Publications by authors named "Jonas Perales"

124 Publications

The resistance of the cowpea cv. BRS Xiquexique to infestation by cowpea weevil is related to the presence of toxic chitin-binding proteins.

Pestic Biochem Physiol 2021 Mar 21;173:104782. Epub 2021 Jan 21.

Laboratório de Química e Função de Proteínas e Peptídeos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro - UENF, Campos dos Goytacazes, RJ 28013-602, Brazil. Electronic address:

The cowpea weevil (Callosobruchus maculatus) is the main pest that attacks cowpea (Vigna unguiculata) seeds during storage, causing nutritional and economic losses in the cowpea crop. Thus, studies aiming to identify resistant cowpea cultivars have been developed. Chitin-binding proteins (CBP), such vicilins and chitinases, have been detected in seeds and related with the toxicity to insects. In this work, we investigated the presence of chitin-binding proteins in the partially resistant cowpea cv. BRS Xiquexique and evaluated their toxicity towards cowpea weevil. The CBP fraction was isolated by chitin affinity chromatography. CBP fraction showed, through 15% SDS PAGE, protein bands with varying molecular masses, mainly below 55 kDa. Proteins present in CBP fraction were identified by Western blotting and mass spectrometry analysis, as vicilins and chitinases. CBP fraction, at 5%, was able to interfere with the development of cowpea weevil, decreasing larval mass and length. A CBV (chitin-binding vicilin) fraction isolated from CBP fraction was toxic, at 2.0%, to C. maculatus, decreasing larval mass and length in 64.3% and 33.23%, respectively. These results suggest that chitin binding proteins, such vicilins and chitinases, may be related to the resistance of cowpea cv. BRS Xiquexique to the infestation by C. maculatus.
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http://dx.doi.org/10.1016/j.pestbp.2021.104782DOI Listing
March 2021

In vitro assessment of the efficacy of protein exudates from seeds against Haemonchus contortus.

Vet Parasitol 2021 Apr 5;292:109399. Epub 2021 Mar 5.

Postgraduate Program in Health Sciences, Federal University of Maranhão, São Luís, MA, Brazil; Laboratory of Parasite Control, Federal University of Maranhão, São Luís, MA, Brazil. Electronic address:

Nematodes develop resistance to the most common commercially available drugs. The aim of this study was to identify and evaluate the action of protein exudates from Mimosa caesalpiniifolia, Leucaena leucocephala, Acacia mangium, and Stylosanthes capitata seeds on the gastrointestinal nematode Haemonchus contortus. The exuded proteins were precipitated, dialyzed, lyophilized, and assessed for their effect on egg hatching and artificial larval exsheathment inhibition. Proteome analysis of the protein extracts was also performed. Although no egg-hatching inhibition was observed, all exudates showed efficacy in inhibiting the larval exsheathment of H. contortus larvae with an EC varying from 0.61 to 0.26 mg P mL. Proteomic analysis revealed the presence of proteases, protease inhibitors, chitinases, and lectins among other proteins in the exudates. Most of the exuded proteins belong to the oxidative stress/plant defense and energy/carbohydrate metabolism functional clusters. This study concluded that the bioactive proteins from different classes exuded by seeds of M. caesalpiniifolia, L. leucocephala, A. mangium, and S. capitata show stage-specific inhibition against H. contortus.
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http://dx.doi.org/10.1016/j.vetpar.2021.109399DOI Listing
April 2021

Proteomic analysis reveals differentially abundant proteins probably involved in the virulence of amastigote and promastigote forms of Leishmania infantum.

Parasitol Res 2021 Feb 8;120(2):679-692. Epub 2021 Jan 8.

Laboratório de Leishmanioses, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, CEP: 31270-901, Brazil.

Owing to the importance and clinical diversity of Leishmania infantum, studying its virulence factors is promising for understanding the relationship between parasites and hosts. In the present study, differentially abundant proteins from strains with different degrees of virulence in promastigote and amastigote forms were compared using two quantitative proteomics techniques, differential gel electrophoresis and isobaric mass tag labeling, followed by identification by mass spectrometry. A total of 142 proteins were identified: 96 upregulated and 46 downregulated proteins in the most virulent strain compared to less virulent. The interaction between the proteins identified in each evolutionary form was predicted. The results showed that in the amastigote form of the most virulent strain, there was a large group of proteins related to glycolysis, heat shock, and ribosomal proteins, whereas in the promastigote form, the group consisted of stress response, heat shock, and ribosomal proteins. In addition, biological processes related to metabolic pathways, ribosomes, and oxidative phosphorylation were enriched in the most virulent strain (BH400). Finally, we noted several proteins previously found to play important roles in L. infantum infection, which showed increased abundance in the virulent strain, such as ribosomal proteins, HSP70, enolase, fructose 1,6-biphosphate aldolase, peroxidoxin, and tryparedoxin peroxidase, many of which interact with each other.
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http://dx.doi.org/10.1007/s00436-020-07020-8DOI Listing
February 2021

Study of the differentially abundant proteins among Leishmania amazonensis, L. braziliensis, and L. infantum.

PLoS One 2020 15;15(10):e0240612. Epub 2020 Oct 15.

Departamento de Parasitologia, Laboratório de Leishmanioses, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

Leishmaniasis has been considered as emerging and re-emerging disease, and its increasing global incidence has raised concerns. The great clinical diversity of the disease is mainly determined by the species. In several American countries, tegumentary leishmaniasis (TL) is associated with both Leishmania amazonensis and L. braziliensis, while visceral leishmaniasis (VL) is associated with L. (L.) infantum. The major molecules that determine the most diverse biological variations are proteins. In the present study, through a DIGE approach, we identified differentially abundant proteins among the species mentioned above. We observed a variety of proteins with differential abundance among the studied species; and the biological networks predicted for each species showed that many of these proteins interacted with each other. The prominent proteins included the heat shock proteins (HSPs) and the protein network involved in oxide reduction process in L. amazonensis, the protein network of ribosomes in L. braziliensis, and the proteins involved in energy metabolism in L. infantum. The important proteins, as revealed by the PPI network results, enrichment categories, and exclusive proteins analysis, were arginase, HSPs, and trypanothione reductase in L. amazonensis; enolase, peroxidoxin, and tryparedoxin1 in L. braziliensis; and succinyl-CoA ligase [GDP -forming] beta-chain and transaldolase in L. infantum.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0240612PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7561129PMC
December 2020

Antimicrobial peptides from Capsicum chinense fruits: agronomic alternatives against phytopathogenic fungi.

