Use of Molecular Genetic Engineering in the Study of Animal Parasites and Their Vectors

Molecular genetics coupled with advances in immunology and parasite culture has become a powerful tool to study animal parasites and their vectors. Recombinant DNA techniques allow one to identify individual genes of DNA probes, amplify the nucleic acid of interest, and use this material to study: the role of the gene product in the biology of the organism; the evolution of parasites and their hosts; heterogeneity between species and within species; taxonomy and develop refined taxonomic tools; the immunology and biochemistry of host-parasite interactions; identification of specific cells or tissues that produce gene products; cytogenetics and localization of genes on chromosome in the study of animal parasites and their vectors will be presented

Molecular Approach to the Study of Trematode Parasites : the Blood Fluke

One important aspect of reproductive development in trematode parasites is the formation of a hardened eggshell which allows the zygote to develop into a miracidium in a hostile environment. The miracidium then can transfer the germline from the vertebrate host to snail intermediate host. Schistosome parasites, unlike other trematodes, have separate sexes and female reproductive development is known to depend on the presence of a male parasite. These facts make the blood flukes ideal material to study the mechanisms that underlie female reproductive development and eggshell formatian. We reasoned that the morphological and biochemical differences between the male and female must be reflected at the molecular level in the differential expression of sexually regulated genes. Radioactive single stranded cDNA was first transcribed from female RNA; and then sequences common to both male and female were removed by hybridization to an excess of male RNA. This probe was used to screen a cDNA library made from mRNA of adult worm paris. One hybridizing clone, pSMf 61-46, was shown to correspond to a 0.9 kilobase mRNA that is present only in mature female worms and is not detectable in female schistosomes from single-sex infections, in male worms or in eggs. Thus expression of the gene was female-specific. During normal bisexual infection this mRNA is first detected 28 days after infection (the time of worm pairing) and increases to a high level at 35 days postinfection, coinciding with egg production. Thus the temporal expression of the gene was dependent on paining with male worm. The nucleotide sequence of the gene shows an open reading frame that encodes a 16 kDA polypeptide that shows strong homology with eggshell proteins on insects. A second female-specific cDNA clone, F-4, represents a 1.6 kilobase mRNA whose expression is also correlated with worm pairing and subsequent egg production, encodes a different putative eggshell component of 44 kDA. The amino acid composition of the 16 kDA and 44 kDA polypeptides show a strong correlation with the actual amino acid composition of the schistosome eggshell. Thus these two polypeptides appear to the major components of the schistosome eggshell. Analysis of the genomic arrangement of the eggshell genes show that pl6 is represented by 5 gene copies and p48 is represented by 2-5 copies. The eggshell genes are expressed in the vitelline cell as recently demonstrated by in hybridization and immunocytochemical localization. The eggshell genes are being expressed in bacteria. The gene products will be used to study the biochemistry of eggshell formation

Evolution of a novel subfamily of nuclear receptors with members that each contain two DNA binding domains

BACKGROUND: Nuclear receptors (NRs) are important transcriptional modulators in metazoans which regulate transcription through binding to the promoter region of their target gene by the DNA binding domain (DBD) and activation or repression of mRNA synthesis through co-regulators bound to the ligand binding domain (LBD). NRs typically have a single DBD with a LBD. RESULTS: Three nuclear receptors named 2DBD-NRs, were identified from the flatworm Schistosoma mansoni that each possess a novel set of two DBDs in tandem with a LBD. They represent a novel NR modular structure: A/B-DBD-DBD-hinge-LBD. The 2DBD-NRs form a new subfamily of NRs, VII. By database mining, 2DBD-NR genes from other flatworm species (Schmidtea mediterranea and Dugesia japonica), from Mollusks (Lottia gigantean) and from arthropods (Daphnia pulex) were also identified. All 2DBD-NRs possess a P-box sequence of CEACKK in the first DBD, which is unique to 2DBD-NRs, and a P-box sequence of CEGCKG in the second DBD. Phylogenetic analyses of both DBD and ligand binding domain sequences showed that 2DBD-NR genes originate from a common two DBD-containing ancestor gene. A single 2DBD-NR orthologue was found in Arthropoda, Platyhelminths and Mollusca. Subsequent 2DBD-NR gene evolution in Mollusks and Platyhelminths involved gene duplication. Chromosome localization of S. mansoni 2DBD-NR genes by Fluorescent in situ hybridization (FISH) suggests that 2DBD-NR genes duplicated on different chromosomes in the Platyhelminths. Dimerization of Sm2DBDα indicates that 2DBD-NRs may act as homodimers, suggesting either that two repeats of a half-site are necessary for each DBD of 2DBD-NRs to bind to its target gene, or that each 2DBD-NR can recognize multiple sites. CONCLUSION: 2DBD-NRs share a common ancestor gene which possessed an extra DBD that likely resulted from a recombination event. After the split of the Arthropods, Mollusks and Platyhelminths, 2DBD-NR underwent a recent duplication in a common ancestor of Mollusks, while two rounds of duplication occurred in a common ancestor of the Platyhelminths. This demonstrates that certain NR gene underwent recent duplication in Prostostome lineages after the split of the Prostostomia and Deuterostomia

