111 results on '"Alves LR"'
Search Results
2. Editorial: Global excellence in fungal pathogenesis: Central and South America.
- Author
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Alves LR, Borges CL, and Almeida F
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- South America, Humans, Central America epidemiology, Fungi pathogenicity, Mycoses microbiology
- Abstract
Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
- Published
- 2024
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3. Prevalence and Risk Factors of Bone and Dental Lesions in Neotropical Deer.
- Author
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Silva TA, Martins ADS, Alves LR, Pereira LWB, Saraiva JR, Duarte JMB, Zanetti EDS, Schweitzer CM, Dutra IS, and Borsanelli AC
- Abstract
Bone and dental lesions have been documented in various deer species globally, affecting the efficiency of ingestion and digestion, consequently influencing their general health and leading to a decline in survival and reproductive performance. The present study aimed to characterize bone and dental lesions in the dry skulls of individual deer, estimate the prevalence of these lesions, and assess potential risk factors associated with the development of bone and dental alterations. This study assessed bone and dental lesions in 180 dry skulls of eleven neotropical deer species, originating from both captivity and wildlife conditions, through direct visual inspection. A high prevalence of bone and dental lesions was observed in all analyzed species. Dental calculus was the most common alteration (96.7%), followed by dental wear (71.1%). Animal age positively correlated with most bone and dental alterations, indicating that older animals showed more lesions. Additionally, the prevalence of these alterations was similar between sexes. Moreover, all lesions were more common in captive-bred animals, likely attributed to their older age and a less diverse diet. Blastocerus dichotomus and Mazama americana were most affected by bone resorption and dental trauma and had the highest dental calculus prevalence, along with Subulo gouazoubira and Passalites nemorivagus . All eleven species evaluated in the present study were susceptible to the occurrence of bone and dental lesions. Therefore, monitoring oral health and diet in captivity are fundamental practices for the conservation of these species.
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- 2024
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4. Comprehensive characterization of extracellular vesicles produced by environmental (Neff) and clinical (T4) strains of Acanthamoeba castellanii .
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Medeiros EG, Valente MR, Honorato L, Ferreira MdS, Mendoza SR, Gonçalves DdS, Martins Alcântara L, Gomes KX, Pinto MR, Nakayasu ES, Clair G, da Rocha IFM, Dos Reis FCG, Rodrigues ML, Alves LR, Nimrichter L, Casadevall A, and Guimarães AJ
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- Humans, Lipid Metabolism genetics, Protozoan Proteins metabolism, Protozoan Proteins genetics, Proteome metabolism, Proteome genetics, Acanthamoeba castellanii metabolism, Acanthamoeba castellanii genetics, Extracellular Vesicles metabolism, Extracellular Vesicles genetics, Proteomics
- Abstract
We conducted a comprehensive comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains, Neff (environmental) and T4 (clinical). Morphological analysis via transmission electron microscopy revealed slightly larger Neff EVs (average = 194.5 nm) compared to more polydisperse T4 EVs (average = 168.4 nm). Nanoparticle tracking analysis (NTA) and dynamic light scattering validated these differences. Proteomic analysis of the EVs identified 1,352 proteins, with 1,107 common, 161 exclusive in Neff, and 84 exclusively in T4 EVs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping revealed distinct molecular functions and biological processes and notably, the T4 EVs enrichment in serine proteases, aligned with its pathogenicity. Lipidomic analysis revealed a prevalence of unsaturated lipid species in Neff EVs, particularly triacylglycerols, phosphatidylethanolamines (PEs), and phosphatidylserine, while T4 EVs were enriched in diacylglycerols and diacylglyceryl trimethylhomoserine, phosphatidylcholine and less unsaturated PEs, suggesting differences in lipid metabolism and membrane permeability. Metabolomic analysis indicated Neff EVs enrichment in glycerolipid metabolism, glycolysis, and nucleotide synthesis, while T4 EVs, methionine metabolism. Furthermore, RNA-seq of EVs revealed differential transcript between the strains, with Neff EVs enriched in transcripts related to gluconeogenesis and translation, suggesting gene regulation and metabolic shift, while in the T4 EVs transcripts were associated with signal transduction and protein kinase activity, indicating rapid responses to environmental changes. In this novel study, data integration highlighted the differences in enzyme profiles, metabolic processes, and potential origins of EVs in the two strains shedding light on the diversity and complexity of A. castellanii EVs and having implications for understanding host-pathogen interactions and developing targeted interventions for Acanthamoeba -related diseases.IMPORTANCEA comprehensive and fully comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains of distinct virulence, a Neff (environmental) and T4 (clinical), revealed striking differences in their morphology and protein, lipid, metabolites, and transcripts levels. Data integration highlighted the differences in enzyme profiles, metabolic processes, and potential distinct origin of EVs from both strains, shedding light on the diversity and complexity of A. castellanii EVs, with direct implications for understanding host-pathogen interactions, disease mechanisms, and developing new therapies for the clinical intervention of Acanthamoeba -related diseases., Competing Interests: The authors declare no conflict of interest.
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- 2024
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5. Unraveling DEHP influence on hemoglobin S polymerization in sickle cell disease: Ex vivo, in vitro and in silico analysis.
- Author
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Camacho RA, Machado AV, de Oliveira Mendonça F, Teixeira-Alves LR, Guimarães-Nobre CC, Mendonça-Reis E, da Silva PF, Cardim-Pires TR, Miranda-Alves L, and Berto-Junior C
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- Humans, Computer Simulation, Anemia, Sickle Cell genetics, Anemia, Sickle Cell metabolism, Hemoglobin, Sickle genetics, Hemoglobin, Sickle metabolism, Polymerization, Erythrocytes drug effects, Erythrocytes metabolism, Diethylhexyl Phthalate toxicity
- Abstract
Sickle cell disease (SCD) is a hereditary hemoglobinopathy, caused by a mutation at position 6 of the β-globin chain and patients are frequently exposed to several blood transfusions in order to maintain physiological function. Transfusion blood bags are composed of PVC and phthalates (as DEHP) are often introduced to the material in order to confer malleability. In this sense, DEHP can easily elute to the blood and cause harmful effects. This study aimed to unravel DEHP effect on SCD patient's hemoglobin function. We found that HbS polymerization using whole erythrocytes is decreased by DEHP in ex vivo experiments and this effect might be mediated by the DEHP-VAL6 interaction, evaluated in silico. Isolated HbS exhibited less polymerization at low DEHP concentrations and increased polymerization rate at higher concentration. When analyzing the propensity to aggregate, HbS is more inclined to aggregate when compared to HbA due to the residue 6 mutation. Circular dichroism showed characteristic hemoglobin peaks for oxygenated HbS that are lost when oxygen is sequestered, and DEHP at higher concentration mildly recovers a peak close to the second hemoglobin one. Finally, by transmission electron microscopy we demonstrated that high DEHP concentration increased polymer formation with a more organized structure. These findings show for the first-time the beneficial effect of low-dose DEHP on HbS polymerization., Competing Interests: Declaration of competing interest There is no conflict of interest., (Copyright © 2024. Published by Elsevier Ltd.)
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- 2024
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6. Density-based lipoprotein depletion improves extracellular vesicle isolation and functional analysis.
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Merij LB, da Silva LR, Palhinha L, Gomes MT, Dib PRB, Martins-Gonçalves R, Toledo-Quiroga K, Raposo-Nunes MA, Andrade FB, de Toledo Martins S, Nascimento ALR, Rocha VN, Alves LR, Bozza PT, de Oliveira Trugilho MR, and Hottz ED
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- Humans, Blood Platelets metabolism, Centrifugation, Density Gradient, Inflammation blood, Proteome, Monocytes metabolism, Extracellular Vesicles metabolism, Proteomics methods, Lipoproteins blood, Chromatography, Gel
- Abstract
Background: Blood plasma is the main source of extracellular vesicles (EVs) in clinical studies aiming to identify biomarkers and to investigate pathophysiological processes, especially regarding EV roles in inflammation and thrombosis. However, EV isolation from plasma has faced the fundamental issue of lipoprotein contamination, representing an important bias since lipoproteins are highly abundant and modulate cell signaling, metabolism, and thromboinflammation., Objectives: Here, we aimed to isolate plasma EVs after depleting lipoproteins, thereby improving sample purity and EV thromboinflammatory analysis., Methods: Density-based gradient ultracentrifugation (G-UC) was used for lipoprotein depletion before EV isolation from plasma through size-exclusion chromatography (SEC) or serial centrifugation (SC). Recovered EVs were analyzed by size, concentration, cellular source, ultrastructure, and bottom-up proteomics., Results: G-UC efficiently separated lipoproteins from the plasma, allowing subsequent EV isolation through SEC or SC. Combined analysis from EV proteomics, cholesterol quantification, and apoB-100 detection confirmed the significant reduction in lipoproteins from isolated EVs. Proteomic analysis identified similar gene ontology and cellular components in EVs, regardless of lipoprotein depletion, which was consistent with similar EV cellular sources, size, and ultrastructure by flow cytometry and transmission electron microscopy. Importantly, lipoprotein depletion increased the detection of less abundant proteins in EV proteome and enhanced thromboinflammatory responses of platelets and monocytes stimulated in vitro with EV isolates., Conclusion: Combination of G-UC+SEC significantly reduced EV lipoprotein contamination without interfering in EV cellular source, gene ontology, and ultrastructure, allowing the recovery of highly pure EVs with potential implications for functional assays and proteomic and lipidomic analyses., Competing Interests: Declaration of competing interests There are no conflicting interests to disclose., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)
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- 2024
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7. TSH Receptor Reduces Hemoglobin S Polymerization and Increases Deformability and Adhesion of Sickle Erythrocytes.
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Mendonça-Reis E, Guimarães-Nobre CC, Teixeira-Alves LR, Miranda-Alves L, and Berto-Junior C
- Abstract
SCD is a hereditary disorder caused by genetic mutation in the beta-globin gene, resulting in abnormal hemoglobin, HbS that forms sickle-shaped erythrocytes under hypoxia. Patients with SCD have endocrine disorders and it was described that 7% of these patients have clinical hypothyroidism. Recent studies have shown that mature erythrocytes possess TSH receptors. Thus, we aimed to assess the effects of TSH on SCD erythrocytes. The experiments were conducted using different concentrations of TSH (1, 2, 3, and 5 mIU/L). In HbS polymerization assay, erythrocytes were exposed to TSH in hypoxia to induce polymerization, and measurements were taken for 30 minutes. The deformability assay was made using Sephacryl-S 500 columns to separate deformable from nondeformable cells. Static adhesion test utilized thrombospondin to assess erythrocyte adhesion in the presence of TSH. TSH at all contractions were able to reduce polymerization of HbS and increase deformability. The static adhesion of erythrocytes at the lowest concentrations of 1 and 2 mIU/L were increased, but at higher contractions of 3 and 5 mIU/L, static adhesion was not modulated. The results suggest that TSH has potential involvement in the pathophysiology of sickle cell disease by inhibiting HbS polymerization, positively modulating deformability and impacting static adhesion to thrombospondin., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2024 Evelyn Mendonça-Reis et al.)
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- 2024
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8. TcZC3HTTP, a regulatory element that contributes to Trypanosoma cruzi cell proliferation.
