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[
International Worm Meeting,
2015]
Soil-transmitted helminth (STH) nematode parasites (hookworms, Ascaris and Trichuris) are key contributors to morbidity and poverty worldwide and infect more than 1 billion people. Few anthelmintics are available for treatment, and only anthelmintic, albendazole, is commonly used in mass drug administrations. New anthelmintics and treatment strategies are greatly needed, in particular as albendazole resistance is inevitable given its current method of usage and given the widespread resistance to benzimidazoles (like albendazole) in veterinary use.Our group has identified Cry5B made by a soil bacterium Bacillus thuringiensis as a promising new anthelmintic for treating STH parasites. Apart from developing Cry5B as a much needed new class of anthelmintic, we are also interested in preserving the potency of Cry5B and other anthelmintics as much as possible-i.e., preventing resistance. To achieve this aim, combination therapies with anthelmintics is an excellent approach.The challenge with anthelmintic combinations is defining a good combination and at what ratio drugs can be productively combined. Although we have published combination studies using the nematode Caenorhabditis elegans, it was not clear how these translate in vivo in infected mammals. By using the hookworm (Ancylostoma ceylanicum) infections in hamster, we establish a new and powerful in vivo paradigm for studying anthelmintics combinations-defining not only how well two drugs combine but also providing some optimization of the ratio for combinations. Here, we will update you our latest results of studying anthelmintic combinatins in vivo and our thoughts on how good C. elegans is for such studies. Our paradigm here provides a powerful means on how to examine and demonstrate the anthelmintic combination therapies.
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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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[
Int J Biochem Cell Biol,
2003]
Kallmann's syndrome (KS) is a genetic condition characterised by hypogonadotrophic hypogonadism (HH) and anosmia; although these are the defining features of the condition, additional neurological and non-neurological sequel may also occur depending on the specific mode of inheritance. KS affects about 1 in 8000 males and 1 in 40,000 females, with most presentations being of the 'sporadic' type. Of the inherited forms, hitherto, only the gene responsible for the X-linked form (X-KS), namely KAL-1, has been identified and the encoded protein, anosmin-1, consists primarily of a whey acidic protein (WAP) and fibronectin-like type III (FnIII) domains which appear to mediate distinctly different protein functions. The WAP/FnIII combination is conserved in anosmins across species and recent studies in rodents and in Caenorhabditis elegans demonstrate that anosmin functions in both axonal targeting and branching. Screening for loci that modify these phenotypes in C. elegans has identified heparan-6-O-sulpbotransferase as a key interactor mediating anosmin-1 function. Furthermore, over-expression and loss of function of the C elegans Kal-1 gene disrupt epidermal morphogenesis, resulting in ventral enclosure and male tail formation defects. These findings provide novel insights into the molecular pathogenesis of X-KS.
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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.