Cold Spring Harbor protocols最新文献_第10页

Manipulating the Maize (Zea mays) Microbiome. 操纵玉米（Zea mays）微生物组。

Cold Spring Harbor protocols Pub Date : 2024-09-05 DOI: 10.1101/pdb.prot108584

Sierra S Raglin, Alonso Favela, Daniel Laspisa, Jason G Wallace

{"title":"Manipulating the Maize (Zea mays) Microbiome.","authors":"Sierra S Raglin, Alonso Favela, Daniel Laspisa, Jason G Wallace","doi":"10.1101/pdb.prot108584","DOIUrl":"https://doi.org/10.1101/pdb.prot108584","url":null,"abstract":"Maize (Zea mays) is a multifaceted cereal grass used globally for nutrition, animal feed, food processing, and biofuels, and a model system in genetics research. Studying the maize microbiome sometimes requires its manipulation to identify the contributions of specific taxa and ecological traits (i.e., diversity, richness, network structure) to maize growth and physiology. Due to regulatory constraints on applying engineered microorganisms in field settings, greenhouse-based experimentation is often the first step for understanding the contribution of root-associated microbiota-whether natural or engineered-to plant phenotypes. In this protocol, we describe methods to inoculate maize with a specific microbiome as a tool for understanding the microbiota's influence on its host plant. The protocol involves removal of the native seed microbiome followed by inoculation of new microorganisms; separate protocols are provided for inoculations from pure culture, from soil slurry, or by mixing in live soil. These protocols cover the most common methods for manipulating the maize microbiome in soil-grown plants in the greenhouse. The methods outlined will ultimately result in rhizosphere microbial assemblages with varying degrees of microbial diversity, ranging from low diversity (individual strain and synthetic community [SynCom] inoculation) to high diversity (percent live inoculation), with the slurry inoculation method representing an \"intermediate diversity\" treatment.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142139527","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

Sampling Root-Associated Microbiome Communities of Maize (Zea mays). 玉米（Zea mays）根相关微生物群落采样。

Cold Spring Harbor protocols Pub Date : 2024-09-05 DOI: 10.1101/pdb.prot108580

Alonso Favela, Sierra Raglin, Jason G Wallace

引用次数: 0

Four Strategies for Whole-Genome Annotation of Transposable Elements and Repeats in Maize. 玉米转座元件和重复序列全基因组注释的四种策略

Cold Spring Harbor protocols Pub Date : 2024-09-05 DOI: 10.1101/pdb.prot108578

Christopher W Benson, Pedro Heringer, Shujun Ou

{"title":"Four Strategies for Whole-Genome Annotation of Transposable Elements and Repeats in Maize.","authors":"Christopher W Benson, Pedro Heringer, Shujun Ou","doi":"10.1101/pdb.prot108578","DOIUrl":"https://doi.org/10.1101/pdb.prot108578","url":null,"abstract":"Transposable elements (TEs) and tandem repeat arrays are ubiquitous components of genomes across all domains of life. Many types of repetitive DNA do not appear to encode for functional proteins, and those that do, typically only code for enzymes involved in their own replication. Nevertheless, repetitive DNA sequences can significantly alter genome structure, and can have a profound impact on an organism's biology at both the molecular and organismal levels. Advances in long-read sequencing technology have enabled the resolution of previously collapsed contigs and scaffolds that are rich in repeats, which has made the accurate annotation of TEs and other repetitive sequences a crucial early step in genome analysis. Here, we provide a detailed tutorial for streamlined annotation of TEs and repeats in the genome of the model plant Zea mays (maize). Maize is ideally suited to illustrate these procedures due to its repeat-rich genome and the volume of publicly available and high-quality genomic resources. We outline four possible approaches for TE and repeat annotation, each aimed at accommodating a different set of scientific interests. Additionally, we demonstrate how to evaluate annotation quality, and provide scripts to help graphically depict TE and repeat landscapes. Although the protocol is tailored for maize, we also offer pointers for researchers working on other systems throughout and expect that these procedures will be broadly applicable to any eukaryotic genome.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142139526","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

Whole-Brain Electrophysiology and Calcium Imaging in Drosophila during Sleep and Wake. 果蝇睡眠和觉醒时的全脑电生理学和钙成像。

