Schistosoma mansoni Tegument Protein Sm29 Is Able to Induce a Th1-Type of Immune Response and Protection against Parasite Infection

Schistosomiasis is the most important human helminth infection in terms of morbidity and mortality. Although the efforts to develop a vaccine against this disease have experienced failures, a new generation of surface antigens revealed by proteomic studies changed this scenario. Our group has characterized the protein Sm29 described previously as one of the most exposed and expressed antigens in the outer tegument of Schistosoma mansoni. Studies in patients living in endemic areas for schistosomiasis revealed high levels of IgG1 and IgG3 anti-Sm29 in resistant individuals. In this study, confocal microscope analysis showed Sm29 present in the surface of lung-stage schistosoluma and adult worms. Recombinant Sm29, when used as vaccine candidate, induced high levels of protection in mice. This protection was associated with a typical Th1 immune response and reduction of worm burden, liver granulomas and in intestinal eggs. Further, microarray analysis of worms recovered from vaccinated mice showed significant down-regulation of several genes encoding previously characterized vaccine candidates and/or molecules exposed on the surface, suggesting an immune evasion strategy of schistosomes under immune attack. These results demonstrated that Sm29 as one of the important antigens with potential to compose a vaccine against schistosomiasis

Effects of proteasome inhibitor MG-132 on the parasite Schistosoma mansoni

Proteasome is a proteolytic complex responsible for intracellular protein turnover in eukaryotes, archaea and in some actinobacteria species. Previous work has demonstrated that in Schistosoma mansoni parasites, the proteasome inhibitor MG-132 affects parasite development. However, the molecular targets affected by MG-132 in S. mansoni are not entirely known. Here, we used expression microarrays to measure the genome-wide changes in gene expression of S. mansoni adult worms exposed in vitro to MG-132, followed by in silico functional analyses of the affected genes using Ingenuity Pathway Analysis (IPA). Scanning electron microscopy was used to document changes in the parasites' tegument. We identified 1,919 genes with a statistically significant (q-value <= 0.025) differential expression in parasites treated for 24 h with MG-132, when compared with control. Of these, a total of 1,130 genes were up-regulated and 790 genes were down-regulated. A functional gene interaction network comprised of MG-132 and its target genes, known from the literature to be affected by the compound in humans, was identified here as affected by MG-132. While MG-132 activated the expression of the 26S proteasome genes, it also decreased the expression of 19S chaperones assembly, 20S proteasome maturation, ubiquitin-like NEDD8 and its partner cullin-3 ubiquitin ligase genes. Interestingly, genes that encode proteins related to potassium ion binding, integral membrane component, ATPase and potassium channel activities were significantly down-regulated, whereas genes encoding proteins related to actin binding and microtubule motor activity were significantly up-regulated. MG132 caused important changes in the worm tegumentpeeling, outbreaks and swelling in the tegument tubercles could be observed, which is consistent with interference on the ionic homeostasis in S. mansoni. Finally, we showed the down-regulation of Bax pro-apoptotic gene, as well as up-regulation of two apoptosis inhibitor genes, IAP1 and BRE1, and in contrast, down-regulation of Apaf-1 apoptotic activator, thus suggesting that apoptosis is deregulated in S. mansoni exposed to MG-132. A considerable insight has been gained concerning the potential of MG-132 as a gene expression modulator, and overall the data suggest that the proteasome might be an important molecular target for the design of new drugs against schistosomiasis.Univ Sao Paulo, Fac Med Ribeirao Preto, Dept Bioquim & Imunol, Ribeirao Preto, SP, BrazilUniv Sao Paulo, Dept Bioquim, Inst Quim, Sao Paulo, SP, BrazilAdolfo Lutz Inst, Ctr Parasitol & Micol, Nucleo Enteroparasitas, Sao Paulo, SP, BrazilUniv Franca, Nucleo Pesquisa Ciencias Exatas & Tecnol, Grp Pesquisa Prod Nat, Franca, SP, BrazilInst Butantan, Lab Expressao Genica Eucariotos, Sao Paulo, SP, BrazilUniv Fed Uberlandia, Inst Genet Bioquim, Campus Patos de Minas, Patos De Minas, MG, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, Discipline Parasitol, Sao Paulo, SP, BrazilUniv Fed Rio de Janeiro, Inst Biol, Ctr Ciencias & Saude, Rio De Janeiro, RJ, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, Discipline Parasitol, Sao Paulo, SP, BrazilWeb of Scienc

Expression analysis and in silico characterization of intronic long noncoding RNAs in renal cell carcinoma: emerging functional associations