Schistosoma mansoni TGF-β Receptor II: Role in Host Ligand-Induced Regulation of a Schistosome Target Gene

Members of transforming growth factor-beta (TGF-β) superfamily play pivotal roles in development in multicellular organisms. We report the functional characterization of the Schistosoma mansoni type II receptor (SmTβRII). Mining of the S. mansoni expressed sequence tag (EST) database identified an EST clone that shows homology to the kinase domain of type II receptors from different species. The amplified EST sequence was used as a probe to isolate a cDNA clone spanning the entire coding region of a type II serine/threonine kinase receptor. The interaction of SmTβRII with SmTβRI was elucidated and shown to be dependent on TGF-β ligand binding. Furthermore, in the presence of human TGF-β1, SmTβRII was able to activate SmTβRI, which in turn activated SmSmad2 and promoted its interaction with SmSmad4, proving the transfer of the signal from the receptor complex to the Smad proteins. Gynaecophoral canal protein (GCP), whose expression in male worms is limited to the gynaecophoric canal, was identified as a potential TGF-β target gene in schistosomes. Knocking down the expression of SmTβRII using short interfering RNA molecules (siRNA) resulted in a concomitant reduction in the expression of GCP. These data provide evidence for the direct involvement of SmTβRII in mediating TGF-β–induced activation of the TGF-β target gene, SmGCP, within schistosome parasites. The results also provide additional evidence for a role for the TGF-β signaling pathway in male-induced female reproductive development

Genomic linkage map of the human blood fluke Schistosoma mansoni

The first genetic linkage map of Schistosoma mansoni reveals insights into higher female recombination, confirms ZW inheritance patterns and recombination hotspots

Protective Potential of Antioxidant Enzymes as Vaccines for Schistosomiasis in a Non-Human Primate Model

Schistosomiasis remains a major cause of morbidity in the world. The challenge today is not so much in the clinical management of individual patients, but rather in population-based control of transmission in endemic areas. Recent large-scale efforts aimed at limiting schistosomiasis have produced limited success. There is an urgent need for complementary approaches, such as vaccines. We demonstrated previously that anti-oxidant enzymes such as Cu-Zn superoxide dismutase (SOD) and glutathione S peroxidase (GPX), when administered as DNA-based vaccines induced significant levels of protection in inbred mice, greater than the target 40% reduction in worm burden compared to controls set as a minimum by the WHO. These results led us to investigate if immunization of non-human primates with antioxidants would stimulate an immune response that could confer protection, as a prelude for human trials. Issues of vaccine toxicity and safety that were difficult to address in mice were also investigated. All baboons in the study were examined clinically throughout the study and no adverse reactions occurred to the immunization. When our outbred baboons were vaccinated with two different formulations of SOD (SmCT-SOD and SmEC-SOD) or one of GPX (SmGPX), they showed a reduction in worm number to varying degrees, when compared with the control group. More pronounced, vaccinated animals showed decreased bloody diarrhea, days of diarrhea and egg excretion (transmission), as well as reduction of eggs in the liver tissue and in the large intestine (pathology) compared to controls. Specific IgG antibodies were present in sera after immunizations and 10 weeks after challenge infection compared to controls. PBMC, mesenteric and inguinal node cells from vaccinated animals proliferated and produced high levels of cytokines and chemokines in response to crude and recombinant antigens compared with controls. These data demonstrate the potential of antioxidants as vaccine candidates

