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Massively Parallel Sequencing and Analysis of the Necator americanus Transcriptome

  • Cinzia Cantacessi,

    Affiliation Department of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia

  • Makedonka Mitreva ,

    robinbg@unimelb.edu.au (RBG); mmitreva@watson.wustl.edu (MM)

    Affiliation Department of Genetics, Genome Sequencing Center, Washington University School of Medicine, St. Louis, Missouri, United States of America

  • Aaron R. Jex,

    Affiliation Department of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia

  • Neil D. Young,

    Affiliation Department of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia

  • Bronwyn E. Campbell,

    Affiliation Department of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia

  • Ross S. Hall,

    Affiliation Department of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia

  • Maria A. Doyle,

    Affiliation Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, Australia

  • Stuart A. Ralph,

    Affiliation Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, Australia

  • Elida M. Rabelo,

    Affiliation Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

  • Shoba Ranganathan,

    Affiliation Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales, Australia

  • Paul W. Sternberg,

    Affiliation Howard Hughes Medical Institute and Division of Biology, California Institute of Technology, Pasadena, California, United States of America

  • Alex Loukas,

    Affiliation James Cook University, Cairns, Queensland, Australia

  • Robin B. Gasser

    robinbg@unimelb.edu.au (RBG); mmitreva@watson.wustl.edu (MM)

    Affiliation Department of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia

Massively Parallel Sequencing and Analysis of the Necator americanus Transcriptome

  • Cinzia Cantacessi, 
  • Makedonka Mitreva, 
  • Aaron R. Jex, 
  • Neil D. Young, 
  • Bronwyn E. Campbell, 
  • Ross S. Hall, 
  • Maria A. Doyle, 
  • Stuart A. Ralph, 
  • Elida M. Rabelo, 
  • Shoba Ranganathan
PLOS
x

Abstract

Background

The blood-feeding hookworm Necator americanus infects hundreds of millions of people worldwide. In order to elucidate fundamental molecular biological aspects of this hookworm, the transcriptome of the adult stage of Necator americanus was explored using next-generation sequencing and bioinformatic analyses.

Methodology/Principal Findings

A total of 19,997 contigs were assembled from the sequence data; 6,771 of these contigs had known orthologues in the free-living nematode Caenorhabditis elegans, and most of them encoded proteins with WD40 repeats (10.6%), proteinase inhibitors (7.8%) or calcium-binding EF-hand proteins (6.7%). Bioinformatic analyses inferred that the C. elegans homologues are involved mainly in biological pathways linked to ribosome biogenesis (70%), oxidative phosphorylation (63%) and/or proteases (60%); most of these molecules were predicted to be involved in more than one biological pathway. Comparative analyses of the transcriptomes of N. americanus and the canine hookworm, Ancylostoma caninum, revealed qualitative and quantitative differences. For instance, proteinase inhibitors were inferred to be highly represented in the former species, whereas SCP/Tpx-1/Ag5/PR-1/Sc7 proteins ( = SCP/TAPS or Ancylostoma-secreted proteins) were predominant in the latter. In N. americanus, essential molecules were predicted using a combination of orthology mapping and functional data available for C. elegans. Further analyses allowed the prioritization of 18 predicted drug targets which did not have homologues in the human host. These candidate targets were inferred to be linked to mitochondrial (e.g., processing proteins) or amino acid metabolism (e.g., asparagine t-RNA synthetase).

Conclusions

This study has provided detailed insights into the transcriptome of the adult stage of N. americanus and examines similarities and differences between this species and A. caninum. Future efforts should focus on comparative transcriptomic and proteomic investigations of the other predominant human hookworm, A. duodenale, for both fundamental and applied purposes, including the prevalidation of anti-hookworm drug targets.

Author Summary

The blood-feeding hookworm Necator americanus infects hundreds of millions of people. To elucidate fundamental molecular biological aspects of this hookworm, the transcriptome of adult Necator americanus was studied using next-generation sequencing and in silico analyses. Contigs (n = 19,997) were assembled from the sequence data; 6,771 of them had known orthologues in the free-living nematode Caenorhabditis elegans, and most encoded proteins with WD40 repeats (10.6%), proteinase inhibitors (7.8%) or calcium-binding EF-hand proteins (6.7%). Bioinformatic analyses inferred that C. elegans homologues are involved mainly in biological pathways linked to ribosome biogenesis (70%), oxidative phosphorylation (63%) and/or proteases (60%). Comparative analyses of the transcriptomes of N. americanus and the canine hookworm, Ancylostoma caninum, revealed qualitative and quantitative differences. Essential molecules were predicted using a combination of orthology mapping and functional data available for C. elegans. Further analyses allowed the prioritization of 18 predicted drug targets which did not have human homologues. These candidate targets were inferred to be linked to mitochondrial metabolism or amino acid synthesis. This investigation provides detailed insights into the transcriptome of the adult stage of N. americanus.

Introduction

Soil-transmitted helminths ( = geohelminths) are responsible for neglected tropical diseases (NTDs) mostly in developing countries [1]. In particular, the blood-feeding hookworms Necator americanus and Ancylostoma duodenale (Nematoda) infect ∼740 million people in rural areas of the tropics and subtropics [2], causing an estimated disease burden of 22 million disability-adjusted life years (DALYs) [3]. Geographically, N. americanus is the most widely distributed hookworm of humans globally [4]. The life cycle is direct, with thin-shelled eggs passed in the faeces from the infected host. Under suitable environmental conditions (e.g., 26°C and 100% humidity; [5]), the eggs hatch and develop through two free-living larval stages to the infective, third-stage (L3; filariform) larvae. The latter larvae penetrate human skin and migrate via the circulatory system and lung to finally reside as adults usually in the duodenum. The adult stages attach by their buccal capsule to the intestinal mucosa, rupture capillaries and feed on blood. The pathogenesis of hookworm disease is mainly a consequence of the blood loss, which occurs during attachment and feeding. The disease ( = necatoriasis) is commonly characterized by iron-deficiency anaemia, which can cause physical and mental retardation and sometimes deaths in children, adverse maternal-foetal outcomes [6][7] and, in chronically infected individuals, can result in a significant alteration of their immune response to helminths [8].

Traditionally, the control of hookworm disease has relied mostly on the treatment of infected individuals with anthelmintics, such as albendazole, mebendazole, pyrantel pamoate and/or levamisole. With mass treatment strategies now in place in a number of countries [9][10], there is an increased potential for hookworms to develop genetic resistance against the compounds administered, if they are used excessively and at suboptimal dosages. Thus, given the experience with drug resistance in parasitic nematodes of livestock [11], it is prudent to maintain a continual focus on the discovery of novel drugs against hookworms of humans. Such a discovery effort could be underpinned by an integrated genomic-bioinformatic approach, using functional genomic and phenomic information available for the free-living nematode Caenorhabditis elegans (see WormBase; www.wormbase.org). This nematode, which is the best characterized metazoan organism [12][13], is considered to be relatively closely related to nematodes of the order Strongylida (to which hookworms belong) [14]. Current evidence indicates that ∼60% of genes in strongylids (or hookworms) have orthologues/homologues in C. elegans [15][16], and that a range of biological pathways is conserved between strongylid nematodes/hookworms and this free-living nematode [17][20]. Therefore, conducting comparative explorations of molecular data sets between these nematodes should identify nematode-specific biological pathways, which, if essential for the development and survival, could provide new targets for nematocidal drugs.

Next generation sequencing technologies, such as ABI-SOLiD, Illumina/Solexa (www.illumina.com; [21]), Helicos (www.helicosbio.com; [22]) and 454/Roche (www.454.com; [23]), together with the recent progress in bioinformatics, are providing unique opportunities for the high-throughput transcriptomic and genomic explorations of nematodes in far more detail than previously possible [24] and at a substantially lower cost than using conventional (Sanger) sequencing. To date, genomic and molecular studies of hookworms have mainly involved the canine hookworm, Ancylostoma caninum [19], [25][27], because of its use as a model for human hookworms [27][28]. In contrast, genomic datasets for N. americanus are scant, representing a major constraint to progress in molecular research of this nematode [4]. In the present study, we (i) conducted a detailed exploration and functional annotation of the transcriptome of the adult stage of N. americanus by 454 sequencing coupled to semi-automated bioinformatic analyses, (ii) compared the transcriptome of N. americanus to currently available transcriptomic data for A. caninum, and (iii) inferred the essentiality of key genes and gene products in order to predict putative drug targets.

Materials and Methods

Accession numbers

The nucleotide sequence data produced for this study are available in the GenBank database under accession SRA012052. The contigs assembled from these data can be requested from the primary author or are available at www.nematode.net.

Parasite material

The “Shanghai strain” of N. americanus (kindly provided by Drs Bin Zhan and Peter Hotez) was produced in golden hamsters (Mesocricetus auratus; infected for 94 days) at the Universidade Federal de Minas Gerais, Brazil. The infection experiment was conducted according the animal ethics guidelines of the Universidade Federal de Minas Gerais.

