Bio-protocol最新文献

{"title":"Live Imaging of the Shoot Apical Meristem of Intact, Soil-Grown, Flowering <i>Arabidopsis</i> Plants.","authors":"Gabriele Bradamante","doi":"10.21769/BioProtoc.5015","DOIUrl":"10.21769/BioProtoc.5015","url":null,"abstract":"<p><p>All aerial organs in plants originate from the shoot apical meristem, a specialized tissue at the tip of a plant, enclosing a few stem cells. Understanding developmental dynamics within this tissue in relation to internal and external stimuli is of crucial importance. Imaging the meristem at the cellular level beyond very early stages requires the apex to be detached from the plant body, a procedure that does not allow studies in living, intact plants over longer periods. This protocol describes a new confocal microscopy method with the potential to image the shoot apical meristem of an intact, soil-grown, flowering Arabidopsis plant over several days. The setup opens new avenues to study apical stem cells, their interconnection with the whole plant, and their responses to environmental stimuli. Key features • Novel dissection and imaging method of the shoot apical meristem of <i>Arabidopsis</i>. • Procedure performed with intact, soil-grown, flowering plants. • Possibility of long-term live imaging of the shoot apical meristem. • Protocol can be adapted to different plant species.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211079/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141473543","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":"A Single-step Generation of AlissAID-based Conditional Knockdown Strains Using Nanobody that Targets GFP or mCherry in Budding Yeast.","authors":"Yoshitaka Ogawa, Taisei P Ueda, Keisuke Obara, Kohei Nishimura, Takumi Kamura","doi":"10.21769/BioProtoc.5019","DOIUrl":"10.21769/BioProtoc.5019","url":null,"abstract":"<p><p>The Auxin-inducible degron (AID) system is a genetic tool that induces rapid target protein depletion in an auxin-dependent manner. Recently, two advanced AID systems-the super-sensitive AID and AID 2-were developed using an improved pair of synthetic auxins and mutated TIR1 proteins. In these AID systems, a nanomolar concentration of synthetic auxins is sufficient as a degradation inducer for target proteins. However, despite these advancements, AID systems still require the fusion of an AID tag to the target protein for degradation, potentially affecting its function and stability. To address this limitation, we developed an affinity linker-based super-sensitive AID (AlissAID) system using a single peptide antibody known as a nanobody. In this system, the degradation of GFP- or mCherry-tagged target proteins is induced in a synthetic auxin (5-Ad-IAA)-dependent manner. Here, we introduce a simple method for generating AlissAID strains targeting GFP or mCherry fusion proteins in budding yeasts. Key features • AlissAID system enables efficient degradation of the GFP or mCherry fusion proteins in a 5-Ad-IAA-depending manner. • Transforming the pAlissAID plasmids into strains with GFP- or mCherry- tagged proteins.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211076/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141473539","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":"Transfection of <i>Babesia duncani</i>: A Genetic Toolbox of this Pathogen to Advance <i>Babesia</i> Biology.","authors":"Sen Wang, Jianyu Wang, Dongfang Li, Fangwei Chen, Wanxin Luo, Junlong Zhao, Lan He","doi":"10.21769/BioProtoc.5016","DOIUrl":"10.21769/BioProtoc.5016","url":null,"abstract":"<p><p>Human babesiosis is a tick-borne disease caused by <i>Babesia</i> pathogens. The disease, which presents with malaria-like symptoms, can be life-threatening, especially in individuals with weakened immune systems and the elderly. The worldwide prevalence of human babesiosis has been gradually rising, prompting alarm among public health experts. In other pathogens, genetic techniques have proven to be valuable tools for conducting functional studies to understand the importance of specific genes in development and pathogenesis as well as to validate novel cellular targets for drug discovery. Genetic manipulation methods have been established for several non-human <i>Babesia</i> and <i>Theileria</i> species and, more recently, have begun to be developed for human Babesia parasites. We have previously reported the development of a method for genetic manipulation of the human pathogen <i>Babesia duncani</i>. This method is based on positive selection using the hDHFR gene as a selectable marker, whose expression is regulated by the ef-1aB promoter, along with homology regions that facilitate integration into the gene of interest through homologous recombination. Herein, we provide a detailed description of the steps needed to implement this strategy in <i>B. duncani</i> to study gene function. It is anticipated that the implementation of this method will significantly improve our understanding of babesiosis and facilitate the development of novel and more effective therapeutic strategies for the treatment of human babesiosis. Key features This protocol provides an effective means of transfection of <i>B. duncani</i>, enabling genetic manipulation and editing to gain further insights into its biology and pathogenesis. The protocol outlined here for the electroporation of <i>B. duncani</i> represents an advancement over previous methods used for <i>B. bovis</i> [1]. Improvements include higher volume of culture used during the electroporation step and an enhancement in the number of electroporation pulses. These modifications likely enhance the efficiency of gene editing in <i>B. duncani</i>, allowing for quicker and more effective selection of transgenic parasites.