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[
Biomed Res Int,
2018]
This study was aimed to evaluate the <i>in vitro</i> lethal activity of the nematophagous fungi <i>Clonostachys rosea</i> against 5 nematodes species belonging to different taxa. Two groups of 35 Petri dishes (PD) each were divided into 5 series of 7 (PD). Group 1 (series 1, 2, 3, 4, and 5) contained only water agar; meanwhile group 2 plates (series 6, 7, 8, 9, and 10) contained <i>C. rosea</i> cultures growth on water agar. Every plate from the two groups was added with 500 nematodes corresponding to the following genera/specie: <i>Haemonchus contortus</i>, <i>Caenorhabditis elegans, Rhabditis</i> sp., <i>Panagrellus redivivus</i>, and <i>Butlerius</i> sp. After 5-day incubation at room temperature, free (nontrapped) larvae were recovered from plates using the Baermann funnel technique. Recovered nematodes were counted and compared with their proper controls. Results shown an important reduction percentage of the nematode population attributed to the fungal lethal activity as follows: <i>H. contortus</i> (L<sub>3</sub>) 87.7%; C. <i>elegans</i> 94.7%; <i>Rhabditis</i> sp. 71.9%; <i>P. redivivus</i> 92.7%; and <i>Butlerius</i> sp. 100% (<i>p</i> 0.05). The activity showed by <i>C. rosea</i> against the <i>H. contortus</i> can be crucial for further studies focused to the biological control of sheep haemonchosis, although the environmental impact against beneficial nematodes should be evaluated.
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[
Zootaxa,
2022]
Rhagovelia medinae sp. nov., of the hambletoni group (angustipes complex), and R. utria sp. nov., of the hirtipes group (robusta complex), are described, illustrated, and compared with similar congeners. Based on the examination of type specimens, six new synonymies are proposed: R. elegans Uhler, 1894 = R. pediformis Padilla-Gil, 2010, syn. nov.; R. cauca Polhemus, 1997 = R. azulita Padilla-Gil, 2009, syn. nov., R. huila Padilla-Gil, 2009, syn. nov., R. oporapa Padilla-Gil, 2009, syn. nov, R. quilichaensis Padilla-Gil, 2011, syn. nov.; and R. gaigei, Drake Hussey, 1947 = R. victoria Padilla-Gil, 2012 syn. nov. The first record from Colombia is presented for R. trailii (White, 1879), and the distributions of the following species are extended in the country: R. cali Polhemus, 1997, R. castanea Gould, 1931, R. cauca Polhemus, 1997, R. gaigei Drake Hussey, 1957, R. elegans Uhler, 1894, R. femoralis Champion, 1898, R. malkini Polhemus, 1997, R. perija Polhemus, 1997, R. sinuata Gould, 1931, R. venezuelana Polhemus, 1997, R. williamsi Gould, 1931, and R. zeteki Drake, 1953.
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[
J Biol Chem,
1990]
The nematode Caenorhabditis elegans (C. elegans) expresses the regulatory subunit (R) of cAMP-dependent protein kinase at a level similar to the levels determined for R subunits in mammalian tissues. Approximately 60% of the C. elegans cAMP-binding protein is tightly associated with particulate structures by noncovalent interactions. Ionic detergents or 7 M urea solubilize particulate R. Solubilized and cytosolic R subunits have apparent Mr values of 52,000 and pI values of 5.5. cDNA and genomic DNA encoding a unique C. elegans R subunit were cloned and sequenced. The derived amino acid sequence contains 375 residues; carboxyl-terminal residues 145-375 are 69% identical with mammalian RI. However, residues 44-145 are markedly divergent from the corresponding regions of all other R sequences. This region might provide sufficient structural diversity to adapt a single R subunit for multiple functional roles in C. elegans. Antibodies directed against two epitopes in the deduced amino acid sequence of C. elegans R avidly bound nematode cytosolic and particulate R subunits on Western blots and precipitated dissociated R subunits and R2C2 complexes from solution. Immunofluorescence analysis revealed that the tip of the head, which contains chemosensory and mechanosensory neurons, and the pharyngeal nerve ring were enriched in R. The R subunit concentration is low during early embryogenesis in C. elegans. A sharp increase (approximately 6-fold) in R content begins several hours before the nematodes hatch and peaks during the first larval stage. Developmental regulation of R expression occurs at translational and/or post-translational levels. The 8-kilobase pair C. elegans R gene is divided into 8 exons by introns ranging from 46 to 4300 base pairs. The 5'-flanking region has no TATA box and contains preferred and minor transcription start sites.
