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[
International Worm Meeting,
2003]
One of the largest families of adhesion molecules with a role in nervous system development is the immunoglobulin-superfamily (IgCAM-SF). IgCAM family members can be grouped according to their modular organisation. The C.elegans genome contains 28 genes encoding transmembrane or GPI-anchored proteins with extracellular IG domains. We are focusing on a core set of 17 IgCAMs, consisting of proteins with Ig domains only (5 genes) or with Ig domains and fibronectin III domains (12 genes). Out of these 17 genes 14 have not been analyzed functionally and ten genes are evolutionary conserved with orthologs in other animals. To identify IgCAM genes with a putative role in axon guidance we started to analyze their expression patterns. Transgenic animals were generated expressing promoter-GFP fusion constructs for individual IgCAMs. Expression typically is dynamic and not confined to a single tissue. Some IgCAMs (e.g. C26G2.1/Nephrin) show predominantly non-neuronal expression. Many others are expressed mainly in particular subsets of neurons. Five IgCAMs (
rig-1,
rig-2/syg-1,
rig-3,
rig-4 and
wrk-1) are expressed in motorneurons or interneurons with axons in the ventral cord. For functional analysis we generated a library of mutagenized worms to isolate deletion alleles. So far we have isolated deletions in ten different IgCAM genes. All mutants are viable and healthy. A detailed phenotypic analysis of these mutants is in progress.
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[
European Worm Meeting,
2002]
Signaling molecules and receptors important for axon guidance and fasciculation are likely to be found among extracellular and cell surface molecules. The complete sequence of the C. elegans genome allows the identification of all members of known receptor and adhesion families. One of the largest families of adhesion molecules with a role in nervous system development is the immunoglobulin-superfamily (IgCAM-SF). IgCAM family members can be grouped according to their modular organisation. 28 genes encode transmembrane or GPI-anchored proteins with extracellular IG modules. We are focusing on a core of 17 IgCAMs. Five of them consist of IG- modules only, the remaining twelve have one or more fibronectin III (F3) modules in addition to IG-modules. For 15 of them no function is known. To identify IgCAMs with a putative role in axonal outgrowth we started to analyze expression patterns for these genes. Transgenic animals were generated expressing promoter-GFP fusion constructs for individual IgCAMs. Expression typically is dynamic and not confined to a single tissue. Some IgCAMs (e.g. C26G2.1) show predominantly non-neuronal expression. Many others are expressed mainly in particular subsets of neurons (see also Supplementary Material to Aurelio O. et al. (2002); Science, 295: 686-690). Five IgCAMS (C53B7.1, F41D9.3, K02E10.8, K09E2.4, Y42H9B.2) are expressed in motorneurons or interneurons with axons in the ventral cord. We are currently isolating deletion alleles in these genes from our deletion library to begin a functional analysis.
-
[
International C. elegans Meeting,
2001]
Our aim is to understand the molecular basis of axon guidance and fasciculation. Signaling molecules and receptors important for axon guidance and fasciculation are likely to be found among extracellular and cell surface molecules. As a first step to identify novel axon guidance genes the C. elegans genome was screened for putative extracellular and cell surface proteins, using representative insect or mammalian proteins or their fragments resulting in a list of several hundred genes. Among them were homologs of known extracellular matrix (ECM) and cell adhesion (CAM) molecules as well as a number of novel proteins forming new families. As a next step to identify axon guidance genes the function for every predicted gene on the list will be investigated by generating transient knockouts using RNAi. In a first approach the quick procedure of direct injection of dsRNAi was performed. The results after dsRNA injection of seven already known axon guidance genes (
unc-5,
unc-6,
unc-40,
unc-44,
unc-51,
unc-129 and
sax-3 ) revealed that direct injection of dsRNA does not work reliably with genes involved in neuronal development. Nevertheless about hundred genes among them members of several CAM families including all IgCAMs and cadherins were injected as dsRNA but the previous results were confirmed as no dramatic effects in the nervous system nor in other tissues could be detected. Therefore we will test different ways of dsRNA introduction into worms to find out the most efficient one. Currently we investigate two methods of RNAi, that have already been successfully used by other labs: feeding worms with dsRNA expressing bacteria and transgenic worms which express dsRNA themselves. To reduce cloning steps we are constructing a vector which can be used for feeding and at the same time for making transgenic worms. The gene of interest is flanked on both sides by a T7 promoter and regulated by the C. elegans
hsp-16 promoter. The T7 promoter gives rise to dsRNA production in the feeding bacteria. If feeding is not efficient the
hsp-16 promoter allows us to induce ectopic transcription of the target gene in transgenic worms. As axon development takes place in a time window of only a few hours during the second have of embryogenesis the
hsp16 promoter enables us to inactivate a gene in a specific developmental stage so that additional phenotypes due to further functions of the investigated genes are minimized. Genes of interest can be inserted into the vector as PCR fragments in sense and antisense orientation via TA-cloning.Transgenic worms carrying the vector produce after heat shock both single stranded RNAs, which anneal to dsRNA causing transient knock out.
