Birchall PS et al. (1993) Worm Breeder's Gazette "FISHing for Complements"

Birchall PS, Albertson DG

[Worm Breeder's Gazette, 1993]

Fluorescence in situ hybridisation (FISH) has been used to detect unc-54 mRNA in post-embryonic stages of N2 . The technique is fast (same-day results) and produces a high resolution signal in about 90% of the sampled nematodes. All washes are done in an eppendorf tube; the worms are spun down, the old wash removed and replaced with 1ml of the next solution. The nematodes are fixed for 6 hours in 3.7% formaldehyde in 0.1M HEPES-KOH (pH 7.5) buffer, permeabilised in 5%- -mercaptoethanol and 50 g/ml Proteinase K and taken into hybridisation buffer (40% formamide, 4xSSC, 0.1M HEPES-KOH (pH 7.0), 80 g/ml sheared salmon sperm DNA, 1.6% SDS and 0.024% SLS). Probes are prepared by one of four methods; PCR, in vitro transcription, nick translation or random priming. In each case, a fluorochrome-conjugated nucleotide is incorporated into the sequence during the polymerase-catalyzed reaction. Hybridization is at 45 C for at least four hours. The nematodes are washed, mounted on a slide, mixed with DABCO (retards fading of the fluorescent signal) and viewed. There is very little background at all (negative controls are hard to find on a slide) and the hybridization signal can be seen with a conventional fluorescence microscope. The nematodes have been simultaneously hybridized with a 1kb ribosomal DNA segment labeled with rhodamine-4-dUTP (Amersham), and with a 1kb unc-54 sequence labeled with fluorescein-12-dUTP (Boehringer Mannheim). In this case, the nucleoli of all the cells appear bright red, the cytoplasm of all the cells appears faintly red and the cytoplasm of the body wall muscle cells appears green, in animals from L1 to adult. These signals are sensitive to RNaseA. An unc-54 -specificsignal is also seen in the vulval and uterine muscles at the L4 /youngadult stage. Embryos are currently being examined. We are extending our investigations to include other genes to gauge the sensitivity and generality of the technique.

Smith RJ et al. (1988) Trop Med Parasitol "Vascular perfusion of Onchocerca volvulus nodules."

Williams JF, Smith RJ, Guderian RH, Cotter TP

[Trop Med Parasitol, 1988]

Perfusion of the vascular bed was achieved in 24 freshly excised nodules of Onchocerca volvulus varying from 0.5 to 2 cm in diameter. India Ink, Microfil polymer, or acrylate perfusates were passed through the vascular supply via cannulation of superficial capsular vessels. After clearing in glycerol or methyl salicylate, or KOH corrosion in the case of the acrylate, nodules were examined microscopically. Small nodules had an extensive blood supply, diffusely distributed throughout the nodule matrix, and in close association with the coils of the worms. In bigger nodules the central area appeared more dense, and intense vascularization appeared to be more peripheral; in the largest nodules the central core was not well vascularized, but a band of heavy vascularization was seen at the margin of the core, fed by superficial vessels and in close contact with worm coils. Very fine branches of the vascular tree were perfused by all three contrast media, but histologically there was evidence of incomplete filling of the smallest vessels. However, there was no extravasation of per-fusates around parasites, even where the approximation between between vessels and parasite surfaces was close. The possibility is considered that O. volvulus may control blood vessel proliferation by release of angiogenesis factors, analogous to rapidly growing solid tumors.

Spasiano D et al. (2016) J Hazard Mater "Removal of benzoylecgonine from water matrices through UV254/H2O2 process: Reaction kinetic modeling, ecotoxicity and genotoxicity assessment."

