Landi Sun, Jana Meissner, Jianfeng He, Lihong Cui, Tobias Fürstenhaupt, Xin Liang
Mechanosensory organelles (MOs) are specialized subcellular entities where force-sensitive channels and supporting structures (e.g., microtubule cytoskeleton) are organized in an orderly manner. The delicate structure of MOs needs to be resolved to understand the mechanisms by which they detect forces and how they are formed. Here, we describe a protocol that allows obtaining detailed information about the nanoscopic ultrastructure of fly MOs by using serial section electron tomography (SS-ET). To preserve fine structural details, the tissues are cryo-immobilized using a high-pressure freezer followed by freeze-substitution at low temperature and embedding in resin at room temperature. Then, sample sections are prepared and used to acquire the dual-axis tilt series images, which are further processed for tomographic reconstruction. Finally, tomograms of consecutive sections are combined into a single larger volume using microtubules as fiducial markers. Using this protocol, we managed to reconstruct the sensory organelles, which provide novel molecular insights as to how fly mechanosensory organelles work and are formed. Based on our experience, we think that, with minimal modifications, this protocol can be adapted to a wide range of applications using different cell and tissue samples. Key features • Resolving the high-resolution 3D ultrastructure of subcellular organelles using serial section electron tomography (SS-ET). • Compared with single-axis tilt series, dual-axis tilt series provides a much wider coverage of Fourier space, improving resolution and features in the reconstructed tomograms. • The use of high-pressure freezing and freeze-substitution maximally preserves the fine structural details.
{"title":"Resolving the In Situ Three-Dimensional Structure of Fly Mechanosensory Organelles Using Serial Section Electron Tomography","authors":"Landi Sun, Jana Meissner, Jianfeng He, Lihong Cui, Tobias Fürstenhaupt, Xin Liang","doi":"10.21769/BioProtoc.4940","DOIUrl":"https://doi.org/10.21769/BioProtoc.4940","url":null,"abstract":"Mechanosensory organelles (MOs) are specialized subcellular entities where force-sensitive channels and supporting structures (e.g., microtubule cytoskeleton) are organized in an orderly manner. The delicate structure of MOs needs to be resolved to understand the mechanisms by which they detect forces and how they are formed. Here, we describe a protocol that allows obtaining detailed information about the nanoscopic ultrastructure of fly MOs by using serial section electron tomography (SS-ET). To preserve fine structural details, the tissues are cryo-immobilized using a high-pressure freezer followed by freeze-substitution at low temperature and embedding in resin at room temperature. Then, sample sections are prepared and used to acquire the dual-axis tilt series images, which are further processed for tomographic reconstruction. Finally, tomograms of consecutive sections are combined into a single larger volume using microtubules as fiducial markers. Using this protocol, we managed to reconstruct the sensory organelles, which provide novel molecular insights as to how fly mechanosensory organelles work and are formed. Based on our experience, we think that, with minimal modifications, this protocol can be adapted to a wide range of applications using different cell and tissue samples. Key features • Resolving the high-resolution 3D ultrastructure of subcellular organelles using serial section electron tomography (SS-ET). • Compared with single-axis tilt series, dual-axis tilt series provides a much wider coverage of Fourier space, improving resolution and features in the reconstructed tomograms. • The use of high-pressure freezing and freeze-substitution maximally preserves the fine structural details.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"16 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139958398","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Inflammatory bowel disease (IBD) is characterized by an aberrant immune response against microbiota. It is well established that T cells play a critical role in mediating the pathology. Assessing the contribution of each subset of T cells in mediating the pathology is crucial in order to design better therapeutic strategies. This protocol presents a method to identify the specific effector T-cell population responsible for intestinal immunopathologies in bone marrow–engrafted mouse models. Here, we used anti-CD4 and anti-CD8β depleting antibodies in bone marrow–engrafted mouse models to identify the effector T-cell population responsible for intestinal damage in a genetic mouse model of chronic intestinal inflammation. Key features • This protocol allows addressing the role of CD4+ or CD8αβ+ in an engrafted model of inflammatory bowel disease (IBD). • This protocol can easily be adapted to address the role of other immune cells or molecules that may play a role in IBD.
