A literature review for a recent ultrastructural study of a trichinelloid eggshell revealed consistently occurring errors in the literature on nematode eggshell anatomy. Examples included nematodes of medical, veterinary, and agricultural importance in several orders. Previous researchers had warned of some of these errors decades ago, but a comprehensive solution was not offered until 2012 when a clarifying new anatomical and developmental interpretation of nematode eggshells was proposed by members of the Caenorhabditis elegans Research Community. However, their findings were explained using arcane acronyms and technical jargon intended for an audience of experimental molecular geneticists, and so their papers have rarely been cited outside the C. elegans community. Herein we (1) provide a critical review of nematode eggshell literature in which we correct errors and relabel imagery in important historical reports; (2) describe common reporting errors and their causes using language familiar to researchers having a basic understanding of microscopy and nematode eggs; (3) recommend a new hexalaminar anatomical and terminological framework for nematode eggshells based on the 2012 C. elegans framework; and (4) recommend new unambiguous terms appropriate for the embryonated/larvated eggs regularly encountered by practicing nematodologists to replace ambiguous or ontogenetically restricted terms in the 2012 C. elegans framework. We also (5) propose a resolution to conflicting claims made by the C. elegans team versus classical literature regarding Layer #3, (6) extend the C. elegans hexalaminar framework to include the polar plugs of trichinelloids, and (7) report new findings regarding trichinelloid eggshell structure.
UNASSIGNED: La coque des œufs des nématodes : un nouveau cadre anatomique et terminologique, avec une revue critique de la littérature pertinente et des lignes directrices suggérées pour l’interprétation et la communication de l’imagerie des coques des œufs.
UNASSIGNED: Une revue de la littérature pour une étude ultrastructurale récente de la coque de l’œuf d’un trichinelloïde a révélé des erreurs récurrentes dans la littérature sur l’anatomie de la coque de l’œuf des nématodes. Les exemples comprenaient des nématodes d’importance médicale, vétérinaire et agricole dans plusieurs ordres. Des chercheurs avaient mis en garde contre certaines de ces erreurs il y a des décennies, mais une solution complète n’a été proposée qu’en 2012, lorsqu’une nouvelle interprétation anatomique et développementale clarifiant la structure des coques des œufs de nématodes a été proposée par des membres de la communauté de recherche de Caenorhabditis elegans. Cependant, leurs découvertes ont été expliquées à l’aide d’acronymes mystérieux et d’un jargon technique destiné à un public de généticiens moléculaires expérimentaux, et leurs articles ont donc rarement été cités en dehors de la communauté de C. elegans. Ici, nous (1) fournissons une revue critique de la littérature sur les coques des œufs de nématodes dans laquelle nous corrigeons les erreurs et réétiquetons les images dans des rapports historiques importants; (2) décrivons les erreurs de description courantes et leurs causes en utilisant un langage familier aux chercheurs ayant une compréhension de base de la microscopie et des œufs de nématodes; (3) recommandons un nouveau cadre anatomique et terminologique hexalaminaire pour les coques des œufs de nématodes basé sur le cadre de C. elegans de 2012; et (4) recommandons de nouveaux termes non ambigus appropriés pour les œufs embryonnés/larvés régulièrement rencontrés par les spécialistes de nématodes en exercice pour remplacer les termes ambigus ou à restriction ontogénétique dans le cadre de C. elegans de 2012. Nous proposons également (5) une résolution des affirmations contradictoires de l’équipe C. elegans par rapport à la littérature classique concernant la couche 3, (6) étendons le cadre hexalaminaire de C. elegans pour inclure les bouchons polaires des trichinelloïdes, et (7) signalons de nouvelles découvertes concernant la structure de la coque des œufs des trichinelloïdes.