- Author
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Romagnoli BAA, Lucena ACR, Freire ER, Munhoz da Rocha IF, Alves LR, and Goldenberg S
- Subjects
- Humans, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, RNA metabolism, RNA, Messenger metabolism, Cell Proliferation, Protozoan Proteins genetics, Protozoan Proteins metabolism, Trypanosoma cruzi, Chagas Disease parasitology
- Abstract
Post-transcriptional regulation of gene expression is a critical process for adapting to and surviving Trypanosoma cruzi , a parasite with a complex life cycle. RNA-binding proteins (RBPs) are key players in this regulation, forming ribonucleoprotein complexes (messenger ribonucleoproteins) and RNA granules that control transcript stability, localization, degradation, and translation modulation. Understanding the specific roles of individual RBPs is crucial for unraveling the details of this regulatory network. In this study, we generated null mutants of the TcZC3HTTP gene, a specific RBP in the Trypanosoma family characterized by a C3H zinc finger and a DNAJ domain associated with RNA and protein binding, respectively. Through cell growth assays, we demonstrated that the absence of TcZC3HTTP or the expression of an additional tagged version impacted epimastigote growth, indicating its contribution to cell proliferation. TcZC3HTTP was found to associate with mRNAs involved in cell cycle and division in epimastigotes, while in nutritionally stressed parasites it exhibited associations with mRNAs coding for other RBPs and rRNA. Furthermore, our analysis identified that TcZC3HTTP protein partners were different during normal growth conditions compared to starvation conditions, with the latter showing enrichment of ribosomal proteins and other RBPs. Therefore, this study provides insights into TcZC3HTTP's role in the post-transcriptional regulation of gene expression during normal growth and nutritional stress in T. cruzi , uncovering its versatile functions in different cellular contexts.IMPORTANCEUnderstanding how Trypanosoma cruzi , the causative agent of Chagas disease, regulates gene expression is crucial for developing targeted interventions. In this study, we investigated the role of TcZC3HTTP, an RNA-binding protein, in post-transcriptional regulation. Our findings demonstrate that TcZC3HTTP is relevant for the growth and proliferation of epimastigotes, a stage of the parasite's life cycle. We identified its associations with specific mRNAs involved in cell cycle and division and its interactions with enzymes and other RNA-binding proteins (RBPs) under normal and starvation conditions. These insights shed light on the regulatory network underlying gene expression in T. cruzi and reveal the multifaceted functions of RBPs in this parasite. Such knowledge enhances our understanding of the parasite's biology and opens avenues for developing novel therapeutic strategies targeting post-transcriptional gene regulation in T. cruzi ., Competing Interests: The authors declare no conflict of interest.
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- 2024
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9. RNA-containing extracellular vesicles in infection.
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Schemiko Almeida K, Rossi SA, and Alves LR
- Subjects
- Humans, Animals, MicroRNAs genetics, MicroRNAs metabolism, RNA metabolism, RNA genetics, Infections metabolism, Cell Communication, Fungi genetics, Fungi metabolism, Extracellular Vesicles metabolism, Host-Pathogen Interactions genetics
- Abstract
Extracellular vesicles (EVs) are membrane-bound particles released by cells that play vital roles in intercellular communication by transporting diverse biologically active molecules, including RNA molecules, including mRNA, miRNA, lncRNA, and other regulatory RNAs. These RNA types are protected within the lipid bilayer of EVs, ensuring their stability and enabling long-distance cellular interactions. Notably, EVs play roles in infection, where pathogens and host cells use EV-mediated RNA transfer to influence immune responses and disease outcomes. For example, bacterial EVs play a crucial role in infection by modulating host immune responses and facilitating pathogen invasion. This review explores the complex interactions between EV-associated RNA and host-pathogen dynamics in bacteria, parasites, and fungi, aiming to uncover molecular mechanisms in infectious diseases and potential therapeutic targets.
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- 2024
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10. Guidelines for the purification and characterization of extracellular vesicles of parasites.
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Fernandez-Becerra C, Xander P, Alfandari D, Dong G, Aparici-Herraiz I, Rosenhek-Goldian I, Shokouhy M, Gualdron-Lopez M, Lozano N, Cortes-Serra N, Karam PA, Meneghetti P, Madeira RP, Porat Z, Soares RP, Costa AO, Rafati S, da Silva AC, Santarém N, Fernandez-Prada C, Ramirez MI, Bernal D, Marcilla A, Pereira-Chioccola VL, Alves LR, Portillo HD, Regev-Rudzki N, de Almeida IC, Schenkman S, Olivier M, and Torrecilhas AC
- Abstract
Parasites are responsible for the most neglected tropical diseases, affecting over a billion people worldwide (WHO, 2015) and accounting for billions of cases a year and responsible for several millions of deaths. Research on extracellular vesicles (EVs) has increased in recent years and demonstrated that EVs shed by pathogenic parasites interact with host cells playing an important role in the parasite's survival, such as facilitation of infection, immunomodulation, parasite adaptation to the host environment and the transfer of drug resistance factors. Thus, EVs released by parasites mediate parasite-parasite and parasite-host intercellular communication. In addition, they are being explored as biomarkers of asymptomatic infections and disease prognosis after drug treatment. However, most current protocols used for the isolation, size determination, quantification and characterization of molecular cargo of EVs lack greater rigor, standardization, and adequate quality controls to certify the enrichment or purity of the ensuing bioproducts. We are now initiating major guidelines based on the evolution of collective knowledge in recent years. The main points covered in this position paper are methods for the isolation and molecular characterization of EVs obtained from parasite-infected cell cultures, experimental animals, and patients. The guideline also includes a discussion of suggested protocols and functional assays in host cells., Competing Interests: No potential conflicts of interest were reported by the authors., (© 2023 The Authors. Journal of Extracellular Biology published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)
- Published
- 2023
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11. The characterization of RNA-binding proteins and RNA metabolism-related proteins in fungal extracellular vesicles.
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Dallastella M, de Oliveira WK, Rodrigues ML, Goldenberg S, and Alves LR
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- Animals, Proteomics, RNA, Messenger metabolism, RNA-Binding Proteins metabolism, Mammals genetics, RNA analysis, Extracellular Vesicles metabolism
- Abstract
RNA-binding proteins (RBPs) are essential for regulating RNA metabolism, stability, and translation within cells. Recent studies have shown that RBPs are not restricted to intracellular functions and can be found in extracellular vesicles (EVs) in different mammalian cells. EVs released by fungi contain a variety of proteins involved in RNA metabolism. These include RNA helicases, which play essential roles in RNA synthesis, folding, and degradation. Aminoacyl-tRNA synthetases, responsible for acetylating tRNA molecules, are also enriched in EVs, suggesting a possible link between these enzymes and tRNA fragments detected in EVs. Proteins with canonical RNA-binding domains interact with proteins and RNA, such as the RNA Recognition Motif (RRM), Zinc finger, and hnRNP K-homology (KH) domains. Polyadenylate-binding protein (PABP) plays a critical role in the regulation of gene expression by binding the poly(A) tail of messenger RNA (mRNA) and facilitating its translation, stability, and localization, making it a key factor in post-transcriptional control of gene expression. The presence of proteins related to the RNA life cycle in EVs from different fungal species suggests a conserved mechanism of EV cargo packing. Various models have been proposed for selecting RNA molecules for release into EVs. Still, the actual loading processes are unknown, and further molecular characterization of these proteins may provide insight into the mechanism of RNA sorting into EVs. This work reviews the current knowledge of RBPs and proteins related to RNA metabolism in EVs derived from distinct fungi species, and presents an analysis of proteomic datasets through GO term and orthology analysis, Our investigation identified orthologous proteins in fungal EVs on different fungal species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Dallastella, Oliveira, Rodrigues, Goldenberg and Alves.)
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- 2023
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12. Modulation of Klebsiella pneumoniae Outer Membrane Vesicle Protein Cargo under Antibiotic Treatment.
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Lucena ACR, Ferrarini MG, de Oliveira WK, Marcon BH, Morello LG, Alves LR, and Faoro H
- Abstract
Klebsiella pneumoniae is a nosocomial pathogen and an important propagator of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. Like other Gram-negative bacteria, they secrete outer membrane vesicles (OMVs) that distribute virulence and resistance factors. Here, we subjected a K . pneumoniae -XDR to subinhibitory concentrations of meropenem, amikacin, polymyxin B, and a combination of these agents to evaluate changes in the protein cargo of OMVs through liquid chromatography-tandem mass spectrometry (LC-MS/MS). Genome sequencing of the clinical isolate K . pneumoniae strain HCD1 (KpHCD1) revealed the presence of 41 resistance genes and 159 virulence factors. We identified 64 proteins in KpHCD1-OMVs modulated with different antibiotic treatments involved in processing genetic information, environmental information, cell envelope formation, energy metabolism, and drug resistance. The OMV proteome expression profile suggests that OMVs may be associated with pathogenicity, survival, stress response, and resistance dissemination.
- Published
- 2023
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13. Modulation of Virulence Factors during Trypanosoma cruzi Differentiation.
- Author
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Oliveira C, Holetz FB, Alves LR, and Ávila AR
- Abstract
Chagas disease is a neglected tropical disease caused by Trypanosoma cruzi . This protozoan developed several mechanisms to infect, propagate, and survive in different hosts. The specific expression of proteins is responsible for morphological and metabolic changes in different parasite stages along the parasite life cycle. The virulence strategies at the cellular and molecular levels consist of molecules responsible for mediating resistance mechanisms to oxidative damage, cellular invasion, and immune evasion, performed mainly by surface proteins. Since parasite surface coat remodeling is crucial to invasion and infectivity, surface proteins are essential virulence elements. Understanding the factors involved in these processes improves the knowledge of parasite pathogenesis. Genome sequencing has opened the door to high-throughput technologies, allowing us to obtain a deeper understanding of gene reprogramming along the parasite life cycle and identify critical molecules for survival. This review therefore focuses on proteins regulated during differentiation into infective forms considered virulence factors and addresses the current known mechanisms acting in the modulation of gene expression, emphasizing mRNA signals, regulatory factors, and protein complexes.
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- 2022
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14. ATR1 Angiotensin II Receptor Reduces Hemoglobin S Polymerization, Phosphatidylserine Exposure, and Increases Deformability of Sickle Cell Disease Erythrocytes.
- Author
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Guimarães-Nobre CC, Mendonça-Reis E, Teixeira-Alves LR, Miranda-Alves L, and Berto-Junior C
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- Acrylates, Angiotensin II metabolism, Angiotensin II pharmacology, Annexin A5, Erythrocytes metabolism, Hemoglobin, Sickle metabolism, Humans, Imidazoles, Phosphatidylserines, Polymerization, Receptor, Angiotensin, Type 1 metabolism, Thiophenes, Anemia, Sickle Cell, Losartan pharmacology
- Abstract
Angiotensin II (Ang II) regulates blood volume and stimulates erythropoiesis through AT1 (ATR1) and AT2 (ATR2) receptors, found in multiple tissues, including erythrocytes. Sickle cell disease (SCD) patients present altered Ang II levels. Hemoglobin S polymerization, deformability and phosphatidylserine translocation are important features of mature erythrocytes, therefore, our hypothesis is Ang II affects these parameters and, if it does, what would be the influence of AT1R and AT2R on these effects. A polymerization assay (PA), deformability, and annexin V binding were performed in SCD erythrocytes samples adding Ang II, ATR1 antagonist (losartan or eprosartan), and ATR2 antagonist (PD123319). Through the PA test, we observed a dose-dependent polymerization inhibition effect when comparing Ang II to control. Losartan did not affect the level or the rate of Ang II inhibition, while PD123319 showed an increased level of protection against polymerization, and eprosartan brought levels back to control. Ang II was able to reduce the translocation of phosphatidylserine from the inner to the outer leaflet, a marker of eryptosis, in the presence of PD123319. Also, ATR1 showed a positive effect increasing deformability. Our data shows that ATR1 is important for maintenance of erythrocyte physiological function in SCD and for prolonging its life., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)
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- 2022
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15. Editorial: Extracellular vesicles in diseases, host-pathogen interaction and therapeutic applications.