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[
Virulence,
2012]
Community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) strains have emerged as major human pathogens. CA-MRSA virulence appears to be distinct from healthcare-associated (HA) MRSA with several factors [-hemolysin (Hla), Panton-Valentine leukocidin (PVL), -type phenol soluble modulins (PSM) and SCCmec IV] postulated to enhance virulence or fitness. Using the Caenorhabditis elegans infection model, we compared the virulence of clinical and laboratory isolates of CA-MRSA and HA-MRSA and explored the contribution of CA-MRSA associated virulence factors to nematode killing. All CA-MRSA strains were highly pathogenic to nematodes, while HA-MRSA strains demonstrated variable nematode killing. Nematode killing by isogenic mutants of hla or the loci for PVL, PSM, PSM, PSM or SCCmec IV was not different than the parental strains. These results demonstrate that CA-MRSA is highly virulent, shows some strains of HA-MRSA are equally virulent toward nematodes and suggests CA-MRSA virulence in C. elegans is not linked to a single virulence factor.
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[
Mol Ther Nucleic Acids,
2016]
Staphylococcus aureus infections present a serious challenge to healthcare practitioners due to the emergence of resistance to numerous conventional antibiotics. Due to their unique mode of action, peptide nucleic acids are novel alternatives to traditional antibiotics to tackle the issue of bacterial multidrug resistance. In this study, we designed a peptide nucleic acid covalently conjugated to the HIV-TAT cell penetrating peptide (GRKKKRRQRRRYK) in order to target the RNA polymerase subunit gene (rpoA) required for bacterial genes transcription. We explored the antimicrobial activity of the anti-rpoA construct (peptide nucleic acid-TAT) against methicillin-resistant S. aureus, vancomycin-intermediate S. aureus, vancomycin-resistant S. aureus, linezolid-resistant S. aureus, and methicillin-resistant S. epidermidis in pure culture, infected mammalian cell culture, and in an in vivo Caenorhabditis elegans infection model. The anti-rpoA construct led to a concentration-dependent inhibition of bacterial growth (at micromolar concentrations) in vitro and in both infected cell culture and in vivo in C. elegans. Moreover, rpoA gene silencing resulted in suppression of its message as well as reduced expression of two important methicillin-resistant S. aureus USA300 toxins (-hemolysin and Panton-Valentine leukocidin). This study confirms that rpoA gene is a potential target for development of novel antisense therapeutics to treat infections caused by methicillin-resistant S. aureus.
-
[
Eur J Clin Microbiol Infect Dis,
2013]
Methicillin-resistant Staphylococcus aureus (MRSA) strains from different geographic areas have different genetic backgrounds, suggesting independent clonal evolutions. To better understand the virulence of MRSA strains and the relationship to their clonal and geographic origins, we undertook an analysis of epidemiologic, molecular, and virulence characteristics of a large number of MRSA isolates from geographically diverse origins, in a Caenorhabditis elegans infection model. A total of 99 MRSA isolates collected between 1993 and 2010 at the Geneva University Hospitals from diverse global origins were characterized with Panton-Valentine leukocidin (PVL), toxic shock syndrome toxin (TSST), accessory gene regulator (agr) group, staphylococcal cassette chromosome mec (SCCmec), S. aureus protein A (spa), multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE) typing. Epidemiologic data were provided from clinical records. The bacterial virulence was tested in a C. elegans host model. The inter-relationships of epidemiological/molecular characteristics in association with nematocidal activities were analyzed with univariate and two-factor analysis of variance (ANOVA). Community-associated MRSA (CA-MRSA) strains were more virulent than hospital-associated MRSA (HA-MRSA), with higher nematocidal activities in CA-MRSA strains (0.776 vs. 0.506, p = 0.0005). All molecular characteristics (PVL, TSST, spa, SCCmec, MLST, and PFGE types) showed a significant association with nematocidal activities on univariate analysis (p < 0.005). PVL was not a significant predictor after adjusting for genomic backgrounds using spa, MLST, or PFGE typing. The dominant CA-MRSA strains in North America showed higher nematocidal activities than strains from other regions (p < 0.0001). Strains with global origins containing distinct genetic backgrounds have different virulence in the C. elegans model. Nematocidal activities were most highly correlated with SCCmec, spa, MLST, and PFGE typing, suggesting that genomic background rather than a single exotoxin characteristic was the most discriminating predictor of virulence.
