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The Gene Ontology (GO) knowledgebase (http://geneontology.org) is a comprehensive resource concerning the functions of genes and gene products (proteins and noncoding RNAs). GO annotations cover genes from organisms across the tree of life as well as viruses, though most gene function knowledge currently derives from experiments carried out in a relatively small number of model organisms. Here, we provide an updated overview of the GO knowledgebase, as well as the efforts of the broad, international consortium of scientists that develops, maintains, and updates the GO knowledgebase. The GO knowledgebase consists of three components: (1) the GO—a computational knowledge structure describing the functional characteristics of genes; (2) GO annotations—evidence-supported statements asserting that a specific gene product has a particular functional characteristic; and (3) GO Causal Activity Models (GO-CAMs)—mechanistic models of molecular “pathways” (GO biological processes) created by linking multiple GO annotations using defined relations. Each of these components is continually expanded, revised, and updated in response to newly published discoveries and receives extensive QA checks, reviews, and user feedback. For each of these components, we provide a description of the current contents, recent developments to keep the knowledgebase up to date with new discoveries, and guidance on how users can best make use of the data that we provide. We conclude with future directions for the project.

The Gene Ontology (GO) knowledgebase (http://geneontology.org) is a comprehensive resource concerning the functions of genes and gene products (proteins and noncoding RNAs). GO annotations cover genes from organisms across the tree of life as well as viruses, though most gene function knowledge currently derives from experiments carried out in a relatively small number of model organisms. Here, we provide an updated overview of the GO knowledgebase, as well as the efforts of the broad, international consortium of scientists that develops, maintains, and updates the GO knowledgebase. The GO knowledgebase consists of three components: (1) the GO-a computational knowledge structure describing the functional characteristics of genes; (2) GO annotations-evidence-supported statements asserting that a specific gene product has a particular functional characteristic; and (3) GO Causal Activity Models (GO-CAMs)-mechanistic models of molecular "pathways" (GO biological processes) created by linking multiple GO annotations using defined relations. Each of these components is continually expanded, revised, and updated in response to newly published discoveries and receives extensive QA checks, reviews, and user feedback. For each of these components, we provide a description of the current contents, recent developments to keep the knowledgebase up to date with new discoveries, and guidance on how users can best make use of the data that we provide. We conclude with future directions for the project.

The Gene Ontology (GO) knowledgebase (http://geneontology.org) is a comprehensive resource concerning the functions of genes and gene products (proteins and noncoding RNAs). GO annotations cover genes from organisms across the tree of life as well as viruses, though most gene function knowledge currently derives from experiments carried out in a relatively small number of model organisms. Here, we provide an updated overview of the GO knowledgebase, as well as the efforts of the broad, international consortium of scientists that develops, maintains, and updates the GO knowledgebase. The GO knowledgebase consists of three components: (1) the GO-a computational knowledge structure describing the functional characteristics of genes; (2) GO annotations-evidence-supported statements asserting that a specific gene product has a particular functional characteristic; and (3) GO Causal Activity Models (GO-CAMs)-mechanistic models of molecular "pathways" (GO biological processes) created by linking multiple GO annotations using defined relations. Each of these components is continually expanded, revised, and updated in response to newly published discoveries and receives extensive QA checks, reviews, and user feedback. For each of these components, we provide a description of the current contents, recent developments to keep the knowledgebase up to date with new discoveries, and guidance on how users can best make use of the data that we provide. We conclude with future directions for the project.

The Alliance of Genome Resources (the Alliance) is a combined effort of 7 knowledgebase projects: Saccharomyces Genome Database, WormBase, FlyBase, Mouse Genome Database, the Zebrafish Information Network, Rat Genome Database, and the Gene Ontology Resource. The Alliance seeks to provide several benefits: better service to the various communities served by these projects; a harmonized view of data for all biomedical researchers, bioinformaticians, clinicians, and students; and a more sustainable infrastructure. The Alliance has harmonized cross-organism data to provide useful comparative views of gene function, gene expression, and human disease relevance. The basis of the comparative views is shared calls of orthology relationships and the use of common ontologies. The key types of data are alleles and variants, gene function based on gene ontology annotations, phenotypes, association to human disease, gene expression, protein–protein and genetic interactions, and participation in pathways. The information is presented on uniform gene pages that allow facile summarization of information about each gene in each of the 7 organisms covered (budding yeast, roundworm Caenorhabditis elegans, fruit fly, house mouse, zebrafish, brown rat, and human). The harmonized knowledge is freely available on the alliancegenome.org portal, as downloadable files, and by APIs. We expect other existing and emerging knowledge bases to join in the effort to provide the union of useful data and features that each knowledge base currently provides.

