Name: the replica set method: a high-throughput approach to quantitatively measure caenorhabditis elegans lifespan
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Funding for this work described in this manuscript was provided by: the University of Rochester Office of The Provost and the School of Medicine and Dentistry Dean&#39;s Office via the Health Sciences Center for Computational Innovation (HSCCI); the Ellison Medical Foundation New Scholars in Aging Fellowship (AG-NS-0681-10) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1. Traditional Method for Scoring C. elegans Longevity
<ol>
	<li>Preparation of reagents
	<ol>
		<li>Identify genes to be inactivated via feeding-based RNAi. Purchase transformed stocks of HT115 E. coli2 containing the RNAi clone of interest. Alternatively, subclone the cDNA of the gene of interest into the multicloning site of the L4440 plasmid. 
		NOTE: HT115 is an RNase III-deficient E. coli strain with IPTG-inducible T7 polymerase activity, wh...

<ol>
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Both the traditional and replica set methods require the synchronization of chronologically aged animals. We include a method that synchronizes animals using hypochlorite treatment of gravid adults, where only fertilized eggs with the gravid adult survive treatment. These embryos hatch in liquid suspension and developmentally arrest at the first larval stage (L1). After seeding L1 animals onto food (e.g. E. coli expressing dsRNA to a gene of interest), animals resume development. Synchronizing L1 animal...

One of the most transformative technological advancements towards understanding the genetic basis of aging was the development of feeding-based RNAi in C. elegans1; prior to the experimental use of RNAi, many phenotypes of aging were not genetically tractable. Feeding-based RNAi is achieved through the production of dsRNA within E. coli that matches an endogenous C. elegans mRNA: IPTG induces bidirectional transcription across an insert of either C. elegans cDNA or a portion of an open reading frame within a plasmid2. When C. elegans feed upon intact E. coli, dsRNA produced by bacteria is transported from the lumen into intestinal cells via the SID-2 transmembrane protein3, and then distributed through the rest of the animal via SID-14. Within each cell, exogenous dsRNA is processed by the Dicer complex into siRNA, which interact with a mature mRNA via complementary base pairing to create a new siRNA-mRNA duplex. This duplex is recognized by the RISC complex and cleaved, thereby degrading the endogenous mRNA5. Thus, by merely changing the plasmid insert, one can inactivate the function of nearly any gene within the C. elegans genome. This discovery led to the creation of several large feeding-based RNAi libraries- collections of transformed E. coli stocks that can be combined to achieve coverage of approximately 86% of known C. elegans genes6,7.
Since the advancement of feeding-based RNAi, comprehensive screens in C. elegans have led to the discovery of more than 900 genes that alter lifespan when inactivated (as evidenced by the RNAi-phenotype associations curated in WormBase), which we refer to as gerogenes. A role for the majority of gerogenes in longevity control was discovered through feeding-based RNAi in just a few seminal reports (see Figure 1A and Supplemental File 1 for details). In some cases, these gerogenes have been identified based on measuring the viability at a single or a few time points, which fails to provide a quantifiable measure of the change in lifespan with RNAi treatment. In other cases, these genes have been quantitatively assessed for changes in lifespan, as well as additional age-associated phenotypes. For instance, we previously identified 159 genes that were necessary for both normal and increased lifespan of animals with decreased insulin/IGF-1 signaling, and quantified changes in healthspan. Of these, 103 gene inactivations result in a progeric phenotype, as loss resulted in one or more signs of premature aging8.
While some gerogenes have been associated with 100 or more studies (e.g. daf-16, daf-2, sir-2.1), over 400 gerogenes have 10 or fewer citations (Figure 1B, and Supplemental File 2). Thus, while comprehensive feeding-based RNAi screens have discovered and cursorily characterized hundreds of putative gerogenes, how these genes function in longevity control, and the genetic interrelationships between these gene products remain poorly studied. Full longitudinal analysis for age-associated phenotypes is a prerequisite for identifying genetic interactions between gerogenes (e.g. epistatic interactions, asynthetic interactions, etc.). Gaining deeper insight into the genetic interrelationships between gerogenes requires a high-throughput quantitative method, which also leverages the advantages of feeding-based RNAi.
The most common surrogate measure of aging is lifespan. The traditional approach for measuring C. elegans mortality tracks the deaths of individual animals over time within a small population sample. A relatively small number of animals are followed over time and periodically are gently prodded with either a platinum wire or eyelash, with movement as an indicator of viability (Figure 2A). This method has been widely used, as it provides straightforward, direct measurements of the average and the maximum lifespan. However, this traditional method is time consuming and relatively low-throughput, which limits the number of animals and conditions that can simultaneously be measured in a controlled manner. A recent simulation study found that many C. elegans lifespan studies do not assay a large enough number of animals to be able to reliably detect small changes between conditions9. Furthermore, this traditional method involves repeatedly handling the same cohort of animals over time, which in turn can introduce contamination, and can damage or kill increasingly fragile, aged animals.
We have developed an alternative &#34;Replica Set&#34; methodology for measuring C. elegans lifespan. To this end, a large population of age-synchronized, isogenic animals are divided into a number of small populations (or replicas). Enough replica samples are generated to cover each time point in the planned experiment. At each observation time point, one of the replicas is scored for the number of living, dead and censored animals, then animals within that replicate are discarded. Thus, over the expected lifespan of the population as a whole, a series of independent subpopulations are periodically sampled (Figure 2B). In using replica sets there is no repeated prodding of animals and no repeated exposure to potential environmental contamination. The viability observed at the one-time point is completely independent of every other observation, which minimizes handling and increases throughput by at least an order of magnitude. This has allowed us to quantitate changes in lifespan for hundreds of RNAi clones simultaneously8,10.
Here we present detailed protocols for conducting C. elegans lifespan via both the Replica Set and traditional methods for scoring C. elegans longevity. We demonstrate that similar results are obtained between the methods. We have developed software to assist in the graphical analysis of lifespan data generated through either approach, which we freely provide under a GPL V3 license (See Table of Materials). &#34;WormLife&#34; is written in R11, and includes a graphical user interface (GUI) for plotting data, which has been tested in Mac OS and Linux. Lastly, we compare and contrast the limitations of each method and highlight other considerations when choosing between approaches to measure quantitative changes in C. elegans lifespan.

