Fabiano Sviatopolk-Mirsky Pais

ZIKV – CDB: A Collaborative Database to Guide Research Linking SncRNAs and ZIKA Virus Disease Symptoms

Background In early 2015, a ZIKA Virus (ZIKV) infection outbreak was recognized in northeast Brazil, where concerns over its possible links with infant microcephaly have been discussed. Providing a causal link between ZIKV infection and birth defects is still a challenge. MicroRNAs (miRNAs) are small noncoding RNAs (sncRNAs) that regulate post-transcriptional gene expression by translational repression, and play important roles in viral pathogenesis and brain development. The potential for flavivirus-mediated miRNA signalling dysfunction in brain-tissue development provides a compelling hypothesis to test the perceived link between ZIKV and microcephaly. Methodology/Principal Findings Here, we applied in silico analyses to provide novel insights to understand how Congenital ZIKA Syndrome symptoms may be related to an imbalance in miRNAs function. Moreover, following World Health Organization (WHO) recommendations, we have assembled a database to help target investigations of the possible relationship between ZIKV symptoms and miRNA-mediated human gene expression. Conclusions/Significance We have computationally predicted both miRNAs encoded by ZIKV able to target genes in the human genome and cellular (human) miRNAs capable of interacting with ZIKV genomes. Our results represent a step forward in the ZIKV studies, providing new insights to support research in this field and identify potential targets for therapy.

Revisitingmolecular serotyping of Streptococcus pneumoniae

BackgroundNinety-two Streptococcus pneumoniae serotypes have been described so far, but the pneumococcal conjugate vaccine introduced in the Brazilian basic vaccination schedule in 2010 covers only the ten most prevalent in the country. Pneumococcal serotype-shifting after massive immunization is a major concern and monitoring this phenomenon requires efficient and accessible serotyping methods. Pneumococcal serotyping based on antisera produced in animals is laborious and restricted to a few reference laboratories. Alternatively, molecular serotyping methods assess polymorphisms in the cps gene cluster, which encodes key enzymes for capsular polysaccharides synthesis in pneumococci. In one such approach, cps-RFLP, the PCR amplified cps loci are digested with an endonuclease, generating serotype-specific fingerprints on agarose gel electrophoresis.MethodsIn this work, in silico and in vitro approaches were combined to demonstrate that XhoII is the most discriminating endonuclease for cps-RFLP, and to build a database of serotype-specific fingerprints that accommodates the genetic diversity within the cps locus of 92 known pneumococci serotypes.ResultsThe expected specificity of cps-RFLP using XhoII was 76% for serotyping and 100% for serogrouping. The database of cps-RFLP fingerprints was integrated to Molecular Serotyping Tool (MST), a previously published web-based software for molecular serotyping. In addition, 43 isolates representing 29 serotypes prevalent in the state of Minas Gerais, Brazil, from 2007 to 2013, were examined in vitro; 11 serotypes (nine serogroups) matched the respective in silico patterns calculated for reference strains. The remaining experimental patterns, despite their resemblance to their expected in silico patterns, did not reach the threshold of similarity score to be considered a match and were then added to the database.ConclusionThe cps-RFLP method with XhoII outperformed the antisera-based and other molecular serotyping methods in regard of the expected specificity. In order to accommodate the genetic variability of the pneumococci cps loci, the database of cps-RFLP patterns will be progressively expanded to include new variant in vitro patterns. The cps-RFLP method with endonuclease XhoII coupled with MST for computer-assisted interpretation of results may represent a relevant contribution to the real time detection of changes in regional pneumococci population diversity in response to mass immunization programs.

