Bio-protocol最新文献

{"title":"Improving Stability of <i>Spiroplasma citri</i> MreB5 Through Purification Optimization and Structural Insights.","authors":"Vani Pande, Pananghat Gayathri","doi":"10.21769/BioProtoc.5086","DOIUrl":"https://doi.org/10.21769/BioProtoc.5086","url":null,"abstract":"<p><p>MreB is a prokaryotic actin homolog. It is essential for cell shape in the majority of rod-shaped cell-walled bacteria. Structural and functional characterization of MreB protein is important to understand the mechanism of ATP-dependent filament dynamics and membrane interaction. In vitro studies on MreBs have been limited due to the difficulty in purifying the homogenous monomeric protein. We have purified MreB from the cell-wall-less bacteria <i>Spiroplasma citri</i>, ScMreB5, using heterologous expression in Escherichia coli. This protocol provides a detailed description of purification condition optimization that led us to obtain high concentrations of stable ScMreB5. Additionally, we have provided a protocol for detecting the presence of monovalent ions in the ScMreB5 AMP-PNP-bound crystal structure. This protocol can be used to obtain a high yield of ScMreB5 for carrying out biochemical and reconstitution studies. The strategies used for ScMreB5 show how optimizing buffer components can enhance the yield and stability of purified protein. Key features • The protocol is a useful approach to standardize purification of nucleotide-dependent cytoskeletal filaments and other nucleotide-binding proteins. • The mechanistic basis of how different ions could stabilize a protein, and hence improve yield in purification, has been demonstrated. • The change in buffer conditions/salt enabled us to get sufficient yield for biochemical and structural characterization.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11540049/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607606","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of the Mammary Gland in Mouse: A Whole-Mount Microscopic Analysis.","authors":"Bo Wang, Yuchen Xie, Zejian Yang, Jingyue Zhang, Huiwen Zhang, Peijun Liu","doi":"10.21769/BioProtoc.5089","DOIUrl":"https://doi.org/10.21769/BioProtoc.5089","url":null,"abstract":"<p><p>The mammary gland undergoes functional, developmental, and structural changes that are essential for lactation and reproductive processes. An overview of such unique tissue can offer clearer insights into mammary development and tumorigenesis. Compared to traditional methods, mouse mammary gland whole mount is a pivotal technique that provides three-dimensional structural perspectives on gland morphology and developmental stages, offering an inexpensive and accessible approach. This protocol outlines the tissue isolation of the mouse mammary gland and provides detailed instructions for whole-mount staining and analysis. Mammary gland tissues are carefully dissected from euthanized mice and stained with Carmine Alum to highlight the ductal structures, enabling detailed visualization of the branching patterns and morphological changes. Light microscopy is used to capture a panoramic image of the stained mammary gland, enabling the quantitative analysis of terminal end buds (TEBs) and bifurcated TEBs to further investigate mammary gland remodeling. This method can provide invaluable insights, particularly in the study of mammary gland morphogenesis and tumorigenesis, underscoring its significance in both basic research and clinical applications. Key features • Monitor mammary gland development within 2 days using whole-mount staining.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11540045/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607585","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Host Receptor Pili for Cryo-EM Single-Particle Reconstruction.","authors":"Ran Meng","doi":"10.21769/BioProtoc.5094","DOIUrl":"https://doi.org/10.21769/BioProtoc.5094","url":null,"abstract":"<p><p>Single-stranded RNA bacteriophages (ssRNA phages) infect their hosts by binding to the host receptor pili. Purification of pili usually involves mechanical shearing of pili from cells followed by precipitation. However, previous methods often result in low efficiency or unstable results due to pili retraction. This protocol presents an optimized method for purifying receptor type IV pili from <i>Acinetobacter genomospecies 16</i> (<i>A. gp16</i>), incorporating enhancements in shearing and collection steps to achieve high yields. We found that repeated passage through syringe needles increases yield, and temperature control is crucial during purification. Additionally, the CsCl density gradient was optimized specifically for this specific strain. The purified type IV pili are suitable for cryogenic electron microscopy (cryo-EM) and various biochemical experiments. Key features • Pili purification for single-particle cryo-electron microscopy (Cryo-EM) analysis • This protocol builds upon the F-pili purification method developed by Costa et al. [1] extending its application to the <i>Acinetobacter genomosp.