Project description:In mammalian systems, extracellular small RNAs can operate in a paracrine manner to communicate information between cells, relying on transport within vesicles. “Foreign” small RNAs derived from bacteria, plants and parasites have also been detected in mammalian body fluids, sparking interest in whether these could mediate inter-species communication. However, there is no mechanistic framework for RNA-mediated interspecies communication and the active movement of RNA via vesicles has not been shown outside of mammals. Here we demonstrate that specific microRNAs and Y RNAs are packaged into vesicles secreted by a gastrointestinal nematode, Heligmosomoides polygyrus, which naturally infects mice. Total RNA was extracted from the secretion product of adult worms and compared to the profile of small RNAs in adult worms, eggs and infective larvae.
Project description:In mammalian systems, extracellular small RNAs can operate in a paracrine manner to communicate information between cells, relying on transport within vesicles. “Foreign” small RNAs derived from bacteria, plants and parasites have also been detected in mammalian body fluids, sparking interest in whether these could mediate inter-species communication. However, there is no mechanistic framework for RNA-mediated interspecies communication and the active movement of RNA via vesicles has not been shown outside of mammals. Here we demonstrate that specific microRNAs and Y RNAs are packaged into vesicles secreted by a gastrointestinal nematode, Heligmosomoides polygyrus, which naturally infects mice. Total RNA was extracted from the serum of mice infected with Litomosoides sigmodontis at 60 days post infection
Project description:We describe the first comprehensive study confirming the existence of DNA methylation, characterising the methylomes of three life stages of the food-borne agent of human trichinellosis, Trichinella spiralis. We further identify sets of genes where the DNA methylation status varied between thedevelopmental stages that are closely related to the parasitism of the organism. Examination of DNA methylation status in three life stages (Adult, muscle larve, new born larve) of Trchinella Spiralis using MethylC-seq.
Project description:Expression profiling of 7,530 Heterodera glycines probesets present on the Affymetrix Soybean Genome Array GeneChip throughout the life cycle of the nematode (egg, infective J2, parasitic J2, J3, J4, adult female).
Project description:Background; Heterodera schachtii is an economically important plant parasitic nematode that forms a syncytium from a cell superficial to the formed vascular bundle by progressive recruitment of other cells into the structure. The pattern of plant gene expression changes dramatically inside the syncytium. The pathogen probably plays a major role in defining the plant response by choice of initial plant cell during precise behaviour in planta and/or by the secretions it releases. The modified plant cells enable a high feeding rate by the female nematode so enhancing its rate of development and subsequent daily egg production. Arabidopsis is widely used as a model plant to characterise molecular responses to nematodes (e.g. Sijmons et al., 1991 Plant J. 1:245-254.). A complete overview of the changes in plant gene expression when sedentary nematodes establish has not yet been gained using Arabidopsis or any other host plant. Experimental Approaches; Our initial studies will focus on the H. schachtii/Arabidopsis interaction. To assure reliable microarray screening care has been taken to minimise extraneous differences between samples (see "Growth conditions" section). At 21 days (Growth stage 3.2-3.5 Boyes et al., 2001 Plant Cell 13:1499-1510) Arabidopsis plants were challenged with rigorously sterilised, infective nematodes of H. schachtii as before (Urwin et al., (1997) Plant Journal 12: 455-461.). 35 sterile J2s were pipetted onto small ~0.5mm2 squares of sterile GF/A filter paper. The GF/A paper was left in direct contact with the zone of elongation on 3 lateral roots per plant for 48 hours. Control plants were mock inoculated with sterile water. Sections of root containing syncytia have been excised from the thin and transparent roots of Arabidopsis and collected into RNAlater solution (Ambion) at 21 days post infection (Growth Stage 6.1 Boyes et al. 2001). The female nematode has been removed with watch-maker's forceps. Equivalent sections of root have been harvested from non-infected plants. Material has been collected from c. 1000 plants for each of the two samples and the uninfected material serves as an internal control. Total RNA has been prepared from the reference and test root material using an RNeasy plant RNA preparation kit (Qiagen) according to methods required by GARNET.Some questions on the form are omitted as we are not using mutant or transgenic lines. This is our first application. Experimenter name = Peter Edward Urwin; Experimenter phone = 0113 343 3035/2909; Experimenter fax = 0113 343 3144; Experimenter address = Centre for Plant Science; Experimenter address = University of Leeds; Experimenter address = Leeds; Experimenter zip/postal_code = LS2 9JT; Experimenter country = UK Experiment Overall Design: 2 samples were used in this experiment
Project description:Despite the clear role of adult secreted and tegumental proteins as well as egg proteins in host-parasite interactions, there has not been any in-depth proteomic analysis of these or other Schistosoma haematobium proteomes. In the current project we have carried out the first comprehensive proteomic analysis of S. haematobium. The characterisation of the molecules playing a key role at the interphase between the host and the parasite is crucial for (i) a better understanding of the parasite’s biology and, (ii) for the development of new control and diagnostic approaches to tackle parasitic infections.
