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Plasmodium parasites cause Malaria disease, which remains a significant threat to global health, affecting 200 million people and causing 400,000 deaths yearly. Plasmodium falciparum and Plasmodium vivax remain the two main malaria species affecting humans. Identifying the malaria disease in blood smears requires years of expertise, even for highly trained specialists. Literature studies have been coping with the automatic identification and classification of malaria. However, several points must be addressed and investigated so these automatic methods can be used clinically in a Computer-aided Diagnosis (CAD) scenario. In this work, we assess the transfer learning approach by using well-known pre-trained deep learning architectures. We considered a database with 6222 Region of Interest (ROI), of which 6002 are from the Broad Bioimage Benchmark Collection (BBBC), and 220 were acquired locally by us at Fundação Oswaldo Cruz (FIOCRUZ) in Porto Velho Velho, Rondônia-Brazil, which is part of the legal Amazon. We exhaustively cross-validated the dataset using 100 distinct partitions with 80% train and 20% test for each considering circular ROIs (rough segmentation). Our experimental results show that DenseNet201 has a potential to identify Plasmodium parasites in ROIs (infected or uninfected) of microscopic images, achieving 99.41% AUC with a fast processing time. We further validated our results, showing that DenseNet201 was significantly better (99% confidence interval) than the other networks considered in the experiment. Our results support claiming that transfer learning with texture features potentially differentiates subjects with malaria, spotting those with Plasmodium even in Leukocytes images, which is a challenge. In Future work, we intend scale our approach by adding more data and developing a friendly user interface for CAD use. We aim at aiding the worldwide population and our local natives living nearby the legal Amazon's rivers.
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Microscopia , Humanos , Microscopia/métodos , Plasmodium falciparum/patogenicidade , Plasmodium vivax , Biologia Computacional/métodos , Malária/parasitologia , Plasmodium , Aprendizado Profundo , Bases de Dados Factuais , Processamento de Imagem Assistida por Computador/métodos , Malária Falciparum/parasitologia , Diagnóstico por Computador/métodosRESUMO
Glucose-6-phosphate dehydrogenase (G6PD) is a pentose phosphate pathway (PPP) enzyme that generates NADPH, which is required for cellular redox equilibrium and reductive biosynthesis. It has been demonstrated that abnormal G6PD activation promotes cancer cell proliferation and metastasis. To date, no G6PD inhibitor has passed clinical testing successfully enough to be launched as a medicine. As a result, in this investigation, cannabinoids were chosen to evaluate their anticancer potential by targeting G6PD. Molecular docking indicated that three molecules, Tetrahydrocannabinolic acid (THCA), Cannabichromenic acid (CBCA), and tetrahydrocannabivarin (THCV), have the highest binding affinities for G6PD of -8.61, - 8.39, and 8.01 Kcal/mol. ADMET analysis found that all of them were safe prospective drug candidates. Molecular dynamics (MD) simulation and MM-PBSA analysis confirm the structural compactness and lower conformational variation of protein-ligand complexes, thereby maintaining structural stability and rigidity. Thus, our in silico investigation exhibited all three cannabinoids as potential competitive inhibitors of G6PD.
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BACKGROUND: The molecular dynamics is an approach to obtain kinetic and thermodynamic characteristics of biomolecular structures. The molecular dynamics simulation softwares are very useful, however, most of them are used in command line form and continue with the same common implementation difficulties that plague researchers who are not computer specialists. RESULTS: Here, we have developed the VisualDynamics-a WEB tool developed to automate biological simulations performed in Gromacs using a graphical interface to make molecular dynamics simulation user-friendly task. In this new application the researcher can submit a simulation of the protein in the free form or complexed with a ligand. Can also download the graphics analysis and log files at the end of the simulation. CONCLUSIONS: VisualDynamics is a tool that will accelerate implementations and learning in the area of molecular dynamics simulation. Freely available at https://visualdynamics.fiocruz.br/login , is supported by all major web browsers. VisualDynamics was developed with Flask, which is a Python-based free and open-source framework for web development. The code is freely available for download at GitHub https://github.com/LABIOQUIM/visualdynamics .