- Author
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Alves LR, Correa A, Guimarães AJ, and Rodrigues ML
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- Host-Pathogen Interactions, Extracellular Vesicles
- Abstract
Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
- Published
- 2022
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16. Caspofungin Affects Extracellular Vesicle Production and Cargo in Candida auris .
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Amatuzzi RF, Zamith-Miranda D, Munhoz da Rocha IF, Lucena ACR, de Toledo Martins S, Streit R, Staats CC, Trentin G, Almeida F, Rodrigues ML, Nosanchuk JD, and Alves LR
- Abstract
Antifungal resistance has become more frequent, either due to the emergence of naturally resistant species or the development of mechanisms that lead to resistance in previously susceptible species. Among these fungal species of global threat, Candida auris stands out for commonly being highly resistant to antifungal drugs, and some isolates are pan-resistant. The rate of mortality linked to C. auris infections varies from 28% to 78%. In this study, we characterized C. auris extracellular vesicles (EVs) in the presence of caspofungin, an echinocandin, which is the recommended first line antifungal for the treatment of infections due to this emerging pathogen. Furthermore, we also analyzed the protein and RNA content of EVs generated by C. auris cultivated with or without treatment with caspofungin. We observed that caspofungin led to the increased production of EVs, and treatment also altered the type and quantity of RNA molecules and proteins enclosed in the EVs. There were distinct classes of RNAs in the EVs with ncRNAs being the most identified molecules, and tRNA-fragments (tRFs) were abundant in each of the strains studied. We also identified anti-sense RNAs, varying from 21 to 55 nt in length. The differentially abundant mRNAs detected in EVs isolated from yeast subjected to caspofungin treatment were related to translation, nucleosome core and cell wall. The differentially regulated proteins identified in the EVs produced during caspofungin treatment were consistent with the results observed with the RNAs, with the enriched terms being related to translation and cell wall. Our study adds new information on how an echinocandin can affect the EV pathway, which is associated with the yeast cell being able to evade treatment and persist in the host. The ability of C. auris to efficiently alter the composition of EVs may represent a mechanism for the fungus to mitigate the effects of antifungal agents.
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- 2022
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17. Selective Loading and Variations in the miRNA Profile of Extracellular Vesicles from Endothelial-like Cells Cultivated under Normoxia and Hypoxia.
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Robert AW, Marcon BH, Angulski ABB, Martins ST, Leitolis A, Stimamiglio MA, Senegaglia AC, Correa A, and Alves LR
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- Cell Proliferation, Endothelial Cells metabolism, Humans, Hypoxia metabolism, Extracellular Vesicles metabolism, MicroRNAs metabolism
- Abstract
Endothelial-like cells may be obtained from CD133
+ mononuclear cells isolated from human umbilical cord blood (hUCB) and expanded using endothelial-inducing medium (E-CD133 cells). Their use in regenerative medicine has been explored by the potential not only to form vessels but also by the secretion of bioactive elements. Extracellular vesicles (EVs) are prominent messengers of this paracrine activity, transporting bioactive molecules that may guide cellular response under different conditions. Using RNA-Seq, we characterized the miRNA content of EVs derived from E-CD133 cells cultivated under normoxia (N-EVs) and hypoxia (H-EVs) and observed that changing the O2 status led to variations in the selective loading of miRNAs in the EVs. In silico analysis showed that among the targets of differentially loaded miRNAs, there are transcripts involved in pathways related to cell growth and survival, such as FoxO and HIF-1 pathways. The data obtained reinforce the pro-regenerative potential of EVs obtained from E-CD133 cells and shows that fine tuning of their properties may be regulated by culture conditions.- Published
- 2022
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18. The RNA Content of Fungal Extracellular Vesicles: At the "Cutting-Edge" of Pathophysiology Regulation.
- Author
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Bitencourt TA, Pessoni AM, Oliveira BTM, Alves LR, and Almeida F
- Subjects
- Humans, Signal Transduction, Extracellular Vesicles metabolism, RNA metabolism
- Abstract
The role of extracellular vesicles (EVs) in interkingdom communication is widely accepted, and their role in intraspecies communication has been strengthened by recent research. Based on the regulation promoted by EV-associated molecules, the interactions between host and pathogens can reveal different pathways that ultimately affect infection outcomes. As a great part of the regulation is ascribable to RNA contained in EVs, many studies have focused on profiling RNAs in fungal and host EVs, tracking their accumulation during infection, and identifying potential target genes. Herein, we overview the main classes of RNA contained in fungal EVs and the biological processes regulated by these molecules, portraying a state-of-the-art picture of RNAs loaded in fungal EVs, while also raising several questions to drive future investigations. Our compiled data show unambiguously that EVs act as key elements in signaling pathways, and play a crucial role in pathosystems. A complete understanding of the processes that govern RNA content loading and trafficking, and its effect on recipient cells, will lead to improved technologies to ward off infectious agents that threaten human health.
- Published
- 2022
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19. Identification of four compounds from the Pharmakon library with antifungal activity against Candida auris and species of Cryptococcus.
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de Oliveira HC, Castelli RF, Alves LR, Nosanchuk JD, Salama EA, Seleem M, and Rodrigues ML
- Subjects
- Animals, Candida, Candida auris, Microbial Sensitivity Tests veterinary, Antifungal Agents pharmacology, Antifungal Agents therapeutic use, Cryptococcus neoformans
- Abstract
There is an urgent need to develop novel antifungals. In this study, we screened 1600 compounds for antifungal activity against Cryptococcus neoformans and Candida auris. We evaluated 4 promising compounds against 24 additional isolates of Cr. neoformans, Ca. auris, Cr. deuterogattii, and Cr. gattii. The four compounds, dequalinium chloride (DQC), bleomycin sulfate (BMS), pentamidine isethionate salt (PIS), and clioquinol (CLQ), varied in their efficacy against these pathogens but were generally more effective against cryptococci. The compounds exerted their antifungal effect via multiple mechanisms, including interference with the capsule of cryptococci and induction of hyphal-like morphology in Ca. auris. Our results indicate that DQC, BMS, PIS, and CLQ represent potential prototypes for the future development of antifungals., Lay Summary: Fungal infections can be lethal and the options to fight them are scarce. We tested 1600 molecules for their ability to control the growth of two important fungal pathogens, namely Candida auris and species of Cryptococcus. Four of these compounds showed promising antifungal activities., (© The Author(s) 2022. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)
- Published
- 2022
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20. Screening of the Pandemic Response Box Reveals an Association between Antifungal Effects of MMV1593537 and the Cell Wall of Cryptococcus neoformans , Cryptococcus deuterogattii , and Candida auris .
- Author
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de Oliveira HC, Castelli RF, Reis FCG, Samby K, Nosanchuk JD, Alves LR, and Rodrigues ML
- Subjects
- Animals, Cell Wall, Chitin, Macrophages, Microbial Sensitivity Tests, Antifungal Agents pharmacology, Candida auris drug effects, Chitinases metabolism, Cryptococcus gattii drug effects, Cryptococcus neoformans drug effects
- Abstract
There is an urgent unmet need for novel antifungals. In this study, we searched for novel antifungal activities in the Pandemic Response Box, a collection of 400 structurally diverse compounds in various phases of drug discovery. We identified five molecules which could control the growth of Cryptococcus neoformans, Cryptococcus deuterogattii, and the emerging global threat Candida auris. After eliminating compounds which demonstrated paradoxical antifungal effects or toxicity to mammalian macrophages, we selected compound MMV1593537 as a nontoxic, fungicidal molecule for further characterization of antifungal activity. Scanning electron microscopy revealed that MMV1593537 affected cellular division in all three pathogens. In Cryptococcus, MMV1593537 caused a reduction in capsular dimensions. Treatment with MMV1593537 resulted in increased detection of cell wall chitooligomers in these three species. Since chitooligomers are products of the enzymatic hydrolysis of chitin, we investigated whether surface chitinase activity was altered in response to MMV1593537 exposure. We observed peaks of enzyme activity in C. neoformans and C. deuterogattii in response to MMV1593537. We did not detect any surface chitinase activity in C. auris. Our results suggest that MMV1593537 is a promising, nontoxic fungicide whose mechanism of action, at least in Cryptococcus spp, requires chitinase-mediated hydrolysis of chitin. IMPORTANCE The development of novel antifungals is a matter of urgency. In this study, we evaluated antifungal activities in a collection of 400 molecules, using highly lethal fungal pathogens as targets. One of these molecules, namely, MMV1593537, was not toxic to host cells and controlled the growth of isolates of Cryptococcus neoformans, C. deuterogattii, C. gattii, Candida auris, C. albicans, C. parapsilosis, and C. krusei. We tested the mechanisms of antifungal action of MMV1593537 in the Cryptococcus and C. auris models and concluded that the compound affects the cell wall, a structure which is essential for fungal life. At least in Cryptococcus, this effect involved chitinase, an enzyme which is required for remodeling the cell wall. Our results suggest that MMV1593537 is a candidate for future antifungal development.
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- 2022
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21. Extracellular Vesicles Regulate Biofilm Formation and Yeast-to-Hypha Differentiation in Candida albicans.
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Honorato L, de Araujo JFD, Ellis CC, Piffer AC, Pereira Y, Frases S, de Sousa Araújo GR, Pontes B, Mendes MT, Pereira MD, Guimarães AJ, da Silva NM, Vargas G, Joffe L, Del Poeta M, Nosanchuk JD, Zamith-Miranda D, Dos Reis FCG, de Oliveira HC, Rodrigues ML, de Toledo Martins S, Alves LR, Almeida IC, and Nimrichter L
- Subjects
- Biofilms, Fatty Acids pharmacology, Hyphae, Saccharomyces cerevisiae, Candida albicans, Extracellular Vesicles
- Abstract
In this study, we investigated the influence of fungal extracellular vesicles (EVs) during biofilm formation and morphogenesis in Candida albicans. Using crystal violet staining and scanning electron microscopy (SEM), we demonstrated that C. albicans EVs inhibited biofilm formation in vitro . By time-lapse microscopy and SEM, we showed that C. albicans EV treatment stopped filamentation and promoted pseudohyphae formation with multiple budding sites. The ability of C. albicans EVs to regulate dimorphism was further compared to EVs isolated from different C. albicans strains, Saccharomyces cerevisiae, and Histoplasma capsulatum. C. albicans EVs from distinct strains inhibited yeast-to-hyphae differentiation with morphological changes occurring in less than 4 h. EVs from S. cerevisiae and H. capsulatum modestly reduced morphogenesis, and the effect was evident after 24 h of incubation. The inhibitory activity of C. albicans EVs on phase transition was promoted by a combination of lipid compounds, which were identified by gas chromatography-tandem mass spectrometry analysis as sesquiterpenes, diterpenes, and fatty acids. Remarkably, C. albicans EVs were also able to reverse filamentation. Finally, C. albicans cells treated with C. albicans EVs for 24 h lost their capacity to penetrate agar and were avirulent when inoculated into Galleria mellonella. Our results indicate that fungal EVs can regulate yeast-to-hypha differentiation, thereby inhibiting biofilm formation and attenuating virulence. IMPORTANCE The ability to undergo morphological changes during adaptation to distinct environments is exploited by Candida albicans and has a direct impact on biofilm formation and virulence. Morphogenesis is controlled by a diversity of stimuli, including osmotic stress, pH, starvation, presence of serum, and microbial components, among others. Apart from external inducers, C. albicans also produces autoregulatory substances. Farnesol and tyrosol are examples of quorum-sensing molecules (QSM) released by C. albicans to regulate yeast-to-hypha conversion. Here, we demonstrate that fungal EVs are messengers impacting biofilm formation, morphogenesis, and virulence in C. albicans. The major players exported in C. albicans EVs included sesquiterpenes, diterpenes, and fatty acids. The understanding of how C. albicans cells communicate to regulate physiology and pathogenesis can lead to novel therapeutic tools to combat candidiasis.