RNA isolation, cDNA synthesis and 454 sequencing

Total RNA from 30 adult worms was prepared using TRIzol Reagent (GibcoBRL, Life Technologies, USA) following the manufacturer's instructions and then treated with Ambion Turbo DNase (Ambion/Applied Biosystems, Austin, TX). The integrity of the RNA was verified using the Bioanalyzer 2100 (Agilent Technologies, USA), and the yield determined using the NanoDrop ND-1000 UV-VIS spectrophotometer v.3.2.1 (NanoDrop Technologies, Wilmington, DE). The cDNA library was constructed using the SMART™ kit (Clontech/Takara Bio, CA) from ∼100ng of total RNA. An optimized PCR cycling protocol (over 20 cycles) was used to amplify full-length cDNAs, employing primers complementary to the SMART IIA-Probe and custom oligo(dT), and the Advantage-HF 2 polymerase mix (Clontech/Takara). The cDNA was normalized by denaturation-reassociation, treated with duplex-specific nuclease (Trimmer kit, Evrogen, CA) and amplified over 11 cycles. Subsequently, the 5′- and 3′- adaptors were removed by digestion with the exonuclease Mme1 and streptavidin-coated paramagnetic beads [29]. The normalized cDNA (500–700 bases) was then amplified using 9 cycles of Long and Accurate (LA)-PCR [30] and then sequenced in a Genome Sequencer™ (GS) Titanium FLX instrument (Roche Diagnostics) employing a standard protocol [23].

Bioinformatic analyses

Expressed sequence tags (ESTs) generated from the normalised cDNA library for N. americanus were assembled and annotated using a standard bioinformatic pipeline [31]. Briefly, sequences were aligned and assembled using the Contig Assembly Program v.3 (CAP3; [32], employing a minimum sequence overlap length of 50 nucleotides and an identity threshold of 95%. ESTs (n = 2,200; www.ncbi.nlm.nih.gov) from adult N. americanus available from previous studies [4], [16], [33], [34] were included for comparative analysis. Following the pre-processing of the ESTs, contigs and singletons from the present dataset were subjected to analysis by BLASTx (NCBI, www.ncbi.nlm.nih.gov) and BLASTn (EMBL-EBI Parasite Genome Blast Server, www.ebi.ac.uk) to identify putative homologues in C. elegans, other nematodes, and organisms other than nematodes (e-value of ≤1e-05). WormBase Release WS200 (www.wormbase.org) was interrogated extensively for relevant information on C. elegans orthologues/homologues, including transcriptomic, proteomic, RNAi phenotypic and interactomic data. Gene ontology (GO) annotations were conducted using BLAST2GO [35]. Peptides were mapped by InterProScan [36] and linked to respective pathways in C. elegans using the KEGG Orthology-Based Annotation System (KOBAS, [37]). The protein sequences inferred from open reading frames (ORFs) of the ESTs with orthologues in C. elegans were also subjected to “secretome analysis” using the program SignalP v.2.0 (available at www.cbs.dtu.dk/services/SignalP/), employing both the neural network and hidden Markov models to predict signal peptides and/or anchors [38][40]. Also, transmembrane domains were inferred using the program TMHMM (www.cbs.dtu.dk/services/TMHMM/; [41][43]). Protein sequences inferred from contigs for N. americanus were compared with those predicted for C. elegans and from a similar-sized, publicly available EST dataset for adult A. caninum produced by 454 sequencing (GenBank accession numbers EW741128-EW744730; EX534506-EX567272); protein similarities were displayed using SimiTri [44].

Prediction of essentiality and drug targets

All protein sequences predicted from contigs for N. americanus were compared with protein sequences available in the OrthoMCL 2.0 database (www.OrthoMCL.org) by BLASTp (e-value cut off of <1e-05). A subset of C. elegans protein homologues was then selected based on: (i) an association with a lethal RNAi phenotype; (ii) the presence/absence of gene paralogues (based on OrthoMCL orthology grouping); and (iii) GO annotation to terms linked to enzyme or G protein-coupled receptor (GPCR) activity (i.e., GO:0003824 or GO:0004930, or a sub-term thereof). The following information was obtained: (i) network connectivity score (cf. http://www.functionalnet.org/wormnet/Wormnet_v1_index.html; see [45]); (ii) presence of mammalian orthologues (based on OrthoMCL orthology grouping (iii) essentiality information (i.e. association with non-wildtype RNAi phenotypes) in other model organisms (including Saccharomyces cerevisiae, Mus musculus and Drosophila melanogaster) based on OrthoMCL groups. Each predicted drug target was selected based on (i) the presence of orthologues linked to non-wildtype RNAi or mutant phenotypes in S. cerevisiae, M. musculus and D. melanogaster, (ii) the absence of orthologues/homologues from the human host and (iii) its network connectivity score [45].

To predict the potential of selected C. elegans orthologues of N. americanus contigs as drug targets ( = “druggability”), the InterPro domains inferred from the predicted proteins were compared with those linked to known small molecular drugs which follow the ‘Lipinsky rule of 5’ regarding bioavailability [46], [47]. Similarly, GO terms inferred from the predicted proteins were mapped to Enzyme Commission (EC) numbers, and a list of enzyme-targeting drugs was compiled based on data available in the BRENDA database (www.brenda-enzymes.info; [48], [49]). The C. elegans orthologues included in the list were ranked according to the ‘severity’ of the non-wild-type RNAi phenotypes (i.e. adult lethal, embryonic and/or larval lethal, sterile and other defects) in C. elegans (cf. www.wormbase.org) defined in previous studies [50], [51].

Results

A total of 116,839 ESTs (287±235 bases in length) was generated by 454 sequencing. After removing the ESTs of <100 bases, 63,523 ESTs were assembled into 19,997 contigs (369 bases±215.31). Of these, 6,771 (33.9%) had known C. elegans orthologues, and 2,287 (11.4%) matched known nucleotide sequences from various nematodes, including Brugia malayi, Haemonchus contortus, Pristionchus pacificus, N. americanus, A. caninum, A. duodenale and Nippostrongylus braziliensis (73.2%), other invertebrates (21.3%) and some vertebrates (5.5%) available in current databases. All of the previously published ESTs for N. americanus (www.ncbi.nlm.nih.gov; [4], [16], [33], [34]) represented a subset (12.4%) of the present dataset (not shown). The number of ORFs in the N. americanus EST data, predicted peptides and their signal, transmembrane and/or InterPro domains as well as the results of GO and KOBAS (pathway mapping) searches are given in Table 1. A total of 12,799 proteins were predicted from the 19,997 contigs, of which 7,214 mapped to known proteins defined by 2,381 different domains (Tables 1 and S1), the most abundant being ‘WD40’ (IPR0011680; 10.6%), ‘proteinase inhibitors’ (IPR000215; 7.8%) and ‘EF-hand’ molecules (IPR018248; 6.7%) (Table 2). The subsequent annotation of the inferred proteins revealed 887 different GO terms, of which 314 were ‘biological process’, 117 ‘cellular component’ and 456 ‘molecular function’ (Tables 3 and S2). The predominant terms were ‘translation’ (GO:0006412, 20.3%) and ‘metabolic process’ (GO:0008152, 14.9%) for ‘biological process’; ‘intracellular’ (GO:0005622, 25.1%) and ‘ribosome’ (GO:0005840, 17%) for ‘cellular component’, and, ‘ATP binding’ (GO:0005524, 18.9%) and ‘structural constituent of ribosome’ (GO:0003735, 17.9%) for ‘molecular function’ (Tables 3 and S2). Proteins inferred from the N. americanus contigs were predicted to be involved in 235 different biological pathways, of which the vast majority represented ‘ribosome biogenesis’ (n = 163, 70%), ‘oxidative phosphorylation’ (n = 148, 63%) and ‘proteases’ (n = 140, 60%) (see Table S3).

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Table 1. Summary of the expressed sequence tag (EST) data for the adult stage of Necator americanus determined following 454 sequencing and detailed bioinformatics annotation and analyses.

https://doi.org/10.1371/journal.pntd.0000684.t001

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Table 2. The thirty most abundant protein domains inferred using the InterProScan software from peptides inferred for Necator americanus and Ancylostoma caninum.

https://doi.org/10.1371/journal.pntd.0000684.t002

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Table 3. The twenty most abundant Gene Ontology (GO) terms (according to the categories ‘biological process’, ‘cellular component’ and ‘molecular function’) for peptides inferred for Necator americanus and Ancylostoma caninum.

https://doi.org/10.1371/journal.pntd.0000684.t003

For comparative analyses, publicly available EST data for the adult stage of A. caninum was included. For this dataset, the same bioinformatic analyses described in the Methods section were conducted. From 15,755 contigs of A. caninum, a total of 12,622 proteins were inferred, of which 4,534 matched those encoded by N. americanus ORFs (Figure 1); 8,650 of these predicted proteins could be mapped to known molecules with 2,546 different motifs (Tables 1 and S1). The protein motifs ‘SCP-like extracellular’ (IPR014044, 9.5%), ‘ankyrin’ (IPR002110, 7%) and ‘allergen V5/Tpx-1 related’ (IPR0011283, 6%) were most commonly recorded in the A. caninum dataset (Table 2). Differences in the numbers of IPR domains identified in the N. americanus and A. caninum predicted peptides were calculated using a Chi-square test (p<0.05) and are indicated in Table 2. GO annotation of the A. caninum predicted peptides revealed 323 different terms for ‘biological process’, 119 for ‘cellular component’ and 500 for ‘molecular function’ (Tables 3 and S2). The terms ‘metabolic process’ (GO:0008152, 7.4%) and ‘proteolysis’ (GO:0006508, 6.6%) had the highest representation for ‘biological process’, as did ‘intracellular’ (GO:0005622, 7.5%) and ‘membrane’ (GO:0016020, 6.6%) for ‘cellular component’; and, ‘ATP binding’ (GO:0005524, 13.4%) and ‘catalytic activity’ (GO:0003824, 9%) for ‘molecular function’ (Tables 3 and S2). Using the protein data, a total of 235 different biological pathways were predicted, of which ‘proteases’ (n = 219, 93%), ‘other enzymes’ (n = 164, 70%) and ‘protein kinases’ (n = 151, 54%) were the most predominant (see Table S3).

