Key Points Hemolytic processes induce rapid systemic and vascular inflammation in C57BL/6 mice that is abolished by a single dose of hydroxyurea (HU). HU exerts some NO-dependent effects and should be investigated as an acute treatment of SCD and for other hemolytic disorders.

No abstract

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 termite's 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).

ABSTRACTas a therapy in SCD. 25 Hydroxyurea is a cytostatic drug that inhibits ribonucleotide reductase, arresting cell division in the S-phase; additionally, data suggest that hydroxyurea can generate nitric oxide following administration. 26 We recently reported that hydroxyurea exerts anti-angiogenic effects in VEGF-dependent EC angiogenic in vitro assays and inhibits VEGF-dependent neovascularization in Matrigel implants in C57BL/6 mice. 27 This anti-angiogenic activity appears to be mediated by the down-regulation of endothelial hypoxia-inducible factor-1α (HIF-1α) expression, and consequent modulation of miRNA 221 expression, leading to the inhibition of EC proliferation and invasion/migration. 27To lend further support to the hypothesis of an unbalanced angiogenic state in SCD, we investigated the in vitro effects of plasma from patients with SCD on key steps of EC angiogenic behavior, namely, proliferative capability, invasive capacity and the formation of capillary-like EC structures. The plasma contents of angiogenic factors were associated with these data, as was the incidence of proliferative retinopathy in HbSC patients, a clinical manifestation thought to reflect augmented angiogenesis. 28The angiogenic activity of plasma from patients on hydroxyurea therapy was also examined. Finally, to confirm that SCD pathophysiology is associated with exacerbated angiogenesis, in vivo neovascularization was examined in a SCD mouse model, compared with non-SCD mice, utilizing a Matrigel plug angiogenesis assay. Methods PatientsPatients with HbSS or HbSC disease (collectively termed SCD) were recruited, during steady-state, at the Hematology Center, UNICAMP (Table 1). Details on the diagnosis of HbSS/HbSC, our definition of steady-state and criteria for commencing hydroxyurea therapy (15-30 mg hydroxyurea/kg/day) are given in the Online Supplementary Information. All patients had undergone ophthalmoscopy and fluorescein angiography for detection of proliferative retinopathy. A total of 29 HbSS patients, who did not have proliferative retinopathy, were recruited into the study. A total of 33 HbSC patients were recruited, of whom 16 did not have proliferative retinopathy (SC group) and 17 did (SCR group). One HbSC patient had been prescribed hydroxyurea following an ischemic stroke. Homozygous hemoglobin A (HbAA) healthy controls were age-and gender-matched, where possible. Informed written consent was obtained from all participants and the study was approved by the UNICAMP Ethics Committee, in accordance with national guidelines. Bioplex assayThe Fluorokine ® MAP, Human Angiogenesis Custom Premix Kit A (R&D Systems, Minneapolis, USA) was used to quantify Ang1, basic fibroblast growth factor (bFGF), PlGF, platelet-derived growth factor (PDGF)-AA, PDGF-BB, VEGF, VEGF-D, endostatin and thrombospondin-2 (TSP-2) in plasma, according to the manufacturer's instructions. The preparation of the plasma for the assay is described in the Online Supplementary Information. In vitro culture of human umbilical vein endothelial c...

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.

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.

No abstract

The lower termite, Coptotermes gestroi (Isoptera: Rhinotermitidae), is originally from Southeast Asia and has become a pest in Brazil. The main goal of this study was to survey C. gestroi transcriptome composition. To accomplish this, we sequenced and analyzed 3003 expressed sequence tags (ESTs) isolated from libraries of worker heads. After assembly, 695 uniESTs were obtained from which 349 have similarity with known sequences. Comparison with insect genomes demonstrated similarity, primarily with genes from Apis mellifera (28%), Tribolium castaneum (28%) and Aedes aegypti (10%). Notably, we identified two endogenous cellulases in the sequences, which may be of interest for biotechnological applications. The results presented in this work represent the first genomic study of the Asian subterranean termite, Coptotermes gestroi.