Biosci Rep 2020 08;40(8)

Laboratório de Fisiologia e Bioquímica de Microrganismos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil.

In recent years, the antimicrobial activity of peptides isolated from a wide variety of organs from plant species has been reported. However, a few studies have investigated the potential of antimicrobial peptides (AMPs) found in fruits, especially Capsicum chinense (pepper). The present study aimed to purify and characterize peptides from Capsicum chinense fruits and evaluate their inhibitory activities against different phytopathogenic fungi and also analyze the possible mechanisms of action involved in microbial inhibition. After fruit protein extraction and high-performance liquid chromatography (HPLC), different fractions were obtained, named F1 to F10. Peptides in the F4 and F5 fractions were sequenced and revealed similarity with the plant antimicrobial peptides like non-specific lipid transfer proteins and defensin-like peptide. The F4 and F5 fractions presented strong antimicrobial activity against the fungus Fusarium solani and Fusarium oxysporum, causing toxic effects on these fungi, leading to membrane permeabilization, endogenous reactive oxygen species increase, activation of metacaspase and loss of mitochondrial function.
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http://dx.doi.org/10.1042/BSR20200950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442975PMC
August 2020

Proteomic analysis of Ascocotyle longa (Trematoda: Heterophyidae) metacercariae.

Mol Biochem Parasitol 2020 09 1;239:111311. Epub 2020 Aug 1.

Laboratório de Avaliação e Promoção da Saúde Ambiental, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil. Electronic address:

Ascocotyle longa is parasitic trematode with wide distribution throughout America, Europe, Africa, and Middle East. Despite the fact that this fish-borne pathogen has been considered an agent of human heterophyiasis in Brazil, the molecules involved in the host-parasite interaction remain unknown. The present study reports the proteome profile of A. longa metacercariae collected from the fish Mugil liza from Brazil. This infective stage for humans, mammals and birds was analyzed using nLC-MS/MS approach. We identified a large repertoire of proteins, which are mainly involved in energy metabolism and cell structure. Peptidases and immunogenic proteins were also identified, which might play roles in host-parasite interface. Our data provided unprecedented insights into the biology of A. longa and represent a first step to understand the natural host-parasite interaction. Moreover, as the first proteome characterized in this trematode, it will provide an important resource for future studies.
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http://dx.doi.org/10.1016/j.molbiopara.2020.111311DOI Listing
September 2020

An immunoproteomics approach to identify proteins to be applied for the diagnosis of visceral leishmaniasis and human immunodeficiency virus co-infection.

Parasitology 2020 08 20;147(9):932-939. Epub 2020 Apr 20.

Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

The co-infection between visceral leishmaniasis (VL) and human immunodeficiency virus (HIV) has increased in several countries in the world. The current serological tests are not suitable since they present low sensitivity to detect the most of VL/HIV cases, and a more precise diagnosis should be performed. In this context, in the present study, an immunoproteomics approach was performed using Leishmania infantum antigenic extracts and VL, HIV and VL/HIV patients sera, besides healthy subjects samples; aiming to identify antigenic markers for these clinical conditions. Results showed that 43 spots were recognized by antibodies in VL and VL/HIV sera, and 26 proteins were identified by mass spectrometry. Between them, β-tubulin was expressed, purified and tested in ELISA experiments as a proof of concept for validation of our immunoproteomics findings and results showed high sensitivity and specificity values to detect VL and VL/HIV patients. In conclusion, the identified proteins in the present work could be considered as candidates for future studies aiming to improvement of the diagnosis of VL and VL/HIV co-infection.
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http://dx.doi.org/10.1017/S0031182020000578DOI Listing
August 2020

The interaction between the natural metalloendopeptidase inhibitor BJ46a and its target toxin jararhagin analyzed by structural mass spectrometry and molecular modeling.

J Proteomics 2020 06 1;221:103761. Epub 2020 Apr 1.

Laboratory of Toxinology, Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, Brazil. Electronic address:

Snakebite envenoming affects millions of people worldwide, being officially considered a neglected tropical disease by the World Health Organization. The antivenom is effective in neutralizing the systemic effects of envenomation, but local effects are poorly neutralized, often leading to permanent disability. The natural resistance of the South American pit viper Bothrops jararaca to its venom is partly attributed to BJ46a, a natural snake venom metalloendopeptidase inhibitor. Upon complex formation, BJ46a binds non-covalently to the metalloendopeptidase, rendering it unable to exert its proteolytic activity. However, the structural features that govern this interaction are largely unknown. In this work, we applied structural mass spectrometry techniques (cross-linking-MS and hydrogen-deuterium exchange MS) and in silico analyses (molecular modeling, docking, and dynamics simulations) to understand the interaction between BJ46a and jararhagin, a metalloendopeptidase from B. jararaca venom. We explored the distance restraints generated from XL-MS experiments to guide the modeling of BJ46a and jararhagin, as well as the protein-protein docking simulations. HDX-MS data pinpointed regions of protection/deprotection at the interface of the BJ46a-jararhagin complex which, in addition to the molecular dynamics simulation data, reinforced our proposed interaction model. Ultimately, the structural understanding of snake venom metalloendopeptidases inhibition by BJ46a could lead to the rational design of drugs to improve anti-snake venom therapeutics, alleviating the high morbidity rates currently observed.
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http://dx.doi.org/10.1016/j.jprot.2020.103761DOI Listing
June 2020

Identification and Characterization of Two Defensins from Capsicum annuum Fruits that Exhibit Antimicrobial Activity.

Probiotics Antimicrob Proteins 2020 09;12(3):1253-1265

Laboratório de Fisiologia e Bioquímica de Microrganismos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, 28013-602, Brazil.

