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[
Nat Struct Mol Biol,
2017]
Although some features underlying replication-origin activation in metazoan cells have been determined, little is known about their regulation during metazoan development. Using the nascent-strand purification method, here we identified replication origins throughout Caenorhabditis elegans embryonic development and found that the origin repertoire is thoroughly reorganized after gastrulation onset. During the pluripotent embryonic stages (pregastrula), potential cruciform structures and open chromatin are determining factors that establish replication origins. The observed enrichment of replication origins in transcription factor-binding sites and their presence in promoters of highly transcribed genes, particularly operons, suggest that transcriptional activity contributes to replication initiation before gastrulation. After the gastrula transition, when embryonic differentiation programs are set, new origins are selected at enhancers, close to CpG-island-like sequences, and at noncoding genes. Our findings suggest that origin selection coordinates replication initiation with transcriptional programs during metazoan development.
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Laromaine A, Bitonto V, Xavier JAM, Munoz-Juan A, Nuez-Martinez M, Pinto CIG, Mendes F, Marques F, Protti N, Guerreiro JF, Crich SG, Teixidor F, Vinas C
[
Cancers (Basel),
2021]
Purpose: The aim of our study was to assess if the sodium salt of cobaltabis(dicarbollide) and its di-iodinated derivative (Na[o-COSAN] and Na[8,8'-I2-o-COSAN]) could be promising agents for dual anti-cancer treatment (chemotherapy + BNCT) for GBM.Methods: The biological activities of the small molecules were evaluated in vitro with glioblastoma cells lines U87 and T98G in 2D and 3D cell models and in vivo in the small model animal Caenorhabditis elegans (C. elegans) at the L4-stage and using the eggs.Results: Our studies indicated that only spheroids from the U87 cell line have impaired growth after treatment with both compounds, suggesting an increased resistance from T98G spheroids, contrary to what was observed in the monolayer culture, which highlights the need to employ 3D models for future GBM studies. In vitro tests in U87 and T98G cells conclude that the amount of 10B inside the cells is enough for BNCT irradiation. BNCT becomes more effective on T98G after their incubation with Na[8,8'-I2-o-COSAN], whereas no apparent cell-killing effect was observed for untreated cells.Conclusions: These small molecules, particularly [8,8'-I2-o-COSAN]-, are serious candidates for BNCT now that the facilities of accelerator-based neutron sources are more accessible, providing an alternative treatment for resistant glioblastoma.
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[
Farmaco,
2005]
Several quinolines were synthesized and evaluated in vitro and in vivo against the nematodes Caenorhabditis elegans, Heligmosomoides polygyrus and the protozoa Trichomonas vaginalis. If some of them have shown in vitro nematocide activity (at 10 muM), however, their trichomonacidal activity reached 50% reduction at only 100 muM. The in vivo activity on Trichinella spiralis model was evaluated for some of the most in vitro active quinolines.
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Silva-Garcia CG, Berson E, Lascarez-Lagunas LI, Colaiacovo MP, Beese-Sims S, Castaner AB, Baran KA, Jordan PW, Martinez-Garcia M, Diaz-Pacheco BN, Shin N, Saito TT, Pacheco S, Naharro PR, Martinez-Perez E, Skinner MW, Nadarajan S
[
PLoS Genet,
2023]
Chromosome movements and licensing of synapsis must be tightly regulated during early meiosis to ensure accurate chromosome segregation and avoid aneuploidy, although how these steps are coordinated is not fully understood. Here we show that GRAS-1, the worm homolog of mammalian GRASP/Tamalin and CYTIP, coordinates early meiotic events with cytoskeletal forces outside the nucleus. GRAS-1 localizes close to the nuclear envelope (NE) in early prophase I and interacts with NE and cytoskeleton proteins. Delayed homologous chromosome pairing, synaptonemal complex (SC) assembly, and DNA double-strand break repair progression are partially rescued by the expression of human CYTIP in
gras-1 mutants, supporting functional conservation. However, Tamalin, Cytip double knockout mice do not exhibit obvious fertility or meiotic defects, suggesting evolutionary differences between mammals.
gras-1 mutants show accelerated chromosome movement during early prophase I, implicating GRAS-1 in regulating chromosome dynamics. GRAS-1-mediated regulation of chromosome movement is DHC-1-dependent, placing it acting within the LINC-controlled pathway, and depends on GRAS-1 phosphorylation at a C-terminal S/T cluster. We propose that GRAS-1 coordinates the early steps of homology search and licensing of SC assembly by regulating the pace of chromosome movement in early prophase I.
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Saito, T., Martinez Garcia, M., Robles Naharro, P., Nadarajan, S., Colaiacovo, M. P., Beese-Sims, S., Shin, N.
