Adult stem cells in the human parasite Schistosoma mansoni
ABSTRACT: RNAseq experiments to identify gene expression changes following gamma irradiation Adult male Schistosoma mansoni (strain: NMRI) were subjected to 200 Gy of gamma irradiation and cultured in vitro for 48hr hours. After 48hr germ line tissues were removed and RNA was extracted from remaining somatic tissues. Samples were processed for RNAseq using Illumina procedures.
Project description:Schistosomes infect more than 200 million of the world's poorest people. These parasites live in the vasculature, producing eggs that spur a variety of chronic, potentially life-threatening, pathologies exacerbated by the long lifespan of schistosomes, that can thrive in the host for decades. How schistosomes maintain their longevity in this immunologically hostile environment is unknown. Here, we demonstrate that somatic stem cells in Schistosoma mansoni are biased towards generating a population of cells expressing factors associated exclusively with the schistosome host-parasite interface, a structure called the tegument. We show cells expressing these tegumental factors are short-lived and rapidly turned over. We suggest that stem cell-driven renewal of this tegumental lineage represents an important strategy for parasite survival in the context of the host vasculature.
Project description:Schistosoma mansoni is a dioecious species, that is, it has two differentiated sexes. Interestingly, this sexual species evolved from a hermaphrodite ancestor. Indeed, most Platyhelminthes are hermaphrodites. Here we characterize the microRNAs of S. mansoni and quantify their differential expression between males and females. Mice were infected with Schistosoma mansoni 1-2 weeks prior to dissection. RNA from two independent samples were extracted and sequenced with Illumina MiSeq technology and AB SOLiD 4 technology. Reads were mapped to the reference genome and microRNA detected and analyzed.
Project description:We report on the small RNA profiles of Schistosoma japonicum (S. japonicum) miRNAs using small RNA deep sequencing in the key stages of male-female pairing, gametogenesis, and egg production. Examination of different miRNAs between males and females in Schistosoma japonicum
Project description:The bystander effect from ionizing radiation consists of cellular responses generated from unirradiated cells to the irradiation of their neighbors. The bystander effect can lead to DNA damage and genomic instability in the affected cells. This non-targeted effect of radiation has received attention due to its potential implications for cancer therapy and radiation protection. Although studied extensively, a complete understanding of its molecular mechanism is the subject of ongoing research. While many studies have targeted specific factors which are suggested to be involved in the bystander effect, few have looked at whole genome gene expression in bystander cells. Furthermore, even fewer studies have looked at the expression in noncancerous human cell lines. In this study we have used a genome-wide microarray approach to investigate transcriptional responses in irradiated and bystander immortalized human fibroblasts following 0.1 Gy α-particle irradiation. Total RNA was isolated from F11hTERT fibroblasts irradiated with 0.1 Gy α-particles and bystander fibroblasts receiving medium from control (sham irradiated) and irradiated cells (0.1 Gy). RNA was isolated 4, 8 and 26 h after irradiation.
Project description:The bystander effect from ionizing radiation consists of cellular responses generated from non-irradiated cells to the irradiation of their neighbors. The bystander effect is predominant at low doses and can lead to DNA damage and genomic instability in the affected cells. This non-targeted effect of radiation has received attention due to its potential implications for cancer therapy and radiation protection. Although studied extensively, a complete understanding of its molecular mechanism is the subject of ongoing research. While many studies have targeted specific factors which are suggested to be involved in the bystander effect, few have looked at whole genome gene expression in bystander cells. Furthermore, even fewer studies have looked at the expression in normal human cell lines. In this study, we have monitored transcriptional responses to γ-radiation in irradiated and bystander normal fibroblasts simultaneously using a genome-wide microarray approach. Bystander fibroblasts incubated in medium from irradiated cells, showed transient enrichment (less than 1.5 fold) in ribosome and oxidative phosphorylation pathways, and neurodegenerative disease pathways associated with mitochondrial dysfunctions. Bystander fibroblasts did not, however, display increases in oxidative stress, a phenomenon often linked with the radiation induced bystander effect. Total RNA was isolated from normal human fibroblasts irradiated with 2.0 Gy and fibroblasts incubated with medium from sham irradiated and irradiated cells 2 h after irradiation. RNA was isolated 4, 8 and 26 h after irradiation and there are 4 replicates for each sample for a total of 36 samples.
Project description:This study establishes a baseline pattern for RNA expression among canonical strains of Toxoplasma gondii grown in tissue culture without perturbation. Parasites cultured within human foreskin fibroblast (HFF) cells grown with D10 media were scraped and harvested ~8-12 hours prior to host cell lysis. RNA was isolated and applied to a T. gondii Affymetrix array.
Project description:Leishmania are unicellular eukaryotes responsible for leishmaniasis in humans. Like other trypanosomatids, leishmania regulate protein coding gene expression almost exclusively at the post-transcriptional level with the help of RNA binding proteins (RBPs). Due to the presence of polycystronic transcription units, leishmania do not regulate RNA polymerase II-dependent transcription initiation. Recent evidence suggests that the main control points in gene expression are mRNA degradation and translation. Protein-RNA interactions are involved in every aspect of RNA biology, such as mRNA splicing, polyadenylation, localization, degradation, and translation. A detailed picture of these interactions would likely prove to be highly informative in understanding leishmania biology and virulence. We developed a strategy involving covalent UV cross-linking of RBPs to mRNA in vivo, followed by interactome capture using oligo(dT) magnetic beads to define comprehensively the mRNA interactome of growing L. donovani amastigotes. The protein mass spectrometry analysis of captured proteins identified 81 mRNA interacting proteins which withstood very stringent washing conditions. Strikingly, we found that 49 of these mRNA interacting proteins had no orthologs or homologs in the human genome. Consequently, these may represent high quality candidates for selective drug targeting leading to novel therapeutics. These results show that this unbiased, systematic strategy has the promise to be applicable to study the mRNA interactome during various biological settings such as metabolic changes, stress (low pH environment, oxidative stress and nutrient deprivation) or drug treatment.
Project description:RNA-binding proteins (RBPs) determine RNA fate from synthesis to decay. Employing two complementary protocols for covalent UV crosslinking of RBPs to RNA, we describe a systematic, unbiased, and comprehensive approach, termed "interactome capture," to define the mRNA interactome of proliferating human HeLa cells. We identify 860 proteins that qualify as RBPs by biochemical and statistical criteria, adding more than 300 RBPs to those previously known and shedding light on RBPs in disease, RNA-binding enzymes of intermediary metabolism, RNA-binding kinases, and RNA-binding architectures. Unexpectedly, we find that many proteins of the HeLa mRNA interactome are highly intrinsically disordered and enriched in short repetitive amino acid motifs. Interactome capture is broadly applicable to study mRNA interactome composition and dynamics in varied biological settings.
Project description:Primary term human trophoblasts were derived from placentas after a healthy pregnancy, and exposed to ionizing irradiation (vs sham) in vitro Primary human trophoblasts were irradiated 24 h after initial plating, defined as time zero. Cells were irradiated at 10 Gy using a Clinac 600C (Varian Medical Systems, Palo Alto, CA) with a 6 MV photon beam and a dose rate of 250 cGy/min. The flasks containing the cells were placed on 1.5 cm of bolus (a tissue equivalent material) since the maximum irradiation depth was 1.5 cm, which corresponded to the plated cell layer. Cells were analyzed 4, 8, and 24 h after irradiation or sham.