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Simulação de Dinâmica Molecular , Software , Proteínas/química , Cinética , NavegadorRESUMO
The inadequacy of available treatments for leishmaniasis has presented up to 40% therapeutic failure. This fact suggests an urgency in the discovery of new drugs or alternative approaches for treating this disease. The objective of this study was to evaluate the antileishmanial activity of combined therapy between crotamine (CTA) from Crotalus durissus terrificus and the pentavalent antimonial Glucantime® (GLU). The assays were in vitro performed measuring the inhibition of Leishmania amazonensis amastigotes, followed by the evaluation of cellular production of cytokines and nitrites. After that, analytical methods were performed in order to characterize the molecules involved in the study by Mass Spectrometry, molecular affinity through an in silico assay and Surface Plasmon Resonance. In vivo experiments with BALB/c mice were performed by analyzing parasitemia, lesion size and immunological mediators. In the in vitro experiments, the pharmacological association improved the inhibition of the amastigotes, modulated the production of cytokines and nitric oxide. The therapy improved the effectiveness of the GLU, demonstrating a decreased parasitemia in the infected tissues. Altogether, the results suggest that the combined approach with CTA and GLU may be a promising alternative for the treatment of cutaneous leishmaniasis.
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Antiprotozoários/uso terapêutico , Venenos de Crotalídeos/uso terapêutico , Crotalus , Leishmania mexicana/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Antimoniato de Meglumina/uso terapêutico , Animais , Antiprotozoários/farmacologia , Venenos de Crotalídeos/farmacologia , Combinação de Medicamentos , Interleucina-12/sangue , Interleucina-12/metabolismo , Leishmania mexicana/isolamento & purificação , Linfonodos/parasitologia , Macrófagos Peritoneais , Espectrometria de Massas , Antimoniato de Meglumina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Óxido Nítrico/metabolismo , Nitritos/análise , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/metabolismoRESUMO
In this report, we describe the semisynthesis of two series of ursolic and betulinic acid derivatives through designed by modifications at the C-3 and C-28 positions and demonstrate their antimalarial activity against chloroquine-resistant P. falciparum (W2 strain). Structural modifications at C-3 were more advantageous to antimalarial activity than simultaneous modifications at C-3 and C-28 positions. The ester derivative, 3ß-butanoyl betulinic acid (7b), was the most active compound (IC50â¯=â¯3.4⯵M) and it did not exhibit cytotoxicity against VERO nor HepG2 cells (CC50â¯>â¯400⯵M), showing selectivity towards parasites (selectivity indexâ¯>â¯117.47). In combination with artemisinin, compound 7b showed an additive effect (CIâ¯=â¯1.14). While docking analysis showed a possible interaction of 7b with the Plasmodium protease PfSUB1, with an optimum binding affinity of -7.02â¯kcal/mol, the rather low inhibition displayed on a Bacillus licheniformis subtilisin A protease activity assay (IC50â¯=â¯93⯵M) and the observed accumulation of ring forms together with a delay of appearance of trophozoites in vitro suggests that the main target of 3ß-butanoyl betulinic acid on Plasmodium may be related to other molecules and processes pertaining to the ring stage. Therefore, compound 7b is the most promising compound for further studies on antimalarial chemotherapy. The results obtained in this study provide suitable information about scaffolds to develop novel antimalarials from natural sources.