{"title":"Addressing the Role of Conventional CD8αβ+ T Cells and CD4+ T Cells in Intestinal Immunopathology Using a Bone Marrow–Engrafted Model","authors":"Amneh Aoudi, Ossama Labiad, Ramdane Igalouzene, Ousséma Mejri, Maxime Sanchez, Saïdi Soudja","doi":"10.21769/bioprotoc.4934","DOIUrl":"https://doi.org/10.21769/bioprotoc.4934","url":null,"abstract":"Inflammatory bowel disease (IBD) is characterized by an aberrant immune response against microbiota. It is well established that T cells play a critical role in mediating the pathology. Assessing the contribution of each subset of T cells in mediating the pathology is crucial in order to design better therapeutic strategies. This protocol presents a method to identify the specific effector T-cell population responsible for intestinal immunopathologies in bone marrow–engrafted mouse models. Here, we used anti-CD4 and anti-CD8β depleting antibodies in bone marrow–engrafted mouse models to identify the effector T-cell population responsible for intestinal damage in a genetic mouse model of chronic intestinal inflammation. Key features • This protocol allows addressing the role of CD4+ or CD8αβ+ in an engrafted model of inflammatory bowel disease (IBD). • This protocol can easily be adapted to address the role of other immune cells or molecules that may play a role in IBD.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"14 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139958406","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Katarina Stoklund Dittlau, Abinaya Chandrasekaran, Kristine Freude, L. Van Den Bosch
Astrocytes are increasingly recognized for their important role in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS). In ALS, astrocytes shift from their primary function of providing neuronal homeostatic support towards a reactive and toxic role, which overall contributes to neuronal toxicity and cell death. Currently, our knowledge on these processes is incomplete, and time-efficient and reproducible model systems in a human context are therefore required to understand and therapeutically modulate the toxic astrocytic response for future treatment options. Here, we present an efficient and straightforward protocol to generate human induced pluripotent stem cell (hiPSC)-derived astrocytes implementing a differentiation scheme based on small molecules. Through an initial 25 days, hiPSCs are differentiated into astrocytes, which are matured for 4+ weeks. The hiPSC-derived astrocytes can be cryopreserved at every passage during differentiation and maturation. This provides convenient pauses in the protocol as well as cell banking opportunities, thereby limiting the need to continuously start from hiPSCs. The protocol has already proven valuable in ALS research but can be adapted to any desired research field where astrocytes are of interest. Key features • This protocol requires preexisting experience in hiPSC culturing for a successful outcome. • The protocol relies on a small molecule differentiation scheme and an easy-to-follow methodology, which can be paused at several time points. • The protocol generates >50 × 106 astrocytes per differentiation, which can be cryopreserved at every passage, ensuring a large-scale experimental output.
{"title":"Generation of Human Induced Pluripotent Stem Cell (hiPSC)-Derived Astrocytes for Amyotrophic Lateral Sclerosis and Other Neurodegenerative Disease Studies","authors":"Katarina Stoklund Dittlau, Abinaya Chandrasekaran, Kristine Freude, L. Van Den Bosch","doi":"10.21769/BioProtoc.4936","DOIUrl":"https://doi.org/10.21769/BioProtoc.4936","url":null,"abstract":"Astrocytes are increasingly recognized for their important role in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS). In ALS, astrocytes shift from their primary function of providing neuronal homeostatic support towards a reactive and toxic role, which overall contributes to neuronal toxicity and cell death. Currently, our knowledge on these processes is incomplete, and time-efficient and reproducible model systems in a human context are therefore required to understand and therapeutically modulate the toxic astrocytic response for future treatment options. Here, we present an efficient and straightforward protocol to generate human induced pluripotent stem cell (hiPSC)-derived astrocytes implementing a differentiation scheme based on small molecules. Through an initial 25 days, hiPSCs are differentiated into astrocytes, which are matured for 4+ weeks. The hiPSC-derived astrocytes can be cryopreserved at every passage during differentiation and maturation. This provides convenient pauses in the protocol as well as cell banking opportunities, thereby limiting the need to continuously start from hiPSCs. The protocol has already proven valuable in ALS research but can be adapted to any desired research field where astrocytes are of interest. Key features • This protocol requires preexisting experience in hiPSC culturing for a successful outcome. • The protocol relies on a small molecule differentiation scheme and an easy-to-follow methodology, which can be paused at several time points. • The protocol generates >50 × 106 astrocytes per differentiation, which can be cryopreserved at every passage, ensuring a large-scale experimental output.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"8 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139958287","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Matteo Manfredi, Castrense Savojardo, P. Martelli, R. Casadio