C. elegans larvae choose between dauer diapause and immediate reproductive development in two phases, the first being a choice between L2 and L2d, and the second between dauer and L3 (Golden and Riddle 1984). We used RNA-seq to study the transcriptomic response of well-fed C. elegans when exposed to a cocktail of diapause-inducing ascarosides (ascr#2, ascr#3, and ascr#8) during the L1 larval stage when they first receive cues from their environment and then adapt via developmental programming (Kaplan et al. 2011). This research was performed in N2 wild-type worms and daf-22(ok693) mutants that lack an enzyme necessary for the biosynthesis of ascaroside pheromones (reviewed by Ludewig and Schroeder 2013). We found that early exposure to ascarosides triggers a pervasive transcriptional response that may drive diapause induction within as little as three hours. In N2, 362 genes were significantly differentially expressed at p = 0.001, while in daf-22, 1,007 genes were significantly differentially expressed at p = 0.001. The daf-22 mutant may have more significantly expressed genes when exposed to ascarosides indicating a stronger response as it has never encountered ascaroside signaling previously, even at low levels.

Many invertebrates are capable of jumping, such as locusts, fleas, mantids, and cockroaches. Some invertebrates can jump without the use of their legs, such as click beetles and springtails (Mo et al., 2020). Nematodes, which are non-segmented roundworms, are capable of a variety of complex behaviors, though jumping is arguably the most visually striking. Jumping is a rare behavior among nematodes, with only a few species in the genus Steinernema being reported as capable of jumping (Campbell and Kaya, 1999). The kinematic performance of S. carpocapsae jumping was first described in 1999, with the peak velocity and peak acceleration reported as 1.13 m/s and 164 g, respectively (Campbell and Kaya, 1999). These data were not calculated based on direct observation and high-speed videography, but were estimated based on the distance, height, and timing of the nematode jumps. This nematode is reported to be capable of jumping a distance of 4.8 mm, and a height of 3.9 mm (Campbell and Kaya, 1999).

The C. elegans vulva is formed from divisions of three vulval precursor cells (VPCs) – P5.p, P6.p, and P7.p – arranged along the anteroposterior axis in the ventral epithelium (Sulston and Horvitz, 1977). Previous analyses show the orientation of P5.p and P7.p descendants is determined by the interaction of multiple Wnt signals. egl-20/Wnt is expressed in the tail (Whangbo and Kenyon, 2000) and forms a posterior-to-anterior concentration gradient (Coudreuse et al., 2006). It has previously been shown that EGL-20 acts instructively during vulva development by imparting directional information, as opposed to being permissive, where it would only be required for polarization (Green et al., 2008; Minor et al., 2013). By moving the source of egl-20 expression from the posterior of the worm to the anchor cell, the axis of symmetry of the developing vulva, we can reorient the daughter cells of P5.p and P7.p toward the center in a wild-type configuration. Expression of egl-20 from the center of the axis of symmetry partially suppresses the lin-17(n671) phenotype (Green et al., 2008; Table 1). To test whether LRP-2 acts downstream of EGL-20, we ectopically expressed egl-20 from the anchor cell in a lin-17(n671); lrp-2(gk272) double mutant background and compared it to a lin-17(n671); lrp-2(+) strain. If LRP-2 acts downstream of EGL-20, then anteriorly-expressed EGL-20 will not be able to suppress the lin-17 phenotype, with is the result observed (Table 1). Thus, like CAM-1 and VANG-1, LRP-2 likely acts downstream of EGL-20.

