Name: tobacco hornworm as an insect model system for cannabinoid pre-clinical studies
Uploaded: 2021-12-29T15:00:00
Description: Watch this Scientific Journal Video about Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies at JoVE.com

This research was supported by the Institute of Cannabis Research at Colorado State University-Pueblo and the Ministry of Science and ICT (2021-DD-UP-0379), and Chuncheon city (Hemp R&#38;D and industrialization, 2020-2021).

1. Hornworm preparation and cannabidiol treatment
<ol>
	<li>Obtain 150-200 viable M. sexta eggs and wheat germ-based artificial diets (see Table of Materials).</li>
	<li>Place the hornworm eggs in a polystyrene Petri dish with a wheat germ-based artificial diet (AD) layer and transfer the eggs to an insect rearing chamber (see Table of Materials) maintained at 25 &#176;C with 40%-60% relative humidity.</li>
	<li>Allow tobacco hornworm eggs for 1-3 day...

<ol>
	<li>Kaplan, J. S., Stella, N., Catterall, W. A., Westenbroek, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabidiol+attenuates+seizures+and+social+deficits+in+a+mouse+model+of+Dravet+syndrome.">Cannabidiol attenuates seizures and social deficits in a mouse model of Dravet syndrome.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (42), 11229-11234 (2017).</li><li>Leehey, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+tolerability+of+cannabidiol+in+Parkinson+Disease:+An+open+label,+dose-escalation+study.">Safety and tolerability of cannabidiol in Parkinson Disease: An open label, dose-escalation study.</a> Cannabis and Cannabinoid Research. 5 (4), 326-336 (2020).</li><li>Al-Ghezi, Z. Z., Miranda, K., Nagarkatti, M., Nagarkatti, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+cannabinoids,+delta+9-+tetrahydrocannabinol+and+cannabidiol,+ameliorates+experimental+multiple+sclerosis+by+suppressing+neuroinflammation+through+regulation+of+miRNA-mediated+signaling+pathways.">Combination of cannabinoids, delta 9- tetrahydrocannabinol and cannabidiol, ameliorates experimental multiple sclerosis by suppressing neuroinflammation through regulation of miRNA-mediated signaling pathways.</a> Frontiers in Immunology. 10, 1921 (2019).</li><li>Seltzer, E. S., Watters, A. K., MacKenzie, D., Granat, L. 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H., Westenbroek, R. E., Stella, N., Catterall, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+antiseizure+efficacy+of+cannabidiol+and+clonazepam+in+a+conditional+mouse+model+of+Dravet+syndrome.">Combined antiseizure efficacy of cannabidiol and clonazepam in a conditional mouse model of Dravet syndrome.</a> Journal of Experimental Neurology. 2 (2), 81-85 (2021).</li><li>Orvos, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electrophysiological+effect+of+cannabidiol+on+hERG+current+and+in+guinea-pig+and+rabbit+cardiac+preparations.">The electrophysiological effect of cannabidiol on hERG current and in guinea-pig and rabbit cardiac preparations.</a> Scientific Reports. 10 (1), 16079 (2020).</li><li>Verrico, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+randomized,+double-blind,+placebo-controlled+study+of+daily+cannabidiol+for+the+treatment+of+canine+osteoarthritis.">A randomized, double-blind, placebo-controlled study of daily cannabidiol for the treatment of canine osteoarthritis.</a> Pain. 161 (9), 2191-2202 (2020).</li><li>Barata, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotection+by+cannabidiol+and+hypothermia+in+a+piglet+model+of+newborn+hypoxic-ischemic+brain+damage.">Neuroprotection by cannabidiol and hypothermia in a piglet model of newborn hypoxic-ischemic brain damage.</a> Neuropharmacology. 146, 1-11 (2019).</li><li>Beardsley, P. M., Scimeca, J. A., Martin, B. 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The feeding study demonstrated that high doses of CBD (2 mM) inhibited the insect&#39;s growth and increased mortality31. The insect model also showed sensitivity to ethanol; however, CBD effectively detoxicated the ethanol toxicity, increasing their survival rate, diet consumption, and food searching behaviors to similar levels to the control group (Figure 3A,B)31. The described insect model system is composed of three critica...