Coiled-coil domains (CCDs) are structural motifs observed in proteins in all organisms that perform several crucial functions. The computational identification of CCD segments over a protein sequence is of great importance for its functional characterization. This task can essentially be divided into three separate steps: the detection of segment boundaries, the annotation of the heptad repeat pattern along the segment, and the classification of its oligomerization state. Several methods have been proposed over the years addressing one or more of these predictive steps. In this protocol, we illustrate how to make use of CoCoNat, a novel approach based on protein language models, to characterize CCDs. CoCoNat is, at its release (August 2023), the state of the art for CCD detection. The web server allows users to submit input protein sequences and visualize the predicted domains after a few minutes. Optionally, precomputed segments can be provided to the model, which will predict the oligomerization state for each of them. CoCoNat can be easily integrated into biological pipelines by downloading the standalone version, which provides a single executable script to produce the output. Key features • Web server for the prediction of coiled-coil segments from a protein sequence. • Three different predictions from a single tool (segment position, heptad repeat annotation, oligomerization state). • Possibility to visualize the results online or to download the predictions in different formats for further processing. • Easy integration in automated pipelines with the local version of the tool.
{"title":"CoCoNat: A Deep Learning–Based Tool for the Prediction of Coiled-coil Domains in Protein Sequences","authors":"Matteo Manfredi, Castrense Savojardo, P. Martelli, R. Casadio","doi":"10.21769/BioProtoc.4935","DOIUrl":"https://doi.org/10.21769/BioProtoc.4935","url":null,"abstract":"Coiled-coil domains (CCDs) are structural motifs observed in proteins in all organisms that perform several crucial functions. The computational identification of CCD segments over a protein sequence is of great importance for its functional characterization. This task can essentially be divided into three separate steps: the detection of segment boundaries, the annotation of the heptad repeat pattern along the segment, and the classification of its oligomerization state. Several methods have been proposed over the years addressing one or more of these predictive steps. In this protocol, we illustrate how to make use of CoCoNat, a novel approach based on protein language models, to characterize CCDs. CoCoNat is, at its release (August 2023), the state of the art for CCD detection. The web server allows users to submit input protein sequences and visualize the predicted domains after a few minutes. Optionally, precomputed segments can be provided to the model, which will predict the oligomerization state for each of them. CoCoNat can be easily integrated into biological pipelines by downloading the standalone version, which provides a single executable script to produce the output. Key features • Web server for the prediction of coiled-coil segments from a protein sequence. • Three different predictions from a single tool (segment position, heptad repeat annotation, oligomerization state). • Possibility to visualize the results online or to download the predictions in different formats for further processing. • Easy integration in automated pipelines with the local version of the tool.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"27 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139958342","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Rahul Chaurasia, M. Ayajuddin, Girish Ratnaparkhi, Shashidhara S. Lingadahalli, S. Yenisetti
Dopaminergic (DAergic) neurodegeneration in the substantia nigra pars compacta of the human brain is the pathological feature associated with Parkinson’s disease (PD). Drosophila also exhibits mobility defects and diminished levels of brain dopamine on exposure to neurotoxicants mimicking PD. Our laboratory demonstrated in a Drosophila model of sporadic PD that there is no decrease in DAergic neuronal number; instead, there is a significant reduction in tyrosine hydroxylase (TH) fluorescence intensity (FI). Here, we present a sensitive assay based on the quantification of FI of the secondary antibody (ab). As the FI is directly proportional to the amount of TH synthesis, its reduction under PD conditions denotes the decrease in the TH synthesis, suggesting DAergic neuronal dysfunction. Therefore, FI quantification is a refined and sensitive method to understand the early stages of DAergic neurodegeneration. FI quantification is performed using the ZEN 2012 SP2 single-user software; a license must be acquired to utilize the imaging system to interactively control image acquisition, image processing, and analysis. This method will be of good use to biologists, as it can also be used with little modification to characterize the extent of degeneration and changes in the level of degeneration in response to drugs in different cell types. Unlike the expensive and cumbersome confocal microscopy, the present method will be an affordable option for fund-constrained neurobiology laboratories. Key features • Allows characterizing the incipient DAergic and other catecholaminergic neurodegeneration, even in the absence of loss of neuronal cell body. • Great alternative for the fund-constrained neurobiology laboratories in developing countries to utilize this method in different cell types and their response to drugs/nutraceuticals.