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[
Worm Breeder's Gazette,
1994]
R-ras I and R-ras 2 (TC21) homologs Per Winge*, Vercna Gobel*+, Stephen Friend*, and John Fleming*+. MGH Cancer Center and +DepL of Pediatrics, Boston, MA. Human r-ras 1 and r-ras 2 (TC21) belong to the closer relatives (>50% amino acid identity) of ras in the ras superfamily of GDP/GTP-binding proteins. They are the first members to exhibit transforming potential when mutated at some which render ras oncogenic and make it insensitive to GAP action (Graham & Der, 1994). These recent findings have led to current investigations of their role-in human cancer. Furthermore, r-ras 1 -- by immunoprecipitation and in the yeast-2-hybrid-system -- was shown to interact with
bc1-2, the human homolog to
ced-9 (Fernandez-Sarabia & Bischoff, 1993) and has thus been implicated as a possible effector of apoptosis. There is evidence that the r-ras proteins participate in some but not all aspects of the ras signal transduction pathway involving upstream tyrosinc kinases and downstream serine/threonine kinases. It has not yet been elucidated in the mammalian system (1) what alternative pathway the r-ras proteins may be utilizing and (2) what functional relevance is represented by the in vitro interaction of r-ras 1 and
bc1-2. We are trying to address these questions in C elegans and have cloned the homologs of r-ras I and r-ras 2 using a degeneratc PCR approach. We have screened c-DNA and genomic libraries and obtamed and sequenced full length c-DNA and genomic clones of r-ras 1 and a full length c-DNA clone of r- ras 2. The genomic sequence of r-ras 2 was recently made available by the genome sequencing project. The amino acid comparison shows high homologyrldentity to thc human proteins for r-ras 1 and r-ras 2 (TC21). R-ras 1 was localizcd to chromosome II ncar
lin-29, and r-ras 2 maps close to embS on chromosome m. To obtain r-ras germline deletions, we have screened a TCl insertion library which we constructed using the mutator strain MT 3126 (protocols kindly proYided by Jocl Rothman, Susan Mango and Ed Maryon), and have isolated transposon insertions in r-ras 1. We are currently in the proccss of sib sclection to purify the strains. To get some first appreciation of a functional role of r-ras towards apoptosis versus growth stimulating propertics, we have also started to inject a r-ras 1 hcat shock promotor expression construct to generatc strains in which r-ras can be overexpressed Ihis additional approach has been choscn since redundancy may be expected in thc ras related protcin familics and thus thc knockout of one of the proteins may not give clear results. We will screen the overexpressing strains for (1) apoptosis and (2) muv phcnotype. In collaboration with Bob Horvitz's laboratory r-ras GST fusion proteins will be generated to test the in vitro interacion with
ccd-9. Finally, we are constructing r-ras 1 and r-ras 2 promotor expression vectors with GFP/betaGAL to define the expression patterns of both genes.
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[
Nat Commun,
2021]
R-bodies are long, extendable protein polymers formed in the cytoplasm of some bacteria; they are best known for their role in killing of paramecia by bacterial endosymbionts. Pseudomonas aeruginosa PA14, an opportunistic pathogen of diverse hosts, contains genes (referred to as the reb cluster) with potential to confer production of R-bodies and that have been implicated in virulence. Here, we show that products of the PA14 reb cluster associate with R-bodies and control stochastic expression of R-body structural genes.PA14 expresses reb genes during colonization of plant and nematode hosts, and R-body production is required for full virulence in nematodes. Analyses of nematode ribosome content and immune response indicate that P. aeruginosa R-bodies act via a mechanism involving ribosome cleavage and translational inhibition. Our observations provide insight into the biology of R-body production and its consequences during P. aeruginosa infection.
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[
Dev Biol,
2024]
While the nervous system of bilaterian animals is mainly left-right (L-R) symmetric at the anatomical level, some molecular and functional L-R asymmetries exist. However, the extent of these molecular asymmetries and their functional consequences remain poorly characterized. C. elegans allows to study L-R asymmetries in the nervous system with single-neuron resolution. We have previously shown that a neural bHLH transcription factor, HLH-16/Olig, is L-R asymmetrically expressed in the AIY neuron lineage and regulates AIY axon projections in a L-R asymmetric manner. Here, by combining a candidate approach and single-cell RNA sequencing data analysis, we identify the ephrin protein EFN-2 and the Flamingo protein FMI-1 as downstream targets of HLH-16 that are L-R asymmetrically expressed in the AIY lineage. We show that EFN-2 and FMI-1 collaborate in the L-R asymmetric regulation of axonal growth. EFN-2 may act via a non-canonical receptor of the L1CAM family, SAX-7. Our study reveals novel molecular L-R asymmetries in the C. elegans nervous system and their functional consequences.