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- 2022
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22. Long-Lasting Efficacy of Radio Electric Asymmetric Conveyer Neuromodulation Treatment on Functional Dysmetria, an Adaptive Motor Behavior.
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Fontani V, Rinaldi A, Rinaldi C, Araldi L, Azzarà A, Carta AM, Casale N, Castagna A, Del Medico M, Di Stasio M, Facchini M, Greco M, LaMarca S, Loro G, Marrone A, Palattella A, Pellegata G, Ruini D, Schmitt C, Vianini F, Maioli M, Ventura C, Caltabiano F, Bueno AJ, Fugino Matuoka A, Massahiro Nabechima E, Bechelli FA, da Silveira Bossi F, Nitschke Fontana GC, Finkielsztejn J, Coelho Pereira JA, Nunes Callegaro J, Vasconcelos Pinheiro K, Ferreira Alves LR, Kodja Daguer M, Marins Martins MC, Bezerra Uliana M, Knop Zisman N, Cezar Schütz P, Fochesato PR, Celso Felipe de Castro P, Tanaka Nabechima RM, Randon RB, and Rinaldi S
- Abstract
Background Fluctuating asymmetry (FA) is widely defined as the deviation from perfect bilateral symmetry and is considered an epigenetic measure of environmental stress. Rinaldi and Fontani hypothesized that the FA morpho-functional changes originate from an adaptive motor behavior determined by functional alterations in the cerebellum and neural circuits, not caused by a lesion, but induced by environmental stress. They called this phenomenon functional dysmetria (FD). On this premise, they developed the radio electric asymmetric conveyer (REAC) technology, a neuromodulation technology aimed at optimizing the best neuro-psycho-motor strategies in relation to environmental interaction. Aims Previous studies showed that specific REAC neuro postural optimization (NPO) treatment can induce stable FD recovery. This study aimed to verify the duration of the NPO effect in inducing the stable FD recovery over time. Materials and methods Data were retrospectively collected from a population of 29,794 subjects who underwent a specific semiological FD assessment and received the NPO treatment, regardless of the pathology referred. Results The analysis of the data collected by the various participants in the study led us to ascertain the disappearance of FD in 100% of the cases treated, with a stability of the result detected up to 18 years after the single administration of the REAC NPO treatment. Conclusions The REAC NPO neurobiological modulation treatment consisting of a single administration surprisingly maintains a very long efficacy in the correction of FD. This effect can be explained as the long-lasting capacity of the NPO treatment to induce greater functional efficiency of the brain dynamics as proven in previous studies., Competing Interests: SR and VF are the authors of the REAC patent.. AR and CR are daughters of SR and VF., (Copyright © 2022, Fontani et al.)
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- 2022
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23. Isolation of Extracellular Vesicles from Candida auris.
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Zamith-Miranda D, Alves LR, Rodrigues ML, Nimrichter L, and Nosanchuk JD
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- Candida auris, Extracellular Vesicles chemistry
- Abstract
Extracellular vesicles (EVs) are structures released by a variety of cells from all kingdoms of life. EVs are typically involved in communication between tissues and organs, between distinct organisms, or inside microbial communities. The plasticity of these structures is reflected in the range of biological effects they are able to induce or inhibit. The study of fungal EVs is relatively new with the first report in 2007, but investigators have already demonstrated in several model systems that fungal EVs significantly modulate the host immune system and that the immunogenic materials in EV can be harnessed as vaccination platforms. This chapter describes the two main procedures used to isolate EVs from an emerging pathogenic fungus, Candida auris., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
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- 2022
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24. Cellular and Extracellular Vesicle RNA Analysis in the Global Threat Fungus Candida auris .
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Munhoz da Rocha IF, Martins ST, Amatuzzi RF, Zamith-Miranda D, Nosanchuk JD, Rodrigues ML, and Alves LR
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- Candida auris genetics, Candidiasis, Invasive drug therapy, Diagnostic Tests, Routine, Fungi genetics, Genetic Techniques, Host-Pathogen Interactions, Humans, Microbial Sensitivity Tests, Antifungal Agents pharmacology, Candida auris drug effects, Candida auris metabolism, Extracellular Vesicles metabolism, RNA metabolism
- Abstract
Emerging and reemerging pathogens are a worldwide concern, and it is predicted that these microbes will cause severe outbreaks. Candida auris affects people with weakened immune systems, particularly those who are hospitalized or are in health care facilities. Extracellular vesicles (EVs) are lipid bilayer structures released by organisms from all domains of life. EVs can deliver functional molecules to target cells, including proteins and nucleic acids, especially RNA molecules. EVs from several pathogenic fungi species play diverse biological roles related to cell-cell communication and pathogen-host interaction. In this study, we describe a data set which we produced by sequencing the RNA content of EVs from C. auris under normal growth conditions and in the presence of the antifungal caspofungin, a first-line drug to treat this fungus. To generate a more complete data set for future comparative studies, we also sequenced the RNA cellular content of EVs under the same conditions. This data set addresses a previously unexplored area of fungal biology regarding cellular small RNA and EV RNA. Our data will provide a molecular basis for the study of the aspects associated with antifungal treatment, gene expression response, and EV composition in C. auris. These data will also allow the exploration of small RNA content in the fungal kingdom and might serve as an informative basis for studies on the mechanisms by which molecules are directed to fungal EVs. IMPORTANCE Candida auris, a relevant emerging human-pathogenic yeast, is the first fungus to be called a global public health threat by the WHO. This is because of its rapid spread on all inhabited continents, together with its extremely high frequency of drug and multidrug resistance. In our study, we generated a large data set for 3 distinct strains of C. auris and obtained cellular small RNA fraction as well as extracellular vesicle RNA (EV-RNA) during normal growth conditions and after treatment with caspofungin, the first-line drug used to treat C. auris infection.
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- 2021
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25. Re-emergence of Gamma-like-II and emergence of Gamma-S:E661D SARS-CoV-2 lineages in the south of Brazil after the 2021 outbreak.
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Oliveira MM, Schemberger MO, Suzukawa AA, Riediger IN, do Carmo Debur M, Becker G, Resende PC, Gräf T, Balsanelli E, de Baura VA, de Souza EM, Pedrosa FO, Alves LR, Blanes L, Nardelli SC, Aguiar AM, Albrecht L, Zanette D, Ávila AR, Morello LG, Marchini FK, Dos Santos HG, Passetti F, Dallagiovanna B, and Faoro H
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- Brazil epidemiology, COVID-19 epidemiology, Disease Outbreaks, Humans, Middle Aged, Mutation, Phylogeny, Population Surveillance, Spike Glycoprotein, Coronavirus chemistry, Spike Glycoprotein, Coronavirus genetics, Whole Genome Sequencing, COVID-19 virology, SARS-CoV-2 genetics, SARS-CoV-2 isolation & purification
- Abstract
Background: We report a genomic surveillance of SARS-CoV-2 lineages circulating in Paraná, southern Brazil, from March 2020 to April 2021. Our analysis, based on 333 genomes, revealed that the first variants detected in the state of Paraná in March 2020 were the B.1.1.33 and B.1.1.28 variants. The variants B.1.1.28 and B.1.1.33 were predominant throughout 2020 until the introduction of the variant P.2 in August 2020 and a variant of concern (VOC), Gamma (P.1), in January 2021. The VOC Gamma, a ramification of the B.1.1.28 lineage first detected in Manaus (northern Brazil), has grown rapidly since December 2020 and was thought to be responsible for the deadly second wave of COVID-19 throughout Brazil., Methods: The 333 genomic sequences of SARS-CoV-2 from March 2020 to April 2021 were generated as part of the genomic surveillance carried out by Fiocruz in Brazil Genomahcov Fiocruz. SARS-CoV-2 sequencing was performed using representative samples from all geographic areas of Paraná. Phylogenetic analyses were performed using the 333 genomes also included other SARS-CoV-2 genomes from the state of Paraná and other states in Brazil that were deposited in the GISAID. In addition, the time-scaled phylogenetic tree was constructed with up to 3 random sequences of the Gamma variant from each state in Brazil in each month of 2021. In this analysis we also added the sequences identified as the B.1.1.28 lineage of the Amazonas state and and the Gamma-like-II (P.1-like-II) lineage identified in different regions of Brazil., Results: Phylogenetic analyses of the SARS-CoV-2 genomes that were previously classified as the VOC Gamma lineage by WHO/PANGO showed that some genomes from February to April 2021 branched in a monophyletic clade and that these samples grouped together with genomes recently described with the lineage Gamma-like-II. Additionally, a new mutation (E661D) in the spike (S) protein has been identified in nearly 10% of the genomes classified as the VOC Gamma from Paraná in March and April 2021.Finally, we analyzed the correlation between the lineage and the Gamma variant frequency, age group (patients younger or older than 60 years old) and the clinical data of 86 cases from the state of Paraná., Conclusions: Our results provided a reliable picture of the evolution of the SARS-CoV-2 pandemic in the state of Paraná characterized by the dominance of the Gamma strain, as well as a high frequencies of the Gamma-like-II lineage and the S:E661D mutation. Epidemiological and genomic surveillance efforts should be continued to unveil the biological relevance of the novel mutations detected in the VOC Gamma in Paraná., (© 2021. The Author(s).)
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- 2021
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26. Transcriptional and translational landscape of Candida auris in response to caspofungin.
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Zamith-Miranda D, Amatuzzi RF, Munhoz da Rocha IF, Martins ST, Lucena ACR, Vieira AZ, Trentin G, Almeida F, Rodrigues ML, Nakayasu ES, Nosanchuk JD, and Alves LR
- Abstract
Candida auris has emerged as a serious worldwide threat by causing opportunistic infections that are frequently resistant to one or more conventional antifungal medications resulting in high mortality rates. Against this backdrop, health warnings around the world have focused efforts on understanding C. auris fungal biology and effective prevention and treatment approaches to combat this fungus. To date, there is little information about the differentially expressed genes when this fungus is treated with conventional antifungals, and caspofungin is a standard echinocandin deployed in the therapy against C. auris . In this work, we treated two distinct strains of C. auris for 24 h with caspofungin, and the cellular responses were evaluated at the morphological, translational and transcriptional levels. We first observed that the echinocandin caused morphological alterations, aggregation of yeast cells, and modifications in the cell wall composition of C. auris . Transcriptomic analysis revealed an upregulation of genes related to the synthesis of the cell wall, ribosome, and cell cycle after exposure to caspofungin. Supporting these findings, the integrated proteomic analysis showed that caspofungin-treated cells were enriched in ribosome-related proteins and cell wall, especially mannoproteins. Altogether, these results provide further insights into the biology of C. auris and expands our understanding regarding the antifungal activity of caspofungin and reveal cellular targets, as the mannose metabolism, that can be further explored for the development of novel antifungals., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)
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- 2021
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27. Analysis of Cryptococcal Extracellular Vesicles: Experimental Approaches for Studying Their Diversity Among Multiple Isolates, Kinetics of Production, Methods of Separation, and Detection in Cultures of Titan Cells.
