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Abstract
Caenorhabditis elegans is now the model organism of choice for a growing number of researchers. A combination of its apparent simplicity, exquisite genetics, the existence of a full molecular toolkit and a complete genome sequence makes it ideal for rapid and effective study of gene function. A survey of the C. elegans genome indicates that this 'simple' worm contains many genes with a high degree of similarity to human disease genes. For many human disease genes it has proven, and will continue to prove, difficult to elucidate their function by direct study. In such cases simpler model organisms may prove to be a more productive starting point. The basic function of a human disease gene may be studied in the background of C. elegans, in which the most important interactions are likely to be conserved, providing an insight into disease process in humans. Here we consider the significance of this modality for human disease processes and discuss how C. elegans may, in some cases, be ideal in the study of the function of human disease genes and act as a model for groups of parasitic nematodes that have a severe impact on world health.
... The genomic comparison of human and C elegans describes that the majority of human disease genes and pathways are found in C. elegans 18 . Approximately 40 -75% of genes linked to human diseases show a certain level of similarity (E < 10 −10 in BLASTP searches) with genes identified in C. elegans [19][20][21][22][23][24] . A comprehensive proteomic analysis of 18,452 C. elegans protein sequences identified human gene homologs for approximately 83% of the C. elegans proteome 25 . ...
... Completion of the C. elegans genome sequence in 1998 59 demonstrated that roughly 38% of worm genes have a human ortholog 60 . The percentage of human disease-related genes that have at least minor similarity (E < 10 −10 on BLASTP searches) with C. elegans genes ranges between 40-75% [19][20][21][22][23][24] . For identifying orthologous genes of most significant human CVDrelated genes, first neighbours of the essential key regulator genes were identified and extracted from the human CVD PPI Network. ...
Cardiovascular disease (CVD) is a collective term for disorders of the heart and blood vessels. The molecular events and biochemical pathways associated with CVD are difficult to study in clinical settings on patients and in vitro conditions. Animal models play a pivotal and indispensable role in cardiovascular disease (CVD) research. Caenorhabditis elegans, a nematode species, has emerged as a prominent experimental organism widely utilised in various biomedical research fields. However, the specific number of CVD-related genes and pathways within the C. elegans genome remains undisclosed to date, limiting its in-depth utilisation for investigations. In the present study, we conducted a comprehensive analysis of genes and pathways related to CVD within the genomes of humans and C. elegans through a systematic bioinformatic approach. A total of 1113 genes in C. elegans orthologous to the most significant CVD-related genes in humans were identified, and the GO terms and pathways were compared to study the pathways that are conserved between the two species. In order to infer the functions of CVD-related orthologous genes in C. elegans, a PPI network was constructed. Orthologous gene PPI network analysis results reveal the hubs and important KRs: pmk-1, daf-21, gpb-1, crh-1, enpl-1, eef-1G, acdh-8, hif-1, pmk-2, andaha-1 in C. elegans. Modules were identified for determining the role of the orthologous genes at various levels in the created network. We also identified 9 commonly enriched pathways between humans and C. elegans linked with CVDs that include autophagy (animal), the ErbB signalling pathway, the FoxO signalling pathway, the MAPK signalling pathway, ABC transporters, the biosynthesis of unsaturated fatty acids, fatty acid metabolism, glutathione metabolism, and metabolic pathways. This study provides the first systematic genomic approach to explore the CVD-associated genes and pathways that are present in C. elegans, supporting the use of C. elegans as a prominent animal model organism for cardiovascular diseases. Keywords: Cardiovascular Disease, Animal models, C. elegans, Orthologous genes, PPI Network
... The free-living nematode C. elegans is a good model organism due to its ease of laboratory maintenance, and it has enough similarities to the parasitic worms to act as an approximate test model [24]. For example, the animal parasitic suborder Strongylida including the human hookworms Ancylostoma and Necator, is closely related to C. elegans and, therefore, the latter is an excellent model organism for these pathogens [25]. The bioassay-guided fractionation of the fruit extract of O. insignis showed that the activity against C. elegans was found in the n-hexane fraction while the ethyl acetate, n-butanol and aqueous fractions have much lower activity against C. elegans. ...
... The free-living nematode C. elegans is a good model organism due to its ease of laboratory maintenance, and it has enough similarities to the parasitic worms to act as an approximate test model [24]. For example, the animal parasitic suborder Strongylida including the human hookworms Ancylostoma and Necator, is closely related to C. elegans and, therefore, the latter is an excellent model organism for these pathogens [25]. The bioassay-guided fractionation of the fruit extract of O. insignis showed that the activity against C. elegans was found in the n-hexane fraction while the ethyl acetate, n-butanol, and aqueous fractions have much lower activity against C. elegans. ...
Ozoroa insignis Del. is an ethnobotanical plant widely used in traditional medicine for various ailments, including schistosomiasis, tapeworm, and hookworm infections. From the so far not investigated fruits of Ozoroa insignis, the anthelmintic principles could be isolated through bioassay-guided isolation using Caenorhabditis elegans and identified by NMR spectroscopic analysis and mass spectrometric studies. Isolated 6-[8(Z)-pentadecenyl] anacardic (1), 6-[10(Z)-heptadecenyl] anacardic acid (2), and 3-[7(Z)-pentadecenyl] phenol (3) were evaluated against the 5 parasitic organisms Schistosoma mansoni (adult and newly transformed schistosomula), Strongyloides ratti, Heligmosomoides polygyrus, Necator americanus, and Ancylostoma ceylanicum, which mainly infect humans and other mammals. Compounds 1–3 showed good activity against Schistosoma mansoni, with compound 1 showing the best activity against newly transformed schistosomula with 50% activity at 1µM. The isolated compounds were also evaluated for their cytotoxic properties against PC-3 (human prostate adenocarcinoma) and HT-29 (human colorectal adenocarcinoma) cell lines, whereby compounds 2 and 3 showed antiproliferative activity in both cancer cell lines, while compound 1 exhibited antiproliferative activity only on PC-3 cells. With an IC50 value of 43.2 µM, compound 3 was found to be the most active of the 3 investigated compounds.
... Many of the basic mechanisms for developing and functioning as a complex organism evolved before nematodes branched away from the rest of the animal kingdom and many signalling pathways are remarkably well conserved from lower organisms to humans. As such research on C. elegans has increased our understanding of many processes relevant to human biology including mechanisms of cell death, development, aging, neurobiology and cell signalling pathways (Ahringer 1997;Aboobaker and Blaxter 2000;Baumeister and Ge 2002). These factors and the high level of conservation of gene and protein sequence and function mean it is increasingly being used as a model system for the study of genes that underlie human genetic disorders (Kuwabara 1997;Ahringer 1997;Culetto and Sattelle 2000;Baumeister and Ge 2002). ...
... Finally, study of C. elegans will increase our understanding of parasitic infections caused by species closely related to C. elegans (Aboobaker and Blaxter 2000;Hashmi et a l 2001). It is also emerging as a useful tool for the study of bacterial and viral infections and innate immunity (Kurz and Ewbank 2003;Sifri et al. 2003;Joshua et al. 2003). ...
The neuronal ceroid lipofuscinoses (Batten disease) are a group of inherited neurological disorders which predominantly affect children. They are characterized by the accumulation of autofluorescent storage material in lysosomes and occur with a frequency between 1 in 12 500 and 1 in 100 000 births. To date, six genes underlying different sub-types of the disease have been cloned and many studies in cell culture and mouse models performed. However the mechanism of disease pathogenesis remains poorly understood. The simple nematode worm, Caenorhabditis elegans, has a fully sequenced genome, completely mapped cell lineage and nervous system, is easy to maintain and manipulate, and is an organism about which much information, including the results of many genome wide studies, is available. It is thus a good model organism for the study of genes that underlie human neurological disorders. The aim of this work was to investigate whether C. elegans could be used as a model system for investigating the function of NCL genes and the pathological mechanisms that underlie disease manifestation. Homologues to PPT1 and CLN3 were identified in C. elegans and their expression confirmed. Mutation of PPT1 underlies the most severe NCL type, infantile NCL (INCL). Further analysis of CePPT-1 determined a high level of sequence and structural similarity to the human enzyme and demonstrated that it could perform the same catalytic reaction under the same conditions. Analysis of a ppt-1 null mutant (MN1) identified a phenotype of developmental delay, defective egg laying and grossly abnormal mitochondrial morphology. A homologue to the enzyme, acyl-protein thioesterase-1 was also identified (ATH-1) and expression confirmed. The role of PPT-1 in the cell and how this may relate to the pathogenesis of INCL and other NCL types is presented.
... elegans dauer entry similarly regulate hookworm L3i arrest [13]. This paradigm has been broadened to suggest that the same pathways regulating C. elegans dauer entry and exit may also regulate L3i arrest and activation in parasitic nematodes generally [24][25][26][27], in what has become known as the "dauer hypothesis." ...
... A similar developmentally arrested stage, the dauer larva, is formed by the free-living nematode Caenorhabditis elegans in response to unfavorable conditions [13,23]. Interestingly, C. elegans dauer larvae and S. stercoralis L3i arrest at the same third larval stage and share similar characteristics of morphology, extended lifespan, stress-resistance, and cessation of feeding [25,108]. Dauer larvae and L3i resume development soon after encountering favorable environmental conditions or the definitive host, respectively [8,30]. ...
Parasitic nematodes inflict a vast global disease burden in humans as well as animals and plants of agricultural importance; understanding how these worms infect their hosts has significant health and economic implications. In humans, soil-transmitted parasitic nematodes cause hookworm disease and strongyloidiasis, and vector-transmitted parasitic nematodes cause filariasis. The infectious form of the species causing these diseases is a developmentally arrested third-stage larva (L3i). Molecular mechanisms governing L3i developmental arrest and activation within a host have been poorly understood. An analogous developmentally arrested third-stage larva—the dauer larva—forms during stressful environmental conditions in the free-living nematode Caenorhabditis elegans and is controlled by four cellular signaling pathways. The "dauer hypothesis" posits that similar mechanisms regulate dauer and L3i development. The parasitic nematode Strongyloides stercoralis was used to test the dauer hypothesis because its life cycle includes both parasitic and free-living forms. To investigate the role of canonical dauer pathway homologs in regulating L3i arrest and activation, this study utilized transcriptome sequencing (RNAseq), transgenesis, and pharmacological studies. Transcripts encoding cyclic guanosine monophosphate (cGMP) pathway components were coordinately up-regulated in L3i. Application of membrane-permeable 8-bromo-cGMP resulted in activation of L3i and modulation of ligand transcripts in other pathways. In comparison to C. elegans, S. stercoralis has few genes encoding insulin/IGF-1 -like signaling (IIS) ligands, several of which have transcripts modulated during L3i development. Application of the phosphatidylinositol-3 kinase inhibitor, LY294002, prevented L3i activation in host-like conditions. The S. stercoralis transcriptome includes seven homologs of the single C. elegans dauer transforming growth factor β (TGFβ) ligand, three of which are only expressed in L3i. Although the C. elegans nuclear hormone receptor ligand delta7-dafachronic acid (DA) stimulates L3i activation, putative DA biosynthetic genes were not coordinately regulated in L3i development. These data demonstrate that S. stercoralis has homologs for nearly every component in the four canonical dauer pathways, that cGMP signaling may transduce host cues during L3i activation, and that IIS regulates L3i arrest and activation. However, dauer TGFβ signaling appears to function in L3i arrest, an opposite role than in C. elegans, and endogenous DA regulation of L3i development remains largely unexplored.
... Nematodes have been in focus since the 1990s when the free-living bacteriovore Caenorhabditis elegansbecame was the first multicellular organism to have a fully sequenced genome (Aboobaker and Blaxter 2000). This provided an important potential for exploring other nematode species, but it was an additional decade before it became accessible for the parasitic nematode Brugia malayi (Aguinaldo et al. 1997). ...
... The tractability and conserved genes of many nematode species have supported the significance of their ultrastructure studies which enable their use as models for diverse biological processes; including human diseases, aging, immunity, development, ecology, evolution, and host-bacterial interactions (Aboobaker and Blaxter 2000;Couillault and Ewbank 2002;Goodrich-Blair and Clarke 2007;Mitreva et al. 2009;Markaki and Tavernarakis 2010;Neher 2010;Xu and Kim 2011). Also, ultrastructural and immunolocalization of two isoenzymes, Glutathione-Stransferase (GST1) and (GST2) were carried out using immunogold electron microscopy. ...
Nematode worms are among the most ubiquitous organisms on earth. They include free-living forms as well as parasites of plants, insects, humans and other animals. Recently, there has been an explosion of interest in nematode biology, including the area of nematode ultrastructure. Nematodes are round with a body cavity. They have one way guts with a mouth at one end and an anus at the other. They have a pseudocoelom that is lined on one side with mesoderm and on the other side with endoderm. It appears that the cuticle is a very complex and evolutionarily plastic feature with important functions involving protection, body movement and maintaining shape. They only have longitudinal muscles so; they seem to thrash back and forth. While nematodes have digestive, reproductive, nervous and excretory systems, they do not have discrete circulatory or respiratory systems. Nematodes use chemosensory and mechanosensory neurons embedded in the cuticle to orient and respond to a wide range of environmental stimuli. Adults are made up of roughly 1000 somatic cells and hundreds of those cells are typically associated with the reproductive systems. Nematodes ultrastructure seeks to provide studies which enable their use as models for diverse biological processes including; human diseases, immunity, host-parasitic interactions and the expression of phylogenomics. The latter has, however, not been brought into a single inclusive entity. Consequently, in the current review we tried to provide a comprehensive approach to the current knowledge available for nematodes ultrastructures.
... The free-living nematode Caenorhabditis elegans is, in many respects, an ideal subject for genetic and molecular biological study, and consequently a relative wealth of basic information on the molecular, cellular, and developmental biology of that organism exists (Epstein and Shakes, 1995; Riddle and Albert, 1997). Caenorhabditis elegans has been proposed by numerous authors as a general model for many aspects of basic molecular, cellular and developmental biology in the less tractable parasitic nematodes (Blaxter, 1998; Burglin et al., 1998; Aboobaker and Blaxter, 2000). One of the most frequent of such analogies is that drawn between the arrest and reactivation of parasitic L3i and the dauer developmental pathway in C. elegans (Hotez et al., 1993). ...