Abstract Background Intronic and intergenic long noncoding RNAs (lncRNAs) are emerging gene expression regulators. The molecular pathogenesis of renal cell carcinoma (RCC) is still poorly understood, and in particular, limited studies are available for intronic lncRNAs expressed in RCC Methods Microarray experiments were performed with custom-designed arrays enriched with probes for lncRNAs mapping to intronic genomic regions. Samples from 18 primary RCC tumors and 11 nontumor adjacent matched tissues were analyzed. Meta-analyses were performed with microarray expression data from three additional human tissues (normal liver, prostate tumor and kidney nontumor samples), and with large-scale public data for epigenetic regulatory marks and for evolutionarily conserved sequences. Results A signature of 29 intronic lncRNAs differentially expressed between RCC and nontumor samples was obtained (false discovery rate (FDR) <5%). A signature of 26 intronic lncRNAs significantly correlated with the RCC five-year patient survival outcome was identified (FDR <5%, p-value ≤0.01). We identified 4303 intronic antisense lncRNAs expressed in RCC, of which 22% were significantly (p <0.05) cis correlated with the expression of the mRNA in the same locus across RCC and three other human tissues. Gene Ontology (GO) analysis of those loci pointed to 'regulation of biological processes’ as the main enriched category. A module map analysis of the protein-coding genes significantly (p <0.05) trans correlated with the 20% most abundant lncRNAs, identified 51 enriched GO terms (p <0.05). We determined that 60% of the expressed lncRNAs are evolutionarily conserved. At the genomic loci containing the intronic RCC-expressed lncRNAs, a strong association (p <0.001) was found between their transcription start sites and genomic marks such as CpG islands, RNA Pol II binding and histones methylation and acetylation. Conclusion Intronic antisense lncRNAs are widely expressed in RCC tumors. Some of them are significantly altered in RCC in comparison with nontumor samples. The majority of these lncRNAs is evolutionarily conserved and possibly modulated by epigenetic modifications. Our data suggest that these RCC lncRNAs may contribute to the complex network of regulatory RNAs playing a role in renal cell malignant transformation.We would like to thank Helder I Nakaya and Tarik A El-Jundi for help with the 44 k microarray experiments and, together with Rodrigo Louro, for helpful discussions. This work was supported by grants from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and from Instituto Nacional de Ciência e Tecnologia em Oncogenômica to SVA and EMR, and by fellowships from FAPESP to AAF, ACT, SAVA and VMC. SVA and EMR received research fellowship awards from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil

Pharmacological inhibition of lysine-specific demethylase 1 (LSD1) induces global transcriptional deregulation and ultrastructural alterations that impair viability in Schistosoma mansoni

Treatment and control of schistosomiasis still rely on only one effective drug, praziquantel (PZQ) and, due to mass treatment, the increasing risk of selecting for schistosome strains that are resistant to PZQ has alerted investigators to the urgent need to develop novel therapeutic strategies. The histone-modifying enzymes (HMEs) represent promising targets for the development of epigenetic drugs against Schistosoma mansoni. In the present study, we targeted the S. mansoni lysine-specific demethylase 1 (SmLSD1), a transcriptional corepressor, using a novel and selective synthetic inhibitor, MC3935, which was used to treat schistosomula and adult worms in vitro. By using cell viability assays and optical and electron microscopy, we showed that treatment with MC3935 affected parasite motility, egg-laying, tegument, and cellular organelle structures, culminating in the death of schistosomula and adult worms. In silico molecular modeling and docking analysis suggested that MC3935 binds to the catalytic pocket of SmLSD1. Western blot analysis revealed that MC3935 inhibited SmLSD1 demethylation activity of H3K4me1/2. Knockdown of SmLSD1 by RNAi recapitulated MC3935 phenotypes in adult worms. RNA-Seq analysis of MC3935-treated parasites revealed significant differences in gene expression related to critical biological processes. Collectively, our findings show that SmLSD1 is a promising drug target for the treatment of schistosomiasis and strongly support the further development and in vivo testing of selective schistosome LSD1 inhibitors

Metagenomic analysis of a tropical composting operation at the São Paulo Zoo Park reveals diversity of biomass degradation functions and organisms.