</i> 16. • It is optimized for higher and more stable pili yields, as well as increased reproducibility. • The method is tested on various bacterial species and can be adapted to purify different types of pili.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11539960/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607601","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Generation and Maintenance of Patient-Derived Endometrial Cancer Organoids.","authors":"Mali Barbi, Divya Gowthaman, Arielle Katcher, Megan Gorman, Brian Yueh, Aaron Nizam, Charlie Chung, Erdogan Oguzhan Akyildiz, Marina Frimer, Gary L Goldberg, Semir Beyaz","doi":"10.21769/BioProtoc.5093","DOIUrl":"https://doi.org/10.21769/BioProtoc.5093","url":null,"abstract":"<p><p>Endometrial cancer (EC) is the leading cause of gynecologic cancer morbidity and mortality in the U.S. Despite advancements in cancer research, EC death rates are increasing, particularly high-grade endometrial cancers. The development of three-dimensional (3D) patient-derived organoid (PDO) models for EC is crucial, as they provide a more accurate representation of the biological and genetic complexity of a patient's tumor compared to traditional 2D cell lines. Here, we describe a protocol for cultivating PDO models from normal endometrium and EC across different EC subtypes. These EC PDO models can be expanded across multiple passages and facilitate the exploration of tumor behavior and drug responses, thereby advancing our understanding of the disease and potentially leading to more effective and individualized novel therapeutic strategies. Key features • Establishment of patient-derived EC and normal endometrium organoids. • Accurate replication of the various histologic and molecular subtypes of EC within the organoids. • Our approach provides a clinically relevant platform for studying EC development, metastasis, progression, and recurrence. • It offers potential for developing targeted therapeutic interventions tailored to specific EC subtypes. Graphical overview Schematic overview of endometrial cancer and normal organoid preparation from patient-derived samples.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11539961/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607598","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Detecting Native Protein-Protein Interactions by APEX2 Proximity Labeling in <i>Drosophila</i> Tissues.","authors":"Jhen-Wei Wu, Chueh-Wen Wang, Wei Yang Hong, Anna C C Jang, Yu-Chiuan Chang","doi":"10.21769/BioProtoc.5090","DOIUrl":"https://doi.org/10.21769/BioProtoc.5090","url":null,"abstract":"<p><p>Enzyme-catalyzed proximity labeling is a potent technique for the discernment of subtle molecular interactions and subcellular localization, furnishing contextual insights into the protein of interest within cells. Although ascorbate peroxidase2 (APEX2) has proven effective in this approach when overexpressed, its compatibility with endogenous proteins remains untested. We improved this technique for studying native protein-protein interactions in live <i>Drosophila</i> ovary tissue. Through CRISPR/Cas9 genome editing, APEX2 was fused with the endogenous dysfusion gene. By pre-treating the tissue with Triton X-100 to enhance biotin-phenol penetration, we successfully identified multiple proteins interacting with the native Dysfusion proteins that reside on the inner nuclear membrane. Our protocol offers a comprehensive workflow for delineating the interactome networks of ovarian components in <i>Drosophila</i>, aiding future studies on endogenous protein-protein interactions in various tissues of other animals. Key features • Elucidating Protein-protein interactions provides deeper insights into the regulation of gene expression in molecular network and complex signaling pathways, advancing protein engineering and drug design. • This protocol utilizes CRISPR/Cas9 knock-in technology to tag endogenous proteins with the APEX2 to label nearby proteins within a 20 nm radius in <i>Drosophila melanogaster</i>. • We optimize APEX2-proximity labeling by using Triton X-100 pre-treatment to enhance biotin-phenol penetration into <i>Drosophila</i> ovaries, enabling endogenous proteins enrichment under native conditions.