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IntroductionExtracellular/environmental stimuli trigger cellular responses to allow Schistosoma sp. parasites adaptation and decide development and survival fate. In this context, signal transduction involving eukaryotic protein kinases (ePKs) has an essential role in regulatory mechanisms. Functional studies had shown the importance of MAPK pathway for Schistosoma mansoni development. In addition, early studies demonstrated that Smp38 MAPK regulates the expression of a large set of genes, among them the hypoxanthine-guanine phosphoribosyl transferase 1 (SmHGPRTase 1, Smp_103560), a key enzyme in the purine salvage pathway that is part of a family comprising five different proteins.MethodsFirst, the regulation of this gene family by the MAPKs pathways was experimentally verified using Smp38-predicted specific inhibitors. In silico analysis showed significant differences in the predicted structure and the domain sequence among the schistosomal HGPRTase family and their orthologs in humans. In order to interrogate the HGPRTases (Smp_103560, Smp_148820, Smp_168500, Smp_312580 and Smp_332640, henceforth SmHGPRTase −1, −2, −3, −4, −5) functional roles, schistosomula, sporocysts, and adult worms were knocked-down using specific dsRNAs.ResultsOur results suggest that SmHGPRTases activity has an essential role in sporocysts and schistosomula development since significant differences in viability, size, and/ or shape were observed after the in vitro knockdown. Also, the knockdown of SmHGPRTases in schistosomula influenced the ovary development and egg maturation in female adult worms during mammalian infection. We also observed alterations in the movement of female adult worms knocked-down in vitro. Most of these results were shown when all gene family members were knocked-down simultaneously, suggesting a redundant function among them.DiscussionThus, this study helps to elucidate the functional roles of the SmHGPRTase gene family in the S. mansoni life cycle and provides knowledge for future studies required for schistosomiasis treatment and control.

Additional file 1: Figure S1. Map of the plates used for the validation assays of antiviral activity against DENV-2 and ZIKV using the MTT method. The validation was performed as per the High Throughput Screening (HTS) protocol described by Iversen et al., 2012 [24]. The model shows a combination of wells that produce the different intercalated signals, namely: uninfected cells (H), infected cells (L) and infected and treated cells (M), suitable for statistical analysis of absorbance readings of the product of the reduction of MTT by the cells. Figure S2. Validation of the antiviral HTS assay against DENV-2 and ZIKV using the MTT method. The validation was performed as per the High Throughput Screening (HTS) protocol described by Iversen et al., 2012 [24]. Raw data values of the plates on the day 3 (endpoint) of plate uniformity study with interleaved distribution of MIN (infected cells), MED (treated and infected cells) and MAX (cell control) signals analyzed by row (A and C) and by column (B and D). Figure S3. CC50 and EC50 titration curves of pretazettine (PTZ),lycorine (LYC), narciclasine (NCL), and narciclasine-4-O-��-D-xylopiranoside (NXP) against DENV-2 and ZIKV. The values were determined by regression curve using GraphPad Prism 5 based on nonlinear logistic regression of the dose-response curves. The values correspond to the average and standard deviation of three independent assays with at least 8 concentrations of the substance. The red dots correspond to the concentration at which the substance has reached host cell toxicity in antiviral assays.

Made available in DSpace on 2016-10-10T03:52:48Z (GMT). No. of bitstreams: 5 Nucleic acid binding properties of SmZF1 a zinc finger protein of Schistosoma mansoni.pdf: 300949 bytes, checksum: 822a3de0faf79e102591fd6230786c99 (MD5) license_url: 52 bytes, checksum: 2f32edb9c19a57e928372a33fd08dba5 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 24623 bytes, checksum: 378d22d8fe50e084ee2f354be78cbe62 (MD5) license.txt: 1887 bytes, checksum: 445d1980f282ec865917de35a4c622f6 (MD5) Previous issue date: 2004 During its life cycle, the flat worm Schistosoma mansoni is exposed to diverse environmental conditions and changes its morphological form. Each change calls for distinct patterns of gene expression. In order to understand the regulation of gene expression, it is necessary to identify regulatory elements in the promoter region of genes, and DNA transacting factors that control transcription. Zinc finger protein domains are responsible for transcription regulation of diverse genes in a wide range of organisms and are also involved in the promotion of protein–protein interactions. A transcript homologous to zinc finger gene sequences was isolated from a S. mansoni adult worm cDNA library and named SmZF1. It codes for a protein of 164 amino acids presenting three C2H2 type zinc finger motifs. The recombinant SmZF1 protein was expressed and used on electrophoretic mobility shift assays to investigate the binding specificity of SmZF1 for DNA and RNA oligonucleotides. Our results demonstrated that SmZF1 binds both ds and ss DNA oligonucleotides, with an apparent preference for the specific D1-3DNA oligonucleotide, and also binds RNA oligonucleotides with lower affinity. Although we found that SmZF1 recognises DNA and RNA oligonucleotides not containing putative target sites, SmZF1 binds preferentially to sequence specific sites. Furthermore, unrelated oligonucleotides are not able to abolish this interaction. In silico studies identified putative SmZF1 binding sites in the complete genome of three model organisms and in partial genome sequences of S. mansoni. Six Drosophila genes presented these binding sites in their promoter region, indicating that they might be controlled by transcription factors containing zinc fingers motifs. Taken together, these results suggest that SmZF1 acts as a putative transcription factor of S. mansoni. Sim Publicado