Effect of human TGF-beta on the gene expression profile of Schistosoma mansoni adult worms

Schistosoma mansoni is responsible for schistosomiasis, a parasitic disease that affects 200 million people worldwide. Molecular mechanisms of host-parasite interaction are complex and involve a crosstalk between host signals and parasite receptors. TGF-beta signaling pathway has been shown to play an important role in S. mansoni development and embryogenesis. In particular human (h) TGF-beta has been shown to bind to a S. mansoni receptor, transduce a signal that regulates the expression of a schistosome target gene. Here we describe 381 parasite genes whose expression levels are affected by in vitro treatment with hTGF-beta. Among these differentially expressed genes we highlight genes related to morphology, development and cell cycle that could be players of cytokine effects on the parasite. We confirm by qPCR the expression changes detected with microarrays for 5 out of 7 selected genes. We also highlight a set of non-coding RNAs transcribed from the same loci of protein-coding genes that are differentially expressed upon hTCF-beta treatment. These datasets offer potential targets to be explored in order to understand the molecular mechanisms behind the possible role of hTGF-beta effects on parasite biology. (C) 2012 Elsevier B.V. All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)NIH [AI 46762]NIHCNPqCNPqNIAIDNIAID [HHSN272201000009I

Observing the Evolution of the Universe

How did the universe evolve? The fine angular scale (l>1000) temperature and polarization anisotropies in the CMB are a Rosetta stone for understanding the evolution of the universe. Through detailed measurements one may address everything from the physics of the birth of the universe to the history of star formation and the process by which galaxies formed. One may in addition track the evolution of the dark energy and discover the net neutrino mass. We are at the dawn of a new era in which hundreds of square degrees of sky can be mapped with arcminute resolution and sensitivities measured in microKelvin. Acquiring these data requires the use of special purpose telescopes such as the Atacama Cosmology Telescope (ACT), located in Chile, and the South Pole Telescope (SPT). These new telescopes are outfitted with a new generation of custom mm-wave kilo-pixel arrays. Additional instruments are in the planning stages.Comment: Science White Paper submitted to the US Astro2010 Decadal Survey. Full list of 177 author available at http://cmbpol.uchicago.ed

Weighing Neutrinos with Cosmic Neutral Hydrogen

We investigate the signatures left by massive neutrinos on the spatial distribution of neutral hydrogen (H I) in the post-reionization era by running hydrodynamic simulations that include massive neutrinos as additional collisionless particles. We find that halos in massive/massless neutrino cosmologies host a similar amount of neutral hydrogen, although for a fixed halo mass, on average, the H I mass increases with the sum of the neutrino masses. Our results show that H I is more strongly clustered in cosmologies with massive neutrinos, while its abundance, Omega(H I) (z), is lower. These effects arise mainly from the impact of massive neutrinos on cosmology: they suppress both the amplitude of the matter power spectrum on small scales and the abundance of dark matter halos. Modeling the H I distribution with hydrodynamic simulations at z > 3 and a simple analytic model at z < 3, we use the Fisher matrix formalism to conservatively forecast the constraints that Phase 1 of the Square Kilometre Array will place on the sum of neutrino masses, M-nu = Sigma m(nu). We find that with 10,000 hr of interferometric observations at 3 less than or similar to z less than or similar to 6 from a deep and narrow survey with SKA1-LOW, the sum of the neutrino masses can be measured with an error sigma(M-nu) less than or similar to 0.3 eV (95% CL). Similar constraints can be obtained with a wide and deep SKA1-MID survey at z less than or similar to 3, using the single-dish mode. By combining data from MID, LOW, and Planck, plus priors on cosmological parameters from a Stage IV spectroscopic galaxy survey, the sum of the neutrino masses can be determined with an error sigma(M-nu) similar or equal to 0.06 eV (95% CL)