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211078/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141473545","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":"A Flow Cytometry-Based Method for Assessing CAR Cell Binding Kinetics Using Stable CAR Jurkat Cells.","authors":"Alex Shepherd, Bigitha Bennychen, Zafer Ahmed, Risini D Weeratna, Scott McComb","doi":"10.21769/BioProtoc.5021","DOIUrl":"10.21769/BioProtoc.5021","url":null,"abstract":"<p><p>Chimeric antigen receptors (CARs) are synthetic fusion proteins that can reprogram immune cells to target specific antigens. CAR-expressing T cells have emerged as an effective treatment method for hematological cancers; despite this success, the mechanisms and structural properties that govern CAR responses are not fully understood. Here, we provide a simple assay to assess cellular avidity using a standard flow cytometer. This assay measures the interaction kinetics of CAR-expressing T cells and targets antigen-expressing target cells. By co-culturing stably transfected CAR Jurkat cells with target positive and negative cells for short periods of time in a varying effector-target gradient, we were able to observe the formation of CAR-target cell doublets, providing a readout of actively bound cells. When using the optimized protocol reported here, we observed unique cellular binding curves that varied between CAR constructs with differing antigen binding domains. The cellular binding kinetics of unique CARs remained consistent, were dependent on specific target antigen expression, and required active biological signaling. While existing literature is not clear at this time whether higher or lower CAR cell binding is beneficial to CAR therapeutic activity, the application of this simplified protocol for assessing CAR binding could lead to a better understanding of the proximal signaling events that regulate CAR functionality. Key features • Determines CAR receptor cellular interaction kinetics using a Jurkat cell model. • Can be used for a wide variety of CAR target antigens, including both hematological and solid tumor targets. • Experiments can be performed in under two hours with no staining using a standard flow cytometer. • Requires stable CAR Jurkat cells and target cells with stable fluorescent marker expression for optimal results.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211074/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141473579","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":"Direct RNA Sequencing of Foot-and-mouth Disease Virus Genome Using a Flongle on MinION.","authors":"Lizhe Xu, Amy Berninger, Steven M Lakin, Vivian O'Donnell, Jim L Pierce, Steven J Pauszek, Roger W Barrette, Bonto Faburay","doi":"10.21769/BioProtoc.5017","DOIUrl":"10.21769/BioProtoc.5017","url":null,"abstract":"<p><p><i>Foot-and-mouth disease (FMD) is a severe and extremely contagious viral disease of cloven-hoofed domestic and wild animals, which leads to serious economic losses to the livestock industry globally. FMD is caused by the FMD virus (FMDV), a positive-strand RNA virus that belongs to the genus</i> Aphthovirus, within the family Picornaviridae. Early detection and characterization of FMDV strains are key factors to control new outbreaks and prevent the spread of the disease. Here, we describe a direct RNA sequencing method using Oxford Nanopore Technology (ONT) Flongle flow cells on MinION Mk1C (or GridION) to characterize FMDV. This is a rapid, low cost, and easily deployed point of care (POC) method for a near real-time characterization of FMDV in endemic areas or outbreak investigation sites. Key features • Saves ~35 min of the original protocol time by omitting the reverse transcription step and lowers the costs of reagents and consumables. • Replaces the GridION flow cell from the original protocol with the Flongle, which saves ~90% on the flow cell cost. • Combines the NGS benchwork with a modified version of our African swine fever virus (ASFV) fast analysis pipeline to achieve FMDV characterization within minutes. Graphical overview Schematic of direct RNA sequencing of foot-and-mouth disease virus (FMDV) process, which takes ~50 min from extracted RNA to final loading, modified from the ONT SQK-RNA002 protocol (Version: DRS_9080_v2_revO_14Aug2019).