Russo D, Andreozzi R, Vaccaro M, Li Puma G, Marotta R, Reis NM, Spasiano D, Guida M, Siciliano A

[J Hazard Mater, 2016]

Benzoylecgonine (BE), the main cocaine metabolite, has been detected in numerous surface water and treatment plants effluents in Europe and there is urgent need for effective treatment methods. In this study, the removal of BE by the UV254/H2O2 process from different water matrices was investigated. By means of competition kinetics method, the kinetic constant of reaction between BE and the photogenerated hydroxyl radicals (OH) was estimated resulting in kOH/BE=5.13x10(9)M(-1)s(-1). By-products and water matrices scavengers effects were estimated by numerical modeling of the reaction kinetics for the UV254/H2O2 process and validated in an innovative microcapillary film (MCF) array photoreactor and in a conventional batch photoreactor. The ecotoxicity of the water before and after treatment was evaluated with four organisms Raphidocelis subcapitata, Daphnia magna, Caenorhabditis elegans, and Vicia faba. The results provided evidence that BE and its transformation by-products do not have significant adverse effects on R. subcapitata, while D. magna underwent an increase of lipid droplets. C. elegans was the most sensitive to BE and its by-products. Furthermore, a genotoxicity assay, using V. faba, showed cytogenic damages during the cell mitosis of primary roots.

Junkum A et al. (2003) Mem Inst Oswaldo Cruz "Comparative studies on the biology and filarial susceptibility of selected blood-feeding and autogenous Aedes togoi sub-colonies."

Boonyatakorn C, Junkum A, Choochote W, Jitpakdi A, Komalamisra N, Leemingsawat S, Jariyapan N

[Mem Inst Oswaldo Cruz, 2003]

Blood-feeding and autogenous sub-colonies were selected from a laboratory, stock colony of Aedes togoi, which was originally collected from Koh Nom Sao, Chanthaburi province, Southeast Thailand. Comparative biology and filarial susceptibility between the two sub-colonies (blood-feeding: F11, F13; autogeny: F38, F40) were investigated to evaluate their viability and vectorial capacity. The results of comparison on biology revealed intraspecific differences, i.e., the average egg deposition/gravid female (F11/F38; F13/F40), embryonation rate (F13/F40), hatchability rate (F11/F38; F13/F40), egg width (F11/F38), wing length of females (F13/F40), and wing length and width of males (F11/F38) in the blood-feeding sub-colony were significantly greater than that in the autogenous sub-colony; and egg length (F11/F38) and width (F13/F40), and mean longevity of adult females (F11/F38) and males (F13/F40) in the blood-feeding sub-colony were significantly less than that in the autogenous sub-colony. The results of comparison on filarial susceptibility demonstrated that both sub-colonies yielded similar susceptibilities to Brugia malayi [blood-feeding/autogeny = 56.7% (F11)/53.3%(F38), 60%(F13)/83.3%(F40)] and Dirofilaria immitis [blood-feeding/autogeny = 85.7%(F11)/75%(F38), 45%(F13)/29.4%(F40)], suggesting autogenous Ae. togoi sub-colony was an efficient laboratory vector in study of filariasis.

Witvliet, D. et al. (2017) International Worm Meeting "The C. elegans connectome consist of invariant, stochastic, and developmentally regulated synapses."

Berger, D.R., Wang, J., Cook, S.J., Holmyard, D., Lichtman, J.W., Neubauer, M., Laskova, V., Zhen, M., Emmons, S.W., Mulcahy, B., Samuel, A.D.T., Kersen, D., Chisholm, A.D., Schalek, R.L., Koh, W.X., Qian, J., Hall, D.H., Mitchell, J.K., Chang, M., Witvliet, D.