... It is possible that cross-talk between insulin-like and glucocorticoid-like regulatory pathways has a regulatory role in dauer arrest in C. elegans and the arrest of L3i in S. stercoralis and other parasitic nematodes, and that the C-terminal domains of forkhead transcription factors like DAF-16 and FKTF-1 may function in this cross-talk. The discovery of FKTF-1, a proposed DAF-16 ortholog in the parasitic nematode S. stercoralis supports the hypothesis that a dauer-like pathway is functional in this parasite and may function in determining any or all of the following: (1) the choice between parasitic and free-living life cycles, (2) developmental arrest and/or extended life span of infective L3i, and (3) reactivation of development after infection of the host (Hotez et al., 1993; Blaxter, 1998; Burglin et al., 1998; Aboobaker and Blaxter, 2000). The C. elegans system has proven an invaluable tool in investigating many aspects of developmental biology that go far beyond the biology of this species. ...
A forkhead transcription factor gene, fktf-1, which we propose to be orthologous to the Caenorhabditis elegans dauer-regulatory gene daf-16 has been discovered in the parasitic nematode Strongyloides stercoralis. Genomic and cDNA sequences from both species predict alternately spliced a and b message isoforms. In contrast to C. elegans, where two a isoforms, daf-16a1 and daf-16a2, are found, a single fktf-1a isoform is found in S. stercoralis. Five of the 10 introns found in the C. elegans gene are found in the proposed S. stercoralis ortholog. Functional motifs common to DAF-16 and several mammalian forkhead transcription factors are conserved in FKTF-1. These include the forkhead DNA binding domain, four Akt/protein kinase B phosphorylation sites and a C-terminal domain that may associate with factors such as the steroid receptor coactivator and other factors necessary for transcriptional regulation. An N-terminal serine-rich domain found in DAF-16A is greatly expanded in FKTF-1A. This domain is missing in DAF-16B, FKTF-1B and all mammalian orthologs. FKTF-1 shows the closest phylogenetic relationship to DAF-16 among all known mammalian and nematode forkhead transcription factors. Like its proposed Caenorhabditis ortholog, the fktf-1 message is expressed at all stages of the life cycle examined thus far. Discovery of fktf-1 indicates the presence of an insulin-like signalling pathway in S. stercoralis similar to that known to regulate dauer development in C. elegans. This pathway is a likely candidate to control infective larval arrest and reactivation as well as regulation of the switch between parasitic and free-living development in the parasite.
... Completion of the C. elegans genome sequence in 1998 18 demonstrated that roughly 38% of worm genes have a human ortholog 58 . The percentage of human disease-related genes that have at least minor similarity (E < 10 −10 on BLASTP searches) with C. elegans genes ranges between 40 and 75% [19][20][21][22][23][24] . For identifying orthologous genes of most significant human CVD-related genes, the first neighbours of the essential key regulator genes were identified and extracted from the human CVD PPI Network. ...
Cardiovascular disease (CVD) is a collective term for disorders of the heart and blood vessels. The molecular events and biochemical pathways associated with CVD are difficult to study in clinical settings on patients and in vitro conditions. Animal models play a pivotal and indispensable role in CVD research. Caenorhabditis elegans, a nematode species, has emerged as a prominent experimental organism widely utilized in various biomedical research fields. However, the specific number of CVD-related genes and pathways within the C. elegans genome remains undisclosed to date, limiting its in-depth utilization for investigations. In the present study, we conducted a comprehensive analysis of genes and pathways related to CVD within the genomes of humans and C. elegans through a systematic bioinformatic approach. A total of 1113 genes in C. elegans orthologous to the most significant CVD-related genes in humans were identified, and the GO terms and pathways were compared to study the pathways that are conserved between the two species. In order to infer the functions of CVD-related orthologous genes in C. elegans, a PPI network was constructed. Orthologous gene PPI network analysis results reveal the hubs and important KRs: pmk-1, daf-21, gpb-1, crh-1, enpl-1,eef-1G, acdh-8, hif-1, pmk-2, and aha-1 in C. elegans. Modules were identified for determining the role of the orthologous genes at various levels in the created network. We also identified 9 commonly enriched pathways between humans and C. elegans linked with CVDs that include autophagy (animal), the ErbB signaling pathway, the FoxO signaling pathway, the MAPK signaling pathway, ABC transporters, the biosynthesis of unsaturated fatty acids, fatty acid metabolism, glutathione metabolism, and metabolic pathways. This study provides the first systematic genomic approach to explore the CVD-associated genes and pathways that are present in C. elegans, supporting the use of C. elegans as a prominent animal model organism for cardiovascular diseases.
... Food digested in the gut is not distributed by any specialized vascular system, and neither is there a respiratory system for the uptake or distribution of oxygen. Rather, nutrients and waste are distributed in the body cavity, whose contents are regulated by an excretory canal along each side of the body (Aboobaker & Blaxter, 2000). Many nematodes are able to suspend their life processes completely when conditions become unfavorable; in these resistant states they can survive extreme drying, heat, or cold, and then return to life when favorable conditions return. ...
Nematodes, like many organisms, are adapted to survive in unfavourable environmental conditions and different strategies are used by different species depending on their ecological niche. The free-living model nematode Caenorhabditis elegans will enter the dauer stage, a larval stage geared for survival, when unfavourable environmental conditions (such as crowding or lack of food) are encountered. Animal and plant-parasitic nematodes frequently use a survival stage in order to synchronize their life cycle with that of their hosts in order to optimize their chances of successful invasion. Animal parasitic nematodes are frequently activated after the parasite is exposed to conditions found in the gut of animals (high pH, increased CO2), while in the case of some plant-parasitic nematodes, hatching is activated by host root diffusates. The sequence of events from perception of the hatching stimulus to hatching varies considerably between species. This linking of host and plant parasite is a form of quiescence and is restricted to species that are host-specific.
... One mg/ml medium of curcumin was fed with standard diet elevated ho mRNA levels (135). Caenorhabditis elegans is a model nematode that is optimal for quick and efficient analysis of gene function because of its evident simplicity, precise genetics, availability of whole genome sequence, and full molecular toolset (142). Therefore, C. elegans has been used extensively to study curcumin's expertise in several parameters. ...
Plant-based natural products are alternative to antibiotics that can be employed as growth promoters in livestock and poultry production and attractive alternatives to synthetic chemical insecticides for insect pest management. Curcumin is a natural polyphenol compound from the rhizomes of turmeric (Curcuma spp.) and has been suggested to have a number of therapeutic benefits in the treatment of human diseases. It is also credited for its nutritional and pesticide properties improving livestock and poultry production performances and controlling insect pests. Recent studies reported that curcumin is an excellent feed additive contributing to poultry and livestock animal growth and disease resistance. Also, they detailed the curcumin's growth-inhibiting and insecticidal activity for reducing agricultural insect pests and insect vector-borne human diseases. This review aims to highlight the role of curcumin in increasing the growth and development of poultry and livestock animals and in controlling insect pests. We also discuss the challenges and knowledge gaps concerning curcumin use and commercialization as a feed additive and insect repellent.
... For the lack of functional genetic and in vitro culture methods, it is unable to detect the functions of Acan-Gal-1 directly in A. cantonensis. Here, C. elegans, proposed by numerous authors as a general model for many aspects of basic molecular, cellular and developmental biology in the less tractable parasitic nematodes [42][43][44], was used to investigate the anatomical expression patterns of Acan-Gal-1 for the closely evolutionary relationship between A. cantonensis and C. elegans, both belonging to Clade V according to Cladistic analysis [42]. Wild type C. elegans (N2 strain) were transformed with the construct pAcan-gal-1::gfp and pCe-lec-1::rfp, respectively (Fig. 1a). ...
Background: Angiostrongylus cantonensis L5, parasitizing in human cerebrospinal fluid, leads to eosinophilic meningitis, which is attributed to tissue inflammatory responses caused primarily by high percentage of eosinophils. Eosinophils are also involved in helminthic killing, using the peroxidative oxidation and hydrogen peroxide (H2O2) generated by dismutation of superoxide produced during respiratory burst. In contrast, helminthic worms have evolved to attenuate eosinophil-mediated tissue inflammatory responses for their survival. In previous study, we have demonstrated the extracellular function of Acan-Gal-1 in inducing the apoptosis of macrophages. And here, the intracellular functions of Acan-Gal-1 were investigated with the aim to further reveal the mechanism of A. cantonensis L5 worms surviving in the central nervous system of human from inflammatory responses.
Methods: Bioinformatics were used to analyse the structural characterisation of Acan-Gal-1; qRT-PCR and microinjection were performed to detect the expression patterns of Acan-gal-1; microinjection was performed to construct transgenic worms; oxidative stress assay and Oil Red O fat staining were used to determine the functions of Acan-Gal-1.
Results: The results showed that Acan-Gal-1 was expressed ubiquitously and mainly localized in cuticle, and it was up-regulated in both L5 and adult worm. N2 worms expressing pCe-Acan-gal-1::Acan-gal-1::rfp, with lipid deposition reduced, were significantly resistant to oxidative stress. lec-1 mutant worms, with lipid deposition increased, showed susceptible to oxidative stress, and this phenotype could be rescued by expressing pCe-Acan-gal-1::Acan-gal-1::rfp. And fat-6;fat-7 double-mutant worms expressing pCe-Acan-gal-1::Acan-gal-1::rfp showed no significant changes in oxidative stress tolerance.
Conclusion: In C. elegans worms, up-regulated Acan-Gal-1 plays a defensive role against damage due to oxidative stress for worm survival through reducing fat deposition. And this might indicate the mechanism of A. cantonensis L5 worms, with Acan-Gal-1 up-regulated, surviving in the central nervous system of human from immune attack of Eosinophil.
... Because of the lack of functional genetic and in vitro culture methods, it is not possible to detect the functions of Acan-Gal-1 directly in A. cantonensis. Here, C. elegans, proposed by numerous authors as a general model for many aspects of basic molecular, cellular and developmental biology in the less tractable parasitic nematodes [41][42][43], was used to investigate the anatomical expression patterns of Acan-Gal-1. ...
Background
Angiostrongylus cantonensis L5, parasitizing human cerebrospinal fluid, causes eosinophilic meningitis, which is attributed to tissue inflammatory responses caused primarily by the high percentage of eosinophils. Eosinophils are also involved in killing helminths, using the peroxidative oxidation and hydrogen peroxide (H 2 O 2 ) generated by dismutation of superoxide produced during respiratory burst. In contrast, helminthic worms have evolved to attenuate eosinophil-mediated tissue inflammatory responses for their survival. In previous study, we demonstrated the extracellular function of Acan -Gal-1 in inducing the apoptosis of macrophages. Here, the intracellular functions of Acan -Gal-1 were investigated, aiming to further reveal the mechanism involved in A. cantonensis L5 worms surviving inflammatory responses in the human central nervous system.
Methods
In this study, a model organism, Caenorhabditis elegans , was used as a surrogate to investigate the intracellular functions of Acan -Gal-1 in protecting the worm from its host’s immune attacks. First, structural characterization of Acan -Gal-1 was analyzed using bioinformatics; second, qRT-PCR was used to monitor the stage specificity of Acan-gal-1 expression in A. cantonensis . Microinjections were performed to detect the tissue specificity of lec-1 expression, the homolog of Acan-gal-1 in C. elegans . Third, microinjection was performed to develop Acan-gal-1::rfp transgenic worms. Then, oxidative stress assay and Oil Red O fat staining were used to determine the functions of Acan -Gal-1 in C. elegans .
Results
The results of detecting the stage specificity of Acan-gal-1 expression showed that Acan -Gal-1 was upregulated in both L5 and adult worms. Detection of the tissue specificity showed that the homolog of Acan-gal-1 in C. elegans , lec-1 was expressed ubiquitously and mainly localized in cuticle. Investigating the intracellular functions of Acan -Gal-1 in the surrogate C. elegans showed that N2 worms expressing pCe-lec-1::Acan-gal-1::rfp , with lipid deposition reduced, were significantly resistant to oxidative stress; lec-1 mutant worms, where lipid deposition increased, showed susceptible to oxidative stress, and this phenotype could be rescued by expressing pCe-lec-1::Acan-gal-1::rfp . Expressing pCe-lec-1::Acan-gal-1::rfp or lec-1 RNAi in fat-6;fat-7 double-mutant worms, where fat stores were reduced, had no significant effect on the oxidative stress tolerance.
Conclusion
In C. elegans worms, upregulated Acan- Gal-1 plays a defensive role against damage due to oxidative stress for worm survival by reducing fat deposition. This might indicate the mechanism by which A. cantonensis L5 worms, with upregulated Acan- Gal-1, survive the immune attack of eosinophils in the human central nervous system.
Graphical Abstract
... For the lack of functional genetic and in vitro culture methods, it is unable to detect the functions of Acan-RPS-30 directly in A. cantonensis. Here, C. elegans, proposed by numerous authors as a general model for many aspects of basic molecular, cellular and developmental biology in the less tractable parasitic nematodes [34][35][36], was used to investigate the anatomical expression patterns of Acan-rps-30 for the closed evolutionary relationship between A. cantonensis and C. elegans, both belonging to Clade V according to Cladistic analysis [34]. Wild type C. elegans (N2 strain) were transformed with the construct pAcan-rps-30::gfp and pCe-rps30::gfp, respectively (Fig. 1a). ...
Background: Eosinophilic meningitis, caused by Angiostrongylus cantonensis L5, is mainly attributed to the Eosinophils, which contribute to tissue inflammatory responses in helminthic infections. Eosinophils are associated with helminthic killing, using the peroxidative oxidation and hydrogen peroxide (H2O2) generated by dismutation of superoxide produced during respiratory burst. In contrast, residing in the host with high level of eosinophils, helminthic worms have evolved to attenuate eosinophil-mediated tissue inflammatory responses for their survival in hosts. Our previous study demonstrated that the expression of Acan-rps-30 was significantly down-regulated in A. cantonensis L5 worms, which reside in the cerebrospinal fluid with high level of Eosinophils. Acan-RPS-30, a homologous protein of human Fau, which plays a pro-apoptotic regulatory role, may function in protecting worms from oxidative stress.
