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[
International Worm Meeting,
2021]
In nature, C. elegans live in a rich 3-dimensional environment. However, their behavior has been assessed almost exclusively on the open, flat surface of NGM (Nematode Growth Medium) plates, the golden standard for C. elegans culture in the lab. We present a method to build 3-dimensional behavioral arenas for C. elegans by directly 3D-printing NGM hydrogel. This is achieved by using a highly customized fused deposition modeling (FDM) 3D-printer, extensively modified to employ NGM hydrogel as ink, i.e., the Parnon Printer. The result is the advancement of 3-dimensional complexity of behavioral assays. To demonstrate the potential of our method, we use the 3D-printed arenas to assess C. elegans physical barrier crossing ability, in the context of aging (young, middle-aged adults), feeding history (fully fed, starved animals) and prior experience (have been or not in the presence of a similar 3D structure before). We also explore the usage of 3D-printed structures to spatially confine C. elegans egg laying behavior. C. elegans behavior in 3-dimensional environments is by definition not possible to explore on standard flat NGM plates. Therefore, the findings reported here would likely not have been brought to light if the Parnon Printer had not been developed. We consider these work a decisive step toward characterizing C. elegans 3-dimensional behavior, an area long overlooked due to technical constrains. We envision our method of 3D-printing NGM arenas as a powerful tool in behavioral neurogenetics and neuroethology.
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[
Cryo Letters,
2013]
Cryopreservation of nematode Caenorhabditis elegans in the adult stage is of importance as the nematode is a powerful research model organism. In this study, we applied the protocol previously established for cryopreservation of the L4 nematode to the adult one, and achieved a survival rate of 84%. When ice seeding was induced with bacteria P. syringae directly added to the nematode suspension instead of using a pre-cooled steel sticking needle, comparable survival rate was obtained after thawing. Moreover, a simple freezing device composed of a polystyrene foam box surrounded by a Dewar vessel put in a deep freezer was developed for a practical use. This simple method obtained a survival rate of 69 +/- 4% for the adult nematode after thawing.
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[
J Nematol,
1970]
The effects of electric shock on Panagrellus redivivus adults and larvae and Meloidogyne incognita acrita larvae were studied. The nematodes were placed in tap water between two stainless steel electrodes, spaced 2 mm apart and cemented to a glass slide. Electric potentials of 1, 5, 10, 15, 20, 30, and 60 vdc/mm and vac/mm were applied for periods of 1 sec to 5 rain at 0.05 to 77 ma. The results demonstrated that ac or dc electric shocks as low as 5 v/mm for larvae and 10 v/mm for adults can be lethal. Some larvae and eggs within the body of P. redivivus females were not affected at 600 v/ram. Potentials of 20 and 60 vdc/mm for 2-sec stimulated hatch of Meloidogyne eggs.
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[
Biomater Sci,
2019]
Biomaterial-associated infections caused by bacterial contamination and the subsequent formation of biofilms on the surfaces are challenges faced by our healthcare system. In this work, povidone-iodine-functionalized fluorinated copolymers with stable antibacterial, antibiofilm, and antifouling activities were designed and prepared by a two-step synthesis. First, a series of poly(hexafluorobutyl methacrylate-co-N-vinyl-2-pyrrolidone), i.e., P(HFBMA-NVP), were synthesized by radical copolymerization at different feed ratios to acquire water insoluble and antifouling copolymers. At the second step, the NVP segments in the copolymer were complexed with iodine to obtain the objective antibacterial and antifouling copolymer P(HFBMA-NVP)-I. The chemical and physical characteristics of the copolymers were investigated using 1H NMR, FTIR, XPS, EDX, UV-Vis, SEM, TEM, elemental analysis, and contact angle measurement. P(HFBMA-NVP)-I exhibited excellent antibacterial activity against both Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus), as well as good biocompatibility towards human hepatocyte cells (L02) and Caenorhabditis elegans. Using the electrospinning or spraying technique, P(HFBMA-NVP)-I was coated on polystyrene slides, medical stainless steel sheets, and cotton fabric, allowing the surfaces to have stable antibacterial and antibiofilm activities against pathogenic bacteria and antifouling capability against foulants and blood, and exhibit excellent self-cleaning properties.