多巴胺能（DAergic）神经变性是帕金森病（PD）的病理特征。果蝇在暴露于模拟帕金森病的神经毒物时也会表现出移动性缺陷和脑多巴胺水平降低。我们的实验室在散发性帕金森病的果蝇模型中证实，多巴胺能神经元的数量并没有减少；相反，酪氨酸羟化酶（TH）的荧光强度（FI）却显著降低。在此，我们提出了一种基于二抗（ab）荧光强度定量的灵敏检测方法。由于 FI 与 TH 的合成量成正比，因此在帕金森病条件下，FI 的降低表示 TH 合成的减少，从而提示 DAergic 神经元功能障碍。因此，FI 定量是了解 DAergic 神经变性早期阶段的一种精细而灵敏的方法。FI定量使用ZEN 2012 SP2单用户软件进行；必须获得许可证才能使用成像系统交互式控制图像采集、图像处理和分析。这种方法对生物学家很有帮助，因为它只需稍加改动就可用于描述不同类型细胞的变性程度以及变性程度对药物反应的变化。与昂贵而繁琐的共聚焦显微镜不同，本方法对于资金有限的神经生物学实验室来说是一种经济实惠的选择。主要特点 - 即使在神经元细胞体没有缺失的情况下，也能描述初生 DA 能和其他儿茶酚胺能神经变性的特征。- 对于发展中国家资金有限的神经生物学实验室来说，利用这种方法研究不同类型的细胞及其对药物/保健品的反应是一个不错的选择。
{"title":"A Simple Immunofluorescence Method to Characterize Neurodegeneration and Tyrosine Hydroxylase Reduction in Whole Brain of a Drosophila Model of Parkinson’s Disease","authors":"Rahul Chaurasia, M. Ayajuddin, Girish Ratnaparkhi, Shashidhara S. Lingadahalli, S. Yenisetti","doi":"10.21769/bioprotoc.4937","DOIUrl":"https://doi.org/10.21769/bioprotoc.4937","url":null,"abstract":"Dopaminergic (DAergic) neurodegeneration in the substantia nigra pars compacta of the human brain is the pathological feature associated with Parkinson’s disease (PD). Drosophila also exhibits mobility defects and diminished levels of brain dopamine on exposure to neurotoxicants mimicking PD. Our laboratory demonstrated in a Drosophila model of sporadic PD that there is no decrease in DAergic neuronal number; instead, there is a significant reduction in tyrosine hydroxylase (TH) fluorescence intensity (FI). Here, we present a sensitive assay based on the quantification of FI of the secondary antibody (ab). As the FI is directly proportional to the amount of TH synthesis, its reduction under PD conditions denotes the decrease in the TH synthesis, suggesting DAergic neuronal dysfunction. Therefore, FI quantification is a refined and sensitive method to understand the early stages of DAergic neurodegeneration. FI quantification is performed using the ZEN 2012 SP2 single-user software; a license must be acquired to utilize the imaging system to interactively control image acquisition, image processing, and analysis. This method will be of good use to biologists, as it can also be used with little modification to characterize the extent of degeneration and changes in the level of degeneration in response to drugs in different cell types. Unlike the expensive and cumbersome confocal microscopy, the present method will be an affordable option for fund-constrained neurobiology laboratories. Key features • Allows characterizing the incipient DAergic and other catecholaminergic neurodegeneration, even in the absence of loss of neuronal cell body. • Great alternative for the fund-constrained neurobiology laboratories in developing countries to utilize this method in different cell types and their response to drugs/nutraceuticals.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139958057","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Shirin Fateh, Reem Alromaihi, Amir Ghaemmaghami, Morgan Alexander
Biomaterials are designed to interact with biological systems to replace, support, enhance, or monitor their function. However, there are challenges associated with traditional biomaterials’ development due to the lack of underlying theory governing cell response to materials’ chemistry. This leads to the time-consuming process of testing different materials plus the adverse reactions in the body such as cytotoxicity and foreign body response. High-throughput screening (HTS) offers a solution to these challenges by enabling rapid and simultaneous testing of a large number of materials to determine their bio-interactions and biocompatibility. Secreted proteins regulate many physiological functions and determine the success of implanted biomaterials through directing cell behaviour. However, the majority of biomaterials’ HTS platforms are suitable for microscopic analyses of cell behaviour and not for investigating non-adherent cells or measuring cell secretions. Here, we describe a multi-well platform adaptable to robotic printing of polymers and suitable for secretome profiling of both adherent and non-adherent cells. We detail the platform's development steps, encompassing the preparation of individual cell culture chambers, polymer printing, and the culture environment, as well as examples to demonstrate surface chemical characterisation and biological assessments of secreted mediators. Such platforms will no doubt facilitate the discovery of novel biomaterials and broaden their scope by adapting wider arrays of cell types and incorporating assessments of both secretome and cell-bound interactions. Key features • Detailed protocols for preparation of substrate for contact printing of acrylate-based polymers including O2 plasma etching, functionalisation process, and Poly(2-hydroxyethyl methacrylate) (pHEMA) dip coating. • Preparations of 7 mm × 7 mm polymers employing pin printing system. • Provision of confined area for each polymer using ProPlate® multi-well chambers. • Compatibility of this platform was validated using adherent cells [primary human monocyte–derived macrophages (MDMs)) and non-adherent cells (primary human monocyte–derived dendritic cells (moDCs)]. • Examples of the adaptability of the platform for secretome analysis including five different cytokines using enzyme-linked immunosorbent assay (ELISA, DuoSet®). Graphical overview
