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[
Cancer Res,
1996]
A second tumor suppressor locus on 17p that is distinct from TP53 has been identified in brain, breast, lung, and ovarian tumors. Using allelic loss mapping and positional cloning methods, we have recently identified two novel genes, which we refer to as OVCA1 and OVCA2, that map to 17p13.3. The two genes are ubiquitously expressed and encode proteins of 443 and 227 amino acids, respectively, with no known functional motifs. Sequence comparison of OVCA1 and OVCA2 revealed extensive sequence identity and similarity to hypothetical proteins from Saccharomyces cerevisiae, Caenorhabditis elegans, and Rattus species. Northern blot analysis reveals that OVCA1 and OVCA2 mRNA were expressed in normal surface epithelial cells of the ovary, but the level of this transcript is significantly reduced or is undetectable in 92% (11/12) of the ovarian tumors and tumor cell lines analyzed. The location, high degree of amino acid conservation, and reduced expression in ovarian tumors and tumor cell lines suggest that decreased expression of these two genes contributes to ovarian tumorigenesis and should be considered candidate tumor suppressor genes.
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[
PLoS One,
2014]
In mammals, Bone Morphogenetic Protein (BMP) pathway signaling is important for the growth and homeostasis of extracellular matrix, including basement membrane remodeling, scarring, and bone growth. A conserved BMP member in Caenorhabditis elegans, DBL-1, regulates body length in a dose-sensitive manner. Loss of DBL-1 pathway signaling also results in increased anesthetic sensitivity. However, the physiological basis of these pleiotropic phenotypes is largely unknown. We created a DBL-1 over-expressing strain and show that sensitivity to anesthetics is inversely related to the dose of DBL-1. Using pharmacological, genetic analyses, and a novel dye permeability assay for live, microwave-treated animals, we confirm that DBL-1 is required for the barrier function of the cuticle, a specialized extracellular matrix. We show that DBL-1 signaling is required to prevent animals from forming tail-entangled aggregates in liquid. Stripping lipids off the surface of wild-type animals recapitulates this phenotype. Finally, we find that DBL-1 signaling affects ultrastructure of the nematode cuticle in a dose-dependent manner, as surface lipid content and cuticular organization are disrupted in animals with genetically altered DBL-1 levels. We propose that the lipid layer coating the nematode cuticle normally prevents tail entanglement, and that reduction of this layer by loss of DBL-1 signaling promotes aggregation. This work provides a physiological mechanism that unites the DBL-1 signaling pathway roles of not only body size regulation and drug responsiveness, but also the novel Hoechst 33342 staining and aggregation phenotypes, through barrier function, content, and organization of the cuticle.
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[
International Worm Meeting,
2013]
Amyotrophic Lateral Sclerosis (ALS) is a lethal neurodegenerative disease with an unknown pathogenesis and limited therapeutic treatments. A major limitation hindering the development of therapeutic treatments is a lack of understanding of the disease's molecular pathways. In humans, a P56S point mutation in the VAPB/ALS8 MSP domain is associated with ALS and late-onset spinal muscular atrophy (SMA) (Funke et al., 2010; Millecamps et al., 2010; Nishimura et al., 2004). The N-terminal MSP domain is cleaved from the C-terminus of the VAPB protein, and is secreted in a cell-type specific manner (Tsuda et al., 2008). However, the P56S mutation inhibits secretion of the MSP domain. Genetic and biochemical evidence support the hypothesis that the MSP domain interacts with the VAB-1 Eph receptor, ROBO/SAX-3 receptor, and CLR-1 Lar-like protein tyrosine phosphatase receptor, which are collectively called growth cone guidance receptors (Miller et al, 2001; Miller et al., 2003; Tsuda et al., 2008; Han et al., 2012). In C. elegans, secreted vMSP acts on CLR-1 and ROBO/SAX-3 receptors expressed in striated muscle, promoting Arp2/3-dependent actin remodeling. This remodeling is critical for proper placement of mitochondria to actin-rich myofilament I-bands (Han et al., 2012). We hypothesize that the vMSP receptors form heteromeric complexes to promote signaling critical for actin remodeling and correct mitochondria placement. To begin testing this hypothesis, I am expressing combinations of VAB-1, SAX-3, and CLR-1 in cultured cells and investigating putative complex formation via co-immunoprecipitation. My preliminary data suggest that SAX-3 complexes with both VAB-1 and CLR-1. Data will also be presented on the role of C. elegans VAPB/VPR-1 in regulating mitochondria in motor neurons. The results could provide insight into growth cone guidance receptor interactions and pathways involved in ALS.
