Cold Spring Harbor protocols最新文献

Optimized Methods for Applying and Assessing Heat, Drought, and Nutrient Stress of Maize Seedlings in Controlled Environment Experiments. 在受控环境实验中应用和评估玉米幼苗热、旱和养分胁迫的优化方法。

Cold Spring Harbor protocols Pub Date : 2024-11-18 DOI: 10.1101/pdb.top108467

Alejandra Quiñones, Leonardo W Lima, Katherine M Murphy, Anna L Casto, Malia A Gehan, Cory D Hirsch

{"title":"Optimized Methods for Applying and Assessing Heat, Drought, and Nutrient Stress of Maize Seedlings in Controlled Environment Experiments.","authors":"Alejandra Quiñones, Leonardo W Lima, Katherine M Murphy, Anna L Casto, Malia A Gehan, Cory D Hirsch","doi":"10.1101/pdb.top108467","DOIUrl":"10.1101/pdb.top108467","url":null,"abstract":"Maize (Zea mays), also known as corn, is an important crop that plays a crucial role in global agriculture. The economic uses of maize are numerous, including for food, feed, fiber, and fuel. It has had a significant historical importance in research as well, with important discoveries made in maize regarding plant domestication, transposons, heterosis, genomics, and epigenetics. Unfortunately, environmental stresses cause substantial yield loss to maize crops each year. Yield losses are predicted to increase in future climate scenarios, posing a threat to food security and other sectors of the global economy. Developing efficient methods to study maize abiotic stress responses is a crucial step toward a more resilient and productive agricultural system. This review describes the importance of and methods for studying the effects of heat, drought, and nutrient deficiency on early developmental stages of maize grown in controlled environments. Studying the early effects of environmental stressors in controlled environments allows researchers to work with a variety of environmental conditions with low environmental variance, which can inform future field-based research. We highlight the current knowledge of physiological responses of maize to heat, drought, and nutrient stress; remaining knowledge gaps and challenges; and information on how standardized protocols can address these issues.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459804","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

Selection of Affibody Molecules Using Phage Display. 利用噬菌体展示选择亲和体分子。

Cold Spring Harbor protocols Pub Date : 2024-11-01 DOI: 10.1101/pdb.prot108399

Linnea Charlotta Hjelm, Charles Dahlsson Leitao, Stefan Ståhl, John Löfblom, Hanna Lindberg

引用次数: 0

Engineering of Affibody Molecules. Affibody 分子工程。

Cold Spring Harbor protocols Pub Date : 2024-11-01 DOI: 10.1101/pdb.top107760

Stefan Ståhl, Hanna Lindberg, Linnea Charlotta Hjelm, John Löfblom, Charles Dahlsson Leitao

引用次数: 0

Activity Monitoring for Analysis of Sleep in Drosophila melanogaster. 通过活动监测分析黑腹果蝇的睡眠情况

Cold Spring Harbor protocols Pub Date : 2024-11-01 DOI: 10.1101/pdb.top108095

Divya Sitaraman, Christopher G Vecsey, Casey Koochagian

引用次数: 0

Selection of Affibody Molecules Using Escherichia coli Display. 利用大肠杆菌显示筛选亲和体分子。

Cold Spring Harbor protocols Pub Date : 2024-11-01 DOI: 10.1101/pdb.prot108400

Charles Dahlsson Leitao, Linnea Charlotta Hjelm, Stefan Ståhl, John Löfblom, Hanna Lindberg

{"title":"Selection of Affibody Molecules Using Escherichia coli Display.","authors":"Charles Dahlsson Leitao, Linnea Charlotta Hjelm, Stefan Ståhl, John Löfblom, Hanna Lindberg","doi":"10.1101/pdb.prot108400","DOIUrl":"10.1101/pdb.prot108400","url":null,"abstract":"Affibody molecules are small (6-kDa) affinity proteins generated by directed evolution for specific binding to various target molecules. The first step in this workflow involves the generation of an affibody library, which can then be used for selection via multiple display methods. This protocol describes selection from affibody libraries by Escherichia coli cell surface display. With this method, high-diversity libraries of 1011 can be displayed on the cell surface. The method involves two steps for selection of binders from high-diversity libraries: magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). MACS is used first to enrich the library in target-binding clones and to decrease diversity to a size that can be effectively screened and sorted in the flow cytometer in a reasonable time (typically <107 cells). The protocol is based on methodology using an AIDA-I autotransporter for display on the outer membrane, but the general procedures can also be adjusted and used for other types of autotransporters or alternative E. coli display methods.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108400"},"PeriodicalIF":0.0,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9924643","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