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Figure 1. Simitri analysis.

Relationships of proteins predicted for Necator americanus with homologues from Ancylostoma caninum and Caenorhabditis elegans, displayed in a SimiTri plot [44]. The description of proteins with most abundant InterPro domains identified in each similarity group is given in the boxes.

https://doi.org/10.1371/journal.pntd.0000684.g001

From the N. americanus dataset, 5,498 proteins matched known proteins encoded by orthologues available in the OrthoMCL 2.0 database (www.OrthoMCL.org); 372 of these proteins had homologues in C. elegans, and 278 (277 enzymes and one G-PCR) of them were linked to adult lethal, embryonic and/or larval lethal and sterile RNAi phenotypes (Table S4). A subset of 18 molecules in N. americanus with homologues in C. elegans but not in humans were defined, also considering RNAi phenotype/s [i.e. adult lethal (n = 2), larval and/or embryonic lethal (n = 16), sterile (n = 4) and other defects (n = 12); cf. Table 4], as drug target candidates. These proteins could be mapped to 54 ‘druggable’ InterPro domains, and 212 EC numbers were linked to ‘druggable’ enzymes; a total of 3,320 effective drugs were predicted (Table S4).

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Table 4. Description of Caenorhabditis elegans orthologues of Necator americanus contigs for which inferred peptides were associated with ‘druggable’ InterPro domains and/or Enzyme Commission (EC) numbers, and examples of candidate nematocidal compounds linked to these domains predicted using the BRENDA database (see Materials and Methods).

https://doi.org/10.1371/journal.pntd.0000684.t004

Discussion

Next-generation sequencing and integrated bioinformatic analyses have provided detailed and biologically relevant insights into the transcriptome of the adult stage of N. americanus. A total of 12,799 ORFs were inferred from the present EST dataset, thus increasing the number of predicted proteins currently available (for this stage/species) in public databases by approximately 27-fold [4]. Amongst the InterPro domains identified, ‘WD40’, ‘proteinase inhibitors’ and ‘EF-hand’ motifs were the most abundant, followed by ‘proteases’ and ‘protein kinases’. WD40 repeats (also known as WD or beta-transducin repeats) are short (∼40 amino acid) motifs found in the proteomes of all eukaryotes and implicated in a variety of functions, ranging from signal transduction and transcription regulation to cell-cycle control and apoptosis [52], [53]. WD40 motifs act as sites for protein-protein interactions; proteins containing WD40 repeats are known to serve as platforms for the assembly of protein complexes or mediators of a transient interplay with other proteins, such as the ubiquitin ligases, involved in the onset of the anaphase during cell mitosis [54]. Similarly, proteins containing ‘EF-hand’ domains are involved in a number of protein-protein interactions regulated by various specialized systems (e.g., Golgi system, voltage-dependent calcium channels and calcium transporters) for the uptake and release of calcium, which acts as a secondary messenger for their activation [55]. In C. elegans, both EF-hand and WD40 proteins are known to be required for the maturation of the nervous system and the formation of ciliated sensory neurons, in particular of the chemoreceptors located in the amphids [8], [56]. The amphids of parasitic nematodes are, besides having the chemoreceptive activity, also known to play a role as secretory organs, primarily to provide an appropriate substrate for the transmission of neuronal potentials [57]. However, in N. americanus, a group of specialized amphidial neuronal cells ( = amphidial glands; [57]) expresses a group of aspartic proteases (i.e. cathepsin D-like Na-APR-1 and Na-APR-2) which are proposed to degrade host haemoglobin and serum proteins in the buccal capsule of adult worms [58]. In the dog hookworm, A. caninum, the amphidial glands have also been shown to produce a proteinase inhibitor (called ‘ancylostomatin’) that acts as an anticoagulant to promote the flow of host blood and tissue fluids into the buccal capsule and the intestine of the parasite [59]. Although proteinase inhibitors, such as the ‘kunitz-type’ molecules, were significantly more abundant in the transcriptome of adult N. americanus [4], [33] than Ancylostoma spp., they have been better characterized in the latter parasites [60][63] for which both single and multiple kunitz-domain proteins have been described [61]. For instance, a cDNA coding a single kunitz-domain proteinase inhibitor (named AceKI-1) was isolated from A. ceylanicum. The corresponding recombinant protein has been shown to act as a tight-binding inhibitor of the serine proteases chymotrypsin, pancreatic elastase, neutrophil elastase and trypsin [60] and confers partial protection against hookworm-associated growth delay in hamsters [62]. Recently, a kunitz-type cDNA was shown to be enriched in the adult male of A. braziliense [63]. Although their precise biological function remains to be determined, kunitz-type proteinase inhibitors of hookworms appear to play pivotal roles in preventing homeostasis and inhibiting host proteases (e.g., pancreatic and intestinal enzymes; [60], [64]).

Proteases were also highly represented in the transcriptome of N. americanus (6.1%) as well as that of A. caninum (4.6%) (see Table 2). These proteases included cysteine, aspartic and metallo- proteases, which are known to function in multi-enzyme cascades to digest haemoglobin and other serum proteins [65], [66]. In N. americanus, cysteine proteases with high sequence homology to the protein cathepsin B were localized to the gut of adult worms and the corresponding mRNAs shown to be upregulated in the adult stage compared with the infective L3 stage, thus strongly suggesting that these enzymes are involved in blood-feeding [67]. In A. caninum, a cysteine protease (Ac-CP-1) with 86% amino acid sequence identity to those characterized in N. americanus, was shown to be expressed in the cephalic and excretory glands [68] and was detected in the excretory/secretory products (ES) [69] of adult worms; thus, it has been proposed that Ac-CP-1 functions as an extracorporeal digestive enzyme at the site of attachment [67]. Another cysteine protease (Ac-CP-2) was localized to the brush border membrane of the intestine and demonstrated to be involved in the digestion of haemoglobin [65]. The N. americanus homologue of Ac-CP-2 (i.e. Na-CP-2) digests haemoglobin [66] and, expressed as a recombinant protein in Escherichia coli and injected subcutaneously into experimental hamsters, has been shown to induce a significant reduction in adult worm burden following challenge infection with L3s of N. americanus [28], suggesting that the immunogenic response directed against this protein severely impairs the digestion of host proteins by the adult worms. However, recently, a cathepsin-like cysteine protease has been isolated and characterized in the human filarial nematode Brugia malayi and shown by double-stranded RNAi to play an essential role in the early development and maturation of embryos of this nematode [70]. Therefore, it is possible that the abundant transcripts encoding proteases in both adult N. americanus and A. caninum also reflect a key role of these enzymes in embryogenesis. Proteases have also been isolated from larval stages of both A. caninum and N. americanus [71], [72]. For instance, a metalloprotease in ES of the activated third-stage larvae (L3) of A. caninum has been characterized and demonstrated to be released specifically in response to stimuli that induce feeding [73]. The corresponding cDNA, isolated from an L3 expression library, encoded a zinc-metalloprotease (Ac-MTP-1) of the astacin family, that has been proposed to (i) regulate developmental changes associated with the transition from the free-living to the parasitic L3 and the subsequent moult to the fourth-stage larva (L4) [72]; (ii) activate host TGF-ß during the infection, which, in turn, could stimulate parasite development directly, determine tissue predilection sites [74] and/or inhibit neutrophil infiltration at the site of penetration [75]; and, (iii) facilitate skin penetration or tissue migration by the invading L3 [72], [76] and/or degrade the cuticular proteins of the sheath surrounding the infective, free-living L3 [77]. In N. americanus, serine proteases have been isolated from ES of the L3 stage and proposed to play a central role in the evasion of the host immune response [71]. Interestingly, a significant number (n = 135, 30%) of N. americanus proteases and protease inhibitors of N. americanus were not predicted to possess signal peptides indicative of secretion (cf. Tables 1 and 2). The likely explanation for this result is technical and would appear to relate to a 3′-bias in sequence reads [78], thus affecting the prediction of ORFs as well as the identification of signal peptide sequences at the 5′-ends.

