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[
PLoS One,
2013]
Soil-transmitted helminths are parasitic nematodes that inhabit the human intestine. These parasites, which include two hookworm species, Ancylostomaduodenale and Necator americanus, the whipworm Trichuristrichiura, and the large roundworm Ascarislumbricoides, infect upwards of two billion people and are a major cause of disease burden in children and pregnant women. The challenge with treating these diseases is that poverty, safety, and inefficient public health policy have marginalized drug development and distribution to control infection in humans. Anthelmintics (anti-worm drugs) have historically been developed and tested for treatment of non-human parasitic nematodes that infect livestock and companion animals. Here we systematically compare the in vitro efficacy of all major anthelmintic classes currently used in human therapy (benzimidazoles, nicotinic acetylcholine receptor agonists, macrocyclic lactones, nitazoxanide) against species closely related to human parasitic nematodes-Ancylostoma ceylanicum, Trichurismuris, and Ascarissuum--- as well as a rodent parasitic nematode used in veterinary drug discovery, Heligmosomoidesbakeri, and the free-living nematode Caenorhabditis elegans. Extensive in vitro data is complemented with single-dose in vivo data in three rodent models of parasitic diseases. We find that the effects of the drugs in vitro and in vivo can vary greatly among these nematode species, e.g., the efficacy of albendazole is strong on A. ceylanicum but weak on H. bakeri. Nonetheless, certain commonalities of the in vitro effects of the drugs can be seen, e.g., nitazoxanide consistently shows an all-or-nothing response. Our in vitro data suggest that further optimization of the clinical efficacy of some of these anthelmintics could be achieved by altering the treatment routine and/or dosing. Most importantly, our in vitro and in vivo data indicate that the hookworm A. ceylanicum is a particularly sensitive and useful model for anthelmintic studies and should be incorporated early on in drug screens for broad-spectrum human soil-transmitted helminth therapies.
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[
J Proteome Res,
2013]
The identification of phosphorylated proteins remains a challenge in proteomics, partially due to the difficulty in assigning tandem mass (MS/MS) spectra to their originating peptide sequences with correct phosphosite localization. Because of its advantages in efficiency and sensitivity, spectral library searching is a promising alternative to conventional sequence database searching. Our work aims to construct the largest collision-induced dissociation (CID) MS/MS spectral libraries of phosphorylated peptides in human (Homo sapiens) and four model organisms (Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, and Mus musculus) to date, to facilitate phosphorylated peptide identification by spectral library searching. We employed state-of-the-art search methods to published data and applied two recently published phosphorylation site localization tools (PhosphoRS and PTMProphet) to ascertain the phosphorylation sites. To further increase the coverage of this library, we predicted "semi-empirical" spectra for peptides containing known phosphorylation sites from the corresponding template unphosphorylated peptide spectra. The performance of the spectral libraries built were evaluated and found to be superior to conventional database searching in terms of sensitivity. Updated spectral libraries of phosphorylated peptides are made freely available for use with the spectral search engine SpectraST. The work flow being developed will be used to continuously update the libraries when new data become available.
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[
Dev Cell,
2002]
Nicastrin is genetically linked to Notch/lin-12 signaling in C. elegans and is part of a large multiprotein complex along with Presenilin. Here we describe the isolation and characterization of Drosophila Nicastrin (Nic) mutants. Nic mutants and tissue clones display characteristic Notch-like phenotypes. Genetic and inhibitor studies indicate a function for Nicastrin in the gamma-secretase step of Notch processing, similar to Presenilin. Further, Nicastrin is genetically required for signaling from membrane-anchored activated Notch. In the absence of Nicastrin, Presenilin is destabilized and mature C-terminal subunits are absent. Nicastrin might recruit gamma-secretase substrates into the proteolytic complex as a prerequisite for Presenilin maturation and active complex assembly.
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[
Elife,
2023]
Imaging endogenous mRNAs in live animals is technically challenging. Here we describe an MS2-based signal amplification with the Suntag system that enables live-cell RNA imaging of high temporal resolution and with 8xMS2 stem-loops, which overcomes the obstacle of inserting a 1,300 nt 24xMS2 into the genome for the imaging of endogenous mRNAs. Using this tool, we were able to image the activation of gene expression and the dynamics of endogenous mRNAs in the epidermis of live C. elegans.