The transcriptome of the mosquito Aedes fluviatilis (Diptera: Culicidae), and transcriptional changes associated with its native Wolbachia infection

BackgroundWolbachia is a bacterial endosymbiont that naturally infects a wide range of insect species, and causes drastic changes to host biology. Stable infections of Wolbachia in mosquitoes can inhibit infection with medically important pathogens such as dengue virus and malaria-causing Plasmodium parasites. However, some native Wolbachia strains can enhance infection with certain pathogens, as is the case for the mosquito Aedes fluviatilis, where infection with Plasmodium gallinaceum is enhanced by the native wFlu Wolbachia strain. To better understand the biological interactions between mosquitoes and native Wolbachia infections, and to investigate the process of pathogen enhancement, we used RNA-Seq to generate the transcriptome of Ae. fluviatilis with and without Wolbachia infection.ResultsIn total, we generated 22,280,160 Illumina paired-end reads from Wolbachia-infected and uninfected mosquitoes, and used these to make a de novo transcriptome assembly, resulting in 58,013 contigs with a median sequence length of 443 bp and an N50 of 2454 bp. Contigs were annotated through local alignments using BlastX, and associated with both gene ontology and KEGG orthology terms. Through baySeq, we identified 159 contigs that were significantly upregulated due to Wolbachia infection, and 98 that were downregulated. Critically, we saw no changes to Toll or IMD immune gene transcription, but did see evidence that wFlu infection altered the expression of several bacterial recognition genes, and immune-related genes that could influence Plasmodium infection. wFlu infection also had a widespread effect on a number of host physiological processes including protein, carbohydrate and lipid metabolism, and oxidative stress. We then compared our data set with transcriptomic data for other Wolbachia infections in Aedes aegypti, and identified a core set of 15 gene groups associated with Wolbachia infection in mosquitoes.ConclusionsWhile the scale of transcriptional changes associated with wFlu infection might be small, the scope is rather large, which confirms that native Wolbachia infections maintain intricate molecular relationships with their mosquito hosts even after lengthy periods of co-evolution. We have also identified several potential means through which wFlu infection might influence Plasmodium infection in Ae. fluviatilis, and these genes should form the basis of future investigation into the enhancement of Plasmodium by Wolbachia.

Evolutionary analysis of the cystatin family in three Schistosoma species

The cystatin family comprises cysteine protease inhibitors distributed in 3 subfamilies (I25A–C). Family members lacking cystatin activity are currently unclassified. Little is known about the evolution of Schistosoma cystatins, their physiological roles, and expression patterns in the parasite life cycle. The present study aimed to identify cystatin homologs in the predicted proteome of three Schistosoma species and other Platyhelminthes. We analyzed the amino acid sequence diversity focused in the identification of protein signatures and to establish evolutionary relationships among Schistosoma and experimentally validated human cystatins. Gene expression patterns were obtained from different developmental stages in Schistosoma mansoni using microarray data. In Schistosoma, only I25A and I25B proteins were identified, reflecting little functional diversification. I25C and unclassified subfamily members were not identified in platyhelminth species here analyzed. The resulting phylogeny placed cystatins in different clades, reflecting their molecular diversity. Our findings suggest that Schistosoma cystatins are very divergent from their human homologs, especially regarding the I25B subfamily. Schistosoma cystatins also differ significantly from other platyhelminth homologs. Finally, transcriptome data publicly available indicated that I25A and I25B genes are constitutively expressed thus could be essential for schistosome life cycle progression. In summary, this study provides insights into the evolution, classification, and functional diversification of cystatins in Schistosoma and other Platyhelminthes, improving our understanding of parasite biology and opening new frontiers in the identification of novel therapeutic targets against helminthiases.

Genome-wide signatures of complex introgression and adaptive evolution in the big cats

Big cat genomes reveal a history of interspecies admixture and adaptive evolution of genes underlying development and sensory perception. The great cats of the genus Panthera comprise a recent radiation whose evolutionary history is poorly understood. Their rapid diversification poses challenges to resolving their phylogeny while offering opportunities to investigate the historical dynamics of adaptive divergence. We report the sequence, de novo assembly, and annotation of the jaguar (Panthera onca) genome, a novel genome sequence for the leopard (Panthera pardus), and comparative analyses encompassing all living Panthera species. Demographic reconstructions indicated that all of these species have experienced variable episodes of population decline during the Pleistocene, ultimately leading to small effective sizes in present-day genomes. We observed pervasive genealogical discordance across Panthera genomes, caused by both incomplete lineage sorting and complex patterns of historical interspecific hybridization. We identified multiple signatures of species-specific positive selection, affecting genes involved in craniofacial and limb development, protein metabolism, hypoxia, reproduction, pigmentation, and sensory perception. There was remarkable concordance in pathways enriched in genomic segments implicated in interspecies introgression and in positive selection, suggesting that these processes were connected. We tested this hypothesis by developing exome capture probes targeting ~19,000 Panthera genes and applying them to 30 wild-caught jaguars. We found at least two genes (DOCK3 and COL4A5, both related to optic nerve development) bearing significant signatures of interspecies introgression and within-species positive selection. These findings indicate that post-speciation admixture has contributed genetic material that facilitated the adaptive evolution of big cat lineages.