Other groups of molecules, such as Ancylostoma-secreted proteins (ASPs), have been proposed to have an immunomodulatory function during the invasion of the host, the migration through tissues, attachment to the intestinal wall and blood-feeding [79]. In the present study, ASPs were amongst the ten most abundant groups of molecules in the N. americanus dataset, and are most abundant in A. caninum (cf. Table 2). ASPs belong to a large group of proteins, the ‘sperm-coating protein (SCP)-like extracellular proteins’, also called SCP/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS; Pfam accession number no. PF00188), characterized by the presence of a single or double ‘SCP-like extracellular domain’ (InterPro: IPR014044). In A. caninum, double and a single SCP-domain ASPs, called Ac-ASP-1 and Ac-ASP-2, respectively, were identified as major components of ES from serum-activated, infective L3s and proposed to be secreted in response to one or more host-specific signals during the infection process [80], [81], as also hypothesized in a transcriptomic analysis of serum-activated L3s [19]. In N. americanus, homologues of Ac-ASP-1 and Ac-ASP-2 (i.e Na-ASP-1 and Na-ASP-2, respectively) have been identified in the L3 stage [82][84]. Results from crystallography [85], combined with the observation that Na-ASP-2 induces neutrophil and monocyte migration [86], suggest that this molecule has a role as an antagonistic ligand of complement receptor 3 (CR3) and alters the immune cascade by preventing the binding of chemotaxin [85]. Because of its immunogenic properties, Na-ASP-2 is under investigation as a vaccine candidate against necatoriasis [7], [28], [81], [87]. In adult A. caninum, at least four other ASPs have been identified to date and named Ac-ASP-3, Ac-ASP-4, Ac-ASP-5 and Ac-ASP-6 [72]. Another SCP/TAPS molecule, designated neutrophil inhibitor factor (NIF), has been isolated and shown to play an immunomodulatory role by blocking the adhesion of activated neutrophils to vascular endothelial cells and the subsequent release of H2O2 from activated neutrophils [88] and by interfering with the function of integrin receptors located on the cell surface, which results in the inhibition of platelet aggregation and adhesion [89]. Subsequently, NIF was shown to be transcribed abundantly in the intestines of both A. caninum and N. americanus [34]. The present study revealed that, although highly represented in the transcriptome of adult N. americanus, ASPs were much more abundant in A. caninum (cf. Results section). One of the possible explanations for this finding is that, although the A. caninum dataset was generated from adult worms recovered from their natural host (i.e. dog), the specimens of N. americanus were recovered from a Chinese strain of the golden hamster (M. auratus), which is not a natural host for this parasite [90], [91]. Indeed, adults of N. americanus recovered from hamsters with patent infections are smaller and less fecund than from the human host [91]. These phenetic differences in this parasite might be associated with variation in transcriptional profiles. However, the difference in prevalence of particular transcripts, such as those of asps, between A. caninum and N. americanus might reflect their distinct roles in the modulation of the host immune response between the two hookworms, an hypothesis that requires testing.

A benefit of investigating the transcriptome of parasitic nematodes using predictive algorithms is that potential drug targets can be inferred and/or prioritized. The present study identified a subset of 278 ‘druggable’ proteins, of which 18 did not match any human homologues (cf. Results section). Of these 18 molecules, mitochondrial-associated proteins were significantly represented (i.e. encoded by the C. elegans orthologues W01a8.4, ucr-1, F26E4.6 and Y71H2aM.4; cf. Table 4). Mitochondria are essential organelles with central roles in diverse cellular processes, such as apoptosis, energy production via oxidative phosphorylation, ion homeostasis, and the synthesis of haeme, lipid, amino acids, and iron-sulfur ions [92]. In C. elegans, defects in the mitochondrial respiratory chain lead to or are associated with a wide variety of abnormalities, including embryonic, larval and adult lethality, sterility and embryonic defects [92]. Despite their essential roles in numerous fundamental biological processes, knowledge of mitochondrial genes and proteins in parasitic nematodes has been utilized mainly to study their systematics, population genetics and ecology [93][95]. However, that some mitochondrial-associated proteins are predicted to be essential in N. americanus and significantly different from human homologues provides a context for the discovery of new drug targets in mitochondrial pathways and chemical compounds that disrupt these pathways [95], [96]. Amongst the other N. americanus orthologues of essential C. elegans genes, nrs-2 encodes an asparaginyl-tRNA synthetase (AsnRS), which is a class II aminoacyl-tRNA synthetase that catalyzes the attachment of asparagine to its cognate tRNA and is required for protein biosynthesis [97]; loss of nrs-2 function via RNAi has been shown to result in a number of phenotypes, including adult and larval lethality and/or larval arrest [97]. In parasitic nematodes, information on amino acid biosynthesis is limited [98]. Although a number of parasitic helminths, including the nematode Heligmosomoides polygyrus [sic. H. bakeri] and the trematode Fasciola hepatica, have been reported to excrete asparagine during in vitro incubation [99], [100], the role of asparagine synthetases in essential biological processes is currently unknown. However, in a study investigating the molecular mechanisms of induced cell differentiation in human pro-myelocytic leukemia, asparagine synthetase transcription was reported to be significantly reduced in maturing monocytes/macrophages [101]; therefore, an active role of asparagine synthetases in the development and growth of cancer cells has been suggested, which led to the hypothesis that the induction of a down-regulation of asparagine synthetases might be a new strategy for the treatment of blast cell leukaemia [102]. This finding raises questions about the role(s) of asparagine synthetases in cell differentiation and maturation in parasitic nematodes and the potential of inhibitors of these enzymes as anti-hookworm drugs.

The present study has provided new insights into the transcriptome of N. americanus, elucidated similarities and differences between the transcriptomes of N. americanus and the related canine hookworm, A. caninum, and predicted a panel of novel drug targets and nematocides. All except one of the essential ‘druggable’ proteins (n = 18) inferred for N. americanus were present in the A. caninum (and C. elegans) but not in the mammalian hosts, suggesting relative sequence conservation for these targets among nematodes. The prediction of such targets is particularly important, considering the risk of emerging drug resistance in parasitic nematodes [102], [103]. Clearly, transcriptomic and genomic studies, such as that carried out here can facilitate and expedite the prevalidation of targets for nematocidal drugs, although the lack of genomic and transcriptomic data for many nematodes, including the human hookworm A. duodenale, impairs the comparative exploration of essential biological pathways in parasitic nematodes of major public health significance [6]. Furthermore, the present analysis has inferred qualitative and quantitative differences in the transcriptome between N. americanus and A. caninum, raising questions as to the suitability of the latter species as a model for the former. Although these differences require experimental validation, there is a need to define the transcriptome of A. duodenale as a foundation for comparative investigations with a perspective on the identification of new and hookworm-specific drug targets.

Supporting Information

Table S1.

InterPro domains identified in the peptides predicted for Necator americanus and Ancylostoma caninum.

https://doi.org/10.1371/journal.pntd.0000684.s001

(0.39 MB XLS)

Table S2.

Gene Ontology (GO) terms (according to the categories ‘biological process’, ‘cellular component’ and ‘molecular function’) linked to peptides predicted for Necator americanus and Ancylostoma caninum.

https://doi.org/10.1371/journal.pntd.0000684.s002

(0.16 MB XLS)

Table S3.

Biological pathways involving key peptides predicted for Necator americanus and Ancylostoma caninum.

https://doi.org/10.1371/journal.pntd.0000684.s003

(0.04 MB XLS)

Table S4.

Description of Caenorhabditis elegans orthologues of Necator americanus contigs for which inferred peptides were associated with ‘druggable’ InterPro domains and/or Enzyme Commission (EC) numbers, and a list of candidate nematocidal compounds linked to these domains predicted using the BRENDA database (see Materials and methods). The presence (&#10003) or absence (X) of known orthologues in Ancylostoma caninum, Haemonchus contortus, Homo sapiens, Drosophila melanogaster and/or Mus musculus is also indicated.

https://doi.org/10.1371/journal.pntd.0000684.s004

(0.23 MB XLS)

Acknowledgments

CC is the grateful recipient of International Postgraduate Research Scholarship (IPRS) from the Australian Government and a fee-remission scholarship through the University of Melbourne as well as the Clunies Ross (2008) and Sue Newton (2009) awards from the School of Veterinary Science of the same university. The contribution of staff at WormBase is gratefully acknowledged.

Author Contributions

Conceived and designed the experiments: CC MM RBG. Performed the experiments: CC MM. Analyzed the data: CC ARJ NDY BEC RSH MAD SAR SR AL RBG. Contributed reagents/materials/analysis tools: MM EMR. Wrote the paper: CC PWS RBG.