Over the past years, public attention has been centered on cannabinoids due to their therapeutic potential, including the treatment of epilepsy1, Parkinson&#39;s disease2, multiple sclerosis3, and various forms of cancer4,5,6 with cannabidiol (CBD). Since Cannabis is legalized as an agricultural commodity in the Agricultural Improvement Act of 2018, Public Law 115-334 (the 2018 Farm Bill), Cannabis and its cannabinoid derivatives in the food, cosmetic, and pharmaceutical industries have exponentially increased. Additionally, clinical-grade isolates of single cannabinoids and cannabinoid mixtures have been successfully tested in human subjects7, cell lines5,8, and diverse animal model systems9,10.
A clinical trial would be ideal for validating the efficacy and adverse effects of cannabinoids on a specific disease. However, there are numerous challenges in clinical trials, including ethical/IRB approval, recruitment, and retention of the subjects11. To overcome these hurdles, various human cell lines were used because human-derived cell lines are cost-effective, easy to handle, can bypass the ethical issues, and provide consistent and reproducible results as the cell lines are a &#39;pure population of cells that have no cross-contamination of other cells and chemicals&#39;12.
Alves et al. (2021)13 tested CBD in a dose-dependent manner in the placental trophoblasts, which are specialized cells of the placenta that play an essential role in embryo implantation and interaction with the decidualized maternal uterus14. Their results showed that CBD caused cell viability loss, cell cycle progression disruption, and apoptosis induction. These observations demonstrate the potential negative impacts of Cannabis use by pregnant women13. Likewise, a series of cell lines were also used to examine the pharmacological effects of CBD in human diseases, in particular, various forms of cancer. The in vitro studies successfully demonstrated anti-cancer effects in pancreatic15, breast8, and colorectal cancer cells16. However, while being widely available and easy to handle, specific cell lines such as HeLa, HEK293 are prone to genetic and phenotypic changes due to alterations in their growth conditions or handling17.
In Cannabis research, various animal model systems, ranging from small animals such as mouse18, guinea-pig19, and rabbit19 to large animals such as canine20, piglet21, monkey22, horse23, have been used to explore unknown therapeutic effects. Mice have been the most preferred animal model system for cannabinoid research due to their anatomical, physiological, and genetic similarity to humans24. Most significantly, mice have CB1/2 receptors in their nervous system, which are present in humans. They also have a shorter life cycle than human subjects, with easier maintenance and abundant genetic resources, thus making it much easier to monitor the effects of cannabinoids throughout an entire life cycle. The mammalian system is widely used and has successfully demonstrated that CBD relieves seizure disorders1, post-traumatic stress disorder9, oral ulcers25, and dementia-like symptoms10. The mouse model has also enabled a social interaction study of individuals within a community which is extremely difficult in large animals and humans26.
Despite all the advantages of the animal model system, it is still costly and requires intensive care during drug administration and data collection. Additionally, there is scrutiny of using mice in research because of irreproducibility and poor recapitulation of human conditions due to limitations in experimental design and rigor27.
With the increasing demand for medical/preclinical studies of cannabinoids, a non-mammalian model system is needed. Invertebrate models traditionally conferred distinctive benefits over vertebrate models. The significant benefits include the ease and low cost of rearing many specimens and enabling researchers to monitor multiple generations of genetically homogeneous populations28. A recent study proved the fruit fly, Drosophila melanogaster, to be an effective insect model system to investigate pharmacological functions of cannabinoids in modulating feeding behaviors29. Among the insect model systems, the authors focused on the tobacco hornworm, Manduca sexta, also known as Carolina sphinx moth or hawk moth, as a novel insect model system for cannabinoid research.
Manduca sexta belongs to the family of Sphingidae. The insect is the most common plant pest in the southern United States, where they feed on solanaceous plants. The insect model has a long history in research in insect physiology, biochemistry, neurobiology, and drug interaction studies. Manduca sexta&#39;s&#160;research portfolio includes a draft genome sequence, allowing for a molecular-level understanding of essential cellular processes30. Another crucial benefit of this model system is its large size, reaching more than 100 mm in length and 10 g in weight in the 18-25 days of larval development. The large size enables researchers to easily monitor morphological and behavioral changes in real-time in response to the CBD treatment. Also, due to the size, electrophysiological responses were examined with the abdominal nervous system, including ganglia dissected from the larvae without high-resolution microscope settings. The unique feature allows researchers to readily investigate acute and long-term responses to the administered cannabinoid(s).
Despite such versatility, M. sexta has only recently been explored for its suitability as an experimental model for Cannabis and cannabinoid&#160;studies. In 2019, the authors used the insect model system for the first time to address the hypothesis that Cannabis has evolved to produce Cannabidiol to protect itself from insect herbivores30,31. The result clearly showed that the plants exploited CBD as a feeding deterrent and inhibited the growth of the pest insect M. sexta caterpillar, as well as causing increased mortality31. The study also demonstrated the rescuing effects of CBD to intoxicated ethanol larvae, identifying the potential vehicle effect of ethanol as a carrier of the CBD. As shown, the insect model system effectively investigated the therapeutic effects of cannabinoids within 3-4 weeks with less labor and costs than other animal systems. Although the insect model lacks cannabinoid receptors (i.e., no CB1/2 receptors), the model system provides a valuable tool to understand the pharmacological roles of cannabinoids through a cannabinoid receptor-independent manner.
The authors of this study have previously worked with the tobacco hornworm as a model system for cannabinoid research31. After careful consideration of the benefits and risks of using M. sexta, we have provided a method involving the proper care and preparation of diet for preclinical trials that allow for opportunities for future preclinical laboratory use.