Long non‐coding RNAs: biomarkers for acute leukaemia subtypes

The analysis of the human genome and transcriptome has revealed a diverse population of DNA sequences that are transcribed into RNAs but do not code for proteins, called non-coding RNAs (ncRNAs) (Kapranov et al, 2010). Deregulation of specific long ncRNAs (lncRNAs – ncRNAs that are longer than 200 nt) has been found in leukaemias (Trimarchi et al, 2014) and this new field of research is starting to be explored. Until recently, Genome-wide Expression Profiling (GEP) studies on ncRNAs in leukaemias focused mainly on microRNAs (miRNAs). However, reports on the study of lncRNAs have emerged in the last two years. Differential expression of lncRNAs among different acute lymphoblastic leukaemia (ALL) subtypes (Nordlund et al, 2012) and between KMT2A (MLL)-rearranged ALL and non-rearranged KMT2A (Fang et al, 2014) were detected. Furthermore, NOTCH-dependent lncRNAs deregulated in T lineage-derived ALL (T-ALL) were also identified (Trimarchi et al, 2014), as well as a significant correlation between the expression of CEBPA and the expression of several lncRNAs in acute myeloid leukaemia (AML) cell lines (Hughes et al, 2014). With the aim of identifying molecular biomarkers of acute leukaemia subtypes, we obtained RNA expression profiles from bone marrow samples of de novo cases of AML (n = 110) and ALL (n = 97), diagnosed according to World Health Organisation classification system (Swerdlow et al, 2008)] using the Sure Print G3 Human GE 8 9 60 K microarray (Agilent Technologies, Santa Clara, CA). Probes with specific expression signatures for each 2-class model were identified by machine learning supervised analysis and the 60 most informative for each model were selected to build a classifier (see Data S1). All programs were run within a local installation of the GenePattern suite (Reich et al, 2006). The classifiers that achieved sensitivity and specificity rates of more than 95%were: AML versus heterogeneous group (the latter group includes control individuals and patients with other haematological diseases); ALL versus heterogeneous group; acute promyelocytic leukaemia (APL) versus other AMLs; AML CBFB-MYH11 versus other AMLs and T-ALL versus B lineagederived ALL (B-ALL) (Tables SI–SIII). Accuracy and precision rates were also high, except CBFB-MYH11 precision (80%). During this process, we realised that a significant number (5– 15%) of the highly informative probes selected for each model were lncRNAs (Table SIV). Most of them belong to the long intergenic ncRNAs class (lincRNAs), three are antisense strand RNA (aRNAs) and four are pseudogenes. We next evaluated expression patterns of four of the ncRNAs by real time quantitative reverse transcription polymerase chain reaction (qRT-PCR), using Taqman Gene Expression Assays (Applied Biosystems, Waltham, MA, USA). The expression of three of the genes studied by qRTPCR (LOC728743, XLOC_009378 and LOC101929333) was significantly different between groups of patients ((Fig 1), although the presence of outliers emphasises sample heterogeneity, a common issue in leukaemia disease. Differential expression of PRKCQ-AS1, selected for the B-ALL/T-ALL model, was not validated by Q-PCR. The PRKCQ protein is a kinase important for T-cell activation and the PRKCQ gene was shown to be up-regulated in T-ALL (Yeoh et al, 2002). It is possible that the PRKCQ-AS1 high expression detected in the microarray is actually an experimental artefact caused by erroneous strand priming during cDNA synthesis and this will be further investigated. LncRNAs were more frequent in the gene signature built to discriminate B-ALL from T-ALL patients, representing 15% of the genes differentially expressed. A non-supervised analysis using only the lncRNAs genes from this signature showed that B-ALL samples clearly clustered separately from T-ALL samples ((Fig 2). The importance of lncRNAs in T cell differentiation has been recognised (Xia et al, 2014). Evidence of their involvement in T-ALL development is also emerging: a NOTCH-regulated lncRNA, LUNAR1, was shown to be required for leukaemia cells growth in vitro and in vivo (Trimarchi et al, 2014). Some of the lncRNAs detected in this work have already been associated with cancer, such as Colorectal Neoplasia Differentially Expressed (CRNDE). Initially identified as an lncRNA whose expression is highly elevated in colorectal cancer, it is also up-regulated in solid tumours and in AMLs, particularly French-American-British (FAB) classification types M2 and M3 (Ellis et al, 2014). In our study, the median CRNDE expression was ten times higher in APL than in other AML subtypes. However, the majority of the lncRNAs described here are transcripts of unknown function and their role in leukemogenesis is still unclear. Our methodological approach did not target lncRNAs specifically, but results showed that they were an important fraction of the biomarkers detected. The data confirm the notion that lncRNA expression may discriminate acute leukaemia molecular subtypes, providing in an additional tool to the classical tests used to categorise leukaemias and stratify Correspondence

