Reviewer
Neelanjan Bose
  • Research scientist, Emery Pharma
Research fields
  • Developmental biology
Calcium Imaging of Neuronal Activity under Gradually Changing Odor Stimulation in Caenorhabditis elegans
Authors:  Yuki Tanimoto and Koutarou D. Kimura, date: 01/05/2021, view: 2993, Q&A: 0

Olfactory behavior is among the most fundamental animal behaviors both in the wild and in the laboratory. To elucidate the neural mechanisms underlying olfactory behavior, it is critical to measure neural responses to odorant concentration changes resembling those that animals actually sense during olfactory behavior. However, reproducing the dynamically changing olfactory stimuli to an animal during such measurements of neural activity is technically challenging. Here, we describe technical details and protocols for odor stimulation during calcium imaging of the sensory neurons of the nematode Caenorhabditis elegans. In this system, the neuronal activity of C. elegans is measured using ratiometric calcium imaging during exposure to quantitatively controlled olfactory stimuli over time. Temporal changes in odor concentrations around the animal are precisely controlled according to a predesigned temporal odor gradient to reproduce a realistic odor concentration change during olfactory behavior in a behavioral arena. By monitoring neural activity in response to the realistic olfactory stimulus, it is possible to elucidate the mechanisms by which olfactory input is processed by neural activities and reflected in behavioral output.

Fluorescence HPLC Analysis of the in-vivo Activity of Glucosylceramide Synthase
Authors:  Kartik R. Roy, Sachin K. Khiste, Zhijun Liu and Yong-Yu Liu, date: 06/20/2019, view: 5178, Q&A: 0
Almost all functions of cells or organs rely on the activities of cellular enzymes. Indeed, the in-vivo activities that directly represent the cellular effects of enzymes in live organs are critical importance to appreciate the roles enzymes play in modulating physiological or pathological processes, although assessments of such in-vivo enzyme activity are more difficult than typical test-tube assays. Recently, we, for the first time, developed a direct and easy-handling method for HPLC analyzing the in-vivo activity of glucosylceramide synthase (GCS). GCS that converts ceramide into glucosylceramide is a limiting-enzyme in the syntheses of glycosphingolipids and is one cause of cancer drug resistance. In our method developed, rubusoside nanomicelles delivers fluorescence N-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoyl]-D-erythro-sphingosine (NBD C6-ceramide) into mice, tissues uptake the cell-permeable substrate, and GCS converts it into NBD C6-glucosylceramide in all organs simultaneously. Further, HPLC analyzes the extracted NBD C6-glucosylceramide to assess alterations of the in-vivo GCS activities in tissues. This method can be broadly used to assess the in-vivo GCS activities in any kind of animal models to appreciate either the role GCS plays in diseases or the therapeutic efficacies of GCS inhibitors.
Microirradiation for Precise, Double-strand Break Induction in vivo in Caenorhabditis elegans
Authors:  Kailey E. Harrell, Emily Koury and Sarit Smolikove, date: 12/20/2018, view: 4415, Q&A: 0
DNA double-strand breaks (DSBs) are toxic lesions that every cell must accurately repair in order to survive. The repair of DSBs is an integral part of a cell life cycle and can lead to lethality if repaired incorrectly. Laser microirradiation is an established technique which has been used in yeast, mammalian cell culture, and Drosophila cell culture to study the regulation of DSB repair. Up to our studies, this method has not been adapted for use in a whole, live, multicellular organism to study this repair in vivo. We have recently shown that this system can be used for study of the recruitment of vital repair proteins to microirradiation-induced breaks in the transparent nematode Caenorhabditis elegans. With the integration of microirradiation and imaging technology, we can precisely induce DSBs in target nuclei and study the recruitment of fluorescently tagged repair proteins from the time of damage induction. Whole, live worms are plated and immobilized for targeting of nuclei, and immediately following induction the targeted region can be imaged for up to an hour and a half post-microirradiation. This method is the first that allows for study of DNA repair protein kinetics in vivo in an intact organism, which can be adapted in numerous ways to allow for study of repair kinetics in various aspects of the repair process.