<table border="0" cellpadding="0" cellspacing="0" width="1120">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">IPTG (isopropyl beta-D-1-thigalactopyranoside)</td>
			<td style="width:108px;">Gold Bio</td>
			<td style="width:119px;">12481C100</td>
			<td style="width:527px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">FuDR (5-Fluoro-2&#39;-deoxyuridine)</td>
			<td style="width:108px;">Alfa Aesar</td>
			<td>L16497</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:367px;">24 Well Culture Plates</td>
			<td style="width:108px;">Greiner Bio-One</td>
			<td style="width:119px;">#662102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Retangular non-treated single-well plate, 128x86mm</td>
			<td style="width:108px;">Thermo-Fisher</td>
			<td style="width:119px;">242811</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">600 &#181;L 96-well plates</td>
			<td style="width:108px;">Greiner Bio-One</td>
			<td style="width:119px;">#786261</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">2mL 96-well plates</td>
			<td style="width:108px;">Greiner Bio-One</td>
			<td style="width:119px;">#780286</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Air-permeable plate seal</td>
			<td style="width:108px;">VWR</td>
			<td style="width:119px;">60941-086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">96-pin plate replicator</td>
			<td style="width:108px;">Nunc</td>
			<td style="width:119px;">250520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">bacto-peptone</td>
			<td style="width:108px;">VWR</td>
			<td style="width:119px;">90000-368</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">bacteriological agar</td>
			<td style="width:108px;">Affymetrix/USB</td>
			<td style="width:119px;">10906</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:367px;">C. elegans RNAi clone library in HT115 bacteria- Ahringer</td>
			<td style="width:108px;">Source Bioscience</td>
			<td style="width:119px;">C. elegans RNAi Collection (Ahringer)</td>
			<td>See also Kamath et. al, Nature 2003.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:367px;">C. elegans RNAi clone library in HT115 bacteria- Vidal</td>
			<td style="width:108px;">Source Bioscience</td>
			<td style="width:119px;">C. elegans ORF-RNAi Resource (Vidal)</td>
			<td>See also Rual et. al, Genome Research 2004. This library is also available from Dharmacon.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">WormLife- Software for Replica Set Survival Analysis</td>
			<td style="width:108px;">Samuelson Lab</td>
			<td style="width:119px;">N/A</td>
			<td>https://github.com/samuelsonlab-urmc/wormlife</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L4440 Empty Vector Plasmid</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">1654</td>
			<td>https://www.addgene.org/1654/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Wormbase</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td>http://www.wormbase.org/&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">OASIS</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td>https://sbi.postech.ac.kr/oasis2/&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Graphpad Prism</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td>https://www.graphpad.com/scientific-software/prism/&#160;</td>
		</tr>
	</tbody>
</table>

the replica set method: a high-throughput approach to quantitatively measure caenorhabditis elegans lifespan

