The World Health Organization (WHO) estimates that approximately 240 million people in 78 countries require treatment for schistosomiasis, an endemic disease caused by trematodes of the genus Schistosoma. In Brazil, Schistosoma mansoni is the only species representative of the genus whose passage through an invertebrate host, snails of the genus Biomphalaria, is obligatory before infecting a mammalian host, including humans. The availability of the genome and transcriptome of B. glabrata makes studying the regulation of gene expression, particularly the regulation of miRNA and piRNA processing pathway genes, possible. This might assist in better understanding the biology of B. glabrata as well as its relationship to the parasite S. mansoni. Some aspects of this interaction are still poorly explored, including the participation of non-coding small RNAs, such as miRNAs and piRNAs, with lengths varying from 18 to 30 nucleotides in mature form, which are potent regulators of gene expression. Using bioinformatics tools and quantitative PCR, we characterized and validated the miRNA and piRNA processing pathway genes in B. glabrata. In silico analyses showed that genes involved in miRNA and piRNA pathways were highly conserved in protein domain distribution, catalytic site residue conservation and phylogenetic analysis. Our study showed differential expression of putative Argonaute, Drosha, Piwi, Exportin-5 and Tudor genes at different snail developmental stages and during infection with S. mansoni, suggesting that the machinery is required for miRNA and piRNA processing in B. glabrata at all stages. These data suggested that the silencing pathway mediated by miRNAs and piRNAs can interfere in snail biology throughout the life cycle of the snail, thereby influencing the B. glabrata/S. mansoni interaction. Further studies are needed to confirm the participation of the small RNA processing pathway proteins in the parasite/host relationship, mainly the effective participation of small RNAs in regulating their target genes.
BACKGROUND Biomphalaria glabrata snails are widely distributed in schistosomiasis endemic areas like America and Caribe, displaying high susceptibility to infection by Schistosoma mansoni. After the availability of B. glabrata genome and transcriptome data, studies focusing on genetic markers and small non-coding RNAs have become more relevant. The small RNAs have been considered important through their ability to finely regulate the gene expression in several organisms, thus controlling the functions like cell growth, metabolism, and susceptibility/resistance to infection. OBJECTIVE The present study aims on identification and characterisation of the repertoire of small non-coding RNAs in B. glabrata (Bgl-small RNAs). METHODS By using small RNA sequencing, bioinformatics tools and quantitative reverse transcription polymerase chain reaction (RT-qPCR), we identified, characterised, and validated the presence of small RNAs in B. glabrata. FINDINGS 89 mature miRNAs were identified and five of them were classified as Mollusk-specific. When compared to model organisms, sequences of B. glabrata miRNAs showed a high degree of conservation. In addition, several target genes were predicted for all the mature miRNAs identified. Furthermore, piRNAs were identified in the genome of B. glabrata for the first time. The B. glabrata piRNAs showed strong conservation of uridine as first nucleotide at 5' end, besides adenine at 10th position. Our results showed that B. glabrata has diverse repertoire of circulating ncRNAs, several which might be involved in mollusk susceptibility to infection, due to their potential roles in the regulation of S. mansoni development. MAIN CONCLUSIONS Further studies are necessary in order to confirm the role of the Bgl-small RNAs in the parasite/host relationship thus opening new perspectives on interference of small RNAs in the organism development and susceptibility to infection.
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 st...
BACKGROUND Biomphalaria glabrata is the major species used for the study of schistosomiasis-related parasite-host relationships, and understanding its gene regulation may aid in this endeavor. The ubiquitin-proteasome system (UPS) performs post-translational regulation in order to maintain cellular protein homeostasis and is related to several mechanisms, including immune responses. OBJECTIVE The aims of this work were to identify and characterise the putative genes and proteins involved in UPS using bioinformatic tools and also their expression on different tissues of B. glabrata . METHODS The putative genes and proteins of UPS in B. glabrata were predicted using BLASTp and as queries reference proteins from model organism. We characterised these putative proteins using PFAM and CDD software describing the conserved domains and active sites. The phylogenetic analysis was performed using ClustalX2 and MEGA5.2. Expression evaluation was performed from 12 snail tissues using RPKM. FINDINGS 119 sequences involved in the UPS in B. glabrata were identified, which 86 have been related to the ubiquitination pathway and 33 to proteasome. In addition, the conserved domains found were associated with the ubiquitin family, UQ_con, HECT, U-box and proteasome. The main active sites were lysine and cysteine residues. Lysines are responsible and the starting point for the formation of polyubiquitin chains, while the cysteine residues of the enzymes are responsible for binding to ubiquitin. The phylogenetic analysis showed an organised distribution between the organisms and the clades of the sequences, corresponding to the tree of life of the animals, for all groups of sequences analysed. The ubiquitin sequence was the only one with a high expression profile found in all libraries, inferring its wide range of performance. MAIN CONCLUSIONS Our results show the presence, conservation and expression profile of the UPS in this mollusk, providing a basis and new knowledge for other studies involving this system. Due to the importance of the UPS and B. glabrata , this work may influence the search for new methodologies for the control of schistosomiasis.
Objective: The objective of this study was to standardize the Ficoll gradient technique for the circulating hematopoietic stem cell (HSC) isolation for the assembly of the peripheral blood mononuclear cell (PBMC) biorepository of breast cancer (BC) patients attended in the Clinical Oncology Service of Instituto Mário Penna. Methods: The study protocol was approved by the Ethics Committee of Instituto Mário Penna (CAEE 82703418.8.0000.5121). In recommended protocols, 15 mL of blood was used. At first, we adapted this volume due to the limited amounts of samples for research available. Blood was collected in a 9-mL sodium heparin tube. The experiments were performed in 50-mL conical tubes, but with reduced blood volume, and no PBMC ring was formed. It was necessary to change to 15 mL conical tubes. Finally, the remaining red blood cells were lysed with ammonium chloride. However, with the reduced volume, this solution lysed the PBMC too. Then, we decided to remove this step from the protocol. Results: We obtained 8.06×106 cells/mm3 with 80% viability. Data were confirmed by a Neubauer camera and an automatic cell counter. The HSCs were labeled with antibodies against CD34 and CD133 by flow cytometry. Conclusion: The characterization of HSCs is important to link tumor-associated HSCs with malignant and immunosuppressive phenotypes. Studies are in progress with this standardization, and they will permit us to perform the HSC characterization of BC patients with a better knowledge of tumor microenvironment.
Objective: The identification and characterization of miRNAs derived from microvesicles of breast cancer patients is the main goal of this study. Methods: A cohort of 28 patients was evaluated, 28.6% are HER2 overexpressed, 46.4% are triple-negative, and 25% are triple-positive. The study protocol was approved by the Ethics Committee of Instituto Mário Penna (CAEE 82703418.8.0000.5121). For this purpose, the peripheral blood was collected in EDTA tubes and obtained the red blood cell-free plasm. The microvesicles were purified from plasm using the microRNeasy kit (Qiagen) with posterior small RNA precipitation, according to manufacturer’s instructions. The small RNA obtained was used for high throughput sequencing using the QIAseq miRNA Library kit (Qiagen) for library construction, according to manufacturer’s instructions. The sequencing was performed by the Illumina NextSeq 550. The sequences obtained were filtered by quality, the adapters were removed, and small RNA patterns were evaluated using the Unitas (version 1.7.8). Results: The three groups of patients showed a significant abundance of miRNA profiles. The triple-negative breast cancer (TNBC) patients showed the highest relative abundance, which can be due to the more intense exocrine activity of this type of tumor. Furthermore, our results highlighted a great abundance of miR-223-3p in the TNBC patient group. Conclusion: Normally, TNBC patients have an aggressive condition of disease, and cell proliferation, migration, and invasion are common events. These characteristics can be regulated by miRNAs exported from tumor cells in microvesicles. Several miRNAs are already related to these events, and this makes them potential therapeutic or diagnostic targets for this disease. miR-223-3p was previously related to epithelial-mesenchymal transition, cell proliferation, and migration. This phenotypic effect is a strong indication that this miRNA could be used as a biomarker in TNBC management and opens great possibilities for further validation of this as a tool for liquid biopsy tests.
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