Exploring TERRA during Leishmania major developmental cycle and continuous in vitro passages.

Telomeres from different eukaryotes, including trypanosomatids, are transcribed into TERRA noncoding RNAs, crucial in regulating chromatin deposition and telomere length. TERRA is transcribed from the C-rich subtelomeric strand towards the 3'-ends of the telomeric array. Using bioinformatics, we confirmed the presence of subtelomeric splice acceptor sites at all L. major chromosome ends. Splice leader sequences positioned 5' upstream of L. major chromosomes subtelomeres were then mapped using SL-RNA-Seq libraries constructed from three independent parasite life stages and helped confirm TERRA expression from several chromosomes ends. Northern blots and RT-qPCR validated the results showing that L. major TERRA is processed by trans-splicing and polyadenylation coupled reactions. The number of transcripts varied with the parasite's life stage and continuous passages, being more abundant in the infective forms. However, no putative subtelomeric promoters involved in TERRA's transcriptional regulation were detected. In contrast, the observed changes in parasite's telomere length during development, suggest that differences in telomeric base J levels may control TERRA transcription in L. major. Also, TERRA-R loops' detection, mainly in the infective forms, was suggestive of TERRA's involvement in telomere protection. Therefore, Leishmania TERRA shares conserved features with other eukaryotes and advances new telomere specific functions in a Public Health-impacting parasite.

SHOC2 scaffold protein modulates daunorubicin-induced cell death through p53 modulation in lymphoid leukemia cells.

SHOC2 scaffold protein has been mainly related to oncogenic ERK signaling through the RAS-SHOC2-PP1 phosphatase complex. In leukemic cells however, SHOC2 upregulation has been previously related to an increased 5-year event-free survival of pediatric pre-B acute lymphoid leukemia, suggesting that SHOC2 could be a potential prognostic marker. To address such paradoxical function, our study investigated how SHOC2 impact leukemic cells drug response. Our transcriptome analysis has shown that SHOC2 can modulate the DNA-damage mediated by p53. Notably, upon genetic inhibition of SHOC2 we observed a significant impairment of p53 expression, which in turn, leads to the blockage of key apoptotic molecules. To confirm the specificity of DNA-damage related modulation, several anti-leukemic drugs has been tested and we did confirm that the proposed mechanism impairs cell death upon daunorubicin-induced DNA damage of human lymphoid cells. In conclusion, our study uncovers new insights into SHOC2 function and reveals that this scaffold protein may be essential to activate a novel mechanism of p53-induced cell death in pre-B lymphoid cells.

Ortholog of the polymerase theta helicase domain modulates DNA replication in Trypanosoma cruzi

DNA polymerase theta (Pol theta), a member of the DNA polymerase family A, exhibits a polymerase C-terminal domain, a central domain, and an N-terminal helicase domain. Pol theta plays important roles in DNA repair via its polymerase domain, regulating genome integrity. In addition, in mammals, Pol theta modulates origin firing timing and MCM helicase recruitment to chromatin. In contrast, as a model eukaryote, Trypanosoma cruzi exhibits two individual putative orthologs of Pol theta in different genomic loci; one ortholog is homologous to the Pol theta C-terminal polymerase domain, and the other is homologous to the Pol theta helicase domain, called Pol theta-polymerase and Pol theta-helicase, respectively. A pull-down assay using the T. cruzi component of the prereplication complex Orc1/Cdc6 as bait captured Pol theta-helicase from the nuclear extract. Orc1/Cdc6 and Pol theta-helicase directly interacted, and Pol theta-helicase presented DNA unwinding and ATPase activities. A T. cruzi strain overexpressing the Pol theta-helicase domain exhibited a significantly decreased amount of DNA-bound MCM7 and impaired replication origin firing. Taken together, these data suggest that Pol theta-helicase modulates DNA replication by directly interacting with Orc1/Cdc6, which reduces the binding of MCM7 to DNA and thereby impairs the firing of replication origins.