Recently, the GAL4-UAS system (cGAL) has been adapted for use in C. elegans for control of gene expression across 15°C - 25°C (Wang et al., 2017). In order to create a desired gene expression pattern, one crosses a transgenic strain containing a driver construct with another strain containing an effector gene. Here we mapped several cGAL driver and effector integrations. We first crossed each of the cGAL driver and effector strains with N2 males, picked the heterozygous male progeny, crossed them with hermaphrodites of the mapping strain (DA438), picked L4 hermaphrodites with the corresponding transgenic marker of the driver or effector strain and scored the progeny in the next generation. The DA438 strain contains six recessive mutations, each of which locates on one of the six chromosomes and produces visible phenotypes (Bli on chromosome I, Rol on II, Vab on III, Unc on IV, Dpy on V, and Lon on X (Avery, 1993). F2 progeny with each of the six phenotypes were selected and examined for the presence or absence of the dominant marker associated with the transgene. In the cases where the dominant transgene marker is unlinked to the recessive phenotypic marker, about three quarters of the F2 progeny will have the dominant marker. If the two markers are linked, very few or no animals are expected to have the dominant transgenic marker. The following tables summarize the mapping results for each cGAL strain, stating the ratios of the F2 mutant progeny with and without the dominant transgenic marker.

cGAL, a recently developed temperature-robust bipartite GAL4-UAS system in C. elegans, consists of two components: a cGAL “driver” that expresses the cGAL protein in specific cells using a promoter (i.e. neuron-specific or tissue-specific), and an “effector” that carries a gene of interest downstream of UAS (Wang et al., 2017). Crossing or combining a driver with an effector leads to the expression of the gene of interest in a cell-specific or tissue-specific manner. Here we report a new cGAL driver for the DVC interneuron. The ceh-63 promoter was chosen due to its restricted expression in the DVC neuron (Feng et al. 2012). The DVC interneuron driver construct containing the ceh-63 promoter (646 bp upstream of ATG translation start site) was injected into N2 and an integrated DVC driver line was generated. When crossed with the UAS-GFP effector strain (PS6843), the ceh-63 cGAL driver dictated GFP expression in the single DVC neuron (Figure 1), in addition to GFP in the coelomocyte from Punc-122::gfp co-injection marker. We did not observe GFP expression in uterus as reported by Feng et al., 2012.

The relatively simple nervous system and facile genetics of Caenorhabditis elegans makes it a potential model organism to study the genetic basis of complex neural diseases, such as autism spectrum disorder (ASD). Our previously published study identified conserved human ASD-missense variants in C. elegans orthologs that have a role on morphology, locomotion or fecundity, suggesting C. elegans as an efficient phenotypic model to study conserved ASD variants (Wong et al., 2019). One of the ASD-associated genes screened in this study is daf-18, an ortholog of PTEN. DAF-18/PTEN is a lipid phosphatase protein that dephosphorylates PIP3, a critical lipid second messenger that mediates downstream kinase cascade signaling of the insulin pathway, that in turn regulates expression of genes involved in C. elegans lifespan and dauer formation (Murphy and Hu, 2013). It is reported that the daf-18(e1375) strain, which has a 30–base pairs insertion in the fourth exon, has a dauer defective phenotype (Ogg and Ruvkun, 1998); however, the effect of daf-18/PTEN single amino acid substitution in dauer formation has not been evaluated before. Here, we investigate the dauer entry abilities of previously published ASD-associated daf-18/PTEN missense variants (Wong et al., 2019). We performed a dauer-entry assay using crude pheromone as a proxy for high population density and heat-killed bacteria as a limited food source. We first determined the crude pheromone concentration required to induce 50% of wild type (N2) strain to enter dauer stage (EC50). Using the calculated EC50value we evaluated each mutant strain in two independent experiments (Table 1). To control day-to-day variations caused by environmental conditions, N2 controls were used in every trail. Additionally, we used the previously reported daf-18 dauer-defective strain (e1375) as a control (Ogg and Ruvkun, 1998). To analyze the obtained data, we performed one-way ANOVA followed by multiple comparisons test (Dunnett test) (Figure 1A). Table 1 Evaluated ASD-associated daf-18/PTEN mutant strains. Strain name PS7439 PS7432 PS7436 PS7430 PS7434 C. elegans allele name daf-18(sy879) daf-18(sy887) daf-18(sy881) daf-18(sy885) daf-18(sy882) C. elegans protein change D66E L115V H138R H168Q T176I Human protein change D22E L70V H93R H123Q T131I As expected, we observed statistically significant difference between our wild type and dauer defective control (e1375). Our results show that H138R (PS7436) and T176I (PS7434) daf-18 mutant strains have a significant difference from the wild type control, and a non-significant difference from the dauer defective control, suggesting these missense alleles cause dauer defective phenotypes to the same level of the e1375 strain. In contrast, we found the D66E (PS7439), L115V (PS7432) and H168Q (PS7430) mutant strains are significantly different from the dauer defective control and not significantly different from the wild type control, suggesting these three missense alleles do not cause the same level of dauer defective phenotype as the defective control does. Altogether, our current data show that H138R and T176I are defective in dauer entry, while D66E, L115V and H168Q daf-18 mutant strains are not (Figure 1A). The Conserved Domain Database (Conserved Domain Search) of NCBI (ID: G5EE01) predicts that four of the five missense variations evaluated in this study are located in the phosphatase domain of DAF-18/PTEN(Figure 1B). It is predicted that amino acid Histidine 138 is located in the active site of the protein, suggesting that H138R (PS7436) amino acid change might have an impact on PIP3 substrate specificity, which could explain the dauer defect phenotype observed. Moreover, it is predicted that both amino acid Histidine 168 and Threonine 176 are located close to the catalytic site of the phosphatase domain. We found a dauer defective phenotype resulting from a T176I substitution (PS7434) but not from a H168Q amino acid substitution (PS7430). These data suggest that certain amino acids in the catalytic and active sites of DAF-18/PTEN decrease the function of the protein, in turn regulating C. elegans dauer development. Future evaluation of PIP3 phosphorylation levels could confirm the effect of these variations on the phosphatase activity. The results of this study contribute to the knowledge of the mechanisms controlling C. elegans dauer entry, and to the C. elegans phenotype-screening of conserved ASD variations.

