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[
International Worm Meeting,
2021]
Across the animal kingdom, ribonucleoprotein condensates called germ granules are required to maintain the immortality and totipotency of the germ lineage. Since their discovery, C. elegans germ granules, called P granules, have been extensively characterized to understand the role of germ granules in metazoan development. However, studies to-date have been impeded by incomplete knowledge of the P granule proteome. To broadly define the proteins localizing to P granules, we employed a proximity-based labeling approach by tagging known P granule proteins with a promiscuous biotin ligase. In our biotinylated protein fraction, we uncovered over 150 novel P granule proteins. Notably, we identified a pair of previously uncharacterized proteins, which we named EGGD-1 and EGGD-2 for Embryonic and Germline P Granules Detached. We find that EGGD-1 is enriched at the base of P granules, and its depletion causes P granules to separate from the nuclear envelope in the adult germline and embryonic primordial germ cells. Further, loss of
eggd-2 exacerbates the P granule mislocalization in
eggd-1 mutant animals. EGGD-1/2 harbor four domains: two intrinsically disordered regions and two putative LOTUS domains. Previous work in Drosophila and mice showed LOTUS domain proteins directly recruit Vasa RNA helicases to organize germ granules, yet the role of LOTUS domain proteins in C. elegans is uncharacterized. Our ongoing work examines the interactions between GLH proteins (C. elegans Vasa orthologs) and EGGD proteins using mutational analysis to determine contributions of individual domains in P granule perinuclear localization. Together, our work defines the P granule proteome and provides novel insights into principles of germ granule formation and perinuclear attachment.
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[
J Appl Microbiol,
2012]
AIMS: Indole is a signalling molecule, produced by a number of Gram-positive and Gram-negative bacteria both in nature as well as clinical environments. Here, we explored the effect of bacterial indole and one of its main derivatives on the virulence of the fungal pathogen Candida albicans. METHODS AND RESULTS: We found that indole and its derivate indole-3-acetonitrile (IAN) did not affect the viability of C. albicans. Interestingly, indole and IAN repressed C. albicans biofilm formation as well as the attachment of C. albicans to intestinal epithelial HT-29 cells and inhibited the ability of the yeast to make filaments that are the main virulence factor of C. albicans. In addition, we used the heterologous model host Caenorhabditis elegans to demonstrate in vivo that the presence of indole or IAN attenuates C. albicans infection (P = 0.0188 and P < 0.0001 for indole and IAN, respectively, compared to worms exposed to C. albicans DAY185 alone) and decreases fungal colonization in the nematode gut. Importantly, quantitative real-time polymerase chain reaction (qRT-PCR) results showed that in C. albicans, indole and IAN strongly stimulated the transcription of NRG1. CONCLUSIONS: Indole and IAN attenuates fungal virulence by regulating the transcription of NRG1, a transcriptional factor that influences filamentation and biofilm formation in C. albicans. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings indicate that the bacterial signalling molecules indole and its derivatives play an inter-kingdom role in dynamic network of microbiota and directly modulate the virulence of fungal C. albicans via NRG1.
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[
International Worm Meeting,
2021]
The Piwi/piRNA pathway suppresses transposable elements and promotes fertility in diverse organisms. Maturation of piRNAs involves pre-piRNA trimming followed by 2′-O-methylation at their 3' termini. We show that 3' termini of C. elegans piRNAs are subject to nontemplated nucleotide addition and piRNAs with 3' addition exhibit extensive base-pairing interaction with their target RNAs. Animals deficient for PARN-1 (pre-piRNA trimmer) and HENN-1 (2′-O-methyltransferase) accumulate piRNAs with 3' nontemplated nucleotides. In
henn-1 mutants piRNAs are shortened prior to 3' addition whereas long isoforms of untrimmed piRNAs are preferentially modified in
parn-1 mutant animals. Loss of either PARN-1 or HENN-1 results in modest reduction in steady-state levels of piRNAs. Deletion of both enzymes leads to depletion of piRNAs, desilencing of piRNA targets, and impaired fecundity. Together, our findings suggest that pre-piRNA trimming and 2′-O-methylation act collaboratively to protect piRNAs from tailing and degradation.
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[
Parasitol Today,
1996]
Historically, peptidergic substances (in the form of neurosecretions) were linked to moulting in nematodes. More recently, there has been a renewal of interest in nematode neurobiology, initially triggered by studies demonstrating the localization of peptide immunoreactivities to the nervous system. Here, David Brownlee, Ian Fairweather, Lindy Holden-Dye and Robert Walker will review progress on the isolation of nematode neuropeptides and efforts to unravel their physiological actions and inactivation mechanisms. Future avenues for research are suggested and the potential exploitation of peptidergic pathways in future therapeutic strategies
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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,
2009]
The Green Fluorescent Protein (GFP) gene from the fluorescent jellyfish Aequorea victoria, and its variants (such as EGFP), are used extensively to study the location and timing of gene expression in transgenic animals and plants (Chalfie et al, 1994). Resultant GFP fusion proteins are especially well-suited to studying in vivo gene expression patterns in the transparent nematode, C. elegans and the transparent larva of the fly D. melanogaster. Perhaps one of the most significant limitations to its use in large-scale genetic screens is the high cost of equipment needed to observe GFP microscopically - namely the Fluorescence Dissecting Stereomicroscope for screening and picking mutants and upright and inverted epi-fluorescent compound microscopes for more detailed studies. Commercially available Fluorescence Dissecting Stereomicroscopes typically sell in the midrange between US$10,000 and US$20,000. They are offered by only the "high-end" microscope companies, based upon their most expensive dissecting scopes, and incorporate their premium-priced mercury arc-lamp illuminators, power supplies and epi-fluorescence modules. This leads us to wonder whether it is possible to cut corners without sacrificing utility, and which corners can be cut. Since the inception of GFP-based expression screens, a variety of researchers have produced custom-made and home-made GFP dissecting scopes. These include, for example, Welcome Bender (Harvard Medical School, pers. comm.) and Ian Chin-Sang (Chin-Sang, 2004). There are several possibilities to consider toward lowering the cost of a Fluorescence Dissecting Stereomicroscope. It may be possible to get sufficient light from relatively inexpensive, long-lived, lower-power-consuming Light Emitting Diodes (LEDs) vs. mercury arc lamps. Depending upon the spectral specificity of the LEDs, filters or dichroic mirrors might be omitted. Getting enough light intensity of the precise wavelengths that excite GFP fluorescence focused onto the sample is important. The exciting beam can be provided directly or focused backward through the microscope using "epi-illumination". We will explore these possibilities and report (and possibly demonstrate) the results. Chin-Sang, Ian. (2004) GFP Stereoscope Using LED light source. Queen''s University, Kingston, ON, Canada.
http://130.15.90.245/gfp_stereoscope.htm. -
[
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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[
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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[
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.