Ortholog of the polymerase theta helicase domain modulates DNA replication in Trypanosoma cruzi

DNA polymerase theta (Pol theta), a member of the DNA polymerase family A, exhibits a polymerase C-terminal domain, a central domain, and an N-terminal helicase domain. Pol theta plays important roles in DNA repair via its polymerase domain, regulating genome integrity. In addition, in mammals, Pol theta modulates origin firing timing and MCM helicase recruitment to chromatin. In contrast, as a model eukaryote, Trypanosoma cruzi exhibits two individual putative orthologs of Pol theta in different genomic loci; one ortholog is homologous to the Pol theta C-terminal polymerase domain, and the other is homologous to the Pol theta helicase domain, called Pol theta-polymerase and Pol theta-helicase, respectively. A pull-down assay using the T. cruzi component of the prereplication complex Orc1/Cdc6 as bait captured Pol theta-helicase from the nuclear extract. Orc1/Cdc6 and Pol theta-helicase directly interacted, and Pol theta-helicase presented DNA unwinding and ATPase activities. A T. cruzi strain overexpressing the Pol theta-helicase domain exhibited a significantly decreased amount of DNA-bound MCM7 and impaired replication origin firing. Taken together, these data suggest that Pol theta-helicase modulates DNA replication by directly interacting with Orc1/Cdc6, which reduces the binding of MCM7 to DNA and thereby impairs the firing of replication origins.

Bartonella rochalimae Detection by a Sensitive and Specific PCR Platform.

Bartonella rochalimae is an emerging zoonotic pathogen present in the United States, South America, and Europe. The molecular detection of B. rochalimae frequently relies on polymerase chain reaction (PCR) assays that target the genus Bartonella coupled with DNA sequencing for species determination. However, the presence of other Bartonella spp. in the sample being tested may result in false-negative results for B. rochalimae, especially when Sanger sequencing is used. We developed a sensitive and specific quantitative PCR platform for B. rochalimae by targeting the intergenic transcribed spacer, gltA, and rpoB genes, which are recommended for subtyping characterization. This PCR platform achieved the limit of detection between five and 10 genomic equivalents per reaction and did not amplify DNA from other Bartonella species or selected hosts. This PCR platform is a fast and cost-effective option to be used in epidemiological evaluations of reservoirs and vectors and in detecting and quantifying B. rochalimae infection in humans.

Molecular Characterization of Tandem Repeat Protein 36 Gene of Ehrlichia canis Detected in Naturally Infected Dogs from Peru.

Ehrlichia spp. are emerging infectious pathogens, especially in the Americas. Although Ehrlichia canis is primarily a parasite of dogs, polymerase chain reaction-confirmed human infections have been reported from Mexico, Venezuela, and Costa Rica. This study reports the presence of E. canis DNA in 13.7% of 205 dogs from urban areas in Peru and of those, five were analyzed for phylogenetic variation using the Tandem Repeat Protein 36 (TRP36) gene. The use of the TRP36 gene for such analysis was validated against 16S rRNA and heat shock protein genes using Shannon's entropy bioinformatic approach. When compared with other E. canis strains previously reported, three unique and novel E. canis strains were detected. In addition, the TRP36 amino acid tandem repeat sequences of the Peruvian strains share close similarity to an E. canis strain detected from four human blood bank samples in Costa Rica. This study reports for the first time domestic dogs infected with E. canis strains closely related to a zoonotic strain, which may be of public health concern as dogs can be chronically infected with this pathogen.

Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism

Background The possibility of emergence of praziquantel-resistant Schistosoma parasites and the lack of other effective drugs demand the discovery of new schistosomicidal agents. In this context the study of compounds that target histone-modifying enzymes is extremely promising. Our aim was to investigate the effect of inhibition of EZH2, a histone methyltransferase that is involved in chromatin remodeling processes and gene expression control; we tested different developmental forms of Schistosoma mansoni using GKS343, a selective inhibitor of EZH2 in human cells. Methodology/Principal findings Adult male and female worms and schistosomula were treated with different concentrations of GSK343 for up to two days in vitro. Western blotting showed a decrease in the H3K27me3 histone mark in all three developmental forms. Motility, mortality, pairing and egg laying were employed as schistosomicidal parameters for adult worms. Schistosomula viability was evaluated with propidium iodide staining and ATP quantification. Adult worms showed decreased motility when exposed to GSK343. Also, an approximate 40% reduction of egg laying by GSK343-treated females was observed when compared with controls (0.1% DMSO). Scanning electron microscopy showed the formation of bulges and bubbles throughout the dorsal region of GSK343-treated adult worms. In schistosomula the body was extremely contracted with the presence of numerous folds, and growth was markedly slowed. RNA-seq was applied to identify the metabolic pathways affected by GSK343 sublethal doses. GSK343-treated adult worms showed significantly altered expression of genes related to transmembrane transport, cellular homeostasis and egg development. In females, genes related to DNA replication and noncoding RNA metabolism processes were downregulated. Schistosomula showed altered expression of genes related to cell adhesion and membrane synthesis pathways. Conclusions/Significance The results indicated that GSK343 presents in vitro activities against S. mansoni, and the characterization of EZH2 as a new potential molecular target establishes EZH2 inhibitors as part of a promising new group of compounds that could be used for the development of schistosomicidal agents. Author summary

Atlas of Schistosoma mansoni long non-coding RNAs and their expression correlation to protein-coding genes

Long non-coding RNAs (lncRNAs) have been widely discovered in several organisms with the help of high-throughput RNA sequencing. LncRNAs are over 200 nt-long transcripts that do not have protein-coding (PC) potential, having been reported in model organisms to act mainly on the overall control of PC gene expression. Little is known about the functionality of lncRNAs in evolutionarily ancient non-model metazoan organisms, like Schistosoma mansoni, the parasite that causes schistosomiasis, one of the most prevalent infectious-parasitic diseases worldwide. In a recent transcriptomics effort, we identified thousands of S. mansoni lncRNAs predicted to be functional along the course of parasite development. Here, we present an online catalog of each of the S. mansoni lncRNAs whose expression is correlated to PC genes along the parasite lifecycle, which can be conveniently browsed and downloaded through a new web resource http://verjolab.usp.br. We also provide access now to navigation on the co-expression networks disclosed in our previous publication, where we correlated mRNAs and lncRNAs transcriptional patterns across five life-cycle stages/forms, pinpointing biological processes where lncRNAs might act upon.

Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism

Background The possibility of emergence of praziquantel-resistant Schistosoma parasites and the lack of other effective drugs demand the discovery of new schistosomicidal agents. In this context the study of compounds that target histone-modifying enzymes is extremely promising. Our aim was to investigate the effect of inhibition of EZH2, a histone methyltransferase that is involved in chromatin remodeling processes and gene expression control; we tested different developmental forms of Schistosoma mansoni using GKS343, a selective inhibitor of EZH2 in human cells. Methodology/Principal findings Adult male and female worms and schistosomula were treated with different concentrations of GSK343 for up to two days in vitro. Western blotting showed a decrease in the H3K27me3 histone mark in all three developmental forms. Motility, mortality, pairing and egg laying were employed as schistosomicidal parameters for adult worms. Schistosomula viability was evaluated with propidium iodide staining and ATP quantification. Adult worms showed decreased motility when exposed to GSK343. Also, an approximate 40% reduction of egg laying by GSK343-treated females was observed when compared with controls (0.1% DMSO). Scanning electron microscopy showed the formation of bulges and bubbles throughout the dorsal region of GSK343-treated adult worms. In schistosomula the body was extremely contracted with the presence of numerous folds, and growth was markedly slowed. RNA-seq was applied to identify the metabolic pathways affected by GSK343 sublethal doses. GSK343-treated adult worms showed significantly altered expression of genes related to transmembrane transport, cellular homeostasis and egg development. In females, genes related to DNA replication and noncoding RNA metabolism processes were downregulated. Schistosomula showed altered expression of genes related to cell adhesion and membrane synthesis pathways. Conclusions/Significance The results indicated that GSK343 presents in vitro activities against S. mansoni, and the characterization of EZH2 as a new potential molecular target establishes EZH2 inhibitors as part of a promising new group of compounds that could be used for the development of schistosomicidal agents. Author summary