Methods: RACE, genome Walking, bioinformatics were used to isolate and analyse the structural characterisation of Acan-RPS-30; qRT-PCR and microinjection was performed to detect the expression patterns of Acan-rps-30; feeding RNAi was used to ced-3 knock-down; microinjection was performed to construct transgenic worms; oxidative stress assay was used to determine the functions of Acan-RPS-30.
Results: Our results showed that Acan-RPS-30 consisted of 130 amino acids, and was grouped into Clade V with C. elegans in phylogenetic analysis. It was expressed ubiquitously in worms and was down-regulated in both L5 and adult A. cantonensis. Worms expressing pCe-rps30::Acan-rps-30::rfp, with the refractile “button-like” apoptotic corpses, were susceptible to oxidative stress. Apoptosis genes ced-3 and ced-4 were both up-regulated in the transgenic worms. And the phenotype susceptible to oxidative stress could be converted with ced-3 defective mutation and RNAi. rps-30–/– mutant worms were resistant to oxidative stress, with ced-3 and ced-4 were both down-regulated. And the oxidative stress resistance phenotype could be rescued and inhibited by expressing pCe-rps30::Acan-rps-30::rfp in rps-30–/– mutant worms.
Conclusion: In C. elegans worms, down-regulated RPS-30 plays a defensive role against damage due to oxidative stress for worm survival by regulating ced-3 down-regulated. And this might indicate the mechanism of A. cantonensis L5 worms, with Acan-RPS-30 down-regulated, surviving in the central nervous system of human from immune attack of Eosinophil.
... Due to the lack of functional genetic and in vitro culture methods, we were unable to detect the functions of Acan-RPS-30 directly in A. cantonensis. In the present study, we used C. elegans, proposed by numerous authors as a general model for many aspects of basic molecular, cellular and developmental biology in the less tractable parasitic nematodes [33][34][35], to investigate the anatomical expression patterns of Acanrps-30 in order to examine the closed evolutionary relationship between A. cantonensis and C. elegans, both of which belong to clade V according to cladistic analysis [33]. Wild-type C. elegans (N2 strain) were transformed with the construct pAcan-rps-30::gfp and pCe-rps30::gfp, respectively (Fig. 1a). ...
Background
Eosinophilic meningitis, caused by fifth-stage larvae of the nematode (roundworm) Angiostrongylus cantonensis, is mainly attributed to the contribution of eosinophils to tissue inflammatory responses in helminthic infections. Eosinophils are associated with the killing of helminths via peroxidative oxidation and hydrogen peroxide generated by the dismutation of superoxide produced during respiratory bursts. In contrast, when residing in the host with high level of eosinophils, helminthic worms have evolved to attenuate eosinophil-mediated tissue inflammatory responses for their survival in the hosts. In a previous study we demonstrated that the expression of the A. cantonensis RPS 30 gene (Acan-rps-30) was significantly downregulated in A. cantonensis L5 roundworms residing in cerebrospinal fluid with a high level of eosinophils. Acan-RPS-30 is a protein homologous to the human Fau protein that plays a pro-apoptotic regulatory role and may function in protecting worms from oxidative stress.
Methods
The isolation and structural characterization of Acan-RPS-30 were performed using rapid amplification of cDNA ends (RACE), genome walking and bioinformatics. Quantitative real-time-PCR and microinjection were used to detect the expression patterns of Acan-rps-30. Feeding RNA interference (RNAi) was used to knockdown the apoptosis gene ced-3. Microinjection was performed to construct transgenic worms. An oxidative stress assay was used to determine the functions of Acan-RPS-30.
Results
Our results showed that Acan-RPS-30 consisted of 130 amino acids. It was grouped into clade V with C. elegans in the phylogenetic analysis. It was expressed ubiquitously in worms and was downregulated in both L5 larvae and adult A. cantonensis. Worms expressing pCe-rps30::Acan-rps-30::rfp, with the refractile “button-like” apoptotic corpses, were susceptible to oxidative stress. Apoptosis genes ced-3 and ced-4 were both upregulated in the transgenic worms. The phenotype susceptible to oxidative stress could be converted with a ced-3 defective mutation and RNAi. rps-30−/− mutant worms were resistant to oxidative stress, with ced-3 and ced-4 both downregulated. The oxidative stress-resistant phenotype could be rescued and inhibited by through the expression of pCe-rps30::Acan-rps-30::rfp in rps-3−/− mutant worms.
Conclusion
In C. elegans worms, downregulated RPS-30 plays a defensive role against damage due to oxidative stress, facilitating worm survival by regulating downregulated ced-3. This observation may indicate the mechanism by which A. cantonensis L5 worms, with downregulated Acan-RPS-30, survive in the central nervous system of humans from the immune response of eosinophils.
Graphic abstract
... For the lack of functional genetic and in vitro culture methods, it is unable to detect the functions of Acan-RPS-30 directly in A. cantonensis. Here, C. elegans, proposed by numerous authors as a general model for many aspects of basic molecular, cellular and developmental biology in the less tractable parasitic nematodes [34][35][36], was used to investigate the anatomical expression patterns of Acan-rps-30 for the closed evolutionary relationship between A. cantonensis and C. elegans, both belonging to Clade V according to Cladistic analysis [34]. Wild type C. elegans (N2 strain) were transformed with the construct pAcan-rps-30::gfp and pCe-rps30::gfp, respectively (Fig. 1A). ...
Background: Eosinophilic meningitis, caused by Angiostrongylus cantonensis L5, is mainly attributed to the Eosinophils, which contribute to tissue inflammatory responses in helminthic infections. Eosinophils are associated with helminthic killing, using the peroxidative oxidation and hydrogen peroxide (H2O2) generated by dismutation of superoxide produced during respiratory burst. In contrast, residing in the host with high level of eosinophils, helminthic worms have evolved to attenuate eosinophil-mediated tissue inflammatory responses for their survival in hosts. Our previous study demonstrated that the expression of Acan-rps-30 was significantly down-regulated in A. cantonensis L5 worms, which reside in the cerebrospinal fluid with high level of Eosinophils. Acan-RPS-30, a homologous protein of human Fau, which plays a pro-apoptotic regulatory role, may function in protecting worms from oxidative stress.
Methods: RACE, genome Walking, bioinformatics were used to isolate and analyse the structural characterisation of Acan-RPS-30; qRT-PCR and microinjection was performed to detect the expression patterns of Acan-rps-30; feeding RNAi was used to ced-3 knock-down; microinjection was performed to construct transgenic worms; oxidative stress assay was used to determine the functions of Acan-RPS-30.
Results: Our results showed that Acan-RPS-30 consisted of 130 amino acids, and was grouped into Clade V with C. elegans in phylogenetic analysis. It was expressed ubiquitously in worms and was down-regulated in both L5 and adult A. cantonensis. Worms expressing pCe-rps30::Acan-rps-30::rfp, with the refractile “button-like” apoptotic corpses, were susceptible to oxidative stress. Apoptosis genes ced-3 and ced-4 were both up-regulated in the transgenic worms. And the phenotype susceptible to oxidative stress could be converted with ced-3 defective mutation and RNAi. rps-30- /- mutant worms were resistant to oxidative stress, with ced-3 and ced-4 were both down-regulated. And the oxidative stress resistance phenotype could be rescued and inhibited by expressing pCe-rps30::Acan-rps-30::rfp in rps-30- /- mutant worms.
Conclusion: In C. elegans worms, down-regulated RPS-30 plays a defensive role against damage due to oxidative stress for worm survival by regulating ced-3 down-regulated. And this might indicate the mechanism of A. cantonensis L5 worms, with Acan-RPS-30 down-regulated, surviving in the central nervous system of human from immune attack of Eosinophil.
... For the lack of functional genetic and in vitro culture methods, it is unable to detect the functions of Acan-RPS-30 directly in A. cantonensis. Here, C. elegans, proposed by numerous authors as a general model for many aspects of basic molecular, cellular and developmental biology in the less tractable parasitic nematodes [34][35][36], was used to investigate the anatomical expression patterns of Acan-rps-30 for the closed evolutionary relationship between A. cantonensis and C. elegans, both belonging to Clade V according to Cladistic analysis [34]. Wild type C. elegans (N2 strain) were transformed with the construct pAcan-rps-30::gfp and pCe-rps30::gfp, respectively (Fig. 1A). ...
Background: Eosinophilic meningitis, caused by Angiostrongylus cantonensis L5, is mainly attributed to the Eosinophils, which contribute to tissue inflammatory responses in helminthic infections. Eosinophils are associated with helminthic killing, using the peroxidative oxidation and hydrogen peroxide (H2O2) generated by dismutation of superoxide produced during respiratory burst. In contrast, residing in the host with high level of eosinophils, helminthic worms have evolved to attenuate eosinophil-mediated tissue inflammatory responses for their survival in hosts. Our previous study demonstrated that the expression of Acan-rps-30 was significantly down-regulated in A. cantonensis L5 worms, which reside in the cerebrospinal fluid with high level of Eosinophils. Acan-RPS-30, a homologous protein of human Fau, which plays a pro-apoptotic regulatory role, may function in protecting worms from oxidative stress.
Methods: RACE, genome Walking, bioinformatics were used to isolate and analyse the structural characterisation of Acan-RPS-30; qRT-PCR and microinjection was performed to detect the expression patterns of Acan-rps-30; feeding RNAi was used to ced-3 knock-down; microinjection was performed to construct transgenic worms; oxidative stress assay was used to determine the functions of Acan-RPS-30.
Results: Our results showed that Acan-RPS-30 consisted of 130 amino acids, and was grouped into Clade V with C. elegans in phylogenetic analysis. It was expressed ubiquitously in worms and was down-regulated in both L5 and adult A. cantonensis. Worms expressing pCe-rps30::Acan-rps-30::rfp, with the refractile “button-like” apoptotic corpses, were susceptible to oxidative stress. Apoptosis genes ced-3 and ced-4 were both up-regulated in the transgenic worms. And the phenotype susceptible to oxidative stress could be converted with ced-3 defective mutation and RNAi. rps-30- /- mutant worms were resistant to oxidative stress, with ced-3 and ced-4 were both down-regulated. And the oxidative stress resistance phenotype could be rescued and inhibited by expressing pCe-rps30::Acan-rps-30::rfp in rps-30- /- mutant worms.
Conclusion: In A. cantonensis L5 worms, down-regulated Acan-RPS-30 plays a defensive role against damage due to oxidative stress for worm survival through inhibiting apoptosis by regulating ced-3 down-regulated.
... For instance, the C. elegans worm has served as an essential model organism for research in developmental biology [1]. Another example are amyloid fibrils of prion proteins, which play an important role in the understanding of neurodegenerative diseases [3]. ...
... Another advantage is its transparent body at all stages, trait that enables optical monitoring and in vivo analysis of fundamental biological processes inside single cells and tissues during aging (Corsi et al., 2015). Also, C. elegans has approximately the same number of genes when compared to humans, most of which are evolutionarily conserved (Aboobaker and Blaxter, 2000). So, investigators can study and understand molecular mechanisms that are conserved in higher eukaryotes, including humans, using a very simple eukaryote instead. ...
... Simple models such as Saccharomyces cerevisiae (Foury, 1997) and Dictyostelium discoideum (Firtel and Chung, 2000;Chung et al., 2001) proved to be very helpful in elucidating the basic mechanisms of eukaryotic cell function, such as the regulation of the cell cycle, the mechanisms of DNA damage and repair, metabolism, and cell signaling. Similarly, invertebrates like Caenorhabditis elegans (Aboobaker and Blaxter, 2000;Culetto and Sattelle, 2000) and Drosophila melanogaster (Bernards and Hariharan, 2001;Reiter et al., 2001;Chien et al., 2002) represent outstanding models to study genes involved in more complex body plans (Bier and McGinnis, 2004). However, their very high evolutionary distance with a low rate of sequence conservation compared to vertebrates and the huge difference in their anatomy and physiology, limit their use in studying vertebratespecific embryonic development and in directly modeling human diseases. ...
Hematopoiesis results in the correct formation of all the different blood cell types. In mammals, it starts from specific hematopoietic stem and precursor cells residing in the bone marrow. Mature blood cells are responsible for supplying oxygen to every cell of the organism and for the protection against pathogens. Therefore, inherited or de novo genetic mutations affecting blood cell formation or the regulation of their activity are responsible for numerous diseases including anemia, immunodeficiency, autoimmunity, hyper- or hypo-inflammation, and cancer. By definition, an animal disease model is an analogous version of a specific clinical condition developed by researchers to gain information about its pathophysiology. Among all the model species used in comparative medicine, mice continue to be the most common and accepted model for biomedical research. However, because of the complexity of human diseases and the intrinsic differences between humans and other species, the use of several models (possibly in distinct species) can often be more helpful and informative than the use of a single model. In recent decades, the zebrafish (Danio rerio) has become increasingly popular among researchers, because it represents an inexpensive alternative compared to mammalian models, such as mice. Numerous advantages make it an excellent animal model to be used in genetic studies and in particular in modeling human blood diseases. Comparing zebrafish hematopoiesis to mammals, it is highly conserved with few, significant differences. In addition, the zebrafish model has a high-quality, complete genomic sequence available that shows a high level of evolutionary conservation with the human genome, empowering genetic and genomic approaches. Moreover, the external fertilization, the high fecundity and the transparency of their embryos facilitate rapid, in vivo analysis of phenotypes. In addition, the ability to manipulate its genome using the last genome editing technologies, provides powerful tools for developing new disease models and understanding the pathophysiology of human disorders. This review provides an overview of the different approaches and techniques that can be used to model genetic diseases in zebrafish, discussing how this animal model has contributed to the understanding of genetic diseases, with a specific focus on the blood disorders.