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Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Triterpenos/farmacologia , Animais , Antimaláricos/síntese química , Antimaláricos/química , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Triterpenos/síntese química , Triterpenos/química , Células VeroRESUMO
This study delves into the functional and structural implications of non-synonymous single nucleotide polymorphisms (nsSNPs) within the Prolactin Receptor (PRLR) gene. Thirteen deleterious nsSNPs were identified through bioinformatics tools, with SIFT predicting 168 out of 395 nsSNPs as detrimental, exhibiting tolerance index (TI) scores ranging from 0 to 0.05. Polyphen2 assigned likelihood scores >0.99 to all 13 nsSNPs, indicating high probability of harm, while Panther scores classified most nsSNPs as 'probably damaging', with specific mutations like W218R scoring 0.74, suggesting a higher impact. Stability analysis using DDG I-Mutant and DDG Mupro consistently predicted decreased stability for all mutations, with CUPSAT indicating mutations like V125G and W218R significantly decreasing stability. Structural analysis through DynaMut predicted destabilization for all mutations except L196I and L292H. MutPred2 highlighted structural alterations for all nsSNPs except L196I, L293V, R315W, and S353N. Domain analysis revealed key mutations within essential functional domains, with five nsSNPs located within Fibronectin type-III domains. Bayesian analysis through ConSurf identified 9 critical residues, with 11 nsSNPs exhibiting notably high conservation. STRING analysis unveiled a complex interaction network, indicating involvement in vital biological processes like lactation. Molecular dynamics (MD) simulations, spanning 100 nanoseconds, elucidated structural dynamics induced by detrimental missense SNPs. Post-translational modification (PTM) analysis identified specific mutations, such as R351, involved in methylation, while S353 was implicated in phosphorylation and glycosylation. These findings offer comprehensive insights into the molecular and phenotypic effects of deleterious nsSNPs in the PRLR gene, crucial for selective breeding.Communicated by Ramaswamy H. Sarma.
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Foot and mouth Disease virus (FMDV) belongs to Picornaviridae family and Aphthovirus genus causing Foot and mouth disease (FMD) in cloven-hoofed animals. FMDV, a prevalent virus induces both acute and chronic infections with high mutation rates resulting in seven primary serotypes, making vaccine development indispensable. Due to time and cost effectiveness of the immunoinformatic approach, we designed in-silico polyepitope vaccine (PEV) for the curtailment of FMDV. Structural and immunogenic parts of FMDV (Viral Protein 1 (VP1), Viral Protein 2 (VP2), Viral Protein 3 (VP3), and Viral Protein 4 (VP4)) were used to design the cytotoxic T Lymphocyte (CTL), Helper T Lymphocyte (HTL), and B-cell epitopes, followed by screening for antigenic, non-allergenic, Interferon (IFN) simulator, and non-toxicity, which narrowed down to 7 CTL, 3 HTL, and 12 B-cell epitopes. These selected epitopes were linked using appropriate linkers and Cholera Toxin B (CTB) adjuvant for immunological modulation. The physiochemical analyses followed by the structure prediction demonstrated the stability, hydrophilicity and solubility of the PEV. The interactions and stability between the vaccine, Toll like Receptor 3 (TLR3) and Toll like receptor 7 (TLR7) were revealed by molecular docking and Molecular Mechanics/Poisson Boltzmann Surface Area (MMPBSA) with high stability and compactness verified by MD simulation. In-silico immune simulation demonstrated a strong immunological response. FMDV-PEV (Poly epitope vaccine) will be effectively produced in an E. coli system, as codon optimization and cloning in an expression vector was performed. The effectiveness, safety, and immunogenicity profile of FMDV-PEV may be confirmed by further experimental validations.Communicated by Ramaswamy H. Sarma.
The structural and immunogenic parts of FMDV were targeted for developing VaccineCTB-adjuvant and appropriate linkers, enhancing the immunogenicity of the PEVMinimal deformability and high stability of Vaccine using immunoinformaticsStrong antigen-specific humoral and cellular immune response of potential vaccineResults indicating the effectiveness, safety, and immunogenicity of the PEV.
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The PfCLAG9 has been extensively studied because their immunogenicity. Thereby, the gene product is important for therapeutics interventions and a potential vaccine candidate. Antibodies against synthetic peptides corresponding to selected sequences of the Plasmodium falciparum antigen PfCLAG9 were found in sera of falciparum malaria patients from Rondônia, in the Brazilian Amazon. Much higher antibody titres were found in semi-immune and immune asymptomatic parasite carriers than in subjects suffering clinical infections, corroborating original findings in Papua Guinea. However, sera of Plasmodium vivax patients from the same Amazon area, in particular from asymptomatic vivax parasite carriers, reacted strongly with the same peptides. Bioinformatic analyses revealed regions of similarity between P. falciparum Pfclag9 and the P. vivax ortholog Pvclag7. Indirect fluorescent microscopy analysis showed that antibodies against PfCLAG9 peptides elicited in BALB/c mice react with human red blood cells (RBCs) infected with both P. falciparum and P. vivax parasites. The patterns of reactivity on the surface of the parasitised RBCs are very similar. The present observations support previous findings that PfCLAG9 may be a target of protective immune responses and raises the possibility that the cross reactive antibodies to PvCLAG7 in mixed infections play a role in regulate the fate of Plasmodium mixed infections.