The protein HSF-1 is the controlling transcription factor of the heat-shock response (HSR). Its binding to the heat-shock elements (HSEs) induces the strong upregulation of conserved heat-shock proteins, including Hsp70s, Hsp40s and small HSPs. Next to these commonly known HSPs, more than 4000 other HSEs are found in the promoter regions of C. elegans genes. In microarray experiments, few of the HSE-containing genes are specifically upregulated during the heat-shock response. Most of the 4000 HSE-containing genes instead are unaffected by elevated temperatures and coexpress with genes unrelated to the HSR. This is also the case for several genes related to the HSP chaperone system, like dnj-12, dnj-13, and hsp-1. Interestingly, several promoters of the dedicated HSR-genes, like F44E5.4p, hsp-16.48p or hsp-16.2p, contain extended HSEs in their promoter region, composed of four or five HSE-elements instead of the common trimeric HSEs. We here aim at understanding how HSF-1 interacts with the different promoter regions. To this end we purify the nematode HSF-1 DBD and investigate the interaction with DNA sequences containing these regions. EMSA assays suggest that the HSF-1 DBD interacts with most of these HSE-containing dsDNAs, but with different characteristics. We employ sedimentation analytical ultracentrifugation (SV-AUC) to determine stoichiometry, affinity, and cooperativity of HSF-1 DBD binding to these HSEs. Interestingly, most HSEs show cooperative binding of the HSF-1 DBD with up to five DBDs being bound. In most cases binding to the HSEs of inducible promoters is stronger, even though the consensus scores are not always higher. The observed high affinity of HSF-1 DBD to the non-inducible HSEs of dnj-12, suggests that constitutive expression may be supported from some promoter regions, a fact that is evident for this transcription factor, that is essential also under non-stress conditions.

Metabolism is one of the attributes of life and supplies energy and building blocks to organisms. Therefore, understanding metabolism is crucial for the understanding of complex biological phenomena. Despite having been in the focus of research for centuries, our picture of metabolism is still incomplete. Metabolomics, the systematic analysis of all small molecules in a biological system, aims to close this gap. In order to facilitate such investigations a blueprint of the metabolic network is required. Recently, several metabolic network reconstructions for the model organism Caenorhabditis elegans have been published, each having unique features. We have established the WormJam Community to merge and reconcile these (and other unpublished models) into a single consensus metabolic reconstruction. In a series of workshops and annotation seminars this model was refined with manual correction of incorrect assignments, metabolite structure and identifier curation as well as addition of new pathways. The WormJam consensus metabolic reconstruction represents a rich data source not only for in silico network-based approaches like flux balance analysis, but also for metabolomics, as it includes a database of metabolites present in C. elegans, which can be used for annotation. Here we present the process of model merging, correction and curation and give a detailed overview of the model. In the future it is intended to expand the model toward different tissues and put special emphasizes on lipid metabolism and secondary metabolism including ascaroside metabolism in accordance to their central role in C. elegans physiology.

The cellular recycling process of autophagy has been extensively characterized with standard assays in yeast and mammalian cell lines. In multicellular organisms, numerous external and internal factors differentially affect autophagy activity in specific cell types throughout the stages of organismal ontogeny, adding complexity to the analysis of autophagy in these metazoans. Here we summarize currently available assays for monitoring the autophagic process in the nematode C. elegans. A combination of measuring levels of the lipidated Atg8 ortholog LGG-1, degradation of well-characterized autophagic substrates such as germline P granule components and the SQSTM1/p62 ortholog SQST-1, expression of autophagic genes and electron microscopy analysis of autophagic structures are presently the most informative, yet steady-state, approaches available to assess autophagy levels in C. elegans. We also review how altered autophagy activity affects a variety of biological processes in C. elegans such as L1 survival under starvation conditions, dauer formation, aging, and cell death, as well as neuronal cell specification. Taken together, C. elegans is emerging as a powerful model organism to monitor autophagy while evaluating important physiological roles for autophagy in key developmental events as well as during adulthood.