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		<tr>
			<td>Analytic balance</td>
			<td>Mettler Instrument Corp.</td>
			<td>AE100S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannabidiol isolate (&#62;99.4%)</td>
			<td>Lilu&#39;s Garden</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cheesecloth</td>
			<td>VWR INTERNATIONAL</td>
			<td>470150-438</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 50mL clear polypropylene (PP) centrifuge tubes</td>
			<td>VWR</td>
			<td>89093-192</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Alcohol, 200 Proof</td>
			<td>Sigma-Aldrich</td>
			<td>EX0276-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fear conditioning chamber</td>
			<td>Coulbourn Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insect rearing chamber</td>
			<td>Darwin Chambers</td>
			<td>INR034</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium chain triglycerides (MCT) oil</td>
			<td>Walmart</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Motion detection software (Actimetrics)</td>
			<td>Coulbourn Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene petri dish (120 mm x 120 mm x 17mm)</td>
			<td>VWR INTERNATIONAL</td>
			<td>688161</td>
			<td></td>
		</tr>
		<tr>
			<td>Tobacco hormworm artificial diet</td>
			<td>Carolina Biological Supply Company</td>
			<td>Item # 143908</td>
			<td>Ready-To-Use-Hornworm-Diet</td>
		</tr>
		<tr>
			<td>Tobacco hormworm eggs</td>
			<td>Carolina Biological Supply Company</td>
			<td>Item # 143880</td>
			<td>Unit of 30-50</td>
		</tr>
	</tbody>
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tobacco hornworm as an insect model system for cannabinoid pre-clinical studies

With increased attention on cannabinoids in medicine, several mammalian model organisms have been used to elucidate their unknown pharmaceutical functions. However, many difficulties remain in mammalian research, which necessitates the development of non-mammalian model organisms for cannabinoid research. The authors suggest the tobacco hornworm Manduca sexta as a novel insect model system. This protocol provides information on preparing the artificial diet with varying amounts of cannabidiol (CBD), setting up a cultivation environment, and monitoring their physiological and behavioral changes in response to CBD treatment. Briefly, upon receiving hornworm eggs, the eggs were allowed 1-3 days at 25 &#176;C on a 12:12 light-dark cycle to hatch before being randomly distributed into control (wheat germ-based artificial diet; AD), vehicle (AD + 0.1% medium-chain triglyceride oil; MCT oil) and treatment groups (AD + 0.1% MCT + 1 mM or 2 mM of CBD). Once the media was prepared, 1st instar larvae were individually placed in a 50 mL test tube with a wooden skewer stick, and then the test tube was covered with a cheesecloth. Measurements were taken in 2-day intervals for physiological and behavioral responses to the CBD administration. This simple cultivation procedure allows researchers to test large specimens in a given experiment. Additionally, the relatively short life cycles enable researchers to study the impact of cannabinoid treatments over multiple generations of a homogenous population, allowing for data to support an experimental design in higher mammalian model organisms.