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

In the development of any new methodology, it is imperative that the new method recapitulates accepted results from previous approaches and meets the standard within a field. We have previously shown empirically that the Replica Set and traditional methods for assaying C. elegans lifespan produce similar results20. Wild-type C. elegans (N2) maintained at 20 &#176;C typically live between 20 and 25 days, which we observed with both the traditional ...

Watch this Scientific Journal Video about The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan at JoVE.com

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

Hundreds of genes have been identified whose function affects how an organism ages. But many of their relationships are poorly understood. This method can help answer a key question in the aging field.Namely, what are the genetic inter-relationships between gerA genes? The main advantage of this technique is an increase in throughput, the number of genetic predebations that can be quantitatively measured simultaneously is increased by at least an order of magnitude. Lifespan data, collected with this procedure can be plotted and analyzed using software we provide with this publication.In addition to provide insight into lifespan, this method can also be applied to other age-related phenotypes, such as stress resistance and changes in health plan. For this procedure, create a fresh stamp of the RNAi sub-library. First, aliquot each well of a 96-well plate, with 200 microliters of LB with ampicillin, using a 12-channel pipetter.Sterilize a 96-pin plate replicator by sequentially immersing the pins in 50%bleach followed by ultra pure water. Then dip the tips in ethanol, and carefully flame the tips. Next, carefully remove adhesive foil cover of a frozen, 96-well glycerol stock library plate, and gently but firmly grind the tips of the sterilized plate replicator into the wells of the still frozen glycerol stocks.Then inoculate the cultures and seal the inoculated plate with the permeable membrane. Allow cultures to grow overnight on the bench top. The next day, sterilize the plate replicator, and carefully remove the seal from the overnight culture, and immerse tips of the replicator pins.Next, inoculate a rectangular LB ampicillin and tetracycline agar plate with the pins. Apply even pressure, and use a small, circular motion to transfer the bacteria without letting the spots get too crowded. Then, incubate the plates overnight at 37 degrees Celsius.The next day, for every 10 replica plates needed, load each well of a deep-welled 96-well plate with 1.5 milliliters of LB with ampicillin. Next, inoculate the wells with the cultured bacteria. Then, seal the plate with a breathable membrane, culture it for 20 hours with shaking.Control wells should be scattered across the 96 wells, such that when the 96-well sub-library plate is divided into groups of 24, each division of 24 wells has one of each of the controls. The following day, load 120 microliter aliquots of the culture, to the 24-well plates, using a six-well multichannel pipette, with adjustable tip spacing. Then, dry plates uncovered in a laminar flow hood until all liquid has been absorbed.Do not let them over dry. Be sure to maintain your focus when aliquoting liquid cultures, to ensure that the proper bacterial culture is put into the proper well in the 24-well plate. Replica set experiments start with the set of independent, 24-well replicate plates, each seeded with the same bacteria RNAi clones.For lifespan experiments, 15 to 20 L1 animals are added to each plate well, and allowed to grow to the L4 stage. At L4, FUdR is added to all wells across replicates. Later in the same day, a replicate plate can be taken from the set and scored as the first time point.To score lifespan, liquid is added to the wells of the plate to induce animal movement. Animals that do not start to move are prodded gently with a worm pick. The total number of animals, and the number of dead animals is then recorded.The plate from the first observation is then discarded. At the next observation time point, another replicate plate is taken from the set, and scored in a similar fashion. This is repeated at the determined scoring interval.Each time with a new plate from the replicate set. Continue scoring a set of plates until no animals remain alive. First, synchronize a population of animals with the same bleach-based prep method used for the traditional lifespan assay.Then, using a six-well adjustable spacing pipette, and reagent reservoir, seed 15 to 20 L1 animals into each well of the prepared 24-well plates. While seeding the plates, periodically mix the L1 animals into solution to maintain even distribution. Now, let