Scientific advances have not been enough to combat the growing resistance to antimicrobial medicines. Antimicrobial peptides (AMPs) are effector molecules of the innate immune defense system in plants and could provide an important source of new antimicrobial drugs. The aim of this work was to extract, purify, characterize, and evaluate the antifungal activities present in fractions obtained from Capsicum annum fruits through reversed-phase chromatography. The fractions named F2 and F3 presented the highest inhibitory activity against Candida and Mycobacterium tuberculosis species. In addition, we identified two sequences of AMPs in the F2 and F3 fractions through mass spectrometry that showed similarity to an already well-characterized family of plant defensins. A plasma membrane permeabilization assay demonstrated that the peptides present in F2, F3, and F4 fractions induced changes in the membrane of some yeast strains, culminating in permeabilization. The production of reactive oxygen species was induced by the fractions in some yeast strains. Fractions F2, F3, and F4 also did not show toxicity in macrophage or monocyte cultures. In conclusion, the obtained data demonstrate that the AMPs, especially those present in the fractions F2 and F3, are promising antimicrobial agents that may be useful to enhance the development of new therapeutic agents for the treatment of diseases.
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http://dx.doi.org/10.1007/s12602-020-09647-6DOI Listing
September 2020

Quantitative Proteomic Map of the Trypanosomatid Strigomonas culicis: The Biological Contribution of its Endosymbiotic Bacterium.

Protist 2019 12 1;170(6):125698. Epub 2019 Nov 1.

Laboratory of Toxinology, IOC, Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, RJ 21040-900, Brazil. Electronic address:

Strigomonas culicis is a kinetoplastid parasite of insects that maintains a mutualistic association with an intracellular symbiotic bacterium, which is highly integrated into the protist metabolism: it furnishes essential compounds and divides in synchrony with the eukaryotic nucleus. The protist, conversely, can be cured of the endosymbiont, producing an aposymbiotic cell line, which presents a diminished ability to colonize the insect host. This obligatory association can represent an intermediate step of the evolution towards the formation of an organelle, therefore representing an interesting model to understand the symbiogenesis theory. Here, we used shotgun proteomics to compare the S. culicis endosymbiont-containing and aposymbiotic strains, revealing a total of 11,305 peptides, and up to 2,213 proteins (2,029 and 1,452 for wild type and aposymbiotic, respectively). Gene ontology associated to comparative analysis between both strains revealed that the biological processes most affected by the elimination of the symbiont were the amino acid synthesis, as well as protein synthesis and folding. This large-scale comparison of the protein expression in S. culicis marks a step forward in the comprehension of the role of endosymbiotic bacteria in monoxenous trypanosomatid biology, particularly because trypanosomatids expression is mostly post-transcriptionally regulated.
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http://dx.doi.org/10.1016/j.protis.2019.125698DOI Listing
December 2019

Glycolytic profile shift and antioxidant triggering in symbiont-free and HO-resistant Strigomonas culicis.

Free Radic Biol Med 2020 01 21;146:392-401. Epub 2019 Nov 21.

Laboratory of Cellular Biology, IOC, Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, RJ, Brazil. Electronic address:

During their life cycle, trypanosomatids are exposed to stress conditions and adapt their energy and antioxidant metabolism to colonize their hosts. Strigomonas culicis is a monoxenous protist found in invertebrates with an endosymbiotic bacterium that completes essential biosynthetic pathways for the trypanosomatid. Our research group previously generated a wild-type HO-resistant (WTR) strain that showed improved mitochondrial metabolism and antioxidant defenses, which led to higher rates of Aedes aegypti infection. Here, we assess the biological contribution of the S. culicis endosymbiont and reactive oxygen species (ROS) resistance to oxidative and energy metabolism processes. Using high-throughput proteomics, several proteins involved in glycolysis and gluconeogenesis, the pentose phosphate pathway and glutathione metabolism were identified. The results suggest that ROS resistance decreases glucose consumption and indicate that the metabolic products from gluconeogenesis are key to supplying the protist with high-energy and reducing intermediates. Our hypothesis was confirmed by biochemical assays showing opposite profiles for glucose uptake and hexokinase and pyruvate kinase activity levels in the WTR and aposymbiotic strains, while the enzyme glucose-6P 1-dehydrogenase was more active in both strains. Regarding the antioxidant system, ascorbate peroxidase has an important role in HO resistance and may be responsible for the high infection rates previously described for A. aegypti. In conclusion, our data indicate that the energy-related and antioxidant metabolic processes of S. culicis are modulated in response to oxidative stress conditions, providing new perspectives on the biology of the trypanosomatid-insect interaction as well as on the possible impact of resistant parasites in accidental human infection.
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http://dx.doi.org/10.1016/j.freeradbiomed.2019.11.025DOI Listing
January 2020

Antioxidant effect of sildenafil: Potential hepatoprotection via differential expression of mitochondrial proteins in apolipoprotein E knockout mice.

Pharmacol Rep 2019 Jun 6;71(3):422-429. Epub 2019 Jan 6.

Laboratory of Translational Physiology, Federal University of Espírito Santo, Vitória, Brazil; Pharmaceutical Sciences Graduate Program, Vila Velha University, Vila Velha, Brazil. Electronic address:

Background: High plasma cholesterol levels are able to trigger several pathophysiological events, including inflammation, cell damage and especially oxidative stress. Previously, studies have shown that sildenafil exhibited antioxidant effects in several experimental models. Here we evaluate the role of sildenafil in liver redox equilibrium of apolipoprotein E knockout (apoE-KO) mice.

Methods: ApoE-KO mice were divided in two groups: one group received the PDE5 inhibitor sildenafil (40 mg/kg/day) for 3 weeks (apoE-KO + Sil) and was compared to a second group of apoE-KO mice, which received only the vehicle (water) for 3 weeks (apoE-KO). Control group (C57 mice) received only a standard chow diet. At the age of 18 weeks, mice livers were collected for the measurement of intracellular ROS levels and apoptotic cells by flow cytometry analysis, and mitochondria isolation for proteomic analysis.

Results: Compared to the control group, liver cells from apoE-KO presented some typical redox imbalance features: higher levels of intracellular ROS (global oxidative stress ˜60%, superoxide anion ˜82%, and peroxynitrite/hydroxyl radical ˜53%), higher amounts of apoptotic cells (up to ˜19%) and higher mitochondrial intensity of catalase (+339%) and transferrin spots (+914%). After treatment with sildenafil, apoE-KO presented ROS levels and the number of apoptotic cells similar to those observed in C57. In addition, when compared to apoE-KO, apoE-KO + Sil showed lower spots volumes of catalase (-23%) and transferrin (-71%) and up-regulation of urate oxidase (+94%).

Conclusion: The treatment with sildenafil is able to induce beneficial changes in liver mitochondrial protein dynamics, which restores the redox homeostasis contributing to a potential hepatoprotection.
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http://dx.doi.org/10.1016/j.pharep.2019.01.005DOI Listing
June 2019

Differential proteomic comparison of breast cancer secretome using a quantitative paired analysis workflow.