[International Worm Meeting, 2017]

When animals are born, neuronal networks are already in place to integrate sensory cues and effect appropriate behavioral or homeostatic responses. During the course of postnatal development, synapses and circuits undergo refinement and remodeling, updating or adapting sensorimotor behaviors. Rules for developmental remodeling are poorly characterized, because a circuit-level analyses at multiple time points, in multiple animals are missing. We use an isogenic C. elegans N2 population to examine how the neural circuit changes during development. At birth, C. elegans has 218 neurons (excluding CAN); 82 new neurons are incorporated into the nervous system before the end of larval development. These include sensory, motor, and interneurons that are located throughout the body, suggesting a system-wide modification of the juvenile circuit during post-embryonic development. Using serial-section electron microscopy, we mapped the synaptic connectivity in the head and tail ganglia for five animals, from hatching to late larval development. These datasets allow us to separate the connectome into core connections, synapses that are present throughout the lifetime, transient connections, synapses that are present only during early or late development, and variable connections, synapses that are not conserved between animals. We propose that the core connections may drive hard-wired behaviors, and developmentally regulated connections exert stage-specific roles, while variable connections may confer individual variability and/or be stochastic in nature. Our current data analyses lead to two notions: First, developmentally regulated connections are enriched for synapses between sensory neurons, and with neuromodulatory neurons, while connections between interneurons are remarkably stable. This implies that for C. elegans, core circuits for decision-making are already established at birth, but their modulation and multi-sensory integration are refined or shaped during development. Second, variable connections, comprising about half of the connection edges in the published adult dataset, are prominent. Stochastic or experience dependent variability in connections may contribute to variability of behaviors set by hard-wired core connections.

Witvliet, D. et al. (2019) International Worm Meeting "Structural plasticity of the C. elegans connectome across development and between individuals."

Neubauer, M., Witvliet, D., Mitchell, J.K., Maeng, S., Meirovitch, Y., Chisholm, A.D., Wang, M.D., Shavit, N., Wang, Z., Ho, C.Y., Samuel, A.D.T., Lichtman, J.W., Koh, W.X., Schalek, R.L., Rehaluk, C., Mulcahy, B., Holmyard, D., Berger, D.R., Zhen, M., Cook, S.J.

[International Worm Meeting, 2019]

Nervous system development is widely thought to be optimized for precise wiring and efficient networks. Maintaining plasticity, however, is necessary for animals to adapt, learn, and evolve. This is especially evident during postembryonic development when newly born neurons are integrated into functioning neurocircuits. Rules for circuit plasticity and variability during and after development are poorly because circuit-level analyses across multiple animals and developmental time points are missing. Using electron microscopy, we mapped the connectome of the central nervous system for eight isogenic C. elegans animals spanning from birth to adulthood. These datasets reveal a highly dynamic circuit architecture, with a five-fold increase in synapse number across postembryonic development. Synapses are built and removed for multiple reasons: (1) to strengthen a remarkably stable core circuitry for decision-making, (2) to fine-tune multi-sensory integration and motor responses by building new unique cell-cell connections, (3) to streamline circuit computations, and (4) to fill connectivity gaps as a result of evolutionary pressures to hatch before initial wiring is complete. This remodeling is driven independently of the contact area between neurons and instead is dependent on neuron class and centrality. Remodeling is overrepresented between connections from sensory neurons to motor and interneurons, while connections between interneurons are remarkably stable. Well-connected, central neurons (typically interneurons), are more heavily regulated than other neurons, but surprisingly this regulation does not originate from other central neurons. Thus, the driving force for synaptic growth is not uniform, and may depend on neuron identity, suggesting an underpinning by genetic or functional identity. Together, our findings show that even in one of the most deterministic animals, nervous system wiring retains a high degree of plasticity. The connectome should be separated into core connections, that are present throughout life, developmentally changing connections, that depend on the stage of development, and variable connections, that are not conserved among individuals. Core connections are the strongest class of connections, but they make up less than half of all connections. The prevalence of wiring plasticity implicates relevance to nervous system adaptation and variability. Our data will be available on nemanode.org before the end of the summer.

Hans S et al. (2019) J Glob Antimicrob Resist "Magnesium deprivation affects cellular circuitry involved in drug resistance and virulence in Candida albicans."

