Cold Spring Harbor protocols最新文献_第9页

Generation of a Phage Display Chicken Single-Chain Variable Fragment Library. 噬菌体展示鸡单链可变片段库的生成。

Cold Spring Harbor protocols Pub Date : 2024-08-16 DOI: 10.1101/pdb.prot108213

Hyunji Yang, Jisu Chae, Hyori Kim, Jinsung Noh, Junho Chung

引用次数: 0

Compositional Analysis of Cutin in Maize Leaves. 玉米叶中 Cutin 的成分分析

Cold Spring Harbor protocols Pub Date : 2024-08-12 DOI: 10.1101/pdb.prot108434

Richard Bourgault, Isabel Molina

{"title":"Compositional Analysis of Cutin in Maize Leaves.","authors":"Richard Bourgault, Isabel Molina","doi":"10.1101/pdb.prot108434","DOIUrl":"https://doi.org/10.1101/pdb.prot108434","url":null,"abstract":"The cuticle is a lipid barrier that covers the air-exposed surfaces of plants. It consists of waxes and cutin, a cell wall-attached lipid polyester of oxygenated fatty acids and glycerol. Unlike waxes, cutin is insoluble in organic solvents, and its composition is typically studied by chemical depolymerization followed by monomer analysis by gas chromatography (GC). Here, we describe a method for the chemical depolymerization of cutin in maize leaves and subsequent compositional analysis of the constituent lipid monomers. The method has been adapted from protocols for cutin analysis developed for Arabidopsis, by both optimizing the amount of leaf tissue used and including a data analysis process specific to the monomers present in maize cutin. The approach uses base-catalyzed transmethylation, which produces fatty acid methyl esters, and silylation, which gives trimethylsilyl ether derivatives of hydroxyl groups for gas chromatographic analysis. For monomer identification, a few representative samples are first analyzed by GC-mass spectrometry (GC-MS). This is then followed by analysis of all replicates by gas chromatography coupled to a flame ionization detector (GC-FID) for monomer quantification, because the flame ionization detector provides a linear response over a wide mass range, is relatively simple to operate, and is more cost-effective to maintain compared to mass spectrometry detectors. Although the protocol bypasses time-consuming cuticle isolation steps by using whole-leaf samples, this means that a fraction of the compounds in the chromatographic profiles do not derive from cutin. Accordingly, we discuss some considerations for the interpretation of the resulting depolymerization products. Our protocol offers specific guidance on preparing maize leaf samples, ensuring reproducible results, and enabling the detection of subtle variations in cutin monomer composition among plant genotypes or developmental stages.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141970789","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}

引用次数: 0

Assaying Nociception Behaviors in Drosophila Larvae During Parasitoid Wasp Attacks. 测定寄生蜂攻击果蝇幼虫时的痛觉行为

Cold Spring Harbor protocols Pub Date : 2024-08-02 DOI: 10.1101/pdb.prot108129

Stephanie E Mauthner, Lydia J Borjon, W Daniel Tracey

{"title":"Assaying Nociception Behaviors in Drosophila Larvae During Parasitoid Wasp Attacks.","authors":"Stephanie E Mauthner, Lydia J Borjon, W Daniel Tracey","doi":"10.1101/pdb.prot108129","DOIUrl":"10.1101/pdb.prot108129","url":null,"abstract":"Nociception in fruit fly (Drosophila melanogaster) larvae is characterized by a stereotyped escape behavior. When a larva encounters a noxious (potentially harmful) stimulus, it responds by curving its body into a c-shape and rolling in a corkscrew-like manner around its long-body axis. This rolling behavior may serve to quickly remove the larva from the source of the noxious stimulus, and is particularly adaptive to escape from a common natural predator of fruit fly larvae: parasitoid wasps (Leptopilina boulardi). L. boulardi completes its life cycle by using fruit fly larvae as hosts for its offspring. Female wasps sting fly larvae with an ovipositor and lay an egg within the larva. The wasp offspring hatches inside the fly larva, consumes the fly tissues during pupation, and eventually emerges from the pupal case as an adult wasp. Fruit fly larvae respond to oviposition attacks by rolling, which causes the long flexible ovipositor to be wound around the larval body like a spool. This dislodges the wasp and allows the larva to attempt to escape by crawling. Rolling behavior is triggered by the activation of sensory neurons (nociceptors) whose function can inform our understanding of the mechanisms of nociception. In this protocol, we describe a simple behavioral assay to test and measure nociceptive responses in Drosophila larvae during oviposition attacks by female parasitoid wasps. First, we discuss parasitoid wasp husbandry and culturing methods in the laboratory. We then describe how to perform the wasp nociception assay on third-instar fruit fly larvae.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141878472","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}