BMC Cancer 2019 Apr 18;19(1):365. Epub 2019 Apr 18.

Laboratory of Toxinology, Oswaldo Cruz Institute, Fiocruz, Av. Brasil 4365, Manguinhos, Rio de Janeiro, 21040-360, Brazil.

Background: Worldwide, breast cancer is the main cause of cancer mortality in women. Most cases originate in mammary ductal cells that produce the nipple aspirate fluid (NAF). In cancer patients, this secretome contains proteins associated with the tumor microenvironment. NAF studies are challenging because of inter-individual variability. We introduced a paired-proteomic shotgun strategy that relies on NAF analysis from both breasts of patients with unilateral breast cancer and extended PatternLab for Proteomics software to take advantage of this setup.

Methods: The software is based on a peptide-centric approach and uses the binomial distribution to attribute a probability for each peptide as being linked to the disease; these probabilities are propagated to a final protein p-value according to the Stouffer's Z-score method.

Results: A total of 1227 proteins were identified and quantified, of which 87 were differentially abundant, being mainly involved in glycolysis (Warburg effect) and immune system activation (activated stroma). Additionally, in the estrogen receptor-positive subgroup, proteins related to the regulation of insulin-like growth factor transport and platelet degranulation displayed higher abundance, confirming the presence of a proliferative microenvironment.

Conclusions: We debuted a differential bioinformatics workflow for the proteomic analysis of NAF, validating this secretome as a treasure-trove for studying a paired-organ cancer type.
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http://dx.doi.org/10.1186/s12885-019-5547-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6474050PMC
April 2019

Biochemical analysis of antimicrobial peptides in two different genotypes after fruit infection by .

Biosci Rep 2019 04 23;39(4). Epub 2019 Apr 23.

Laboratório de Fisiologia e Bioquímica de Microrganismos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil

There are several phytosanitary problems that have been causing serious damage to the crops, including anthracnose. Upon attack by certain pathogens, various protein molecules are produced, which are known as proteins related to pathogenesis (PR proteins), including antimicrobial peptides such as protease inhibitors, defensins and lipid transfer proteins (LTPs). The objective of this work is to identify antimicrobial proteins and/or peptides of two genotypes from fruits infected with The fungus was inoculated into fruits by the deposition of a spore suspension (10 conidia ml), and after 24 and 48 h intervals, the fruits were removed from the humid chamber and subjected to a protein extraction process. Protein analysis of the extracts was performed by tricine gel electrophoresis and Western blotting. The distinctive bands between genotypes in the electrophoresis profiles were subjected to mass spectrometry sequencing. Trypsin inhibition assays, reverse zymographic detection of protease inhibition and β-1,3-glucanase activity assays were also performed and extracts were also tested for their ability to inhibit the growth of fungi ' There were several low molecular weight proteins in all treated samples, and some treatments in which antimicrobial peptides such as defensin, lipid transfer protein (LTP) and protease inhibitor have been identified. It was shown that the green fruits are more responsive to infection, showing the production of antimicrobial peptides in response to injury and inoculation of the fungus, what did not occur in ripe fruits under any treatment.
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http://dx.doi.org/10.1042/BSR20181889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481241PMC
April 2019

Data on antigen recognition hindrance by antibodies covalently immobilized to Protein G magnetic beads by dimethyl pimelimidate (DMP) cross-linking.

Data Brief 2019 Feb 21;22:516-521. Epub 2018 Dec 21.

Laboratório de Biologia Celular, IOC, Fiocruz, Rio de Janeiro, RJ, Brazil.

The data presented herein is related to the article entitled " immunoproteome: calpain-like CAP5.5 differentially detected throughout distinct stages of human Chagas disease cardiomyopathy" [1]. Electrophoretic analyses under denaturing and reducing conditions indicate that covalent immobilization of human IgG to Protein G magnetic beads by cross-linking with 50 mM dimethyl pimelimidate hinders the recognition of antigens in immunoprecipitation assays.
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http://dx.doi.org/10.1016/j.dib.2018.12.057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6327068PMC
February 2019

Trypanosoma cruzi immunoproteome: Calpain-like CAP5.5 differentially detected throughout distinct stages of human Chagas disease cardiomyopathy.

J Proteomics 2019 03 29;194:179-190. Epub 2018 Nov 29.

Laboratório de Biologia Celular, IOC, Fiocruz, Rio de Janeiro, RJ, Brazil. Electronic address:

Chagas disease, caused by the protozoan Trypanosoma cruzi, affects millions of people worldwide, especially in Latin America. Approximately 30% of the cases evolve to the chronic symptomatic stage due to cardiac and/or digestive damage, generally accompanied by nervous system impairment. Given the higher frequency and severity of clinical manifestations related to cardiac tissue lesion, the goal of this study was the identification of proteins associated with the disease progression towards its cardiac form. Thus, T. cruzi bloodstream trypomastigotes proteins were submitted to immunoprecipitation using antibodies from patients with the asymptomatic or cardiac (stages B1 and C) forms of the disease and from healthy donors as control. Immunoreactive proteins were identified and quantified based on mass spectrometry analysis and shifts in the recognition profile were further evaluated. Compared to asymptomatic samples, IgG from stage C patients predominantly detected the I/6 autoantigen, whereas IgG from B1 patients resulted in higher yield of dihydrolipoamide acetyltransferase precursor, calpain cysteine peptidase, and two variants of CAP5.5. In this work, CAP5.5 recognition by serum immunoglobulin from patients with early cardiomyopathy generated a 23-fold abundance variation when compared to samples from asymptomatic patients, highlighting the participation of this protein in cardiac form progression of the disease. SIGNIFICANCE: While T. cruzi has become the major cause of infectious cardiomyopathy in Latin America, research groups have been struggling to find alternative treatment, vaccine candidates, and improved diagnostic tests. In addition, the absence of adequate biomarkers to assess cure and progression of disease is a major setback for clinical trials and patients monitoring. Therefore, our findings may contribute to a better understanding of T. cruzi pathogenesis and evaluation of suitable candidates for vaccine and diagnostic tests, besides the clinical applicability of the potential biomarkers for patient follow-up and prognosis. Finally, the identification of T. cruzi proteins recognized by IgG from healthy donors may contribute for the understanding and discovery of epitope conservation among a broad range of pathogens.
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http://dx.doi.org/10.1016/j.jprot.2018.11.019DOI Listing
March 2019

A cysteine protease from the latex of Ficus benjamina has in vitro anthelmintic activity against Haemonchus contortus.

