Schistosomiasis: Setting Routes for Drug Discovery

Schistosomiasis is the second most prevalent parasitic disease in the world. Currently, the treatment of this disease relies on a single drug, praziquantel, and due to the identification of resistant parasites, the development of new drugs is urged. The demand for the development of robust high‐throughput parasite screening techniques is increasing as drug discovery research in schistosomiasis gains significance. Here, we review the most common methods used for compound screening in the parasites life stages and also summarize some of the methods that have been recently developed. In addition, we reviewed the methods most commonly implemented to search for promising targets and how they have been used to validate new targets against the parasite Schistosoma mansoni. We also review some promising targets in this parasite and show the main approaches and the major advances that have been achieved by those studies. Moreover, we share our experiences in schistosomiasis drug discovery attained with our S. mansoni drug screening platform establishment

DNA barcoding as a valuable tool for delimiting mollusk species of the genus Biomphalaria Preston, 1910 (Gastropoda: Planorbidae)

IntroductionThe genus Biomphalaria in Brazil includes 11 species and one subspecies, three of which are intermediate hosts of Schistosoma mansoni. Due to the recent evolution of this group, some species are difficult to identify based on morphological characters, making the use of genetic markers necessary for species identification. This study aimed to evaluate the use of partial sequences of the cytochrome c oxidase I (coi) gene for the identification of Biomphalaria species using phylogenetic reconstruction and species delimitation algorithms. The study tested the use of DNA barcoding technique for species delimitation within the genus.MethodsDNA barcoding was performed by sequencing a partial region of the coi gene from specimens, and the sequences were analyzed using phylogenetic reconstruction and algorithms to delimit Operational Taxonomic Units (OTUs).ResultsThe study found that the use of the coi gene in the reconstruction of the phylogeny of the genus might be an alternative for understanding the evolution and dispersion of species. However, this marker alone is not enough to solve complex taxonomic problems within the genus. A total of 223 sequences were analyzed, 102 of which could be separated using the barcode gap, enabling the correct identification of seven taxa.DiscussionThe study demonstrated that accurate mollusk identification is necessary for effective schistosomiasis control. The DNA barcoding methodology was found to be promising for accurate mollusk identification, which is crucial for concentrating schistosomiasis control efforts in places where it is needed

Smp38 MAP Kinase Regulation in Schistosoma mansoni: Roles in Survival, Oviposition, and Protection Against Oxidative Stress

Eukaryotic protein kinases (ePKs) are good medical targets for drug development in different biological systems. ePKs participate in many cellular processes, including the p38 MAPK regulation of homeostasis upon oxidative stress. We propose to assess the role of Smp38 MAPK signaling pathway in Schistosoma mansoni development and protection against oxidative stress, parasite survival, and also to elucidate which target genes have their expression regulated by Smp38 MAPK. After a significant reduction of up to 84% in the transcription level by Smp38 MAPK gene knockdown, no visible phenotypic changes were reported in schistosomula in culture. The development of adult worms was tested in vivo in mice infected with the Smp38 knocked-down schistosomula. It was observed that Smp38 MAPK has an essential role in the transformation and survival of the parasites as a low number of adult worms was recovered. Smp38 knockdown also resulted in decreased egg production, damaged adult worm tegument, and underdeveloped ovaries in females. Furthermore, only ~13% of the eggs produced developed into mature eggs. Our results suggest that inhibition of the Smp38 MAPK activity interfere in parasites protection against reactive oxygen species. Smp38 knockdown in adult worms resulted in 80% reduction in transcription levels on the 10th day, with consequent reduction of 94.4% in oviposition in vitro. In order to search for Smp38 MAPK pathway regulated genes, we used an RNASeq approach and identified 1,154 DEGs in Smp38 knockdown schistosomula. A substantial proportion of DEGs encode proteins with unknown function. The results indicate that Smp38 regulates essential signaling pathways for the establishment of parasite homeostasis, including genes related to antioxidant defense, structural composition of ribosomes, spliceosomes, cytoskeleton, as well as, purine and pyrimidine metabolism pathways. Our data show that the Smp38 MAPK signaling pathway is a critical route for parasite development and may present attractive therapeutic targets for the treatment and control of schistosomiasis