{"title":"Unlocking Bio-Instructive Polymers: A Novel Multi-Well Screening Platform Based on Secretome Sampling","authors":"Shirin Fateh, Reem Alromaihi, Amir Ghaemmaghami, Morgan Alexander","doi":"10.21769/BioProtoc.4939","DOIUrl":"https://doi.org/10.21769/BioProtoc.4939","url":null,"abstract":"Biomaterials are designed to interact with biological systems to replace, support, enhance, or monitor their function. However, there are challenges associated with traditional biomaterials’ development due to the lack of underlying theory governing cell response to materials’ chemistry. This leads to the time-consuming process of testing different materials plus the adverse reactions in the body such as cytotoxicity and foreign body response. High-throughput screening (HTS) offers a solution to these challenges by enabling rapid and simultaneous testing of a large number of materials to determine their bio-interactions and biocompatibility. Secreted proteins regulate many physiological functions and determine the success of implanted biomaterials through directing cell behaviour. However, the majority of biomaterials’ HTS platforms are suitable for microscopic analyses of cell behaviour and not for investigating non-adherent cells or measuring cell secretions. Here, we describe a multi-well platform adaptable to robotic printing of polymers and suitable for secretome profiling of both adherent and non-adherent cells. We detail the platform's development steps, encompassing the preparation of individual cell culture chambers, polymer printing, and the culture environment, as well as examples to demonstrate surface chemical characterisation and biological assessments of secreted mediators. Such platforms will no doubt facilitate the discovery of novel biomaterials and broaden their scope by adapting wider arrays of cell types and incorporating assessments of both secretome and cell-bound interactions. Key features • Detailed protocols for preparation of substrate for contact printing of acrylate-based polymers including O2 plasma etching, functionalisation process, and Poly(2-hydroxyethyl methacrylate) (pHEMA) dip coating. • Preparations of 7 mm × 7 mm polymers employing pin printing system. • Provision of confined area for each polymer using ProPlate® multi-well chambers. • Compatibility of this platform was validated using adherent cells [primary human monocyte–derived macrophages (MDMs)) and non-adherent cells (primary human monocyte–derived dendritic cells (moDCs)]. • Examples of the adaptability of the platform for secretome analysis including five different cytokines using enzyme-linked immunosorbent assay (ELISA, DuoSet®). Graphical overview","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"10 10","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139958146","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Structural and functional changes in vascular networks play a vital role during development, causing or contributing to the pathophysiology of injury and disease. Current methods to trace and image the vasculature in laboratory settings have proven inconsistent, inaccurate, and labor intensive, lacking the inherent three-dimensional structure of vasculature. Here, we provide a robust and highly reproducible method to image and quantify changes in vascular networks down to the capillary level. The method combines vasculature tracing, tissue clearing, and three-dimensional imaging techniques with vessel segmentation using AI-based convolutional reconstruction to rapidly process large, unsectioned tissue specimens throughout the body with high fidelity. The practicality and scalability of our protocol offer application across various fields of biomedical sciences. Obviating the need for sectioning of samples, this method will expedite qualitative and quantitative analyses of vascular networks. Preparation of the fluorescent gel perfusate takes < 30 min per study. Transcardiac perfusion and vasculature tracing takes approximately 20 min, while dissection of tissue samples ranges from 5 to 15 min depending on the tissue of interest. The tissue clearing protocol takes approximately 24–48 h per whole-tissue sample. Lastly, three-dimensional imaging and analysis can be completed in one day. The entire procedure can be carried out by a competent graduate student or experienced technician. Key features • This robust and highly reproducible method allows users to image and quantify changes in vascular networks down to the capillary level. • Three-dimensional imaging techniques with vessel segmentation enable rapid processing of large, unsectioned tissue specimens throughout the body. • It takes approximately 2–3 days for sample preparation, three-dimensional imaging, and analysis. • The user-friendly pipeline can be completed by experienced and non-experienced users.