-
[
Parasitol Res,
2012]
The need for new anthelmintic with no chemical residues is becoming urgent. In a program aiming at the evaluation of plant as sources of new active molecules, the anthelmintic activities of the essential oils (EOs) obtained from either Zanthoxylum zanthoxyloides seeds or Newbouldia laevis leaves were evaluated against Strongyloides ratti by analyzing the results of two in vitro bioassays. These two plants and their tested parts were retained after an ethnopharmacology survey that confirmed their use by small-scale farmers for treatment of small ruminants affected by digestive helminths. The plants were harvested in Benin, and their EO were obtained by hydrodistillation. The EO yield of extraction was 0.65% (w/w) of for Z. zanthoxyloides seeds and 0.05% (w/w) for N. laevis. The chemical compositions of the two EOs were analyzed by gas chromatography coupled with mass spectrometry. The major constituents of the EO from Z. zanthoxyloides consisted of the following compounds: -terpinene (18 %), undecane (15 %), valencene (8.3 %), decanal (8.3 %), and 3-carene (6.7 %). In contrast, the major constituents of the EO from N. laevis leaves consisted of the following compounds: -caryophyllene (36 %) and eugenol (5.8 %). An egg-hatching inhibition (EHI) assay was developed and a larval migration inhibition assay was used on S. ratti to examine the effects of the EOs and to evidence their inhibitory concentrations (IC(50) and IC(90)) values on this nematode. Furthermore, the toxicity of the two EOs on Vero cell line was evaluated. When tested on S. ratti egg hatching, the two EOs resulted in similar IC(50) values (19.5 and 18.2 g/ml for Z. zanthoxyloides and N. laevis, respectively), which were about sevenfold higher than that of the control (thiabendazole, IC(50)=2.5 g/ml). Larval migration was inhibited at similar concentrations for: Z. zanthoxyloides (IC(50)=46 g/ml), N. laevis (IC(50)=51 g/ml), and the control [levamisole (IC(50)=36 g/ml)]. No cytotoxicity was found on Vero cells because both EOs had IC(50) values higher than 50 g/ml. Therefore, we have concluded that the EOs from two plants, used in folk medicine, may contain compounds with anthelmintic activity and could be used as improved traditional medicines or, at least, as food additives in a combined treatment for the control of helminth infections.
-
Pennington PR, Heistad RM, Nyarko JNK, Barnes JR, Bolanos MAC, Parsons MP, Knudsen KJ, De Carvalho CE, Leary SC, Mousseau DD, Buttigieg J, Maley JM, Quartey MO
[
Sci Rep,
2021]
The pool of -Amyloid (A) length variants detected in preclinical and clinical Alzheimer disease (AD) samples suggests a diversity of roles for A peptides. We examined how a naturally occurring variant, e.g. A(1-38), interacts with the AD-related variant, A(1-42), and the predominant physiological variant, A(1-40). Atomic force microscopy, Thioflavin T fluorescence, circular dichroism, dynamic light scattering, and surface plasmon resonance reveal that A(1-38) interacts differently with A(1-40) and A(1-42) and, in general, A(1-38) interferes with the conversion of A(1-42) to a -sheet-rich aggregate. Functionally, A(1-38) reverses the negative impact of A(1-42) on long-term potentiation in acute hippocampal slices and on membrane conductance in primary neurons, and mitigates an A(1-42) phenotype in Caenorhabditis elegans. A(1-38) also reverses any loss of MTT conversion induced by A(1-40) and A(1-42) in HT-22 hippocampal neurons and APOE 4-positive human fibroblasts, although the combination of A(1-38) and A(1-42) inhibits MTT conversion in APOE 4-negative fibroblasts. A greater ratio of soluble A(1-42)/A(1-38) [and A(1-42)/A(1-40)] in autopsied brain extracts correlates with an earlier age-at-death in males (but not females) with a diagnosis of AD. These results suggest that A(1-38) is capable of physically counteracting, potentially in a sex-dependent manner, the neuropathological effects of the AD-relevant A(1-42).
-
[
Worm Breeder's Gazette,
2003]
Wormgenes is a new resource for C.elegans offering a detailed summary about each gene and a powerful query system.
-
[
Front Pharmacol,
2020]
Oligomeric assembly of Amyloid- (A) is the main toxic species that contribute to early cognitive impairment in Alzheimer's patients. Therefore, drugs that reduce the formation of A oligomers could halt the disease progression. In this study, by using transgenic <i>Caenorhabditis elegans</i> model of Alzheimer's disease, we investigated the effects of frondoside A, a well-known sea cucumber <i>Cucumaria frondosa</i> saponin with anti-cancer activity, on A aggregation and proteotoxicity. The results showed that frondoside A at a low concentration of 1 M significantly delayed the worm paralysis caused by A aggregation as compared with control group. In addition, the number of A plaque deposits in transgenic worm tissues was significantly decreased. Frondoside A was more effective in these activities than ginsenoside-Rg3, a comparable ginseng saponin. Immunoblot analysis revealed that the level of small oligomers as well as various high molecular weights of A species in the transgenic <i>C. elegans</i> were significantly reduced upon treatment with frondoside A, whereas the level of A monomers was not altered. This suggested that frondoside A may primarily reduce the level of small oligomeric forms, the most toxic species of A. Frondoside A also protected the worms from oxidative stress and rescued chemotaxis dysfunction in a transgenic strain whose neurons express A. Taken together, these data suggested that low dose of frondoside A could protect against A-induced toxicity by primarily suppressing the formation of A oligomers. Thus, the molecular mechanism of how frondoside A exerts its anti-A aggregation should be studied and elucidated in the future.