Flávia Costa Leonardo

Acute hemolytic vascular inflammatory processes are prevented by nitric oxide replacement or a single dose of hydroxyurea

Hemolysis and consequent release of cell-free hemoglobin (CFHb) impair vascular nitric oxide (NO) bioavailability and cause oxidative and inflammatory processes. Hydroxyurea (HU), a common therapy for sickle cell disease (SCD), induces fetal Hb production and can act as an NO donor. We evaluated the acute inflammatory effects of intravenous water-induced hemolysis in C57BL/6 mice and determined the abilities of an NO donor, diethylamine NONOate (DEANO), and a single dose of HU to modulate this inflammation. Intravenous water induced acute hemolysis in C57BL/6 mice, attaining plasma Hb levels comparable to those observed in chimeric SCD mice. This hemolysis resulted in significant and rapid systemic inflammation and vascular leukocyte recruitment within 15 minutes, accompanied by NO metabolite generation. Administration of another potent NO scavenger (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) to C57BL/6 mice induced similar alterations in leukocyte recruitment, whereas hemin-induced inflammation occurred over a longer time frame. Importantly, the acute inflammatory effects of water-induced hemolysis were abolished by the simultaneous administration of DEANO or HU, without altering CFHb, in an NO pathway-mediated manner. In vitro, HU partially reversed the Hb-mediated induction of endothelial proinflammatory cytokine secretion and adhesion molecule expression. In summary, pathophysiological levels of hemolysis trigger an immediate inflammatory response, possibly mediated by vascular NO consumption. HU presents beneficial anti-inflammatory effects by inhibiting rapid-onset hemolytic inflammation via an NO-dependent mechanism, independently of fetal Hb elevation. Data provide novel insights into mechanisms of hemolytic inflammation and further support perspectives for the use of HU as an acute treatment for SCD and other hemolytic disorders.

Functional characterization and target discovery of glycoside hydrolases from the digestome of the lower termite Coptotermes gestroi

BackgroundLignocellulosic materials have been moved towards the forefront of the biofuel industry as a sustainable resource. However, saccharification and the production of bioproducts derived from plant cell wall biomass are complex and lengthy processes. The understanding of termite gut biology and feeding strategies may improve the current state of biomass conversion technology and bioproduct production.ResultsThe study herein shows comprehensive functional characterization of crude body extracts from Coptotermes gestroi along with global proteomic analysis of the termites digestome, targeting the identification of glycoside hydrolases and accessory proteins responsible for plant biomass conversion. The crude protein extract from C. gestroi was enzymatically efficient over a broad pH range on a series of natural polysaccharides, formed by glucose-, xylose-, mannan- and/or arabinose-containing polymers, linked by various types of glycosidic bonds, as well as ramification types. Our proteomic approach successfully identified a large number of relevant polypeptides in the C. gestroi digestome. A total of 55 different proteins were identified and classified into 29 CAZy families. Based on the total number of peptides identified, the majority of components found in the C. gestroi digestome were cellulose-degrading enzymes. Xylanolytic enzymes, mannan- hydrolytic enzymes, pectinases and starch-degrading and debranching enzymes were also identified. Our strategy enabled validation of liquid chromatography with tandem mass spectrometry recognized proteins, by enzymatic functional assays and by following the degradation products of specific 8-amino-1,3,6-pyrenetrisulfonic acid labeled oligosaccharides through capillary zone electrophoresis.ConclusionsHere we describe the first global study on the enzymatic repertoire involved in plant polysaccharide degradation by the lower termite C. gestroi. The biochemical characterization of whole body termite extracts evidenced their ability to cleave all types of glycosidic bonds present in plant polysaccharides. The comprehensive proteomic analysis, revealed a complete collection of hydrolytic enzymes including cellulases (GH1, GH3, GH5, GH7, GH9 and CBM 6), hemicellulases (GH2, GH10, GH11, GH16, GH43 and CBM 27) and pectinases (GH28 and GH29).

