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FREP3 is a determinant of resistance to schistosome infection in snails (part 2)

ABSTRACT: Transcriptional profile of BS-90 snails injected with a cocktail of four FREP3 specific 27-mer DSiRNA oligos and two hours later exposed to S. mansoni miricidia. Compared to BS-90 snails injected with a cocktail of three GFP specific DSiRNA oligos and two hours later exposed to S. mansoni miricidia. Experiments were done over the course of 49 days. Snails were collected (10each) at 2 and 4 dpe to S. mansoni for comparison.

ORGANISM(S): Biomphalaria glabrata

PROVIDER: GSE33525 | GEO | 2012/04/01

SECONDARY ACCESSION(S): PRJNA148911

REPOSITORIES: GEO

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Project description:STUDY THE EFFECT OF TGF beta ON SCHISTOSOME ADULT WORMSGoal: Primary: Assess the effects of TGF beta on transcription of adult male and female schistosomes. Secondary: Identify the pathways regulated by TGF beta in worm pairs, mature males, mature females, immature males and immature females.METHODS:Parasite: Schistosoma mansoni employed is the NMRI strain that is maintained in a Puerto Rican Biomphalaria glabrata and Golden Syrian hamsters. The life cycle is maintained in the LoVerde lab.Cercariae obtained from infected snails were used to infect experimental animals. The cercariae wre shed from snails exposed to multiple miracidia for mixed infections or from snails exposed to a single miracidia for single-sex infections. Schistosome worms were recovered from hamsters by perfusion (Duvall and DeWitt, 1967) 45 days post infection. Freshly perfused adult worms containing mixed-sex or single-sex infections were collected from infected golden hamsters, washed and incubated for overnight at 37C, 5% CO2 in MEM medium supplemented with 1 mM sodium pyruvate, 1 X non-essential amino acid solution, 1 X amino acid solution, 100 U ml penicillin, 100 ug ml streptomycin 1 ug ml amphotericin B (antibiotic antimycotic solution) and 2 mM Glutamax-I. Recombinant human TGF- beta1 was added individually to about 25 pairs or 30 individual schistosomes to a final concentrations of 1.0 nM and incubation was continued for another 24 hr. The controls were treated as above except no TGF-beta1 was added. The worms were placed in RNAlater and sent to WTSI (Anna) for RNA extraction and sequencing.Duvall RH, Dewitt WD. An improved perfusion technique for recovering adult schistosomes from laboratory animals. Am J Trop Med Hyg 1967;16:483-486.. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

Project description:Schistosoma mansoni is one of the most common etiological agents responsible for the disease schistosomiasis. More than 200 million people suffer from this disease making it the most severe tropical disease after malaria in terms of morbidity. Praziquantel (PZQ) is the treatment of choice for schistosomiasis and has been used almost exclusively to treat the disease since the 1970s. However, while the drug is lethal for sexually mature schistosomes, it is ineffective against juveniles. Thus, while morbidity can be eased, a cure is difficult to achieve. As a result there is an urgent need to develop a new generation of anti-schistosomal drugs, a task that will be made easier by understanding the mechanism of action of PZQ. As yet, neither the molecule to which PZQ binds nor the means by which it kills mature schistosomes is known. The overarching aim of this study was to understand the molecular basis of PZQ sensitivity in S. mansoni. We believe that juvenile worms survive PZQ treatment in vivo due to the induction of, as yet, unidentified protective molecular pathways. To address this hypothesis juvenile and adult PR1 S. mansoni were treated in vitro with sub-lethal concentrations of PZQ. mRNA was extracted from replicate samples, cRNA prepagreen and labeled with cyanine dyes for analysis using a 44K S. mansoni microarray. The data was then analyzed using Genespring. Our findings suggest that a number of genes associated with drug transport, iron homeostasis and apoptosis are induced in juvenile but not adult schistosomes and that this allows the juvenile worms to protect themselves against the lethal effects of PZQ long enough for the drug to be metabolized by the human host. 42 days post exposure (DPE) male schistosomes were exposed to 0 and 1 µg/mL praziquantel (PZQ) for 1 or 20 h and 10 µg/mL PZQ for 1 h. 42 DPE female schistosomes were exposed to 0, 1 and 10 µg/mL PZQ for 20 h. 28 DPE mixed-sex schistosomes were exposed to 0, 1 and 10 µg/mL PZQ for 1 and 20 h. All experiments were performed in biological triplicates. The 0 µg/mL PZQ exposures were used as controls to the appropriate groups and a universal reference was used in all experiments that was comprised of RNA derived from 100 mixed sex, unexposed 42 DPE schistosomes spiked with 0.5 % (v/v) RNA isolated from 42 DPE mixed sex worms exposed to 10 µg/mL PZQ, 0.5 % (v/v) RNA isolated from 28 DPE worms exposed to 10 µg/mL PZQ and 0.5 % (v/v) RNA isolated from 28 DPE worms unexposed to PZQ. Transcriptional response to praziquantel exposure by time and concentration

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FREP3 is a determinant of resistance to schistosome infection in snails (part 2)

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