{"title":"A Versatile Pipeline for High-fidelity Imaging and Analysis of Vascular Networks Across the Body","authors":"Stephen Vidman, Elliot Dion, Andrea Tedeschi","doi":"10.21769/BioProtoc.4938","DOIUrl":"https://doi.org/10.21769/BioProtoc.4938","url":null,"abstract":"Structural and functional changes in vascular networks play a vital role during development, causing or contributing to the pathophysiology of injury and disease. Current methods to trace and image the vasculature in laboratory settings have proven inconsistent, inaccurate, and labor intensive, lacking the inherent three-dimensional structure of vasculature. Here, we provide a robust and highly reproducible method to image and quantify changes in vascular networks down to the capillary level. The method combines vasculature tracing, tissue clearing, and three-dimensional imaging techniques with vessel segmentation using AI-based convolutional reconstruction to rapidly process large, unsectioned tissue specimens throughout the body with high fidelity. The practicality and scalability of our protocol offer application across various fields of biomedical sciences. Obviating the need for sectioning of samples, this method will expedite qualitative and quantitative analyses of vascular networks. Preparation of the fluorescent gel perfusate takes < 30 min per study. Transcardiac perfusion and vasculature tracing takes approximately 20 min, while dissection of tissue samples ranges from 5 to 15 min depending on the tissue of interest. The tissue clearing protocol takes approximately 24–48 h per whole-tissue sample. Lastly, three-dimensional imaging and analysis can be completed in one day. The entire procedure can be carried out by a competent graduate student or experienced technician. Key features • This robust and highly reproducible method allows users to image and quantify changes in vascular networks down to the capillary level. • Three-dimensional imaging techniques with vessel segmentation enable rapid processing of large, unsectioned tissue specimens throughout the body. • It takes approximately 2–3 days for sample preparation, three-dimensional imaging, and analysis. • The user-friendly pipeline can be completed by experienced and non-experienced users.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"12 5","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139958211","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
An Hsieh, Sean Reardon, Jubilee Munozvilla-Cabellon, Jiayu Shen, Smita Patel, Tatiana Mishanina
Human mitochondrial DNA (mtDNA) encodes several components of oxidative phosphorylation responsible for the bulk of cellular energy production. The mtDNA is transcribed by a dedicated human mitochondrial RNA polymerase (POLRMT) that is structurally distinct from its nuclear counterparts, instead closely resembling the single-subunit viral RNA polymerases (e.g., T7 RNA polymerase). The initiation of transcription by POLRMT is aided by two initiation factors: transcription factor A, mitochondrial (TFAM), and transcription factor B2, mitochondrial (TFB2M). Although many details of human mitochondrial transcription initiation have been elucidated with in vitro biochemical and structural studies, much remains to be addressed relating to the mechanism and regulation of transcription. Studies of such mechanisms require reliable, high-yield, and high-purity methods for protein production, and this protocol provides the level of detail and troubleshooting tips that are necessary for a novice to generate meaningful amounts of proteins for experimental work. The current protocol describes how to purify recombinant POLRMT, TFAM, and TFB2M from Escherichia coli using techniques such as affinity column chromatography (Ni2+ and heparin), how to remove the solubility tags with TEV protease and recover untagged proteins of interest, and how to overcome commonly encountered challenges in obtaining high yield of each protein. Key features • This protocol builds upon purification methods developed by Patel lab (Ramachandran et al., 2017) and others with greater detail than previously published works. • The protocol requires several days to complete as various steps are designed to be performed overnight. • The recombinantly purified proteins have been successfully used for in vitro transcription experiments, allowing for finer control of experimental components in a minimalistic system.
