Trypsin-like serine peptidase profiles in the egg, larval, and pupal stages of Aedes albopictus

BACKGROUND: Aedes albopictus, a ubiquitous mosquito, is one of the main vectors of dengue and yellow fever, representing an important threat to public health worldwide. Peptidases play key roles in processes such as digestion, oogenesis, and metamorphosis of insects. However, most of the information on the proteolytic enzymes of mosquitoes is derived from insects in the adult stages and is often directed towards the understanding of blood digestion. The aim of this study was to investigate the expression of active peptidases from the preimaginal stages of Ae. albopictus. METHODS: Ae. albopictus eggs, larvae, and pupae were analyzed using zymography with susbtrate-SDS-PAGE. The pH, temperature and peptidase inhibitor sensitivity was evaluated. In addition, the proteolytic activities of larval instars were assayed using the fluorogenic substrate Z-Phe-Arg-AMC. RESULTS: The proteolytic profile of the larval stage was composed of 8 bands ranging from 17 to 130 kDa. These enzymes displayed activity in a broad range of pH values, from 5.5 to 10.0. The enzymatic profile of the eggs was similar to that of the larvae, although the proteolytic bands of the eggs showed lower intensities. The pupal stage showed a complex proteolytic pattern, with at least 6 bands with apparent molecular masses ranging from 30 to 150 kDa and optimal activity at pH 7.5. Peptidases from larval instars were active from 10°C to 60°C, with optimal activity at temperatures between 37°C and 50°C. The proteolytic profile of both the larval and pupal stages was inhibited by phenyl-methyl sulfonyl-fluoride (PMSF) and Nα-Tosyl L-lysine chloromethyl ketone hydrochloride (TLCK), indicating that the main peptidases expressed during these developmental stages are trypsin-like serine peptidases. CONCLUSION: The preimaginal stages of Ae. albopictus exhibited a complex profile of trypsin-like serine peptidase activities. A comparative analysis of the active peptidase profiles revealed differential expression of trypsin-like isoforms among the preimaginal stages, suggesting that some of these enzymes are stage specific. Additionally, a comparison of the peptidase expression between larvae from eggs collected in the natural environment and larvae obtained from the eggs of female mosquitoes maintained in colonies for a long period of time demonstrated that the proteolytic profile is invariable under such conditions

ATLANTIC EPIPHYTES: a data set of vascular and non-vascular epiphyte plants and lichens from the Atlantic Forest

Epiphytes are hyper-diverse and one of the frequently undervalued life forms in plant surveys and biodiversity inventories. Epiphytes of the Atlantic Forest, one of the most endangered ecosystems in the world, have high endemism and radiated recently in the Pliocene. We aimed to (1) compile an extensive Atlantic Forest data set on vascular, non-vascular plants (including hemiepiphytes), and lichen epiphyte species occurrence and abundance; (2) describe the epiphyte distribution in the Atlantic Forest, in order to indicate future sampling efforts. Our work presents the first epiphyte data set with information on abundance and occurrence of epiphyte phorophyte species. All data compiled here come from three main sources provided by the authors: published sources (comprising peer-reviewed articles, books, and theses), unpublished data, and herbarium data. We compiled a data set composed of 2,095 species, from 89,270 holo/hemiepiphyte records, in the Atlantic Forest of Brazil, Argentina, Paraguay, and Uruguay, recorded from 1824 to early 2018. Most of the records were from qualitative data (occurrence only, 88%), well distributed throughout the Atlantic Forest. For quantitative records, the most common sampling method was individual trees (71%), followed by plot sampling (19%), and transect sampling (10%). Angiosperms (81%) were the most frequently registered group, and Bromeliaceae and Orchidaceae were the families with the greatest number of records (27,272 and 21,945, respectively). Ferns and Lycophytes presented fewer records than Angiosperms, and Polypodiaceae were the most recorded family, and more concentrated in the Southern and Southeastern regions. Data on non-vascular plants and lichens were scarce, with a few disjunct records concentrated in the Northeastern region of the Atlantic Forest. For all non-vascular plant records, Lejeuneaceae, a family of liverworts, was the most recorded family. We hope that our effort to organize scattered epiphyte data help advance the knowledge of epiphyte ecology, as well as our understanding of macroecological and biogeographical patterns in the Atlantic Forest. No copyright restrictions are associated with the data set. Please cite this Ecology Data Paper if the data are used in publication and teaching events. © 2019 The Authors. Ecology © 2019 The Ecological Society of Americ

Expression of active trypsin-like serine peptidases in the midgut of sugar-feeding female Anopheles aquasalis

