Brady, S.C. et al. (2017) International Worm Meeting "Identification of the gene underlying variation in Caenorhabditis elegans bleomycin sensitivity."

Zdraljevic, S., Andersen, E.C., Tanny, R.E., Cook, D.E., Brady, S.C.

[International Worm Meeting, 2017]

Individual cancer patients exhibit differential responses to anti-neoplastic treatments. In many cases, a portion of this variability is attributable to genetic factors that impact drug responses. Bleomycin is an effective anti-tumor antibiotic that causes double-stranded DNA breaks and subsequent cytotoxicity. However, patients with increased bleomycin sensitivity experience potentially lethal pulmonary toxicity and other less severe side effects. Although bleomycin sensitivity is heritable, genetic variants that underlie differences in bleomycin response have not been discovered yet. By leveraging genetic diversity in Caenorhabditis elegans and an established high-throughput fitness assay, we identified a quantitative trait locus (QTL) that is highly correlated with bleomycin response. Within the confidence interval of this QTL, we identified a single candidate gene that contains one non-synonymous variant between sensitive and resistant strains. We will present evidence from CRISPR/Cas9-generated loss-of-function alleles and reciprocal allele replacements to causally connect this gene to the differences in bleomycin sensitivity.

Brady SC et al. (2019) Genetics "A Novel Gene Underlies Bleomycin-Response Variation in Caenorhabditis elegans."

Zdraljevic S, Tanny RE, Cook DE, Bisaga KW, Andersen EC, Lee D, Brady SC, Wang Y

[Genetics, 2019]

Bleomycin is a powerful chemotherapeutic drug used to treat a variety of cancers. However, individual patients vary in their responses to bleomycin. The identification of genetic differences that underlie this response variation could improve treatment outcomes by tailoring bleomycin dosages to each patient. We used the model organism <i>Caenorhabditis elegans</i> to identify genetic determinants of bleomycin-response differences by performing linkage mapping on recombinants derived from a cross between the laboratory strain (N2) and a wild strain (CB4856). This approach identified a small genomic region on chromosome V that underlies bleomycin-response variation. Using near-isogenic lines and strains with CRISPR-Cas9 mediated deletions and allele replacements, we discovered that a novel nematode-specific gene (<i>scb-1</i>) is required for bleomycin resistance. Although the mechanism by which this gene causes variation in bleomycin responses is unknown, we suggest that a rare variant present in the CB4856 strain might cause differences in the potential stress-response function of <i>scb-1</i> between the N2 and CB4856 strains, thereby leading to differences in bleomycin resistance.

Evans, K.S. et al. (2017) International Worm Meeting "Leveraging the power of Caenorhabditis elegans to dissect quantitative traits across a diverse collection of drug responses."

Andersen, E.C., Evans, K.S., Cook, D.E., Brady, S.C.

[International Worm Meeting, 2017]

Within natural populations, most phenotypes are determined by the contributions of multiple genetic loci and/or interactions with the environment. Additionally, genetic factors can act independently and/or interact to control trait differences across populations. Studies of yeast and Arabidopsis found that the majority of phenotypic variation is attributable to independent additive quantitative trait loci (QTL). By contrast, studies of Drosophila and humans suggest that most traits are controlled by interacting genetic loci. Here, we leverage the genetic power of a panel of 265 recombinant inbred advanced intercross lines (RIAILs) of Caenorhabditis elegans, derived from the Bristol strain (N2) and a wild isolate from Hawaii (CB4856), to reconcile this debate in a highly powered statistical study. For this panel of strains, we use a high-throughput fitness assay to analyze strain responses to 16 different conditions, including heavy metals, antineoplastic drugs, pesticides, and neuroactive compounds. Using linkage mapping, we identify a diverse set of genomic regions that underlie differences in responses to these compounds. We will present the relative contributions of additive QTL and higher-order genetic interactions across various growth parameters in responses to these 16 drugs. Additionally, we identified four genomic regions that impact responses to multiple classes of compounds. We generated chromosome-substitution strains (CSSs) and near-isogenic lines (NILs) to experimentally validate three of these "QTL hotspots". For many conditions, these strains recapitulate the phenotypic effect predicted using the recombinant inbred lines, validating the power of our efforts. Furthermore, the discovery of these QTL hotspots may be indicative of pleiotropic loci that control responses to multiple conditions.