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[
Environ Toxicol Chem,
2018]
Engineered nanoparticles entering the environment are subject to various transformations that in turn influence how particles are presented to, and taken up by organisms. To understand the effect of soil properties on the toxicity of nanosilver to C. elegans toxicity assays were performed in pore water extracts from natural soils with varying organic matter content and pH using 3-8nm un-functionalised (Ag 3-8Unf), 52nm PVP-coated silver nanoparticles (Ag 52PVP) and AgNO<sub>3</sub> as ionic silver. Effects on nanoparticle agglomeration and stability were investigated using UV-vis spectroscopy and asymmetric flow field-flow fractionation (AF4). Ag<sup>+</sup> showed greater overall toxicity than nanosilver with little difference between the nanoparticle types. Increasing soil organic matter content significantly decreased the toxicity of Ag 3-8Unf while it increased that of AgNO<sub>3</sub> . The toxicity of all silver treatments significantly decreased with increasing pore water pH. Dissolution of both nanoparticles in the pore water extracts was too low to have contributed to their observed toxic effects. UV-vis spectroscopy revealed low levels of agglomeration/aggregation independent of soil properties for Ag 3-8Unf, while higher organic matter as well as low pH appeared to stabilise Ag 52PVP. Overall both soil organic matter content and pH affected nanoparticle fate as well as toxicity to C. elegans, however, there appears to be no clear connection between the measured particle characteristics and their effect. This article is protected by copyright. All rights reserved.
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[
Proc Biol Sci,
2016]
The effects from multigenerational exposures to engineered nanoparticles (ENPs) in their pristine and transformed states are currently unknown despite such exposures being an increasingly common scenario in natural environments. Here, we examine how exposure over 10 generations affects the sensitivity of the nematode Caenorhabditis elegans to pristine and sulfidized Ag ENPs and AgNO3 We also include populations that were initially exposed over six generations but kept unexposed for subsequent four generations to allow recovery from exposure. Toxicity of the different silver forms decreased in the order AgNO3, Ag ENPs and Ag2S ENPs. Continuous exposure to Ag ENPs and AgNO3 caused pronounced sensitization (approx. 10-fold) in the F2 generation, which was sustained until F10. This sensitization was less pronounced for Ag2S ENP exposures, indicating different toxicity mechanisms. Subtle changes in size and lifespan were also measured. In the recovery populations, the sensitivity to Ag ENPs and AgNO3 resulting from the initial multigenerational exposure persisted. Their response sensitivity for all endpoints was most closely related to the last ancestral exposed generation (F5), rather than unexposed controls. The mechanisms of transgenerational transfer of sensitivity are probably organized through the epigenome, and we encourage others to investigate such effects as a priority for mechanistic toxicology.
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[
International Worm Meeting,
2013]
Transforming Growth Factor-b (TGF-b) is a family of secreted cell signaling ligands. DBL-1, a C. elegans TGF-b superfamily member, is secreted from nervous tissue, but must be trafficked to the epidermis where it binds to receptors. Alteration of the DBL-1 pathway generates distinct, dose-dependent phenotypes, where animals with increased or decreased signaling are long or small respectively. However, how body size is altered in
dbl-1 pathway variants is still poorly understood. The goals of this project are to determine subcellular localization of secreted DBL-1, analyze its regulation between secreting and receiving cells, and elucidate the molecular and physiological underpinnings of body length regulation by DBL-1 pathway signaling.
We found that that secreted GFP-tagged DBL-1 localizes in a discrete punctate pattern along the dorsal and ventral nerve cords. Using a whole-mount microwave-based immunofluorescence method we developed, we found that mouse BMP4, a DBL-1 homolog, rescues body size defects in C. elegans caused by loss of DBL-1 pathway signaling, but also co-localizes with GFP-tagged DBL-1. Immunocytochemical studies also show that these punctae co-localize with cell-cell contact sites and caveolar bodies in the ventral nerve cord. These studies are the first to decipher the subcellular localization of DBL-1, expanding the knowledge of TGF-b trafficking in C. elegans. Microarray analyses have revealed DBL-1 signaling regulates transcription of cuticular components, which may together affect body size. Our genetic and pharmacological studies reveal that DBL-1 signaling affects cuticular permeability, altering sensitivity to soluble anesthetics based on the dose of DBL-1. Using transmission electron microscopy, we found that ultrastructural composition of the cuticle is affected in a dose-dependent manner in
dbl-1 variants.