References

  1. 1. Hotez PJ (2007) Neglected diseases and poverty in “The other America”: The Greatest Health Disparity in the United States? PLoS Negl Trop Dis 1: e149.PJ Hotez2007Neglected diseases and poverty in “The other America”: The Greatest Health Disparity in the United States?PLoS Negl Trop Dis1e149
  2. 2. de Silva NR, Brooker S, Hotez PJ, Montresor A, Engels D, et al. (2003) Soil-transmitted helminth infections: updating the global picture. Trends Parasitol 19: 547–551.NR de SilvaS. BrookerPJ HotezA. MontresorD. Engels2003Soil-transmitted helminth infections: updating the global picture.Trends Parasitol19547551
  3. 3. Hotez PJ, Bethony J, Bottazzi ME, Brooker S, Diemert D, et al. (2006) New technologies for the control of human hookworm infection. Trends Parasitol 22: 327–331.PJ HotezJ. BethonyME BottazziS. BrookerD. Diemert2006New technologies for the control of human hookworm infection.Trends Parasitol22327331
  4. 4. Rabelo EM, Hall RS, Loukas A, Cooper L, Hu M, et al. (2009) Improved insights into the transcriptomes of the human hookworm Necator americanus–fundamental and biotechnological implications. Biotechnol Adv 27: 122–132.EM RabeloRS HallA. LoukasL. CooperM. Hu2009Improved insights into the transcriptomes of the human hookworm Necator americanus–fundamental and biotechnological implications.Biotechnol Adv27122132
  5. 5. Matthews BE (1985) The influence of temperature and osmotic stress on the development and eclosion of hookworm eggs. J Helminthol 59: 217–224.BE Matthews1985The influence of temperature and osmotic stress on the development and eclosion of hookworm eggs.J Helminthol59217224
  6. 6. Bethony J, Brooker S, Albonico M, Geiger SM, Loukas A, et al. (2006) Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm. Lancet 367: 1521–1532.J. BethonyS. BrookerM. AlbonicoSM GeigerA. Loukas2006Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm.Lancet36715211532
  7. 7. Loukas A, Bethony J, Brooker S, Hotez P (2006) Hookworm vaccines: past, present, and future. Lancet Infect Dis 6: 733–741.A. LoukasJ. BethonyS. BrookerP. Hotez2006Hookworm vaccines: past, present, and future.Lancet Infect Dis6733741
  8. 8. Fujiwara RT, Cançado GG, Freitas PA, Santiago HC, Massara CL, et al. (2009) Necator americanus infection: a possible cause of altered dendritic cell differentiation and eosinophil profile in chronically infected individuals. PLoS Negl Trop Dis 3: e399.RT FujiwaraGG CançadoPA FreitasHC SantiagoCL Massara2009Necator americanus infection: a possible cause of altered dendritic cell differentiation and eosinophil profile in chronically infected individuals.PLoS Negl Trop Dis3e399
  9. 9. Bungiro R, Cappello M (2004) Hookworm infection: new developments and prospects for control. Curr Opin Infect Dis 17: 421–426.R. BungiroM. Cappello2004Hookworm infection: new developments and prospects for control.Curr Opin Infect Dis17421426
  10. 10. Diemert DJ, Bethony JM, Hotez PJ (2008) Hookworm vaccines. Clin Infect Dis 46: 282–288.DJ DiemertJM BethonyPJ Hotez2008Hookworm vaccines.Clin Infect Dis46282288
  11. 11. Wolstenholme AJ, Fairweather I, Prichard R, von Samson-Himmelstjerna G, Sangster NC (2004) Drug resistance in veterinary helminths. Trends Parasitol 20: 469–476.AJ WolstenholmeI. FairweatherR. PrichardG. von Samson-HimmelstjernaNC Sangster2004Drug resistance in veterinary helminths.Trends Parasitol20469476
  12. 12. Riddle DL, Blumenthal T, Meyer BJ, Priess JR, editors. (1997) C. elegans II. DL RiddleT. BlumenthalBJ MeyerJR Priess1997C. elegans II.Cold Spring Harbor Laboratory Press, New York, USA. Cold Spring Harbor Laboratory Press, New York, USA.
  13. 13. Sugimoto A (2004) High-throughput RNAi in Caenorhabditis elegans: genome-wide screens and functional genomics. Differentiation 72: 81–91.A. Sugimoto2004High-throughput RNAi in Caenorhabditis elegans: genome-wide screens and functional genomics.Differentiation728191
  14. 14. Blaxter ML, De Ley P, Garey JR, Liu LX, Scheldeman P, et al. (1998) A molecular evolutionary framework for the phylum Nematoda. Nature 392: 71–75.ML BlaxterP. De LeyJR GareyLX LiuP. Scheldeman1998A molecular evolutionary framework for the phylum Nematoda.Nature3927175
  15. 15. Bürglin TR, Lobos E, Blaxter ML (1998) Caenorhabditis elegans as a model for parasitic nematodes. Int J Parasitol 28: 395–411.TR BürglinE. LobosML Blaxter1998Caenorhabditis elegans as a model for parasitic nematodes.Int J Parasitol28395411
  16. 16. Parkinson J, Mitreva M, Whitton C, Thomson M, Daub J, et al. (2004) A transcriptomic analysis of the phylum Nematoda. Nat Genet 36: 1259–1267.J. ParkinsonM. MitrevaC. WhittonM. ThomsonJ. Daub2004A transcriptomic analysis of the phylum Nematoda.Nat Genet3612591267
  17. 17. Nikolaou S, Gasser RB (2006) Extending from PARs in Caenorhabditis elegans to homologues in Haemonchus contortus and other parasitic nematodes. Parasitology 134: 461–482.S. NikolaouRB Gasser2006Extending from PARs in Caenorhabditis elegans to homologues in Haemonchus contortus and other parasitic nematodes.Parasitology134461482
  18. 18. Campbell BE, Nagaraj SH, Hu M, Zhong W, Sternberg PW, et al. (2008) Gender-enriched transcripts in adult Haemonchus contortus – predicted functions and genetic interactions based on comparative analyses with Caenorhabditis elegans. Int J Parasitol 38: 65–83.BE CampbellSH NagarajM. HuW. ZhongPW Sternberg2008Gender-enriched transcripts in adult Haemonchus contortus – predicted functions and genetic interactions based on comparative analyses with Caenorhabditis elegans.Int J Parasitol386583
  19. 19. Datu BJ, Gasser RB, Nagaraj SH, Ong EK, O'Donoghue P, et al. (2008) Transcriptional changes in the hookworm, Ancylostoma caninum, during the transition from a free-living to a parasitic larva. PLoS Negl Trop Dis 2: e130.BJ DatuRB GasserSH NagarajEK OngP. O'Donoghue2008Transcriptional changes in the hookworm, Ancylostoma caninum, during the transition from a free-living to a parasitic larva.PLoS Negl Trop Dis2e130
  20. 20. Cantacessi C, Loukas A, Campbell BE, Mulvenna J, Ong EK, et al. (2009) Exploring transcriptional conservation between Ancylostoma caninum and Haemonchus contortus by oligonucleotide microarray and bioinformatic analyses. Mol Cell Probes 23: 1–9.C. CantacessiA. LoukasBE CampbellJ. MulvennaEK Ong2009Exploring transcriptional conservation between Ancylostoma caninum and Haemonchus contortus by oligonucleotide microarray and bioinformatic analyses.Mol Cell Probes2319
  21. 21. Bentley DR, Balasubramanian S, Swerdlow HP, Smith GP, Milton J, et al. (2008) Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456: 53–59.DR BentleyS. BalasubramanianHP SwerdlowGP SmithJ. Milton2008Accurate whole human genome sequencing using reversible terminator chemistry.Nature4565359
  22. 22. Harris TD, Buzby PR, Babcock H, Beer E, Bowers J, et al. (2008) Single-molecule DNA sequencing of a viral genome. Science 320: 106–109.TD HarrisPR BuzbyH. BabcockE. BeerJ. Bowers2008Single-molecule DNA sequencing of a viral genome.Science320106109
  23. 23. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, et al. (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437: 376–380.M. MarguliesM. EgholmWE AltmanS. AttiyaJS Bader2005Genome sequencing in microfabricated high-density picolitre reactors.Nature437376380
  24. 24. Droege M, Hill B (2008) The Genome Sequencer FLX System–longer reads, more applications, straight forward bioinformatics and more complete data sets. J Biotechnol 136: 3–10.M. DroegeB. Hill2008The Genome Sequencer FLX System–longer reads, more applications, straight forward bioinformatics and more complete data sets.J Biotechnol136310
  25. 25. Mitreva M, McCarter JP, Arasu P, Hawdon J, Martin J, et al. (2005) Investigating hookworm genomes by comparative analysis of two Ancylostoma species. BMC Genomics 26: 58.M. MitrevaJP McCarterP. ArasuJ. HawdonJ. Martin2005Investigating hookworm genomes by comparative analysis of two Ancylostoma species.BMC Genomics2658
  26. 26. Moser JM, Freitas T, Arasu P, Gibson G (2005) Gene expression profiles associated with the transition to parasitism in Ancylostoma caninum larvae. Mol Biochem Parasitol 143: 39–48.JM MoserT. FreitasP. ArasuG. Gibson2005Gene expression profiles associated with the transition to parasitism in Ancylostoma caninum larvae.Mol Biochem Parasitol1433948
  27. 27. Abubucker S, Martin J, Yin Y, Fulton L, Yang SP, et al. (2008) The canine hookworm genome: analysis and classification of Ancylostoma caninum survey sequences. Mol Biochem Parasitol 157: 187–192.S. AbubuckerJ. MartinY. YinL. FultonSP Yang2008The canine hookworm genome: analysis and classification of Ancylostoma caninum survey sequences.Mol Biochem Parasitol157187192