Murthy PK et al. (1992) Folia Parasitol (Praha) "Brugia malayi in Mastomys natalensis: efficacy of mebendazole in combination with Freund's complete adjuvant."

Tyagi K, Chatterjee RK, Murthy PK

[Folia Parasitol (Praha), 1992]

The chemotherapeutic efficacy of mebendazole given in combination with Freund's complete adjuvant (FCA) against Brugia malayi in multimammate rat was evaluated. Animals treated with mebendazole, orally at 200 mg/kg x 5 consecutive days along with FCA given subcutaneously (s.c.) on day -10, day 0 and day +15 of the drug treatment killed 48.51% of the adult worms. This drug given alone at the same regimen and by the same route showed only 18.7% mortality rate on adults. Mebendazole given intraperitoneally along with FCA given s.c., however, was four times more efficacious as filaricide than mebendazole alone. Nevertheless, the animals receiving FCA alone also revealed 23.5% mortality rate of adult worms. The animals receiving a combination therapy or FCA alone showed significant increase in antibody titre to the filariae which however decreased in the later stages. No enhancement of antibody level could be detected in animals treated with mebendazole alone. The non-specific immunopotentiation induced by FCA appeared to play a major role in enhancing the activity of mebendazole.

Zdraljevic, S. et al. (2017) International Worm Meeting "Multiple interacting loci contribute to topoisomerase II poison responses in Caenorhabditis elegans."

Kim, T.W., Cook, D.E., Andersen, E.C., Brady, S.C., Kameny, L.P., Hippleheuser, S.W., Zdraljevic, S.

[International Worm Meeting, 2017]

Interacting genetic and environmental factors influence responses to anti-neoplastic treatments causing large differences in efficacy among individuals in the human population. Understanding the causes of these population-wide differences is a critical step towards the elucidation of markers that are predictive of patient responses in the clinic to personalize treatments and improve existing medications. In an attempt to disentangle this complexity, we used an unbiased linkage mapping approach in Caenorhabditis elegans to identify genetic determinants underlying response to a broadly administered class of anti-neoplastic compounds that poison the activity of topoisomerase II (TOP-2) enzymes. We identified numerous quantitative trait loci (QTL) that explain phenotypic variation among a panel of recombinant inbred advanced intercross lines generated between the Bristol and Hawaiian strains in response to amsacrine, dactinomycin, and etoposide. Interestingly, multiple overlapping QTL located on the center of chromosome V explain phenotypic variation in response to these three poisons, suggesting a common mechanism underlying the responses to these compounds. We constructed reciprocal Bristol and Hawaiian near-isogenic lines (NILs) that encompass all six chrV QTL. Interestingly, these NILs only recapitulated the etoposide and dactinomycin QTL effects, but failed to recapitulate the amsacrine QTL effects. We used these NILs to generate 80 recombinant NILs with smaller introgressed regions to separate a possible amsacrine-specific multi-locus interaction within the NIL region and to narrow the QTL confidence intervals associated with responses to all three drugs. Phenotypic analysis of these sub-interval NILs isolated the two etoposide and dactinomycin-specific QTL within the original NIL region and uncoupled an amsacrine-specific incompatibility locus from an amsacrine-specific QTL. These results highlight the complexity of genetic factors that influence metazoan responses to anti-neoplastic compounds.

Kawanabe M et al. (1988) Parasitol Res "Transmammary transmission of Strongyloides ratti."

Uchikawa R, Kawanabe M, Nojima H

[Parasitol Res, 1988]

The rate of transmammary transmission of Stronglyloides ratti was examined in albino rats in terms of the route of subcutaneous (s.c.) migration from the infection site (the skin) to the cranium. Inoculation sites nearer the cranium resulted in less frequent transmammary infection. The maximum number of adult worms was recovered from the sucklings when the mother was inoculated in her hindquarter and sucklings were allowed to feed for 30-36 h after inoculation (AI). Few worms were recovered from sucklings when they were allowed to nurse during periods of less than 24 h AI or greater than 42 h AI. In lactating mothers, larval infection of the mammary glands was commonly observed, and these larvae showed an increased esophagus length. In nonlactating mothers, most larvae completed their migration to the cranium within 36 h AI.