Autoinflammatory diseases (AIDs) and autoimmune diseases (ADs) are characterized by an aberrant chronic activation of the immune system which causes tissue inflammation and damage in genetically predisposed individuals. Pathogenetic mechanisms underlying this damage differ between these two types of diseases; in AIDs, the innate immune system is directly responsible for tissue inflammation, while in ADs it works by activating the adaptive immune system, which becomes the main effector of the inflammatory process. Despite the fact that AIDs have only been recently defined, they are older than ADs. The innate immune system is found in plants and animals, and it developed earlier than the adaptive immune system, which first appeared in jawed vertebrates. According to genetic background and clinical, serological, and radiological findings, AIDs and ADs might be considered as a single spectrum of disorders, with a wide range of manifestations. Indeed, autoinflammatory-like diseases have been reported in simple organisms such as Drosophila melanogaster and Caenorhabditis elegans. We analyzed here the main pathogenetic and clinical features of these two groups of diseases mostly dealing with their similarities and differences.

An increasing number of SH3 domain-ligand interactions continue to be described that involve the conserved peptide-binding surface of SH3, but structurally deviate substantially from canonical docking of consensus motif-containing SH3 ligands. Indeed, it appears that that the relative frequency and importance of these types of interactions may have been underestimated. Instead of atypical, we propose referring to such peptides as type I or II (depending on the binding orientation) non-consensus ligands. Here we discuss the structural basis of non-consensus SH3 ligand binding and the dominant role of the SH3 domain specificity zone in selective target recognition, and review some of the best-characterized examples of such interactions.

BACKGROUND: STAR/GSG proteins regulate gene expression in metazoans by binding consensus sites in the 5\' or 3\' UTRs of target mRNA transcripts. Owing to the high degree of homology across the STAR domain, most STAR proteins recognize similar RNA consensus sequences. Previously, the consensus for a number of well-characterized STAR proteins was defined as a hexameric sequence, referred to as the SBE, for STAR protein binding element. C. elegans GLD-1 and mouse Quaking (Qk-1) are two representative STAR proteins that bind similar consensus hexamers, which differ only in the preferred nucleotide identities at certain positions. Earlier reports also identified partial consensus elements located upstream or downstream of a canonical consensus hexamer in target RNAs, although the relative contribution of these sequences to the overall binding energy remains less well understood. Additionally, a recently identified STAR protein called STAR-2 from C. elegans is thought to bind target RNA consensus sites similar to that of GLD-1 and Qk-1.
RESULTS: Here, a combination of fluorescence-polarization and gel mobility shift assays was used to demonstrate that STAR-2 binds to a similar RNA consensus as GLD-1 and Qk-1. These assays were also used to further delineate the contributions of each hexamer consensus nucleotide to high-affinity binding by GLD-1, Qk-1 and STAR-2 in a variety of RNA contexts. In addition, the effects of inserting additional full or partial consensus elements upstream or downstream of a canonical hexamer in target RNAs were also measured to better define the sequence elements and RNA architecture recognized by different STAR proteins.
CONCLUSIONS: The results presented here indicate that a single hexameric consensus is sufficient for high-affinity RNA binding by STAR proteins, and that upstream or downstream partial consensus elements may alter binding affinities depending on the sequence and spacing. The general requirements determined for high-affinity RNA binding by STAR proteins will help facilitate the identification of novel regulatory targets in vivo.

All organisms except the nematode Caenorhabditis elegans have been shown to possess an import system for peroxisomal proteins containing a peroxisome targeting signal type 2 (PTS2). The currently accepted consensus sequence for this amino-terminal nonapeptide is -(R/K)(L/V/I)X(5)(H/Q)(L/A)-. Some C.elegans proteins contain putative PTS2 motifs, including the ortholog (CeMeK) of human mevalonate kinase, an enzyme known to be targeted by PTS2 to mammalian peroxisomes. We cloned the gene for CeMeK (open reading frame Y42G9A.4) and examined the subcellular localization of CeMeK and of two other proteins with putative PTS2s at their amino termini encoded by the open reading frames D1053.2 and W10G11.11. All three proteins localized to the cytosol, confirming and extending the finding that C.elegans lacks PTS2-dependent peroxisomal protein import. The putative PTS2s of the proteins encoded by D1053.2 and W10G11.11 did not function in targeting to peroxisomes in yeast or mammalian cells, suggesting that the current PTS2 consensus sequence is too broad. Analysis of available experimental data on both functional and nonfunctional PTS2s led to two re-evaluated PTS2 consensus sequences: -R(L/V/I/Q)XX(L/V/I/H)(L/S/G/A)X(H/Q)(L/A)-, describes the most common variants of PTS2, while -(R/K)(L/V/I/Q)XX(L/V/I/H/Q)(L/S/G/A/K)X(H/Q)(L/A/F)-, describes essentially all variants of PTS2. These redefined PTS2 consensus sequences will facilitate the identification of proteins of unknown cellular localization as possible peroxisomal proteins.