A new allele of pkd-2 was isolated in a behavioral genetic screen for male mating defects, and found to result in a substitution of Arginine for Glycine in the equivalent of human PKD2 alanine 615. The C. elegans ortholog of polcystin-2 is encoded by pkd-2 (Barr et al., 2001). From an EMS screen of a plg-1; him-5 strain for male mating defective mutants and a secondary behavioral screen for defects in discrete steps of male mating behavior, namely response to contact to hermaphrodites and vulval location (described in Schindelman et al., 2006), we identified a new allele of pkd-2 based on mapping and complementation. sy680 fails to complement pkd-2(sy606) for defects in response to contact with hermaphrodite and vulval location. Here we report the sequence of this allele. PCR amplification and sequencing of pkd-2 exons indicated that there was a c-->t transition in the transcribed DNA strand (g-->a in the pkd-2 sense strand; Figure 1A). This change leads to an altered codon, a Glycine to Arginine substitution the PKD-2 protein. This position corresponds to A615 of the human protein (Figure 1B).

ina-1 is expressed throughout uterine toroids. At mid L4, expression is seen in uterine toroid 1 and 4 (ut1, ut4) and in the anterior uterine toroids 2 and 3 (ut2, ut3). In addition, ina-1::GFP is detected in the spermathecal-uterine junction and spermatheca. At the L4 lethargus stage, bright expression is observed in uterine toroids 1 and 4, the spermathecal-uterine junction, and spermatheca. Dimmer expression is present in uterine toroid 2 and posterior toroid 3. Fluorescence is also detected in vulE. Reagents: gmIs5

The bacterium Xenorhabdus nematophila is a mutualist of entomopathogenic Steinernema carpocapsae nematodes and facilitates infection of insect hosts. X. nematophila colonizes the intestine of S. carpocapsae which carries it between insects. In the X. nematophila colonization-defective mutant nilD6::Tn5, the transposon is inserted in a region lacking obvious coding potential. We demonstrate that the transposon disrupts expression of a single CRISPR RNA, NilD RNA. A variant NilD RNA also is expressed by X. nematophila strains from S. anatoliense and S. websteri nematodes. Only nilD from the S. carpocapsae strain of X. nematophila rescued the colonization defect of the nilD6::Tn5 mutant, and this mutant was defective in colonizing all three nematode host species. NilD expression depends on the presence of the associated Cas6e but not Cas3, components of the Type I-E CRISPR-associated machinery. While cas6e deletion in the complemented strain abolished nematode colonization, its disruption in the wild-type parent did not. Likewise, nilD deletion in the parental strain did not impact colonization of the nematode, revealing that the requirement for NilD is evident only in certain genetic backgrounds. Our data demonstrate that NilD RNA is conditionally necessary for mutualistic host colonization and suggest that it functions to regulate endogenous gene expression.