Deciphering the synergism of endogenous glycoside hydrolase families 1 and 9 from Coptotermes gestroi

Termites can degrade up to 90% of the lignocellulose they ingest using a repertoire of endogenous and symbiotic degrading enzymes. Termites have been shown to secrete two main glycoside hydrolases, which are GH1 (EC 3.2.1.21) and GH9 (EC 3.2.1.4) members. However, the molecular mechanism for lignocellulose degradation by these enzymes remains poorly understood. The present study was conducted to understand the synergistic relationship between GH9 (CgEG1) and GH1 (CgBG1) from Coptotermes gestroi, which is considered the major urban pest of São Paulo State in Brazil. The goal of this work was to decipher the mode of operation of CgEG1 and CgBG1 through a comprehensive biochemical analysis and molecular docking studies. There was outstanding degree of synergy in degrading glucose polymers for the production of glucose as a result of the endo-β-1,4-glucosidase and exo-β-1,4-glucosidase degradation capability of CgEG1 in concert with the high catalytic performance of CgBG1, which rapidly converts the oligomers into glucose. Our data not only provide an increased comprehension regarding the synergistic mechanism of these two enzymes for cellulose saccharification but also give insight about the role of these two enzymes in termite biology, which can provide the foundation for the development of a number of important applied research topics, such as the control of termites as pests as well as the development of technologies for lignocellulose-to-bioproduct applications.

Key endothelial cell angiogenic mechanisms are stimulated by the circulating milieu in sickle cell disease and attenuated by hydroxyurea

As hypoxia-induced inflammatory angiogenesis may contribute to the manifestations of sickle cell disease, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from patients with steady-state sickle cell anemia contained elevated concentrations of pro-angiogenic factors (angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly increasing endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice than in non-sickle cell disease mice, consistent with an up-regulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy had a pro-angiogenic profile and more significant effects on endothelial cell proliferation and capillary formation than plasma from patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factors and inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, individuals with sickle cell anemia or hemoglobin SC disease with retinopathy present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy to prevent the progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the benefits and drawbacks of the potent anti-angiogenic effects of hydroxyurea should be clarified.

Reduced rate of sickle-related complications in Brazilian patients carrying HbF-promoting alleles at the BCL11A and HMIP-2 loci

The presence of high levels of fetal haemoglobin (HbF) provides well‐validated clinical benefits to patients with sickle cell anaemia (SCA). Nevertheless it has been difficult to show clear direct effects of the known genetic HbF modifiers, such as the enhancer polymorphisms for haematopoietic transcription factors BCL11A and MYB, on SCA severity. Investigating SCA patients from Brazil, with a high degree of European genetic admixture, we have detected strong effects of these variants on HbF levels. Critically, we have shown, for the first time, that the presence of such HbF‐promoting variants leads to a reduced rate of SCA complications, especially stroke.

Expanding the Knowledge on Lignocellulolytic and Redox Enzymes of Worker and Soldier Castes from the Lower Termite Coptotermes gestroi

Termites are considered one of the most efficient decomposers of lignocelluloses on Earth due to their ability to produce, along with its microbial symbionts, a repertoire of carbohydrate-active enzymes (CAZymes). Recently, a set of Pro-oxidant, Antioxidant, and Detoxification enzymes (PAD) were also correlated with the metabolism of carbohydrates and lignin in termites. The lower termite Coptotermes gestroi is considered the main urban pest in Brazil, causing damage to wood constructions. Recently, analysis of the enzymatic repertoire of C. gestroi unveiled the presence of different CAZymes. Because the gene profile of CAZy/PAD enzymes endogenously synthesized by C. gestroi and also by their symbiotic protists remains unclear, the aim of this study was to explore the eukaryotic repertoire of these enzymes in worker and soldier castes of C. gestroi. Our findings showed that worker and soldier castes present similar repertoires of CAZy/PAD enzymes, and also confirmed that endo-glucanases (GH9) and beta-glucosidases (GH1) were the most important glycoside hydrolase families related to lignocellulose degradation in both castes. Classical cellulases such as exo-glucanases (GH7) and endo-glucanases (GH5 and GH45), as well as classical xylanases (GH10 and GH11), were found in both castes only taxonomically related to protists, highlighting the importance of symbiosis in C. gestroi. Moreover, our analysis revealed the presence of Auxiliary Activity enzyme families (AAs), which could be related to lignin modifications in termite digestomes. In conclusion, this report expanded the knowledge on genes and proteins related to CAZy/PAD enzymes from worker and soldier castes of lower termites, revealing new potential enzyme candidates for second-generation biofuel processes.

