"2024-03-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140140484","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 : 2024-03-12DOI: 10.1038/s41596-024-00960-w
Nolan K. Newman, Matthew S. Macovsky, Richard R. Rodrigues, Amanda M. Bruce, Jacob W. Pederson, Jyothi Padiadpu, Jigui Shan, Joshua Williams, Sankalp S. Patil, Amiran K. Dzutsev, Natalia Shulzhenko, Giorgio Trinchieri, Kevin Brown, Andrey Morgun
We present Transkingdom Network Analysis (TkNA), a unique causal-inference analytical framework that offers a holistic view of biological systems by integrating data from multiple cohorts and diverse omics types. TkNA helps to decipher key players and mechanisms governing host–microbiota (or any multi-omic data) interactions in specific conditions or diseases. TkNA reconstructs a network that represents a statistical model capturing the complex relationships between different omics in the biological system. It identifies robust and reproducible patterns of fold change direction and correlation sign across several cohorts to select differential features and their per-group correlations. The framework then uses causality-sensitive metrics, statistical thresholds and topological criteria to determine the final edges forming the transkingdom network. With the subsequent network’s topological features, TkNA identifies nodes controlling a given subnetwork or governing communication between kingdoms and/or subnetworks. The computational time for the millions of correlations necessary for network reconstruction in TkNA typically takes only a few minutes, varying with the study design. Unlike most other multi-omics approaches that find only associations, TkNA focuses on establishing causality while accounting for the complex structure of multi-omic data. It achieves this without requiring huge sample sizes. Moreover, the TkNA protocol is user friendly, requiring minimal installation and basic familiarity with Unix. Researchers can access the TkNA software at https://github.com/CAnBioNet/TkNA/ . Transkingdom Network Analysis (TkNA) is a unique analytical framework for inferring causal factors underlying host–microbiota and other multi-omic interactions, by integrating data from multiple cohorts and diverse omics types.
{"title":"Transkingdom Network Analysis (TkNA): a systems framework for inferring causal factors underlying host–microbiota and other multi-omic interactions","authors":"Nolan K. Newman, Matthew S. Macovsky, Richard R. Rodrigues, Amanda M. Bruce, Jacob W. Pederson, Jyothi Padiadpu, Jigui Shan, Joshua Williams, Sankalp S. Patil, Amiran K. Dzutsev, Natalia Shulzhenko, Giorgio Trinchieri, Kevin Brown, Andrey Morgun","doi":"10.1038/s41596-024-00960-w","DOIUrl":"10.1038/s41596-024-00960-w","url":null,"abstract":"We present Transkingdom Network Analysis (TkNA), a unique causal-inference analytical framework that offers a holistic view of biological systems by integrating data from multiple cohorts and diverse omics types. TkNA helps to decipher key players and mechanisms governing host–microbiota (or any multi-omic data) interactions in specific conditions or diseases. TkNA reconstructs a network that represents a statistical model capturing the complex relationships between different omics in the biological system. It identifies robust and reproducible patterns of fold change direction and correlation sign across several cohorts to select differential features and their per-group correlations. The framework then uses causality-sensitive metrics, statistical thresholds and topological criteria to determine the final edges forming the transkingdom network. With the subsequent network’s topological features, TkNA identifies nodes controlling a given subnetwork or governing communication between kingdoms and/or subnetworks. The computational time for the millions of correlations necessary for network reconstruction in TkNA typically takes only a few minutes, varying with the study design. Unlike most other multi-omics approaches that find only associations, TkNA focuses on establishing causality while accounting for the complex structure of multi-omic data. It achieves this without requiring huge sample sizes. Moreover, the TkNA protocol is user friendly, requiring minimal installation and basic familiarity with Unix. Researchers can access the TkNA software at https://github.com/CAnBioNet/TkNA/ . Transkingdom Network Analysis (TkNA) is a unique analytical framework for inferring causal factors underlying host–microbiota and other multi-omic interactions, by integrating data from multiple cohorts and diverse omics types.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140110685","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 : 2024-03-11DOI: 10.1038/s41596-024-00965-5