daf-16 is a forkhead-type transcription factor, functioning downstream of insulin-like signals, and is known to be critical to the regulation of life span in Caenorhabditis elegans. Mammalian DAF-16 homologues include AFX, FKHR and FKHRL1, which contain a conserved forkhead domain and three putative phosphorylation sites for the Ser/Thr kinase Akt/protein kinase B (PKB), as well as for DAF-16. To assess the function of the homologues, we examined tissue distribution patterns of mRNAs for DAF-16 homologues in mice. In the embryos, expressions of AFX, FKHR and FKHRL1 mRNAs were complementary to each other and were highest in muscle, adipose tissue and embryonic liver. The characteristic expression pattern remained in the adult, except that signals of FKHRL1 became evident in more tissues, including the brain. In order to clarify whether each DAF-16 homologue had different target genes, we determined the consensus sequences for the binding of DAF-16 and the mouse homologues. The binding sequences for all four proteins shared a core sequence, TTGTTTAC, daf-16 family protein-binding element (DBE) binding protein. However, electrophoretic mobility shift assay showed that the binding affinity of DAF-16 homologues to the core sequence was stronger than that to the insulin-responsive element in the insulin-like growth factor binding protein-1 promoter region, which has been identified as a binding sequence for them. We identified one copy of the DBE upstream of the first exon of sod-3 by searching the genomic database of C. elegans. Taken together, DAF-16 homologues can fundamentally regulate the common target genes in insulin-responsive tissues and the specificity to target genes of each protein is partially determined by the differences in their expression patterns.

The small- and large-subunit mitochondrial ribosomal RNA genes (mt-s-rRNA and mt-l-rRNA) of the nematode worms Caenorhabditis elegans and Ascaris suum encode the smallest rRNAs so far reported for metazoa. These size reductions correlate with the previously described, smaller, structurally anomalous mt-tRNAs of C. elegans and A. suum. Using primer extension analysis, the 5\' end nucleotides of the mt-s-rRNA and mt-l-rRNA genes were determined to be adjacent to the 3\' end nucleotides of the tRNA(Glu) and tRNA(His) genes, respectively. Detailed, consensus secondary-structure models were constructed for the mt-s-rRNA genes and the 3\' 64% of mt-l-rRNA genes of the two nematodes. The mt-s-rRNA secondary-structure model bears a remarkable resemblance to the previously defined universal core structure of E. coli 16S rRNA: most of the nucleotides that have been classified as variable or semiconserved in the E. coli model appear to have been eliminated from the C. elegans and A. suum sequences. Also, the secondary structure model constructed for the 3\' 64% of the mt-l-rRNA is similar to the corresponding portion of the previously defined E. coli 23S rRNA core secondary structure. The proposed C. elegans/A. suum mt-s-rRNA and mt-l-rRNA models include all of the secondary-structure element-forming sequences that in E. coli rRNAs contain nucleotides important for A-site and P-site (but not E-site) interactions with tRNAs. Sets of apparently homologous sequences within the mt-s-rRNA and mt-l-rRNA core structures, derived by alignment of the C. elegans and A. suum mt-rRNAs to the corresponding mt-rRNAs of other eukaryotes, and E. coli rRNAs were used in maximum-likelihood analyses. The patterns of divergence of metazoan phyla obtained show considerable agreement with the most prevalent metazoan divergence patterns derived from more classical, morphological, and developmental data.