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Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Moléculas de Adesão Celular/imunologia , Malária Falciparum/imunologia , Malária Vivax/imunologia , Plasmodium falciparum/imunologia , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Animais , Brasil , Portador Sadio , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Eritrócitos/parasitologia , Feminino , Humanos , Malária Falciparum/parasitologia , Malária Vivax/parasitologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Bothrops atrox venom comprises several types of bioactive molecules, enzymatic and non-enzymatic, among those, Batroxrhagin is the most predominant SVMP P-III enzyme, which are responsible for induction of local and systemic hemorrhage and muscle fibers damage, impairing regeneration. Due to great difficulties in establishing an antibothropic drug, new strategies must be addressed to achieve a more effective and efficient treatment. There are no studies of specific catalytic inhibitors of Batroxrhagin. However, there are in vitro studies that have described similar metalloprotease inhibitors. The inhibitor batimastat was used as a leading compound for the search and selection of similar candidates. This molecule is widely cited as a metalloprotease inhibitor and as an antimetastatic. In addition to batimastat-like molecules, four other reported metalloprotease inhibitors were included to compose the study's positive control group. Hence, 580 molecules were tested. The three-dimensional structure of B. atrox Batroxrhagin was predicted based on homologous structures using Modeller 9.20. Molecular docking calculation was performed using Autodock 4.2 and molecular surfaces and interactions were analyzed using Biovia/Discovery Studio 2017. Among 576 molecules, 42 similar to batismast resulted in a better energy of interaction than all positive controls, including batimastat itself. The batimastat-like molecules with lowest energy and positive controls were subjected to molecular dynamics for 30 ns in Gromacs 2019.4. This batimastat-like molecule produced better stability among all the Batroxrhagin-ligand complexes analyzed. Overall, the proposed compounds present justifiable evidence for future in vitro tests aiming to inhibit Batroxrhagin. Communicated by Ramaswamy H. Sarma.
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Bothrops , Venenos de Crotalídeos , Animais , Simulação de Acoplamento Molecular , MetaloproteasesRESUMO
Background: Cathepsin D (CatD) is a lysosomal proteolytic enzyme expressed in almost all tissues and organs. This protease is a multifunctional enzyme responsible for essential biological processes such as cell cycle regulation, differentiation, migration, tissue remodeling, neuronal growth, ovulation, and apoptosis. The overexpression and hypersecretion of CatD have been correlated with cancer aggressiveness and tumor progression, stimulating cancer cell proliferation, fibroblast growth, and angiogenesis. In addition, some studies report its participation in neurodegenerative diseases and inflammatory processes. In this regard, the search for new inhibitors from natural products could be an alternative against the harmful effects of this enzyme. Methods: An investigation was carried out to analyze CatD interaction with snake venom toxins in an attempt to find inhibitory molecules. Interestingly, human CatD shows the ability to bind strongly to snake venom phospholipases A2 (svPLA2), forming a stable muti-enzymatic complex that maintains the catalytic activity of both CatD and PLA2. In addition, this complex remains active even under exposure to the specific inhibitor pepstatin A. Furthermore, the complex formation between CatD and svPLA2 was evidenced by surface plasmon resonance (SPR), two-dimensional electrophoresis, enzymatic assays, and extensive molecular docking and dynamics techniques. Conclusion: The present study suggests the versatility of human CatD and svPLA2, showing that these enzymes can form a fully functional new enzymatic complex.