Rev Bras Parasitol Vet 2018 Oct-Dec;27(4):473-480. Epub 2018 Nov 8.

Laboratório de Controle de Parasitos, Departamento de Patologia, Centro de Ciências Biológicas e da Saúde, Universidade Federal do Maranhão - UFMA, São Luís, MA, Brasil.

Haemonchus contortus is a gastrointestinal nematode that is responsible for high mortality rates in ruminant herds. The resistance of nematodes to synthetic anthelmintics is widespread and requires a continuous search for new bioactive molecules, such as proteins. The objective of this study was to evaluate the anthelmintic potential of a protease purified from the latex of Ficus benjamina against H. contortus . Fresh latex was collected from plants via small incisions in the green stems, the rubber was removed by centrifugation, and the latex protein extract (LPE) was obtained. After LPE fractionation with ammonium sulfate and chromatography of the fraction containing the highest proteolytic activity on CM-cellulose, a cysteine protease (FbP) was purified. FbP has a molecular mass of approximately 23.97 kDa, and its proteolytic activity was stable between pH 6.0 and pH 10 and over a broad temperature range, with optimum activity at 60 °C. FbP inhibited both the development and exsheathment of H. contortus larvae, with 50% effective concentrations of 0.26 and 0.79 mg/mL, respectively. We conclude that this cysteine protease from F. benjamina latex with anthelmintic activity against H. contortus could be a promising alternative for the development of products for use in parasite control programmes.
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http://dx.doi.org/10.1590/S1984-296120180070DOI Listing
June 2019

Proteomic Deep Mining the Venom of the Red-Headed Krait, .

Toxins (Basel) 2018 09 13;10(9). Epub 2018 Sep 13.

School of Biological Sciences, University of Northern Colorado, 501 20th St., CB 92, Greeley, CO 80639-0017, USA.

The use of -omics technologies allows for the characterization of snake venom composition at a fast rate and at high levels of detail. In the present study, we investigated the protein content of Red-headed Krait () venom. This analysis revealed a high diversity of snake venom protein families, as evidenced by high-throughput mass spectrometric analysis. We found all six venom protein families previously reported in a transcriptome study of the venom gland of , including phospholipases A₂ (PLA₂s), Kunitz-type serine proteinase inhibitors (KSPIs), three-finger toxins (3FTxs), cysteine-rich secretory proteins (CRISPs), snaclecs, and natriuretic peptides. A combined approach of automated database searches and de novo sequencing of tandem mass spectra, followed by sequence similarity searches, revealed the presence of 12 additional toxin families. De novo sequencing alone was able to identify 58 additional peptides, and this approach contributed significantly to the comprehensive description of the venom. Abundant protein families comprise 3FTxs (22.3%), KSPIs (19%), acetylcholinesterases (12.6%), PLA₂s (11.9%), venom endothelial growth factors (VEGFs, 8.4%), nucleotidases (4.3%), and C-type lectin-like proteins (snaclecs, 3.3%); an additional 11 toxin families are present at significantly lower concentrations, including complement depleting factors, a family not previously detected in venoms. The utility of a multifaceted approach toward unraveling the proteome of snake venoms, employed here, allowed detection of even minor venom components. This more in-depth knowledge of the composition of venom facilitates a better understanding of snake venom molecular evolution, in turn contributing to more effective treatment of krait bites.
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http://dx.doi.org/10.3390/toxins10090373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162843PMC
September 2018

Proteomic profile, biological activities and antigenic analysis of the venom from Bothriopsis bilineata smaragdina ("loro machaco"), a pitviper snake from Peru.

J Proteomics 2018 09 23;187:171-181. Epub 2018 Jul 23.

Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901, Belo Horizonte, Minas Gerais, Brasil. Electronic address:

In order to determine Bothriopsis bilineata smaragdina venom (BbsV) composition, proteomic approaches were performed. Venom components were analyzed by RP-HPLC, SDS- PAGE and nano LC on line with LTQ Orbitrap XL. Results showed a total of 189 identified proteins, grouped into 11 different subgroups, which include snake venom metalloproteinases (SVMPs, 54.67%), snake C-type lectins (Snaclecs, 15.78%), snake venom serine proteinases (SVSPs, 14.69%), cystein-rich secretory proteins (CRISP, 2.61%), phospholipases A (PLA, 1.14%), phosphodiesterase (PDE, 1.17%), venom endothelial growth factor (VEGF, 1.06%) 5'nucleotidases (0.33%), L-amino acid oxidases (LAAOs, 0.28%) and other proteins. In vitro enzymatic activities (SVMP, SVSP, LAAO, Hyal and PLA) of BbsV were also analyzed. BbsV showed high SVSP activity but low PLA activity, when compared to other Bothrops venoms. In vivo, BbsV induced hemorrhage and edema in mice and showed intraperitoneal median lethal dose (LD) of 92.74 (± 0.15) μg/20 g of mice. Furthermore, BbsV reduced cell viability when incubated with VERO cells. Peruvian and Brazilian bothropic antivenoms recognize BbsV proteins, as detected by ELISA and Western Blotting. Both antivenoms were able to neutralize in vivo edema and hemorrhage.

Significance: In Peru, snakebite is a public health problem, especially in the rain forest, as a result of progressive colonization of this geographical area. This country is the second in Latin America, after Brazil, to exhibit the largest variety of venomous snakes. B. atrox and B. b. smaragdina snakes are sympatric species in Peruvian Amazon region and are responsible for approximately 95% of the envenomings reported in this region. B. b. smaragdina may cause a smaller share (3 to 38%) of those accidents, due to its arboreal habits, that make human encounters with these snakes less likely to happen. Despite B. b. smaragdina recognized medical importance, its venom composition and biological activities have been poorly studied. Furthermore, BbsV is not a component of the antigenic pool used to produce the corresponding Peruvian bothropic antivenom (P-BAV). Our results not only provide new insights on BbsV composition and biological activity, but also demonstrate that both P-BAV and B-BAV polyvalent antivenoms have a considerable recognition of proteins from BbsV and, more importantly, neutralized hemorrhage and edema, the main local effects of bothropic envenomation.
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http://dx.doi.org/10.1016/j.jprot.2018.07.016DOI Listing
September 2018

Myracrodruon urundeuva seed exudates proteome and anthelmintic activity against Haemonchus contortus.

PLoS One 2018 19;13(7):e0200848. Epub 2018 Jul 19.