人类线粒体DNA (mtDNA)编码氧化磷酸化的几个组成部分，负责大部分细胞能量的产生。mtDNA是由一种专门的人类线粒体RNA聚合酶(POLRMT)转录的，该酶在结构上与其核对应物不同，而与单亚基病毒RNA聚合酶(如T7 RNA聚合酶)非常相似。POLRMT启动转录需要两个启动因子:转录因子A，线粒体(TFAM)和转录因子B2，线粒体(TFB2M)。尽管体外生化和结构研究已经阐明了人类线粒体转录起始的许多细节，但与转录的机制和调控有关的许多问题仍有待解决。这种机制的研究需要可靠，高产，高纯度的蛋白质生产方法，本协议提供的细节和故障排除提示的水平，是必要的新手产生有意义的蛋白质的实验工作。目前的方案描述了如何使用亲和柱层析(Ni2+和肝素)等技术从大肠杆菌中纯化重组POLRMT, TFAM和TFB2M，如何使用TEV蛋白酶去除溶解度标签并回收未标记的感兴趣蛋白，以及如何克服在获得每种蛋白高产率方面常见的挑战。•本协议建立在Patel实验室(Ramachandran et al.， 2017)和其他比以前发表的作品更详细的纯化方法的基础上。•该方案需要几天才能完成，因为各种步骤都是在一夜之间完成的。•重组纯化蛋白已成功用于体外转录实验，允许在极简系统中对实验成分进行更精细的控制。
{"title":"Expression and Purification of Recombinant Human Mitochondrial RNA Polymerase (POLRMT) and the Initiation Factors TFAM and TFB2M","authors":"An Hsieh, Sean Reardon, Jubilee Munozvilla-Cabellon, Jiayu Shen, Smita Patel, Tatiana Mishanina","doi":"10.21769/bioprotoc.4892","DOIUrl":"https://doi.org/10.21769/bioprotoc.4892","url":null,"abstract":"Human mitochondrial DNA (mtDNA) encodes several components of oxidative phosphorylation responsible for the bulk of cellular energy production. The mtDNA is transcribed by a dedicated human mitochondrial RNA polymerase (POLRMT) that is structurally distinct from its nuclear counterparts, instead closely resembling the single-subunit viral RNA polymerases (e.g., T7 RNA polymerase). The initiation of transcription by POLRMT is aided by two initiation factors: transcription factor A, mitochondrial (TFAM), and transcription factor B2, mitochondrial (TFB2M). Although many details of human mitochondrial transcription initiation have been elucidated with in vitro biochemical and structural studies, much remains to be addressed relating to the mechanism and regulation of transcription. Studies of such mechanisms require reliable, high-yield, and high-purity methods for protein production, and this protocol provides the level of detail and troubleshooting tips that are necessary for a novice to generate meaningful amounts of proteins for experimental work. The current protocol describes how to purify recombinant POLRMT, TFAM, and TFB2M from Escherichia coli using techniques such as affinity column chromatography (Ni2+ and heparin), how to remove the solubility tags with TEV protease and recover untagged proteins of interest, and how to overcome commonly encountered challenges in obtaining high yield of each protein. Key features • This protocol builds upon purification methods developed by Patel lab (Ramachandran et al., 2017) and others with greater detail than previously published works. • The protocol requires several days to complete as various steps are designed to be performed overnight. • The recombinantly purified proteins have been successfully used for in vitro transcription experiments, allowing for finer control of experimental components in a minimalistic system.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"87 9","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138600184","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Recent advancements in chemogenetic tools, such as designer receptors exclusively activated by designer drugs (DREADDs), allow the simultaneous manipulation of activity over a specific, broad brain region in nonhuman primates. However, the introduction of DREADDs into large and complexly shaped cortical sulcus regions of macaque monkeys is technically demanding; previously reported methods are time consuming or do not allow the spatial range of expression to be controlled. In the present report, we describe the procedure for an adeno-associated viral vector (AAV2.1) delivery via handheld injections into the dorsolateral prefrontal cortex (Brodmann’s area 9/46) of macaque monkeys, with reference to pre-scanned anatomical magnetic resonance images. This procedure allows the precise delivery of DREADDs to a specific cortical region. Key features • This article describes the procedures for injecting viral vectors encoding functional proteins for chemogenetic manipulation into targeted cortical sulcus regions. • The protocol requires magnetic resonance imaging for the accurate estimation of the injection sites prior to surgery. • Viral vector solutions are injected using a handheld syringe under microscopic guidance. • This protocol allows for the precise introduction of designer receptors exclusively activated by designer drugs (DREADDs) to large and complex cortical regions.