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[
Exp Gerontol,
1975]
A technique is described for the continuous growth of an age-synchronized population of Caenrohabditis elegans. After initial isolation of newly hatched individuals from a mixed age culture, synchrony through the reproductive phase was sufficiently well maintained to ensure that over 90% of the post-reproductive population belonged to the original generation. The technique is based on culturing the nematodes on the upper surface of a fine stainless steel mesh through which only larvae, but not adults, are small enough to burrow. It largely overcomes serious disadvantages of the two previously used methods for maintaining synchrony which were based on the use of inhibitors or the manipulation of individual worms. Supplementary techniques are described for the handling and initial synchronization of the worms which are cultured in a sterile completely defined medium. General features of the development and senescence of the worms under our conditions are reported. Mean life span (50% survival) was approx. 58 days and maximum longevity in excess of 80 days. Reproduction lasted from the 5th to about the 26th day of age and its termination coincided with a short period of high mortality. Growth in size of the worms ceased during the reproductive period but recommenced for about 25 days in worms surviving the post-reproductive mortality.
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[
Pathog Dis,
2016]
The current study was intentionally focused on cyclo(L-leucyl- L-prolyl) (CLP)-a cyclic dipeptide with myriad pharmaceutical significance, to explore its antivirulence efficacy against the predominant food-borne pathogen-Listeria monocytogenes (LM). Minimum inhibitory concentration (MIC) of CLP against LM ATCC 19111 was found to be 512 g mL(-1). CLP at sub-MICs (64,128, 256 g mL(-1)) demonstrated a profound non-bactericidal dose-dependent antibiofilm efficacy (on polystyrene and glass) against LM, which was further confirmed through confocal and scanning electron microscopic analysis (on stainless steel surface). In vitro bioassays divulged the phenomenal inhibitory efficacy of CLP towards various virulence traits of LM, specifically its overwhelming suppression of swimming and swarming motility. Data of in vivo assay using Caenorhabditis elegans signified that the plausible mechanism of CLP could be by impeding the pathogen's initial adhesion and thereby attenuating the biofilm assemblage and its associated virulence. This was further confirmed by significant decrease in exopolymeric substance, auto-aggregation, hydrophobicity index and extracellular DNA (eDNA) of the CLP treated-LM cells. Collectively, the current study unveils the antivirulence efficacy of CLP against the Gram-positive food borne-pathogen and the strain Bacillus amyloliquifaciens augurs well to be a promising probiotic in controlling infections associated with LM.
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Toscanesi M, Tommasi F, Oral R, Mozzillo M, Gravina M, D'Ambra L, Siciliano A, Pagano G, Trifuoggi M, Thomas PJ, Lyons DM, Palumbo A, Crisci A, Guida M, Buric P
[
Environ Monit Assess,
2018]
Adverse environmental conditions in the Taranto area (southern Italy) were investigated in studies of air, marine sediment, and human health. The present study aimed at providing unprecedented information on soil pollution and toxicity in a set of sites around recognized pollution sources in the Taranto area, since previous studies were focused on marine or air pollution, or on human health effects. The investigated area included a steel foundry and a power plant, as well as some sites located in an adjacent neighborhood. Surface soil samples and urban dust were collected and submitted to inorganic and organic analyses and tested for toxicity in two invertebrate bioassay models; a sea urchin (Sphaerechinus granularis) and an annelid (Caenorhabditis elegans). Inorganic analysis was carried out using ICP-MS for elemental composition for a total of 34 elements, whose levels were evaluated as a function of bioassay data analyzed through principal component analysis (PCA). Other analyses included asbestos search by powder X-ray diffraction (PXRD) and organic analysis for polycyclic aromatic hydrocarbons (PAHs) and aliphatic compounds (C10-C40). Toxicity bioassays were carried out on a sea urchin (Sphaerechinus granularis), and an annelid (Caenorhabditis elegans). Sea urchin bioassays evaluated effects of topsoil or street dust sample exposures (0.1 to 0.5% dry wt/vol) on developing embryos and on sperm, and scored as (a) % developmental defects, (b) inhibition of fertilization success and offspring damage, and (c) frequencies of mitotic aberrations. C. elegans mortality assay displayed significant toxicity associated with soil samples. The overall effects of samples showed very high toxicity at four out of nine sites. These effects were consistent with the highest levels measured for metals and PAHs. Further studies of health effects related to dust exposures in residential areas are warranted. Graphical abstract .