... The simplicity, tractability, and conserved genes of many nematode species have supported their use as models for diverse biological processes, including human diseases, aging, immunity, development, ecology, evolution, and hostbacterial interactions (Aboobaker and Blaxter, 2000;Couillault and Ewbank, 2002;Goodrich-Blair, 2007;Markaki and Tavernarakis, 2010;Neher, 2010;Xu and Kim, 2011). This last phenomenon-the intimate associations between two of the most speciose organisms on the planet-is the focus of the remainder of this review. ...
Nematodes are ubiquitous organisms that have a significant global impact on ecosystems, economies, agriculture, and human health. The applied importance of nematodes and the experimental tractability of many species have promoted their use as models in various research areas, including developmental biology, evolutionary biology, ecology, and animal-bacterium interactions. Nematodes are particularly well suited for the investigation of host associations with bacteria because all nematodes have interacted with bacteria during their evolutionary history and engage in a variety of association types. Interactions between nematodes and bacteria can be positive (mutualistic) or negative (pathogenic/parasitic) and may be transient or stably maintained (symbiotic). Furthermore, since many mechanistic aspects of nematode-bacterium interactions are conserved, their study can provide broader insights into other types of associations, including those relevant to human diseases. Recently, genome-scale studies have been applied to diverse nematode-bacterial interactions and have helped reveal mechanisms of communication and exchange between the associated partners. In addition to providing specific information about the system under investigation, these studies also have helped inform our understanding of genome evolution, mutualism, and innate immunity. In this review we discuss the importance and diversity of nematodes, “omics”’ studies in nematode-bacterial systems, and the wider implications of the findings.
... Novel Therapeutic Targets The free living nematode, Caenorhabditis elegans (C. elegans), offers a number of distinct advantages like anatomical simplicity, small size, large brood size, short generation time, ease of maintenance, transparence, opportunity to visualise cell migration, apoptosis in the living animal 53 . The ease to develop mutants (more than 8000 genetic strains) and carryout forward genetics has made C. elegans a model of choice for medium-to-high throughput screening of potential therapeutic agents using a variety of end points 54 . ...
Appropriate models are essential for making the transition from scientific discoveries to meaningful applications of the knowledge for human use. Acute as well as delayed effects of ionising radiation to the biological systems develop hierarchically starting from damage to the vital macromolecules up to the disturbances caused at the whole organism level. In vitro models like bacteria, yeast, various mammalian cells cultured as monolayers (2-D) and spheroids (3-D) as well as cells with specific genetic alterations have provided insight into the complex relationships between damage induction and various signal transduction pathways, allowing identification of molecular and sub-cellular targets vital to the fate of irradiated cells. On the other hand, in vivo models (multicellular whole organisms), ranging from simple worms to non-human primates, have been gainfully employed to evaluate efficacy as well as toxicity of potential countermeasure agents (molecules, combinations and formulated preparations) facilitating their deployment in human subjects. This review provides a brief account of the efforts with various in vitro and in vivo models for understanding the biological basis of radiation damage as well as the development of radiation countermeasures, viz., protectors, mitigators and therapeutics.
... Novel Therapeutic Targets The free living nematode, Caenorhabditis elegans (C. elegans), offers a number of distinct advantages like anatomical simplicity, small size, large brood size, short generation time, ease of maintenance, transparence, opportunity to visualise cell migration, apoptosis in the living animal 53 . The ease to develop mutants (more than 8000 genetic strains) and carryout forward genetics has made C. elegans a model of choice for medium-to-high throughput screening of potential therapeutic agents using a variety of end points 54 . ...
Appropriate models are essential for making the transition from scientific discoveries to meaningful applications of the knowledge for human use. Acute as well as delayed effects of ionising radiation to the biological systems develop hierarchically starting from damage to the vital macromolecules up to the disturbances caused at the whole organism level. In vitro models like bacteria, yeast, various mammalian cells cultured as monolayers (2-D) and spheroids (3-D) as well as cells with specific genetic alterations have provided insight into the complex relationships between damage induction and various signal transduction pathways, allowing identification of molecular and sub-cellular targets vital to the fate of irradiated cells. On the other hand, in vivo models (multicellular whole organisms), ranging from simple worms to non-human primates, have been gainfully employed to evaluate efficacy as well as toxicity of potential countermeasure agents (molecules, combinations and formulated preparations) facilitating their deployment in human subjects. This review provides a brief account of the efforts with various in vitro and in vivo models for understanding the biological basis of radiation damage as well as the development of radiation countermeasures, viz., protectors, mitigators and therapeutics.
... Two extremely specialized modes of gut development are observed in the terrestrial nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, the most widely used invertebrate model systems in developmental biology and biomedical research [11][12][13]. Both the nematode and the fruit fly belong to the Ecdysozoa (molting animals) [14] ( Figure 1A), which is one of the three main animal lineages that form the Protostomia, together with the Spiralia and the enigmatic Chaetognatha [15][16][17]. ...
The digestive systems of animals can become highly specialized in response to their exploration and occupation of new ecological niches. Although studies on different animals have revealed commonalities in gut formation, the model systems Caenorhabditis elegans and Drosophila melanogaster, which belong to the invertebrate group Ecdysozoa, exhibit remarkable deviations in how their intestines develop. Their morphological and developmental idiosyncrasies have hindered reconstructions of ancestral gut characters for the Ecdysozoa, and limit comparisons with vertebrate models. In this respect, the phylogenetic position, and slow evolving morphological and molecular characters of marine priapulid worms advance them as a key group to decipher evolutionary events that occurred in the lineages leading to C. elegans and D. melanogaster.
In the priapulid Priapulus caudatus, the gut consists of an ectodermal foregut and anus, and a mid region of at least partial endodermal origin. The inner gut develops into a 16-cell primordium devoid of visceral musculature, arranged in 3 mid tetrads and 2 posterior duplets. The mouth invaginates ventrally and shifts to a terminal anterior position as the ventral anterior ectoderm differentially proliferates. Contraction of the musculature occurs as the head region retracts into the trunk and resolves the definitive larval body plan. Despite obvious developmental differences with C. elegans and D. melanogaster, the expression in P. caudatus of the gut-related candidate genes NK2.1, foxQ2, FGF8/17/16, GATA456, HNF4, wnt1 and evx demonstrate three distinct evolutionarily conserved molecular profiles that correlate with morphologically identified subregions of the gut.
The comparative analysis of priapulid development suggests that a midgut formed by a single endodermal population of vegetal cells, a ventral mouth, and the blastoporal origin of the anus are ancestral features in the Ecdysozoa. Our molecular data on P. caudatus reveal a conserved ecdysozoan gut patterning program and demonstrates that extreme morphological divergence has not been accompanied by major molecular innovations in transcriptional regulators during digestive system evolution in the Ecdysozoa. Our data help to understand the origins of the ecdysozoan body plan, including those of C. elegans and D. melanogaster, and this is critical for comparisons between these two prominent model systems and their vertebrate counterparts.
... Die Rekombinationsrate bei der Meiose ist hoch und vergleichbar mit der von Hefe (Francis, 1998). Unter ungünstigen Witterungs-und Ernährungsbedingungen bevorzugt der Organismus jedoch einen vegetativen Zyklus (Raper, 1941 (Gottmann and Weijer, 1986;Rietdorf et al., 1996) (Aboobaker and Blaxter, 2000;Reiter et al., 2001;Resor et al., 2001;Sturley, 2000 Entwicklungsvorgängen eingesetzt Escalante and Vicente, 2000;Firtel and Meili, 2000;Kay, 2000 (Linskens et al., 1999;Malnasi-Csizmadia et al., 2000). ...
... We are particularly interested in the estimation of population statistics in a fast, reliable and highly automatic way. These statistics are needed in biology, drug discovery (Aziz & Blaxter, 2000), toxicology and crop protection (Kaletta & Hengartner, 2006) research. To obtain statistics, skilled technicians identify and measure a number of specimens. ...
In this work, we discuss the problems of automatic segmentation of Arabidopsis thaliana epidermal cell patterning in images captured using a Differential Interference Contrast (DIC) microscope. These images are difficult to analyze due to the non-linear nature of DIC optics. The border of objects appear as a combination of bright and dark shadows with variable thickens and contrast levels. Our segmentation approach exploits prior knowledge on the optical properties of A. thaliana cell walls. A set of matching filters and a scale space line detector are used to generate an enhanced image that shows a single response at the location of cellular walls. To reduce the uncertainty in low contrast cellular walls several images are captured at different orientations. After image fusion, we obtain a single image that can be segmented using well established algorithms. Experiments on a manually annotated DIC image data set demonstrated the effectiveness of the proposed scheme.
... A similar developmentally arrested stage, the dauer larva, is formed by the free-living nematode Caenorhabditis elegans in response to unfavorable conditions [11,12]. Interestingly, C. elegans dauer larvae and S. stercoralis L3i arrest at the same third larval stage and share similar characteristics of morphology, extended lifespan, stress-resistance, and cessation of feeding [13,14]. Dauer larvae and L3i resume development soon after encountering favorable environmental conditions or the definitive host, respec- tively [10,15]. ...
Infective third-stage larvae (L3i) of the human parasite Strongyloides stercoralis share many morphological, developmental, and behavioral attributes with Caenorhabditis elegans dauer larvae. The ‘dauer hypothesis’ predicts that the same molecular genetic mechanisms control both dauer larval development in C. elegans and L3i morphogenesis in S. stercoralis. In C. elegans, the phosphatidylinositol-3 (PI3) kinase catalytic subunit AGE-1 functions in the insulin/IGF-1 signaling (IIS) pathway to regulate formation of dauer larvae. Here we identify and characterize Ss-age-1, the S. stercoralis homolog of the gene encoding C. elegans AGE-1. Our analysis of the Ss-age-1 genomic region revealed three exons encoding a predicted protein of 1,209 amino acids, which clustered with C. elegans AGE-1 in phylogenetic analysis. We examined temporal patterns of expression in the S. stercoralis life cycle by reverse transcription quantitative PCR and observed low levels of Ss-age-1 transcripts in all stages. To compare anatomical patterns of expression between the two species, we used Ss-age-1 or Ce-age-1 promoter::enhanced green fluorescent protein reporter constructs expressed in transgenic animals for each species. We observed conservation of expression in amphidial neurons, which play a critical role in developmental regulation of both dauer larvae and L3i. Application of the PI3 kinase inhibitor LY294002 suppressed L3i in vitro activation in a dose-dependent fashion, with 100 µM resulting in a 90% decrease (odds ratio: 0.10, 95% confidence interval: 0.08–0.13) in the odds of resumption of feeding for treated L3i in comparison to the control. Together, these data support the hypothesis that Ss-age-1 regulates the development of S. stercoralis L3i via an IIS pathway in a manner similar to that observed in C. elegans dauer larvae. Understanding the mechanisms by which infective larvae are formed and activated may lead to novel control measures and treatments for strongyloidiasis and other soil-transmitted helminthiases.
... Unicellular organisms such as yeast (Saccharomyces) (Foury, 1997) and the facultatively colonial slime mold (Dictyostelium) (Firtel and Chung, 2000;Chung et al., 2001) can be used to analyze phenomena that involve important basic eukaryotic cell functions, such as metabolism, regulation of the cell cycle, membrane targeting and dynamics, protein folding, and DNA repair. Simple invertebrate systems such as Drosophila (Bernards and Hariharan, 2001;Reiter et al., 2001;Chien et al., 2002) or Caenorhabditis elegans (Aboobaker and Blaxter, 2000;Culetto and Sattelle, 2000) are excellent models for examining the coordinated actions of genes that function as components of a common molecular machine such as a signal-transduction pathway or a complex of physically interacting proteins. These proteins may or may not have highly related sequences in yeast, but if so, the value of the invertebrate system would be most pronounced if the human disease condition involved a tissue-specific requirement for the protein in question (e.g.. metabolic disorders resulting in neurological phenotypes). ...
he past two decades have brought major breakthroughs in our un- derstanding of the molecular and genetic circuits that control a myriad of developmental events in vertebrates and invertebrates. These detailed studies have revealed surprisingly deep similarities in the mechanisms underlying developmental processes across a wide range of bilaterally symmetric metazoans (bilateralia). Such phyloge- netic comparisons have defined a common core of genetic pathways guiding development and have made it possible to reconstruct many features of the most recent common ancestor of all bilateral animals, which most likely lived 600-800 million years ago (Shubin et al., 1997; Knoll and Carroll, 1999). As flushed out in more detail below and reiterated as a major unifying theme throughout the book, the com- mon metazoan ancestor already had in place many of the genetic path- ways that are present in modern-day vertebrates and invertebrates. This ancestor can be imagined as an advanced worm-like or primitive shrimp-like creature which had a few distinct body specializations along the nose-to-tail axis and was subdivided into three distinct germ layers (ectoderm, mesoderm, and endoderm). It also had evolved an inductive signaling system to partition the ectoderm into neural ver- sus nonneural components and is likely to have possessed appendages or outgrowths from its body wall with defined anterior-posterior, dor- sal-ventral, and proximo-distal axes, as well as light-sensitive organs, a sensory system for detecting vibrations, a rudimentary heart, a mo- lecular guidance system for initiating axon outgrowth to the midline of the nervous system, ion channels for conducting electrical impulses, synaptic machinery required for neural transmission, trachea, germ cells, and an innate immune system. The fact that the ancestor of vertebrate and invertebrate model or- ganisms was a highly evolved creature which had already invented complex interacting systems controlling development, physiology, and behavior has profound implications for medical genetics. The central points that we explore in this chapter can be broadly put into two cat- egories: (1) the great advantages of model organisms for identifying and understanding genes that are altered in heritable human diseases and (2) the functions of many of those genes and the evidence that they were present in the ancestral bilateral organisms and have re- mained largely intact in both vertebrate and invertebrate lineages dur- ing the ensuing course of evolution. In the course of discussing these points, we review the compelling evidence that developmentally im- portant genes have been phylogenetically conserved and the likelihood that developmental disorders in humans will often involve genes con- trolling similar morphogenetic processes in vertebrates and inverte- brates. A systematic analysis of human disease gene homologs in Drosophila supports this view since 75% of human disease genes are structurally related to genes present in Drosophila and more than a third of these human genes are highly related to their fruit fly coun- terparts (Bernards and Hariharan, 2001; Reiter et al., 2001; Chien et process, the emphasis in human genetics is shifting to understanding the function of these disease genes. An obvious avenue for functional analysis of disease genes is to study them in the closely related mouse using gene knockout techniques to assess the effects of either elimi- nating the gene's function or inducing specific disease-causing muta- tions. In some cases, this type of analysis has resulted in excellent mouse models for diseases that have phenotypes very similar to human dis- eases. In other cases, mouse knockout mutations have been less in- formative than hoped, either because the greater genetic redundancy in vertebrates masks the effect of mutations in single genes or because the mutations of interest are lethal at an early embryonic stage. Since there are limitations to the mouse system and there are deep ancestrally de- rived commonalities in the body plan organization and physiology of vertebrate and invertebrate model organisms, particularly flies and ne- matodes for which there are well-developed and powerful molecular genetic tools, these organisms are likely to play an increasingly im- portant role in the functional analysis of human disease genes. This chapter also compares the strengths and weaknesses of several well-de- veloped model systems, ranging from single-cell eukaryotes to pri- mates, as tools for dissecting the function of human disease genes. We propose that multiple model systems can be employed in cross-genomic analysis of human disease genes to address different kinds of issues, such as basic eukaryotic cellular functions (e.g., yeast and slime molds), assembly of genes into various types of molecular machines and path- ways (e.g., flies and nematodes), and accurate models of human dis- ease processes (e.g., vertebrates such as zebrafish and mice).