Hameed S, Fatima Z, Hans S

[J Glob Antimicrob Resist, 2019]

OBJECTIVES: Candida albicans has to struggle for the limited micronutrients present in the hostile niche. One such significant environmental factor that C. albicans must surmount is magnesium (Mg) limitation. Herein, we aimed to study the effect of Mg deprivation on drug resistance mechanisms and virulence traits of C. albicans. METHODS: Drug susceptibilities against C. albicans SC5314was measured by broth microdilution and spot assay. Drug efflux pump activity was measured by substrate R6G efflux. Membrane intactness was studied by propidium iodide influx and ergosterol levels by alcoholic KOH method. Metabolic flexibility was studied by enzymatic activities of glyoxylate cycle. Virulence factors were assessed by yeast to hyphae, biofilm formation and cell adherence. In-vivo study was performed by Caenorhabditis elegans infection model. RESULTS: We found that Mg chelation leads to potentiation of membrane targeting antifungals. We explored the role of Mg on membrane homeostasis and found significant differences in ergosterol levels. Interestingly we also explored that Mg deprivation impedes metabolic flexibility of C. albicans SC5314 by inhibiting glyoxylate cycle enzymes. Furthermore, Mg deprivation inhibited potential virulence traits including morphological transition, biofilm formation and buccal epithelial cell adherence. All the disrupted targets were validated by RT-PCR which corroborates our findings. Lastly, we demonstrated the enhanced survival of C. albicans SC5314 infected Caenorhabditis elegans model under Mg deprivation. CONCLUSION: In view of the restricted growth of C. albicans in Mg deficient environment, approaches could be utilized to boost the effectiveness of existing antifungals thereby improving the management of fungal infections.

Leung, K.L. et al. (2017) International Worm Meeting "Hydrolysis of fluorescent 7-hydroxy-4-trifluoromethylcoumarin to non-fluorescent metabolite in C. elegans."

Leung, K.L., Wong, G., Juvonen, R.O.

[International Worm Meeting, 2017]

During aging and stress, nematodes may encounter a variety of xenobiotics in their environment. Organisms such as C. elegans protect themselves against xenobiotics by transforming them to water soluble excretable metabolites. A sensitive and convenient method to study this phenomenon is to use fluorescent or pro-fluorescent model xenobiotics. The aim of this study was to determine if fluorescent 7-hydroxy-4-trifluoromethylcoumarin (HFC) is transformed to a non-fluorescent metabolite by C. elegans. When 6.25 M HFC was incubated with live and frozen C. elegans in M9 solution, the fluorescence decreased immediately in incubation with the live worms and after six to eight hours after melting and keeping the frozen worms at room temperature. The decrease of fluorescence was enzyme dependent, since heating the worms at 100 deg C for 20 min prevented the reaction. The magnitude of the decrease of fluorescence depended on time and number of worms. In 10 mM KOH alkaline condition the fluorescence of HFC disappeared in one hour. We could detect 3-(2,4-dihydroxyphenyl)-4,4,4-trifluorobut-2-enoic acid (DiHPFEacid) in the incubates with both alkaline solution and C. elegans. Therefore we conclude that lactone of HFC is hydrolyzed to DiHPFEacid via an enzymatic reaction in C. elegans. The enzyme responsible for this reaction in C. elegans remains unknown, however western blot data suggests the presence of an ortholog of CYP2A6, an enzyme which catalyzes this reaction in higher organisms. Taken together, these results provide a convenient whole animal model system to study fluorescent based xenobiotic metabolism.

Anna Elisabetta Salcini et al. (2006) European Worm Meeting "C. elegans Intersectin: A Synaptic Adaptor Protein Coupling Endocytic Membrane Traffic to Exocytosis"

Anna Elisabetta Salcini, Simon Rose, Maria Grazia Malabarba, Pier Paolo Di Fiore

[European Worm Meeting, 2006]