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[
Worm Breeder's Gazette,
1977]
For several years we have been studying age-related changes in enzymes isolated from the free-living nematode Turbatrix aceti. This has required the large-scale cultivation, synchronization and aging of nematodes in axenic, chemically defined media. Organisms are grown in one liter Roux bottles containing 50 ml of medium for 14 to 16 days ant then harvested by filtration with a fritted glass Buechner funnel. Small individuals are separated by 'screening' through stainless steel filter cloth and transferred to a medium containing fluorodeoxyuridine (FUDR) plus uridine (100 g/ml and 150 g/ml, respectively). FUDR inhibits reproduction, but not the life-span of the nematodes and thereby permits the development and ageing of cultures without contamination by newborn organisms. Growth of the organisms in length is nearly normal during the aging process. Turbatrix can also be synchronized by increasing the incubation temperature to 36 C or by repeated removal of small larvae from cultures. Biochemical characteristics of nematodes obtained by these methods appear to be similar to those of FUDR-treated worms. The yield of organisms in mixed culture on a wet weight basis is approximately 2 grams per 50 ml of medium in a period of two weeks. Our maximum incubator capacity is 500 Roux bottles, resulting in a potential production of a kilogram of nematodes per fortnight. Quantities available from aged worms are only 10 to 20% of this figure due to losses in screening and to the limitations of the worm concentration which is permissible in 'ageing' cultures. Based on our experience with Turbatrix and Caenorhabditis briggsae and various methods in the published literature, it is reasonable to conclude that C. elegans can be synchronized and aged under similar conditions. An advantage to using this organism is its ability to grow in cultures which are aerated by mechanical agitation. The use of rotary shaker cultures permits a greater yield of organisms and more economy of space than static cultures. Unfortunately, T. aceti will not grow under the former conditions. The culture of nematodes in completely defined medium offers an advantage for certain biological studies. However, C. elegans has a requirement for an unidentified growth factor present in soy-peptone. Elucidation of the structure of this material is therefore of importance if we are to fully utilize this organism for ageing studies. We also believe that the cultivation of C. elegans in an axenic system has definite potential for developmental biology and genetics.