Submitted by sandra infurna ([email protected]) on 2016-03-29T10:48:31Z No. of bitstreams: 1 geovane_lopes_etal_IOC_2015.pdf: 2716068 bytes, checksum: d03ae7889799d83f7377bcb234f3f9b2 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-03-29T11:04:46Z (GMT) No. of bitstreams: 1 geovane_lopes_etal_IOC_2015.pdf: 2716068 bytes, checksum: d03ae7889799d83f7377bcb234f3f9b2 (MD5)Made available in DSpace on 2016-03-29T11:04:46Z (GMT). No. of bitstreams: 1 geovane_lopes_etal_IOC_2015.pdf: 2716068 bytes, checksum: d03ae7889799d83f7377bcb234f3f9b2 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Leishmaniose. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Departamento de Química. Laboratório de Química de Proteínas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Leishmaniose. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil / Universidade Federal de São João Del Rey. Faculdade de Medicina. Departamento de Medicina. São João del Rey, MG, Brasil.Background: Anopheles aquasalis is a dipteran of the family Culicidae that is widely distributed in the coastal regions of South and Central America. This species acts as a vector of Plasmodium vivax, an important etiological agent of malaria, which represents a serious public health problem. In mosquitoes, trypsin-like serine proteases are important in blood meal digestion, immune responses and reproductive functions. The study of peptidases expressed in the mosquito midgut is essential to understanding the mechanisms of parasite-host interaction and the physiological process of nutrient digestion. Methods: Our study aimed to identify and characterize the proteolytic activities in the midgut of sugar-fed An. aquasalis females using zymographic analyses (substrate-SDS-PAGE), in-solution assays and mass spectrometry. Results: Here, we used a zymographic analysis to further biochemically characterize the proteolytic profile of the midgut of sugar-feeding An. aquasalis females. The trypsin peptidases migrated between ~17 and ~76 kDa and displayed higher proteolytic activities between pH 7.5 and 10 and at temperatures between 37 °C and 50 °C. Four putative trypsin-like serine peptidases were identified using mass spectrometry and data mining. The molecular masses of these peptidases were similar to those observed using zymography, which suggested that these peptidases could be responsible for some of the observed proteolytic bands. Conclusions: Taken together, our results contribute to the gene annotation of the unknown genome of this species, to the tissue location of these peptidases, and to the functional prediction of these crucial enzymes, which all impact further studies of this species

In-depth characterization of trypsin-like serine peptidases in the midgut of the sugar fed Culex quinquefasciatus

Submitted by sandra infurna ([email protected]) on 2016-04-07T15:42:04Z No. of bitstreams: 1 leonardo_vahia_etal_IOC_2015.pdf: 3145632 bytes, checksum: f7c63cd91874d18f27549c49ba79df45 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-04-07T15:55:39Z (GMT) No. of bitstreams: 1 leonardo_vahia_etal_IOC_2015.pdf: 3145632 bytes, checksum: f7c63cd91874d18f27549c49ba79df45 (MD5)Made available in DSpace on 2016-04-07T15:55:39Z (GMT). No. of bitstreams: 1 leonardo_vahia_etal_IOC_2015.pdf: 3145632 bytes, checksum: f7c63cd91874d18f27549c49ba79df45 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Leishmaniose. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Química. Laboratório de Química de Proteínas. Unidade de Proteômica. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Leishmaniose. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil / Universidade Federal de São João del Rei. Faculdade de Medicina. Departamento de Medicina. São João del Rey, MG, Brasil.Background: Culex quinquefasciatus is a hematophagous insect from the Culicidae family that feeds on the blood of humans, dogs, birds and livestock. This species transmits a wide variety of pathogens between humans and animals. The midgut environment is the first location of pathogen-vector interactions for blood-feeding mosquitoes and the expression of specific peptidases in the early stages of feeding could influence the outcome of the infection. Trypsin-like serine peptidases belong to a multi-gene family that can be expressed in different isoforms under distinct physiological conditions. However, the confident assignment of the trypsin genes that are expressed under each condition is still a challenge due to the large number of trypsin-coding genes in the Culicidae family and most likely because they are low abundance proteins. Methods: We used zymography for the biochemical characterization of the peptidase profile of the midgut from C. quinquefasciatus females fed on sugar. Protein samples were also submitted to SDS-PAGE followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis for peptidase identification. The peptidases sequences were analyzed with bioinformatics tools to assess their distinct features. Results: Zymography revealed that trypsin-like serine peptidases were responsible for the proteolytic activity in the midgut of females fed on sugar diet. After denaturation in SDS-PAGE, eight trypsin-like serine peptidases were identified by LC-MS/MS. These peptidases have structural features typical of invertebrate digestive trypsin peptidases but exhibited singularities at the protein sequence level such as: the presence of different amino acids at the autocatalytic motif and substrate binding regions as well as different number of disulfide bounds. Data mining revealed a group of trypsin-like serine peptidases that are specific to C. quinquefasciatus when compared to the culicids genomes sequenced so far. Conclusion: We demonstrated that proteomics approaches combined with bioinformatics tools and zymographic analysis can lead to the functional annotation of trypsin-like serine peptidases coding genes and aid in the understanding of the complexity of peptidase expression in mosquitoes