... Analogous effective methodologies have been established for MS analysis of affinitypurified protein complexes in the multicellular nematode worm Caenorhabditis elegans [49,50]. The utility and evolutionary conservation of interaction networks in these and other genetically tractable metazoan organisms is well established, making them powerful models for exploring human biology and disease mechanisms [52][53][54][55]. ...
Protein-protein interactions (PPIs) and multi-protein complexes perform central roles in the cellular systems of all living organisms. In humans, disruptions of the normal patterns of PPIs and protein complexes can be causative or indicative of a disease state. Recent developments in the biological applications of mass spectrometry (MS)-based proteomics have expanded the horizon for the application of systematic large-scale mapping of physical interactions to probe disease mechanisms. In this review, we examine the application of MS-based approaches for the experimental analysis of PPI networks and protein complexes, focusing on the different model systems (including human cells) used to study the molecular basis of common diseases such as cancer, cardiomyopathies, diabetes, microbial infections, and genetic and neurodegenerative disorders.
... The simplicity, tractability, and conserved genes of many nematode species have supported their use as models for diverse biological processes, including human diseases, aging, immunity, development, ecology, evolution, and hostbacterial interactions (Aboobaker and Blaxter, 2000;Couillault and Ewbank, 2002;Goodrich-Blair, 2007;Markaki and Tavernarakis, 2010;Neher, 2010;Xu and Kim, 2011). This last phenomenon-the intimate associations between two of the most speciose organisms on the planet-is the focus of the remainder of this review. ...
Nematodes are ubiquitous organisms that have a significant global impact on ecosystems, economies, agriculture, and human health. The applied importance of nematodes and the experimental tractability of many species have promoted their use as models in various research areas, including developmental biology, evolutionary biology, ecology, and animal-bacterium interactions. Nematodes are particularly well suited for the investigation of host associations with bacteria because all nematodes have interacted with bacteria during their evolutionary history and engage in a variety of association types. Interactions between nematodes and bacteria can be positive (mutualistic) or negative (pathogenic/parasitic) and may be transient or stably maintained (symbiotic). Furthermore, since many mechanistic aspects of nematode-bacterium interactions are conserved, their study can provide broader insights into other types of associations, including those relevant to human diseases. Recently, genome-scale studies have been applied to diverse nematode-bacterial interactions and have helped reveal mechanisms of communication and exchange between the associated partners. In addition to providing specific information about the system under investigation, these studies also have helped inform our understanding of genome evolution, mutualism, and innate immunity. In this review we discuss the importance and diversity of nematodes, "omics"' studies in nematode-bacterial systems, and the wider implications of the findings.
... Human disease genes are difficult to elucidate functionally by direct study. For many types of molecular functions, it is more fruitful to study their orthologs in model organisms [17]. To assist such studies, a number of databases have been developed to map disease genes to orthologs. ...
Orthology is one of the most important tools available to modern biology, as it allows making inferences from easily studied model systems to much less tractable systems of interest, such as ourselves. This becomes important not least in the study of genetic diseases. We here review work on the orthology of disease-associated genes and also present an updated version of the InParanoid-based disease orthology database and web site OrthoDisease, with 14-fold increased species coverage since the previous version. Using this resource, we survey the taxonomic distribution of orthologs of human genes involved in different disease categories. The hypothesis that paralogs can mask the effect of deleterious mutations predicts that known heritable disease genes should have fewer close paralogs. We found large-scale support for this hypothesis as significantly fewer duplications were observed for disease genes in the OrthoDisease ortholog groups.
... C. elegans is well-suited for studies of nervous system function in general, and synaptic transmission in particular. Although the advantages of C. elegans as a genetic system are now widely known and have been extolled elsewhere (Riddle et al., 1988; Ahringer, 1997; Aboobaker and Blaxter, 2000), these strengths also confer specific benefits for studying nervous system function ( Koushika and Nonet, 2000). ...
Thesis (Ph. D.)--Dept. of Biology, University of Utah, 2001. Includes bibliographical references.
... All of these features may be indicative of problems with muscular and/or neuronal function; both of these tissues are highly dependent on mitochondrial respiration. The nuo-1 and atp-2 mutations reproduce some of the features of mitochondrial diseases, suggesting that C. elegans may be a good model system for studying mitochondrial disorders (54,55). ...
The growth and development of Caenorhabditis elegans are energy-dependent and rely on the mitochondrial respiratory chain (MRC) as the major source of ATP. The MRC is composed of approximately 70 nuclear and 12 mitochondrial gene products. Complexes I and V are multisubunit proteins of the MRC. The nuo-1 gene encodes the NADH- and FMN-binding subunit of complex I, the NADH-ubiquinone oxidoreductase. The atp-2 gene encodes the active-site subunit of complex V, the ATP synthase. The nuo-1(ua1) and atp-2(ua2) mutations are both lethal. They result in developmental arrest at the third larval stage (L3), arrest of gonad development at the second larval stage (L2), and impaired mobility, pharyngeal pumping, and defecation. Surprisingly, the nuo-1 and atp-2 mutations significantly lengthen the life spans of the arrested animals. When MRC biogenesis is blocked by chloramphenicol or doxycycline (inhibitors of mitochondrial translation), a quantitative and homogeneous developmental arrest as L3 larvae also results. The common phenotype induced by the mutations and drugs suggests that the L3-to-L4 transition may involve an energy-sensing developmental checkpoint. Since approximately 200 gene products are needed for MRC assembly and mtDNA replication, transcription, and translation, we predict that L3 arrest will be characteristic of mutations in these genes.
... The latter invertebrate is particularly well suited for studies of such pathways because of the ease of propagation and culture, the ability to disrupt developmental pathways through genetic manipulation, and the detection of resultant phenotypic effects on the nematode (Bürglin et al., 1998;Plasterk, 1999;Aboobaker and Blaxter, 2000). ...
A putative serine/threonine protein kinase (HcSTK) from the parasitic nematode Haemonchus contortus was characterised at the mRNA and amino acid levels. HcSTK displays a high level of identity (85-93% in the catalytic domain) with proteins of the PAR-1/MARK serine/threonine protein kinase (STK) subfamily, which represent signal transduction molecules involved in establishing and maintaining polarity in proliferating and differentiating cells. The transcript of hcstk is expressed in different developmental stages (second-, third-, fourth-stage larvae and adults) and various organs (muscle, intestine and reproductive) of H. contortus. In addition, there are several isoforms which appear to relate to a single gene. The expression profile of hcstk is similar to that of Caenorhabditis elegans PAR-1, and the level of sequence identity among members of the PAR-1/MARK STK subfamily, representing a range of species of vertebrates (e.g. humans and rodents), invertebrates (e.g. insects and C. elegans) and yeast, suggests that HcSTK may be involved in a conserved signal transduction pathway.
... Although the dauer stage is facultative in C. elegans, it is often obligatory in other species. As parasitic nematodes can infect humans and agricultural crops, a fundamental understanding of the mechanisms that underlie the dauer state in C. elegans might illuminate methods for control of nematodes that are pests (Aboobaker and Blaxter, 2000;Blaxter and Bird, 1997). Analysis of dauer recovery in C. elegans could help define a conserved developmental transition for all nematodes. ...
The dauer is a developmental stage in C. elegans that exhibits increased longevity, stress resistance, nictation and altered metabolism compared with normal worms. We have used DNA microarrays to profile gene expression differences during the transition from the dauer state to the non-dauer state and after feeding of starved L1 animals, and have identified 1984 genes that show significant expression changes. This analysis includes genes that encode transcription factors and components of signaling pathways that could regulate the entry to and exit from the dauer state, and genes that encode components of metabolic pathways important for dauer survival and longevity. Homologs of C. elegans dauer-enriched genes may be involved in the disease process in parasitic nematodes.
Inflammatory bowel disease (IBD) research often relies on animal models to study the etiology, pathophysiology, and management of IBD. Among these models, rats and mice are frequently employed due to their practicality and genetic manipulability. However, for studies aiming to closely mimic human pathology, non-human primates such as monkeys and dogs offer valuable physiological parallels. Guinea pigs, while less commonly used, present unique advantages for investigating the intricate interplay between neurological and immunological factors in IBD. Additionally, New Zealand rabbits excel in endoscopic biopsy techniques, providing insights into mucosal inflammation and healing processes. Pigs, with their physiological similarities to humans, serve as ideal models for exploring the complex relationships between nutrition, metabolism, and immunity in IBD. Beyond mammals, non-mammalian organisms including zebrafish, Drosophila melanogaster, and nematodes offer specialized insights into specific aspects of IBD pathology, highlighting the diverse array of model systems available for advancing our understanding of this multifaceted disease. In this review, we conduct a thorough analysis of various animal models employed in IBD research, detailing their applications and essential experimental parameters. These include clinical observation, Disease Activity Index score, pathological assessment, intestinal barrier integrity, fibrosis, inflammatory markers, intestinal microbiome, and other critical parameters that are crucial for evaluating modeling success and drug efficacy in experimental mammalian studies. Overall, this review will serve as a valuable resource for researchers in the field of IBD, offering insights into the diverse array of animal models available and their respective applications in studying IBD.
The bulk of information about the phylum Nematoda is concerned with the thousands of parasitic species, particularly those of medical, veterinary, and entomopathogenic significance, and the free-living Caenorhabditis elegans . Longevity information about free-living nematodes is lacking, but males are believed to be much shorter lived than females. Feeding by free-living nematodes exploits many different food habits and the animals may be selective or nonselective feeders. The hydrostatic skeleton is a result of the nematode pseudocoel. The nervous system of nematodes is quite simple and consists of only a few hundred neurons, allowing for at least three sensory responses: chemical, mechanical, and thermal. Most protozoan parasites of nematodes are associated with animal or plant parasitic species. Nutritional diseases are observed in free-living nematodes as a retardation of the normal life cycle. Metabolic diseases or disease conditions have been observed by the incorporation of inhibitors into culture media.
The insulin-like signaling (IIS) pathway is considered to be significant in regulating fat metabolism, dauer formation, stress response and longevity in Caenorhabditis elegans. “Dauer hypothesis” indicates that similar IIS transduction mechanism regulates dauer development in free-living nematode C. elegans and the development of infective third-stage larvae (iL3) in parasitic nematodes, and this is bolstered by a few researches on structures and functions of the homologous genes in the IIS pathway cloned from several parasitic nematodes. In this study, we identified the insulin-like receptor encoding gene, Acan-daf-2, from the parasitic nematode Angiostrongylus cantonensis, and determined the genomic structures, transcripts and functions far more thorough in longevity, stress resistance and dauer formation. The sequence of Acan-DAF-2, consisting of 1413 amino acids, contained all of the characteristic domains of insulin-like receptors from other taxa. The expression patterns of Acan-daf-2 in the C. elegans surrogate system showed that pAcan-daf-2:gfp was only expressed in intestine, compared with the orthologue in C. elegans, Ce-daf-2 in both intestine and neurons. In addition to the similar genomic organization to Ce-daf-2, Acan-DAF-2 could also negatively regulate Ce-DAF-16A through nuclear/cytosolic translocation and partially restore the C. elegans daf-2(e1370) mutation in longevity, dauer formation and stress resistance. These findings provided further evidence of the functional conservation of DAF-2 between parasitic nematodes and the free-living nematode C. elegans, and might be significant in understanding the developmental biology of nematode parasites, particularly in the infective process and the host-specificity.
Fork head box transcription factors subfamily O (FoxO) is regarded to be significant in cell-cycle control, cell differentiation, ageing, stress response, apoptosis, tumour formation and DNA damage repair. In the free-living nematode Caenorhabditis elegans, the FoxO transcription factor is encoded by Ce-daf-16, which is negatively regulated by insulin-like signaling (IIS) and involved in promoting dauer formation through bringing about its hundreds of downstream genes expression. In nematode parasites, orthologues of daf-16 from several species have been identified, with functions in rescue of dauer phenotypes determined in a surrogate system C. elegans. In this study, we identified the FoxO encoding gene, Acan-daf-16, from the parasitic nematode Angiostrongylus cantonensis, and determined the genomic structures, transcripts and functions far more thorough in longevity, stress resistance and dauer formation. Acan-daf-16 encodes two proteins, Acan-DAF-16A and Acan-DAF-16B, consisting of 555 and 491 amino acids, respectively. Both isoforms possess the highly conserved fork head domains. Acan-daf-16A and Acan-daf-16B are expressed from distinct promoters. The expression patterns of Acan-daf-16 isoforms in the C. elegans surrogate system showed that p Acan-daf-16a:gfp was expressed in all cells of C. elegans, including the pharynx, and the expression of p Acan-daf-16b:gfp was restricted to the pharynx. In addition to the same genomic organization to the orthologue in C. elegans, Ce-daf-16, both Acan-DAF-16 isoforms could restore the C. elegans daf-16(mg54) mutation in longevity, dauer formation and stress resistance, in spite of the partial complementation of Acan-DAF-16B isoform in longevity. These findings provide further evidence of the functional conservation of DAF-16s between parasitic nematodes and the free-living nematode C. elegans.
