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[
International Worm Meeting,
2021]
In 2016, nearly 800 million tablets of ivermectin were distributed to countries for use in elimination programs for human filarial diseases. Despite its widespread use, the mode of action of ivermectin against filarial nematodes is not well understood, and its in vivo potency cannot be replicated in vitro. To better understand how ivermectin affects filarial worms, our lab previously performed a transcriptomics study to identify differently expressed genes (DEG) in Brugia malayi adults and microfilariae after treatment of infected gerbils. Forty-four of these DEG had C. elegans orthologs available as mutant strains through the C. elegans Genetics Center. We have assayed these mutant strains for differential sensitivity to ivermectin by measuring three phenotypes affected by ivermectin: egg production, development, and motility. We have identified several resistant and hypersensitive strains of C. elegans as well as differences between responses to the three assays. Mutations conferring resistance included those in
che-12 (
e1812), a gene involved in chemotaxis, cilium assembly, and hyperosmotic response; and
inx-14 (
ag17), which is predicted to have gap junction hemi-channel activity and is expressed in the muscular, nervous, and reproductive systems. The
che-12 mutants are additionally resistant to ivermectin's effect on pharyngeal pumping, while
inx-14 mutants are not different from control. Overall, twenty-three genes, with eleven strong candidate genes, have been identified as altering ivermectin sensitivity in at least one assay, supporting the validity of the overall approach. These may give insight into how ivermectin acts against filarial parasites as well as potential mechanisms of resistance. These results are currently being used as the basis of an RNAi screen in B. malayi to identify the effect of these candidate genes on ivermectin sensitivity in filarial parasites.
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[
MicroPubl Biol,
2024]
High-quality DNA extraction from organoids is an important step in molecular genetics research. Here, we show that a lysis buffer from the field of <i>Caenorhabditis elegans</i> research, called Single Worm Lysis Buffer (SWLB), is a low-cost, yet reliable method for DNA extraction from mammalian organoids. SWLB is superior in terms of price, storage, hands-on time and sustainability compared to current standardized DNA extraction protocols, while equally effective. This work indicates that it is useful to compare methods from different model systems, such as mammalian organoids and invertebrate nematodes, to find useful alternatives for research methodologies.
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[
International Worm Meeting,
2015]
Investigation of the neuronal basis of economic decisions would be accelerated by establishing decision making paradigms in simple, genetically tractable organisms, such as the nematode Caenorhabditis elegans. For an organism to be a valid model of economic decision making its choice behavior must be sensitive to: (i) the difference between high and low quality goods, and (ii) the relative cost of those options.Previous work has shown that the nematode worm C. elegans quickly learns to feed on those foods (species of bacteria) that promote higher rates of growth and reproduction. Worms spend more time foraging in patches of Good bacteria (high worm growth rate) versus Mediocre bacteria (moderate growth rate) when equally abundant. Until now, however, it has not been possible to simultaneously present two food choices of different quality and cost. To that end, we have developed an electro-microfluidic device in which a semi-restrained worm forages between contiguous yet discrete fluid streams containing good and mediocre quality food. This arrangement constitutes a two-alternative forced-choice task, analogous to those used in behavioral economics. Electrodes inserted into the device monitor muscular impulses associated with individual swallowing events. Relative consumption of Good and Mediocre food is measured by counting the number of swallowing events in the respective fluid streams. The fraction of total swallowing events in Good vs Mediocre food serves as an index of food preference. Importantly, we can alter the effective prices of the two foods by adjusting the concentration of the bacteria, with price being inversely related to concentration.Here we present behavioral data delineating preference for Good vs Mediocre food across a range of relative prices. We find that worms exposed to the two species of bacteria at equal prices prefer Good bacteria, indicating that feeding preferences are normal in the device. Worms respond to price adjustments as predicted by economic theory in that increasing the relative price of a food leads to a decline in its consumption. In addition, we present calcium-imaging data from sensory neurons showing that they respond to transitions between Good and Mediocre foods, and the amplitude of calcium signal scales with relative food preference. These results show that C. elegans forages in an economic manner, and that relative value is represented at the level of the sensory neurons.