Project description:In nature, animals often face feast or famine conditions. We aimed to identify the miRNAs of Caenorhabditis elegans that changed their expression under starvation conditions in stage L4 larvae. Our results highlight 14 miRNAs that show differential expression in starved versus well-fed larvae. In particular, miRNAs of the miR-35-3p/miR-41-3p family were upregulated 6-20 fold upon starvation. We verified the upregulation of miR-35-3p with qPCR. Additionally, we showed that the expression of gld-1, important in ovogenesis, and a validated target of miR-35-3p, was downregulated when the expression of miR-35-3p was higher. This study represents a starting point for a more comprehensive understanding of the role of miRNAs during starvation in C. elegans. Illumina small RNA sequencing of starved and well-fed L4 worms.
Project description:Effective silencing by RNA-interference (RNAi) depends on mechanisms that amplify and propagate the silencing signal. In some organisms, small-interfering (si) RNAs are amplified from target mRNAs by RNA-dependent RNA polymerase (RdRP). Both RdRP recruitment and mRNA silencing require Argonaute proteins, which are generally thought to degrade RNAi targets by directly cleaving them. However in C. elegans, the enzymatic activity of the primary Argonaute, RDE-1, is not required for silencing activity. We show that RDE-1 can instead recruit an endoribonuclease, RDE-8, to target RNA. RDE-8 can cleave RNA in vitro and is needed for the production of 3′ uridylated fragments of target mRNA in vivo. We also find that RDE-8 promotes RdRP activity, thereby ensuring amplification of siRNAs. Together, our findings suggest a model in which RDE-8 cleaves target mRNAs to mediate silencing, while generating 3’ uridylated mRNA fragments to serve as templates for the RdRP-directed amplification of the silencing signal. We examined the role of rde-8 in C. elegans small RNA biogenesis pathways, including endogenous and exogenous RNAi pathways. We performed 3' RACE seq from the sel-1 target mRNA and correlate with small RNAs from wild type, rde-8 and rde-8 transgenic strains after sel-1(RNAi) for different lengths of time.
Project description:Trichinellosis of human and other mammals was caused through the ingestion of the parasite，Trichinella spiralis，contaminated meat. It is a typical zoonotic disease that affects more than 10 million people world-wide. Parasites of Trichinella genus are unique intracellular pathogens. Adult Trichinella parasites directly release newborn larvae which invade striated muscle cells and causes diseases. In this study, we profiled the global transcriptome in the three developmental stages of T. spiralis. The transcriptomic analysis revealed the global gene expression patterns from newborn larval stage through muscle larval stage to adults. Thousands of genes with stage-specific transcriptional patterns were described and novel genes involving host-parasite interaction were identified. More than 45% of the protein-coding genes showed evidence of transcription from both sense and antisense strands which suggests the importance of RNA-mediated gene regulation in the parasite. This study presents a first deep analysis of the transcriptome of T. spiralis, providing insight information of the parasite biology. Messenger RNA from three developmental stages of T. spiralis was selectively purified from total RNA using oligo-(dT) conjugated magnetic beads. Complementary DNA (cDNA) was synthesized guided by oligo-(dT) as a primer.
Project description:Gastrointestinal nematode (GIN) is a major economic and health concern is sheep farming. Sheep breeds such as Texel are relatively resistant to GIN than the Suffolk. With the objective to understand the underlying genetic mechanism of resistance and susceptibility at the transcriptomic level, two groups of animal from both the breed were artificially (orally) infected with 30,000 L3 larvae of prominent GIN Teladorsagia circumcincta. Subgroups of animals from each breed were slaughtered on day 0, 3, 7, 14 and 21 of post infection (p.i.). Transcriptomic profiling of abomasal lymph node was performed using RNA-seq. The perturbations in gene expression profiles in both the breeds were evident and Texel showed a more tightly regulated immune response than the Suffolk. The number of differentially expressed (DE) genes between the breeds was highest (437) on un-infected control (day 0) and lowest (173) on day 7 p.i.. Pathway analysis of DE genes identified 3 significant pathways, which involved only more highly expressed genes of Suffolk breed on day 0 and only more highly expressed genes of Texel (with one exception) on day 7 p.i.. The Th1, Th2 and Treg response was evident in response to GIN in Texel and was synchronized, while in Suffolk Th1 response was reduced after infection and pronounced Th2 and Treg was not evident. The study suggests maximum level of transcriptional activity in both breeds on day 7 p.i. and there was a shift of transcriptional activity from Suffolk on day 0 to Texel on day 7 p.i.. Suffolk had a reduced Th1 response with less pronounced Th2 and Treg immune response, while Texel had an active and synchronized Th1/Th2/Treg immune response in response to GIN infection. Abomasal lymph node tissue was taken from control (n=10) and experimentally infected (with T. circumcincta) lambs (n=36) from Texel and Suffolk breed on day 0, 3, 7, 14 and 21 post infection.