The goal of this chapter is to identify and prioritize species of animals that can serve as surrogates for environmental and human health in comparative toxicogenomics studies. By characterizing current research needs and by deriving a set of criteria for selecting appropriate surrogate species, we will accomplish this goal. These criteria will be applied in reviewing the species currently used in toxicological testing and research, ecological monitoring and research, and the genomic sciences. The review includes a short discussion on why a community-based approach is used (in one case) to promote a model species for this emerging field and will conclude by identifying a suite of species that could serve as an initial starting point for a multinational comparative toxicogenomics research program. © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC).
Caenorhabditis elegans is a free-living nematode cultured in an axenic medium, the Caenorhabditis elegans Maintenance Medium (CeMM), which contains B-vitamins, salts, amino acids, nucleic acid substituents, growth factors and glucose as an energy source. After initial experiments established that either pantothenate or pantethine would satisfy the vitamin B5 requirement in C. elegans, reproduction in the nematodes was measured in eight equimolar concentrations of calcium pantothenate, pantethine or coenzyme A. The optimal levels for pantothenate were found to be 7.5, 30 and 120 μg ml−1. The optimal levels for pantethine and coenzyme A were found to be 35 μg ml−1 and 100 μg ml−1, respectively. Among the three compounds, coenzyme A (at 100 μg/ml) supported a significantly greater population growth and, perhaps, is a more metabolically active form. Mild toxicity was demonstrated for pantothenate at 480μg ml−1, pantethine at 560 and 140 μg ml−1, and coenzyme A was found to exhibit toxicity at 410 and 1700 μg ml−1. Based on our results, we recommend that in the future the CeMM could be supplemented with pantothenate (7.5 μg ml−1) alone.
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To investigate the toxicological effects of nanomaterials, experimental studies on the absorption and accumulation in organisms are of broad interest. In the present study, Caenorhabditis elegans (C. elegans) was used as a ''model'' organism to investigate the bioaccumulation and toxicological effects of engineered copper nanoparticles with a scanning technique of microbeam synchrotron radiation X-ray fluorescence (m-SRXRF). The adult hermaphrodite is anatomically simple with 959 somatic cells and 1 mm in length. The mapping results of the whole organism indicate that the exposure to copper nanoparticles can result in an obvious elevation of Cu and K levels, and a change of bio-distribution of Cu in nematodes. Accumulation of Cu occurs in the head and at a location 1/3 of the way up the body from the tail compared to the un-exposed control. In contrast, a higher amount of Cu was detected in other portion of worm body, especially in its excretory cells and intestine when exposed to Cu 2+ . The results compared well with total Cu levels in nematodes, which were 4.10 AE 0.54, 12.32 AE 0.49 and 5.22 AE 0.63 mg g À1 dry weight for the PBS, Cu 2+ and Cu nanoparticle groups, respectively, measured by ICP-MS. The nondestructive and multi-elemental m-SRXRF provides an important tool for mapping the elemental distribution in the whole body of a single tiny nematode at lower levels.
Diseases of intestinal inflammation, including Crohn's disease, ulcerative colitis and necrotizing enterocolitis, cause substantial acute and chronic disability in a large proportion of the population. Crohn's disease and ulcerative colitis, which are collectively referred to as inflammatory bowel disease (IBD), lead to recurrent episodes of intestinal dysfunction and systemic illness, whereas necrotizing enterocolitis is characterized by the development of dramatic and all too often fatal intestinal necrosis in infants. To determine the molecular underpinnings of these disorders, investigators have explored a variety of animal models that vary widely in their complexity. These experimental systems include the invertebrate nematode Caenorhabditis elegans, the more complex invertebrate Drosophila melanogaster, and vertebrate systems including mice, rats and other mammals. This review explores the experimental models that are used to mimic and evaluate the pathogenic mechanisms leading to these diseases of intestinal inflammation. We then highlight, as an example, how the use of different experimental models that focus on the role of Toll-like receptor 4 (TLR4) signaling in the gut has revealed important distinctions between the pathogenesis of IBD and necrotizing enterocolitis. Specifically, TLR4-mediated signaling plays a protective role in the development of Crohn's disease and ulcerative colitis, whereas this signaling pathway plays a causative role in the development of necrotizing enterocolitis in the newborn small intestine by adversely affecting intestinal injury and repair mechanisms.
Experiments on model organisms are used to extend the understanding of complex biological processes. In Caenorhabditis elegans studies, populations of specimens are sampled to measure certain morphological properties and a population is characterized based on statistics extracted from such samples. Automatic detection of C. elegans in such culture images is a difficult problem. The images are affected by clutter, overlap and image degradations. In this paper, we exploit shape and appearance differences between C. elegans and non-C. elegans segmentations. Shape information is captured by optimizing a parametric open contour model on training data. Features derived from the contour energies are proposed as shape descriptors and integrated in a probabilistic framework. These descriptors are evaluated for C. elegans detection in culture images. Our experiments show that measurements extracted from these samples correlate well with ground truth data. These positive results indicate that the proposed approach can be used for quantitative analysis of complex nematode images.
LET-721 is the Caenorhabditis elegans ortholog of electron-transferring flavoprotein dehydrogenase (ETFDH). We are studying this protein in C. elegans in order to establish a tractable model system for further exploration of ETFDH structure and function. ETFDH is an inner mitochondrial membrane localized enzyme that plays a key role in the beta-oxidation of fatty acids and catabolism of amino acids and choline. ETFDH accepts electrons from at least twelve mitochondrial matrix flavoprotein dehydrogenases via an intermediate dimer protein and transfers the electrons to ubiquinone. In humans, ETFDH mutations result in the autosomal recessive metabolic disorder, multiple acyl-CoA dehydrogenase deficiency. Mutants of let-721 in C. elegans are either maternal effect lethals or semi-sterile. let-721 is transcribed in the pharynx, body wall muscle, hypoderm, intestine and somatic gonad. In addition, the subcellular localization of LET-721 agrees with predictions that it is localized to mitochondria. We identified and confirmed three cis-regulatory sequences (pha-site, rep-site, and act-site). Phylogenetic footprinting of each site indicates that they are conserved between four Caenorhabditis species. The pha-site mapped roughly 1,300 bp upstream of let-721's translational start site and is necessary for expression in pharyngeal tissues. The rep-site mapped roughly 830 bp upstream of the translational start site and represses expression of LET-721 within pharyngeal tissues. The act-site mapped roughly 800 bp upstream of the translational start site and is required for expression within spermatheca, body wall muscle, pharynx, and intestine. Taken together, we find that LET-721 is a mitochondrially expressed protein that is under complex transcriptional controls.
As an experimental system, Caenorhabditis elegans offers a unique opportunity to interrogate in vivo the genetic and molecular functions of human disease-related genes. For example, C. elegans has provided crucial insights into fundamental biologic processes, such as cell death and cell fate determinations, as well as pathologic processes such as neurodegeneration and microbial susceptibility. The C. elegans model has several distinct advantages, including a completely sequenced genome that shares extensive homology with that of mammals, ease of cultivation and storage, a relatively short lifespan and techniques for generating null and transgenic animals. However, the ability to conduct unbiased forward and reverse genetic screens in C. elegans remains one of the most powerful experimental paradigms for discovering the biochemical pathways underlying human disease phenotypes. The identification of these pathways leads to a better understanding of the molecular interactions that perturb cellular physiology, and forms the foundation for designing mechanism-based therapies. To this end, the ability to process large numbers of isogenic animals through automated work stations suggests that C. elegans, manifesting different aspects of human disease phenotypes, will become the platform of choice for in vivo drug discovery and target validation using high-throughput/content screening technologies.
The soil nematode Caenorhabditis elegans is one of the simplest animals having the status of a laboratory model. Its genome contains 80 cytochrome P450 genes (CYP). In order to study CYP gene expression in C. elegans mixed stages and synchronized hermaphrodites were exposed to 18 known xenobiotic cytochrome P450 inducers. Messenger RNA expression was detected by DNA arrays and semiquantitative RT-PCR. Using subfamily-specific primers, a pooled set of exon-rich CYP fragments could be amplified. In this way it was possible to systematically check the influence of different inducers on CYP expression at the same time. The well-known CYP1A inducers beta-naphthoflavone, PCB52, and lansoprazol were the most active and in particular they strongly induced almost all CYP35 isoforms. A few number of further CYP forms were found to be inducible by other xenobiotics like phenobarbital, atrazine, and clofibrate. In addition, a transgenic C. elegans line expressing GFP under control of the CYP35A2 promoter showed a strong induction of the fusion by beta-naphthoflavone in the intestine.
We describe the successful use of the reverse genetic technique RNA interference (RNAi) to investigate gene function in the human filarial nematode parasite Brugia malayi. We used fluorescently labelled double stranded RNA (dsRNA) to demonstrate that 300 bp molecules are able to enter adult females in culture while they remain excluded from microfilariae (mf). We have developed an optimised microvolume culture system to allow the exposure of parasites to high concentrations of dsRNA for extended periods. Culturing of adult female parasites in this system for 24h does not significantly reduce parasite lifespan or mf release in culture. Three B. malayi genes, beta-tubulin (Bm-tub-1), RNA polymerase II large subunit (Bm-ama-1) and B. malayi mf sheath protein 1/mf22 (Bm-shp-1) were targeted by soaking adult female B. malayi in dsRNA complementary to these transcripts in the optimised culture system. Targeting of the two housekeeping genes Bm-tub-1 and Bm-ama-1 led to a reduction in the levels of their transcripts, as assessed by reverse transcriptase coupled PCR (RT-PCR), and resulted in parasite death in culture. In contrast, targeting of the Bm-shp-1 gene was not lethal to adult females in culture. A marked reduction in mf release was observed for shp-1 RNAi parasites compared to controls and in addition 50% of mf released did not have fully elongated sheaths. This "short" phenotype correlated with the loss of the stockpiled shp-1 transcript from developing mf in treated adult female gonads. From these data we conclude that RNAi may be a useful method for assessment of drug target potential of genes identified in filarial gene discovery projects.
International efforts are underway that aim at determining the complete genome sequence of the social amoeba Dictyostelium discoideum. As strategy, a whole chromosome shotgun (WCS) approach was chosen and each of the six Dictyostelium chromosomes was assigned to project partners. The project is well advanced, chromosome 2 was recently published, and it is expected that the sequences of chromosomes 1 and 6 and a gene catalogue for the complete genome will be available at the end of this year. The genome sequence, together with powerful molecular genetic tools, will undoubtedly further accelerate Dictyostelium research into a number of fundamental biological processes that are common to a wide range of eukaryotes. Furthermore, it will constitute the basis for genome-wide functional analyses. The integration of results from these studies should ultimately lead to a better understanding of the complex networks that govern cellular behavior and development.
The 97-megabase genomic sequence of the nematode Caenorhabditis elegans reveals over 19,000 genes. More than 40 percent of the predicted protein products find significant matches in other organisms.
There is a variety of repeated sequences, both local and dispersed. The distinctive distribution of some repeats and highly
conserved genes provides evidence for a regional organization of the chromosomes.
Genetic linkage studies place a gene causing early onset familial Alzheimer's disease (FAD) on chromosome 14q24.3 (refs 1−4). Five mutations within the S182 (Presenilin 1: PS−1) gene, which maps to this region, have recently been reported in several early onset FAD kindreds5. We have localized the PS-1 gene to a 75 kb region and present the structure of this gene, evidence for alternative splicing and describe six novel mutations in early onset FAD pedigrees all of which alter residues conserved in the STM2
6 (Presenilin 2: PS-2) gene.
Nematodes are important: parasitic nematodes threaten the health of plants, animals and humans on a global scale; interstitial nematodes pervade sediment and soil ecosystems in overwhelming numbers; and Caenorhabditis elegans is a favourite experimental model system. A lack of clearly homologous characters and the absence of an informative fossil record have prevented us from deriving a consistent evolutionary framework for the phylum. Here we present a phylogenetic analysis, using 53 small subunit ribosomal DNA sequences from a wide range of nematodes. With this analysis, we can compare animal-parasitic, plant-parasitic and free-living taxa using a common measurement. Our results indicate that convergent morphological evolution may be extensive and that present higher-level classification of the Nematoda will need revision. We identify five major clades within the phylum, all of which include parasitic species. We suggest that animal parasitism arose independently at least four times, and plant parasitism three times. We clarify the relationship of C. elegans to major parasitic groups; this will allow more effective exploitation of our genetic and biological knowledge of this model species.
The limb-girdle muscular dystrophies are a genetically heterogeneous group of inherited progressive muscle disorders that affect mainly the proximal musculature, with evidence for at least three autosomal dominant and eight autosomal recessive loci. The latter mostly involve mutations in genes encoding components of the dystrophin-associated complex; another form is caused by mutations in the gene for the muscle-specific protease calpain 3. Using a positional cloning approach, we have identified the gene for a form of limb-girdle muscular dystrophy that we previously mapped to chromosome 2p13 (LGMD2B). This gene shows no homology to any known mammalian gene, but its predicted product is related to the C. elegans spermatogenesis factor fer-1. We have identified two homozygous frameshift mutations in this gene, resulting in muscular dystrophy of either proximal or distal onset in nine families. The proposed name 'dysferlin' combines the role of the gene in producing muscular dystrophy with its C. elegans homology.
