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[
Front Physiol,
2013]
A rich chapter in the history of insect endocrinology has focused on hormonal control of diapause, especially the major roles played by juvenile hormones (JHs), ecdysteroids, and the neuropeptides that govern JH and ecdysteroid synthesis. More recently, experiments with adult diapause in Drosophila melanogaster and the mosquito Culex pipiens, and pupal diapause in the flesh fly Sarcophaga crassipalpis provide strong evidence that insulin signaling is also an important component of the regulatory pathway leading to the diapause phenotype. Insects produce many different insulin-like peptides (ILPs), and not all are involved in the diapause response; ILP-1 appears to be the one most closely linked to diapause in C. pipiens. Many steps in the pathway leading from perception of daylength (the primary environmental cue used to program diapause) to generation of the diapause phenotype remain unknown, but the role for insulin signaling in mosquito diapause appears to be upstream of JH, as evidenced by the fact that application of exogenous JH can rescue the effects of knocking down expression of ILP-1 or the Insulin Receptor. Fat accumulation, enhancement of stress tolerance, and other features of the diapause phenotype are likely linked to the insulin pathway through the action of a key transcription factor, FOXO. This review highlights many parallels for the role of insulin signaling as a regulator in insect diapause and dauer formation in the nematode Caenorhabditis elegans.
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[
Ther Deliv,
2020]
<b>Aim:</b> The aim of this study is to prepare and characterize simvastatin-loaded nanoemulsions (SIM-LN) as well as evaluate their physicochemical properties and toxicity. <b>Methodology & results:</b> The SIM-LN were prepared, their characteristics evaluated for 30days, and after that, the SIM-LN toxicity was evaluated using Vero cell culture and the <i>in vivo</i> model of <i>Caenorhabditis elegans</i>. The prepared SIM-LN had an average droplet size of 139+/-22nm, with high encapsulation rate (>98.4%). The storage at room temperature proved to be the most optimal condition. Toxicity assays demonstrated no toxicity. <b>Conclusion:</b> It was demonstrated that the surfactants used as emulsifiers optimized the properties without side effects, because no toxicity was measured in preliminary tests.
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[
Proc Natl Acad Sci U S A,
2016]
In directed graphs, relationships are asymmetric and these asymmetries contain essential structural information about the graph. Directed relationships lead to a new type of clustering that is not feasible in undirected graphs. We propose a spectral co-clustering algorithm called di-sim for asymmetry discovery and directional clustering. A Stochastic co-Blockmodel is introduced to show favorable properties of di-sim To account for the sparse and highly heterogeneous nature of directed networks, di-sim uses the regularized graph Laplacian and projects the rows of the eigenvector matrix onto the sphere. A nodewise asymmetry score and di-sim are used to analyze the clustering asymmetries in the networks of Enron emails, political blogs, and the Caenorhabditis elegans chemical connectome. In each example, a subset of nodes have clustering asymmetries; these nodes send edges to one cluster, but receive edges from another cluster. Such nodes yield insightful information (e.g., communication bottlenecks) about directed networks, but are missed if the analysis ignores edge direction.
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[
Cytoskeleton (Hoboken),
2017]
We used structured illumination microscopy (SIM) to obtain super-resolution images of muscle attachment structures in C. elegans striated muscle. SIM imaging of M-line components revealed two patterns: PAT-3 (-integrin) and proteins that interact in a complex with the cytoplasmic tail of -integrin and localize to the basal muscle cell membrane (UNC-112 (kindlin), PAT-4 (ILK), UNC-97 (PINCH), PAT-6 (-parvin) and UNC-95), are found in discrete, angled segments with gaps. In contrast, proteins localized throughout the depth of the M-line (UNC-89 (obscurin) and UNC-98) are imaged as continuous lines. Systematic immunostaining of muscle cell boundaries revealed that dense body components close to the basal muscle cell membrane also localize at cell boundaries. SIM imaging of muscle cell boundaries reveal "zipper-like" structures. Electron micrographs reveal electron dense material similar in appearance to dense bodies located adjacent to the basolateral cell membranes of adjacent muscle cells separated by ECM. Moreover, by EM, there are a variety of features of the muscle cell boundaries that help explain the zipper-like pattern of muscle protein localization observed by SIM. Short dense bodies in
atn-1 mutants that are null for -actinin and lack the deeper extensions of dense bodies, showed "zipper-like" structures by SIM similar to cell boundary structures, further indicating that the surface-proximal components of dense bodies form the "zipper-like" structures at cell boundaries. Moreover, mutants in thin and thick filament components do not have "dot-like" dense bodies, suggesting that myofilament tension is required for assembly or maintenance of proper dense body shape. This article is protected by copyright. All rights reserved.