Atlas of Schistosoma mansoni long non-coding RNAs and their expression correlation to protein-coding genes

Long non-coding RNAs (lncRNAs) have been widely discovered in several organisms with the help of high-throughput RNA sequencing. LncRNAs are over 200 nt-long transcripts that do not have protein-coding (PC) potential, having been reported in model organisms to act mainly on the overall control of PC gene expression. Little is known about the functionality of lncRNAs in evolutionarily ancient non-model metazoan organisms, like Schistosoma mansoni, the parasite that causes schistosomiasis, one of the most prevalent infectious-parasitic diseases worldwide. In a recent transcriptomics effort, we identified thousands of S. mansoni lncRNAs predicted to be functional along the course of parasite development. Here, we present an online catalog of each of the S. mansoni lncRNAs whose expression is correlated to PC genes along the parasite lifecycle, which can be conveniently browsed and downloaded through a new web resource http://verjolab.usp.br. We also provide access now to navigation on the co-expression networks disclosed in our previous publication, where we correlated mRNAs and lncRNAs transcriptional patterns across five life-cycle stages/forms, pinpointing biological processes where lncRNAs might act upon.

The DNA methyltransferase inhibitor zebularine exerts antitumor effects and reveals BATF2 as a poor prognostic marker for childhood medulloblastoma.

Medulloblastoma (MB) is the most common solid tumor among pediatric patients and corresponds to 20 % of all pediatric intracranial tumors in this age group. Its treatment currently involves significant side effects. Epigenetic changes such as DNA methylation may contribute to its development and progression. DNA methyltransferase (DNMT) inhibitors have shown promising anticancer effects. The agent Zebularine acts as an inhibitor of DNA methylation and shows low toxicity and high efficacy, being a promising adjuvant agent for anti-cancer chemotherapy. Several studies have reported its effects on different types of tumors; however, there are no studies reporting its effects on MB. We analyzed its potential anticancer effects in four pediatric MB cell lines. The treatment inhibited proliferation and clonogenicity, increased the apoptosis rate and the number of cells in the S phase (p < 0.05), as well as the expression of p53, p21, and Bax, and decreased cyclin A, Survivin and Bcl-2 proteins. In addition, the combination of zebularine with the chemotherapeutic agents vincristine and cisplatin resulted in synergism and antagonism, respectively. Zebularine also modulated the activation of the SHH pathway, reducing SMO and GLI1 levels and one of its targets, PTCH1, without changing SUFU levels. A microarray analysis revealed different pathways modulated by the drug, including the Toll-Like Receptor pathway and high levels of the BATF2 gene. The low expression of this gene was associated with a worse prognosis in MB. Taken together, these data suggest that Zebularine may be a potential drug for further in vivo studies of MB treatment.

The linked human imprintome v1.0: over 120 genes confirmed as imprinted impose a major review on previous censuses.

The whole set of human imprinted genes, termed imprintome, is here analysed by means of a reasonable, valid application of the Semantic Web and Linked Data approaches to a few structured datasets in order to provide a comprehensive collection of imprinted genes in the human genome. Thus, we have stored, organised, filtered, and analysed massive amounts of existing data on human imprinted genes towards compiling, structuring and linking data to comprise a sharing resource for genome and epigenome interrogated studies. Our datasets of linked data are the actual research outcome of this human imprintome analysis because as genomics become more and more data intensive, due to huge amounts of biological data, so does our needs for more structured data to be easier mined and shared. We present the resulting first version of the Linked Human Imprintome as a project within Linked Open Data (LOD) initiative (http://lod-cloud.net/) through Data Hub (http:// thedatahub.org/en/dataset/a-draft-version-of-the-linked-human-imprintome).

A novel A2 allele found in Leishmania (Leishmania) infantum chagasi.

Visceral leishmaniasis (VL) is a widely spread zoonotic disease. In Brazil the disease is caused by Leishmania (Leishmania)infantum chagasi. Peridomestic sandflies acquire the etiological agent by feeding on blood of infected reservoir animals, such as dogs or wildlife. The disease is endemic in Brazil and epidemic foci have been reported in densely populated cities all over the country. Many clinical features of Leishmania infection are related to the host-parasite relationship, and many candidate virulence factors in parasites that cause VL have been studied such as A2 genes. The A2 gene was first isolated in 1994 and then in 2005 three new alleles were described in Leishmania (Leishmania) infantum. In the present study we amplified by polymerase chain reaction (PCR) and sequenced the A2 gene from the genome of a clonal population of L. (L.) infantum chagasi VL parasites. The L. (L.) infantum chagasi A2 gene was amplified, cloned, and sequenced in. The amplified fragment showed approximately 90% similarity with another A2 allele amplified in Leishmania (Leishmania) donovani and in L. (L.) infantum described in literature. However, nucleotide translation shows differences in protein amino acid sequence, which may be essential to determine the variability of A2 genes in the species of the L. (L.) donovani complex and represents an additional tool to help understanding the role this gene family may have in establishing virulence and immunity in visceral leishmaniasis. This knowledge is important for the development of more accurate diagnostic tests and effective tools for disease control.

A novel A2 allele found in Leishmania (Leishmania) infantum chagasi / Novo alelo do gene A2 descrito em Leishmania (Leishmania) infantum chagasi

Visceral leishmaniasis (VL) is a widely spread zoonotic disease. In Brazil the disease is caused by Leishmania (Leishmania) infantum chagasi. Peridomestic sandflies acquire the etiological agent by feeding on blood of infected reservoir animals, such as dogs or wildlife. The disease is endemic in Brazil and epidemic foci have been reported in densely populated cities all over the country. Many clinical features of Leishmania infection are related to the host-parasite relationship, and many candidate virulence factors in parasites that cause VL have been studied such as A2 genes. The A2 gene was first isolated in 1994 and then in 2005 three new alleles were described in Leishmania (Leishmania) infantum. In the present study we amplified by polymerase chain reaction (PCR) and sequenced the A2 gene from the genome of a clonal population of L. (L.) infantum chagasi VL parasites. The L. (L.) infantum chagasi A2 gene was amplified, cloned, and sequenced in. The amplified fragment showed approximately 90 percent similarity with another A2 allele amplified in Leishmania (Leishmania) donovani and in L.(L.) infantum described in literature. However, nucleotide translation shows differences in protein amino acid sequence, which may be essential to determine the variability of A2 genes in the species of the L. (L.) donovani complex and represents an additional tool to help understanding the role this gene family may have in establishing virulence and immunity in visceral leishmaniasis. This knowledge is important for the development of more accurate diagnostic tests and effective tools for disease control.

A leishmaniose visceral (LV) é uma zoonose amplamente disseminada, causada no Brasil pela Leishmania (Leishmania) infantum chagasi. Flebotomíneos vetores adquirem o agente etiológico, alimentando-se do sangue de animais contaminados, como cachorros ou animais selvagens. A doença é endêmica no Brasil, e focos de epidemia são relatados em cidades densamente povoadas por todo o país. Muitas manifestações clínicas relacionadas à infecção por Leishmania estão ligadas à relação parasito-hospedeiro, e vários possíveis fatores de virulência dos parasitas, que causam a LV, são alvos de estudo, tais como os genes A2. O gene A2 foi isolado pela primeira vez em 1994 e, em seguida, em 2005, três novos alelos foram descritos em Leishmania (Leishmania) infantum. No presente estudo, um fragmento do gene A2 de uma população clonal de L.(L.) infantum chagasi foi amplificado por PCR e sua sequência de nucleotídeos determinada. O fragmento mostrou 90 por cento de similaridade com alelos do gene A2 de Leishmania (Leishmania) donovani e de L. (L.) infantum, descritos na literatura. Entretanto, a tradução da sequência de nucleotídeos mostra diferenças na sequência de aminoácidos da proteína, que podem ser essenciais em determinar a variabilidade do gene A2 em espécies do complexo L. (L.) donovani e representa uma ferramenta adicional na compreenssão do papel dessa família de genes na virulência e imunidade da leishmaniose visceral. O conhecimento dessa variação é importante para o desenvolvimento de testes diagnósticos mais precisos e ferramentas mais eficazes no controle da doença.