Positional cloning has already produced the sequences of more than 70 human genes associated with specific diseases. In addition to their medical importance, these genes are of interest as a set of human genes isolated solely on the basis of the phenotypic effect of the respective mutations. We analyzed the protein sequences encoded by the positionally cloned disease genes using an iterative strategy combining several sensitive computer methods. Comparisons to complete sequence databases and to separate databases of nematode, yeast, and bacterial proteins showed that for most of the disease gene products, statistically significant sequence similarities are detectable in each of the model organisms. Only the nematode genome encodes apparent orthologs with conserved domain architecture for the majority of the disease genes. In yeast and bacterial homologs, domain organization is typically not conserved, and sequence similarity is limited to individual domains. Generally, human genes complement mutations only in orthologous yeast genes. Most of the positionally cloned genes encode large proteins with several globular and nonglobular domains, the functions of some or all of which are not known. We detected conserved domains and motifs not described previously in a number of proteins encoded by disease genes and predicted functions for some of them. These predictions include an ATP-binding domain in the product of hereditary nonpolyposis colon cancer gene (a MutL homolog), which is conserved in the HS90 family of chaperone proteins, type II DNA topoisomerases, and histidine kinases, and a nuclease domain homologous to bacterial RNase D and the 3′-5′ exonuclease domain of DNA polymerase I in the Werner syndrome gene product.
A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
The Tc1 transposable element family of the nematode Caenorhabditis elegans consists primarily of 1.6-kb size elements. This uniformity of size is in contrast to P in Drosophila and Ac/Ds in maize. Germline transposition and excision of Tc1 are detectable in the Bergerac (BO) strain, but not in the commonly used Bristol (N2) strain. A previous study suggested that multiple genetic components are responsible for the germline Tc1 activity of the BO strain. To analyze further this mutator activity, we derived hybrid strains between the BO strain and the N2 strain. One of the hybrid strains exhibits a single locus of mutator activity, designated mut-4, which maps to LGI. Two additional mutators, mut-5 II and mut-6 IV, arose spontaneously in mut-4 harboring strains. This spontaneous appearance of mutator activity at new sites suggests that the mutator itself transposes. The single mutator-harboring strains with low Tc1 copy number generated in this study should be useful in investigations of the molecular basis of mutator activity. As a first step toward this goal, we examined the Tc1 elements in these low copy number strains for elements consistently co-segregating with mutator activity. Three possible candidates were identified: none was larger than 1.6 kb.
The sex myoblasts (SMs) in C. elegans hermaphrodites undergo anteriorly directed cell migrations that allow for the proper localization of the egg-laying muscles. These migrations are controlled in part by a signal emanating from gonadal cells that allows the SMs to be attracted to their precise final positions flanking the center of the gonad. Mutations in egl-15 alter the nature of the interaction between the gonad and the SMs, resulting in the posterior displacement of the SMs. Here we show that egl-15 encodes a receptor tyrosine kinase of the fibroblast growth factor receptor (FGFR) subfamily with multiple roles in development. Three genes were identified that behave genetically as activators or mediators of egl-15 activity. One of these genes, sem-5, encodes an adaptor molecule that transduces signals from a variety of receptor tyrosine kinases. Like egl-15 and sem-5, the other two genes may similarly act in FGFR signaling pathways in C. elegans.
Although transforming growth factor beta (TGF-beta) superfamily ligands play critical roles in diverse developmental processes, how cells transduce signals from these ligands is still poorly understood. Cell surface receptors for these ligands have been identified, but their cytoplasmic targets are unknown. We have identified three Caenorhabditis elegans genes, sma-2, sma-3, and sma-4, that have mutant phenotypes similar to those of the TGF-beta-like receptor gene daf-4, indicating that they are required for daf-4-mediated developmental processes. We show that sma-2 functions in the same cells as daf-4, consistent with a role in transducing signals from the receptor. These three genes define a protein family, the dwarfins, that includes the Mad gene product, which participates in the decapentaplegic TGF-beta-like pathway in Drosophila [Sekelsky, J. J., Newfeld, S. J., Raftery, L. A., Chartoff, E. H. & Gelbart, W. M. (1995) Genetics 139, 1347-1358]. The identification of homologous components of these pathways in distantly related organisms suggests that dwarfins may be universally required for TGF-beta-like signal transduction. In fact, we have isolated highly conserved dwarfins from vertebrates, indicating that these components are not idiosyncratic to invertebrates. These analyses suggest that dwarfins are conserved cytoplasmic signal transducers.
The genome of the yeast Saccharomyces cerevisiae has been completely sequenced through a worldwide collaboration. The sequence of 12,068 kilobases defines 5885 potential
protein-encoding genes, approximately 140 genes specifying ribosomal RNA, 40 genes for small nuclear RNA molecules, and 275
transfer RNA genes. In addition, the complete sequence provides information about the higher order organization of yeast's
16 chromosomes and allows some insight into their evolutionary history. The genome shows a considerable amount of apparent
genetic redundancy, and one of the major problems to be tackled during the next stage of the yeast genome project is to elucidate
the biological functions of all of these genes.
We provide evidence that normal human presenilins can substitute for Caenorhabditis elegans SEL-12 protein in functional assays in vivo. In addition, six familial Alzheimer disease-linked mutant human presenilins were tested and found to have reduced ability to rescue the sel-12 mutant phenotype, suggesting that they have lower than normal presenilin activity. A human presenilin 1 deletion variant that fails to be proteolytically processed and a mutant SEL-12 protein that lacks the C terminus display considerable activity in this assay, suggesting that neither presenilin proteolysis nor the C terminus is absolutely required for normal presenilin function. We also show that sel-12 is expressed in most neural and nonneural cell types in all developmental stages. The reduced activity of mutant presenilins and as yet unknown gain-of-function properties may be a contributing factor in the development of Alzheimer disease.
The arthropods constitute the most diverse animal group, but, despite their rich fossil record and a century of study, their phylogenetic relationships remain unclear. Taxa previously proposed to be sister groups to the arthropods include Annelida, Onychophora, Tardigrada and others, but hypotheses of phylogenetic relationships have been conflicting. For example, onychophorans, like arthropods, moult periodically, have an arthropod arrangement of haemocoel, and have been related to arthropods in morphological and mitochondrial DNA sequence analyses. Like annelids, they possess segmental nephridia and muscles that are a combination of smooth and obliquely striated fibres. Our phylogenetic analysis of 18S ribosomal DNA sequences indicates a close relationship between arthropods, nematodes and all other moulting phyla. The results suggest that ecdysis (moulting) arose once and support the idea of a new clade, Ecdysozoa, containing moulting animals: arthropods, tardigrades, onychophorans, nematodes, nematomorphs, kinorhynchs and priapulids. No support is found for a clade of segmented animals, the Articulata, uniting annelids with arthropods. The hypothesis that nematodes are related to arthropods has important implications for developmental genetic studies using as model systems the nematode Caenorhabditis elegans and the arthropod Drosophila melanogaster, which are generally held to be phylogenetically distant from each other.
The 4,639,221–base pair sequence of Escherichia coliK-12 is presented. Of 4288 protein-coding genes annotated, 38 percent have no attributed function. Comparison with five other
sequenced microbes reveals ubiquitous as well as narrowly distributed gene families; many families of similar genes within
E. coli are also evident. The largest family of paralogous proteins contains 80 ABC transporters. The genome as a whole is strikingly
organized with respect to the local direction of replication; guanines, oligonucleotides possibly related to replication and
recombination, and most genes are so oriented. The genome also contains insertion sequence (IS) elements, phage remnants,
and many other patches of unusual composition indicating genome plasticity through horizontal transfer.
Caenorhabditis elegans has become a popular model system for genetic and molecular research, since it is easy to maintain and has a very fast life-cycle. Its genome is small and a virtually complete physical map in the form of cosmids and YAC clones exists. Thus it was chosen as a model system by the Genome Project for sequencing, and it is expected that by 1998 the complete sequence (100 million bp) will be available. The accumulated wealth of information about C. elegans should be a boon for nematode parasitologists, as many aspects of gene regulation and function can be studied in this simple model system. A large array of techniques is available to study many aspects of C. elegans biology. In combination with genome projects for parasitic nematodes, conserved genes can be identified rapidly. We expect many new areas of fertile research that will lead to new insights in helminth parasitology, which are based not only on the information gained from C. elegans per se, but also from its use as a heterologous system to study parasitic genes.
Receptor tyrosine phosphatases have been implicated in playing important roles in cell signaling events by their ability to regulate the level of protein tyrosine phosphorylation. Although the catalytic activity of their phosphatase domains has been well established, the biological roles of these molecules are, for the most part, not well understood. Here we show that the Caenorhabditis elegans protein CLR-1 (CLeaR) is a receptor tyrosine phosphatase (RTP) with a complex extracellular region and two intracellular phosphatase domains. Mutations in clr-1 result in a dramatic Clr phenotype that we have used to study the physiological requirements for the CLR-1 RTP. We show that the phosphatase activity of the membrane-proximal domain is essential for the in vivo function of CLR-1. By contrast, we present evidence that the membrane-distal domain is not required to prevent the Clr phenotype in vivo. The Clr phenotype of clr-1 mutants is mimicked by activation of the EGL-15 fibroblast growth factor receptor (FGFR) and is suppressed by mutations that reduce or eliminate the activity of egl-15. Our data strongly indicate that CLR-1 attenuates the action of an FGFR-mediated signaling pathway by dephosphorylation.
A maternally expressed Caenorhabditis elegans gene called cyk-1 is required for polar body extrusion during meiosis and for a late step in cytokinesis during embryonic mitosis. Other microfilament- and microtubule-dependent processes appear normal in cyk-1 mutant embryos, indicating that cyk-1 regulates a specific subset of cytoskeletal functions. Because cytokinesis initiates normally and cleavage furrows ingress extensively in cyk-1 mutant embryos, we propose that the wild-type cyk-1 gene is required for a late step in cytokinesis. Cleavage furrows regress after completion of mitosis in cyk-1 mutants, leaving multiple nuclei in a single cell. Positional cloning and sequence analysis of the cyk-1 gene reveal that it encodes an FH protein, a newly defined family of proteins that appear to interact with the cytoskeleton during cytokinesis and in the regulation of cell polarity. Consistent with cyk-1 function being required for a late step in embryonic cytokinesis, we show that the CYK-1 protein co-localizes with actin microfilaments as a ring at the leading edge of the cleavage furrow, but only after extensive furrow ingression. We discuss our findings in the context of other studies suggesting that FH genes in yeast and insects function early in cytokinesis to assemble a cleavage furrow.
The stereotyped mating behaviour of the Caenorhabditis elegans male is made up of several substeps: response, backing, turning, vulva location, spicule insertion and sperm transfer. The complexity of this behaviour is reflected in the sexually dimorphic anatomy and nervous system¹. Behavioural functions have been assigned to most of the male-specific sensory neurons by means of cell ablations; for example, the hook sensory neurons HOA and HOB are specifically required for vulva location². We have investigated how sensory perception of the hermaphrodite by the C. elegans male controls mating behaviours. Here we identify a gene, lov-1 (for location of vulva), that is required for two male sensory behaviours: response and vulva location. lov-1 encodes a putative membrane protein with a mucin-like, serine–threonine-rich amino terminus³ followed by two blocks of homology to human polycystins, products of the autosomal dominant polycystic kidney-disease loci PKD1 and PKD2 (ref 4). LOV-1 is the closest C. elegans homologue of PKD1. lov-1 is expressed in adult males in sensory neurons of the rays, hook and head, which mediate response, vulva location, and potentially chemotaxis to hermaphrodites, respectively2, ⁵. PKD-2, the C. elegans homologue of PKD2, is localized to the same neurons as LOV-1, suggesting that they function in the same pathway.
Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.
This chapter provides a practical guide to immunofluorescence microscopy of Caenorhabditis elegans. In this method, fixed tissue is stained with a fluorescently labeled antibody, and visualized with the light microscope. The antibody ensures that staining is limited to the location of the antigen, and the microscope provides a magnified image of the fluorescent area. Immunofluorescence microscopy is an especially powerful tool for studies of C. elegans. Immunofluorescence staining can provide essential information for the analysis of mutant phenotypes. Mutations that alter developmental programs or the expression of particular proteins can be examined in situ and described at the level of individual cells or cell lineages. Immunofluorescence microscopy can be used to detect the expression of transgenes in C. elegans. Specific epitope tags can be incorporated into a transgene, such that the epitope-tagged protein can be detected with a specific antibody that does not react with the endogenous protein. Epitope tagging is particularly useful for cases in which a null mutation in the endogenous gene is either not available or desirable in the transgenic animal.
This chapter discusses the whole-mount in situ hybridization for the detection of RNA in Caenorhabditis elegans embryos. In situ hybridization to RNA is an effective tool for the analysis of gene expression during development. This technique is particularly important for Caenorhabditis elegans, as isolation of RNA from specific tissues or developmental stages is generally not possible in this organism. The availability of the complete cell lineage and the reproducibility of cell positions from one animal to the next allow RNA expression patterns to be analyzed at the level of individual cells. A number of in situ hybridization protocols have been developed for the detection of RNA in squashed, dissected, or sectioned tissues of C. elegans. More recently, protocols using whole-mount preparations of C. elegans have also been described. By preserving the three-dimensional structure of the specimen, whole-mount preparations facilitate the identification of specific cells and the analysis of complex expression patterns. This chapter describes a protocol for detection of RNA in whole-mount C. elegans embryos. This procedure is based on protocols for Drosophila, which makes use of highly sensitive digoxigenin-labeled probes.
Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.
The tremendous value--both intellectual and commercial--of genomic sequence is now generally recognized. The publicly funded International Human Genome Project must deliver the human sequence as rapidly as possible, while retaining its standards of high accuracy and complete mapping. The pressure is being met by increasing capacity to accelerate the release of unfinished data and to bring forward the target date for completion.
Hookworms are gut-dwelling, blood-feeding nematodes that infect hundreds of millions of people, particularly in the
tropics. As part of a program aiming to define novel drug targets and vaccine candidates for human parasitic nematodes,
genes expressed in adults of the human hookworm Necator americanus were surveyed by the expressed sequence tag
approach. In total 161 new hookworm genes were identified. For the majority of these, a function could be assigned by
homology. The dataset includes proteases, protease inhibitors, a lipid binding protein, C-type lectins, an anti-bacterial
factor, globins and other genes of interest from a drug or vaccine development viewpoint. Three different classes of small,
secreted proteins were identified that may be involved in the host–parasite interaction, including potential potassium
channel blocking peptides. One third of the genes were novel. These included highly expressed, secreted (glyco)proteins
which may be part of the excretory–secretory products of these important pathogens. Of particular interest are a family
of 9 genes with similarity to the immunomodulatory protein, neutrophil inhibitory factor, that may play a role in
establishing an immunocompromised niche for this successful parasite.