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[
International Worm Meeting,
2017]
Value-based decision making - choices driven by subjective assessments of utility - is a central function of the brain and the focus of intensive study in mammals. Until now, evidence that nematodes are capable of value-based decision making has mainly been suggestive. However, economists have developed formal procedures for determining whether a consumer's decisions are based on subjective value as opposed to random or capricious impulses. We recently developed microfluidic devices that enable such tests to be performed on nematodes for the first time. The worm is held at the confluence of contiguous streams of high and low quality bacterial food leaving its head free to move. Bacteria concentrations are adjusted by the experimenter to change the relative "prices" of the two foods in terms of number of bacteria consumed per pharyngeal pump. Food concentrations can also be adjusted in tandem to increase or decrease the worm's overall consumption possibilities, i.e. "budget." Consumption is measured by counting pharyngeal pumps recorded electrically. Worms typically fed in both streams, consuming a mixture of high and low quality food that was unique for each combination of price and budget. We found that worms make globally rational choices in that they obey transitivity. That is, for all sets of food mixtures A, B, and C, if A is preferred to B, and B to C, then A is preferred to C. As transitivity is the necessary and sufficient condition for value maximization, these data provide formal evidence that C. elegans exhibits value-based decision making. Further, we found that the olfactory neuron AWC, known to be activated by the sudden absence of food, is required for intact food choice behavior. Surprisingly, however, we found that AWC is also activated by the switch from high quality food to low quality food, even when the two foods are at the same concentration (price). Thus, subjective value may be represented at the level of individual olfactory neurons. Our behavioral and neuronal data are consistent with a model in which olfactory neurons represent the subjective value of the local environment to direct behavior toward preferable mixtures of particular foods. To our knowledge, this is the first formal demonstration of value-based decision making in a genetically tractable model organism with a simple nervous system, opening the door to the discovery of conserved genes and neural circuits for rational decision making.
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[
International Worm Meeting,
2009]
Behavioral and developmental choices made by any animal represent a strategy for surviving and reproducing in its environment. The essence of strategy is making decisions on the basis of incomplete information, decisions whose costs and benefits can''t be accurately determined at the time they must be made. Worms make many such choices: the decision to leave low-quality food in search of higher quality, the decision to lay eggs or allow them to hatch internally, and the decision to become a dauer or remain a dauer are examples. Not coincidentally, most of these decisions involve food availability, perhaps the most important environmental variable to a worm. I am trying to quantitatively model such decisions, using mathematical tools developed for financial markets. The L2/L2d decision is particularly interesting, because it appears to be unnecessary. An L2d can become either an L3 or a dauer, while an L2 can only become an L3. Since the L2d can do everything the L2 can do and more, why does the L2 exist? A likely answer is suggested by the work of Golden and Riddle. They showed that L1 L2d L3 pathway takes a few hours more than L1 L2 L3. Under ideal conditions a worm population doubles in 10-11 hours. (This number is calculated from published life-history traits, and is in approximate accord with lab experience.) A delay of 7 hours, therefore, reduces fitness by a factor of 27/10.5 = 0.62. Thus a worm that becomes an L2d pays a price of about 40% of its fitness for the option of eventually becoming a dauer. A worm should become an L2d if it can confidently predict that conditions will be so bad in the future as to cause a decrease of fitness of this magnitude. Most of what we know about dauer formation concerns how the worm evaluates environmental conditions. However, the L2d should also be preferred in highly uncertain environments, since it postpones the dauer decision into the future, when more accurate information will be available. This effect can be modeled using the tools of stochastic calculus, used to price options in financial markets. They predict that the L2/L2d decision should be strongly influenced not only by how good the environment is, but also by how volatile it is.
