Anti-schistosomal intervention targets identified by lifecycle transcriptomic analyses

BACKGROUND: Novel methods to identify anthelmintic drug and vaccine targets are urgently needed, especially for those parasite species currently being controlled by singular, often limited strategies. A clearer understanding of the transcriptional components underpinning helminth development will enable identification of exploitable molecules essential for successful parasite/host interactions. Towards this end, we present a combinatorial, bioinformatics-led approach, employing both statistical and network analyses of transcriptomic data, for identifying new immunoprophylactic and therapeutic lead targets to combat schistosomiasis. METHODOLOGY/PRINCIPAL FINDINGS: Utilisation of a Schistosoma mansoni oligonucleotide DNA microarray consisting of 37,632 elements enabled gene expression profiling from 15 distinct parasite lifecycle stages, spanning three unique ecological niches. Statistical approaches of data analysis revealed differential expression of 973 gene products that minimally describe the three major characteristics of schistosome development: asexual processes within intermediate snail hosts, sexual maturation within definitive vertebrate hosts and sexual dimorphism amongst adult male and female worms. Furthermore, we identified a group of 338 constitutively expressed schistosome gene products (including 41 transcripts sharing no sequence similarity outside the Platyhelminthes), which are likely to be essential for schistosome lifecycle progression. While highly informative, statistics-led bioinformatics mining of the transcriptional dataset has limitations, including the inability to identify higher order relationships between differentially expressed transcripts and lifecycle stages. Network analysis, coupled to Gene Ontology enrichment investigations, facilitated a re-examination of the dataset and identified 387 clusters (containing 12,132 gene products) displaying novel examples of developmentally regulated classes (including 294 schistosomula and/or adult transcripts with no known sequence similarity outside the Platyhelminthes), which were undetectable by the statistical comparisons. CONCLUSIONS/SIGNIFICANCE: Collectively, statistical and network-based exploratory analyses of transcriptomic datasets have led to a thorough characterisation of schistosome development. Information obtained from these experiments highlighted key transcriptional programs associated with lifecycle progression and identified numerous anti-schistosomal candidate molecules including G-protein coupled receptors, tetraspanins, Dyp-type peroxidases, fucosyltransferases, leishmanolysins and the netrin/netrin receptor complex

Excreted/secreted Schistosoma mansoni venom allergen-like 9 (SmVAL9) modulates host extracellular matrix remodelling gene expression

AbstractThe Schistosoma mansoni venom allergen-like (SmVAL) protein family consists of 29 members, each possessing a conserved α-β-α sandwich tertiary feature called the Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain. While the SmVALs have been found in both excretory/secretory (E/S) products and in intra/sub-tegumental (non-E/S) fractions, the role(s) of this family in host/parasite relationships or schistosome developmental processes remains poorly resolved. In order to begin quantifying SmVAL functional diversity or redundancy, dissecting the specific activity (ies) of individual family members is necessary. Towards this end, we present the characterisation of SmVAL9; a protein previously found enriched in both miracidia/sporocyst larval transformation proteins and in egg secretions. While our study confirms that SmVAL9 is indeed found in soluble egg products and miracidia/sporocyst larval transformation proteins, we find it to be maximally transcribed/translated in miracidia and subsequently down-regulated during in vitro sporocyst development. SmVAL9 localisation within sporocysts appears concentrated in parenchymal cells/vesicles as well as associated with larval germinal cells. Furthermore, we demonstrate that egg-derived SmVAL9 carries an N-linked glycan containing a schistosome-specific difucosyl element and is an immunogenic target during chronic murine schistosomiasis. Finally, we demonstrate that recombinant SmVAL9 affects the expression of extracellular matrix, remodelling matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase (TIMP) gene products in both Biomphalaria glabrata embryonic cell (BgMMP1) and Mus musculus bone marrow-derived macrophage (MmMMP2, MmMMP9, MmMMP12, MmMMP13, MmMMP14, MmMMP28, TIMP1 and TIMP2) in vitro cultures. These findings importantly suggest that excreted/secreted SmVAL9 participates in tissue reorganisation/extracellular matrix remodelling during intra-mammalian egg translocation, miracidia infection and intra-molluscan sporocyst development/migration