[
International Worm Meeting,
2019]
Meiosis is a specialized cell division program resulting in the production of haploid gametes (eggs and sperm) from diploid germ cells. Errors in achieving accurate chromosome segregation during meiosis can result in infertility and birth defects such as Down syndrome. Therefore, understanding the mechanisms underlying accurate meiotic chromosome segregation is of tremendous importance for human reproductive health. A zipper-like structure that assembles between homologous chromosomes during meiosis from yeast to humans, known as the synaptonemal complex (SC), assures accurate chromosome segregation. However, despite its importance, very little is known about how its assembly is regulated. Through combined genetic, cytological, molecular biology and biochemical approaches we have uncovered a role for GRAS-1, the worm homolog of mammalian GRASP and CYTIP proteins, in coordinating SC assembly with cytoskeletal forces outside the nucleus. A role for GRAS-1/GRASP/CYTIP during meiosis has not been previously demonstrated. During early prophase I, GRAS-1 localizes in close proximity to the nuclear envelope-associated protein SUN-1, promotes SC assembly, and physically interacts with SC structural components.
gras-1 mutants exhibit a delay in achieving homologous pairing, the formation of large aggregates of SC proteins that persist into pachytene while chromosome axes appear unaltered, and impaired DNA double-strand break repair progression. Our analysis indicates that GRAS-1 undergoes phosphorylation, which may be CHK-1/2-dependent. We hypothesize that GRAS-1 serves as a scaffold for a multi-protein complex consisting of central region components of the SC during meiosis, participating in licensing synapsis initiation. Such a "scaffold activity" for SC formation has not yet been identified in any organism.
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[
Biochemistry,
2012]
Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.
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[
J Infect Dis,
2015]
BACKGROUND: Elimination of onchocerciasis and lymphatic filariasis is targeted for 2020. Given the coincident Loa loa infections in Central Africa and the potential for drug resistance development, the need for new microfilaricides and macrofilaricides has never been greater. With the genomes of L. loa, Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi available, new drug targets have been identified. METHODS: The effects of the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib on B. malayi adult males, adult females, L3 larvae, and microfilariae were assessed using a wide dose range (0-100 M) in vitro. RESULTS: For microfilariae, median inhibitory concentrations (IC50 values) on day 6 were 6.06 M for imatinib, 3.72 M for dasatinib, and 81.35 M for nilotinib; for L3 larvae, 11.27 M, 13.64 M, and 70.98 M, respectively; for adult males, 41.6 M, 3.87 M, and 68.22 M, respectively; and for adult females, 42.89 M, 9.8 M, and >100 M, respectively. Three-dimensional modeling suggests how these tyrosine kinase inhibitors bind and inhibit filarial protein activity. CONCLUSIONS: Given the safety of imatinib in humans, plans are underway for pilot clinical trials to assess its efficacy in patients with filarial infections.
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[
International Worm Meeting,
2017]
Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.
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[
Worm Breeder's Gazette,
1976]
We have studied maternal effects in 23 zyg ts mutants to estimate the times of expression of genes whose products are required in embryogenesis. We have used the following three tests, called arbitrarily A, B, and C. A test: Heterozygous (m/+) L4's are shifted to 25 C and allowed to self-fertilize. If 100% of their eggs yield larvae (25% of which express the mutant phenotype as adults), then the mutant is scored as maternal (M). If 25% of the F1 eggs fail to hatch, then the mutant is scored as non-maternal (N). An M result indicates that expression of the + allele in the parent allows m/m zygotes to hatch and grow to adulthood. A result of N indicates the opposite: that the + allele must be expressed in the zygote for hatching to occur. Out of 23 zyg mutants tested, 3 were scored N and 20 were scored M in the A test. Therefore, for most of the genes defined by these mutants, expression in the parent is sufficient for zygote survival, even if the gene is not expressed in the zygote. B test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with N2 (+/+) males. If eggs fail to hatch at 25 C, but mated hermaphrodites shifted to 16 C produce cross progeny to give proof of mating, then the mutant is scored M. If cross progeny appear in the 25 C mating, then the mutant is scored N. An M result indicates that expression of the + allele in the zygote is not sufficient to allow m/+ progeny of an m/m hermaphrodite to survive. Conversely an N result indicates either that zygotic expression of the + allele is sufficient for survival, or that a sperm function or factor needed for early embryogenesis can be supplied paternally (see C test below). Out of the 23 zyg mutants tested, 11 were scored M and 12 were scored N. The combined results of A and B tests and their simplest interpretation are as follows. Ten mutants are M,M; the genes defined by these mutants must be expressed in the hermaphrodite parent for the zygote to survive. Ten mutants are M,N; these genes can be expressed either in the parent or in the zygote. Two mutants are N,N; these genes must be expressed in the zygote. One mutant is N,M; this gene must be expressed both in the maternal parent and in the zygote. C test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with heterozygous (m/+) males. If rescue by a +/+ male in the B test depends on the + allele, then only half the cross progeny zygotes of a C test mating (m/+ male x m/m hermaphrodite) should survive. However, if rescue depends on a function or cytoplasmic component from the male sperm, then all the cross progeny zygotes in a C test should survive. Of the 10 M,N mutants, 6 have been C tested; one exhibited paternal rescue independent of the + allele. The A and B tests also were carried out on 16 mutants that arrest before the L3 molt (acc mutants). In the A test on 2 of these mutants, all m/m progeny of m/+ parents grew to adulthood at 25 C. Therefore, parental contributions are sufficient to overcome a progeny mutational block as late as the L2 stage. All 16 acc mutants scored N in the B test.
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[
Worm Breeder's Gazette,
1994]
cej-1 Encodes a Novel Protein with Poly-Threonine Motif M. L. A. Khanl, M. Tabish, T. Fukushigel1 S. Tsukita2, M. Itoh , Sh. Tsukita , and S. S. Siddiqui. (1): Lab. of Molecular Biology, Dept of Ecological Engg. Toyohashi Univ. Technology, Toyohashi 441, and (2). National Institute for Physiological Sciences, Okazaki 444, Japan.