Schistosoma mansoni coactivator associated arginine methyltransferase 1 (SmCARM1) effect on parasite reproduction

IntroductionThe human blood fluke parasite Schistosoma mansoni relies on diverse mechanisms to adapt to its diverse environments and hosts. Epigenetic mechanisms play a central role in gene expression regulation, culminating in such adaptations. Protein arginine methyltransferases (PRMTs) promote posttranslational modifications, modulating the function of histones and non-histone targets. The coactivator-associated arginine methyltransferase 1 (CARM1/PRMT4) is one of the S. mansoni proteins with the PRMT core domain.MethodsWe carried out in silico analyses to verify the expression of SmPRMTs in public datasets from different infection stages, single-sex versus mixed-worms, and cell types. The SmCARM1 function was evaluated by RNA interference. Gene expression levels were assessed, and phenotypic alterations were analyzed in vitro, in vivo, and ex vivo.ResultsThe scRNAseq data showed that SmPRMTs expression is not enriched in any cell cluster in adult worms or schistosomula, except for Smcarm1 expression which is enriched in clusters of ambiguous cells and Smprmt1 in NDF+ neurons and stem/germinal cells from schistosomula. Smprmt1 is also enriched in S1 and late female germ cells from adult worms. After dsRNA exposure in vitro, we observed a Smcarm1 knockdown in schistosomula and adult worms, 83 and 69%, respectively. Smcarm1-knockdown resulted in reduced oviposition and no significant changes in the schistosomula or adult worm phenotypes. In vivo analysis after murine infection with Smcarm1 knocked-down schistosomula, showed no significant change in the number of worms recovered from mice, however, a significant reduction in the number of eggs recovered was detected. The ex vivo worms presented a significant decrease in the ovary area with a lower degree of cell differentiation, vitelline glands cell disorganization, and a decrease in the testicular lobe area. The worm tegument presented a lower number of tubercles, and the ventral sucker of the parasites presented a damaged tegument and points of detachment from the parasite body.DiscussionThis work brings the first functional characterization of SmCARM1 shedding light on its roles in S. mansoni biology and its potential as a drug target. Additional studies are necessary to investigate whether the reported effects of Smcarm1 knockdown are a consequence of the SmCARM1-mediated methylation of histone tails involved in DNA packaging or other non-histone proteins