  28. 28. Xiao S, Zhan B, Xue J, Goud GN, Loukas A, et al. (2008) The evaluation of recombinant hookworm antigens as vaccines in hamsters (Mesocricetus auratus) challenged with human hookworm, Necator americanus. Exp Parasitol 118: 32–40.S. XiaoB. ZhanJ. XueGN GoudA. Loukas2008The evaluation of recombinant hookworm antigens as vaccines in hamsters (Mesocricetus auratus) challenged with human hookworm, Necator americanus.Exp Parasitol1183240
  29. 29. Mitreva M, Mardis ER (2009) Large-scale sequencing and analytical processing of ESTs. Methods Mol Biol 533: 153–187.M. MitrevaER Mardis2009Large-scale sequencing and analytical processing of ESTs.Methods Mol Biol533153187
  30. 30. Barnes WM (1994) PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci U S A 91: 2216–2220.WM Barnes1994PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates.Proc Natl Acad Sci U S A9122162220
  31. 31. Ranganathan S, Menon R, Gasser RB (2009) Advanced in silico analysis of expressed sequence tag (EST) data for parasitic nematodes of major socio-economic importance–fundamental insights toward biotechnological outcomes. Biotechnol Adv 27: 439–448.S. RanganathanR. MenonRB Gasser2009Advanced in silico analysis of expressed sequence tag (EST) data for parasitic nematodes of major socio-economic importance–fundamental insights toward biotechnological outcomes.Biotechnol Adv27439448
  32. 32. Huang X, Madan A (1999) CAP3: A DNA sequence assembly program. Genome Res 9: 868–877.X. HuangA. Madan1999CAP3: A DNA sequence assembly program.Genome Res9868877
  33. 33. Daub J, Loukas A, Pritchard DI, Blaxter M (2000) A survey of genes expressed in adults of the human hookworm, Necator americanus. Parasitology 120: 171–184.J. DaubA. LoukasDI PritchardM. Blaxter2000A survey of genes expressed in adults of the human hookworm, Necator americanus.Parasitology120171184
  34. 34. Ranjit N, Jones MK, Stenzel DJ, Gasser RB, Loukas A (2006) A survey of the intestinal transcriptomes of the hookworms, Necator americanus and Ancylostoma caninum, using tissues isolated by laser microdissection microscopy. Int J Parasitol 36: 701–710.N. RanjitMK JonesDJ StenzelRB GasserA. Loukas2006A survey of the intestinal transcriptomes of the hookworms, Necator americanus and Ancylostoma caninum, using tissues isolated by laser microdissection microscopy.Int J Parasitol36701710
  35. 35. Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, et al. (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21: 3674–3676.A. ConesaS. GötzJM García-GómezJ. TerolM. Talón2005Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research.Bioinformatics2136743676
  36. 36. Hunter S, Apweiler R, Attwood TK, Bairoch A, Bateman A, et al. (2009) InterPro: the integrative protein signature database. Nucleic Acids Res 37: D211–D215.S. HunterR. ApweilerTK AttwoodA. BairochA. Bateman2009InterPro: the integrative protein signature database.Nucleic Acids Res37D211D215
  37. 37. Wu J, Mao X, Cai T, Luo J, Wei L (2006) KOBAS server: a web-based platform for automated annotation and pathway identification. Nucleic Acids Res 34: W720–W724.J. WuX. MaoT. CaiJ. LuoL. Wei2006KOBAS server: a web-based platform for automated annotation and pathway identification.Nucleic Acids Res34W720W724
  38. 38. Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Prot Eng 10: 1–6.H. NielsenJ. EngelbrechtS. BrunakG. von Heijne1997Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites.Prot Eng1016
  39. 39. Nielsen H, Krogh A (1998) Prediction of signal peptides and signal anchors by a hidden Markov model. pp. 122–130.H. NielsenA. Krogh1998Prediction of signal peptides and signal anchors by a hidden Markov model.122130Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology (ISMB 6), AAAI Press, Menlo Park, California. Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology (ISMB 6), AAAI Press, Menlo Park, California.
  40. 40. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340: 783–795.JD BendtsenH. NielsenG. von HeijneS. Brunak2004Improved prediction of signal peptides: SignalP 3.0.J Mol Biol340783795
  41. 41. Sonnhammer ELL, von Heijne G, Krogh A (1998) A hidden Markov model for predicting transmembrane helices in protein sequences. pp. 175–182.ELL SonnhammerG. von HeijneA. Krogh1998A hidden Markov model for predicting transmembrane helices in protein sequences.175182Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology, Menlo Park, CA, AAAI;. Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology, Menlo Park, CA, AAAI;.
  42. 42. Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305: 567–580.A. KroghB. LarssonG. von HeijneEL Sonnhammer2001Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.J Mol Biol305567580
  43. 43. Moller S, Croning MDR, Apweiler R (2001) Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 17: 646–653.S. MollerMDR CroningR. Apweiler2001Evaluation of methods for the prediction of membrane spanning regions.Bioinformatics17646653
  44. 44. Parkinson J, Blaxter M (2003) SimiTri–visualizing similarity relationships for groups of sequences. Bioinformatics 19: 390–395.J. ParkinsonM. Blaxter2003SimiTri–visualizing similarity relationships for groups of sequences.Bioinformatics19390395
  45. 45. Lee I, Lehner B, Crombie C, Wong W, Fraser AG, et al. (2008) A single gene network accurately predicts phenotypic effects of gene perturbation in Caenorhabditis elegans. Nat Genet 40: 181–188.I. LeeB. LehnerC. CrombieW. WongAG Fraser2008A single gene network accurately predicts phenotypic effects of gene perturbation in Caenorhabditis elegans.Nat Genet40181188
  46. 46. Lipinski C, Lombardo F, Dominy B, Feeney P (1997) Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev 23: 3–25.C. LipinskiF. LombardoB. DominyP. Feeney1997Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings.Adv Drug Deliv Rev23325
  47. 47. Hopkins AL, Groom CR (2002) The druggable genome. Nat Rev Drug Discov 1: 727–730.AL HopkinsCR Groom2002The druggable genome.Nat Rev Drug Discov1727730
  48. 48. Robertson JG (2005) Mechanistic basis of enzyme-targeted drugs. Biochemistry 44: 5561–5571.JG Robertson2005Mechanistic basis of enzyme-targeted drugs.Biochemistry4455615571
  49. 49. Chang A, Scheer M, Grote A, Schomburg I, Schomburg D (2009) BRENDA, AMENDA and FRENDA the enzyme information system: new content and tools in 2009. Nucleic Acids Res 37: D588–D592.A. ChangM. ScheerA. GroteI. SchomburgD. Schomburg2009BRENDA, AMENDA and FRENDA the enzyme information system: new content and tools in 2009.Nucleic Acids Res37D588D592
  50. 50. Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, et al. (2003) Systemic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421: 231–237.RS KamathAG FraserY. DongG. PoulinR. Durbin2003Systemic functional analysis of the Caenorhabditis elegans genome using RNAi.Nature421231237
  51. 51. Sönnichsen B, Koski LB, Walsh A, Marschall P, Neumann B, et al. (2005) Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans. Nature 434: 462–469.B. SönnichsenLB KoskiA. WalshP. MarschallB. Neumann2005Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans.Nature434462469
  52. 52. Wolf DA, Jackson PK (1998) Cell cycle: oiling the gears of anaphase. Curr Biol 8: R636–R639.DA WolfPK Jackson1998Cell cycle: oiling the gears of anaphase.Curr Biol8R636R639
  53. 53. Leipe DD, Koonin EV, Aravind L (2004) STAND, a class of P-loop NTPases including animal and plant regulators of programmed cell death: multiple, complex domain architectures, unusual phyletic patterns, and evolution by horizontal gene transfer. J Mol Biol 343: 1–28.DD LeipeEV KooninL. Aravind2004STAND, a class of P-loop NTPases including animal and plant regulators of programmed cell death: multiple, complex domain architectures, unusual phyletic patterns, and evolution by horizontal gene transfer.J Mol Biol343128
  54. 54. Yu H (2007) Cdc20: a WD40 activator for a cell cycle degradation machine. Mol Cell 27: 3–16.H. Yu2007Cdc20: a WD40 activator for a cell cycle degradation machine.Mol Cell27316
  55. 55. Nagamune K, Moreno SN, Chini EN, Sibley LD (2008) Calcium regulation and signaling in apicomplexan parasites. Subcell Biochem 47: 70–81.K. NagamuneSN MorenoEN ChiniLD Sibley2008Calcium regulation and signaling in apicomplexan parasites.Subcell Biochem477081
  56. 56. Estevez AO, Cowie RH, Gardner KL, Estevez M (2006) Both insulin and calcium channel signaling are required for developmental regulation of serotonin synthesis in the chemosensory ADF neurons of Caenorhabditis elegans. Dev Biol 298: 32–44.AO EstevezRH CowieKL GardnerM. Estevez2006Both insulin and calcium channel signaling are required for developmental regulation of serotonin synthesis in the chemosensory ADF neurons of Caenorhabditis elegans.Dev Biol2983244