Anna Elisabetta Salcini1, Simon Rose1, Maria Grazia Malabarba2, Pier Paolo Di Fiore2 . Intersectin family proteins are scaffold proteins believed to play a role in endocytosis. Intersectins have a modular organization with two N-terminal EH domains (Eps15 homology), a central coiled-coil region and five SH3 domains in the C-terminal region. A long, brain-specific isoform contains at the C-terminal an additional DH domain (Dbl homology), a PH (Pleckstrin homology) and a C2 domain. Intersectin proteins were identified by several groups based on their ability to bind proteins involved in a variety of processes, such as endocytosis, exocytosis, signal transduction pathways and actin remodeling. Recently, in vivo studies in Drosophila melanogaster indicated a function for Dap160/Intersectin in synaptic development and in the stabilization of an endocytic macromolecular complex, since the levels of several endocytic molecules such as dynamin, synaptojanin and endophilin are severely reduced in dap160 mutant NMJs (Marie et al. 2004; Koh et al., 2004). We will present the analysis of Y116A8C.36 gene, encoding for ITSN-1 protein, the unique orthologue of the intersectin family in C. elegans. We will discuss our data showing that 1) ITSN-1, differently then in Drosophila melanogaster, is not an essential gene in C.elegans. 2) ITSN-1 protein is mainly expressed in neuronal cells and it is localized in synaptic vesicle-rich regions. 3) ITSN-1 plays a role in synaptic transmission, controlling acetylcholine release. 4) itsn-1 genetically interacts with dyn-1 and ehs-1 genes, both implicated in compensatory endocytosis at the synapses. 5) ITSN-1 protein interacts in vivo with EHS-1 and Dynamin proteins.. Our in vivo studies challenge the participation of ITSN-1 protein in exocytic processes and in the control of synaptic vesicle cycling suggesting a role for ITSN-1 in temporal and spatial coordination of exocytosis and endocytosis.

Maduro MF et al. (1998) West Coast Worm Meeting "GOTTA LOTTA GATAs: THE DIVERSE ROLES OF GATA FACTORS IN C. ELEGANS EMBRYOS"

Rothman JH, Koh K, Newman-Smith ED, Maduro MF, Strohmaier K

[West Coast Worm Meeting, 1998]

Zinc-finger transcription factors of the GATA type (which bind the consensus sequence WGATAR) are known in fungi, worms, flies and vertebrates. In animals, GATA factors are implicated in regulating differentiation of a range of different cell types. Six GATA factors, each with two fingers, have been identified in vertebrates, three of which are involved in hematopoesis, and three others in formation of organs such as heart and endodermal organs. To evaluate the range of developmental events that GATA factors might direct in an animal, we are analyzing the expression and function of a number of C. elegans GATA factors. From the C. elegans genomic sequence, we can now deduce how many GATA factors are required to make a metazoan. Five were known from genetic and molecular studies: ELT-1 and ELT-3 are involved in development of the embryonic epidermis (Page et al. 1997; Gilleard and Shafi, wm97e186) and ELT-2, END-1 and END-3 regulate specification of the endoderm and differentiation of the gut (Fukushige et al. wm97e169; Zhu et al. 1997; Maduro et al. this meeting). Blast searches of the C. elegans sequence database led us to identify at least four more apparent GATA factors, tentatively called ELT-4 through 7. All appear to be single-finger GATA factors. elt-4 and elt-5, adjacent genes on the left end of IV, appear to correspond to a polycistronic unit; these genes are expressed in, and appear to be involved in the development of, seam cells among other cell types (see abstract by Koh et al.). Although GFP constructs with elt-6 X (T24D3.1) have been uninformative, and no phenotype was observed by RNAi, ectopic expression of ELT-6 shows that it can promote pharynx development at the expense of other cell types: hs-elt-6 embryos often arrest with large numbers of pharynx muscles and no intestine (i.e., the reciprocal effect of the endoderm-specific GATA factors). Analysis of elt-7 V (cosmid C18G1) is in progress. Our tentative conclusion is that most, if not all GATA factors in C. elegans, function in distinct embryonic specification and/or differentiation events. References: Page et al. (1997) Genes & Dev. 11: 1651-1661. Zhu et al. (1997) Genes & Dev. 11: 2883-2896.