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The coronavirus disease COVID-19 has been the cause of millions of deaths worldwide. Among the SARS-CoV-2 proteins, the non-structural protein 1 (NSP1) has great importance during the virus infection process and is present in both alpha and beta-CoVs. Therefore, monitoring of NSP1 polymorphisms is crucial in order to understand their role during infection and virus-induced pathogenicity. Herein, we analyzed how mutations detected in the circulating SARS-CoV-2 in the population of the city of Manaus, Amazonas state, Brazil could modify the tertiary structure of the NSP1 protein. Three mutations were detected in the SARS-CoV-2 NSP1 gene: deletion of the amino acids KSF from positions 141 to 143 (delKSF), SARS-CoV-2, lineage B.1.195; and two substitutions, R29H and R43C, SARS-CoV-2 lineage B.1.1.28 and B.1.1.33, respectively. The delKSF was found in 47 samples, whereas R29H and R43C were found in two samples, one for each mutation. The NSP1 structures carrying the mutations R43C and R29H on the N-terminal portion (e.g. residues 10 to 127) showed minor backbone divergence compared to the Wuhan model. However, the NSP1 C-terminal region (residues 145 to 180) was severely affected in the delKSF and R29H mutants. The intermediate variable region (residues 144 to 148) leads to changes in the C-terminal region, particularly in the delKSF structure. New investigations must be carried out to analyze how these changes affect NSP1 activity during the infection. Our results reinforce the need for continuous genomic surveillance of SARS-CoV-2 to better understand virus evolution and assess the potential impact of the viral mutations on the approved vaccines and future therapies.
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COVID-19/epidemiologia , SARS-CoV-2/genética , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos/genética , Substituição de Aminoácidos/genética , Brasil/epidemiologia , Humanos , Polimorfismo Genético/genética , Deleção de Sequência/genéticaRESUMO
There is a growing need for research on new antimalarial agents against Plasmodium falciparum infection, especially in regards to planning molecular architecture for specific molecular targets of the parasite. Thus, a metalloprotease from Bothrops moojeni, known as BmooMPα-I, was explored in this study, through in silico assays, aiming at the development of a peptide generated from this molecule with potential inhibitory action on PfPNP, an enzyme necessary for the survival of the parasite. In order to isolate BmooMPα-I, cation exchange and reverse phase chromatographies were performed, followed by in vitro assays of antiparasitic activity against the W2 strain of P. falciparum. The interactions between BmooMPα-I and PfPNP were evaluated via docking, and the resulting peptide, described as Pep1 BM, was selected according to the BmooMPα-I region demonstrating the best interaction score with the target of interest. The values for the specific activities of the PfPNP reaction were measured using the inorganic phosphate substrate and MESG. The fraction corresponding to BmooMPα-I was identified as fraction 4 in the cation exchange chromatography step, due to proteolytic activity on casein and the presence of a major band at â 23â¯kDa. BmooMPα-I was able to inhibit in vitro growth of W2 P. falciparum, with an IC50 value of 16.14⯵g/mL. Virtual screening with Pep1 BM demonstrated two PfPNP target binding regions, with ΔG values at the interaction interface of -10.75â¯kcal/mol and -11.74â¯kcal/mol. A significant reduction in the enzymatic activity of PfPNP was observed in the presence of Pep 1 BM when compared to the assay in the absence of this possible inhibitor. BmooMPα-I showed activity in vitro against W2 P. falciparum. By means of in silico techniques, the Pep 1 BM was identified as having potential binding affinity to the catalytic site of PfPNP and of inhibiting its catalytic activity in vitro.