the animals develop to L4.The timing of L4 development must be known for this protocol.Periodically monitor the development of the worms. When animals reach L4, add 24 microliters of 50x FUdR to each well, and return the plates to the incubator. Using L4 animals is critical.If hermaphrodites reach reproductive maturity before adding FUdR, their progeny will survive and eat the food. Conversely, adding FUdR too early leads to ruptured animals. At the desired time points, use one replicate plate to score viability.To do so, flood the well with M9 solution. Before scoring viability each day, sensor any wells that do not have food, appear contaminated, or have animals with morphological or developmental defects. For each well, record the total animals per well, and check the status of non-moving worms, by gently touching each on the head with a worm pick.After counting all the non-moving dead in a plate, discard the plate. For this procedure, grow transformed e. Coli overnight in LB with ampicillin.The next day, concentrate the bacteria by centrifugation at 3, 000 G's for 15 to 20 minutes. Resuspend the bacteria in 1/10 of the starting volume. Next, aliquot 200 microliters of the 10x bacteria on to six-centimeter plates.Prepare three or four plates per test condition, and include two extra plates in case of contamination. Dry the plates in a laminar flow batch, until all the liquid has been absorbed or evaporated. Once dried, store the plates in a plastic box overnight at room temperature to induce the double-stranded RNA production.Then, store the plates for up to two weeks at four degrees Celsius. For the animals, collect gravid, adult animals in M9.Wash them twice with M9.Repeating the spin at aspiration. Then, resuspend C.elegans in four milliliters of M9, and add two milliliters of hypochlorite solution.Now, immediately vortex the worms for three minutes, with periodic, vigorous shaking. Then, under a dissecting microscope, look for a cloud of eggs. If the cloud isn't seen, vortex the worms for an additional 10 to 20 seconds.Next, quickly wash the eggs with M9, two times, as before. And resuspend the washed eggs in three milliliters M9.Then, transfer the eggs to a new, 15-milliliter tube, and incubate tube at 20 degrees Celsius with gentle rotation, and allow the embryos to hatch overnight. The next day, determine the density of the prepared L1 solution by calculating the average number of L1s per microliter in three, 10-microliter drops.Load the prepared bacteria seeded plates with 50 L1 animals, each. Set up the plates in a 20 degree Celsuis incubator, until the embryos develop to the L4 stage. Normal development from L1 to L4 takes about 40 hours.Then, to prevent progeny production, add 160 microliters of 50x FUdR to each plate. Then remove the males, as lifespan is typically measured only with hermaphrodites. Once completed, repack the plates into the 20 degrees Celsuis incubator.Until all the worms are dead, score their viability on a daily basis. To do so, gently touch each non-moving nematode on the head with a platinum wire or eyelash. Non-reactive animals are dead, and must be removed from the plate.Take note of any anomalies during this routine. Replica set and traditional methods for assaying C.elegans lifespan produce similar results with wild-type N2 animals. For example, feeding-based RNAi knockdown of mml-1 or mxl-2 significantly decreased normal lifespans.This was measured by both a traditional lifespan assay, and by the replica set assay. Extended longevity can also be detected. Seen here, RNAi for mdl-1 or mxl-1 significantly increased the lifespans of C.Elegans when measured by either methodology.Using the replica set method, it is possible to simultaneously quantify changes in lifespans across more than 100 conditions, which would not be reasonable with a more traditional approach. For example, a genome-wide feeding-based RNAi screen identified 159 genes necessary for the increased lifespan conferred by decrease daf-2 insulin-like signaling. The relationship between insulin-like signaling and these genes was further described by measuring the health span of the animals.Health was indicated by c. Elegans thrashing when suspended in solution. After watching this video, you should have a good understanding of how to use feeding-based RNAi to measure lifespan of C.Elegans.But RNAi is versatile and can be applied for many different phenotypes. Once mastered, the replica set method allows lifespan to be assessed for over 100 RNAi clones each day, if it is performed properly. Early and late time points can be scored quickly.All the animals in a well will be either alive and moving, or dead. Scoring takes longest when animals are starting to die. Stagger the set up of independent trials with this in mind.