引用次数: 0

Mechanical Nociception Assay in Drosophila Larvae. 果蝇幼虫的机械痛觉试验

Cold Spring Harbor protocols Pub Date : 2024-08-02 DOI: 10.1101/pdb.prot108125

Stephanie E Mauthner, W Daniel Tracey

{"title":"Mechanical Nociception Assay in Drosophila Larvae.","authors":"Stephanie E Mauthner, W Daniel Tracey","doi":"10.1101/pdb.prot108125","DOIUrl":"10.1101/pdb.prot108125","url":null,"abstract":"The nervous system of animals can sense and respond to noxious stimuli, which include noxious thermal, chemical, or mechanical stimuli, through a process called nociception. Here, we describe a simple behavioral assay to measure mechanically induced nociceptive responses in Drosophila larvae. This assay tests larval mechanosensitivity to noxious force with calibrated von Frey filaments. First, we explain how to construct and calibrate the customizable von Frey filaments that can be used to deliver reproducible stimuli of a defined force or pressure. Next, we describe how to perform the mechanical nociception assay on third-instar larvae. Through comparison of the responses of genotypes of interest, this assay can be useful for investigation of molecular, cellular, and circuit mechanisms of mechanical nociception. At the molecular level, prior studies have identified the importance of sensory ion channels such as Pickpocket/Balboa, Piezo, dTRPA1, and Painless. At the cellular level, the class IV multidendritic arborizing (md-da) neurons are the main mechanical nociceptor neurons of the peripheral system, but class III and class II md-da have been found to also play a role. At the circuit level, studies have shown that mechanical nociception relies on interneurons of the abdominal ganglia that integrate inputs from these various md-da neuron classes.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141878473","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}

引用次数: 0

Optogenetic Stimulation of Nociceptive Escape Behaviors in Drosophila Larvae. 光遗传刺激果蝇幼虫的痛觉逃逸行为

Cold Spring Harbor protocols Pub Date : 2024-08-02 DOI: 10.1101/pdb.prot108128

Stephanie E Mauthner, W Daniel Tracey

{"title":"Optogenetic Stimulation of Nociceptive Escape Behaviors in Drosophila Larvae.","authors":"Stephanie E Mauthner, W Daniel Tracey","doi":"10.1101/pdb.prot108128","DOIUrl":"10.1101/pdb.prot108128","url":null,"abstract":"In animals, noxious stimuli activate a neural process called nociception. Drosophila larvae perform a rolling escape locomotion behavior in response to nociceptive sensory stimuli. Noxious mechanical, thermal, and chemical stimuli each trigger this same escape response in larvae. The polymodal sensory neurons that initiate the rolling response have been identified based on the expression patterns of genes that are known to be required for nociception responses. The synaptic output of these neurons, known as class IV multidendritic sensory neurons, is required for behavioral responses to thermal, mechanical, and chemical triggers of the rolling escape locomotion. Importantly, optogenetic stimulation of the class IV multidendritic neurons has also shown that the activation of those cells is sufficient to trigger nociceptive rolling. Optogenetics uses light-activated ion channels expressed in neurons of interest to bypass the normal physiological transduction machinery so that the cell may be activated in response to light that is applied by the investigator. This protocol describes an optogenetic technique that uses channelrhodopsin-2 (ChR2) to activate larval nociceptors and trigger nociceptive rolling. First, we explain how to set up the necessary genetic crosses and culture the larval progeny. Next, we describe how to perform the optogenetic nociception assay on third-instar larvae.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141878475","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}