</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211080/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141473540","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":"Measuring Sleep and Activity Patterns in Adult Zebrafish.","authors":"Fusun Doldur-Balli, Amber J Zimmerman, Christoph Seiler, Olivia Veatch, Allan I Pack","doi":"10.21769/BioProtoc.5014","DOIUrl":"10.21769/BioProtoc.5014","url":null,"abstract":"<p><p>Sleep is an essential behavior that is still poorly understood. Sleep abnormalities accompany a variety of psychiatric and neurological disorders, and sleep can serve as a modifiable behavior in the treatment of these disorders. Zebrafish (<i>Danio rerio</i>) has proven to be a powerful model organism to study sleep and the interplay between sleep and these disorders due to the high conservation of the neuro-modulatory mechanisms that control sleep and wake states between zebrafish and humans. The zebrafish is a diurnal vertebrate with a relatively simple nervous system compared to mammalian models, exhibiting conservation of sleep ontogeny across different life stages. Zebrafish larvae are an established high-throughput model to assess sleep phenotypes and the biological underpinnings of sleep disturbances. To date, sleep measurement in juvenile and adult zebrafish has not been performed in a standardized and reproducible manner because of the relatively low-throughput nature in relation to their larval counterparts. This has left a gap in understanding sleep across later stages of life that are relevant to many psychiatric and neurodegenerative disorders. Several research groups have used homemade systems to address this gap. Here, we report employing commercially available equipment to track activity and sleep/wake patterns in juvenile and adult zebrafish. The equipment allows researchers to perform automated behavior assays in an isolated environment with light/dark and temperature control for multiple days. We first explain the experimental procedure to track the sleep and activity of adult zebrafish and then validate the protocol by measuring the effects of melatonin and DMSO administration. Key features • Allows an isolated and controllable environment to carry out activity and sleep assays in juvenile and adult zebrafish. • Measures activity of zebrafish in life stages later than early development, which requires feeding animals during the assay. • Requires use of a commercially available equipment system and six tanks. • The activity of zebrafish can be tracked for five days including an acclimation step.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211082/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141473544","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":"Isolation and Characterization of Extracellular Vesicles Derived from Ex Vivo Culture of Visceral Adipose Tissue.","authors":"Ankita Arora, Vinit Sharma, Rajesh Gupta, Anjali Aggarwal","doi":"10.21769/BioProtoc.5011","DOIUrl":"10.21769/BioProtoc.5011","url":null,"abstract":"<p><p>Extracellular vesicles (EVs) are a heterogeneous group of nanoparticles possessing a lipid bilayer membrane that plays a significant role in intercellular communication by transferring their cargoes, consisting of peptides, proteins, fatty acids, DNA, and RNA, to receiver cells. Isolation of EVs is cumbersome and time-consuming due to their nano size and the co-isolation of small molecules along with EVs. This is why current protocols for the isolation of EVs are unable to provide high purity. So far, studies have focused on EVs derived from cell supernatants or body fluids but are associated with a number of limitations. Cell lines with a high passage number cannot be considered as representative of the original cell type, and EVs isolated from those can present distinct properties and characteristics. Additionally, cultured cells only have a single cell type and do not possess any cellular interactions with other types of cells, which normally exist in the tissue microenvironment. Therefore, studies involving the direct EVs isolation from whole tissues can provide a better understanding of intercellular communication in vivo. This underscores the critical need to standardize and optimize protocols for isolating and characterizing EVs from tissues. We have developed a differential centrifugation-based technique to isolate and characterize EVs from whole adipose tissue, which can be potentially applied to other types of tissues. This may help us to better understand the role of EVs in the tissue microenvironment in both diseased and normal conditions. Key features • Isolation of tissue-derived extracellular vesicles from ex vivo culture of visceral adipose tissue or any whole tissue. • Microscopic visualization of extracellular vesicles' morphology without dehydration steps, with minimum effect on their shape. • Flow cytometry approach to characterize the extracellular vesicles using specific protein markers, as an alternative to the time-consuming western blot.