Vias de sinalização de proteínas MAPKS em Schistosoma mansoni

Submitted by Maria Gomes ([email protected]) on 2019-10-30T21:12:37Z No. of bitstreams: 1 Tese_SandraGava_final.pdf: 52810684 bytes, checksum: eabef99bd3268ea8c5b4f60ed6323643 (MD5)Approved for entry into archive by Eliane Andrade ([email protected]) on 2019-10-31T14:14:49Z (GMT) No. of bitstreams: 1 Tese_SandraGava_final.pdf: 52810684 bytes, checksum: eabef99bd3268ea8c5b4f60ed6323643 (MD5)Made available in DSpace on 2019-10-31T15:23:44Z (GMT). No. of bitstreams: 1 Tese_SandraGava_final.pdf: 52810684 bytes, checksum: eabef99bd3268ea8c5b4f60ed6323643 (MD5) Previous issue date: 2017-04-27FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas GeraisCAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorA identificação e caracterização dos mecanismos e moléculas envolvidos em sinalização celular são essenciais para o entendimento da biologia parasitária de S. mansoni. Proteínas quinases desempenham papel chave em vias de sinalização e têm sido propostas como potenciais alvos para o desenvolvimento de novas drogas. O genoma de S. mansoni codifica 252 proteínas quinases eucarióticas (ePKs), entretanto, apenas 32 possuem alguma evidência funcional experimental. Nosso grupo demonstrou por estudos funcionais que MAPKs estão envolvidas no desenvolvimento, reprodução e/ou sobrevivência do parasito e podem, portanto, serem consideradas alvos potenciais para o desenvolvimento e reprodução de S. mansoni. Para isso, as ePKs em S. mansoni, o presente trabalho tem como motivação ´principal contribuir para sua caracterização experimental correlacionando dados de expressão com o desenvolvimento de novas drogas. Em virtude da escassez de dados sobre a função das ePKs em S. mansoni, o presente trabalho tem como motivação principal contribuir para sua caracterização experimental correlacionando dados de expressão com o desenvolvimento e reprodução de S. mansoni. Para isso, as ePKs SmCaMK2, SmERK-1, SmERK-2, SmJNK e Smp38 foram silenciadas em esquistossômulos por RNA de interferência, incluindo três réplicas biológicas. Após dois dias foi realizada a extração de RNA total e a avaliação dos níveis de transcrição por RT-qPCR. Para todos os genes, com exceção de SmERK-2, foi observada uma redução média de 75% nos níveis de transcrito dos alvos silenciados. O RNA extraído foi utilizado para construção de bibliotecas do tipo fragmentado, sequenciadas na plataforma Illumina HiSeq 2500. As sequências obtidas foram mapeadas na última versão do genoma de referência de S. mansoni e os genes diferencialmente expressos (DEGS) foram identificados pela comparação das amostras de parasitos expostos aos dsRNAs específicos em relação aos parasitos não tratados. Foram identificados 606 DEGs em esquistossômulos SmJNK knockdown e 1154 DEGs em esquistossômulos Smp38 knockdown. Grande parte dos DEGs (505) encontrados codificam proteínas ainda sem função conhecida. O silenciamento de SmJNK promove diminuição de expressão de genes relacionados à defesa antioxidante, à composição estrutural de ribossomos e do citoesqueleto e dos spliceossomos. O silenciamento de Smp38 MAPK promove diminuição da expressão de genes relacionados à defesa antioxidante, à composição estrutural de ribossomos e do citoesqueleto e dos spliceossomos. O silenciamento de Smp38 MAPK promove diminuição da expressão de genes relacionados à composição estrutural de ribossomos, do citoesqueleto, dos spliceossomos e dos fagossomos, além da diminuição da expressão de genes das vias de fosforilação oxidativa e metabolismo de purinas. Este trabalho possibilitou uma melhor compreensão das vias de sinalização de SmJNK e Smp38, elucidando papéis funcionais dessas MAPKs e dos alvos por elas regulados

Estudo funcional da proteína quinase JNK de Schistosoma mansoniatravés de expressão heteróloga no organismo modelo Caenorhabditis elegans