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Foos M, Liu D, Rabinow L, Chung V, Gnad F, Sopko R, Perrimon N, Studer RA, Landry SD, Hu Y, Beltrao P
[
G3 (Bethesda),
2018]
Post-translational modification (PTM) serves as a regulatory mechanism for protein function, influencing their stability, interactions, activity and localization, and is critical in many signaling pathways. The best characterized PTM is phosphorylation, whereby a phosphate is added to an acceptor residue, most commonly serine, threonine and tyrosine in metazoans. As proteins are often phosphorylated at multiple sites, identifying those sites that are important for function is a challenging problem. Considering that any given phosphorylation site might be non-functional, prioritizing evolutionarily conserved phosphosites provides a general strategy to identify the putative functional sites. To facilitate the identification of conserved phosphosites, we generated a large-scale phosphoproteomics dataset from <i>Drosophila</i> embryos collected from six closely-related species. We built iProteinDB (https://www.flyrnai.org/tools/iproteindb/), a resource integrating these data with other high-throughput PTM datasets, including vertebrates, and manually curated information for <i>Drosophila</i> At iProteinDB, scientists can view the PTM landscape for any <i>Drosophila</i> protein and identify predicted functional phosphosites based on a comparative analysis of data from closely-related <i>Drosophila</i> species. Further, iProteinDB enables comparison of PTM data from <i>Drosophila</i> to that of orthologous proteins from other model organisms, including human, mouse, rat, <i>Xenopus tropicalis</i>, <i>Danio rerio,</i> and <i>Caenorhabditis elegans.</i>
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[
Appl Environ Microbiol,
2013]
Soil-transmitted helminths (hookworms, whipworms, and large roundworms) are agents of intestinal roundworm diseases of poverty that infect upwards of 2 billion people worldwide. A great challenge in treating these diseases is the development of anthelmintic therapeutics that are inexpensive, can be produced in great quantity, and are capable of delivery under varied and adverse environmental conditions. A potential solution to this challenge is the use of live bacteria that are acceptable for human consumption, e.g., Bacillus subtilis, and that can be engineered with therapeutic properties. In this study, we expressed the Bacillus thuringiensis anthelmintic protein Cry5B in a bacterial strain that has been used as a model for live bacterial therapy, Bacillus subtilis PY79. PY79 transformed with a Cry5B expression plasmid (PY79-Cry5B) is able to express Cry5B from the endogenous B. thuringiensis
cry5B promoter. During sporulation of PY79-Cry5B, Cry5B is packaged as a crystal. Furthermore, Cry5B produced in PY79 is bioactive, with a 50% lethal concentration (LC50) of 4.3 g/ml against the roundworm Caenorhabditis elegans. PY79-Cry5B was a significantly effective therapeutic in experimental Ancylostoma ceylanicum hookworm infections of hamsters. A single 10-mg/kg (0.071 mol/kg of body weight) dose of Cry5B administered as a Cry5B-PY79 spore crystal lysate achieved a 93% reduction in hookworm burdens, which is superior on a molar level to reductions seen with clinically used anthelmintics. Given that a bacterial strain such as this one can be produced cheaply in massive quantities, our results demonstrate that the engineering and delivery of live bacterial strains have great potential to treat a significant contributor to poverty worldwide, namely, hookworm disease and other soil-transmitted helminthiasis.
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[
Proc Natl Acad Sci U S A,
2010]
The soil-transmitted helminths or nematodes (hookworms, whipworms, and Ascaris) are roundworms that infect more than 1 billion of the poorest peoples and are leading causes of morbidity worldwide. Few anthelmintics are available for treatment, and only one is commonly used in mass drug administrations. New anthelmintics are urgently needed, and crystal (Cry) proteins made by Bacillus thuringiensis are promising new candidates. Combination drug therapies are considered the ideal treatment for infectious diseases. Surprisingly, little work has been done to define the characteristics of anthelmintic combinations. Here, by means of quantitative assays with wild-type and mutants of the roundworm Caenorhabditis elegans, we establish a paradigm for studying anthelmintic combinations using Cry proteins and nicotinic acetylcholine receptor (nAChR) agonists, e.g., tribendimidine and levamisole. We find that nAChR agonists and Cry proteins, like Cry5B and Cry21A, mutually display what is known in the HIV field as hypersusceptibility--when the nematodes become resistant to either class, they become hypersensitive to the other class. Furthermore, we find that when Cry5B and nAChR agonists are combined, their activities are strongly synergistic, producing combination index values as good or better than seen with antitumor, anti-HIV, and insecticide combinations. Our study provides a powerful means by which anthelmintic combination therapies can be examined and demonstrate that the combination of nAChR agonists and Cry proteins has excellent properties and is predicted to give improved cure rates while being recalcitrant to the development of parasite resistance.