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[
Worm Breeder's Gazette,
1992]
After tabulating the results of the Worm Plate Survey. we have come up with some interesting results. Most notably. the high variability in prices that labs are paying for their plates, even for the exact same plates from the same supplier, and the fact that most plates are marked up considerably over the actual cost. The replies can be separated into 4 categories: Labs that get plates from Fisher ($29-$58). but wish they had non-vented plates Labs that get non-vented plates via Applied Scientific (~$38) Labs that get plates from Falcon (vented) or Nunc (non-vented) and pay much more Most labs' plates were "slipable" or "semi-stackable", but all labs wanted plates that stack well for easy manual pouring, seeding, carrying, and using. Everyone wanted plates with shallow lids such that the bottoms can be lifted out of the tops for inverted use. Some labs expressed an interest in plates slightly smaller than "60 mm". That number is in quotes because all of the companies' plates have bottoms smaller than 60 mm (e.g. Fisher -54 x 14 mm). We have negotiated with the plastic companies that really make the plates for Fisher, Applied Scientific, etc. (that actually just resell them to you). I have come to the conclusion that we can provide you with better worm plates, the same worm plates cheaper, or in most cases better worm plates cheaper. This is true for every lab. The bottom line is that we can get you top quality non-vented "60 mm" plates (like Applied Scientific's, except fully stackable) for about $29 per 500 case INCLUDING shipping depending on your usage and how many cases you can receive at one time. Several labs have found the non-vented plates last longer without drying out or getting contaminated, compared with normal vented plates, so you should save that way, too. We offer full service shipping (e.g. standing orders and same-day telephone orders, free. Similarly low prices are available on 100 mm and 150 mm plates that exceed industry standards for flatness (reducing media usage) and clarity. The 100 mm are about $27 per 500 case plus shipping; The 150 mm dishes (good for DNA & RNA preps and library platings, with more than 2.25x the surface area of 100 mm dishes) are made thicker and deeper than industry standards and are about $21.50 per 100 case plus shipping. The shipping charge is very low for labs, or groups of labs in one city, that can take delivery of many cases in a single shipment. You can even suggest that your stockroom order plates from us. Call us for an exact price quote depending on your usage and how many cases you can receive at one time. In any case, we'll work things out to save you money. In the future, we can offer inexpensive 35 mm dishes if the community at large can order about 2000 cases per year, so let me know about your needs for other sizes. The response was very mixed about pre-poured plates. We may set that up later, but for now we can help the most by saving you lots on empty petri dishes (and later, maybe media .supplies). We are happy to send out free samples so you can examine the dishes. If we haven't contacted you yet, just give us a call. Respondents: 38 (including 5 anonymous) "Winners": Horvitz = 550, Meyer = 400, Thomas = 400, Greenwald = 300 200-299 cases 8 labs 100-199 cases 7 labs 4-99 cases 19 labs Highest price per case: US = 118.75, Canada = $117 (non-vented) Lowest price per case: US = $29, Canada = $25 (vented) Farthest away response: Malta! No responses from MRC or anyone else in Europe or Asia. It is possible that we can save money and/or provide better plates for these labs, including, shipping, too. Let us know.
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Filippidis, G, Papazoglou, TG, Voglis, G, Kapsokalyvas, D, Tavernarakis, N, Kouloumentas, C
[
J Phys D Appl Phys,
2005]
Two-photon excitation fluorescence (TPEF) and second-harmonic generation (SHG) are relatively new promising tools for the imaging and mapping of biological structures and processes at the microscopic level. The combination of the two image-contrast modes in a single instrument can provide unique and complementary information concerning the structure and the function of tissues and individual cells. The extended application of this novel, innovative technique by the biological community is limited due to the high price of commercial multiphoton microscopes. In this study, a compact, inexpensive and reliable setup utilizing ferntosecond pulses for excitation was developed for the TPEF and SHG imaging of biological samples. Specific cell types of the nematode Caenorhabditis elegans were imaged. Detection of the endogenous structural proteins of the worm, which are responsible for observation of SHG signals, was achieved. Additionally, the binding of different photosensitizers in the HL-60 cell line was investigated, using non-linear microscopy. The sub-cellular localization of photosensitizers of a new generation, very promising for photodynamic therapy (PDT), (Hypericum perforatum L. extracts) was achieved. The sub-cellular localization of these novel photosensitizers was linked with their photodynamic action during PDT, and the possible mechanisms for cell killing have been elucidated.