Duration of adjuvant chemotherapy for stage III colon cancer

BACKGROUND Since 2004, a regimen of 6 months of treatment with oxaliplatin plus a fluoropyrimidine has been standard adjuvant therapy in patients with stage III colon cancer. However, since oxaliplatin is associated with cumulative neurotoxicity, a shorter duration of therapy could spare toxic effects and health expenditures. METHODS We performed a prospective, preplanned, pooled analysis of six randomized, phase 3 trials that were conducted concurrently to evaluate the noninferiority of adjuvant therapy with either FOLFOX (fluorouracil, leucovorin, and oxaliplatin) or CAPOX (capecitabine and oxaliplatin) administered for 3 months, as compared with 6 months. The primary end point was the rate of disease-free survival at 3 years. Noninferiority of 3 months versus 6 months of therapy could be claimed if the upper limit of the two-sided 95% confidence interval of the hazard ratio did not exceed 1.12. RESULTS After 3263 events of disease recurrence or death had been reported in 12,834 patients, the noninferiority of 3 months of treatment versus 6 months was not confirmed in the overall study population (hazard ratio, 1.07; 95% confidence interval [CI], 1.00 to 1.15). Noninferiority of the shorter regimen was seen for CAPOX (hazard ratio, 0.95; 95% CI, 0.85 to 1.06) but not for FOLFOX (hazard ratio, 1.16; 95% CI, 1.06 to 1.26). In an exploratory analysis of the combined regimens, among the patients with T1, T2, or T3 and N1 cancers, 3 months of therapy was noninferior to 6 months, with a 3-year rate of disease-free survival of 83.1% and 83.3%, respectively (hazard ratio, 1.01; 95% CI, 0.90 to 1.12). Among patients with cancers that were classified as T4, N2, or both, the disease-free survival rate for a 6-month duration of therapy was superior to that for a 3-month duration (64.4% vs. 62.7%) for the combined treatments (hazard ratio, 1.12; 95% CI, 1.03 to 1.23; P=0.01 for superiority). CONCLUSIONS Among patients with stage III colon cancer receiving adjuvant therapy with FOLFOX or CAPOX, noninferiority of 3 months of therapy, as compared with 6 months, was not confirmed in the overall population. However, in patients treated with CAPOX, 3 months of therapy was as effective as 6 months, particularly in the lower-risk subgroup. (Funded by the National Cancer Institute and others.

Will all scientists working on snails and the diseases they transmit please stand up?

Copyright © 2012 Adema et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.No abstract available

Gene drives for schistosomiasis transmission control.

Schistosomiasis is one of the most important and widespread neglected tropical diseases (NTD), with over 200 million people infected in more than 70 countries; the disease has nearly 800 million people at risk in endemic areas. Although mass drug administration is a cost-effective approach to reduce occurrence, extent, and severity of the disease, it does not provide protection to subsequent reinfection. Interventions that target the parasites' intermediate snail hosts are a crucial part of the integrated strategy required to move toward disease elimination. The recent revolution in gene drive technology naturally leads to questions about whether gene drives could be used to efficiently spread schistosome resistance traits in a population of snails and whether gene drives have the potential to contribute to reduced disease transmission in the long run. Responsible implementation of gene drives will require solutions to complex challenges spanning multiple disciplines, from biology to policy. This Review Article presents collected perspectives from practitioners of global health, genome engineering, epidemiology, and snail/schistosome biology and outlines strategies for responsible gene drive technology development, impact measurements of gene drives for schistosomiasis control, and gene drive governance. Success in this arena is a function of many factors, including gene-editing specificity and efficiency, the level of resistance conferred by the gene drive, how fast gene drives may spread in a metapopulation over a complex landscape, ecological sustainability, social equity, and, ultimately, the reduction of infection prevalence in humans. With combined efforts from across the broad global health community, gene drives for schistosomiasis control could fortify our defenses against this devastating disease in the future

Role of the Endogenous Antioxidant System in the Protection of Schistosoma mansoni Primary Sporocysts against Exogenous Oxidative Stress