Selection of Affibody Molecules Using Staphylococcal Display. 利用葡萄球菌显示筛选亲和体分子。

Cold Spring Harbor protocols Pub Date : 2024-11-01 DOI: 10.1101/pdb.prot108401

John Löfblom, Linnea Charlotta Hjelm, Charles Dahlsson Leitao, Stefan Ståhl, Hanna Lindberg

{"title":"Selection of Affibody Molecules Using Staphylococcal Display.","authors":"John Löfblom, Linnea Charlotta Hjelm, Charles Dahlsson Leitao, Stefan Ståhl, Hanna Lindberg","doi":"10.1101/pdb.prot108401","DOIUrl":"10.1101/pdb.prot108401","url":null,"abstract":"Affibody molecules are small (6-kDa) affinity proteins generated by directed evolution for specific binding to various target molecules. The first step in this workflow involves the generation of an affibody library, which can then be used for biopanning using multiple display methods. This protocol describes selection from affibody libraries using display on Staphylococcus carnosus Display of affibodies on staphylococci is very efficient and straightforward because of the single cell membrane and the use of a construct with a constitutive promoter. The workflow involves display of affibody libraries on the surface of S. carnosus cells, followed by screening and selection of binders using fluorescence-activated cell sorting (FACS). The transformation of DNA libraries into S. carnosus is less efficient and more complicated than for Escherichia coli. Because of this, staphylococcal display is suitable for affinity maturation or other protein-engineering efforts that are not dependent on very high diversity, and thus magnetic-activated cell sorting (MACS) is often not required before FACS. However, MACS is an option, and MACS procedures used for E. coli can easily be adapted for use in S. carnosus if needed.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108401"},"PeriodicalIF":0.0,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9924641","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

Neural Stimulation during Drosophila Activity Monitor (DAM)-Based Studies of Sleep and Circadian Rhythms in Drosophila melanogaster. 基于果蝇活动监测器（DAM）的黑腹果蝇睡眠和昼夜节律研究中的神经刺激。

Cold Spring Harbor protocols Pub Date : 2024-11-01 DOI: 10.1101/pdb.prot108180

Christopher G Vecsey, Casey Koochagian, Martin Reyes, Divya Sitaraman

{"title":"Neural Stimulation during Drosophila Activity Monitor (DAM)-Based Studies of Sleep and Circadian Rhythms in Drosophila melanogaster.","authors":"Christopher G Vecsey, Casey Koochagian, Martin Reyes, Divya Sitaraman","doi":"10.1101/pdb.prot108180","DOIUrl":"10.1101/pdb.prot108180","url":null,"abstract":"Sleep is a fundamental feature of life for virtually all multicellular animals, but many questions remain about how sleep is regulated by circadian rhythms, homeostatic sleep drive that builds up with wakefulness, and modifying factors such as hunger or social interactions, as well as about the biological functions of sleep. Substantial headway has been made in the study of both circadian rhythms and sleep in the fruit fly Drosophila melanogaster, much of it through studies of individual fly activity using Drosophila activity monitors (DAMs). Here, we describe approaches for the activation of specific neurons of interest using optogenetics (involving genetic modifications that allow for light-based neuronal activation) and thermogenetics (involving genetic modifications that allow for temperature-based neuronal activation) so that researchers can evaluate the roles of those neurons in controlling rest and activity behavior. In this protocol, we describe how to set up a rig for simultaneous optogenetic or thermogenetic stimulation and activity monitoring for analysis of sleep and circadian rhythms in Drosophila, how to raise appropriate flies, and how to perform the experiment. This protocol will allow researchers to assess the causative role in the regulation of sleep and activity rhythms of any genetically tractable subset of cells.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108180"},"PeriodicalIF":0.0,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139711754","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

