The transcriptional response of Caenorhabditis elegans to ivermectin exposure identifies novel genes involved in the response to reduced food intake

We have examined the transcriptional response of Caenorhabditis elegans following exposure to the anthelmintic drug ivermectin (IVM) using whole genome microarrays and real-time QPCR. Our original aim was to identify candidate molecules involved in IVM metabolism and/or excretion. For this reason the IVM tolerant strain, DA1316, was used to minimise transcriptomic changes related to the phenotype of drug exposure. However, unlike equivalent work with benzimidazole drugs, very few of the induced genes were members of xenobiotic metabolising enzyme families. Instead, the transcriptional response was dominated by genes associated with fat mobilization and fatty acid metabolism including catalase, esterase, and fatty acid CoA synthetase genes. This is consistent with the reduction in pharyngeal pumping, and consequential reduction in food intake, upon exposure of DA1316 worms to IVM. Genes with the highest fold change in response to IVM exposure, cyp-37B1, mtl-1 and scl-2, were comparably up-regulated in response to short–term food withdrawal (4 hr) independent of IVM exposure, and GFP reporter constructs confirm their expression in tissues associated with fat storage (intestine and hypodermis). These experiments have serendipitously identified novel genes involved in an early response of C. elegans to reduced food intake and may provide insight into similar processes in higher organisms

Glutamate-Gated Chloride Channels of Haemonchus contortus Restore Drug Sensitivity to Ivermectin Resistant Caenorhabditis elegans

Anthelmintic resistance is a major problem in livestock farming, especially of small ruminants, but our understanding of it has been limited by the difficulty in carrying out functional genetic studies on parasitic nematodes. An important nematode infecting sheep and goats is Haemonchus contortus; in many parts of the world this species is resistant to almost all the currently available drugs, including ivermectin. It is extremely polymorphic and to date it has proved impossible to relate any sequence polymorphisms to its ivermectin resistance status. Expression of candidate drug-resistance genes in Caenorhabditis elegans could provide a convenient means to study the effects of polymorphisms found in resistant parasites, but may be complicated by differences between the gene families of target and model organisms. We tested this using the glutamate-gated chloride channel (GluCl) gene family, which forms the ivermectin drug target and are candidate resistance genes. We expressed GluCl subunits from C. elegans and H. contortus in a highly resistant triple mutant C. elegans strain (DA1316) under the control of the avr-14 promoter; expression of GFP behind this promoter recapitulated the pattern previously reported for avr-14. Expression of ivermectin-sensitive subunits from both species restored drug sensitivity to transgenic worms, though some quantitative differences were noted between lines. Expression of an ivermectin-insensitive subunit, Hco-GLC-2, had no effect on drug sensitivity. Expression of a previously uncharacterised parasite-specific subunit, Hco-GLC-6, caused the transgenic worms to become ivermectin sensitive, suggesting that this subunit also encodes a GluCl that responds to the drug. These results demonstrate that both orthologous and paralogous subunits from C. elegans and H. contortus are able to rescue the ivermectin sensitivity of mutant C. elegans, though some quantitative differences were observed between transgenic lines in some assays. C. elegans is a suitable system for studying parasitic nematode genes that may be involved in drug resistance

The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy

Abnormal uterine activity in pregnancy causes a range of important clinical disorders, including preterm birth, dysfunctional labour and post-partum haemorrhage. Uterine contractile patterns are controlled by the generation of complex electrical signals at the myometrial smooth muscle plasma membrane. To identify novel targets to treat conditions associated with uterine dysfunction, we undertook a genome-wide screen of potassium channels that are enriched in myometrial smooth muscle. Computational modelling identified Kir7.1 as potentially important in regulating uterine excitability during pregnancy. We demonstrate Kir7.1 current hyper-polarizes uterine myocytes and promotes quiescence during gestation. Labour is associated with a decline, but not loss, of Kir7.1 expression. Knockdown of Kir7.1 by lentiviral expression of miRNA was sufficient to increase uterine contractile force and duration significantly. Conversely, overexpression of Kir7.1 inhibited uterine contractility. Finally, we demonstrate that the Kir7.1 inhibitor VU590 as well as novel derivative compounds induces profound, long-lasting contractions in mouse and human myometrium; the activity of these inhibitors exceeds that of other uterotonic drugs. We conclude Kir7.1 regulates the transition from quiescence to contractions in the pregnant uterus and may be a target for therapies to control uterine contractility