  57. 57. Ashton FT, Li J, Schad GA (1999) Chemo- and thermosensory neurons: structure and function in animal parasitic nematodes. Vet Parasitol 84: 297–316.FT AshtonJ. LiGA Schad1999Chemo- and thermosensory neurons: structure and function in animal parasitic nematodes.Vet Parasitol84297316
  58. 58. Williamson AL, Brindley PJ, Abbenante G, Datu BJ, Prociv P, et al. (2003) Hookworm aspartic protease, Na-APR-2, cleaves human hemoglobin and serum proteins in a host-specific fashion. J Infect Dis 187: 484–494.AL WilliamsonPJ BrindleyG. AbbenanteBJ DatuP. Prociv2003Hookworm aspartic protease, Na-APR-2, cleaves human hemoglobin and serum proteins in a host-specific fashion.J Infect Dis187484494
  59. 59. Eiff JA (1966) Nature of an anticoagulant from the cephalic glands of Ancylostoma caninum. J Parasitol 52: 833–843.JA Eiff1966Nature of an anticoagulant from the cephalic glands of Ancylostoma caninum.J Parasitol52833843
  60. 60. Milstone AM, Harrison LM, Bungiro RD, Kuzmic P, Cappello M (2000) A broad spectrum kunitz type serine protease inhibitor secreted by the hookworm Ancylostoma ceylanicum. J Biol Chem 275: 29391–29399.AM MilstoneLM HarrisonRD BungiroP. KuzmicM. Cappello2000A broad spectrum kunitz type serine protease inhibitor secreted by the hookworm Ancylostoma ceylanicum.J Biol Chem2752939129399
  61. 61. Hawdon JM, Datu B, Crowell M (2003) Molecular cloning of a novel multidomain kunitz-type proteinase inhibitor from the hookworm Ancylostoma caninum. J Parasitol 89: 402–407.JM HawdonB. DatuM. Crowell2003Molecular cloning of a novel multidomain kunitz-type proteinase inhibitor from the hookworm Ancylostoma caninum.J Parasitol89402407
  62. 62. Chu D, Bungiro RD, Ibanez M, Harrison LM, Campodonico E, et al. (2004) Molecular characterization of Ancylostoma ceylanicum Kunitz-type serine protease inhibitor: evidence for a role in hookworm-associated growth delay. Infect Immunol 72: 2214–2221.D. ChuRD BungiroM. IbanezLM HarrisonE. Campodonico2004Molecular characterization of Ancylostoma ceylanicum Kunitz-type serine protease inhibitor: evidence for a role in hookworm-associated growth delay.Infect Immunol7222142221
  63. 63. Costa AF, Gasser RB, Dias SR, Rabelo EM (2009) Male-enriched transcription of genes encoding ASPs and Kunitz-type protease inhibitors in Ancylostoma species. Mol Cell Probes 23: 298–303.AF CostaRB GasserSR DiasEM Rabelo2009Male-enriched transcription of genes encoding ASPs and Kunitz-type protease inhibitors in Ancylostoma species.Mol Cell Probes23298303
  64. 64. Furmidge BA, Horn LA, Pritchard DI (1996) The anti-haemostatic strategies of the human hookworm Necator americanus. Parasitology 112: 81–87.BA FurmidgeLA HornDI Pritchard1996The anti-haemostatic strategies of the human hookworm Necator americanus.Parasitology1128187
  65. 65. Williamson AL, Lecchi P, Turk BE, Choe Y, Hotez PJ, et al. (2004) A multi-enzyme cascade of hemoglobin proteolysis in the intestine of blood-feeding hookworms. J Biol Chem 279: 35950–35957.AL WilliamsonP. LecchiBE TurkY. ChoePJ Hotez2004A multi-enzyme cascade of hemoglobin proteolysis in the intestine of blood-feeding hookworms.J Biol Chem2793595035957
  66. 66. Ranjit N, Zhan B, Hamilton B, Stenzel D, Lowther J, et al. (2009) Proteolytic degradation of hemoglobin in the intestine of the human hookworm Necator americanus. J Infect Dis 199: 904–912.N. RanjitB. ZhanB. HamiltonD. StenzelJ. Lowther2009Proteolytic degradation of hemoglobin in the intestine of the human hookworm Necator americanus.J Infect Dis199904912
  67. 67. Ranjit N, Zhan B, Stenzel DJ, Mulvenna J, Fujiwara R, et al. (2008) A family of cathepsin B cysteine proteases expressed in the gut of the human hookworm, Necator americanus. Mol Biochem Parasitol 160: 90–99.N. RanjitB. ZhanDJ StenzelJ. MulvennaR. Fujiwara2008A family of cathepsin B cysteine proteases expressed in the gut of the human hookworm, Necator americanus.Mol Biochem Parasitol1609099
  68. 68. Loukas A, Bethony JM, Williamson AL, Goud GN, Mendez S, et al. (2004) Vaccination of dogs with a recombinant cysteine protease from the intestine of canine hookworms diminishes the fecundity and growth of worms. J Infect Dis 189: 1952–1961.A. LoukasJM BethonyAL WilliamsonGN GoudS. Mendez2004Vaccination of dogs with a recombinant cysteine protease from the intestine of canine hookworms diminishes the fecundity and growth of worms.J Infect Dis18919521961
  69. 69. Mulvenna J, Hamilton B, Nagaraj SH, Smyth D, Loukas A, et al. (2009) Proteomics analysis of the excretory/secretory component of the blood-feeding stage of the hookworm, Ancylostoma caninum. Mol Cell Proteomics 8: 109–121.J. MulvennaB. HamiltonSH NagarajD. SmythA. Loukas2009Proteomics analysis of the excretory/secretory component of the blood-feeding stage of the hookworm, Ancylostoma caninum.Mol Cell Proteomics8109121
  70. 70. Ford L, Zhang J, Liu J, Hashmi S, Fuhrman JA, et al. (2009) Functional analysis of the cathepsin-like cysteine protease genes in adult Brugia malayi using RNA interference. PLoS Negl Trop Dis 3: e377.L. FordJ. ZhangJ. LiuS. HashmiJA Fuhrman2009Functional analysis of the cathepsin-like cysteine protease genes in adult Brugia malayi using RNA interference.PLoS Negl Trop Dis3e377
  71. 71. Kumar S, Pritchard DI (1992) The partial characterization of proteases present in the excretory/secretory products and exsheathing fluid of the infective (L3) larva of Necator americanus. Int J Parasitol 22: 563–572.S. KumarDI Pritchard1992The partial characterization of proteases present in the excretory/secretory products and exsheathing fluid of the infective (L3) larva of Necator americanus.Int J Parasitol22563572
  72. 72. Zhan B, Liu Y, Badamchian M, Williamson A, Feng J, et al. (2003) Molecular characterization of the Ancylostoma-secreted protein family from the adult stage of Ancylostoma caninum. Int J Parasitol 33: 897–907.B. ZhanY. LiuM. BadamchianA. WilliamsonJ. Feng2003Molecular characterization of the Ancylostoma-secreted protein family from the adult stage of Ancylostoma caninum.Int J Parasitol33897907
  73. 73. Hawdon JM, Jones BF, Perregaux MA, Hotez PJ (1995) Ancylostoma caninum: metalloprotease release coincides with activation of infective larvae in vitro. Exp Parasitol 80: 205–211.JM HawdonBF JonesMA PerregauxPJ Hotez1995Ancylostoma caninum: metalloprotease release coincides with activation of infective larvae in vitro.Exp Parasitol80205211
  74. 74. Rajan TV (1998) A hypothesis for the tissue specificity of nematode parasites. Exp Parasitol 89: 140–142.TV Rajan1998A hypothesis for the tissue specificity of nematode parasites.Exp Parasitol89140142
  75. 75. Clark DA, Coker R (1998) Transforming growth factor-beta (TGF-beta). Inter J Biochem Cell Biol 30: 293–298.DA ClarkR. Coker1998Transforming growth factor-beta (TGF-beta).Inter J Biochem Cell Biol30293298
  76. 76. Williamson AL, Lustigman S, Oksov Y, Deumic V, Plieskatt J, et al. (2006) Ancylostoma caninum MTP-1, an astacin-like metalloprotease secreted by infective hookworm larvae, is involved in tissue migration. Infect Immun 74: 961–967.AL WilliamsonS. LustigmanY. OksovV. DeumicJ. Plieskatt2006Ancylostoma caninum MTP-1, an astacin-like metalloprotease secreted by infective hookworm larvae, is involved in tissue migration.Infect Immun74961967
  77. 77. Rogers WP, Sommerville RI (1957) Physiology of exsheathment in nematodes and its relation to parasitism. Nature 179: 619–621.WP RogersRI Sommerville1957Physiology of exsheathment in nematodes and its relation to parasitism.Nature179619621
  78. 78. Shin H, Hirst M, Bainbridge MN, Magrini V, Mardis E, et al. (2008) Transcriptome analysis for Caenorhabditis elegans based on novel expressed sequence tags. BMC Biol 6: 30.H. ShinM. HirstMN BainbridgeV. MagriniE. Mardis2008Transcriptome analysis for Caenorhabditis elegans based on novel expressed sequence tags.BMC Biol630
  79. 79. Loukas A, Constant SL, Bethony JM (2005) Immunobiology of hookworm infection. FEMS Immunol Med Microbiol 43: 115–124.A. LoukasSL ConstantJM Bethony2005Immunobiology of hookworm infection.FEMS Immunol Med Microbiol43115124
  80. 80. Hawdon JM, Jones BF, Hoffman DR, Hotez PJ (1996) Cloning and characterization of Ancylostoma-secreted protein. A novel protein associated with the transition to parasitism by infective hookworm larvae. J Biol Chem 271: 6672–6678.JM HawdonBF JonesDR HoffmanPJ Hotez1996Cloning and characterization of Ancylostoma-secreted protein. A novel protein associated with the transition to parasitism by infective hookworm larvae.J Biol Chem27166726678