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11540050/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607568","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Single Cell Isolation from Human Diabetic Fibrovascular Membranes for Single-Cell RNA Sequencing.","authors":"Katia Corano Scheri, Thomas Tedeschi, Amani A Fawzi","doi":"10.21769/BioProtoc.5096","DOIUrl":"https://doi.org/10.21769/BioProtoc.5096","url":null,"abstract":"<p><p>Single-cell transcriptomic analyses have emerged as very powerful tools to query the gene expression changes at the single-cell level in physiological and pathological conditions. The quality of the analysis is heavily dependent on tissue digestion protocols, with the goal of preserving thousands of single live cells to submit to the subsequent processing steps and analysis. Multiple digestion protocols that use different enzymes to digest the tissues have been described. Harsh digestion can damage certain cell types, but this might be required to digest especially fibrotic tissue as in our experimental condition. In this paper, we summarize a collagenase type I digestion protocol for preparing the single-cell suspension from fibrovascular tissues surgically removed from patients with proliferative diabetic retinopathy (PDR) for single-cell RNA sequencing (scRNA-Seq) analyses. We also provide a detailed description of the data analysis that we implemented in a previously published study. Key features • Single-cell suspension from fibrovascular membranes isolated from PDR patients. • Single-cell RNA sequencing analyses performed using Seurat package in RStudio. • Trajectory analyses or pseudotime analyses to study the trajectory over (pseudo)time of specific cell types. • This protocol requires Illumina HiSEQ4000 instrument and knowledge of R and RStudio language for the analyses.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11540046/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607615","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Chemogenetic Silencing of Neonatal Spontaneous Activity of Projection Neurons in the Dorsal Striatum of Mice.","authors":"Bojana Kokinovic, Maria Ryazantseva, Svetlana Molchanova","doi":"10.21769/BioProtoc.5088","DOIUrl":"https://doi.org/10.21769/BioProtoc.5088","url":null,"abstract":"<p><p>Neuroscience incorporates manipulating neuronal circuitry to enhance the understanding of intricate brain functions. An effective strategy to attain this objective entails utilizing viral vectors to induce varied gene expression by delivering transgenes into brain cells. Here, we combine the use of transgenic mice, neonatal transduction with adeno-associated viral constructs harboring inhibitory designer receptor exclusively activated by designer drug (DREADD) gene, and the DREADD agonist clozapine N-oxide (CNO). In this way, a chemogenetic approach is employed to suppress neuronal activity in the region of interest during a critical developmental window, with subsequent investigation into its effects on the neuronal circuitry in adulthood. Key features • Comprehensive protocol for newborn viral transduction in the dorsal striatum of mice • Uses a viral construct encoding inhibitory DREADD under the control of Cre recombinase to attenuate the activity of specific cell types in the brain.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11539959/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607436","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative Analysis of Fish Morphology Through Landmark and Outline-based Geometric Morphometrics with Free Software.","authors":"Du Luo","doi":"10.21769/BioProtoc.5087","DOIUrl":"https://doi.org/10.21769/BioProtoc.5087","url":null,"abstract":"<p><p>Morphology underpins key biological and evolutionary processes that remain elusive. This is in part due to the limitations in robustly and quantitatively analyzing shapes within and between groups in an unbiased and high-throughput manner. Geometric morphometrics (GM) has emerged as a widely employed technique for studying shape variation in biology and evolution. This study presents a comprehensive workflow for conducting geometric morphometric analysis of fish morphology. The step-by-step manual provides detailed instructions for using popular free software, such as the TPS series, MorphoJ, ImageJ, and R, to carry out generalized Procrustes analysis (GPA), principal component analysis (PCA), discriminant function analysis (DFA), canonical variate analysis (CVA), mean shape analysis, and thin plate spline analysis (TPS). The Momocs package in R is specifically utilized for in-depth analysis of fish outlines. In addition, selected functions from the dplyr package are used to assist in the analysis. The full process of fish outline analysis is covered, including extracting outline coordinates, converting and scaling data, defining landmarks, creating data objects, analyzing outline differences, and visualizing results. In conclusion, the current protocol compiles a detailed method for evaluating fish shape variation based on landmarks and outlines. As the field of GM continues to evolve and related software develops rapidly, the limitations associated with morphological analysis of fish are expected to decrease. Interoperable data