These results show that DBL-1-mediated body size differences directly correlate with changes in cuticular organization. These studies expand our understanding of the molecular mechanisms involved in regulation of body size by the DBL-1 pathway.
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[
J Vis Exp,
2012]
The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal(1-4). The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility(4-6). Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer(7). Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development(8). Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages(2,9). Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology(5,6,10,11). While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression(12), antibody staining(13), and electron microscopy(1). Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures(14). Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail(15). We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.
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[
International Worm Meeting,
2011]
Body size development is strictly regulated in C. elegans through genetic and environmental components. The most well studied of these genetic regulators is the TGF-b DBL-1 pathway. Loss of DBL-1 signaling leads to decreased body size as well as abnormal development of male-specific structures. Mutants lacking DYF-7 display a small body size and do not properly sense their environment. DYF-7 is a transmembrane zona pellucida (ZP) domain-containing protein expressed in head and tail neurons as well as seam cells. Previous studies have shown that DYF-7 is involved in neural tip anchoring, which explains its sensing defect, but its role in body size development has remained unexplored. To determine if DYF-7 regulates body size though DBL-1 signaling, we performed epistasis and expression studies. Our epistasis analyses between
dyf-7 and
dbl-1 pathway members reveal that double mutants are significantly smaller than either single mutant, indicating DYF-7 regulates body length independent from the TGF-b DBL-1 pathway. We also found GFP-tagged DBL-1 is properly expressed and localized in animals lacking DYF-7 signaling. Further, male-specific structures appear normal in
dyf-7 mutants. An additional mode of body size regulation is through organization of the cuticle, which surrounds the exterior of the animal. The cuticle in
dyf-7 mutants has morphological defects that could contribute to the small body size phenotype. These results indicate that DYF-7 acts through cuticle organization, independent of the TGF-b pathway, to regulate body size.
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[
Development,
2017]
The major sperm protein domain (MSPd) has an extracellular signaling function implicated in amyotrophic lateral sclerosis. Secreted MSPds derived from the C. elegans VAPB homolog VPR-1 promote mitochondrial localization to actin-rich I-bands in body wall muscle. Here we show that the nervous system and germ line are key MSPd secretion tissues. MSPd signals are transduced through the CLR-1 Lar-like tyrosine phosphatase receptor. We show that CLR-1 is expressed throughout the muscle plasma membrane, where it is accessible to MSPd within the pseudocoelomic fluid. MSPd signaling is sufficient to remodel the muscle mitochondrial reticulum during adulthood. An RNAi suppressor screen identified survival of motor neuron 1 (SMN-1) as a downstream effector. SMN-1 acts in muscle, where it colocalizes at myofilaments with ARX-2, a component of the Arp2/3 actin-nucleation complex. Genetic studies suggest that SMN-1 promotes Arp2/3 activity important for localizing mitochondria to I-bands. Our results support the model that VAPB homologs are circulating hormones that pattern the striated muscle mitochondrial reticulum. This function is crucial in adults and requires SMN-1 in muscle, likely independent of its role in pre-mRNA splicing.
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[
Environ Sci Technol,
2021]
To better understand nanoplastic effects, the potential for surface functionalization and dissolve organic matter eco-corona formation to modify the mechanisms of action and toxicity of different nanoplastics needs to be established. Here, we assess how different surface charges modifying functionalization (postive (+ve) aminated; neutral unfunctionalized; negative (-ve) carboxylated) altered the toxicity of 50 and 60 nm polystyrene nanoplastics to the nematode <i>Caenorhabditis elegans</i>. The potency for effects on survival, growth, and reproduction reduced in the order +ve aminated > neutral unfunctionalized -ve carboxylated with toxicity >60-fold higher for the +ve than -ve charged forms. Toxicokinetic-toxicodynamic modeling (DEBtox) showed that the charge-related potency was primarily linked to differences in effect thresholds and dose-associated damage parameters, rather than to toxicokinetic parameters. This suggests that surface functionalization may change the nature of nanoplastic interactions with membrane and organelles leading to variations in toxicity. Eco-corona formation reduced the toxicity of all nanoplastics indicating that organic molecule associations may passivate surfaces. Between particles, eco-corona interactions resulting in more equivalent effects; however, even despite these changes, the order of potency of the charged forms was retained. These results have important implications for the development of future grouping approaches.