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[
Comput Struct Biotechnol J,
2022]
Paralogs are genes which arose via gene duplication, and when such paralogs retain overlapping or redundant function, this poses a challenge to functional genetics research. Recent technological advancements have made it possible to systematically probe gene function for redundant genes using dual or multiplex gene perturbation, and there is a need for a simple bioinformatic tool to identify putative paralogs of a gene(s) of interest. We have developed Paralog Explorer (https://www.flyrnai.org/tools/paralogs/), an online resource that allows researchers to quickly and accurately identify candidate paralogous genes in the genomes of the model organisms D. melanogaster, C. elegans, D. rerio, M. musculus, and H. sapiens. Paralog Explorer deploys an effective between-species ortholog prediction software, DIOPT, to analyze within-species paralogs. Paralog Explorer allows users to identify candidate paralogs, and to navigate relevant databases regarding gene co-expression, protein-protein and genetic interaction, as well as gene ontology and phenotype annotations. Altogether, this tool extends the value of current ortholog prediction resources by providing sophisticated features useful for identification and study of paralogous genes.
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[
PLoS Negl Trop Dis,
2009]
BACKGROUND: Intestinal parasitic nematodes such as hookworms, Ascaris lumbricoides, and Trichuris trichiura are amongst most prevalent tropical parasites in the world today. Although these parasites cause a tremendous disease burden, we have very few anthelmintic drugs with which to treat them. In the past three decades only one new anthelmintic, tribendimidine, has been developed and taken into human clinical trials. Studies show that tribendimidine is safe and has good clinical activity against Ascaris and hookworms. However, little is known about its mechanism of action and potential resistance pathway(s). Such information is important for preventing, detecting, and managing resistance, for safety considerations, and for knowing how to combine tribendimidine with other anthelmintics. METHODOLOGY/PRINCIPAL FINDINGS: To investigate how tribendimidine works and how resistance to it might develop, we turned to the genetically tractable nematode, Caenorhabditis elegans. When exposed to tribendimidine, C. elegans hermaphrodites undergo a near immediate loss of motility; longer exposure results in extensive body damage, developmental arrest, reductions in fecundity, and/or death. We performed a forward genetic screen for tribendimidine-resistant mutants and obtained ten resistant alleles that fall into four complementation groups. Intoxication assays, complementation tests, genetic mapping experiments, and sequencing of nucleic acids indicate tribendimidine-resistant mutants are resistant also to levamisole and pyrantel and alter the same genes that mutate to levamisole resistance. Furthermore, we demonstrate that eleven C. elegans mutants isolated based on their ability to resist levamisole are also resistant to tribendimidine. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that the mechanism of action of tribendimidine against nematodes is the same as levamisole and pyrantel, namely, tribendimidine is an L-subtype nAChR agonist. Thus, tribendimidine may not be a viable anthelmintic where resistance to levamisole or pyrantel already exists but could productively be used where resistance to benzimidazoles exists or could be combined with this class of anthelmintics.
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[
PLoS Negl Trop Dis,
2012]
BACKGROUND: Hookworm infections are one of the most important parasitic infections of humans worldwide, considered by some second only to malaria in associated disease burden. Single-dose mass drug administration for soil-transmitted helminths, including hookworms, relies primarily on albendazole, which has variable efficacy. New and better hookworm therapies are urgently needed. Bacillus thuringiensis crystal protein Cry5B has potential as a novel anthelmintic and has been extensively studied in the roundworm Caenorhabditis elegans. Here, we ask whether single-dose Cry5B can provide therapy against a hookworm infection and whether C. elegans mechanism-of-action studies are relevant to hookworms. METHODOLOGY/PRINCIPAL FINDINGS: To test whether the C. elegans invertebrate-specific glycolipid receptor for Cry5B is relevant in hookworms, we fed Ancylostoma ceylanicum hookworm adults Cry5B with and without galactose, an inhibitor of Cry5B-C. elegans glycolipid interactions. As with C. elegans, galactose inhibits Cry5B toxicity in A. ceylanicum. Furthermore,
p38 mitogen-activated protein kinase (MAPK), which controls one of the most important Cry5B signal transduction responses in C. elegans, is functionally operational in hookworms. A. ceylanicum hookworms treated with Cry5B up-regulate
p38 MAPK and knock down of
p38 MAPK activity in hookworms results in hypersensitivity of A. ceylanicum adults to Cry5B attack. Single-dose Cry5B is able to reduce by >90% A. ceylanicum hookworm burdens from infected hamsters, in the process eliminating hookworm egg shedding in feces and protecting infected hamsters from blood loss. Anthelmintic activity is increased about 3-fold, eliminating >97% of the parasites with a single 3 mg dose (30 mg/kg), by incorporating a simple formulation to help prevent digestion in the acidic stomach of the host mammal. CONCLUSIONS/SIGNIFICANCE: These studies advance the development of Cry5B protein as a potent, safe single-dose anthelmintic for hookworm therapy and make available the information of how Cry5B functions in C. elegans in order to study and improve Cry5B function against hookworms.