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Reis FCG, Gimenez B, Jozefowicz LJ, Castelli RF, Martins ST, Alves LR, de Oliveira HC, and Rodrigues ML
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- Cryptococcus classification, Cryptococcus genetics, Cryptococcus isolation & purification, Culture Media chemistry, Culture Media metabolism, Extracellular Vesicles metabolism, Extracellular Vesicles ultrastructure, Humans, Kinetics, Microscopy, Electron, Transmission, Cryptococcosis microbiology, Cryptococcus chemistry, Extracellular Vesicles chemistry
- Abstract
Extracellular vesicles (EVs) produced by members of the Cryptococcus genus are associated with fundamental processes of fungal physiology and virulence. However, several questions about the properties of cryptococcal EVs remain unanswered, mostly because of technical limitations. We recently described a fast and efficient protocol of high-yield EV isolation from solid medium. In this study, we aimed at using the solid medium protocol to address some of the open questions about EVs, including the kinetics of EV production, the diversity of EVs produced by multiple isolates under different culture conditions, the separation of vesicles in a density gradient followed by the recovery of functional EVs, the direct detection of EVs in culture supernatants, and the production of vesicles in solid cultures of Titan cells. Our results indicate that the production of EVs is directly impacted by the culture medium and time of growth, resulting in variable detection of EVs per cell and a peak of EV detection at 24 h of growth. Nanoparticle tracking analysis (NTA) of EV samples revealed that multiple isolates produce vesicles with variable properties, including particles of diverging dimensions. EVs were produced in the solid medium in amounts that were separated on a centrifugation density gradient, resulting in the recovery of functional EVs containing the major cryptococcal capsular antigen. We also optimized the solid medium protocol for induction of the formation of Titan cells, and analyzed the production of EVs by NTA and transmission electron microscopy. This analysis confirmed that EVs were isolated from solid cultures of cryptococcal enlarged cells. With these approaches, we expect to implement simple methods that will facilitate the analysis of EVs produced by fungal cells. IMPORTANCE Fungal extracellular vesicles (EVs) are considered to be important players in the biology of fungal pathogens. However, the limitations in the methodological approaches to studying fungal EVs impair the expansion of knowledge in this field. In the present study, we used the Cryptococcus genus as a model for the study of EVs. We explored the simplification of protocols for EV analysis, which helped us to address some important, but still unanswered, questions about fungal EVs.
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- 2021
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28. Comparative Molecular and Immunoregulatory Analysis of Extracellular Vesicles from Candida albicans and Candida auris.
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Zamith-Miranda D, Heyman HM, Couvillion SP, Cordero RJB, Rodrigues ML, Nimrichter L, Casadevall A, Amatuzzi RF, Alves LR, Nakayasu ES, and Nosanchuk JD
- Abstract
Candida auris is a recently described multidrug-resistant pathogenic fungus that is increasingly responsible for health care-associated outbreaks across the world. Bloodstream infections of this fungus cause death in up to 70% of cases. Aggravating this scenario, the disease-promoting mechanisms of C. auris are poorly understood. Fungi release extracellular vesicles (EVs) that carry a broad range of molecules, including proteins, lipids, carbohydrates, pigments, and RNA, many of which are virulence factors. Here, we carried out a comparative molecular characterization of C. auris and Candida albicans EVs and evaluated their capacity to modulate effector mechanisms of host immune defense. Using proteomics, lipidomics, and transcriptomics, we found that C. auris released EVs with payloads that were significantly different from those of EVs released by C. albicans. EVs released by C. auris potentiated the adhesion of this yeast to an epithelial cell monolayer, while EVs from C. albicans had no effect. C. albicans EVs primed macrophages for enhanced intracellular yeast killing, whereas C. auris EVs promoted survival of the fungal cells. Moreover, EVs from both C. auris and C. albicans induced the activation of bone marrow-derived dendritic cells. Together, our findings show distinct profiles and properties of EVs released by C. auris and by C. albicans and highlight the potential contribution of C. auris EVs to the pathogenesis of this emerging pathogen. IMPORTANCE Candida auris is a recently described multidrug-resistant pathogenic fungus that is responsible for outbreaks across the globe, particularly in the context of nosocomial infections. Its virulence factors and pathogenesis are poorly understood. Here, we tested the hypothesis that extracellular vesicles (EVs) released by C. auris are a disease-promoting factor. We describe the production of EVs by C. auris and compare their biological activities against those of the better-characterized EVs from C. albicans. C. auris EVs have immunoregulatory properties, of which some are opposite those of C. albicans EVs. We also explored the cargo and structural components of those vesicles and found that they are remarkably distinct compared to EVs from C. auris's phylogenetic relative Candida albicans.
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- 2021
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29. Cryptococcus extracellular vesicles properties and their use as vaccine platforms.
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Rizzo J, Wong SSW, Gazi AD, Moyrand F, Chaze T, Commere PH, Novault S, Matondo M, Péhau-Arnaudet G, Reis FCG, Vos M, Alves LR, May RC, Nimrichter L, Rodrigues ML, Aimanianda V, and Janbon G
- Subjects
- Amino Acid Motifs, Animals, Antigens, Fungal immunology, Antigens, Fungal metabolism, Cryoelectron Microscopy, Cryptococcosis immunology, Extracellular Vesicles microbiology, Female, Fungal Proteins immunology, Fungal Proteins metabolism, Mice, Mice, Inbred BALB C, Proteome, Proteomics methods, Cryptococcus neoformans immunology, Cryptococcus neoformans metabolism, Extracellular Vesicles immunology, Extracellular Vesicles metabolism, Membrane Proteins immunology, Membrane Proteins metabolism, Vaccines immunology
- Abstract
Whereas extracellular vesicle (EV) research has become commonplace in different biomedical fields, this field of research is still in its infancy in mycology. Here we provide a robust set of data regarding the structural and compositional aspects of EVs isolated from the fungal pathogenic species Cryptococcus neoformans, C. deneoformans and C. deuterogattii . Using cutting-edge methodological approaches including cryogenic electron microscopy and cryogenic electron tomography, proteomics, and flow cytometry, we revisited cryptococcal EV features and suggest a new EV structural model, in which the vesicular lipid bilayer is covered by mannoprotein-based fibrillar decoration, bearing the capsule polysaccharide as its outer layer. About 10% of the EV population is devoid of fibrillar decoration, adding another aspect to EV diversity. By analysing EV protein cargo from the three species, we characterized the typical Cryptococcus EV proteome. It contains several membrane-bound protein families, including some Tsh proteins bearing a SUR7/PalI motif. The presence of known protective antigens on the surface of Cryptococcus EVs, resembling the morphology of encapsulated virus structures, suggested their potential as a vaccine. Indeed, mice immunized with EVs obtained from an acapsular C. neoformans mutant strain rendered a strong antibody response in mice and significantly prolonged their survival upon C. neoformans infection., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)
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- 2021
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30. Omics Approaches for Understanding Biogenesis, Composition and Functions of Fungal Extracellular Vesicles.
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Zamith-Miranda D, Peres da Silva R, Couvillion SP, Bredeweg EL, Burnet MC, Coelho C, Camacho E, Nimrichter L, Puccia R, Almeida IC, Casadevall A, Rodrigues ML, Alves LR, Nosanchuk JD, and Nakayasu ES
- Abstract
Extracellular vesicles (EVs) are lipid bilayer structures released by organisms from all kingdoms of life. The diverse biogenesis pathways of EVs result in a wide variety of physical properties and functions across different organisms. Fungal EVs were first described in 2007 and different omics approaches have been fundamental to understand their composition, biogenesis, and function. In this review, we discuss the role of omics in elucidating fungal EVs biology. Transcriptomics, proteomics, metabolomics, and lipidomics have each enabled the molecular characterization of fungal EVs, providing evidence that these structures serve a wide array of functions, ranging from key carriers of cell wall biosynthetic machinery to virulence factors. Omics in combination with genetic approaches have been instrumental in determining both biogenesis and cargo loading into EVs. We also discuss how omics technologies are being employed to elucidate the role of EVs in antifungal resistance, disease biomarkers, and their potential use as vaccines. Finally, we review recent advances in analytical technology and multi-omic integration tools, which will help to address key knowledge gaps in EVs biology and translate basic research information into urgently needed clinical applications such as diagnostics, and immuno- and chemotherapies to fungal infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zamith-Miranda, Peres da Silva, Couvillion, Bredeweg, Burnet, Coelho, Camacho, Nimrichter, Puccia, Almeida, Casadevall, Rodrigues, Alves, Nosanchuk and Nakayasu.)
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- 2021
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31. Characterization of the RNA-Binding Protein TcSgn1 in Trypanosoma cruzi .
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Oliveira C, Gerber AP, Goldenberg S, and Alves LR
- Abstract
RNA-binding proteins (RBPs) participate in several steps of post-transcriptional regulation of gene expression, such as splicing, messenger RNA transport, mRNA localization, and translation. Gene-expression regulation in trypanosomatids occurs primarily at the post-transcriptional level, and RBPs play important roles in the process. Here, we characterized the RBP TcSgn1, which contains one RNA recognition motif (RRM). TcSgn1 is a close ortholog of yeast Saccharomyces cerevisiae protein ScSgn1, which plays a role in translational regulation in the cytoplasm. We found that TcSgn1 in Trypanosoma cruzi is localized in the nucleus in exponentially growing epimastigotes. By performing immunoprecipitation assays of TcSgn1, we identified hundreds of mRNAs associated with the protein, a significant fraction of them coding for nucleic acids binding, transcription, and endocytosis proteins. In addition, we show that TcSgn1 is capable of interacting directly with the poly(A) tail of the mRNAs. The study of parasites under nutritional stress showed that TcSgn1 was localized in cytoplasmic granules in addition to localizing in the nucleus. Similar to ScSgn1, we observed that TcSgn1 also interacts with the PABP1 protein, suggesting that this protein may play a role in regulating gene expression in T. cruzi . Taken together, our results show that RNA-binding protein TcSgn1 is part of ribonucleoprotein complexes associated with nuclear functions, stress response, and RNA metabolism.
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- 2021
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32. Lessons Learned from Studying Histoplasma capsulatum Extracellular Vesicles.
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Zamith-Miranda D, Alves LR, Nakayasu ES, and Nosanchuk JD
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- Histoplasma, Humans, Virulence, Extracellular Vesicles, Mycoses
- Abstract
Histoplasma capsulatum is a major endemic mycosis. Our laboratories have demonstrated that H. capsulatum produces extracellular vesicles (EV) that are loaded with diverse compounds that influence virulence. We have further shown that H. capsulatum dynamically regulates the loading and release of fungal EV in response to stimuli and growth conditions. This chapter details the current knowledge of EV biology in H. capsulatum and the impact of this information on our understanding of this important process that is closely linked to pathogenesis., (© 2021. Springer Nature Switzerland AG.)
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- 2021
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33. Biogenesis of Fungal Extracellular Vesicles: What Do We Know?