Sjoberg HT et al. (2019) PLoS Negl Trop Dis "Short-course, oral flubendazole does not mediate significant efficacy against Onchocerca adult male worms or Brugia microfilariae in murine infection models."

Engelen M, Taylor MJ, Pionnier N, Lachaud S, Tayong DB, Fombad FF, Chounna PWN, Turner JD, Quirynen L, Njouendou AJ, Wanji S, Steven A, Gandjui NVT, Ward SA, Chunda VC, Tekle F, Ndzeshang BL, Baeten B, Aljayyoussi G, Akumtoh DN, Sjoberg HT, Metuge HM

[PLoS Negl Trop Dis, 2019]

The Onchocerca ochengi adult implant and Brugia malayi microfilariemic Severe-Combined Immunodeficient (SCID) mouse models are validated screens to measure macrofilaricidal and microfilaricidal activities of candidate onchocerciasis drugs. The purpose of this study was to assess whether 5 daily sub-cutaneous (s.c.) injections of standard flubendazole (FBZ) suspension (10mg/kg), a single s.c. injection (10mg/kg) or 5 daily repeated oral doses of FBZ amorphous solid dispersion (ASD) formulation (0.2, 1.5 or 15mg/kg) mediated macrofilaricidal efficacy against O. ochengi male worms implanted into SCID mice. The direct microfilaricidal activity against circulating B. malayi microfilariae of single dose FBZ ASD formulation (2 or 40 mg/kg) was also evaluated and compared against the standard microfilaricide, ivermectin (IVM). Systemic exposures of FBZ/FBZ metabolites achieved following dosing were measured by pharmacokinetic (PK) bioanalysis. At necropsy, five weeks following start of FBZ SC injections, there were significant reductions in burdens of motile O. ochengi worms following multiple injections (93%) or single injection (82%). Further, significant proportions of mice dosed following multiple injections (5/6; 83%) or single injection (6/10; 60%) were infection negative (drug-cured). In comparison, no significant reduction in recovery of motile adult O. ochengi adult worms was obtained in any multiple-oral dosage group. Single oral-dosed FBZ did not mediate any significant microfilaricidal activity against circulating B. malayi mf at 2 or 7 days compared with &gt;80% efficacy of single dose IVM. In conclusion, multiple oral FBZ formulation doses, whilst achieving substantial bioavailability, do not emulate the efficacy delivered by the parenteral route in vivo against adult O. ochengi. PK analysis determined FBZ efficacy was related to sustained systemic drug levels rather than achievable Cmax. PK modelling predicted that oral FBZ would have to be given at low dose for up to 5 weeks in the mouse model to achieve a matching efficacious exposure profile.

Tan PL et al. (2007) Proc Natl Acad Sci U S A "Loss of Bardet Biedl syndrome proteins causes defects in peripheral sensory innervation and function."

Bradley BA, Watnick T, Garcia-Gonzalez MA, Albrecht PJ, Germino GG, Inglis PN, Beales PL, Rice FL, Katsanis N, Tan PL, Caterina MJ, Barr T, Huang SM, Leroux MR, Coforio S, Mitsuma N

[Proc Natl Acad Sci U S A, 2007]

Reception and interpretation of environmental stimuli is critical for the survival of all organisms. Here, we show that the ablation of BBS1 and BBS4, two genes mutated in Bardet-Biedl syndrome and that encode proteins that localize near the centrioles of sensory neurons, leads to alterations of s.c. sensory innervation and trafficking of the thermosensory channel TRPV1 and the mechanosensory channel STOML3, with concomitant defects in peripheral thermosensation and mechanosensation. The thermosensory phenotype is recapitulated in Caenorhabditis elegans, because BBS mutants manifest deficient thermosensory responses at both physiological and nociceptive temperatures and defective trafficking of OSM-9, a polymodal sensory channel protein and a functional homolog of TRPV1 or TRPV4. Our findings suggest a hitherto unrecognized, but essential, role for mammalian basal body proteins in the acquisition of mechano- and thermosensory stimuli and highlight potentially clinical features of ciliopathies in humans.