Laboratory of Parasite Control, Department of Pathology, Center for Biological and Health Sciences, Federal University of Maranhão, São Luís, Maranhão, Brazil.

Seed exudates are plant-derived natural bioactive compounds consisting of a complex mixture of organic and inorganic molecules. Plant seed exudates have been poorly studied against parasite nematodes. This study was undertaken to identify proteins in the Myracrodruon urundeuva seed exudates and to assess the anthelmintic activity against Haemonchus contortus, an important parasite of small ruminants. M. urundeuva seed exudates (SEX) was obtained after immersion of seeds in sodium acetate buffer. SEX was fractionated with ammonium sulfate at 0-90% concentration to generate the ressuspended pellet (SEXF1) and the supernatant (SEXF2). SEX, SEXF1, and SEXF2 were exhaustively dialyzed against distilled water (cut-off: 12 kDa) and the protein contents determined. Mass spectrometry analyses of SEX, SEXF1, and SEXF2 were done to identify proteins and secondary metabolites. The seed exudates contained protease, protease inhibitor, peptidase, chitinase, and lipases as well as the low molecular weight secondary compounds ellagic acid and quercetin rhamnoside. SEX inhibited H. contortus larval development (LDA) (IC50 = 0.29 mg mL-1), but did not affect larval exsheathment (LEIA). On the other hand, although SEXF1 and SEXF2 inhibited H. contortus LEIA (IC50 = 1.04 and 0.93 mg mL-1, respectively), they showed even greater inhibition efficiency of H. contortus larval development (IC50 = 0.29 and 0.42 mg mL-1, respectively). To the best of our knowledge, this study is the first to show the anthelmintic activity of plant exudates against a gastrointestinal nematode. Moreover, it suggests the potential of exuded proteins as candidates to negatively interfere with H. contortus life cycle.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0200848PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053183PMC
January 2019

Ferritin from the haemolymph of adult ants: an extraction method for characterization and a ferromagnetic study.

Eur Biophys J 2018 Sep 28;47(6):641-653. Epub 2018 Mar 28.

Coordenação de Materiais, Física Aplicada e Nanociências, Centro Brasileiro de Pesquisas Físicas, R. Dr. Xavier Sigaud 150, Rio de Janeiro, RJ, 22290-180, Brazil.

Ferritin has been studied in many animals, plants and bacteria. The main functions of ferritin in mammals are iron concentration and stabilization, protection against oxidants and iron storage for later developmental or iron-dependent activities. Although insect ferritin plays a key role in iron transport, only a few studies to date have examined its properties and function. Ferritin isolation from the haemolymph of adult Camponotus sericeiventris ants involved heating at 75 °C, followed by protein fractionation with 3.2 M KBr gradients and ferritin sedimentation with KBr. Protein identification was performed using high-resolution proteomics techniques. SDS-PAGE revealed three subunits with molecular weights (MW) of 26, 28 and 31 kDa. Native PAGE indicated a MW higher than 669 kDa. Proteomic analysis strongly suggested the 26 and 31 kDa bands as F2LCH and F1HCH subunits of ferritin, respectively. Ferromagnetic resonance (FMR) at 100 K showed, at low field, a characteristic broad component of the ferritin iron core, suggesting that its distribution was shifted to values greater than 3000, a higher content than in mammals. The protein yield and MW were comparable to those reported in other studies of insects. To the best of our knowledge, this is the first report on ferritin extracted from adult ants to date. These results are discussed on the basis of the protein structure-function relation of secreted insect and mammal ferritins. This purification method will allow the use of magnetic techniques, which are relevant for understanding the role of ferritin in the biomineralization of magnetic nanoparticles in insects.
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http://dx.doi.org/10.1007/s00249-018-1293-3DOI Listing
September 2018

Blood coagulation abnormalities in multibacillary leprosy patients.

PLoS Negl Trop Dis 2018 03 22;12(3):e0006214. Epub 2018 Mar 22.

Lab. of Cellular Microbiology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.

Background: Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae infection. In 2016, more than 200,000 new cases of leprosy were detected around the world, representing the most frequent cause of infectious irreversible deformities and disabilities.

Principal Findings: In the present work, we demonstrate a consistent procoagulant profile on 40 reactional and non-reactional multibacillary leprosy patients. A retrospective analysis in search of signs of coagulation abnormalities among 638 leprosy patients identified 35 leprosy patients (5.48%) which displayed a characteristic lipid-like clot formed between blood clot and serum during serum harvesting, herein named 'leprosum clot'. Most of these patients (n = 16, 45.7%) belonged to the lepromatous leprosy pole of the disease. In addition, formation of the leprosum clot was directly correlated with increased plasma levels of soluble tissue factor and von Willebrand factor. High performance thin layer chromatography demonstrated a high content of neutral lipids in the leprosum clot, and proteomic analysis demonstrated that the leprosum clot presented in these patients is highly enriched in fibrin. Remarkably, differential 2D-proteomics analysis between leprosum clots and control clots identified two proteins present only in leprosy patients clots: complement component 3 and 4 and inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP). In agreement with those observations we demonstrated that M. leprae induces hepatocytes release of IHRP in vitro.

Conclusions: We demonstrated that leprosy MB patients develop a procoagulant status due to high levels of plasmatic fibrinogen, anti-cardiolipin antibodies, von Willebrand factor and soluble tissue factor. We propose that some of these components, fibrinogen for example, presents potential as predictive biomarkers of leprosy reactions, generating tools for earlier diagnosis and treatment of these events.
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http://dx.doi.org/10.1371/journal.pntd.0006214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5863944PMC
March 2018

The Primary Duct of Bothrops jararaca Glandular Apparatus Secretes Toxins.

Toxins (Basel) 2018 03 13;10(3). Epub 2018 Mar 13.

Laboratório de Farmacologia, Instituto Butantan, São Paulo SP 05503-900, Brazil.