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de Oliveira HC, Kato AF, Sena BAG, Duarte I, Jozefowicz LJ, Castelli RF, Kuczera D, Reis FCG, Alves LR, and Rodrigues ML
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- Cell Membrane, Fungi genetics, Exosomes, Extracellular Vesicles
- Abstract
So far, extracellular vesicles (EVs) have been described in 15 genera of fungi. They carry molecules that contribute to the interaction of fungal cells with the host. Although the number of studies on fungal EVs has increased, the mechanisms involved in their biogenesis are still poorly understood. The current knowledge of EV biogenesis shows us that they can originate both in the cytoplasm and at the plasma membrane. In this chapter, we will focus on these two cellular sites to review what is known about the biogenesis of fungal EVs., (© 2021. Springer Nature Switzerland AG.)
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- 2021
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34. Extracellular Vesicles in Viral Infections: Two Sides of the Same Coin?
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Martins ST and Alves LR
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- Biological Transport, Cell Communication, Humans, Extracellular Vesicles metabolism, Virus Diseases metabolism, Viruses
- Abstract
Extracellular vesicles are small membrane structures containing proteins and nucleic acids that are gaining a lot of attention lately. They are produced by most cells and can be detected in several body fluids, having a huge potential in therapeutic and diagnostic approaches. EVs produced by infected cells usually have a molecular signature that is very distinct from healthy cells. For intracellular pathogens like viruses, EVs can have an even more complex function, since the viral biogenesis pathway can overlap with EV pathways in several ways, generating a continuum of particles, like naked virions, EVs containing infective viral genomes and quasi-enveloped viruses, besides the classical complete viral particles that are secreted to the extracellular space. Those particles can act in recipient cells in different ways. Besides being directly infective, they also can prime neighbor cells rendering them more susceptible to infection, block antiviral responses and deliver isolated viral molecules. On the other hand, they can trigger antiviral responses and cytokine secretion even in uninfected cells near the infection site, helping to fight the infection and protect other cells from the virus. This protective response can also backfire, when a massive inflammation facilitated by those EVs can be responsible for bad clinical outcomes. EVs can help or harm the antiviral response, and sometimes both mechanisms are observed in infections by the same virus. Since those pathways are intrinsically interlinked, understand the role of EVs during viral infections is crucial to comprehend viral mechanisms and respond better to emerging viral diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Martins and Alves.)
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- 2020
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35. Cross-Kingdom Extracellular Vesicles EV-RNA Communication as a Mechanism for Host-Pathogen Interaction.
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Munhoz da Rocha IF, Amatuzzi RF, Lucena ACR, Faoro H, and Alves LR
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- Communication, Fungi, Host-Pathogen Interactions, Extracellular Vesicles, RNA
- Abstract
The extracellular vesicle (EVs) traffic has been highlighted as a very important pathway of cellular communication. EVs are produced by prokaryotes and eukaryotes organisms and can carry molecules to help maintain homeostasis, responding to general disbalance, infections, and allowing rapid modulation of the immune system. In the context of infection, EVs from both the host and the pathogen have been identified as playing roles in the recruitment of immunological molecules that can lead to the resolution of the infection or the host's defeat. Bacterial vesicles RNA cargo play roles in the host cell by regulating gene expression and modulating immune response. In fungi the RNA molecules present in EVs are diverse and participate in communication between the host and pathogenic fungi. Little is known about how cross-kingdom sRNA trafficking occurs, although in recent years, there has been an increase in studies that relate EV participation in sRNA delivery. This review aims to elucidate and update the reader concerning the role of extracellular vesicles, with emphasis in the RNA content. We describe the EVs during infection from the host point-of-view, as well as the bacteria and fungi pathogens producing EVs that help the establishment of the disease., (Copyright © 2020 Munhoz da Rocha, Amatuzzi, Lucena, Faoro and Alves.)
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- 2020
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36. Mechanisms of cadmium-stress avoidance by selenium in tomato plants.
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Alves LR, Prado ER, de Oliveira R, Santos EF, Lemos de Souza I, Dos Reis AR, Azevedo RA, and Gratão PL
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- Antioxidants, Glutathione, Hydrogen Peroxide, Oxidation-Reduction, Plant Leaves, Plant Roots, Cadmium toxicity, Solanum lycopersicum physiology, Oxidative Stress physiology, Selenium metabolism, Soil Pollutants toxicity
- Abstract
Cadmium (Cd) is probably the most damaging metal to plant species; with a long biological half-life, it can be taken up by plants, disrupting the cell homeostasis and triggering several metabolic pathways. Selenium (Se) improves plant defence systems against stressful conditions, but the biochemical antioxidant responses to Cd stress in tomato plants is poorly understood. To further address the relationship of Cd-stress responses with Se mineral uptake, Cd and Se concentration, proline content, MDA and H
2 O2 production, and the activity of SOD, APX, CAT and GR enzymes were analyzed in Micro-Tom (MT) plants submitted to 0.5 mM Cd. The results revealed different responses according to Se combination and Cd application. For instance, roots and leaves of MT plants treated with Se exhibited an increase in dry mass and nutritional status, exhibited lower proline content and higher APX and GR activities when compared with plants with no Se application. Plants submitted to 0.5 mM Cd, irrespective of Se exposure, exhibited lower proline, MDA and H2 O2 content and higher SOD, CAT and GR activities. Selenium may improve tolerance against Cd, which allowed MT plants exhibited less oxidative damage to the cell, even under elevated Cd accumulation in their tissues. The results suggest that Se application is an efficient management technique to alleviate the deleterious effects of Cd-stress, enhancing the nutritional value and activity of ROS-scavenging enzymes in tomato plants.- Published
- 2020
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37. Comparative Genomics of Acinetobacter baumannii Clinical Strains From Brazil Reveals Polyclonal Dissemination and Selective Exchange of Mobile Genetic Elements Associated With Resistance Genes.
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Leal NC, Campos TL, Rezende AM, Docena C, Mendes-Marques CL, de Sá Cavalcanti FL, Wallau GL, Rocha IV, Cavalcanti CLB, Veras DL, Alves LR, Andrade-Figueiredo M, de Barros MPS, de Almeida AMP, de Morais MMC, Leal-Balbino TC, Xavier DE, and de-Melo-Neto OP
- Abstract
Acinetobacter baumannii is an opportunistic bacterial pathogen infecting immunocompromised patients and has gained attention worldwide due to its increased antimicrobial resistance. Here, we report a comparative whole-genome sequencing and analysis coupled with an assessment of antibiotic resistance of 46 Acinetobacter strains (45 A. baumannii plus one Acinetobacter nosocomialis ) originated from five hospitals from the city of Recife, Brazil, between 2010 and 2014. An average of 3,809 genes were identified per genome, although only 2,006 genes were single copy orthologs or core genes conserved across all sequenced strains, with an average of 42 new genes found per strain. We evaluated genetic distance through a phylogenetic analysis and MLST as well as the presence of antibiotic resistance genes, virulence markers and mobile genetic elements (MGE). The phylogenetic analysis recovered distinct monophyletic A. baumannii groups corresponding to five known (ST1, ST15, ST25, ST79, and ST113) and one novel ST (ST881, related to ST1). A large number of ST specific genes were found, with the ST79 strains having the largest number of genes in common that were missing from the other STs. Multiple genes associated with resistance to β-lactams, aminoglycosides and other antibiotics were found. Some of those were clearly mapped to defined MGEs and an analysis of those revealed known elements as well as a novel Tn 7 -Tn 3 transposon with a clear ST specific distribution. An association of selected resistance/virulence markers with specific STs was indeed observed, as well as the recent spread of the OXA-253 carbapenemase encoding gene. Virulence genes associated with the synthesis of the capsular antigens were noticeably more variable in the ST113 and ST79 strains. Indeed, several resistance and virulence genes were common to the ST79 and ST113 strains only, despite a greater genetic distance between them, suggesting common means of genetic exchange. Our comparative analysis reveals the spread of multiple STs and the genomic plasticity of A. baumannii from different hospitals in a single metropolitan area. It also highlights differences in the spread of resistance markers and other MGEs between the investigated STs, impacting on the monitoring and treatment of Acinetobacter in the ongoing and future outbreaks., (Copyright © 2020 Leal, Campos, Rezende, Docena, Mendes-Marques, de Sá Cavalcanti, Wallau, Rocha, Cavalcanti, Veras, Alves, Andrade-Figueiredo, de Barros, de Almeida, de Morais, Leal-Balbino, Xavier and de-Melo-Neto.)
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- 2020
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38. Brazilian guidelines for the pharmacological treatment of idiopathic pulmonary fibrosis. Official document of the Brazilian Thoracic Association based on the GRADE methodology.
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Baddini-Martinez J, Ferreira J, Tanni S, Alves LR, Cabral Junior BF, Carvalho CRR, Cezare TJ, Costa CHD, Gazzana MB, Jezler S, Kairalla RA, Kawano-Dourado L, Lima MS, Mancuzo E, Moreira MAC, Rodrigues MP, Rodrigues SCS, Rubin AS, Rufino RL, Steidle LJM, Storrer K, and Baldi BG
- Subjects
- Acetylcysteine therapeutic use, Aged, Anti-Inflammatory Agents therapeutic use, Brazil, Humans, Indoles therapeutic use, Male, Pyridones therapeutic use, Idiopathic Pulmonary Fibrosis drug therapy, Practice Guidelines as Topic
- Abstract
Idiopathic pulmonary fibrosis (IPF) is a form of chronic interstitial lung disease of unknown cause, which predominantly affects elderly men who are current or former smokers. Even though it is an uncommon disease, it is of great importance because of its severity and poor prognosis. In recent decades, several pharmacological treatment modalities have been investigated for the treatment of this disease, and the classic concepts have therefore been revised. The purpose of these guidelines was to define evidence-based recommendations regarding the use of pharmacological agents in the treatment of IPF in Brazil. We sought to provide guidance on the practical issues faced by clinicians in their daily lives. Patients of interest, Intervention to be studied, Comparison of intervention and Outcome of interest (PICO)-style questions were formulated to address aspects related to the use of corticosteroids, N-acetylcysteine, gastroesophageal reflux medications, endothelin-receptor antagonists, phosphodiesterase-5 inhibitors, pirfenidone, and nintedanib. To formulate the PICO questions, a group of Brazilian specialists working in the area was assembled and an extensive review of the literature on the subject was carried out. Previously published systematic reviews with meta-analyses were analyzed for the strength of the compiled evidence, and, on that basis, recommendations were developed by employing the Grading of Recommendations Assessment, Development and Evaluation approach. The authors believe that the present document represents an important advance to be incorporated in the approach to patients with IPF, aiming mainly to improve its management, and can become an auxiliary tool for defining public policies related to IPF.
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- 2020
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39. Selenium improves photosynthesis and induces ultrastructural changes but does not alleviate cadmium-stress damages in tomato plants.
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Alves LR, Rossatto DR, Rossi ML, Martinelli AP, and Gratão PL
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- Selenium pharmacology, Cadmium adverse effects, Solanum lycopersicum chemistry, Photosynthesis physiology, Selenium therapeutic use
- Abstract
The application of Se to plants growing under Cd contamination may become an alternative strategy to minimize Cd damage. However, there is no specific information available regarding whether Se can affect the anatomical structure and photosynthetic rates of plants under Cd stress. To address questions related to Se-protective responses under Cd stress, we evaluated the structural and ultrastructural aspects, photosynthetic rates and growth of tomato cv. Micro-Tom plants. Plants were exposed to 0.5 mM CdCl
2 and further supplemented with 1.0 μM of selenite or selenate. The overall results revealed different trends according to the Se source and Cd application. Both Se sources improved growth, photosynthesis, leaf characteristics and middle lamella thickness between mesophyll cells. In contrast, Cd caused decreases in photosynthesis and growth and damage to the ultrastructure of the chloroplast. The number of mitochondria, peroxisomes, starch grains and plastogloboli and the disorganization of the thylakoids and the middle lamella in plants increased in the presence of Cd or Cd + Se. Se plays an important role in plant cultivation under normal conditions. This finding was corroborated by the identification of specific structural changes in Se-treated plants, which could benefit plant development. However, a reversal of Cd stress effects was not observed in the presence of Se.- Published
- 2020
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40. RNA Binding Proteins and Gene Expression Regulation in Trypanosoma cruzi .