Manduca sexta as a model system to examine cannabinoids toxicity 
Figure 1 depicts the key components of the CBD experiment using tobacco hornworm Manduca sexta. Large numbers of insects (&#62;20) were individually reared at 25 &#176;C on a 12 h:12 h = light: dark cycle. The insects&#39; size, weight, and mortality were measured at 2-day intervals to monitor for short-and long-term responses after high-dose CBD (2 mM) treatment.
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Watch this Scientific Journal Video about Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies at JoVE.com

Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies

The present protocol provides instructional information for using tobacco hornworm Manduca sexta in cannabinoid research. The method described here includes all necessary supplies and protocols to monitor physiological and behavioral changes of the insect model in response to cannabidiol (CBD) treatment.

With increased attention on cannabinoids in medicine, several mammalian model organisms have been used to elucidate their unknown pharmaceutical ...

The protocol proposes using tobacco hornworm as an insect model system for cannabinoid preclinical studies. This system allows researchers to investigate acute and long-term responses to cannabinoids readily. The insect model system enables researchers to investigate cannabinoid therapeutic effects in a large population within three to four weeks with less labor and costs than other animal systems.Obtain 150 to 200 viable M.sexta eggs and wheat germ-based artificial diets. Place the hornworm eggs in a polystyrene Petri dish with a wheat germ-based artificial diet layer and transfer the eggs to an insect rearing chamber maintained at 25 degree Celsius with 40 to 60%relative humidity. Allow the eggs to hatch for one to three days inside the insect rearing chamber.To prepare a 200 millimolar cannabidiol stock solution, add 1.26 grams of greater than 98%purity cannabidiol isolate in 20 milliliters of ethanol or 100%medium chain triglyceride oil. Add five milliliters and 10 milliliters of the 200 millimolar cannabidiol stock solution to the 1, 000 grams of an artificial diet for one millimolar and two millimolar of cannabidiol, respectively. Dispense 20 grams of control vehicle and cannabidiol-containing media to the bottom of the 50-milliliter tube.Randomly distribute approximately two millimeter-long first instar larvae individually in a 50-milliliter test tube and cover with a perforated lid or cheesecloth. Grow them inside an insect rearing chamber maintained at 25 degree Celsius with a 12-hours light-dark cycle. To measure the larval growth, record each group's initial mass and size with an analytical balance and ruler, respectively, and introduce the larvae to their respective diets.Subtract the mass of the larvae at each measurement at two-day intervals from the initial mass to determine mass gains between larvae developmental stages. Also, record the number of days between the instar developmental stages to understand differences in the developmental timeframe until pupation. To measure the diet consumption, record the initial grams of diet at the beginning at the experiment and subtract it from the remaining amount of diet when the larvae entered the complete pupation stage.For the larval mobility measurement, allow the subjected insect to acclimate to the fear conditioning chamber for at least five minutes and track the distances the three groups of fifth instar insects traveled for five minutes. Then, run the motion detection software to analyze the mobility response. The insects reared on an artificial diet showed the best growth performance.The vehicle control that used 0.1%medium chain triglyceride oil as a dissolving agent for CBD isolate also showed normal growth without any detrimental effects. However, a high dose of CBD of two millimeter induced weight loss and led to a higher mortality rate than control and vehicle groups. M.sexta larvae reared on AD with one millimeter CBD consumed at least 3.1 times greater diet than those reared on an ethanol-added diet.However, the diet consumption of the insects reared on two millimolar CBD-added media was not significantly different from the larvae reared on ethanol-only diets. The mobile index of M.sexta larva reared on different conditions shows that 1%ethanol and one millimolar CBD-treated larvae did not affect mobility. The 2%ethanol treatments turned out to be lethal.Adding a high dose of CBD into AD containing 2%ethanol restored low mobility. Once CBD is added to diets, completely homogenize the diet CBD mixture and grow insects in the insect rearing chamber maintained at 25 degrees Celsius with 40 to 60%humidity to avoid a fungal outbreak. Electrophysiological responses can be also monitored with the ventral chain ganglia dissected from Manduca sexta to determine whether CBD affects the tobacco hornworm's central nervous system.The insect model system will enable researchers to explore the pharmaceutical roles of other major and minor cannabinoids by testing over multiple generations, allowing for an experimental design in higher mammals.

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Biology

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3.
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp. Several species of African grasses of the genus Brachiaria are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5.

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

This video explains mechanisms of host plant resistance to herbivory and demonstrates a no-choice test that estimates the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp.

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...