Despite numerous studies concerning morphology and venom production and secretion in the main venom gland (and some data on the accessory gland) of the venom glandular apparatus of Viperidae snakes, the primary duct has been overlooked. We characterized the primary duct of the snake by morphological analysis, immunohistochemistry and proteomics. The duct has a pseudostratified epithelium with secretory columnar cells with vesicles of various electrondensities, as well as mitochondria-rich, dark, basal, and horizontal cells. Morphological analysis, at different periods after venom extraction, showed that the primary duct has a long cycle of synthesis and secretion, as do the main venom and accessory glands; however, the duct has a mixed mode venom storage, both in the lumen and in secretory vesicles. Mouse anti- venom serum strongly stained the primary duct's epithelium. Subsequent proteomic analysis revealed the synthesis of venom toxins-mainly C-type lectin/C-type lectin-like proteins. We propose that the primary duct's toxin synthesis products complement the final venom bolus. Finally, we hypothesize that the primary duct and the accessory gland (components of the venom glandular apparatus) are part of the evolutionary path from a salivary gland towards the main venom gland.
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http://dx.doi.org/10.3390/toxins10030121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5869409PMC
March 2018

Identification of the alpha-enolase P46 in the extracellular membrane vesicles of Bacteroides fragilis.

Mem Inst Oswaldo Cruz 2018 Mar;113(3):178-184

Universidade Federal do Rio de Janeiro, Instituto de Microbiologia Paulo de Góes, Departamento de Microbiologia Médica, Laboratório de Biologia de Anaeróbios, Rio de Janeiro, RJ, Brasil.

Background: Members of the Bacteroides fragilis group are the most important components of the normal human gut microbiome, but are also major opportunistic pathogens that are responsible for significant mortality, especially in the case of bacteraemia and other severe infections, such as intra-abdominal abscesses. Up to now, several virulence factors have been described that might explain the involvement of B. fragilis in these infections. The secretion of extracellular membrane vesicles (EMVs) has been proposed to play a role in pathogenesis and symbiosis in gram-negative bacteria, by releasing soluble proteins and other molecules. In B. fragilis, these vesicles are known to have haemagglutination and sialidosis activities, and also contain a capsular polysaccharide (PSA), although their involvement in virulence is still not clear.

Objective: The aim of this study was to identify proteins in the EMV of the 638R B. fragilis strain by mass spectrometry, and also to assess for the presence of Bfp60, a surface plasminogen (Plg) activator, previously shown in B. fragilis to be responsible for the conversion of inactive Plg to active plasmin, which can also bind to laminin-1.

Methods: B. fragilis was cultured in a minimum defined media and EMVs were obtained by differential centrifugation, ultracentrifugation, and filtration. The purified EMVs were observed by both transmission electron microscopy (TEM) and immunoelectron microscopy (IM). To identify EMV constituent proteins, EMVs were separated by 1D SDS-PAGE and proteomic analysis of proteins sized 35 kDa to approximately 65 kDa was performed using mass spectrometry (MALDI-TOF MS).

Findings: TEM micrographs proved the presence of spherical vesicles and IM confirmed the presence of Bfp60 protein on their surface. Mass spectrometry identified 23 proteins with high confidence. One of the proteins from the B. fragilis EMVs was identified as an enolase P46 with a possible lyase activity.

Main Conclusions: Although the Bfp60 protein was not detected by proteomics, α-enolase P46 was found to be present in the EMVs of B. fragilis. The P46 protein has been previously described to be present in the outer membrane of B. fragilis as an iron-regulated protein.
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http://dx.doi.org/10.1590/0074-02760170340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5804310PMC
March 2018

Bothrops jararaca accessory venom gland is an ancillary source of toxins to the snake.

J Proteomics 2018 04 8;177:137-147. Epub 2018 Jan 8.

Laboratório de Farmacologia, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900 São Paulo, Brazil; Instituto Nacional de Ciência e Tecnologia em Toxinas (INCTTox/CNPq), Brazil. Electronic address:

In Viperidae snakes, it has been attributed to the main venom gland, a component of the venom gland apparatus, the function of synthesizing all venom toxins and storing them inside a basal-central lumen. However, the role of the accessory gland is still unknown. Here, we analyzed the proteome and the transcriptome of the accessory gland during venom production and secretion cycle. We showed that the accessory gland expresses and synthesizes toxins that are similar to those produced by the main venom gland such as C-type lectin/C-type lectin-like proteins, metalloproteinase, phospholipase A, cysteine rich secretory protein, nerve growth factor, vascular endothelial growth factor, serine proteinase, and l-amino acid oxidase. Our data have shown that toxin synthesis in the accessory gland is asynchronous when compared to the same process in the venom gland. Moreover, this gland also expresses inhibitors of venom phospholipases A and metalloproteinases. Transcriptome analysis showed that the transcripts that correspond to toxins in the accessory gland have a good correlation to the main venom gland transcripts. Therefore, it is proposed that the accessory gland is an ancillary source of toxins to the snake, and provides inhibitors that could control venom toxicity (and integrity) during storage.

Significance: In this study, we propose that the accessory venom gland acts as an important ancillary source of toxins to the snake, in lieu of a depleted main venom gland, and provides inhibiting agents that control venom toxicity (and integrity) during its storage.
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http://dx.doi.org/10.1016/j.jprot.2017.12.009DOI Listing
April 2018

Soybean seed coat chitinase as a defense protein against the stored product pest Callosobruchus maculatus.

Pest Manag Sci 2018 Jun 8;74(6):1449-1456. Epub 2018 Feb 8.

Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro-UENF, Campos dos Goytacazes, RJ, Brazil.

Background: Chitinases (EC 3.2.1.14) are enzymes involved in the breaking of the β-1,4-glycosidic linkages of chitin. In insects, chitin is present mainly in the cuticle and in peritrophic membranes and peritrophic gel. Enzymes with the potential to damage peritrophic membranes and gel, such as chitinase, have been associated with plant defense systems. Identification and characterization of seed coat chitinase as a plant defense molecule may indicate a more effective target for manipulation strategies, which may lead to the prevention of consumption of embryonic tissues by larvae and consequently minimization of seed damage.

Results: We studied the efficiency of soybean seed coat chitinase as a defense molecule against the insect Callosobruchus maculatus. The seed coat chitinase was isolated and identified by mass spectrometry, immunoreacted with an anti-chitinase antibody and shown to have activity against chitin azure and 4-methylumbelliferyl β-D-N,N',N''-triacetylchitotrioside. A chitinase fraction incorporated in artificial cotyledons at 0.1% reduced larval survival by approximately 77%, and at 0.5%, the reduction in larval mass was 60%. Fluorescein isothiocyanate (FITC)-labeled chitinase was detected in the guts and feces of larvae. At 25% in thick artificial seed coats, chitinase showed a high toxicity to larvae, with mortality of 90% and a reduction of larval mass of 87%.