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Antimaláricos/farmacologia , Venenos de Crotalídeos/enzimologia , Metaloendopeptidases/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Purina-Núcleosídeo Fosforilase/metabolismo , Animais , Antimaláricos/química , Bothrops/metabolismo , Domínio Catalítico , Venenos de Crotalídeos/química , Venenos de Crotalídeos/farmacologia , Cinética , Malária Falciparum/tratamento farmacológico , Metaloendopeptidases/química , Simulação de Acoplamento Molecular/métodos , Peptídeos/química , Especificidade por SubstratoRESUMO
Background Cathepsin D (CatD) is a lysosomal proteolytic enzyme expressed in almost all tissues and organs. This protease is a multifunctional enzyme responsible for essential biological processes such as cell cycle regulation, differentiation, migration, tissue remodeling, neuronal growth, ovulation, and apoptosis. The overexpression and hypersecretion of CatD have been correlated with cancer aggressiveness and tumor progression, stimulating cancer cell proliferation, fibroblast growth, and angiogenesis. In addition, some studies report its participation in neurodegenerative diseases and inflammatory processes. In this regard, the search for new inhibitors from natural products could be an alternative against the harmful effects of this enzyme. Methods An investigation was carried out to analyze CatD interaction with snake venom toxins in an attempt to find inhibitory molecules. Interestingly, human CatD shows the ability to bind strongly to snake venom phospholipases A2 (svPLA2), forming a stable muti-enzymatic complex that maintains the catalytic activity of both CatD and PLA2. In addition, this complex remains active even under exposure to the specific inhibitor pepstatin A. Furthermore, the complex formation between CatD and svPLA2 was evidenced by surface plasmon resonance (SPR), two-dimensional electrophoresis, enzymatic assays, and extensive molecular docking and dynamics techniques. Conclusion The present study suggests the versatility of human CatD and svPLA2, showing that these enzymes can form a fully functional new enzymatic complex.
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Catepsina D/análise , Venenos Elapídicos/química , Fosfolipases A2/análise , Complexos Multienzimáticos/químicaRESUMO
Purine nucleoside phosphorylase (PNP) (EC.2.4.2.1) is an enzyme that catalyzes the cleavage of N-ribosidic bonds of the purine ribonucleosides and 2-deoxyribonucleosides in the presence of inorganic orthophosphate as a second substrate. This enzyme is involved in purine-salvage pathway and has been proposed as a promising target for design and development of antimalarial and antibacterial drugs. Recent elucidation of the three-dimensional structure of PNP by X-ray protein crystallography left open the possibility of structure-based virtual screening initiatives in combination with molecular dynamics simulations focused on identification of potential new antimalarial drugs. Most of the previously published molecular dynamics simulations of PNP were carried out on human PNP, a trimeric PNP. The present article describes for the first time molecular dynamics simulations of hexameric PNP from Plasmodium falciparum (PfPNP). Two systems were simulated in the present work, PfPNP in ligand free form, and in complex with immucillin and sulfate. Based on the dynamical behavior of both systems the main results related to structural stability and protein-drug interactions are discussed.
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Simulação de Dinâmica Molecular , Plasmodium falciparum/enzimologia , Multimerização Proteica , Purina-Núcleosídeo Fosforilase/química , Sítios de Ligação , Biocatálise , Estabilidade Enzimática , Fosfatos/metabolismo , Maleabilidade , Estrutura Terciária de Proteína , Nucleosídeos de Purina/química , Nucleosídeos de Purina/metabolismo , Purina-Núcleosídeo Fosforilase/metabolismo , Pirimidinonas/química , Pirimidinonas/metabolismo , Sulfatos/metabolismo , Fatores de TempoRESUMO
The PfCLAG9 has been extensively studied because their immunogenicity. Thereby, the gene product is important for therapeutics interventions and a potential vaccine candidate. Antibodies against synthetic peptides corresponding to selected sequences of the Plasmodium falciparum antigen PfCLAG9 were found in sera of falciparum malaria patients from Rondônia, in the Brazilian Amazon. Much higher antibody titres were found in semi-immune and immune asymptomatic parasite carriers than in subjects suffering clinical infections, corroborating original findings in Papua Guinea. However, sera of Plasmodium vivax patients from the same Amazon area, in particular from asymptomatic vivax parasite carriers, reacted strongly with the same peptides. Bioinformatic analyses revealed regions of similarity between P. falciparum Pfclag9 and the P. vivax ortholog Pvclag7. Indirect fluorescent microscopy analysis showed that antibodies against PfCLAG9 peptides elicited in BALB/c mice react with human red blood cells (RBCs) infected with both P. falciparum and P. vivax parasites. The patterns of reactivity on the surface of the parasitised RBCs are very similar. The present observations support previous findings that PfCLAG9 may be a target of protective immune responses and raises the possibility that the cross reactive antibodies to PvCLAG7 in mixed infections play a role in regulate the fate of Plasmodium mixed infections.