Morphologic study of the effect of iron on pseudocyst formation in Trichomonas vaginalis and its interaction with human epithelial cells

Submitted by Sandra Infurna ([email protected]) on 2017-11-09T11:11:13Z No. of bitstreams: 1 geovane_lopes_etal_IOC_2017.pdf: 2178070 bytes, checksum: a46da62a76d42d8f194ab2e1ec37016c (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-11-09T11:22:04Z (GMT) No. of bitstreams: 1 geovane_lopes_etal_IOC_2017.pdf: 2178070 bytes, checksum: a46da62a76d42d8f194ab2e1ec37016c (MD5)Made available in DSpace on 2017-11-09T11:22:04Z (GMT). No. of bitstreams: 1 geovane_lopes_etal_IOC_2017.pdf: 2178070 bytes, checksum: a46da62a76d42d8f194ab2e1ec37016c (MD5) Previous issue date: 2017Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Leishmanioses. Rio de Janeiro, RJ, Brasil.Universidade Federal de São João Del Rei. Faculdade de Medicina. Departamento de Medicina. São João Del Rei, MG, Brasil.Universidade Federal de São João Del Rei. Faculdade de Medicina. Departamento de Medicina. São João Del Rei, MG, Brasil.Instituto de Ensino Superior Presidente Tancredo de Almeida Neves. São João Del Rei, MG, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Ultraestrutura Celular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Universidade Estadual do Norte Fluminense Darcy Ribeiro. Centro de Biociências e Biotecnologia. Laboratório de Biologia Celular e Tecidual. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Leishmanioses. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil / Universidade Federal de São João Del Rei. Faculdade de Medicina. Departamento de Medicina. São João Del Rei, MG, Brasil.Trichomonas vaginalis is the aetiological agent of human trichomoniasis, which is one of the most prevalent sexually transmitted diseases in humans. Iron is an important element for the survival of this parasite and the colonisation of the host urogenital tract

Morphologic study of the effect of iron on pseudocyst formation in Trichomonas vaginalis and its interaction with human epithelial cells

BACKGROUND Trichomonas vaginalis is the aetiological agent of human trichomoniasis, which is one of the most prevalent sexually transmitted diseases in humans. Iron is an important element for the survival of this parasite and the colonisation of the host urogenital tract. OBJECTIVES In this study, we investigated the effects of iron on parasite proliferation in the dynamics of pseudocyst formation and morphologically characterised iron depletion-induced pseudocysts. METHODS We performed structural and ultrastructural analyses using light microscopy, scanning electron microscopy and transmission electron microscopy. FINDINGS It was observed that iron depletion (i) interrupts the proliferation of T. vaginalis, (ii) induces morphological changes in typical multiplicative trophozoites to spherical non-proliferative, non-motile pseudocysts, and (iii) induces the arrest of cell division at different stages of the cell cycle; (iv) iron is the fundamental element for the maintenance of typical trophozoite morphology; (v) pseudocysts induced by iron depletion are viable and reversible forms; and, finally, (vi) we demonstrated that pseudocysts induced by iron depletion are able to interact with human epithelial cells maintaining their spherical forms. MAIN CONCLUSIONS Together, these data suggest that pseudocysts could be induced as a response to iron nutritional stress and could have a potential role in the transmission and infection of T. vaginalis

Insights into the proteomic profile and gene expression of Lutzomyia longipalpis-derived lulo cell line

Lutzomyia longipalpis-derived cell line (Lulo), has been suggested as a model for studies of interaction between sand flies and Leishmania. Here, we present data of proteomic and gene expression analyses of Lulo cell related to interactions with L. (V.) braziliensis. Lulo cell protein extracts were analysed through a combination of two-dimensional gel electrophoresis and mass spectrometry and resulting spots were further investigated in silico to identify proteins, using Mascot search, and, afterwards, resulting sequences were applied for analysis with VectorBase. Sixty-four spots were identified showing similarities to other proteins registered in the databases and could be classified according to their biological function, such as ion-binding proteins (23 %), proteases (14 %), cytoskeletal proteins (11 %) and interactive membrane proteins (9.5 %). Effects of interaction with L. (V.) braziliensis with the expression of three genes (enolase, tubulin, and vacuolar transport protein) were observed after an 8-hour timeframe and compared to culture without parasites, and demonstrated the impact of parasite interaction with the expression of the following genes: LLOJ000219 (1.69-fold), LLOJ000326 (1.43-fold) and LLOJ006663 (2.41-fold). This set of results adds relevant information regarding the usefulness of the Lulo cell line for studies with Leishmania parasites that indicate variations of protein expression