Conclusion: Seed coat chitinase is an important seed defense molecule not only in the cotyledons but also in seed coats, acting as part of the array of defense mechanisms against Callosobruchus maculatus. © 2017 Society of Chemical Industry.
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http://dx.doi.org/10.1002/ps.4832DOI Listing
June 2018

In vitro anthelmintic effects of Spigelia anthelmia protein fractions against Haemonchus contortus.

PLoS One 2017 15;12(12):e0189803. Epub 2017 Dec 15.

Laboratory of Parasite Control, Department of Pathology, Center for Biological and Health Sciences, Federal University of Maranhão, São Luíz MA, Brazil.

Gastrointestinal nematodes are a significant concern for animal health and well-being, and anthelmintic treatment is mainly performed through the use of chemical products. However, bioactive compounds produced by plants have shown promise for development as novel anthelmintics. The aim of this study is to assess the anthelmintic activity of protein fractions from Spigelia anthelmia on the gastrointestinal nematode Haemonchus contortus. Plant parts were separated into leaves, stems and roots, washed with distilled water, freeze-dried and ground into a fine powder. Protein extraction was performed with sodium phosphate buffer (75 mM, pH 7.0). The extract was fractionated using ammonium sulfate (0-90%) and extensively dialyzed. The resulting fractions were named LPF (leaf protein fraction), SPF (stem protein fraction) and RPF (root protein fraction), and the protein contents and activities of the fractions were analyzed. H. contortus egg hatching (EHA), larval exsheathment inhibition (LEIA) and larval migration inhibition (LMIA) assays were performed. Proteomic analysis was conducted, and high-performance liquid chromatography (HPLC) chromatographic profiles of the fractions were established to identify proteins and possible secondary metabolites. S. anthelmia fractions inhibited H. contortus egg hatching, with LPF having the most potent effects (EC50 0.17 mg mL-1). During LEIA, SPF presented greater efficiency than the other fractions (EC50 0.25 mg mL-1). According to LMIA, the fractions from roots, stems and leaves also reduced the number of larvae, with EC50 values of 0.11, 0.14 and 0.21 mg mL-1, respectively. Protein analysis indicated the presence of plant defense proteins in the S. anthelmia fractions, including protease, protease inhibitor, chitinase and others. Conversely, secondary metabolites were absent in the S. anthemia fractions. These results suggest that S. anthelmia proteins are promising for the control of the gastrointestinal nematode H. contortus.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0189803PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5731696PMC
January 2018

Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris.

PLoS Negl Trop Dis 2017 Jul 31;11(7):e0005829. Epub 2017 Jul 31.

Laboratory of Toxinology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, RJ, Brazil.

Snakebite envenomation is a neglected condition that constitutes a public health problem in tropical and subtropical countries, including Brazil. Interestingly, some animals are resistant to snake envenomation due to the presence of inhibitory glycoproteins in their serum that target toxic venom components. DM64 is an acidic glycoprotein isolated from Didelphis aurita (opossum) serum that has been characterized as an inhibitor of the myotoxicity induced by bothropic toxins bearing phospholipase A2 (PLA2) structures. This antitoxic protein can serve as an excellent starting template for the design of novel therapeutics against snakebite envenomation, particularly venom-induced local tissue damage. Therefore, the aim of this work was to produce a recombinant DM64 (rDM64) in the methylotrophic yeast Pichia pastoris and to compare its biological properties with those of native DM64. Yeast fermentation in the presence of Pefabloc, a serine protease inhibitor, stimulated cell growth (~1.5-fold), increased the rDM64 production yield approximately 10-fold and significantly reduced the susceptibility of rDM64 to proteolytic degradation. P. pastoris fermentation products were identified by mass spectrometry and Western blotting. The heterologous protein was efficiently purified from the culture medium by affinity chromatography (with immobilized PLA2 myotoxin) and/or an ion exchange column. Although both native and recombinant DM64 exhibit different glycosylation patterns, they show very similar electrophoretic mobilities after PNGase F treatment. rDM64 formed a noncovalent complex with myotoxin II (Lys49-PLA2) from Bothrops asper and displayed biological activity that was similar to that of native DM64, inhibiting the cytotoxicity of myotoxin II by 92% at a 1:1 molar ratio.
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http://dx.doi.org/10.1371/journal.pntd.0005829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5552330PMC
July 2017

Identification of potential biomarkers for systemic lupus erythematosus diagnosis using two-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry.

Autoimmunity 2017 Jun 4;50(4):247-256. Epub 2017 Jul 4.

a Department of Clinical and Toxicological Analysis, Faculty of Pharmacy , Universidade Federal de Minas Gerais , Belo Horizonte , Minas Gerais , Brazil.

Systemic lupus erythematosus (SLE) is an autoimmune disease of the connective tissue with a large spectrum of clinical manifestations. Immune deregulation leads to autoantibody and immune complexes overproduction, complement activation, and persistent tissue inflammation. Considering that the current diagnosis depends on the interpretation of the complex criteria established by the American College of Rheumatology and that the disease course is characterized by unpredictable activations and remissions, each patient develops different manifestations, and therefore, the discovery of specific biomarkers is urgently required. Therefore, this study aimed to identify putative biomarkers for active and inactive SLE potentially capable in distinguishing laboratorial SLE from other autoimmune diseases. The 2D-DIGE proteomics technique was used to evaluate the differential abundance of proteins between patients with active SLE, inactive SLE, patients with other autoimmune disease, and healthy individuals. Six proteins showed increased abundance in active SLE (A) and inactive SLE (I) compared to the C and O groups, but not between groups A and I. There were two transthyretin (TTR) fragments or proteins with a structure similar to TTR (accession numbers: PDB: 1GKO_A and 2PAB_A), retinol-binding protein 4 (RBP4) isoform X1 (no information in databases such as UNIPROT), and antibody fragments. Two proteins, APO-AIV and SP-40,40, were upregulated in group A than in O and C and in group I versus C, but not in group I versus O. Therefore, we suggest these proteins to be considered as candidates for the diagnosis of SLE.
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http://dx.doi.org/10.1080/08916934.2017.1344975DOI Listing
June 2017

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue.

PLoS Pathog 2017 May 19;13(5):e1006385. Epub 2017 May 19.

Laboratório de Toxinologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.

Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue, and sheds light on new mechanisms of platelet activation and platelet-mediated immune and inflammatory responses.
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http://dx.doi.org/10.1371/journal.ppat.1006385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453622PMC
May 2017