Chronic treatment with resveratrol improves overactive bladder in obese mice via antioxidant activity

The objective of the present work was to evaluate whether oral intake with resveratrol ameliorates overactive bladder in high-fat fed mice. Male C57BL6 mice fed with standard chow or high-fat diet to induce obesity received a two-week therapy with resveratrol (100mg/kg, given as a daily gavage). Weight and metabolic profile, together with cystometry and in vitro bladder contractions were evaluated. Measurements of gp91phox and SOD1 mRNA expressions and reactive-oxygen species (ROS) in bladder tissues, and serum TBARS were performed. Obese mice exhibited increases in body weight and epididymal fat mass, which were significantly reduced by oral treatment with resveratrol. Cystometric study in obese mice showed increases in non-voiding contractions, post-voiding pressure and voiding frequency that were reversed by resveratrol treatment. Likewise, the in vitro bladder overactivity in response to electrical-field stimulation (80V, 1-32Hz) or carbachol (1nM to 10mM) were normalized by resveratrol. The gp91phox and SOD1 mRNA expressions in bladder tissues were markedly higher in obese mice compared with lean group. In addition, ROS levels in bladder tissues and serum lipid peroxidation (TBARS assay) were markedly higher in obese compared with lean mice, all of which were reduced by resveratrol treatment. In lean group, resveratrol had no effect in any parameter evaluated. Our results show that two-week therapy of obese mice with resveratrol reduces the systemic and bladder oxidative stress, and greatly ameliorated the cystometry alterations and in vitro bladder overactivity. Resveratrol treatment could be an option to prevent obesity-associated overactive bladder.

On the Apterous Line of the Termite Velocitermes heteropterus (Isoptera: Termitidae): Developmental Pathways and Cellulose Digestion

Termites are social insects with an extraordinary ability to digest cellulose. Termite societies are structured into castes, and patterns of postembryonic development vary between different termite species. The apterous line may exhibit polymorphism (“physical castes”), in which workers are dimorphic and soldiers can be either dimorphic or trimorphic. We examined the occurrence of polymorphism in the apterous line of Velocitermes heteropterus and determined the developmental pathways for this termite species. We also investigated the expression of the cellulase genes encoding &bgr;-glucosidase and endo-&bgr;-1,4-glucanase among the castes to determine whether there is a difference in digestion and, consequently, a possible division of labor with respect to this activity among the worker castes. The apterous line of V. heteropterus presents individuals of both sexes with two larval instars. The female larvae become major workers, and the male larvae become minor workers and soldiers. The expression of &bgr;-glucosidase was similar within the castes, but the expression of endo-&bgr;-1,4-glucanase was higher in workers than in soldiers. No significant differences were found between minor and major workers. These results suggest that there is no division of labor between the minors and majors with regard to cellulose digestion, with both workers contributing similarly to this process.

Reduced plasma angiotensin II levels are reversed by hydroxyurea treatment in mice with sickle cell disease

AIMS Sickle cell disease (SCD) pathogenesis leads to recurrent vaso-occlusive and hemolytic processes, causing numerous clinical complications including renal damage. As vasoconstrictive mechanisms may be enhanced in SCD, due to endothelial dysfunction and vasoactive protein production, we aimed to determine whether the expression of proteins of the renin-angiotensin system (RAS) may be altered in an animal model of SCD. MAIN METHODS Plasma angiotensin II (Ang II) was measured in C57BL/6 (WT) mice and mice with SCD by ELISA, while quantitative PCR was used to compare the expressions of the genes encoding the angiotensin-II-receptors 1 and 2 (AT1R and AT2R) and the angiotensin-converting enzymes (ACE1 and ACE2) in the kidneys, hearts, livers and brains of mice. The effects of hydroxyurea (HU; 50-75mg/kg/day, 4weeks) treatment on these parameters were also determined. KEY FINDINGS Plasma Ang II was significantly diminished in SCD mice, compared with WT mice, in association with decreased AT1R and ACE1 expressions in SCD mice kidneys. Treatment of SCD mice with HU reduced leukocyte and platelet counts and increased plasma Ang II to levels similar to those of WT mice. HU also increased AT1R and ACE2 gene expression in the kidney and heart. SIGNIFICANCE Results indicate an imbalanced RAS in an SCD mouse model; HU therapy may be able to restore some RAS parameters in these mice. Further investigations regarding Ang II production and the RAS in human SCD may be warranted, as such changes may reflect or contribute to renal damage and alterations in blood pressure.