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Zullini, A, Tatti, F, Batani, D, Drobne, D, Poletti, G, Orsini, F, Milani, M, Zrimec, A
[
Scanning,
2005]
A novel focused ion beam-based technique is presented for the read-out of microradiographs of Caenorhabditis elegans nematodes generated by soft x-ray contact microscopy (SXCM). In previous studies, the read-out was performed by atomic force microscopy (AFM), but in our work SXCM microradiographs were imaged by scanning ion microscopy (SIM) in a focused ion beam/scanning electron microscope (FIB/SEM). It allows an ad libitum selection of a sample region for gross morphologic to nanometric investigations, with a sequence of imaging and cutting. The FIB/SEM is less sensitive to height variation of the relief, and sectioning makes it possible to analyse the sample further. The SXCM can be coupled to SIM in a more efficient and faster way than to AFM. Scanning ion microscopy is the method of choice for the read-out of microradiographs of small multicellular organisms.
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[
Trop Med Parasitol,
1989]
Free ecdysteroids were detected in Onchocerca gibsoni, in tissues constituting O. volvulus and O. gibsoni nodules and in unrelated bovine tissues. Ecdysone and 20-hydroxyecdysone were identified by HPLC-RIA and GC/MS(SIM). The concentration of free ecdysteroids in the nodule tissue immediately surrounding the parasites was at least an order of magnitude higher than that detected in the worms themselves, or in adjacent nodular tissues or other bovine tissues.
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[
Mol Cell,
2016]
During Caenorhabditis elegans oocyte meiosis, a multi-protein ring complex (RC) localized between homologous chromosomes, promotes chromosome congression through the action of the chromokinesin KLP-19. While some RC components are known, the mechanism of RC assembly has remained obscure. We show that SUMO E3 ligase GEI-17/PIAS is required for KLP-19 recruitment to the RC, and proteomic analysis identified KLP-19 as a SUMO substrate invivo. Invitro analysis revealed that KLP-19 is efficiently sumoylated in a GEI-17-dependent manner, while GEI-17 undergoes extensive auto-sumoylation. GEI-17 and another RC component, the kinase BUB-1, contain functional SUMO interaction motifs (SIMs), allowing them to recruit SUMO modified proteins, including KLP-19, into the RC. Thus, dynamic SUMO modification and the presence of SIMs in RC components generate a SUMO-SIM network that facilitates assembly of the RC. Our results highlight the importance of SUMO-SIM networks in regulating the assembly of dynamic protein complexes.