Analysis of Positional Preference in Drosophila Using Multibeam Activity Monitors. 利用多波束活动监测器分析果蝇的位置偏好。

Cold Spring Harbor protocols Pub Date : 2024-11-01 DOI: 10.1101/pdb.prot108181

Maria T Porter, Gregg Roman, Christopher G Vecsey

{"title":"Analysis of Positional Preference in Drosophila Using Multibeam Activity Monitors.","authors":"Maria T Porter, Gregg Roman, Christopher G Vecsey","doi":"10.1101/pdb.prot108181","DOIUrl":"10.1101/pdb.prot108181","url":null,"abstract":"The positional preference of an animal can be very informative regarding the choices it makes about how to interact with its environment. The fruit fly Drosophila melanogaster has been used as a robust system for examining neurobiological mechanisms underlying behavior. Fruit fly positional preference can be gathered from TriKinetics Drosophila activity monitors (DAMs), which contain four infrared beams, allowing for tracking the position of individual flies along the length of a tube. Here, we describe a method for using DAM5Ms to examine food preference. Specifically, we show an example in which circadian changes in food preference are compared between different Drosophila species. More information about the evolution of behavior can be gathered by measuring feeding preference relative to time of day. Noni, fruit from Morinda citrifolia, contains octanoic acid, a chemical toxic to many species of Drosophila D. melanogaster and D. simulans, both food generalists, show high sensitivity to octanoic acid, whereas D. sechellia, a specialist, can tolerate high concentrations. When two different food substrates are provided at each end of a tube, food preference can be inferred at various times of the day, using the sleep and circadian analysis MATLAB program (SCAMP) to extract and analyze positional data from DAM5Ms. Data gathered from these analyses can be used to compare avoidance or attraction to nutrients, tastants, or odors between species and genotypes or after specific different treatments. Additionally, such data can be examined as a function of time of day.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108181"},"PeriodicalIF":0.0,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11467806/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139711752","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}

引用次数: 0

Analysis of Sleep and Circadian Rhythms from Drosophila Activity-Monitoring Data Using SCAMP. 利用 SCAMP 从果蝇活动监测数据中分析睡眠和昼夜节律

Cold Spring Harbor protocols Pub Date : 2024-11-01 DOI: 10.1101/pdb.prot108182

Christopher G Vecsey, Casey Koochagian, Maria T Porter, Gregg Roman, Divya Sitaraman

{"title":"Analysis of Sleep and Circadian Rhythms from Drosophila Activity-Monitoring Data Using SCAMP.","authors":"Christopher G Vecsey, Casey Koochagian, Maria T Porter, Gregg Roman, Divya Sitaraman","doi":"10.1101/pdb.prot108182","DOIUrl":"10.1101/pdb.prot108182","url":null,"abstract":"Sleep is a fundamental feature of life for virtually all multicellular animals, but many questions remain about how sleep is regulated and what biological functions it plays. Substantial headway has been made in the study of both circadian rhythms and sleep in the fruit fly Drosophila melanogaster, much of it through studies of individual fly activity using beam break counts from Drosophila activity monitors (DAMs). The number of laboratories worldwide studying sleep in Drosophila has grown from only a few 20 years ago to hundreds today. The utility of these studies is limited by the quality of the metrics that can be extracted from the data. Many software options exist to help analyze DAM data; however, these are often expensive or have significant limitations. Therefore, we describe here a method for analyzing DAM-based data using the sleep and circadian analysis MATLAB program (SCAMP). This user-friendly software has an advantage of combining several analyses of both sleep and circadian rhythms in one package and produces graphical outputs as well as spreadsheets of the outputs for further statistical analysis. The version of SCAMP described here is also the first published software package that can analyze data from multibeam DAM5Ms, enabling determination of positional preference over time.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108182"},"PeriodicalIF":0.0,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11552080/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139711753","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}

引用次数: 0

Cloning of Affibody Libraries for Display Methods. 克隆用于显示方法的 Affibody 库。

Cold Spring Harbor protocols Pub Date : 2024-11-01 DOI: 10.1101/pdb.prot108398

Stefan Ståhl, Linnea Charlotta Hjelm, Charles Dahlsson Leitao, John Löfblom, Hanna Lindberg

引用次数: 0