{"title":"Targeted Delivery of Chemogenetic Adeno-Associated Viral Vectors to Cortical Sulcus Regions in Macaque Monkeys by Handheld Injections","authors":"Kei Oyama, Yuji Nagai, T. Minamimoto","doi":"10.21769/BioProtoc.4897","DOIUrl":"https://doi.org/10.21769/BioProtoc.4897","url":null,"abstract":"Recent advancements in chemogenetic tools, such as designer receptors exclusively activated by designer drugs (DREADDs), allow the simultaneous manipulation of activity over a specific, broad brain region in nonhuman primates. However, the introduction of DREADDs into large and complexly shaped cortical sulcus regions of macaque monkeys is technically demanding; previously reported methods are time consuming or do not allow the spatial range of expression to be controlled. In the present report, we describe the procedure for an adeno-associated viral vector (AAV2.1) delivery via handheld injections into the dorsolateral prefrontal cortex (Brodmann’s area 9/46) of macaque monkeys, with reference to pre-scanned anatomical magnetic resonance images. This procedure allows the precise delivery of DREADDs to a specific cortical region. Key features • This article describes the procedures for injecting viral vectors encoding functional proteins for chemogenetic manipulation into targeted cortical sulcus regions. • The protocol requires magnetic resonance imaging for the accurate estimation of the injection sites prior to surgery. • Viral vector solutions are injected using a handheld syringe under microscopic guidance. • This protocol allows for the precise introduction of designer receptors exclusively activated by designer drugs (DREADDs) to large and complex cortical regions.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"139 28","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138598636","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The recent surge in plant genomic and transcriptomic data has laid a foundation for reconstructing evolutionary scenarios and inferring potential functions of key genes related to plants’ development and stress responses. The classical scheme for identifying homologous genes is sequence similarity–based searching, under the crucial assumption that homologous sequences are more similar to each other than they are to any other non-homologous sequences. Advances in plant phylogenomics and computational algorithms have enabled us to systemically identify homologs/orthologs and reconstruct their evolutionary histories among distantly related lineages. Here, we present a comprehensive pipeline for homologous sequences identification, phylogenetic relationship inference, and potential functional profiling of genes in plants. Key features • Identification of orthologs using large-scale genomic and transcriptomic data. • This protocol is generalized for analyzing the evolution of plant genes.
{"title":"Phylogenetic Inference of Homologous/Orthologous Genes Among Distantly Related Plants","authors":"Zilong Xu, Wenyan Sun, Ziqiang Zhu, Bojian Zhong, Zhenhua Zhang","doi":"10.21769/BioProtoc.4893","DOIUrl":"https://doi.org/10.21769/BioProtoc.4893","url":null,"abstract":"The recent surge in plant genomic and transcriptomic data has laid a foundation for reconstructing evolutionary scenarios and inferring potential functions of key genes related to plants’ development and stress responses. The classical scheme for identifying homologous genes is sequence similarity–based searching, under the crucial assumption that homologous sequences are more similar to each other than they are to any other non-homologous sequences. Advances in plant phylogenomics and computational algorithms have enabled us to systemically identify homologs/orthologs and reconstruct their evolutionary histories among distantly related lineages. Here, we present a comprehensive pipeline for homologous sequences identification, phylogenetic relationship inference, and potential functional profiling of genes in plants. Key features • Identification of orthologs using large-scale genomic and transcriptomic data. • This protocol is generalized for analyzing the evolution of plant genes.","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"109 19","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138599988","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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