  81. 81. Bethony J, Loukas A, Smout M, Brooker S, Mendez S, et al. (2005) Antibodies against a secreted protein from hookworm larvae reduce the intensity of hookworm infection in humans and vaccinated laboratory animals. FASEB J 19: 1743–1745.J. BethonyA. LoukasM. SmoutS. BrookerS. Mendez2005Antibodies against a secreted protein from hookworm larvae reduce the intensity of hookworm infection in humans and vaccinated laboratory animals.FASEB J1917431745
  82. 82. Zhan B, Hawdon J, Qiang S, Hainan R, Huiqing Q, et al. (1999) Ancylostoma secreted protein 1 (ASP-1) homologues in human hookworms. Mol Biochem Parasitol 98: 143–149.B. ZhanJ. HawdonS. QiangR. HainanQ. Huiqing1999Ancylostoma secreted protein 1 (ASP-1) homologues in human hookworms.Mol Biochem Parasitol98143149
  83. 83. Goud GN, Zhan B, Ghosh K, Loukas A, Hawdon J, et al. (2004) Cloning, yeast expression, isolation, and vaccine testing of recombinant Ancylostoma-secreted protein (ASP)-1 and ASP-2 from Ancylostoma ceylanicum. J Infect Dis 189: 19–29.GN GoudB. ZhanK. GhoshA. LoukasJ. Hawdon2004Cloning, yeast expression, isolation, and vaccine testing of recombinant Ancylostoma-secreted protein (ASP)-1 and ASP-2 from Ancylostoma ceylanicum.J Infect Dis1891929
  84. 84. Goud GN, Bottazzi ME, Zhan B, Mendez S, Deumic V, et al. (2005) Expression of the Necator americanus hookworm larval antigen Na-ASP-2 in Pichia pastoris and purification of the recombinant protein for use in human clinical trials. Int J Parasitol 35: 303–313.GN GoudME BottazziB. ZhanS. MendezV. Deumic2005Expression of the Necator americanus hookworm larval antigen Na-ASP-2 in Pichia pastoris and purification of the recombinant protein for use in human clinical trials.Int J Parasitol35303313
  85. 85. Asojo OA, Loukas A, Inan M, Barent R, Huang J, et al. (2005) Crystallization and preliminary X-ray analysis of Na-ASP-1, a multi-domain pathogenesis-related-1 protein from the human hookworm parasite Necator americanus. Acta Crystallogr Sect F Struct Biol Cryst Commun 61: 391–394.OA AsojoA. LoukasM. InanR. BarentJ. Huang2005Crystallization and preliminary X-ray analysis of Na-ASP-1, a multi-domain pathogenesis-related-1 protein from the human hookworm parasite Necator americanus.Acta Crystallogr Sect F Struct Biol Cryst Commun61391394
  86. 86. Bower MA, Constant SL, Mendez S (2008) Necator americanus: the Na-ASP-2 protein secreted by the infective larvae induces neutrophil recruitment in vivo and in vitro. Exp Parasitol 118: 569–575.MA BowerSL ConstantS. Mendez2008Necator americanus: the Na-ASP-2 protein secreted by the infective larvae induces neutrophil recruitment in vivo and in vitro.Exp Parasitol118569575
  87. 87. Mendez S, D' Samuel A, Antoine AD, Ahn S, Hotez P (2008) Use of the air pouch model to investigate immune responses to a hookworm vaccine containing the Na-ASP-2 protein in rats. Parasite Immunol 30: 53–56.S. MendezA. D' SamuelAD AntoineS. AhnP. Hotez2008Use of the air pouch model to investigate immune responses to a hookworm vaccine containing the Na-ASP-2 protein in rats.Parasite Immunol305356
  88. 88. Moyle M, Foster DL, McGrath DE, Brown SM, Laroche Y, et al. (1994) A hookworm glycoprotein that inhibits neutrophil function is a ligand of the integrin CD11b/CD18. J Biol Chem 269: 10008–10015.M. MoyleDL FosterDE McGrathSM BrownY. Laroche1994A hookworm glycoprotein that inhibits neutrophil function is a ligand of the integrin CD11b/CD18.J Biol Chem2691000810015
  89. 89. Del Valle A, Jones BF, Harrison LM, Chadderdon RC, Cappello M (2003) Isolation and molecular cloning of a secreted hookworm platelet inhibitor from adult Ancylostoma caninum. Mol Biochem Parasitol 129: 167–177.A. Del ValleBF JonesLM HarrisonRC ChadderdonM. Cappello2003Isolation and molecular cloning of a secreted hookworm platelet inhibitor from adult Ancylostoma caninum.Mol Biochem Parasitol129167177
  90. 90. Jian X, Sen L, Hui-Qin Q, Hai-Nan R, Tie-Hua L, et al. (2003) Necator americanus: maintenance through one hundred generations in golden hamsters (Mesocricetus auratus). I. Host sex-associated differences in hookworm burden and fecundity. Exp Parasitol 104: 62–66.X. JianL. SenQ. Hui-QinR. Hai-NanL. Tie-Hua2003Necator americanus: maintenance through one hundred generations in golden hamsters (Mesocricetus auratus). I. Host sex-associated differences in hookworm burden and fecundity.Exp Parasitol1046266
  91. 91. Jian X, Shu-Hua X, Hui-Qing Q, Sen L, Hotez P, et al. (2003) Necator americanus: maintenance through one hundred generations in golden hamsters (Mesocricetus auratus). II. Morphological development of the adult and its comparison with humans. Exp Parasitol 105: 192–200.X. JianX. Shu-HuaQ. Hui-QingL. SenP. Hotez2003Necator americanus: maintenance through one hundred generations in golden hamsters (Mesocricetus auratus). II. Morphological development of the adult and its comparison with humans.Exp Parasitol105192200
  92. 92. Grad LI, Sayles LC, Lemire BD (2007) Isolation and functional analysis of mitochondria from the nematode Caenorhabditis elegans. Methods Mol Biol 372: 51–66.LI GradLC SaylesBD Lemire2007Isolation and functional analysis of mitochondria from the nematode Caenorhabditis elegans.Methods Mol Biol3725166
  93. 93. Hu M, Chilton NB, Gasser RB (2002) The mitochondrial genomes of the human hookworms, Ancylostoma duodenale and Necator americanus (Nematoda: Secernentea). Int J Parasitol 32: 145–158.M. HuNB ChiltonRB Gasser2002The mitochondrial genomes of the human hookworms, Ancylostoma duodenale and Necator americanus (Nematoda: Secernentea).Int J Parasitol32145158
  94. 94. Hu M, Gasser RB, Abs El-Osta YG, Chilton NB (2003) Structure and organization of the mitochondrial genome of the canine heartworm, Dirofilaria immitis. Parasitology 127: 37–51.M. HuRB GasserYG Abs El-OstaNB Chilton2003Structure and organization of the mitochondrial genome of the canine heartworm, Dirofilaria immitis.Parasitology1273751
  95. 95. Hu M, Zhong W, Campbell BE, Sternberg PW, Pellegrino MW, et al. (2010) Elucidating ANTs in worms using genomic and bioinformatic tools - Biotechnological prospects? Biotechnol Adv 28: 49–60.M. HuW. ZhongBE CampbellPW SternbergMW Pellegrino2010Elucidating ANTs in worms using genomic and bioinformatic tools - Biotechnological prospects?Biotechnol Adv284960
  96. 96. Bos DH, Mayfield C, Minchella DJ (2009) Analysis of regulatory protease sequences identified through bioinformatic data mining of the Schistosoma mansoni genome. BMC Genomics 10: 488.DH BosC. MayfieldDJ Minchella2009Analysis of regulatory protease sequences identified through bioinformatic data mining of the Schistosoma mansoni genome.BMC Genomics10488
  97. 97. Kamath RS, Ahringer J (2003) Genome-wide RNAi screening in Caenorhabditis elegans. Methods 30: 313–321.RS KamathJ. Ahringer2003Genome-wide RNAi screening in Caenorhabditis elegans.Methods30313321
  98. 98. Grantham BD, Barrett J (1988) Glutamine and asparagine synthesis in the nematodes Heligmosomoides polygyrus and Panagrellus redivivus. J Parasitol 74: 1052–1053.BD GranthamJ. Barrett1988Glutamine and asparagine synthesis in the nematodes Heligmosomoides polygyrus and Panagrellus redivivus.J Parasitol7410521053
  99. 99. Myers RF, Krusberg LR (1965) Organic substances discharged by plant-parasitic nematodes. Phytopathology 55: 429–437.RF MyersLR Krusberg1965Organic substances discharged by plant-parasitic nematodes.Phytopathology55429437
  100. 100. Locatelli A, Camerini E (1969) Chromato-graphic studies of amino acids released in the in-cubation media of Fasciola hepatica. Ital J Biochem 18: 376–381.A. LocatelliE. Camerini1969Chromato-graphic studies of amino acids released in the in-cubation media of Fasciola hepatica.Ital J Biochem18376381
  101. 101. Hashimoto K, Suzuki F, Sakagami H (2009) Declined asparagine synthetase mRNA expression and enhanced sensitivity to asparaginase in HL-60 cells committed to monocytic differentiation. Anticancer Res 29: 1303–1308.K. HashimotoF. SuzukiH. Sakagami2009Declined asparagine synthetase mRNA expression and enhanced sensitivity to asparaginase in HL-60 cells committed to monocytic differentiation.Anticancer Res2913031308
  102. 102. Coles GC, Jackson F, Pomroy WE, Prichard RK, von Samson-Himmelstjerna G, et al. (2006) The detection of anthelmintic resistance in nematodes of veterinary importance. Vet Parasitol 136: 167–185.GC ColesF. JacksonWE PomroyRK PrichardG. von Samson-Himmelstjerna2006The detection of anthelmintic resistance in nematodes of veterinary importance.Vet Parasitol136167185
  103. 103. Gilleard JS, Beech RN (2007) Population genetics of anthelmintic resistance in parasitic nematodes. Parasitology 134: 1133–1147.JS GilleardRN Beech2007Population genetics of anthelmintic resistance in parasitic nematodes.Parasitology13411331147