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[
Neurotoxicology,
2012]
Manganese (Mn) is a well established neurotoxin associated with specific damage to the basal ganglia in humans. The phenotype associated with Mn neurotoxicity was first described in two workers with occupational exposure to Mn oxide (Couper, 1837). Although the description did not use modern clinical terminology, a parkinsonian illness characterized by slowness of movement (bradykinesia), masked facies, and gait impairment (postural instability) appears to have predominated. Nearly 100 years later an outbreak of an atypical parkinsonian illness in a Chilean Mn mine provided a phenotypic description of a fulminant neurologic disorder with parkinsonism, dystonia, and neuropsychiatric symptoms (Rodier, 1955). Exposures associated with this syndrome were massive and an order of magnitude greater than modern exposures (Rodier, 1955; Hobson et al., 2011). The clinical syndrome associated with Mn neurotoxicity has been called manganism. Modern exposures to Mn occur primarily through occupations in the steel industry and welding. These exposures are often chronic and varied, occurring over decades in the healthy workforce. Although the severe neurologic disorder described by Rodier and Couper are no longer seen, several reports have suggested a possible increased risk of neurotoxicity in these workers (Racette et al., 2005b; Bowler et al., 2007; Harris et al., 2011). Based upon limited prior imaging and pathologic investigations into the pathophysiology of neurotoxicity in Mn exposed workers (Huang et al., 2003), many investigators have concluded that the syndrome spares the dopamine system distinguishing manganism from Parkinson disease (PD), the most common cause of parkinsonism in the general population, and a disease with characteristic degenerative changes in the dopaminergic system (Jankovic, 2005). The purpose of this symposium was to highlight recent advances in the understanding of the pathophysiology of Mn associated neurotoxicity from Caenorhabditis elegans to humans. Dr. Aschner's presentation discussed mechanisms of dopaminergic neuronal toxicity in C. elegans and demonstrates a compelling potential role of Mn in dopaminergic degeneration. Dr. Guilarte's experimental, non-human primate model of Mn neurotoxicity suggests that Mn decreases dopamine release in the brain without loss of neuronal integrity markers, including dopamine. Dr. Racette's presentation demonstrates a unique pattern of dopaminergic dysfunction in active welders with chronic exposure to Mn containing welding fumes. Finally, Dr. Dydak presented novel magnetic resonance (MR) spectroscopy data in Mn exposed smelter workers and demonstrated abnormalities in the thalamus and frontal cortex for those workers. This symposium provided some converging evidence of the potential neurotoxic impact of Mn on the dopaminergic system and challenged existing paradigms on the pathophysiology of Mn in the central nervous system.
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[
International Worm Meeting,
2021]
The platinum worm pick, a fixture in C. elegans laboratories for decades, has two drawbacks: (1) the high cost of platinum, a significant problem in many educational settings, and (2) the reliance on an open flame for sterilization, which presents safety hazards. To address the first drawback, we evaluated whether platinum could be replaced with an alternative metal. An ideal worm pick cools quickly after heating, withstands high temperature without degradation, can be flattened and shaped easily, and is inexpensive. With these criteria in mind, we compared 90% platinum, 10% iridium wire (PT9010) with 5 alternatives: stainless steel (SS), Nickel 200, two nickel chromium (Nichrome) alloys, and iron-chromium-aluminum (FeCrAl). To measure cooling rate we built a circuit to resistively heat wires (all 255 microm in diameter) to 800 C and measured the time it took them to cool to 25 C. We found that PT9010 and FeCrAl cooled more rapidly (6-7 s) than the other metals tested (8-9 s). To assay heat resistance we conducted a bending test after 3000 heating cycles of duration 4 s at 800 C. All materials except SS showed good heat resistance, withstanding >50 bends after 3000 heating cycles. SS exhibited poor heat resistance, breaking spontaneously after ~300 cycles. All materials could be easily flattened using standard tools. With regard to cost, all alternative materials were < 0.20 USD/m, as compared to 140 USD/m for PT9010. These results show that all metal alloys tested except for SS represent reasonable, economical alternatives for worm picks. The most promising is FeCrAl which cools as rapidly as platinum, exhibits good heat resistance, and is available at a fraction of the cost. Next, to explore an alternative to flame sterilization, we designed an electric worm pick consisting of a loop of PT9010 or FeCrAl wire attached to a handle containing a rechargeable battery and circuit board. Depressing a button causes current to flow through the loop, heating it to about 800 C within 2 s. A battery charge lasts for ~500 sterilizations. Worm researchers who tested the device reported that the wire loop could be used similar to a worm pick and that electric sterilization promoted faster work since no movements to a flame were necessary. Our device represents a convenient and safer alternative to flame-sterilized worm picks. We are using a similar loop-based worm picking technique in our automated worm picking system (see abstract by Zihao Li et al).