Pleiotropic alterations in gene expression in Latin American Fasciola hepatica isolates with different susceptibility to drugs

BackgroundFasciola hepatica is the main agent of fasciolosis, a zoonotic disease affecting livestock worldwide, and an emerging food-borne disease in humans. Even when effective treatments are available, drugs are costly and can result in tolerance, liver damage and normally they do not prevent reinfection. Drug-resistant strains in livestock have been reported in various countries and, more worryingly, drug resistance in human cases has emerged in South America. The present study aims to characterize the transcriptome of two South American resistant isolates, the Cajamarca isolate from Peru, resistant to both triclabendazole and albendazole (TCBZR/ABZR) and the Rubino isolate from Uruguay, resistant to ABZ (TCBZS/ABZR), and compare them to a sensitive strain (Cenapa, Mexico, TCBZS/ABZS) to reveal putative molecular mechanisms leading to drug resistance.ResultsWe observed a major reduction in transcription in the Cajamarca TCBZR/ABZR isolate in comparison to the other isolates. While most of the differentially expressed genes are still unannotated, several trends could be detected. Specific reduction in the expression levels of cytoskeleton proteins was consistent with a role of tubulins as putative targets of triclabendazole (TCBZ). A marked reduction of adenylate cyclase might be underlying pleiotropic effects on diverse metabolic pathways of the parasite. Upregulation of GST mu isoforms suggests this detoxifying mechanism as one of the strategies associated with resistance.ConclusionsOur results stress the value of transcriptomic approaches as a means of providing novel insights to advance the understanding of drug mode of action and drug resistance. The results provide evidence for pleiotropic variations in drug-resistant isolates consistent with early observations of TCBZ and ABZ effects and recent proteomic findings.

Evolutionary relationships among protein lysine deacetylases of parasites causing neglected diseases

The availability of the genomic data of diverse parasites provides an opportunity to identify new drug candidates against neglected tropical diseases affecting people worldwide. Histone modifying enzymes (HMEs) are potential candidates since they play key roles in the regulation of chromatin modifications, thus globally regulating gene expression. Furthermore, aberrant epigenetic states are often associated with human diseases, leading to great interest in HMEs as therapeutic targets. Our work focused on two families of protein lysine deacetylases (HDACs and sirtuins). First, we identified potential homologues in the predicted proteomes of selected taxa by using hidden Markov model profiles. Then, we reconstructed the evolutionary relationships of protein sequences by Bayesian inference and maximum likelihood method. In addition, we constructed homology models for five parasite HDACs to provide information for experimental validation and structure-based optimization of inhibitors. Our results showed that parasite genomes code for diverse HDACs and sirtuins. The evolutionary pattern of protein deacetylases with additional experimental data points to these enzymes as common drug targets among parasites. This work has improved the functional annotation of approximately 63% HDACs and 51% sirtuins in the selected taxa providing insights for experimental design. Homology models pointed out structural conservation and differences among parasite and human homologues and highlight potential candidates for further inhibitor development. Some of these parasite proteins are undergoing RNA interference or knockout analyses to validate the function of their corresponding genes. In the future, we will investigate the main functions performed by these proteins, related phenotypes, and their potential as therapeutic targets.