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Research

JoVE Journal

Genetics

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

At the neuromuscular junction (NMJ), the binding of the excitatory neurotransmitter acetylcholine (ACh) to postsynaptic receptors leads to muscle contraction. As in vertebrate skeletal muscle, cholinergic signaling in the body wall muscles of the model organism Caenorhabditis elegans is required for locomotion. Exposure to levamisole, a pharmacological agonist of one class of ACh receptors on the body wall muscles, causes time-dependent paralysis of wild-type animals. Altered sensitivity to levamisole suggests defects in signaling at the NMJ or muscle function. Here, a protocol for a liquid levamisole assay performed on C. elegans grown in 24-well plates is presented. Vigorous swimming of the animals in liquid allows for the assessment and quantitation of levamisole-induced paralysis in hundreds of worms over a one-hour time period without requiring physical manipulation. This procedure can be used with both wild-type and mutants that have altered sensitivity to levamisole to demonstrate the functional consequences of altered signaling at the NMJ.

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

The present protocol describes an assay to determine response to levamisole, a pharmacological agonist of one class of Caenorhabditis elegans acetylcholine receptors. In this liquid levamisole swim assay, researchers visually observe and quantitate the time-dependent paralysis of animals cultivated in 24-well plates.

At the neuromuscular junction (NMJ), the binding of the excitatory neurotransmitter acetylcholine (ACh) to postsynaptic receptors leads to muscle ...

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as self-fertilizing hermaphrodites, which produce large broods of progeny-when males are present, they can produce even larger broods of cross progeny. Errors in meiosis, fertilization, and embryogenesis can be rapidly assessed as phenotypes of sterility, reduced fertility, or embryonic lethality. This article describes a method to determine embryonic viability and brood size in C. elegans. We demonstrate how to set up this assay by picking a worm onto an individual Modified Youngren's, Only Bacto-peptone (MYOB) plate, establish the appropriate timeframe to count viable progeny and non-viable embryos, and explain how to accurately count live worm specimens. This technique can be used to determine viability in self-fertilizing hermaphrodites as well as cross-fertilization by mating pairs. These relatively simple experiments are easily adoptable for new researchers, such as undergraduate students and first-year graduate students.

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Here, we present a general method to determine the embryonic viability and total number of embryos produced (brood) using the model organism C. elegans.

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as ...

The Serial Anesthesia Array for the High-Throughput Investigation of Volatile Agents Using Drosophila melanogaster

Volatile general anesthetics (VGAs) are used worldwide on millions of people of all ages and medical conditions. High concentrations of VGAs (hundreds of micromolar to low millimolar) are necessary to achieve a profound and unphysiological suppression of brain function presenting as "anesthesia" to the observer. The full spectrum of the collateral effects triggered by such high concentrations of lipophilic agents is not known, but interactions with the immune-inflammatory system have been noted, although their biological significance is not understood.
To investigate the biological effects of VGAs in animals, we developed a system termed the serial anesthesia array (SAA) to exploit the experimental advantages offered by the fruit fly (Drosophila melanogaster). The SAA consists of eight chambers arranged in series and connected to a common inflow. Some parts are available in the lab, and others can be easily fabricated or purchased. A vaporizer, which is necessary for the calibrated administration of VGAs, is the only commercially manufactured component. VGAs constitute only a small percentage of the atmosphere flowing through the SAA during operation, as the bulk (typically over 95%) is carrier gas; the default carrier is air. However, oxygen and any other gases can be investigated. 
The SAA's principal advantage over prior systems is that it allows the simultaneous exposure of multiple cohorts of flies to exactly titrable doses of VGAs. Identical concentrations of VGAs are achieved within minutes in all the chambers, thus providing indistinguishable experimental conditions. Each chamber can contain from a single fly to hundreds of flies. For example, the SAA can simultaneously examine eight different genotypes or four genotypes with different biological variables (e.g., male vs. female, old vs. young). We have used the SAA to investigate the pharmacodynamics of VGAs and their pharmacogenetic interactions in two experimental fly models associated with neuroinflammation-mitochondrial mutants and traumatic brain injury (TBI).

The Serial Anesthesia Array for the High-Throughput Investigation of Volatile Agents Using Drosophila melanogaster

The fruit fly (Drosophila melanogaster) is widely used for biological and toxicological research. To expand the utility of flies, we developed an instrument, the serial anesthesia array, that simultaneously exposes multiple fly samples to volatile general anesthetics (VGAs), making it possible to investigate the collateral effects (toxic and protective) of VGAs.

Volatile general anesthetics (VGAs) are used worldwide on millions of people of all ages and medical conditions. High concentrations of VGAs (hundreds of ...