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Romagnoli BAA, Holetz FB, Alves LR, and Goldenberg S
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- Gene Expression Regulation, Protozoan Proteins genetics, Protozoan Proteins metabolism, RNA, Messenger genetics, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Trypanosoma cruzi genetics, Trypanosoma cruzi metabolism
- Abstract
The regulation of gene expression in trypanosomatids occurs mainly at the post-transcriptional level. In the case of Trypanosoma cruzi , the characterization of messenger ribonucleoprotein (mRNP) particles has allowed the identification of several classes of RNA binding proteins (RBPs), as well as non-canonical RBPs, associated with mRNA molecules. The protein composition of the mRNPs as well as the localization and functionality of the mRNAs depend on their associated proteins. mRNPs can also be organized into larger complexes forming RNA granules, which function as stress granules or P-bodies depending on the associated proteins. The fate of mRNAs in the cell, and consequently the genes expressed, depends on the set of proteins associated with the messenger molecule. These proteins allow the coordinated expression of mRNAs encoding proteins that are related in function, resulting in the formation of post-transcriptional operons. However, the puzzle posed by the combinatorial association of sets of RBPs with mRNAs and how this relates to the expressed genes remain to be elucidated. One important tool in this endeavor is the use of the CRISPR/CAS system to delete genes encoding RBPs, allowing the evaluation of their effect on the formation of mRNP complexes and associated mRNAs in the different compartments of the translation machinery. Accordingly, we recently established this methodology for T. cruzi and deleted the genes encoding RBPs containing zinc finger domains. In this manuscript, we will discuss the data obtained and the potential of the CRISPR/CAS methodology to unveil the role of RBPs in T. cruzi gene expression regulation., (Copyright © 2020 Romagnoli, Holetz, Alves and Goldenberg.)
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- 2020
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41. Analysis of the In Vivo Translation Process in Trypanosoma cruzi Using Ribosome Profiling.
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Poubel SB, Holetz FB, Romagnoli BAA, Goldenberg S, and Alves LR
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- Alternative Splicing genetics, Base Sequence genetics, Codon Usage genetics, Gene Library, Parasitology methods, RNA, Messenger genetics, RNA, Messenger isolation & purification, RNA, Messenger metabolism, RNA, Protozoan genetics, RNA, Protozoan isolation & purification, RNA, Protozoan metabolism, Ribosomes metabolism, High-Throughput Nucleotide Sequencing, Peptide Chain Initiation, Translational genetics, Ribosomes genetics, Trypanosoma cruzi genetics
- Abstract
The technique of ribosome profiling is based on the isolation of sequences around 30 nucleotides in size protected by mRNA-associated ribosomes, following digestion with specific nucleases, generating a footprint. After isolation and purification, these 30-nucleotide sequences are converted to a cDNA library and analyzed by deep sequencing, providing a high-precision picture of the translation process in vivo. In addition, this powerful technique allows for the study of several biological phenomena such as alternative splicing, alternative codon usage and initiation of translation by non-AUG codons. Furthermore, the ribosome footprinting technique has proved to be very efficient for studies of ribosome pause sites on mRNAs, which could act as key regulators in the translation process. Here we describe a modified protocol of the ribosome footprinting technique for translation efficiency analysis in Trypanosoma cruzi.
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- 2020
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42. Immunoprecipitation for the Analysis of Macromolecular Complexes in Trypanosoma cruzi.
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Romagnoli BAA, Goldenberg S, and Alves LR
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- Macromolecular Substances metabolism, Mass Spectrometry methods, Parasitology methods, Protein Interaction Mapping, Protozoan Proteins isolation & purification, Protozoan Proteins metabolism, RNA, Protozoan isolation & purification, RNA, Protozoan metabolism, Immunomagnetic Separation methods, Immunoprecipitation methods, Macromolecular Substances isolation & purification, Trypanosoma cruzi physiology
- Abstract
Immunoprecipitation is a helpful tool to assess interactions between proteins and proteins or nucleic acids (DNA or RNA). Its principle consists in capturing and enriching one or multiple target proteins from a complex sample with a specific antibody conjugated to a solid matrix and isolating the RNA and/or protein molecules associated to those target(s) group of proteins that can be further identified by advanced techniques such as RNA-seq and/or mass spectrometry. Since this technique allows for identifying, mapping, and checking new protein-protein and protein-RNA interactions, its use is very convenient in situations where many proteins remain with their functions uncharacterized, as is the case of the protozoan Trypanosoma cruzi. Here we describe a protocol that is based on the cryogrinding method for cell lysis and the use of antibodies conjugated to magnetic beads to capture and purify protein complexes in a robust and efficient way.
- Published
- 2020
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43. New insights into cadmium stressful-conditions: Role of ethylene on selenium-mediated antioxidant enzymes.
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Alves LR, Rodrigues Dos Reis A, Prado ER, Lavres J, Pompeu GB, Azevedo RA, and Gratão PL
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- Adaptation, Physiological, Ascorbate Peroxidases metabolism, Cadmium metabolism, Catalase metabolism, Environmental Exposure, Glutathione metabolism, Glutathione Reductase metabolism, Humans, Hydrogen Peroxide metabolism, Solanum lycopersicum metabolism, Mutation, Oxidation-Reduction, Plant Leaves metabolism, Selenic Acid pharmacology, Selenious Acid pharmacology, Selenium metabolism, Superoxide Dismutase metabolism, Antioxidants metabolism, Cadmium adverse effects, Ethylenes metabolism, Solanum lycopersicum drug effects, Oxidative Stress drug effects, Selenium pharmacology
- Abstract
Cadmium (Cd) contamination has generated an environmental problem worldwide, leading to harmful effects on human health and damages to plant metabolism. Selenium (Se) is non essential for plants, however it can improve plant growth and reduce the adverse effects of abiotic stress. In addition, ethylene may interplay the positive effects of Se in plants. In order to investigate the role of ethylene in Se-modulation of antioxidant defence system in response to Cd-stress, we tested the hormonal mutant Epinastic (epi) with a subset of constitutive activation of the ethylene response and Micro-Tom (MT) plants. For this purpose, Se mineral uptake, Cd and Se concentrations, pigments, malondialdeyde (MDA) and hydrogen peroxide (H
2 O2 ) contents, ethylene production, glutathione (GSH) compound, and superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GSH-Px) activities were analysed in MT and epi plants submitted to 0.5 mM CdCl2 and 1 μM of selenate or selenite. MT plants treated with both Se forms increased growth in the presence or not of 0.5 mM CdCl2 , but not change epi growth. Both Se forms reduced Cd uptake in MT plants and cause reverse effect in epi plants. P, Mg, S, K and Zn uptake increased in epi plants with Se application, irrespective to Cd exposure. Chlorophylls and carotenoids contents decreased in both genotypes under Cd exposure, in contrast to what was observed in epi leaves in the presence of Se. When antioxidant enzymes activities were concerned, Se application increased Mn-SOD, Fe-SOD and APX activities. In the presence of Cd, MT and epi plants exhibited decreased SOD activity and increased CAT, APX and GR activities. MT and epi plants with Se supply exhibited increased APX and GR activities in the presence of Cd. Overall, these results suggest that ethylene may be involved in Se induced-defence responses, that triggers a positive response of the antioxidant system and improve growth under Cd stress. These results showed integrative roles of ethylene and Se in regulating the cell responses to stressful-conditions and, the cross-tolerance to stress could be used to manipulate ethylene regulated gene expression to induce heavy metal tolerance., (Copyright © 2019 Elsevier Inc. All rights reserved.)- Published
- 2019
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44. Contributions and challenges of hospital nursing management: scientific evidence.
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Ferreira VHS, Teixeira VM, Giacomini MA, Alves LR, Gleriano JS, and Chaves LDP
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- Hospital Administrators, Humans, Bibliometrics, Health Facility Administration, Nurse Administrators, Nursing Service, Hospital
- Abstract
Aim: Analyze the scientific evidence, national and international, about contributions and challenges of nursing management in hospital care., Method: Integrative literature review, with guiding question: What are the scientific evidence about nursing management in hospital care? Data was collected in LILACS, PubMed, Scopus, CINAHL and EMBASE databases. The final sample of 14 articles resulted in two categories: "Contributions" and "Challenges.", Results: Contributions refer to aspects that facilitate the development and organization of work from a technical-political perspective, by qualifying the productive processes. The challenges were related to professional development, work satisfaction, overload, quality of service, conflict resolution and teamwork., Conclusion: Management and care processes are inseparable, requires adequate and up-to-date knowledge to provide a better care experience for the health services user and coordinate team actions.
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- 2019
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45. Comparison of the RNA Content of Extracellular Vesicles Derived from Paracoccidioides brasiliensis and Paracoccidioides lutzii .
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Peres da Silva R, Longo LGV, Cunha JPCD, Sobreira TJP, Rodrigues ML, Faoro H, Goldenberg S, Alves LR, and Puccia R
- Subjects
- Animals, Cells, Cultured, Virulence, Extracellular Vesicles genetics, Paracoccidioides genetics, Paracoccidioides pathogenicity, Paracoccidioidomycosis microbiology, RNA genetics
- Abstract
Paracoccidioides brasiliensis and P. lutzii cause human paracoccidioidomycosis. We have previously characterized the <200-nt RNA sub-populations contained in fungal extracellular vesicles (EVs) from P. brasiliensis Pb18 and other pathogenic fungi. We have presently used the RNA-seq strategy to compare the <200- and >200-nt RNA fractions contained in EVs isolated from culture supernatants of P. brasiliensis Pb18, Pb3, and P. lutzii Pb01. Shared mRNA sequences were related to protein modification, translation, and DNA metabolism/biogenesis, while those related to transport and oxidation-reduction were exclusive to Pb01. The presence of functional full-length mRNAs was validated by in vitro translation. Among small non-coding (nc)RNA, 15 were common to all samples; small nucleolar (sno)RNAs were enriched in P. brasiliensis EVs, whereas for P. lutzii there were similar proportions of snoRNA, rRNA, and tRNA. Putative exonic sRNAs were highly abundant in Pb18 EVs. We also found sRNA sequences bearing incomplete microRNA structures mapping to exons. RNA-seq data suggest that extracellular fractions containing Pb18 EVs can modulate the transcriptome of murine monocyte-derived dendritic cells in a transwell system. Considering that sRNA classes are involved in transcription/translation modulation, our general results may indicate that differences in virulence among fungal isolates can be related to their distinct EV-RNA content.
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- 2019
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46. Unveiling the partners of the DRBD2-mRNP complex, an RBP in Trypanosoma cruzi and ortholog to the yeast SR-protein Gbp2.