Cold Spring Harbor protocols Pub Date : 2024-09-03 DOI: 10.1101/pdb.top108394

Matthew Van De Poll, Lucy Tainton-Heap, Michael Troup, Bruno van Swinderen

{"title":"Whole-Brain Electrophysiology and Calcium Imaging in Drosophila during Sleep and Wake.","authors":"Matthew Van De Poll, Lucy Tainton-Heap, Michael Troup, Bruno van Swinderen","doi":"10.1101/pdb.top108394","DOIUrl":"10.1101/pdb.top108394","url":null,"abstract":"Sleep is likely a whole-brain phenomenon, with most of the brain probably benefiting from this state of decreased arousal. Recent advances in our understanding of some potential sleep functions, such as metabolite clearance and synaptic homeostasis, make it evident why the whole brain is likely impacted by sleep: All neurons have synapses, and all neurons produce waste metabolites. Sleep experiments in the fly Drosophila melanogaster suggest that diverse sleep functions appear to be conserved across all animals. Studies of brain activity during sleep in humans typically involve multidimensional data sets, such as those acquired by electroencephalograms (EEGs) or functional magnetic resonance imaging (fMRI), and these whole-brain read-outs often reveal important qualities of different sleep stages, such as changes in frequency dynamics or connectivity. Recently, various techniques have been developed that allow for the recording of neural activity simultaneously across multiple regions of the fly brain. These whole-brain-recording approaches will be important for better understanding sleep physiology and function, as they provide a more comprehensive view of neural dynamics during sleep and wake in a relevant model system. Here, we present a brief summary of some of the findings derived from sleep activity recording studies in sleeping Drosophila flies and discuss the value of electrophysiological versus calcium imaging techniques. Although these involve very different preparations, they both highlight the value of multidimensional data for studying sleep in this model system, like the use of both EEG and fMRI in humans.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top108394"},"PeriodicalIF":0.0,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139039622","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

Validating Single-Guide RNA for Aedes aegypti Gene Editing. 埃及伊蚊基因编辑的单引导RNA验证。

Cold Spring Harbor protocols Pub Date : 2024-09-03 DOI: 10.1101/pdb.prot108340

Ivan Hok Yin Lo, Benjamin J Matthews

引用次数: 0

Design and Validation of Guide RNAs for CRISPR-Cas9 Genome Editing in Mosquitoes. 蚊子CRISPR-Cas9基因组编辑指南RNA的设计和验证。

Cold Spring Harbor protocols Pub Date : 2024-09-03 DOI: 10.1101/pdb.top107688

Ivan Hok Yin Lo, Benjamin J Matthews

引用次数: 0

Taste Sensory Responses in Mosquitoes. 蚊子的味觉反应

Cold Spring Harbor protocols Pub Date : 2024-09-03 DOI: 10.1101/pdb.top107680

Adriana Medina Lomelí, Anupama Arun Dahanukar

{"title":"Taste Sensory Responses in Mosquitoes.","authors":"Adriana Medina Lomelí, Anupama Arun Dahanukar","doi":"10.1101/pdb.top107680","DOIUrl":"10.1101/pdb.top107680","url":null,"abstract":"Analysis of taste sensory responses has been a powerful approach for understanding principles of taste detection and coding. The shared architecture of external taste sensing units, called sensilla, in insects opened up the study of tastant-evoked responses in any model of choice using a single-sensillum tip recording method that was developed in the mid-1900s. Early studies in blowflies were instrumental for identifying distinct taste neurons based on their responses to specific categories of chemicals. Broader system-wide analyses of whole organs have since been performed in the genetic model insect Drosophila melanogaster, revealing principles of stereotypical organization and function that appear to be evolutionarily conserved. Although limited in scope, investigations of taste sensory responses in mosquitoes showcase conservation in sensillar organization, as well as in groupings of functionally distinct taste neurons in each sensillum. The field is now poised for more thorough dissections of mosquito taste function, which should be of immense value in understanding close-range chemosensory interactions of mosquitoes with their hosts and environment. Here, we provide an introduction to the basic structure of a taste sensillum and functional analysis of the chemosensory neurons within it.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top107680"},"PeriodicalIF":0.0,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10433980","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

Single-Sensillum Taste Recordings in Mosquitoes. 蚊子的单感受器味觉记录

Cold Spring Harbor protocols Pub Date : 2024-09-03 DOI: 10.1101/pdb.prot108195

Adriana Medina Lomelí, Anupama Arun Dahanukar

{"title":"Single-Sensillum Taste Recordings in Mosquitoes.","authors":"Adriana Medina Lomelí, Anupama Arun Dahanukar","doi":"10.1101/pdb.prot108195","DOIUrl":"10.1101/pdb.prot108195","url":null,"abstract":"In insects, gustatory neurons sense chemicals upon contact and directly inform many behaviors critical for survival and reproduction, including biting, feeding, mating, and egg laying. However, the taste sensory system is underexplored in many anthropophilic disease vectors such as mosquitoes, which acquire and transmit human pathogens during blood feeding from human hosts. This results in a big gap in vector biology-the study of organisms that spread disease by transmitting pathogens-because insect vectors closely interact with humans while selecting suitable individuals and appropriate bite sites for blood meals. Human sweat and skin-associated chemistries are rich in nonvolatile compounds that can be sensed by the mosquito's taste system when she lands on the skin. Taste sensory units, called sensilla, are distributed in many organs across the mosquito body, including the mouthparts, legs, and ovipositors (female-specific structures used to lay eggs). Each sensillum is innervated by as many as five taste neurons, which allow detection and discrimination between various tastants such as water, sugars, salts, amino acids, and plant-derived compounds that taste bitter to humans. Single-sensillum recordings provide a robust way to survey taste responsiveness of individual sensilla to various diagnostic and ecologically relevant chemicals. Such analyses are of immense value for understanding links between mosquito taste responses and behaviors to specific chemical cues and can provide insights into why mosquitoes prefer certain hosts. The results can also aid development of strategies to disrupt close-range mosquito-human interactions to control disease transmission. Here we describe a protocol that is curated for electrophysiological recordings from taste sensilla in mosquitoes and sure to yield exciting results for the field.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108195"},"PeriodicalIF":0.0,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10433984","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