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11166539/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141319329","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":"CRISPR-Cas9 Protocol for Efficient Gene Knockout and Transgene-free Plant Generation.","authors":"Enzo A Perk, Ana M Laxalt, Ignacio Cerrudo","doi":"10.21769/BioProtoc.5012","DOIUrl":"10.21769/BioProtoc.5012","url":null,"abstract":"<p><p>Gene editing technologies have revolutionized plant molecular biology, providing powerful tools for precise gene manipulation for understanding function and enhancing or modifying agronomically relevant traits. Among these technologies, the CRISPR-Cas9 system has emerged as a versatile and widely accepted strategy for targeted gene manipulation. This protocol provides detailed, step-by-step instructions for implementing CRISPR-Cas9 genome editing in tomato plants, with a specific focus in generating knockout lines for a target gene. For that, the guide RNA should preferentially be designed within the first exon downstream and closer to the start codon. The edited plants obtained are free of transgene cassette for expression of the CRISPR-Cas9 machinery. Key features • Two sgRNAs employed. • Takes 6-12 months to have an edited transgene-free plant. • Setup in tomato.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11166535/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141319318","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":"A Multi-Color Immunofluorescence Assay to Distinguish Intracellular From External <i>Leishmania</i> Parasites.","authors":"Arani Datta, Umaru Barrie, Dawn M Wetzel","doi":"10.21769/BioProtoc.5009","DOIUrl":"10.21769/BioProtoc.5009","url":null,"abstract":"<p><p>Leishmaniasis, a neglected tropical disease, is caused by the intracellular protozoan parasite <i>Leishmania</i>. Upon its transmission through a sandfly bite, <i>Leishmania</i> binds and enters host phagocytic cells, ultimately resulting in a cutaneous or visceral form of the disease. The limited therapeutics available for leishmaniasis, in combination with this parasite's techniques to evade the host immune system, call for exploring various methods to target this infection. To this end, our laboratory has been characterizing how <i>Leishmania</i> is internalized by phagocytic cells through the activation of multiple host cell signaling pathways. This protocol, which we use routinely for our experiments, delineates how to infect mammalian macrophages with either promastigote or amastigote forms of the <i>Leishmania</i> parasite. Subsequently, the number of intracellular parasites, external parasites, and macrophages can be quantified using immunofluorescence microscopy and semi-automated analysis protocols. Studying the pathways that underlie <i>Leishmania</i> uptake by phagocytes will not only improve our understanding of these host-pathogen interactions but may also provide a foundation for discovering additional treatments for leishmaniasis. Key features • This protocol visualizes and quantifies multiple intracellular forms of <i>Leishmania</i>. • It offers flexibility at various points for researchers to introduce modifications according to their study needs.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11166538/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141319316","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":"Fast, Easy, and Comprehensive Techniques for Microscopic Observations of Fungal and Oomycete Organisms Inside the Roots of Herbaceous and Woody Plants.","authors":"Tomáš Toma, Ján Kováč, Jaroslav Ďurkovič","doi":"10.21769/BioProtoc.5013","DOIUrl":"10.21769/BioProtoc.5013","url":null,"abstract":"<p><p>The roots of herbaceous and woody plants growing in soil are complex structures that are affected by both natural and artificial fungal colonization to various extents. To obtain comprehensive information about the overall distribution of fungi or oomycetes inside a plant root system, rapid, effective, and reliable screening methods are required. To observe both fine roots, i.e., a common site for penetration of fungi and oomycetes, and mature roots, different techniques are required to overcome visual barriers, such as root browning or tissue thickening. In our protocol, we propose using fast, cost-effective, and non-harmful methods to localize fungal or oomycete structures inside plant roots. Root staining with a fluorescent dye provides a quick initial indication of the presence of fungal structures on the root surfaces. The protocol is followed by clearing and staining steps, resulting in a deeper insight into the root tissue positioning, abundance, and characteristic morphological/reproductive features of fungal or oomycete organisms. If required, the stained samples can be prepared by using freeze-drying for further observations, including advanced microscopic techniques. Key features • The protocol enhances tissue-clearing techniques employing KOH or NaOH and is applicable to a broad range of roots from different plant species. • Hydroxides are mixed with hydrogen peroxide to obtain an efficient bleaching solution, which effectively clears roots without causing significant tissue damage. • The protocol could also be used for staining of fungi or oomycetes localized both on the root surface or inside the root tissues. • Simple combination of non-fluorescent methyl blue and fluorescent solophenyl flavine dyes allows the observation of fungal organisms in both brightfield and fluorescence microscopy.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11166536/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141319328","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}