Joseph SK et al. (2011) Acta Trop "Sensitization with anti-inflammatory BmAFI of Brugia malayi allows L3 development in the hostile peritoneal cavity of Mastomys coucha."

Joseph SK, Murthy PK, Verma SK, Dixit S, Kushwaha V, Saxena JK, Sharma A, Verma AK, Sahoo MK, Saxena K

[Acta Trop, 2011]

Filarial parasites survive by inducing tolerance in host but the antigens and mechanisms involved are not clear. Recently we found that BmAFI, a Sephadex G-200 eluted fraction of Brugia malayi adult worm extract, stimulates IL-10 release from THP-1 cells. In the present study, we determined the SDS-PAGE profile of BmAFI and infective 3rd stage larva (L3), investigated the effect of pre-sensitization of host with BmAFI on the survival and development of L3 in the non-permissive peritoneal cavity (p.c.) of the permissive host Mastomys coucha and in the p.c. of non-permissive Swiss mice, and studied immunological correlates for the observed effects. The parasite development and burden in p.c., was determined in sensitized infected M. coucha and Swiss mice and the release of TGF-, IL-4, IL-10, IL-13, IFN- and NO, cellular proliferative response to Con A and BmAFI and levels of IgG subclasses and IgE were determined in sensitized infected M. coucha. Cellular proliferative response to Con A and BmAFI, mRNA expression of GATA-3, CTLA-4 and T-bet were determined in sensitized Swiss mice. In addition, the parasitological parameter was also studied in BmAFI-sensitized M. coucha exposed to the infection by standard subcutaneous (s.c.) route to assess whether sensitization enhances the intensity of infection. BmAFI-sensitization permitted survival of L3 and their development to adult stage by day 60 p.i. in the p.c. of M. coucha; in non-sensitized animals L3 could molt to L4 only and no parasite could be recovered beyond day 30 p.i. In M. coucha that received infection by s.c. route, pre-sensitization with BmAFI enhanced the microfilaraemia and adult worm recovery. In sensitized Swiss mice L3 could successfully molt to L4 in p.c. with improved recovery of parasite. BmAFI sensitization upregulated TGF- and IL-10 release, IgG1 and IgG2b levels, GATA-3 and CTLA-4 mRNA expression, suppressed the cellular proliferative response and downregulated Con A stimulated response, IgE, IL-13, IFN- and NO responses. Immunoblot analysis showed that the BmAFI antiserum also strongly reacts with some L3 molecules. The results show, for the first time, that sensitization with the anti-inflammatory BmAFI which shares some of its molecules with those in L3, facilitates parasite survival in the non-permissive p.c. of the permissive host M. coucha, render a non-permissive Swiss mouse partially permissive to infection and enhances parasite load in M. coucha receiving the infection through permissive s.c. route by evoking a modified Th2 type of response and anti-inflammatory milieu. In conclusion, the findings suggest that the anti-inflammatory BmAFI fraction facilitates survival of B. malayi infection even in non-permissive environment.

Shownkeen R et al. (1995) International C. elegans Meeting "THE CAENORHABDITIS ELEGANS PHYSICAL MAP"

Chissoe SL, Waterston RH, Fraser A, Sulston JE, Shownkeen R, Coulson AR

[International C. elegans Meeting, 1995]

Twelve contigs of cosmids and yeast artificial chromosomes (YACs) span more than 95Mb of the 100Mb C.elegans genome. 650 markers link the physical and genetic maps.Hybridisation of tag-sequenced cDNA clones to a map-representative set of YACs indicates that the map incorporates in excess of 99.8% of genes. The map is accessible in ACeDB. We (S.C.) are investigating the representation by bacterial artificial chromosomes (BACs) of regions of the genome not represented by cosmids. Two grids of YACs, of 958 clones ('Poly2') and 223 clones ('Suppoly') are available on request. The latter represents regions of the genome that have been characterised or better defined since the selection of clones for the former. Cosmid clones and YAC grids are available from the Sanger Centre (requests to alan@sanger.ac.uk; FAX 01223 494919). YAC clones and 'cm' series cDNA clones are available from the Sanger Centre or Washington University (rw@nematode. wustl.edu; FAX 314 362 2985).