Submitted by Nuzia Santos ([email protected]) on 2013-11-20T18:45:22Z No. of bitstreams: 1 Dissertacao-SandraGrossiGava_Final.pdf: 2345943 bytes, checksum: 542baff1ce20827a286564ee6746784b (MD5)Made available in DSpace on 2013-11-20T18:45:22Z (GMT). No. of bitstreams: 1 Dissertacao-SandraGrossiGava_Final.pdf: 2345943 bytes, checksum: 542baff1ce20827a286564ee6746784b (MD5) Previous issue date: 2013Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brasil.A identificação e caracterização dos mecanismos e moléculas envolvidos em sinalização celular são essenciais para o entendimento da biologia parasitária do S. mansoni. Proteína quinases desempenham papel chave em vias de sinalização e tem sido propostas como potenciais alvos para o desenvolvimento de novas drogas anti-Schistosoma. Visto que a caracterização funcional em S. mansoni é dificultada por limitações nos métodos de transformação genética deste parasito, o presente estudo propõe o uso de C. elegans como um modelo para a expressão heteróloga de genes de S. mansonique codificam proteínas quinases. Genes que codificam proteína quinases em S. mansoni, homólogos aos identificados em C. elegans, foram selecionados pelo nosso grupo a partir do proteoma do parasito através de uma abordagem filogenômica. Inicialmente, foi selecionada a proteína quinase JNK, que participa da via de sinalização das MAP quinases para a realização das análises experimentais. Em C. elegans, a proteína JNK está relacionada ao aumento de longevidade e da resistência aos estresses térmico e oxidativo. Oligonucleotídeos específicos foram desenhados para amplificar a região promotora do gene em C. elegans bem como as regiões codificantes (CDS) em ambos os organismos. A região promotora foi amplificada a partir do DNA genômico extraído de C. elegans adultos. O RNA total foi extraído de esquistossômulos e C. elegans adultos. As CDS foram amplificadas a partir do cDNA sintetizado e os fragmentos de DNA resultantes foram clonados em E. coliDH5α. As construções obtidas foram digeridas com enzimas de restrição selecionadas de forma a linearizar o vetor contendo a região promotora e recuperar as CDS. Posteriormente, foram realizadas subclonagens através da ligação das CDS de C. elegans e S. mansoni com a construção contendo a região promotora. C. elegans N2 receberam as construções finais através de microinjeção. Foram obtidas três linhagens que expressam Ce_JNK-1, denominadas N2 Ex01[Ce_jnk-1], N2 Ex02[Ce_jnk-1]e N2 Ex03[Ce_jnk-1]e duas linhagens expressando Sm_JNK, denominadas N2 Ex04[Sm_jnk] e N2 Ex05[Sm_jnk]. Os níveis de expressão de Sm_JNK e Ce_JNK-1 nas linhagens transgênicas foram avaliados por RT-PCR quantitativo. Apesar do aumento da expressão de JNK observado nas linhagens transgênicas, não houve aumento na longevidade das mesmas. Embora esta seja a primeira utilização de expressão heteróloga em C. elegans para investigar a função de genes de S. mansoni, esta técnica tem sido utilizada com sucesso para nematóides parasitos e pode tornar-se uma abordagem alternativa para os estudos funcionais em outros parasitos.The identification and characterization ofmechanisms and molecules involved in cell signaling are essential to the understanding of the S. mansoni parasite’s biology. Protein kinases play key roles in signaling pathways and have been proposed as potential targets for the development of new anti-schistosome drugs. Since functional characterization in S. mansoni is hampered by limitations in methods for genetic transformation in this parasite, the present study proposes to use C. elegans as a model for heterologous expression of S. mansoni genes encoding protein kinases. S. mansoniprotein kinase coding genes orthologous to those identified in C. elegans were selected from the parasite’s proteome by using a phylogenomic approach. Initially, the protein quinase JNK that participates in the MAP kinases signaling pathwaywas selected to perform the experimental analyses. In C. elegans, JNK is related to increased longevity and resistance to oxidative and thermal stress. Specific primers were designed to amplify promoter regions of the JNK gene in C. elegans as well as the protein coding regions (CDS) in both organisms. The promoter region was amplified from the adult nematode’s DNA. Total RNA was extracted from schistosomula and mature C. elegans. CDS were amplified from synthesized cDNA and the resulting DNA fragments were cloned in E. coli DH5α. The construction obtained was digested with restriction enzymes to linearize the vector containing the promoter region and recover the CDS. Subsequently, subcloning was performed by ligation of C. elegansand S. mansoni CDS with the final construct containing the promoter region. C. elegansN2 received the final constructions through microinjections. Weobtained three lineages that expresses Ce_JNK-1, named N2 Ex01[Ce_jnk-1], N2 Ex02[Ce_jnk-1]and N2 Ex03[Ce_jnk-1] and two line ages that express Sm_JNK, named N2 Ex04[Sm_jnk] and N2 Ex05[Sm_jnk]. The expression levels of Ce_JNK-1 and Sm_JNK in transgenic lineages were measured by quantitative RT-PCR. Despite the increased expression of the JNK gene in the transgenic lineages, increased longevity was not observed in these organisms. Although this is the first use of heterologous expression in C. elegans to investigate gene functions in S. mansoni, it has been used successfully for nematode parasites and may become an alternative approach to functional studies in other parasites