Rafael Tapia-Rojo, Marc Mora, Sergi Garcia-Manyes
The reversible unfolding and refolding of proteins is a regulatory mechanism of tissue elasticity and signalling used by cells to sense and adapt to extracellular and intracellular mechanical forces. However, most of these proteins exhibit low mechanical stability, posing technical challenges to the characterization of their conformational dynamics under force. Here, we detail step-by-step instructions for conducting single-protein nanomechanical experiments using ultra-stable magnetic tweezers, which enable the measurement of the equilibrium conformational dynamics of single proteins under physiologically relevant low forces applied over biologically relevant timescales. We report the basic principles determining the functioning of the magnetic tweezer instrument, review the protein design strategy and the fluid chamber preparation and detail the procedure to acquire and analyze the unfolding and refolding trajectories of individual proteins under force. This technique adds to the toolbox of single-molecule nanomechanical techniques and will be of particular interest to those interested in proteins involved in mechanosensing and mechanotransduction. The procedure takes 4 d to complete, plus an additional 6 d for protein cloning and production, requiring basic expertise in molecular biology, surface chemistry and data analysis. Ultra-stable magnetic tweezers allow measuring individual protein dynamics in equilibrium under physiologically relevant pulling forces and over timescales of days to weeks, enabling high-precision molecular studies in mechanobiology.
{"title":"Single-molecule magnetic tweezers to probe the equilibrium dynamics of individual proteins at physiologically relevant forces and timescales","authors":"Rafael Tapia-Rojo, Marc Mora, Sergi Garcia-Manyes","doi":"10.1038/s41596-024-00965-5","DOIUrl":"10.1038/s41596-024-00965-5","url":null,"abstract":"The reversible unfolding and refolding of proteins is a regulatory mechanism of tissue elasticity and signalling used by cells to sense and adapt to extracellular and intracellular mechanical forces. However, most of these proteins exhibit low mechanical stability, posing technical challenges to the characterization of their conformational dynamics under force. Here, we detail step-by-step instructions for conducting single-protein nanomechanical experiments using ultra-stable magnetic tweezers, which enable the measurement of the equilibrium conformational dynamics of single proteins under physiologically relevant low forces applied over biologically relevant timescales. We report the basic principles determining the functioning of the magnetic tweezer instrument, review the protein design strategy and the fluid chamber preparation and detail the procedure to acquire and analyze the unfolding and refolding trajectories of individual proteins under force. This technique adds to the toolbox of single-molecule nanomechanical techniques and will be of particular interest to those interested in proteins involved in mechanosensing and mechanotransduction. The procedure takes 4 d to complete, plus an additional 6 d for protein cloning and production, requiring basic expertise in molecular biology, surface chemistry and data analysis. Ultra-stable magnetic tweezers allow measuring individual protein dynamics in equilibrium under physiologically relevant pulling forces and over timescales of days to weeks, enabling high-precision molecular studies in mechanobiology.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140102049","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 : 2024-03-05DOI: 10.1038/s41596-024-00982-4
G Heinrich, M Kondratiuk, L J Gooßen, M P Wiesenfeldt
{"title":"Publisher Correction: Rapid reaction optimization by robust and economical quantitative benchtop <sup>19</sup>F NMR spectroscopy.","authors":"G Heinrich, M Kondratiuk, L J Gooßen, M P Wiesenfeldt","doi":"10.1038/s41596-024-00982-4","DOIUrl":"10.1038/s41596-024-00982-4","url":null,"abstract":"","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140039882","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}