Staining the Germline in Live Caenorhabditis elegans: Overcoming Challenges by Applying a Fluorescent-dye Feeding Strategy
Author:  K. Adam Bohnert, date: 11/05/2018, view: 6070, Q&A: 0
C. elegans provides a tractable model organism for studying germline cell biology. Microscopy experiments are relatively facile, as this worm is transparent and germline development can be observed in real-time using DIC microscopy and/or fluorescent transgenes. Despite these many tools, robust staining techniques for imaging germ cells in live worms have been more elusive, due to the tough outer cuticle of the worm, which impairs staining efficiency. This limitation has restricted the spectrum of probes that can be used to investigate reproductive cell biology in C. elegans. Building on previous approaches, I recently applied a fluorescent-dye feeding strategy to reproducibly label organelles and monitor physiological changes in germlines of living C. elegans. In this approach, fluorescent dyes are initially introduced into the agar plates and bacterial lawns on which worms are subsequently cultured. After worms are grown on the dyed plates, oocytes show staining patterns consistent with verified transgenic markers. Thus, this approach offers an effective solution for labeling difficult-to-stain tissues in live worms, and establishes an entry point for incorporating new probes and sensors into analyses of C. elegans germline biology.
Stable-isotope Labeled Metabolic Analysis in Drosophila melanogaster: From Experimental Setup to Data Analysis
Authors:  Yuping Cai, Nan Liu and Zheng-Jiang Zhu, date: 09/20/2018, view: 5981, Q&A: 0
Stable-isotope labeled metabolic analysis is an essential methodology to characterize metabolic regulation during biological processes. However, the method using stable-isotope-labeled tracer (e.g., 13C-glucose) in live animal is only beginning to be developed. Here, we contribute a qualitative metabolic labeling experiment protocol in Drosophila melanogaster using stable-isotope-labeled 13C-glucose tracer followed by liquid chromatography-mass spectrometry (LC-MS) analysis. Detailed experimental setup, data acquisition and analysis are provided to facilitate the application of in vivo metabolic labeling analysis that might be applied in a wide range of biological studies.
Quantitative ChIP-seq by Adding Spike-in from Another Species
Authors:  Kongyan Niu, Rui Liu and Nan Liu, date: 08/20/2018, view: 20362, Q&A: 0
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a routine procedure in the lab; however, epigenome-wide quantitative comparison among independent ChIP-seq experiments remains a challenge. Here, we contribute an experimental protocol combined with a computational workflow allowing quantitative and comparative assessment of epigenome using animal tissues.
Measuring Spatiotemporal Dynamics of Odor Gradient for Small Animals by Gas Chromatography
Authors:  Akiko Yamazoe-Umemoto, Yuishi Iwasaki and Koutarou D. Kimura, date: 04/05/2018, view: 6452, Q&A: 0
Odor is the most fundamental chemical stimulus that delivers information regarding food, mating partners, enemies, and danger in the surrounding environment. Research on odor response in animals is widespread, although studies on experimental systems in which the gradient of odor concentration is quantitatively measured has been quite limited. Here, we describe a method for measuring a gradient of odor concentration established by volatilization and diffusion in a relatively small enclosed space, which has been used widely in laboratories to analyze small model animals such as the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. We first vaporized known amounts of a liquid odorant 2-nonanone in a tank and subjected them to gas chromatographic analysis to obtain a calibration curve. Then, we aspirated a small amount of gas phase from a small hole on an agar plate and measured the odor concentration. By repeating this at different spatial and temporal points, we were able to detect a gradient of the odor concentration that increased over time. Furthermore, by applying these measured values to mathematical models of volatilization and diffusion, we were able to visualize an estimated dynamic change in odor concentration over an agar plate. Combining monitoring of odor concentration change in an agar plate with behavioral monitoring by machine vision will allow us to estimate how the brain computes information regarding odor concentration change in order to regulate behavior.