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[
Commun Integr Biol,
2011]
The development of bilateral symmetry during the evolution of species probably 600 million years ago brought about several important innovations: It fostered efficient locomotion, streamlining and favored the development of a central nervous system through cephalization. However, to increase their functional capacities, many organisms exhibit chirality by breaking their superficial left-right (l-r) symmetry, which manifests in the lateralization of the nervous system or the l-r asymmetry of internal organs. In most bilateria, the mechanisms that maintain consistent l-r asymmetry throughout development are poorly understood. This review highlights insights into mechanisms that couple early embryonic l-r symmetry breaking to subsequent l-r patterning in the roundworm Caenorhabditis elegans. A recently identified strategy for l-r patterning in the early C. elegans embryo is discussed, the spatial separation of midline and anteroposterior axis, which relies on a rotational cellular rearrangement and non-canonical Wnt signaling. Evidence for a general relevance of rotational/torsional rearrangements during organismal l-r patterning and for non-canonical Wnt signaling/planar cell polarity as a common signaling mechanism to maintain l-r asymmetry is presented.
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Ragle, J.M., Martinez, R., Johnsen, L., Ward, J., Saeger, H., Ashley, G.
[
International Worm Meeting,
2019]
Circadian clocks ensure cellular and physiological processes are coordinated with respect to a 24-hour solar day. These clocks have very ancient roots and the circuitry is conserved from bacteria to plants and humans, though specific components within the clock differ. Aberrations in clock function are also linked to cancers, highlighting the importance of circadian timing in human health. The mammalian testes appears to lack a clock. Canonical clock genes are expressed in mammalian testes, but do not oscillate as in somatic tissues. Clock gene expression is critical in testes: mice lacking the clock protein Bmal have lower sperm counts, as do rats depleted for RORa in specific testes cells. The C. elegans molt cycle is oscillatory, similar to mammalian circadian rhythms, and is regulated by homologs of mammalian clock proteins, such as NHR-23 (homolog of mammalian RORa). Studying the role of NHR-23 in molting, we observed that NHR-23 does not oscillate in primary spermatocytes and is essential for spermatogenesis: germline-specific NHR-23 depletion in hermaphrodites causes infertility rescuable by mating with wild-type males. Depleting NHR-23 in the germline, sperm are fated and pachytene chromosomes have wildtype morphology. Chromosomes condense and align on the metaphase plate in meiosis I and are marked with Separase, the protease which triggers anaphase. However, cells never progress to anaphase, instead remaining arrested in metaphase I. Supernumerary gamma-tubulin foci are observed in the arrested spermatocytes and aberrant spindles form. We observe defects in fibrous body membranous organelles, the compartment that sequesters and transports Major Sperm Protein to maturing spermatids. Membranous organelles form without fibrous bodies, producing large vacuoles similar to other spermatogenesis mutants. Additionally, RNA-FISH experiments suggest that a negative feedback loop regulates NHR-23 expression in primary spermatocytes, providing a potential mechanism for how NHR-23 can be expressed constitutively. NHR-23 thus offers a model to understand how developmental timers oscillate in the soma to coordinate cyclic physiological processes and how they are continuously expressed in the germline to produce gametes.
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[
MicroPubl Biol,
2020]
The Auxin-Inducible Degradation (AID) system is a powerful technique in the C. elegans toolkit that enables conditional and reversible protein depletion with high temporal and spatial specificity (Zhang et al. 2015; Martinez et al. 2020; Ashley et al. 2020; Martinez and Matus 2020). This system relies on tagging a gene of interest with a short AID degron sequence and transgenic expression of TIR1, an inducible E3 ubiquitin ligase normally found only in plants (Nishimura et al. 2009; Zhang et al. 2015). Upon exposure to the plant-derived hormone Auxin, TIR1 is activated and targets AID-tagged proteins for proteasomal degradation (Nishimura et al. 2009; Zhang et al. 2015) (Figure 1A). While there are qualitative reports that Auxin does not overtly affect the morphology or behavior of wild-type C. elegans (Zhang et al. 2015), this has not been quantitatively assessed. Determining whether Auxin significantly affects C. elegans morphology and behavior, even in subtle ways, is important given the C. elegans communitys rapid uptake of the AID system (Kasimatis et al. 2018; Nance and Frkjr-Jensen 2019; Ashley et al. 2020; McDiarmid et al. 2020). Here, we use our high-throughput machine vision tracking system, the Multi-Worm Tracker (MWT) (Swierczek et al. 2011), to investigate whether exposure to Auxin affects a suite of morphological, locomotor, mechanosensory, and short-term habituation learning phenotypes in our labs derivate of Bristol N2 wild-type worms and the CGC wild-type reference strain, PD1074 (Yoshimura et al. 2019) (Figure 1B).
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[
J Biol Chem,
2007]
The biological methyl donor, S adenosylmethionine (AdoMet), can exist in two diastereoisomeric states with respect to its sulfonium ion. The "S" configuration, (S,S)AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the "R" form, producing (R,S)AdoMet. As of yet, (R,S)AdoMet has no known physiological function and may inhibit cellular reactions. In this study, two enzymes have been found in Saccharomyces cerevisiae that are capable of recognizing (R,S)AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, previously identified as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine respectively. We find here that Sam4 recognizes both (S,S) and (R,S)AdoMet, but its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet while no activity is seen with the S,S form. R,S-specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)AdoMet in these organisms.