Antioxidants produced by the parasite Schistosoma mansoni are believed to be involved in the maintenance of cellular redox balance, thus contributing to larval survival in their intermediate snail host, Biomphalaria glabrata. Here, we focused on specific antioxidant enzymes, including glutathione-S-transferases 26 and 28 (GST26 and 28), glutathione peroxidase (GPx), peroxiredoxin 1 and 2 (Prx1 and 2) and Cu/Zn superoxide dismutase (SOD), known to be involved in cellular redox reactions, in an attempt to evaluate their endogenous antioxidant function in the early-developing primary sporocyst stage of S. mansoni. Previously we demonstrated a specific and consistent RNA interference (RNAi)-mediated knockdown of GST26 and 28, Prx1 and 2, and GPx transcripts, and an unexpected elevation of SOD transcripts in sporocysts treated with gene-specific double-stranded (ds)RNA. In the present followup study, in vitro transforming sporocysts were exposed to dsRNAs for GST26 and 28, combined Prx1/2, GPx, SOD or green-fluorescent protein (GFP, control) for 7 days in culture, followed by assessment of the effects of specific dsRNA treatments on protein levels using semi-quantitative Western blot analysis (GST26, Prx1/2 only), and larval susceptibility to exogenous oxidative stress in in vitro killing assays. Significant decreases (80% and 50%) in immunoreactive GST26 and Prx1/2, respectively, were observed in sporocysts treated with specific dsRNA, compared to control larvae treated with GFP dsRNA. Sporocysts cultured with dsRNAs for GST26, GST28, Prx1/2 and GPx, but not SOD dsRNA, were significantly increased in their susceptibility to H2O2 oxidative stress (60–80% mortalities at 48 hr) compared to GFP dsRNA controls (∼18% mortality). H2O2-mediated killing was abrogated by bovine catalase, further supporting a protective role for endogenous sporocyst antioxidants. Finally, in vitro killing of S. mansoni sporocysts by hemocytes of susceptible NMRI B. glabrata snails was increased in larvae treated with Prx1/2, GST26 and GST28 dsRNA, compared to those treated with GFP or SOD dsRNAs. Results of these experiments strongly support the hypothesis that endogenous expression and regulation of larval antioxidant enzymes serve a direct role in protection against external oxidative stress, including immune-mediated cytotoxic reactions. Moreover, these findings illustrate the efficacy of a RNAi-type approach in investigating gene function in larval schistosomes

Modern microwave methods in solid state inorganic materials chemistry: from fundamentals to manufacturing

No abstract available

Phenotypic Screen of Early-Developing Larvae of the Blood Fluke, Schistosoma mansoni, using RNA Interference

RNA interference (RNAi) represents the only method currently available for manipulating gene-specific expression in Schistosoma spp., although application of this technology as a functional genomic profiling tool has yet to be explored. In the present study 32 genes, including antioxidants, transcription factors, cell signaling molecules and metabolic enzymes, were selected to determine if gene knockdown by RNAi was associated with morphologically definable phenotypic changes in early intramolluscan larval development. Transcript selection was based on their high expression in in vitro cultured S. mansoni primary sporocysts and/or their potential involvement in developmental processes. Miracidia were allowed to transform to sporocysts in the presence of synthesized double-stranded RNAs (dsRNAs) and cultivated for 7 days, during which time developing larvae were closely observed for phenotypic changes including failure/delay in transformation, loss of motility, altered growth and death. Of the phenotypes evaluated, only one was consistently detected; namely a reduction in sporocyst size based on length measurements. The size-reducing phenotype was observed in 11 of the 33 (33%) dsRNA treatment groups, and of these 11 phenotype-associated genes (superoxide dismutase, Smad1, RHO2, Smad2, Cav2A, ring box, GST26, calcineurin B, Smad4, lactate dehydrogenase and EF1α), only 6 demonstrated a significant and consistent knockdown of specific transcript expression. Unexpectedly one phenotype-linked gene, superoxide dismutase (SOD), was highly induced (∼1600-fold) upon dsRNA exposure. Variation in dsRNA-mediated silencing effects also was evident in the group of sporocysts that lacked any definable phenotype. Out of 22 nonphenotype-expressing dsRNA treatments (myosin, PKCB, HEXBP, calcium channel, Sma2, RHO1, PKC receptor, DHHC, PepcK, calreticulin, calpain, Smeg, 14.3.3, K5, SPO1, SmZF1, fibrillarin, GST28, GPx, TPx1, TPx2 and TPx2/TPx1), 12 were assessed for the transcript levels. Of those, 6 genes exhibited consistent reductions in steady-state transcript levels, while expression level for the rest remained unchanged. Results demonstrate that the efficacy of dsRNA-treatment in producing consistent phenotypic changes and/or altered gene expression levels in S. mansoni sporocysts is highly dependent on the selected gene (or the specific dsRNA sequence used) and the timing of evaluation after treatment. Although RNAi holds great promise as a functional genomics tool for larval schistosomes, our finding of potential off-target or nonspecific effects of some dsRNA treatments and variable efficiencies in specific gene knockdown indicate a critical need for gene-specific testing and optimization as an essential part of experimental design, execution and data interpretation