- Author
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Wippel HH, Malgarin JS, Inoue AH, Leprevost FDV, Carvalho PC, Goldenberg S, and Alves LR
- Subjects
- Gene Expression Regulation, Protein Binding, Protein Domains, Proteomics, Protozoan Proteins chemistry, Protozoan Proteins genetics, Protozoan Proteins metabolism, RNA-Binding Proteins metabolism, Saccharomyces cerevisiae Proteins genetics, Sequence Analysis, RNA, Sequence Homology, Amino Acid, Trypanosoma cruzi genetics, RNA-Binding Proteins chemistry, RNA-Binding Proteins genetics, Ribonucleoproteins metabolism, Trypanosoma cruzi metabolism
- Abstract
Background: RNA-binding proteins (RBPs) are well known as key factors in gene expression regulation in eukaryotes. These proteins associate with mRNAs and other proteins to form mRNP complexes that ultimately determine the fate of target transcripts in the cell. This association is usually mediated by an RNA-recognition motif (RRM). In the case of trypanosomatids, these proteins play a paramount role, as gene expression regulation is mostly posttranscriptional. Despite their relevance in the life cycle of Trypanosoma cruzi, the causative agent of Chagas' disease, to date, few RBPs have been characterized in this parasite., Results: We investigated the role of DRBD2 in T. cruzi, an RBP with two RRM domains that is associated with cytoplasmic translational complexes. We show that DRBD2 is an ortholog of the Gbp2 in yeast, an SR-rich protein involved in mRNA quality control and export. We used an immunoprecipitation assay followed by shotgun proteomics and RNA-seq to assess the interaction partners of the DRBD2-mRNP complex in epimastigotes. The analysis identified mostly proteins involved in RNA metabolism and regulation, such as ALBA1, ALBA3, ALBA4, UBP1, UBP2, DRBD3, and PABP2. The RNA-seq results showed that most of the transcripts regulated by the DRBD2 complex mapped to hypothetical proteins related to multiple processes, such as to biosynthetic process, DNA metabolic process, protein modification, and response to stress., Conclusions: The identification of regulatory proteins in the DRBD2-mRNP complex corroborates the important role of DRBD2 in gene expression regulation in T. cruzi. We consider these results an important contribution to future studies regarding gene expression regulation in T. cruzi, especially in the field of RNA-binding proteins.
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- 2019
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47. Extracellular Vesicle-Mediated RNA Release in Histoplasma capsulatum .
- Author
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Alves LR, Peres da Silva R, Sanchez DA, Zamith-Miranda D, Rodrigues ML, Goldenberg S, Puccia R, and Nosanchuk JD
- Subjects
- Fungal Proteins genetics, Gene Expression Profiling, Gene Expression Regulation, Fungal, High-Throughput Nucleotide Sequencing, MicroRNAs genetics, RNA, Messenger genetics, Stress, Physiological genetics, Extracellular Vesicles metabolism, Histoplasma genetics, RNA, Fungal genetics
- Abstract
Eukaryotic cells, including fungi, release extracellular vesicles (EVs). These lipid bilayered compartments play essential roles in cellular communication and pathogenesis. EV composition is complex and includes proteins, glycans, pigments, and RNA. RNAs with putative roles in pathogenesis have been described in EVs produced by fungi. Here we describe the RNA content in EVs produced by the G186AR and G217B strains of Histoplasma capsulatum , an important human-pathogenic fungal pathogen. A total of 124 mRNAs were identified in both strains. In this set of RNA classes, 93 transcripts were enriched in EVs from the G217B strain, whereas 31 were enriched in EVs produced by the G186AR strain. This result suggests that there are important strain-specific properties in the mRNA composition of fungal EVs. We also identified short fragments (25 to 40 nucleotides in length) that were strain specific, with a greater number identified in EVs produced by the G217B strain. Remarkably, the most highly enriched processes were stress responses and translation. Half of these fragments aligned to the reverse strand of the transcript, suggesting the occurrence of microRNA (miRNA)-like molecules in fungal EVs. We also compared the transcriptome profiles of H. capsulatum with the RNA composition of EVs, and no correlation was observed. Taking the results together, our study provided information about the RNA molecules present in H. capsulatum EVs and about the differences in composition between the strains. In addition, we found no correlation between the most highly expressed transcripts in the cell and their presence in the EVs, reinforcing the idea that the RNAs were directed to the EVs by a regulated mechanism. IMPORTANCE Extracellular vesicles (EVs) play important roles in cellular communication and pathogenesis. The RNA molecules in EVs have been implicated in a variety of processes. EV-associated RNA classes have recently been described in pathogenic fungi; however, only a few reports of studies describing the RNAs in fungal EVs are available. Improved knowledge of EV-associated RNA will contribute to the understanding of their role during infection. In this study, we described the RNA content in EVs produced by two isolates of Histoplasma capsulatum Our results add this important pathogen to the current short list of fungal species with the ability to use EVs for the extracellular release of RNA., (Copyright © 2019 Alves et al.)
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- 2019
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48. A Novel Protocol for the Isolation of Fungal Extracellular Vesicles Reveals the Participation of a Putative Scramblase in Polysaccharide Export and Capsule Construction in Cryptococcus gattii .
- Author
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Reis FCG, Borges BS, Jozefowicz LJ, Sena BAG, Garcia AWA, Medeiros LC, Martins ST, Honorato L, Schrank A, Vainstein MH, Kmetzsch L, Nimrichter L, Alves LR, Staats CC, and Rodrigues ML
- Subjects
- Biological Transport, Cryptococcus gattii genetics, Cryptococcus neoformans cytology, Cryptococcus neoformans genetics, Extracellular Vesicles ultrastructure, Microscopy, Electron, Transmission, Polysaccharides genetics, Polysaccharides isolation & purification, Cryptococcus gattii enzymology, Extracellular Vesicles chemistry, Fungal Polysaccharides chemistry, Fungal Proteins genetics, Mycology methods
- Abstract
Regular protocols for the isolation of fungal extracellular vesicles (EVs) are time-consuming, hard to reproduce, and produce low yields. In an attempt to improve the protocols used for EV isolation, we explored a model of vesicle production after growth of Cryptococcus gattii and Cryptococcus neoformans on solid media. Nanoparticle tracking analysis in combination with transmission electron microscopy revealed that C. gattii and C. neoformans produced EVs in solid media. The properties of cryptococcal vesicles varied according to the culture medium used and the EV-producing species. EV detection was reproduced with an acapsular mutant of C. neoformans , as well as with isolates of Candida albicans , Histoplasma capsulatum , and Saccharomyces cerevisiae Cryptococcal EVs produced in solid media were biologically active and contained regular vesicular components, including the major polysaccharide glucuronoxylomannan (GXM) and RNA. Since the protocol had higher yields and was much faster than the regular methods used for the isolation of fungal EVs, we asked if it would be applicable to address fundamental questions related to cryptococcal secretion. On the basis that polysaccharide export in Cryptococcus requires highly organized membrane traffic culminating with EV release, we analyzed the participation of a putative scramblase (Aim25; CNBG_3981) in EV-mediated GXM export and capsule formation in C. gattii EVs from a C. gattii aim25 Δ strain differed from those obtained from wild-type (WT) cells in physical-chemical properties and cargo. In a model of surface coating of an acapsular cryptococcal strain with vesicular GXM, EVs obtained from the aim25 Δ mutant were more efficiently used as a source of capsular polysaccharides. Lack of the Aim25 scramblase resulted in disorganized membranes and increased capsular dimensions. These results associate the description of a novel protocol for the isolation of fungal EVs with the identification of a previously unknown regulator of polysaccharide release. IMPORTANCE Extracellular vesicles (EVs) are fundamental components of the physiology of cells from all kingdoms. In pathogenic fungi, they participate in important mechanisms of transfer of antifungal resistance and virulence, as well as in immune stimulation and prion transmission. However, studies on the functions of fungal EVs are still limited by the lack of efficient methods for isolation of these compartments. In this study, we developed an alternative protocol for isolation of fungal EVs and demonstrated an application of this new methodology in the study of the physiology of the fungal pathogen Cryptococcus gattii Our results describe a fast and reliable method for the study of fungal EVs and reveal the participation of scramblase, a phospholipid-translocating enzyme, in secretory processes of C. gattii ., (Copyright © 2019 Reis et al.)
- Published
- 2019
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49. The Nuclear RNA-binding Protein RBSR1 Interactome in Trypanosoma cruzi.
- Author
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Wippel HH, Malgarin JS, Martins ST, Vidal NM, Marcon BH, Miot HT, Marchini FK, Goldenberg S, and Alves LR
- Subjects
- Amino Acid Sequence, Phylogeny, Protozoan Proteins metabolism, RNA-Binding Proteins metabolism, Trypanosoma cruzi metabolism, Protozoan Proteins genetics, RNA-Binding Proteins genetics, Trypanosoma cruzi genetics
- Abstract
Trypanosoma cruzi, the etiological agent of Chagas disease, has been widely studied, reflecting both its medical importance and the particular features that make this pathogen an attractive model for basic biological studies. The repression of transcripts by messenger ribonucleoprotein (mRNP) complexes is an important pathway of post-transcriptional regulation in eukaryotes, including T. cruzi. RBSR1 is a serine-arginine (SR)-rich RNA-binding protein (RBP) in T. cruzi that contains one RNA-recognition motif (RRM); this protein has a primarily nuclear localization and is developmentally regulated, not being detected in metacyclic trypomastigotes. RBSR1 interacts with other RBPs, such as UBP1 and UBP2, and the nuclear SR-protein TRRM1. Phylogenetic analysis indicated that RBSR1 is orthologous to the human splicing factor SRSF7, what might indicate its possible involvement in pre-RNA processing. Accordingly, ribonomics data showed the enrichment of snoRNAs and snRNAs in the RBSR1 immunoprecipiatation complex, hence reinforcing the supposition that this protein might be involved in RNA processing in the nucleus., (© 2018 International Society of Protistologists.)
- Published
- 2019
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50. Maturation-associated gene expression profiles during normal human bone marrow erythropoiesis.
- Author
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Mello FV, Land MGP, Costa ES, Teodósio C, Sanchez ML, Bárcena P, Peres RT, Pedreira CE, Alves LR, and Orfao A
- Abstract
Erythropoiesis has been extensively studied using in vitro and in vivo animal models. Despite this, there is still limited data about the gene expression profiles (GEP) of primary (ex vivo) normal human bone marrow (BM) erythroid maturation. We investigated the GEP of nucleated red blood cell (NRBC) precursors during normal human BM erythropoiesis. Three maturation-associated populations of NRBC were identified and purified from (fresh) normal human BM by flow cytometry and the GEP of each purified cell population directly analyzed using DNA-oligonucleotide microarrays. Overall, 6569 genes (19% of the genes investigated) were expressed in ≥1 stage of BM erythropoiesis at stable (e.g., genes involved in DNA process, cell signaling, protein organization and hemoglobin production) or variable amounts (e.g., genes related to cell differentiation, apoptosis, metabolism), the latter showing a tendency to either decrease from stage 1 to 3 (genes associated with regulation of erythroid differentiation and survival, e.g., SPI1 , STAT5A ) or increase from stage 2 to stage 3 (genes associated with autophagy, erythroid functions such as heme production, e.g., ALAS1 , ALAS2), iron metabolism (e.g., ISCA1, SLC11A2 ), protection from oxidative stress (e.g., UCP2 , PARK7 ), and NRBC enucleation (e.g., ID2 , RB1 ). Interestingly, genes involved in apoptosis (e.g., CASP8, P2RX1 ) and immune response (e.g., FOXO3, TRAF6 ) were also upregulated in the last stage (stage 3) of maturation of NRBC precursors. Our results confirm and extend on previous observations and providing a frame of reference for better understanding the critical steps of human erythroid maturation and its potential alteration in patients with different clonal and non-clonal erythropoietic disorders., Competing Interests: The authors declare that they have no conflict of interest.
- Published
- 2019
- Full Text
- View/download PDF
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