The fluorescence microscope is one of the most important tools in modern clinical diagnosis and biological science. However, its expense, size and limited field-of-view (FOV) are becoming bottlenecks in key applications such as large-scale phenotyping and low-resource-setting diagnostics. Here we report a low-cost, compact chip-scale fluorescence-imaging platform, termed the Fluorescence Talbot Microscopy (FTM), which utilizes the Talbot self-imaging effect to enable efficient fluorescence imaging over a large and directly-scalable FOV. The FTM prototype has a resolution of 1.2 microns and an FOV of 3.9 mm x 3.5 mm. We demonstrate the imaging capability of FTM on fluorescently labeled breast cancer cells (SK-BR-3) and HEK cells expressing green fluorescent protein.

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/PrincipalFindings: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.

Protein kinases are key enzymes that regulate a wide range of cellular processes, including cell-cycle progression, transcription, DNA replication and metabolic functions. These enzymes catalyse the transfer of phosphates to serine, threonine and tyrosine residues, thus playing functional roles in reversible protein phosphorylation. There are two main groups, namely eukaryotic protein kinases (ePKs) and atypical protein kinases (aPKs); RIO kinases belong to the latter group. While there is some information about RIO kinases and their roles in animals, nothing is known about them in parasites. This is the first study to characterise a RIO1 kinase from any parasite. A full-length cDNA (Tv-rio-1) encoding a RIO1 protein kinase (Tv-RIO1) was isolated from the economically important parasitic nematode Trichostrongylus vitrinus (Order Strongylida). The uninterrupted open reading frame (ORF) of 1476 nucleotides encoded a protein of 491 amino acids, containing the characteristic RIO1 motif LVHADLSEYNTL. Tv-rio-1 was transcribed at the highest level in the third-stage larva (L3), and a higher level in adult females than in males. Comparison with homologues from other organisms showed that protein Tv-RIO1 had significant homology to related proteins from a range of metazoans and plants. Amino acid sequence identity was most pronounced in the ATP-binding motif, active site and metal binding loop. Phylogenetic analyses of selected amino acid sequence data revealed Tv-RIO1 to be most closely related to the proteins in the species of Caenorhabditis. A structural model of Tv-RIO1 was constructed and compared with the published crystal structure of RIO1 of Archaeoglobus fulgidus (Af-Rio1). This study provides the first insights into the RIO1 protein kinases of nematodes, and a foundation for further investigations into the biochemical and functional roles of this molecule in biological processes in parasitic nematodes.

The order Strongylida (clade V) represents parasites most closely related to C. elegans, and the group most likely to benefit from the comparative value of its genome sequence. C. elegans has already served as an essential guide for exploring the genomes of other nematode species. However, transcriptomic data from parasitic nematode species, generated by EST approaches, have already served as a boon for C. elegans biology, as they confirm gene predictions and add depth to analyses of structure-function. Here, we propose sequencing the genomes of 20 species, members of the order Strongylida (‘strongylids’). Strongylids, including human hookworms and many intestinal parasites of livestock, are relatively closely related to one another and may have resulted from a recent radiation. To advance and facilitate molecular studies of strongylid parasites, an expressed sequence tag (EST)-based gene discovery program has been carried out, as a part of a broad study of the transcriptomes of members of the phylum Nematoda. More than 60,000 ESTs have been generated for key parasites of human and domestic animals, and for model parasitic species belonging to order Strongylida (comprising 83% of all clade V non-Caenorhabditis ESTs), and deposited in the public databases. Although the transcriptomic data hold promise for the development of novel control strategies for these important pathogens, emerging whole genome data will be pivotal in a more comprehensive approach to identify alternative control strategies, which is increasingly pressing as levels of parasite resistance to available drugs is rising, making some treatment strategies that worked just 5 years ago, ineffective. Also, some drug treatments and environmental nematocideecological contamination.