Composting operations are a rich source for prospection of biomass degradation enzymes. We have analyzed the microbiomes of two composting samples collected in a facility inside the Sao Paulo Zoo Park, in Brazil. All organic waste produced in the park is processed in this facility, at a rate of four tons/day. Total DNA was extracted and sequenced with Roche/454 technology, generating about 3 million reads per sample. To our knowledge this work is the first report of a composting whole-microbial community using high-throughput sequencing and analysis. The phylogenetic profiles of the two microbiomes analyzed are quite different, with a clear dominance of members of the Lactobacillus genus in one of them. We found a general agreement of the distribution of functional categories in the Zoo compost metagenomes compared with seven selected public metagenomes of biomass deconstruction environments, indicating the potential for different bacterial communities to provide alternative mechanisms for the same functional purposes. Our results indicate that biomass degradation in this composting process, including deconstruction of recalcitrant lignocellulose, is fully performed by bacterial enzymes, most likely by members of the Clostridiales and Actinomycetales orders.FAPESP 2009/52030-5RCNPqCAPE

Influence of Ecto-Nucleoside Triphosphate Diphosphohydrolase Activity on Trypanosoma cruzi Infectivity and Virulence

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, an endemic zoonosis present in some countries of South and Central Americas. The World Health Organization estimates that 100 million people are at risk of acquiring this disease. The infection affects mainly muscle tissues in the heart and digestive tract. There are no vaccines or effective treatment, especially in the chronic phase when most patients are diagnosed, which makes a strong case for the development of new drugs to treat the disease. In this work we evaluate a family of proteins called Ecto-Nucleoside-Triphosphate-Diphosphohydrolase (Ecto-NTPDase) as new chemotherapy target to block T. cruzi infection in mammalian cells and in mice. We have used inhibitors and antibodies against this protein and demonstrated that T. cruzi Ecto-NTPDases act as facilitators of infection in mammalian cells and virulence factors in mice model. Two of the drugs used in this study (Suramin and Gadolinium) are currently used for other diseases in humans, supporting the possibility of their use in the treatment of Chagas disease

Exploring the Schistosoma mansoni adult male transcriptome using RNA-seq

Schistosoma mansoni is one of the agents of schistosomiasis, a chronic and debilitating disease. Here we, present a transcriptome-wide characterization of adult S. mansoni males by high-throughput RNA-sequencing. We obtained 1,620,432 high-quality ESTs from a directional strand-specific cDNA library, resulting in a 26% higher coverage of genome bases than that of the public ESTs available at NCBI. With a 15 x-deep coverage of transcribed genomic regions, our data were able to (i) confirm for the first time 990 predictions without previous evidence of transcription; (ii) correct gene predictions; (iii) discover 989 and 1196 RNA-seq contigs that map to intergenic and intronic genomic regions, respectively, where no gene had been predicted before. These contigs could represent new protein-coding genes or non-coding RNAs (ncRNAs). Interestingly, we identified 11 novel Micro-exon genes (MEGs). These data reveal new features of the S. mansoni transcriptional landscape and significantly advance our understanding of the parasite transcriptome. (c) 2011 Elsevier Inc. 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)FAPESP young researcher grantFAPESP young researcher grantFinanciadora de Estudos e Projetos (FINEP)Financiadora de Estudos e Projetos (FINEP)FP-7 European Community, SEtTReND Grant [241865]FP7 European Community, SEtTReND GrantFAPESPFAPESPConselho Nacional de Desenvolvimento Cientifico e Tecnologico (Brazil)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazi

Screening the Schistosoma mansoni transcriptome for genes differentially expressed in the schistosomulum stage in search for vaccine candidates

Schistosomiasis affects more than 200 million people worldwide; another 600 million are at risk of infection. The schistosomulum stage is believed to be the target of protective immunity in the attenuated cercaria vaccine model. In an attempt to identify genes up-regulated in the schistosomulum stage in relation to cercaria, we explored the Schistosoma mansoni transcriptome by looking at the relative frequency of reads in EST libraries from both stages. The 400 genes potentially up-regulated in schistosomula were analyzed as to their Gene Ontology categorization, and we have focused on those encoding-predicted proteins with no similarity to proteins of other organisms, assuming they could be parasite-specific proteins important for survival in the host. Up-regulation in schistosomulum relative to cercaria was validated with real-time reverse transcription polymerase chain reaction (RT-PCR) for five out of nine selected genes (56%). We tested their protective potential in mice through immunization with DNA vaccines followed by a parasite challenge. Worm burden reductions of 16-17% were observed for one of them, indicating its protective potential. Our results demonstrate the value and caveats of using stage-associated frequency of ESTs as an indication of differential expression coupled to DNA vaccine screening in the identification of novel proteins to be further investigated as potential vaccine candidates.FAPESP, Fundacao de Amparo a Pesquisa do Estado de Sao Paulo[04/12.872-3]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CNPq, Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brasil[506638/2004-9]Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CAPES, Coordenacao de Aperfeicoamento de Pessoal de Nivel SuperiorCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES