DEFAR - Departamento de Farmácia
URI permanente desta comunidadehttp://www.hml.repositorio.ufop.br/handle/123456789/530
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Item In vitro and in silico evaluation of the schistosomicidal activity of eugenol derivatives using biochemical, molecular, and morphological tools.(2022) Souza, Isabella Maria Monteiro de; Novaes, Rômulo Dias; Gonçalves, Reggiani Vilela; Fialho, Felipe Leonardo Bley; Carvalho, Diogo Teixeira; Souza, Thiago Belarmino de; Dias, Danielle Ferreira; Lavorato, Stefânia Neiva; Souza, Raquel Lopes Martins; Marques, Marcos José; Castro, Aline PereiraBackground: Eugenol shows both antibacterial and antiparasitic activities, suggesting that it might be evaluated as an option for the treatment of praziquantel-resistant schistosome. Methods: The in vitro activities of three eugenol derivatives (FB1, FB4 and FB9) on adult worms from Schistosoma mansoni were examined by fluorescence and scanning electron microscopy to analyze effects on the excretory system and integument damage, respectively. Biochemical tests with verapamil (a calcium channel antagonist) and ouabain (a Na+ /K+-ATPase pump inhibitor) were used to characterize eugenol derivative interactions with calcium channels and the Na+/K+-ATPase, while in silico analysis identified potential Na+/K+-ATPase binding sites. Results: The compounds showed effective doses (ED50) of 0.324 mM (FB1), 0.167 mM (FB4), and 0.340 mM (FB9). In addition, FB4 (0.322 mM), which showed the lowest ED50, ED90 and ED100 (p < 0.05), caused the most damage to the excretory system and integument, according to both fluorescence and scanning electron microscopy analysis. The death of adult worms was delayed by ouabain treatment plus FB1 (192 versus 72 hours) and FB9 (192 versus 168 hours), but the response to FB4 was the same in the presence or absence of ouabain. Besides, no changes were noted when all of the eugenol derivatives were combined with verapamil. Moreover, FB1 and FB9 inhibited Na+/K+-ATPase activity according to in silico analysis but FB4 did not show a time- dependent relationship and may act on targets other than the parasite Na+/K+-ATPase. Conclusion: Eugenol derivatives, mainly FB4 when compared to FB1 and FB9, seem to act more effectively on the integument of adult S. mansoni worms.Item Deep sequencing of small RNAs reveals the repertoire of miRNAs and piRNAs in Biomphalaria glabrata.(2020) Queiroz, Fábio Ribeiro; Portilho, Laysa Gomes; Jeremias, Wander de Jesus; Babá, Élio Hideo; Amaral, Laurence Rodrigues do; Silva, Luciana Maria; Coelho, Paulo Marcos Zech; Caldeira, Roberta Lima; Gomes, Matheus de SouzaBACKGROUND 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.Item Genome-wide identification, characterisation and expression profiling of the ubiquitin-proteasome genes in Biomphalaria glabrata.(2019) Portilho, Laysa Gomes; Duarte, Bruna Custódio Dias; Queiroz, Fábio Ribeiro; Ribeiro, Thales Henrique Cherubino; Jeremias, Wander de Jesus; Babá, Élio Hideo; Coelho, Paulo Marcos Zech; Morais, Enyara Rezende; Cabral, Fernanda Janku; Caldeira, Roberta Lima; Gomes, Matheus de SouzaBACKGROUND 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.Item Enhanced schistosomicidal efficacy of tartar emetic encapsulated in pegylated liposomes.(2003) Melo, Alan Lane de; Barcellos, Neila Marcia Silva; Demicheli, Cynthia Peres; Frezard, Frederic Jean GeorgesThe aim of the present study was to evaluate the ability of liposomes to improve the efficacy of tartar emetic (TA) against established Schistosoma mansoni infection. TA was used as a schistosomicidal drug model and both conventional liposomes (CL) and long-circulating pegylated liposomes (LCL) were evaluated. In the first experiment, TA, either free or encapsulated within CL or LCL, was given intraperitoneally (i.p.) as a single dose of 11 mg Sb/kg to mice experimentally infected with S. mansoni. Only the group treated with LCL showed a significant (55%) reduction in the worm burden, compared to the control groups (untreated or treated with empty LCL). In the second experiment, the efficacy of TA-containing LCL was evaluated at a higher dose (27 mg Sb/kg) by both subcutaneous (s.c.) and i.p. routes. Reduction levels of 67 and 82% were achieved by s.c. and i.p. routes, respectively. Strikingly, all mice survived to this high dose of antimony. This is in contrast with free TA that was lethal in 100% of mice at the same dose. The present work demonstrates that LCL reduce the acute toxicity of TA and effectively deliver this drug to S. mansoni during the late stages of parasite infection.