The Biomphalaria glabrata DNA methylation machinery displays spatial tissue expression, is differentially active in distinct snail populations and is modulated by interactions with Schistosoma mansoni

BBSRC Grant (BB/K005448/1)Background The debilitating human disease schistosomiasis is caused by infection with schistosome parasites that maintain a complex lifecycle alternating between definitive (human) and intermediate (snail) hosts. While much is known about how the definitive host responds to schistosome infection, there is comparably less information available describing the snail?s response to infection. Methodology/Principle findings Here, using information recently revealed by sequencing of the Biomphalaria glabrata intermediate host genome, we provide evidence that the predicted core snail DNA methylation machinery components are associated with both intra-species reproduction processes and inter-species interactions. Firstly, methyl-CpG binding domain protein (Bgmbd2/3) and DNA methyltransferase 1 (Bgdnmt1) genes are transcriptionally enriched in gonadal compared to somatic tissues with 5-azacytidine (5-AzaC) treatment significantly inhibiting oviposition. Secondly, elevated levels of 5-methyl cytosine (5mC), DNA methyltransferase activity and 5mC binding in pigmented hybrid- compared to inbred (NMRI)- B. glabrata populations indicate a role for the snail?s DNA methylation machinery in maintaining hybrid vigour or heterosis. Thirdly, locus-specific detection of 5mC by bisulfite (BS)-PCR revealed 5mC within an exonic region of a housekeeping protein-coding gene (Bg14-3-3), supporting previous in silico predictions and whole genome BS-Seq analysis of this species? genome. Finally, we provide preliminary evidence for parasite-mediated host epigenetic reprogramming in the schistosome/snail system, as demonstrated by the increase in Bgdnmt1 and Bgmbd2/3 transcript abundance following Bge (B. glabrata embryonic cell line) exposure to parasite larval transformation products (LTP). Conclusions/Significance The presence of a functional DNA methylation machinery in B. glabrata as well as the modulation of these gene products in response to schistosome products, suggests a vital role for DNA methylation during snail development/oviposition and parasite interactions. Further deciphering the role of this epigenetic process during Biomphalaria/Schistosoma co-evolutionary biology may reveal key factors associated with disease transmission and, moreover, enable the discovery of novel lifecycle intervention strategiespublishersversionPeer reviewe

Duration of adjuvant doublet chemotherapy (3 or 6 months) in patients with high-risk stage II colorectal cancer

PURPOSE: As oxaliplatin results in cumulative neurotoxicity, reducing treatment duration without loss of efficacy would benefit patients and healthcare providers. PATIENTS AND METHODS: Four of the six studies in the International Duration of Adjuvant Chemotherapy (IDEA) collaboration included patients with high-risk stage II colon and rectal cancers. Patients were treated (clinician and/or patient choice) with either fluorouracil, leucovorin, and oxaliplatin (FOLFOX) or capecitabine and oxaliplatin (CAPOX) and randomly assigned to receive 3- or 6-month treatment. The primary end point is disease-free survival (DFS), and noninferiority of 3-month treatment was defined as a hazard ratio (HR) of < 1.2- v 6-month arm. To detect this with 80% power at a one-sided type one error rate of 0.10, a total of 542 DFS events were required. RESULTS: 3,273 eligible patients were randomly assigned to either 3- or 6-month treatment with 62% receiving CAPOX and 38% FOLFOX. There were 553 DFS events. Five-year DFS was 80.7% and 83.9% for 3-month and 6-month treatment, respectively (HR, 1.17; 80% CI, 1.05 to 1.31; P [for noninferiority] .39). This crossed the noninferiority limit of 1.2. As in the IDEA stage III analysis, the duration effect appeared dependent on the chemotherapy regimen although a test of interaction was negative. HR for CAPOX was 1.02 (80% CI, 0.88 to 1.17), and HR for FOLFOX was 1.41 (80% CI, 1.18 to 1.68). CONCLUSION: Although noninferiority has not been demonstrated in the overall population, the convenience, reduced toxicity, and cost of 3-month adjuvant CAPOX suggest it as a potential option for high-risk stage II colon cancer if oxaliplatin-based chemotherapy is suitable. The relative contribution of the factors used to define high-risk stage II disease needs better understanding