Mammalian cells sense and react to the mechanics of their immediate microenvironment. Therefore, the characterization of the biomechanical properties of tissues with high spatial resolution provides valuable insights into a broad variety of developmental, homeostatic and pathological processes within living organisms. The biomechanical properties of the basement membrane (BM), an extracellular matrix (ECM) substructure measuring only ∼100–400 nm across, are, among other things, pivotal to tumor progression and metastasis formation. Although the precise assignment of the Young’s modulus E of such a thin ECM substructure especially in between two cell layers is still challenging, biomechanical data of the BM can provide information of eminent diagnostic potential. Here we present a detailed protocol to quantify the elastic modulus of the BM in murine and human lung tissue, which is one of the major organs prone to metastasis. This protocol describes a streamlined workflow to determine the Young’s modulus E of the BM between the endothelial and epithelial cell layers shaping the alveolar wall in lung tissues using atomic force microscopy (AFM). Our step-by-step protocol provides instructions for murine and human lung tissue extraction, inflation of these tissues with cryogenic cutting medium, freezing and cryosectioning of the tissue samples, and AFM force-map recording. In addition, it guides the reader through a semi-automatic data analysis procedure to identify the pulmonary BM and extract its Young’s modulus E using an in-house tailored user-friendly AFM data analysis software, the Center for Applied Tissue Engineering and Regenerative Medicine processing toolbox, which enables automatic loading of the recorded force maps, conversion of the force versus piezo-extension curves to force versus indentation curves, calculation of Young’s moduli and generation of Young’s modulus maps, where the pulmonary BM can be identified using a semi-automatic spatial filtering tool. The entire protocol takes 1–2 d. Atomic force microscopy can be used to determine the stiffness of materials. This protocol describes how to measure and quantify the Young’s modulus E of pulmonary mouse and human basement membranes with atomic force microscopy and the Center for Applied Tissue Engineering and Regenerative Medicine processing toolbox.
{"title":"Profiling native pulmonary basement membrane stiffness using atomic force microscopy","authors":"Bastian Hartmann, Lutz Fleischhauer, Monica Nicolau, Thomas Hartvig Lindkær Jensen, Florin-Andrei Taran, Hauke Clausen-Schaumann, Raphael Reuten","doi":"10.1038/s41596-024-00955-7","DOIUrl":"10.1038/s41596-024-00955-7","url":null,"abstract":"Mammalian cells sense and react to the mechanics of their immediate microenvironment. Therefore, the characterization of the biomechanical properties of tissues with high spatial resolution provides valuable insights into a broad variety of developmental, homeostatic and pathological processes within living organisms. The biomechanical properties of the basement membrane (BM), an extracellular matrix (ECM) substructure measuring only ∼100–400 nm across, are, among other things, pivotal to tumor progression and metastasis formation. Although the precise assignment of the Young’s modulus E of such a thin ECM substructure especially in between two cell layers is still challenging, biomechanical data of the BM can provide information of eminent diagnostic potential. Here we present a detailed protocol to quantify the elastic modulus of the BM in murine and human lung tissue, which is one of the major organs prone to metastasis. This protocol describes a streamlined workflow to determine the Young’s modulus E of the BM between the endothelial and epithelial cell layers shaping the alveolar wall in lung tissues using atomic force microscopy (AFM). Our step-by-step protocol provides instructions for murine and human lung tissue extraction, inflation of these tissues with cryogenic cutting medium, freezing and cryosectioning of the tissue samples, and AFM force-map recording. In addition, it guides the reader through a semi-automatic data analysis procedure to identify the pulmonary BM and extract its Young’s modulus E using an in-house tailored user-friendly AFM data analysis software, the Center for Applied Tissue Engineering and Regenerative Medicine processing toolbox, which enables automatic loading of the recorded force maps, conversion of the force versus piezo-extension curves to force versus indentation curves, calculation of Young’s moduli and generation of Young’s modulus maps, where the pulmonary BM can be identified using a semi-automatic spatial filtering tool. The entire protocol takes 1–2 d. Atomic force microscopy can be used to determine the stiffness of materials. This protocol describes how to measure and quantify the Young’s modulus E of pulmonary mouse and human basement membranes with atomic force microscopy and the Center for Applied Tissue Engineering and Regenerative Medicine processing toolbox.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140009082","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}