Overexpression of eukaryotic initiation factor 5A (eIF5A) affects susceptibility to benznidazole in Trypanosoma cruzi populations

Eukaryotic initiation factor 5A (eIF5A) is a conserved protein with an essential role in translation elongation. Using one and two-dimensional western blotting, we showed that the eIF5A protein level was 2-fold lower in benznidazole (BZ)-resistant (BZR and 17LER) Trypanosoma cruzi populations than in their respective susceptible counterparts (BZS and 17WTS). To confirm the role of eIF5A in BZ resistance, we transfected BZS and 17WTS with the wild-type eIF5A or mutant eIF5A-S2A (in which serine 2 was replaced by alanine). Upon overexpressing eIF5A, both susceptible lines became approximately 3- and 5-fold more sensitive to BZ. In contrast, the eIF5A-S2A mutant did not alter its susceptibility to BZ. These data suggest that BZ resistance might arise from either decreasing the translation of proteins that require eIF5A, or as a consequence of differential levels of precursors for the hypusination reactions (e.g., spermidine and trypanothione), both of which alter BZ’s effects in the parasite.

Comparative sequence analysis reveals regulation of genes in developing schistosomula of Schistosoma mansoni exposed to host portal serum

Once inside a vertebrate host after infection, individual schistosomula of the parasite Schistosoma mansoni find a new and complex environment, which requires quick adjustments for survival, such as those that allow it to avoid the innate immune response of the host. Thus, it is very important for the parasite to remain within the skin after entering the host for a period of about 3 days, at which time it can then reach the venous system, migrate to the lungs and, by the end of eighth day post-infection, it reach the portal venous system, while undergoing minimal changes in morphology. However, after just a few days in the portal blood system, the parasite experiences an extraordinary increase in biomass and significant morphological alterations. Therefore, determining the constituents of the portal venous system that may trigger these changes that causes the parasite to consolidate its development inside the vertebrate host, thus causing the disease schistosomiasis, is essential. The present work simulated the conditions found in the portal venous system of the vertebrate host by exposing schistosomula of S. mansoni to in vitro culture in the presence of portal serum of the hamster, Mesocricetus auratus. Two different incubation periods were evaluated, one of 3 hours and one of 12 hours. These time periods were used to mimic the early contact of the parasite with portal serum during the course of natural infection. As a control, parasites were incubated in presence of hamster peripheral serum, in order to compare gene expression signatures between the two conditions. The mRNA obtained from parasites cultured under both conditions were submitted to a whole transcriptome library preparation and sequenced with a next generation platform. On average, nearly 15 million reads were produced per sample and, for the purpose of gene expression quantification, only reads mapped to one location of the transcriptome were considered. After statistical analysis, we found 103 genes differentially expressed by schistosomula cultured for 3 hours and 12 hours in the presence of hamster portal serum. After the subtraction of a second list of genes, also differentially expressed between schistosomula cultured for 3 hours and 12 hours in presence of peripheral serum, a set of 58 genes was finally established. This pattern was further validated for a subset of 17 genes, by measuring gene expression through quantitative real time polymerase chain reaction (qPCR). Processes that were activated by the portal serum stimulus include response to stress, membrane transport, protein synthesis and folding/degradation, signaling, cytoskeleton arrangement, cell adhesion and nucleotide synthesis. Additionally, a smaller number of genes down-regulated under the same condition act on cholinergic signaling, inorganic cation and organic anion membrane transport, cell adhesion and cytoskeleton arrangement. Considering the role of these genes in triggering processes that allow the parasite to quickly adapt, escape the immune response of the host and start maturation into an adult worm after contact with the portal serum, this work may point to unexplored molecular targets for drug discovery and vaccine development against schistosomiasis.