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[
European Worm Meeting,
2006]
Yohann Duverger1, Jrme Reboul2, Daniel Wong1 and Jonathan Ewbank1 For the last three years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is equipped with a Zymark twister robot, allowing automated analysis of multiple 96 well plates. We recently upgraded the machine to include the Profiler II that generates >1000 individual measurements per worm simultaneously for up to 4 channels (including 2 fluorescent). This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented.. This platform is part of a fully integrated functional genomics facility open to the academic community (see
http://www.ciml.univ-mrs.fr/EWBANK_jonathan /RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays free of charge to the French C.elegans community and at cost price to academic researchers in Europe.. This French functional genomics platform has been made possible through funding from the National Genopole network, Marseille-Nice genopole, the CNRS and support from Union Biometrica.
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[
MicroPubl Biol,
2021]
During the process of cell differentiation, specific cytoskeletal proteins can sequentially assemble into a wide variety of diverse molecular superstructures. Nematode spermatogenesis provides a powerful system for studying these transitions since sperm-specific transcription ceases prior to the meiotic divisions and translation ceases shortly thereafter (Chu and Shakes, 2013). Therefore, structural transitions that follow the meiotic divisions must be carried out by the remodeling of already synthesized proteins. The Major Sperm Protein (MSP) is a nematode-specific cytoskeletal element whose polymerization dynamics drive the pseudopod-based motility of the activated sperm (Roberts, 2005). In C. elegans, MSP additionally functions as the extracellular signaling molecule for triggering both ovulation and oocyte maturation (Miller et al., 2003). MSP is highly abundant in sperm, where it reaches 10-15% of total and 40% of soluble cellular protein (Roberts 2005). Within developing spermatocytes, MSP is packaged into fibrous bodymembranous organelle (FB-MO) complexes (Fig. 1A, Roberts et al., 1986). By assembling into paracrystalline FBs, MSP is both sequestered away from the critical meiotic processes of chromosome segregation and cytokinesis while also being packaged for efficient segregation into spermatids during the post-meiotic partitioning process (Chu and Shakes 2013, Nishimura and LHernault, 2010, Price et al., 2021). Following the meiotic divisions and sperm individualization, FBs disassemble, and MSP disperses as dimers throughout the spermatid cytoplasm (Fig. 1A). When sperm activate to form motile spermatozoa, MSP polymerization within the pseudopod drives the motility of the crawling sperm (Chu and Shakes, 2013). Thus, MSP exists in at least three distinct molecular states: 1) in highly organized paracrystalline FBs within developing spermatocytes 2) as unpolymerized dimers within spermatids, and 3) in dynamically polymerizing filaments and fibers within crawling spermatozoa.
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[
Development & Evolution Meeting,
2008]
Aging is a complex biological process characterized by a decline in the general vitality and functioning of an organism over time. Despite the ubiquity of aging, many open questions remain with regard to the genetic and physiological mechanisms that underlie the aging process, as well as the selective pressures that sculpt natural rates of aging. A leading hypothesis to explain the universality of aging and associated degenerative diseases is the "free radical hypothesis", which states that physical deterioration is a direct result of accumulated cellular and genetic damage caused by reactive oxygen species (ROS), the primary source of which is leakage from the mitochondrial electron transport chain (ETC). As part of our goal to develop a natural model system for studying the genetic and physiological determinants of aging, we have surveyed the pattern of decline in pharyngeal pumping rates among natural isolates of Caenorhabditis briggsae. D. Howe and D. Denver (Oregon State Univ.) recently showed that these isolates harbor variable amounts of non-coding mitochondrial DNA and associated deletions of the NADH dehydrogenase 5 (ND5) gene, the wildtype product of which functions as a central component of complex I of the ETC. Based on recent findings that partial disruption of respiratory-chain components leads to smaller yet longer-lived C. elegans, we expect C. briggsae isolates with high ND5 deletion frequencies to exhibit reduced respiration rates and extended lifespans. Preliminary findings support this expectation and further suggest that worms pay a price for living long with a substantial trade-off in reproductive output. To understand the degree to which extended lifespan in these strains is associated with slowed rates of aging; i.e., prolonged physical health, we have characterized among-isolate variation in rates of pharyngeal pumping decline. We expect that a slowed rate of decline in pharyngeal pumping will accompany extended lifespan in C. briggsae isolates harboring a greater proportion of ND5 deletion-bearing genomes. These data will be combined with life-history, morphological and biochemical data to better understand the relationship between mitochondrial functioning, ROS production, aging and lifespan.