Mono-dimensional fiber-based electronics can effectively address the growing demand for improved wearable electronic devices because of their exceptional flexibility and stretchability. For practical applications, functional fiber electronic devices need to be integrated into more powerful and versatile systems to execute complex tasks that cannot be completed by single-fiber devices. Existing techniques, such as printing and sintering, reduce the flexibility and cause low connection strength of fiber-based electronic devices because of the high curvature of the fiber. Here, we outline a twisting fabrication process for fiber electrodes, which can be woven into functional threads and integrated within textiles. The design of the twisted thread structure for fiber devices ensures stable interfacing and good flexibility, while the textile structure features easily accessible, interlaced points for efficient circuit connections. Electronic textiles can be customized to act as displays, health monitors and power sources. We detail three main fabrication sections, including the fabrication of the fiber electrodes, their twisting into electronic threads and their assembly into functional textile-based devices. The procedures require ~10 d and are easily reproducible by researchers with expertise in fabricating energy and electronic devices. We provide a twisting fabrication process for fiber electrodes that can be assembled into electronic threads and then integrated in electronic textile-based wearables.
单维纤维电子器件因其卓越的柔韧性和伸展性，可有效满足对改进型可穿戴电子设备日益增长的需求。在实际应用中，功能性纤维电子器件需要集成到功能更强大、用途更广泛的系统中，以执行单纤维器件无法完成的复杂任务。现有的技术，如印刷和烧结，会降低纤维电子器件的柔韧性，并由于纤维的高曲率而导致连接强度低。在此，我们概述了纤维电极的加捻制造工艺，这种工艺可将纤维电极编织成功能线，并集成到纺织品中。用于纤维设备的捻线结构设计可确保稳定的接口和良好的灵活性，而纺织品结构的特点是易于接触、交错点多，可实现高效的电路连接。电子纺织品可定制为显示器、健康监测器和电源。我们详细介绍了三个主要制造环节，包括纤维电极的制造、将其捻成电子线缆以及将其组装成基于纺织品的功能器件。这些步骤需要约 10 d 的时间，对于具有制造能源和电子设备专业知识的研究人员来说很容易复制。
{"title":"Design and fabrication of wearable electronic textiles using twisted fiber-based threads","authors":"Kailin Zhang, Xiang Shi, Haibo Jiang, Kaiwen Zeng, Zihao Zhou, Peng Zhai, Lihua Zhang, Huisheng Peng","doi":"10.1038/s41596-024-00956-6","DOIUrl":"10.1038/s41596-024-00956-6","url":null,"abstract":"Mono-dimensional fiber-based electronics can effectively address the growing demand for improved wearable electronic devices because of their exceptional flexibility and stretchability. For practical applications, functional fiber electronic devices need to be integrated into more powerful and versatile systems to execute complex tasks that cannot be completed by single-fiber devices. Existing techniques, such as printing and sintering, reduce the flexibility and cause low connection strength of fiber-based electronic devices because of the high curvature of the fiber. Here, we outline a twisting fabrication process for fiber electrodes, which can be woven into functional threads and integrated within textiles. The design of the twisted thread structure for fiber devices ensures stable interfacing and good flexibility, while the textile structure features easily accessible, interlaced points for efficient circuit connections. Electronic textiles can be customized to act as displays, health monitors and power sources. We detail three main fabrication sections, including the fabrication of the fiber electrodes, their twisting into electronic threads and their assembly into functional textile-based devices. The procedures require ~10 d and are easily reproducible by researchers with expertise in fabricating energy and electronic devices. We provide a twisting fabrication process for fiber electrodes that can be assembled into electronic threads and then integrated in electronic textile-based wearables.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140013018","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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