引用次数: 0

Nociception in Drosophila Larvae. 果蝇幼虫的痛觉感应

Cold Spring Harbor protocols Pub Date : 2024-08-02 DOI: 10.1101/pdb.top108172

Lydia J Borjon, Stephanie E Mauthner, W Daniel Tracey

引用次数: 0

Collecting, Storing, and Hatching Aedes aegypti Eggs. 收集、储存和孵化埃及伊蚊卵。

Cold Spring Harbor protocols Pub Date : 2024-08-01 DOI: 10.1101/pdb.prot108183

Noah H Rose, John J Shepard, Diego Ayala

引用次数: 0

Structure, Biology, and Applications of Filamentous Bacteriophages. 噬丝状细菌病毒的结构、生物学和应用。

Cold Spring Harbor protocols Pub Date : 2024-08-01 DOI: 10.1101/pdb.over107754

Jasna Rakonjac, Vicki A M Gold, Rayén I León-Quezada, Catherine H Davenport

{"title":"Structure, Biology, and Applications of Filamentous Bacteriophages.","authors":"Jasna Rakonjac, Vicki A M Gold, Rayén I León-Quezada, Catherine H Davenport","doi":"10.1101/pdb.over107754","DOIUrl":"10.1101/pdb.over107754","url":null,"abstract":"The closely related Escherichia coli Ff filamentous phages (f1, fd, and M13) have taken a fantastic journey over the past 60 years, from the urban sewerage from which they were first isolated, to their use in high-end technologies in multiple fields. Their relatively small genome size, high titers, and the virions that tolerate fusion proteins make the Ffs an ideal system for phage display. Folding of the fusions in the oxidizing environment of the E. coli periplasm makes the Ff phages a platform that allows display of eukaryotic surface and secreted proteins, including antibodies. Resistance of the Ffs to a broad range of pH and detergents facilitates affinity screening in phage display, whereas the stability of the virions at ambient temperature makes them suitable for applications in material science and nanotechnology. Among filamentous phages, only the Ffs have been used in phage display technology, because of the most advanced state of knowledge about their biology and the various tools developed for E. coli as a cloning host for them. Filamentous phages have been thought to be a rather small group, infecting mostly Gram-negative bacteria. A recent discovery of more than 10 thousand diverse filamentous phages in bacteria and archaea, however, opens a fascinating prospect for novel applications. The main aim of this review is to give detailed biological and structural information to researchers embarking on phage display projects. The secondary aim is to discuss the yet-unresolved puzzles, as well as recent developments in filamentous phage biology, from a viewpoint of their impact on current and future applications.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.over107754"},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9830045","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}

引用次数: 0

Wind Tunnels and Airflow-Driven Assays: Methods for Establishing the Cues and Orientation Mechanisms That Modulate Female Mosquito Attraction to Human Hosts. 风洞和气流驱动试验：确定调节雌蚊吸引人类宿主的线索和定向机制的方法。

Cold Spring Harbor protocols Pub Date : 2024-08-01 DOI: 10.1101/pdb.over107675

Ring T Cardé

{"title":"Wind Tunnels and Airflow-Driven Assays: Methods for Establishing the Cues and Orientation Mechanisms That Modulate Female Mosquito Attraction to Human Hosts.","authors":"Ring T Cardé","doi":"10.1101/pdb.over107675","DOIUrl":"10.1101/pdb.over107675","url":null,"abstract":"Understanding how female mosquitoes find a prospective host is crucial to developing means that can interfere with this process. Many methods are available to researchers studying cues and orientation mechanisms that modulate female mosquito attraction to hosts. Behaviors that can be monitored with these assays include activation, taking flight, upwind flight along an odor plume (optomotor anemotaxis), close approach to the stimulus (including hovering), and landing. Video recording can three-dimensionally document flight tracks and can correlate overall distribution patterns and moment-to-moment movements with odor contact and the presence of nearby cues such as a visual target. Here, we introduce mosquito host-seeking behaviors and methods to study them: wind tunnels (which allow orientation in free-flight), airflow-driven assays (using either tethered mosquitoes or small assay chambers that permit flight but also often dictate walking orientation), and still-air assays (wherein in odor concentration and spatial distribution are the orientation cues). We also describe factors that affect the assays and provide assay design considerations.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.over107675"},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139402196","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}

引用次数: 0

The pComb3 Phagemid Family of Phage Display Vectors. 噬菌体展示载体 pComb3 Phagemid 系列。

Cold Spring Harbor protocols Pub Date : 2024-08-01 DOI: 10.1101/pdb.over107756

Christoph Rader

引用次数: 0