Chapter Schistosomiasis: Setting Routes for Drug Discovery

Air quality sampling campaigns in three European subway systems (Barcelona, Athens and Oporto) were conducted in order to characterise particulate matter (PM) to better understand the main factors controlling it. PM mass concentrations varied among the European subway platforms, and also within the same underground system, this being mainly associated to differences in the design of the stations and tunnels, system age, train frequency, ventilation and air‐conditioning systems, commuter's density, rails geometry and outdoor air quality. PM concentrations displayed clear diurnal patterns, depending largely on the operation and frequency of the trains and the ventilation system. Chemically, subway PM2.5 on the platforms consisted of iron, carbonaceous material, crustal matter, secondary inorganic compounds, insoluble sulphate, halite and trace elements. Fe was the most abundant element, accounting for 19–46% of the bulk PM2.5, which is generated mainly from mechanical wear at rail‐wheel‐brake interfaces. A source apportionment analysis allowed the identification of outdoor (sea salt, fuel‐oil combustion and secondary aerosol) and subway sources on platforms. The use of air‐conditioning inside the trains was an effective approach to reduce exposure concentrations, being more efficient removing coarser particles. PM concentrations inside the trains were greatly affected by the surrounding (i.e. platforms and tunnels) air quality conditions

Heterologous expression in Caenorhabditis elegans as an alternative approach to functional studies in Schistosoma mansoni.

The lack of an accurate diagnosis has been a serious obstacle to the advancement of the anti-Trypanosoma cruzi chemotherapy and long-term infection can result in different health risks to human. PCRs are alternative methods, more sensitive than conventional parasitological techniques, which due to their low sensitivities are considered unsuitable for these purposes. The aim of this study was to investigate a sensitive diagnostic strategy to quantify blood and cardiac tissues parasites based on real-time PCR tools during acute and chronic phases of murine Chagas disease, as well as to monitor the evolution of infection in those mice under specific treatment. In parallel, fresh blood examination, immunological analysis and quantification of cardiac inflammation were also performed to confront and improve real-time PCR data. Similar profiles of parasitemia curves were observed in both quantification techniques during the acute phase of the infection. In contrast, parasites could be quantified only by real-time PCR at 60 and 120 days of infection. In cardiac tissue, real-time PCR detected T. cruzi DNA in 100% of infected mice, and using this tool a significant Pearson correlation between parasite load in peripheral blood and in cardiac tissue during acute and chronic phases was observed. Levels of serum CCL2, CCL5 and nitric oxide were coincident with parasite load but focal and diffuse mononuclear infiltrates was observed, even with significant (p < 0.05) reduction of parasitism after 60 days of infection. Later, this methodology was used to monitor the evolution of infection in animals treated with itraconazole (Itz). Itz-treatment induced a reduction of parasite load in both blood and cardiac muscle at the treatment period, but after the end of chemotherapy an increase of parasitism was detected. Interestingly, inflammatory mediators levels and heart inflammation intensity had similar evolution to the parasite load, in the group of animals treated. Taken together, our data show that real-time PCR strategy used was suitable for studies of murine T. cruzi infection and may prove useful in investigations involving experimental chemotherapy of the disease and the benefits of treatment in relation to parasitism and inflammatory respons

Landscape of the spliced leader trans-splicing mechanism in Schistosoma mansoni

Abstract Spliced leader dependent trans-splicing (SLTS) has been described as an important RNA regulatory process that occurs in different organisms, including the trematode Schistosoma mansoni. We identified more than seven thousand putative SLTS sites in the parasite, comprising genes with a wide spectrum of functional classes, which underlines the SLTS as a ubiquitous mechanism in the parasite. Also, SLTS gene expression levels span several orders of magnitude, showing that SLTS frequency is not determined by the expression level of the target gene, but by the presence of particular gene features facilitating or hindering the trans-splicing mechanism. Our in-depth investigation of SLTS events demonstrates widespread alternative trans-splicing (ATS) acceptor sites occurring in different regions along the entire gene body, highlighting another important role of SLTS generating alternative RNA isoforms in the parasite, besides the polycistron resolution. Particularly for introns where SLTS directly competes for the same acceptor substrate with cis-splicing, we identified for the first time additional and important features that might determine the type of splicing. Our study substantially extends the current knowledge of RNA processing by SLTS in S. mansoni, and provide basis for future studies on the trans-splicing mechanism in other eukaryotes

Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries

Background Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks. Methods The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned. Results A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P &lt; 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31). Conclusion Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)