Church/state relationships and Roman Catholic schools in Northern Ireland 1922-1996

In 2 vols.Available from British Library Document Supply Centre-DSC:DXN012638 / BLDSC - British Library Document Supply CentreSIGLEGBUnited Kingdo

An Ivermectin-Sensitive Glutamate-Gated Chloride Channel from the Parasitic Nematode Haemonchus contortus

Nematode glutamate-gated chloride channels are targets of the macrocyclic lactones, the most important group of anthelmintics available. In Xenopus laevis oocytes, channels formed by the GluClα3B subunit from the parasite Haemonchus contortus were more sensitive to l-glutamate (EC50 = 27.6 ± 2.7 μM) than those formed by the homologous subunit from Caenorhabditis elegans (EC50 = 2.2 ± 0.12 mM). Ibotenate was a partial agonist (EC50 = 87.7 ± 3.5 μM). The H. contortus channels responded to low concentrations of ivermectin (estimated EC50 =∼0.1 ± 1.0 nM), opening slowly and irreversibly in a highly cooperative manner: the rate of channel opening was concentration-dependent. Responses to glutamate and ivermectin were inhibited by picrotoxinin and fipronil. Mutating an N-terminal domain amino acid, leucine 256, to phenylalanine increased the EC50 for l-glutamate to 92.2 ± 3.5 μM, and reduced the Hill number from 1.89 ± 0.35 to 1.09 ± 0.16. It increased the Kd for radiolabeled ivermectin binding from 0.35 ± 0.1 to 2.26 ± 0.78 nM. Two other mutations (E114G and V235A) had no effect on l-glutamate activation or ivermectin binding: one (T300S) produced no detectable channel activity, but ivermectin binding was similar to wild-type. The substitution of any aromatic amino acid for Leu256 had similar effects in the radioligand binding assay. Molecular modeling studies suggested that the GluCl subunits have a fold similar to that of other Cys-loop ligand-gated ion channels and that amino acid 256 was unlikely to play a direct role in ligand binding but may be involved in mediating the allosteric properties of the receptor

Teladorsagia circumcincta: molecular characterisation of the avr-14B subunit and its relatively minor role in ivermectin resistance

Individual mutations (e.g. L256F) and polymorphisms in the avr-14B gene, a glutamate-gated chloride channel subunit, have been associated with ivermectin (IVM) resistance in Caenorhabditis elegans and Cooperia oncophora. The aim of the present study was to determine the full-length coding sequence of the avr-14B subunit homologue in Teladorsagia circumcincta and determine the presence/absence of the putative L256F SNP or any other potential SNPs of interest. Subsequently, we investigated sequence polymorphisms and transcription patterns between four different T. circumcincta isolates: two from Scotland (MTci1 susceptible and MTci5 triple resistant to benzimidazoles, levamisole and IVM) and two from Spain (S-Sp susceptible and R-Sp double resistant to levamisole and IVM). The complete amino acid sequence of the T. circumcincta avr-14B subunit comprises 438 amino acids. Pyrosequencing analysis failed to detect the presence of the L256F mutation in any of the MTci5 or Sp-R samples tested. However, we revealed significant allele frequency changes by means of SSCP analysis of a 106 bp region encompassing the L256F SNP. Allele E showed the greatest change, following IVM exposure in vitro and in vivo, although sequence analysis did not reveal any coding changes. Sequence analysis of the full-length avr-14B coding sequence showed that two SNPs exclusively found in the resistant strain McTi5 (I270F and T305A) are situated in codons involved in the interaction of the receptor with IVM. Moreover, other potentially significant SNPs (K361E and L364M) were identified between transmembrane regions 3 and 4. However, due to the low frequency of all these SNPs, we cannot conclude they confer IVM resistance in T. circumcincta. Moreover, a modest increase in expression of the avr-14B in both resistant isolates has been shown although these differences were not sufficiently great to consider avr-14B to be the sole or even a major determinant of IVM resistance in this species