Whole-Brain Electrophysiology in Drosophila during Sleep and Wake. 果蝇睡眠和觉醒时的全脑电生理学

Cold Spring Harbor protocols Pub Date : 2024-09-03 DOI: 10.1101/pdb.prot108418

Matthew Van De Poll, Bruno van Swinderen

{"title":"Whole-Brain Electrophysiology in Drosophila during Sleep and Wake.","authors":"Matthew Van De Poll, Bruno van Swinderen","doi":"10.1101/pdb.prot108418","DOIUrl":"10.1101/pdb.prot108418","url":null,"abstract":"Sleep studies in Drosophila melanogaster rely mostly on behavioral read-outs to support molecular or circuit-level investigations in this model. Electrophysiology can provide an additional level of understanding in these studies to, for example, investigate changes in brain activity associated with sleep manipulations. In this protocol, we describe a procedure for performing multichannel local field potential (LFP) recordings in the fruit fly, with a flexible system that can be adapted to different experimental paradigms and situations. The approach uses electrodes containing multiple recording sites (16), allowing the acquisition of large amounts of neuronal activity data from a transect through the brain while flies are still able to sleep. The approach starts by tethering the fly, followed by positioning it on an air-supported ball. A multichannel silicon probe is then inserted laterally into the fly brain via one eye, allowing for recording of electrical signals from the retina through to the central brain. These recordings can be acquired under spontaneous conditions or in the presence of visual stimuli, and the minimal surgery promotes long-term recordings (e.g., overnight). Sleep and wake can be tracked using infrared cameras, which allow for the measurement of locomotive activity as well as microbehaviors such as proboscis extensions during sleep. The protocol has been optimized to promote subject survivability, which is an important factor when performing long-term (∼16-h) recordings. The approach described here uses specific recording probes, data acquisition devices, and analysis tools. Although it is expected that some of these items might need to be adapted to the equipment available in different laboratories, the overall aim is to provide an overview on how to record electrical activity across the brain of behaving (and sleeping) flies using this kind of approach and technology.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108418"},"PeriodicalIF":0.0,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139039623","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

Whole-Brain Calcium Imaging in Drosophila during Sleep and Wake. 果蝇睡眠和觉醒时的全脑钙成像

Cold Spring Harbor protocols Pub Date : 2024-09-03 DOI: 10.1101/pdb.prot108419

Lucy Tainton-Heap, Michael Troup, Matthew Van De Poll, Bruno van Swinderen

{"title":"Whole-Brain Calcium Imaging in Drosophila during Sleep and Wake.","authors":"Lucy Tainton-Heap, Michael Troup, Matthew Van De Poll, Bruno van Swinderen","doi":"10.1101/pdb.prot108419","DOIUrl":"10.1101/pdb.prot108419","url":null,"abstract":"Genetically encoded calcium indicators (GECIs) allow for the noninvasive evaluation of neuronal activity in vivo, and imaging GECIs in Drosophila has become commonplace for understanding neural functions and connectivity in this system. GECIs can also be used as read-outs for studying sleep in this model organism. Here, we describe a methodology for tracking the activity of neurons in the fly brain using a two-photon (2p) microscopy system. This method can be adapted to perform functional studies of neural activity in Drosophila under both spontaneous and evoked conditions, as well as during spontaneous or induced sleep. We first describe a tethering and surgical procedure that allows survival under the microscopy conditions required for long-term recordings. We then outline the steps and reagents required for optogenetic activation of sleep-promoting neurons while simultaneously recording neural activity from the fly brain. We also describe the procedure for recording from two different locations-namely, the top of the head (e.g., to record mushroom body calyx activity) or the back of the head (e.g., to record central complex activity). We also provide different strategies for recording from GECIs confined to the cell body versus the entire neuron. Finally, we describe the steps required for analyzing the multidimensional data that can be acquired. In all, this protocol shows how to perform calcium imaging experiments in tethered flies, with a focus on acquiring spontaneous and induced sleep data.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108419"},"PeriodicalIF":0.0,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139039621","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