The structure and connectivity of the nervous system of the nematode Caenorhabditis elegans has been deduced from reconstructions of electron micrographs of serial sections. The hermaphrodite nervous system has a total complement of 302 neurons, which are arranged in an essentially invariant structure. Neurons with similar morphologies and connectivities have been grouped together into classes; there are 118 such classes. Neurons have simple morphologies with few, if any, branches. Processes from neurons run in defined positions within bundles of parallel processes, synaptic connections being made en passant. Process bundles are arranged longitudinally and circumferentially and are often adjacent to ridges of hypodermis. Neurons are generally highly locally connected, making synaptic connections with many of their neighbours. Muscle cells have arms that run out to process bundles containing motoneuron axons. Here they receive their synaptic input in defined regions along the surface of the bundles, where motoneuron axons reside. Most of the morphologically identifiable synaptic connections in a typical animal are described. These consist of about 5000 chemical synapses, 2000 neuromuscular junctions and 600 gap junctions.
High density arrays of DNA fragments on a solid surface allow the expression of thousands of genes to be assessed in a single experiment. The development of this 'gene chip' technique heralds a new era of studies that promises to provide an integrated view of the expression of all genes of an organism.
We describe a series of plasmid vectors which contain modular features particularly useful for studying gene expression in eukaryotic systems. The vectors contain the Escherichia coli beta-galactosidase (beta Gal)-encoding region (the lacZ gene) flanked by unique polylinker segments on the 5' and 3' ends, and several combinations of a variety of modules: a selectable marker (an amber suppressor tRNA), a translational initiation region, a synthetic intron segment, the early polyadenylation signal from SV40, and 3' regions from two nematode genes. A segment encoding the nuclear localization peptide from the SV40 T antigen is incorporated into many of the constructs, leading to beta Gal accumulation in nuclei, which can facilitate identification of producing cells in complex tissues. To make functional beta Gal fusions to secreted proteins, we constructed plasmids with an alternate module encoding a synthetic transmembrane domain upstream from lacZ. This domain is designed to stop transfer of secreted proteins across the membrane during secretion, allowing the beta Gal domain of the fusion polypeptide to remain in the cytoplasm and thus function in enzymatic assays. We have used the vectors to analyze expression of several genes in the nematode Caenorhabditis elegans, and have demonstrated in these studies that lacZ can be expressed in a wide variety of different tissues and cell types. These vectors should be useful in studying gene expression both in C. elegans and in other experimental systems.
The genetic activity of transposable elements is tightly controlled in many species. Transposons that are relatively quiescent under certain circumstances can excise or transpose at greatly increased rates under other circumstances. For example, 'genomic shock' can activate quiescent maize transposons, 'cytotype' and tissue-specific splicing regulate Drosophila P factors, copy number controls Tn5 transposition in bacteria, and developmental timing affects the production of transposon-like intracisternal A-particles in mouse embryos. The Caenorhabditis elegans transposable element Tc1 is subject to both strain-specific and tissue-specific control. Multiple copies of Tc1 are present in the genome of all C. elegans strains collected from nature. However, these elements are genetically active in only certain isolates. For example, in C. elegans variety Bristol transposition and excision of Tc1 are undetectable, but in variety Bergerac transposition and excision are frequent. Moreover, in variety Bergerac, Tc1 is about 1,000-fold more active in somatic cells than in germ cells. We have investigated the genetic basis for the germ/soma regulation of Tc1 activity. We have isolated mutants that exhibit increased frequencies of Tc1 excision in the germ line. The frequencies of Tc1 excision in the soma are unaltered in these mutants. These mutants also exhibit high frequencies of Tc1 germ-line transposition, and this results in a mutator phenotype. Nearly all mutator-induced mutations are caused by insertion of Tc1.
The embryonic cell lineage of Caenorhabditis elegans has been traced from zygote to newly hatched larva, with the result that the entire cell lineage of this organism is now known. During embryogenesis 671 cells are generated; in the hermaphrodite 113 of these (in the male 111) undergo programmed death and the remainder either differentiate terminally or become postembryonic blast cells. The embryonic lineage is highly invariant, as are the fates of the cells to which it gives rise. In spite of the fixed relationship between cell ancestry and cell fate, the correlation between them lacks much obvious pattern. Thus, although most neurons arise from the embryonic ectoderm, some are produced by the mesoderm and a few are sisters to muscles; again, lineal boundaries do not necessarily coincide with functional boundaries. Nevertheless, cell ablation experiments (as well as previous cell isolation experiments) demonstrate substantial cell autonomy in at least some sections of embryogenesis. We conclude that the cell lineage itself, complex as it is, plays an important role in determining cell fate. We discuss the origin of the repeat units (partial segments) in the body wall, the generation of the various orders of symmetry, the analysis of the lineage in terms of sublineages, and evolutionary implications.
The Pax-6 transcription-factor gene, containing a paired domain and a paired-type homeodomain, is conserved in structure and ubiquitously present among Metazoa. It is required for development of the central nervous system, and is mutated in human aniridia, mouse and rat small eye and Drosophila eyeless. We identified the Pax-6 gene of the nematode Caenorhabditis elegans in genetic studies of male tail morphology. C. elegans Pax-6 encodes at least two independent genetic functions. One, like other Pax-6 genes, contains paired and homeodomains; this constitutes the genetic locus vab-3. The other, described here, is expressed from an internal promoter and contains only the homeodomain portion; this constitutes the genetic locus mab-18. The mab-18 form of the gene is expressed in a peripheral sense organ and is necessary for specification of sense-organ identity. Its function in this context could be to regulate the expression of cell recognition and adhesion proteins required for sense-organ assembly.
Resistance to antimitotic chemotherapeutics in pathogenic nematodes, fungi and mammalian cells is closely associated with structural changes in cytoskeletal beta-tubulin. We investigated the possibility of using the well-characterised free-living nematode Caenorhabditis elegans as a model for studying the mechanism of resistance against benzimidazole (BZ) drugs in the parasitic nematode Haemonchus contortus. Functional analysis of a conserved beta-tubulin isotype (tub-1) mutation near GTP-binding domain II, which is linked to BZ resistance, was carried out in C. elegans by heterologous expression of: (1) parasite BZ-sensitive alleles; (2) BZ-resistant alleles; and (3) in vitro mutagenised beta-tubulin gene constructs. The injected heterologous gene constructs were not only stably maintained, but also expressed as shown by reverse transcriptase-polymerase chain reaction analysis. The degree of BZ drug susceptibility of the transformants was assayed and quantified by incubation with both benomyl and thiabendazol. All H. contortus tub-1 constructs, which encoded Phe at position 200, conferred susceptibility to thiabendazole in BZ-resistant C. elegans ben-1 mutants. In contrast, constructs carrying Tyr200 did not alter the BZ drug phenotype. From these experiments we conclude that: (1) C. elegans can be used as an expression host, since injected parasite genes were biologically active; and (2) the single Phe to Tyr mutation at position 200 in beta-tubulin isotype 1 is the cause of BZ resistance in H. contortus.
Implicit in the characterization of a model organism is the hope that insights into its biology can be extended to other species. For this hope to be fulfilled, the phylogenetic position of the model organism within a larger evolutionary framework must be known. We focus here on major model organisms of developmental genetics and cell biology. We first consider the positions of the nematode Caenorhabditis elegans and the arthropod Drosophila melanogaster within a phylogeny of the major advanced metazoan groups. Then we consider the evolutionary relationships between fungi (represented by Saccharomyces cerevisiae and Schizosaccharomyces pombe), plants, and animals.
We show, by a direct comparison with small subunit ribosomal RNA (18 S rRNA), that RNA polymerase II is an appropriate molecule for addressing the phylogenetic branchings in the early evolution of eukaryotes. The results from the analyses of newly determined and previously published sequences of the two largest subunits of RNA polymerase II suggest the following. Firstly, that plants and animals share a last common ancestor that excludes fungi, the lineage of which originated earlier. Secondly, that the lineage leading to the nematode Caenorhabditis elegans diverged earlier from the Metazoa than the lineages of arthropods, deuterostomes, annelids and molluscs. Finally, that deuterostomes arose from within protostomes.
RNA polymerase II is well-suited for the elucidation of the evolutionary relationships among eukaryotes. We emphasize the implications of our results for other biological disciplines in addition to molecular evolution, as a phylogenetic framework allows predictions and inferences to be made about the existence of fundamental biological mechanisms elucidated in model organisms.
A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.
We used the polymerase chain reaction to detect insertions of the transposon Tc1 into mlc-2, one of two Caenorhabditis elegans regulatory myosin light chain genes. Our goals were to develop a general method to identify mutations in any sequenced gene and to establish the phenotype of mlc-2 loss-of-function mutants. The sensitivity of the polymerase chain reaction allowed us to identify nematode populations containing rare Tc1 insertions into mcl-2. mlc-2::Tc1 mutants were subsequently isolated from these populations by a sib selection procedure. We isolated three mutants with Tc1 insertions within the mlc-2 third exon and a fourth strain with Tc1 inserted in nearby noncoding DNA. To demonstrate the generality of our procedure, we isolated two additional mutants with Tc1 insertions within hlh-1, the C. elegans MyoD homolog. All of these mutants are essentially wild type when homozygous. Despite the fact that certain of these mutants have Tc1 inserted within exons of the target gene, these mutations may not be true null alleles. All three of the mlc-2 mutants contain mlc-2 mRNA in which all or part of Tc1 is spliced from the pre-mRNA, leaving small in-frame insertions or deletions in the mature message. There is a remarkable plasticity in the sites used to splice Tc1 from these mlc-2 pre-mRNAs; certain splice sites used in the mutants are very different from typical eukaryotic splice sites.
To understand how genotype determines the phenotype of the animal Caenorhabditis elegans, one ideally needs to know the complete sequence of the genome and the contribution of genes to phenotype, which requires an efficient strategy for reverse genetics. We here report that the Tc1 transposon induces frequent deletions of flanking DNA, apparently resulting from Tc1 excision followed by imprecise DNA repair. We use this to inactivate genes in two steps. (i) We established a frozen library of 5000 nematode lines mutagenized by Tc1 insertion, from which insertion mutants of genes of interest can be recovered. Their address within the library is determined by PCR. (ii) Animals are then screened, again by PCR, to detect derivatives in which Tc1 and 1000-2000 base pairs of flanking DNA are deleted, and thus a gene of interest is inactivated. We have thus far isolated Tc1 insertions in 16 different genes and obtained deletion derivatives of 6 of those.
The bakers' yeast, Saccharomyces cerevisiae, a microorganism of major importance for bioindustries, and one of the favored model organisms for basic biological research, is the first eukaryote whose genome is entirely sequenced. Beyond the wealth of novel biological information, it is the extent of what remains to be understood in the genome of a simple unicellular organism that is the most striking result: a significant proportion of yeast genes are orphans of unpredictable function. Offering the possibility of large-scale reverse genetics, yeast will be a powerful model for post-sequencing studies. But geneticists are now faced with the difficulty of asking novel questions.
We have isolated 165 Caenorhabditis elegans mutants, representing 21 genes, that are resistant to inhibitors of cholinesterase (Ric mutants). Since mutations in 20 of the genes appear not to affect acetylcholine reception, we suggest that reduced acetylcholine release contributes to the Ric phenotype of most Ric mutants. Mutations in 15 of the genes lead to defects in a gamma-aminobutyric acid-dependent behavior; these genes are likely to encode proteins with general, rather than cholinergic-specific, roles in synaptic transmission. Ten of the genes have been cloned. Seven encode homologs of proteins that function in the synaptic vesicle cycle: two encode cholinergic-specific proteins, while five encode general presynaptic proteins. Two other Ric genes encode homologs of G-protein signaling molecules. Our assessment of synaptic function in Ric mutants, combined with the homologies of some Ric mutants to presynaptic proteins, suggests that the analysis of Ric genes will continue to yield insights into the regulation and functioning of synapses.
Caenorhabditis elegans will be the first multicellular animal to have its entire genome sequenced. This is not just good news for those currently working in the field, but also for those trying to understand the biology of more complex animals, including humans. C elegans is a relatively simple animal that is amenable to studies of genetics and developmental processes that are common to all animals, making this an attractive model in which to study basic processes that are altered in human disease. Powerful forward and reverse genetics mean that virtually any gene of interest can be studied at the functional level.
Traditional reverse genetics on yeast, mice and other organisms uses homologous recombination with transgenic DNA to interrupt a target gene. Here we report that target-selected gene inactivation can be be achieved in Caenorhabditis elegans with the use of chemical mutagens. We use PCR to selectively visualize deletions in genes of interest; the method is sensitive enough to permit detection of a single mutant among more than 15,000 wild types. A permanent frozen mutant collection of more than a million mutagenized animals has been established, and deletion mutants of several G-protein genes were isolated from it. The approach is suitable to be scaled up for systematic inactivation of all 17,000 C. elegans genes. Because it requires no transgenesis or cell culturing, it may also be applicable to small organisms usually considered to be outside the realm of reverse genetics (for example, other nematodes and insects). Any sequenced gene in any organism that can be handled in very large numbers can possibly be targeted in this way.
Several loci have been identified in the nematode worm Caenorhabditis elegans that, when mutated, can increase life span. Three of these genes, age-1, daf-2 and clk-1, have now been cloned. Mutations in these three genes are highly pleiotropic and affect many aspects of worm development and behaviour, age-1 and daf-2 act in the same genetic pathway and have similar effects on the worm, age-1 encodes a homologue of the p110 subunit of phosphatidylinositol 3-kinase and daf-2 encodes an insulin receptor family member, clk-1 encodes a protein of unknown biochemical function similar to the yeast metabolic regulator Cat5p/Coq7p. The implications of these findings for our understanding of organismal ageing are discussed.
Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.