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[
Anal Chem,
2013]
Under favorable conditions, Caenorhabditis elegans larvae grow into reproductive adults after a series of molting cycles. When environmental conditions are harsh, they arrest as dauer larvae. Dafachronic acid (DA), a C. elegans steroid hormone, is required for reproductive development. Here, we report a mass spectrometry (MS) method for absolute quantitation of DA in C. elegans. The extraction of DA from C. elegans was optimized to achieve a recovery rate of greater than 83%. The MS sensitivity to DA increased 100-fold after carboxyl group derivatization with 2-picolylamine. High-resolution selected ion monitoring (HR-SIM) on a Q-Orbitrap mass spectrometer Q Exactive outperformed targeted-MS2 on the same instrument and selected reaction monitoring (SRM) on a triple-quadrupole mass spectrometer TSQ Quantum Discovery. With a limit of quantification as low as 1 pg of DA, the HR-SIM method enables absolute quantification of endogenous DA during the reproductive development of C. elegans. We found that in wild-type (WT) worms, DA increases from 0.04 +/- 0.02 ng/mg protein in the L1 larval stage to 1.21 +/- 0.67 ng/mg protein in the L2 larval stage and decreases again after the L3 stage. In comparison, four genetic mutants that have a constitutive dauer-formation phenotype due to disrupted insulin, TGF-, or cGMP signaling all have a very low DA level in the L2 stage (below 15% of the WT). These mutants are able to escape the dauer fate and most of them grow into fertile adults when supplied with exogenous DA. Therefore, a DA spike in the L2 stage is critical for the reproductive development of C. elegans.
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[
Methods Mol Biol,
2022]
Studies of C. elegans will benefit from a powerful method for super-resolution imaging of proteins and mRNAs at any 3-D locations throughout the entire animal. Conventional methods of super-resolution imaging in C. elegans, such as STORM, PALM, SR-SIM and STED, are limited by imaging depths that are insufficient to map the entire depth of adult worms, and involve hardware that may not be accessible to all labs. We recently developed expansion of C. elegans (ExCel), a method for physically magnifying fixed whole animals of C. elegans with high isotropy, which provides effective resolutions finer than the diffraction limit, across the entire animal, on conventional confocal microscopes. In this chapter, we present a family of three detailed ExCel protocols. The standard ExCel protocol features simultaneous readout of diverse molecules (fluorescent proteins, RNA, DNA, and general anatomy), all at ~70nm resolution (~3.5x linear expansion). The epitope-preserving ExCel protocol enables imaging of endogenous proteins with off-the-shelf antibodies, at a~100nm resolution (~2.8x linear expansion). The iterative ExCel protocol allows readout of fluorescent proteins at ~25nm resolution (~20x linear expansion). The protocols described here comprise a versatile toolbox for super-resolution imaging of C. elegans.
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Hadian K, Maiser A, Schneider S, Hofmeister O, Wagner A, Schmitt S, Schorpp K, Conradt B, Zischka H, Aasumets K, Wolf A, Leonhardt H, Gerhold JM, Feuchtinger A, Rolland SG
[
J Cell Sci,
2019]
The Fe(II) and 2-oxoglutarate dependent oxygenase Alkb homolog 1 (Alkbh1) has been shown to act on a wide range of substrates, like DNA, tRNA or histones. Thereby different enzymatic activities have been identified including, amongst others demethylation of <i>N</i><sup>3</sup>-methylcytosine (m<sup>3</sup>C) in RNA- and single-stranded DNA-oligonucleotides, demethylation of <i>N</i><sup>1</sup>-methyladenosine (m<sup>1</sup>A) in tRNA or formation of 5-formyl cytosine (f<sup>5</sup>C) in tRNA. In accordance with the different substrates Alkbh1 has also been proposed to reside in distinct cellular compartments in human and mouse cells, including the nucleus, cytoplasm and mitochondria. Here we describe further evidence for a role of human Alkbh1 in regulation of mitochondrial protein biogenesis, including localisation of Alkbh1 into mitochondrial RNA granules with super-resolution 3D SIM microscopy. Electron microscopy and high-resolution respirometry analyses revealed an impact of Alkbh1 level on mitochondrial respiration, but not on mitochondrial structure. Downregulation of Alkbh1 impacts cell growth in Hela cells and delays development in <i>C. elegans</i>, where the mitochondrial role of Alkbh1 seems to be conserved. Alkbh1 knockdown, but not Alkbh7 knockdown, triggers mitochondrial unfolded protein response (UPR<sup>mt</sup>) in <i>C. elegans</i>.