Measurement of Intracellular ROS in Caenorhabditis elegans Using 2’,7’-Dichlorodihydrofluorescein Diacetate
Authors:  Dong Suk Yoon, Myon-Hee Lee and Dong Seok Cha, date: 03/20/2018, view: 14745, Q&A: 1
Reactive oxygen species (ROS) are generated during normal metabolic processes under aerobic conditions. Since ROS production initiates harmful radical chain reactions on cellular macromolecules, including lipid peroxidation, DNA mutation, and protein denaturation, it has been implicated in a wide spectrum of diseases such as cancer, cardiovascular disease, ischemia-reperfusion and aging. Over the past several decades, antioxidants have received explosive attention regarding their protective potential against these deleterious reactions. Accordingly, many analytical methodologies have been developed for the evaluation of the antioxidant capacity of compounds or complex biological samples. Herein, we introduce a simple and convenient method to detect in vivo intracellular ROS levels photometrically in Caenorhabditis elegans using 2’,7’-dichlorofluorescein diacetate (H2DCFDA), a cell permeant tracer.
Extraction and Analysis of Pan-metabolome Polar Metabolites by Ultra Performance Liquid Chromatography–Tandem Mass Spectrometry (UPLC-MS/MS)
Authors:  Dania M. Malik, Seth Rhoades and Aalim Weljie, date: 02/05/2018, view: 8334, Q&A: 0
Modern triple quadrupole mass spectrometers provide the ability to detect and quantify a large number of metabolites using tandem mass spectrometry (MS/MS). Liquid chromatography (LC) is advantageous, as it does not require derivatization procedures and a large diversity in physiochemical characteristics of analytes can be accommodated through a variety of column chemistries. Recently, the comprehensive optimization of LC-MS metabolomics using design of experiments (COLMeD) approach has been described and used by our group to develop robust LC-MS workflows (Rhoades and Weljie, 2016). The optimized LC-MS/MS method described here has been utilized extensively for metabolomics analysis of polar metabolites. Typically, tissue or biofluid samples are extracted using a modified Bligh-Dyer protocol (Bligh and Dyer, 1959; Tambellini et al., 2013). The protocol described herein describes this workflow using targeted polar metabolite multiple reaction monitoring (MRM) from tissues and biofluids via ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). This workflow has been utilized extensively for chronometabolic analysis (Krishnaiah et al., 2017), with applications generalized to other types of analyses as well (Sengupta et al., 2017; Sivanand et al., 2017).
Identification and Quantification of Secondary Metabolites by LC-MS from Plant-associated Pseudomonas aurantiaca and Pseudomonas chlororaphis
Authors:  Izzah Shahid, Muhammad Rizwan and Samina Mehnaz, date: 01/20/2018, view: 11057, Q&A: 0
Increased antibiotic resistance of plants and human pathogens and continuous use of chemical fertilizers has pushed microbiologists to explore new microbial sources as potential antagonists. In this study, eight strains of Pseudomonas aurantiaca and Pseudomonas chlororaphis, have been isolated from different plant sources and screened for their antagonistic and plant growth promoting potential (Shahid et al., 2017). All strains were compared with reference strain PB-St2 and their secondary metabolites were isolated by the use of solvent partitioning and subjected to LC/ESI/MS for confirmation of compounds. The ESI-mass spectra obtained were used to characterize the surfactants ionization behavior and [M + H]+ and [M + Na]+ ions were monitored for phenazines, derivatives of lahorenoic acid and cyclic lipopeptide (WLIP). LC-MS and HPLC methods were developed to see the elution of dominant metabolites in a single run to avoid the labor and separate methods of detection for all compounds. The method was found suitable and distinctively separated the compounds at different retention times in gradient flow. This method can be helpful to explore the metabolome of Pseudomonas sp. overall and in identification and quantification of strain specific metabolites.
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