formats and analytical methods may facilitate the sharing of morphological data and help resolve related scientific problems. The convenience of this protocol allows for fast and effective morphological analysis. Furthermore, this detailed protocol could be adapted to assess image-based differences across a broader range of species or to analyze morphological data of the same species from different origins. Key features • This protocol provides a comprehensive set of commonly used GM-analyzing methods and visualizing skills plus supporting information to help assess the appropriate analysis method • By incorporating both landmarks and outlines, this protocol facilitates a thorough analysis of two-dimensional shape variation in fish, covering a wide range of morphological features • The simplified workflow and detailed procedures make it accessible for non-experienced users to successfully complete the analysis while also providing valuable insights for experienced users Graphical overview <b>Workflow for conducting geometric morphometrics analysis on fish.</b> The steps include image acquisition as data sources, digitization of fish morphology using landmark-based methods, analysis of shape variation characteristics, and visualization of the results in relation to biological interpretation. Largemouth bass (<i>Micropterus salmoides</i>) is used as an example in the schematic representation.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11540051/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607609","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Chromogranin B Purification for Condensate Formation and Client Partitioning Assays In Vitro.","authors":"Anup Parchure, Julia Von Blume","doi":"10.21769/BioProtoc.5095","DOIUrl":"https://doi.org/10.21769/BioProtoc.5095","url":null,"abstract":"<p><p>Chromogranin B and other members of the granin protein family form condensates that recruit clients like proinsulin. The condensation in the lumen of <i>trans</i>-Golgi network (TGN) is critical for the biogenesis of secretory granules. Here, we describe a protocol to purify the tagged version of chromogranin B close to its native form at the TGN, which can then be utilized for microscopy-based assays to monitor condensate formation in vitro and client partitioning depending on the material properties of chromogranin B assemblies. Key features • First instance of purification of full-length and tagged version of members of the chromogranin family of proteins. • Allows purification of proteins with post-translational modifications that are acquired en route in the secretory pathway, thus closely resembling their native form at the TGN.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11540053/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607564","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Metabolic RNA Labeling and Translating Ribosome Affinity Purification for Measurement of Nascent RNA Translation.","authors":"Hirotatsu Imai, Akio Yamashita","doi":"10.21769/BioProtoc.5091","DOIUrl":"https://doi.org/10.21769/BioProtoc.5091","url":null,"abstract":"<p><p>Regulation of gene expression in response to various biological processes, including extracellular stimulation and environmental adaptation, requires nascent mRNA synthesis and translation. Simultaneous analysis of the coordinated regulation of dynamic mRNA synthesis and translation using the same experiment remains a major challenge in the field. Here, we describe a step-by-step protocol for the simultaneous measurement of transcription of nascent mRNA and its translation at the gene level during the acute unfolded protein response (UPR) in HEK293 cells by combining 4-thiouridine metabolic mRNA labeling with translational ribosome affinity purification (TRAP) using a monoclonal antibody against evolutionarily conserved ribosomal P-stalk proteins (P-TRAP). Since P-TRAP captures full-length RNAs bound to ribosomes, it is compatible with 3' mRNA-seq, which analyzes the uridine-rich 3' UTRs of polyadenylated RNAs, allowing robust quantification of T>C conversions. Our nascent P-TRAP (nP-TRAP) method, in which P-TRAP is combined with metabolic mRNA labeling, can serve as a simple and powerful tool to analyze the coordinated regulation of transcription and translation of individual genes in cultured cells. Key features • Simple and retriable analysis of nascent mRNA synthesis and its translation in cultured cells • Combination of 4-thiouridine metabolic RNA labeling with translating ribosome affinity purification (TRAP) • Ribosomal P-stalk-mediated TRAP (P-TRAP) allows single-step and efficient purification of non-tagged ribosomes and translated mRNAs.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11540052/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607943","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}