Alpha-Glucosidase Promotes Hemozoin Formation in a Blood-Sucking Bug: An Evolutionary History

Background

Hematophagous insects digest large amounts of host hemoglobin and release heme inside their guts. In Rhodnius prolixus, hemoglobin-derived heme is detoxified by biomineralization, forming hemozoin (Hz). Recently, the involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated.

Methodology/Principal Findings

Hz formation activity of an α-glucosidase was investigated. Hz formation was inhibited by specific α-glucosidase inhibitors. Moreover, Hz formation was sensitive to inhibition by Diethypyrocarbonate, suggesting a critical role of histidine residues in enzyme activity. Additionally, a polyclonal antibody raised against a phytophagous insect α-glucosidase was able to inhibit Hz formation. The α-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that α-glucosidase should act in the nucleation step of Hz formation. Hz formation was seen to be dependent on the substrate-binding site of enzyme, in a way that maltose, an enzyme substrate, blocks such activity. dsRNA, constructed using the sequence of α-glucosidase gene, was injected into R. prolixus females'' hemocoel. Gene silencing was accomplished by reduction of both α-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of α-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of α-glucosidase shows a high similarity to the insect α-glucosidases, with critical histidine and aspartic residues conserved among the enzymes.

Conclusions/Significance

Herein the Hz formation is shown to be associated to an α-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that α-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance.     

Molasses as an external carbon source for anaerobic treatment of sulphite evaporator condensate

Failures in stability and COD removal performance often occurred in full-scale anaerobic reactors treating the evaporator condensate from a sulphite pulp mill due to substrate inhibition and occasional contaminations with red liquor (wood cooking liquor). With this work, the beneficial effect provided by the continuous addition of an external carbon source (sugarcane molasses) on the overall performance and stability of the biological process was evaluated. With a moderate addition of molasses the inhibition was mitigated which led to an increase of the COD removal rate from 52% to 77% and a methane production increase from 460 to 1650 ml d^?1 at an organic loading rate of 2.61 g COD l^?1 d^?1. A similar conclusion can be drawn for the case when contamination with red liquor occurs. These results suggest that sugarcane molasses addition may be regarded as a low-cost operational strategy for the anaerobic treatment of sulphite evaporator condensate.     

p63 Immunoexpression in lip carcinogenesis

Actinic cheilitis (AC) is a potentially malignant disorder, which can present degrees of epithelial dysplasia, and may even evolve into lip squamous cell carcinoma (LSCC). Since p63 is a protein homologous to p53, which can be associated with tumorigenesis in epithelial tissues, this study aims to evaluate it in AC and LSCC, in the hopes to estimate the biological behavior of these lesions. Forty AC lesions and sixty-five cases of LSCC were quantitatively analyzed by immunohistochemistry, using anti-p63 antibody with ten cases of normal lip mucosa used as a control group. In all AC and LSCC cases studied, it was possible to detect the presence of the p63 protein. There was no statistically significant difference between immunostained cells and degree of epithelial dysplasias, nor between the LSCC grading malignancy. Nevertheless, p63 immunoexpression showed to be significantly correlated with AC and LSCC lesions as compared to normal lip epithelium. The results indicate that p63 protein is consistently expressed in AC and LSCC, and might be of help in the differential diagnosis between normal and dysplastic/neoplastic epithelium, although the evaluation using a primary antibody to all isotypes did not prove to be a risk biomarker during lip carcinogenesis. Thus, the production of antibodies for the six different p63 isotypes is urged, since in isolation they can have predictive value, mainly the ΔNp63 isoforms.     

Improved Enrichment and Isolation of Polycyclic Aromatic Hydrocarbons (PAH)-Degrading Microorganisms in Soil Using Anthracene as a Model PAH

Lack of attention to soil and microbial characteristics that influence PAHs degradation has been a leading cause of failures in isolation of efficient PAH degraders and bioaugumentation processes with microbial consortia. This study compared the classic method of isolation of PAHs-degraders with a modified method employing a pre-enrichment respirometric analysis. The modified enrichment of PAH degrading microorganisms using in vitro microcosm resulted to reduced enrichment period and more efficient PAH-degrading microbial consortia. Results indicate that natural soils with strong heterotrophic microbial activity determined through pre-enrichment analysis, are better suited for the isolation of efficient PAH degrading microorganisms with significant reduction of the enrichment period.     

Archaeal and bacterial communities of heavy metal contaminated acidic waters from zinc mine residues in Sepetiba Bay

Mining of metallic sulfide ore produces acidic water with high metal concentrations that have harmful consequences for aquatic life. To understand the composition and structure of microbial communities in acid mine drainage (AMD) waters associated with Zn mine tailings, molecular diversity of 16S genes was examined using a PCR, cloning, and sequencing approach. A total of 78 operational taxonomic units (OTUs) were obtained from samples collected at five different sites in and around mining residues in Sepetiba Bay, Brazil. We analyzed metal concentration, physical, chemical, and microbiological parameters related to prokaryotic diversity in low metal impacted compared to highly polluted environments with Zn at level of gram per liter and Cd–Pb at level of microgram per liter. Application of molecular methods for community structure analyses showed that Archaea and Bacteria groups present a phylogenetic relationship with uncultured environmental organisms. Phylogenetic analysis revealed that bacteria present at the five sites fell into seven known divisions, α-Proteobacteria (13.4%), β-Proteobacteria (16.3%), γ-Proteobacteria (4.3%), Sphingobacteriales (4.3%), Actinobacteria (3.2%) Acidobacteria (2.1%), Cyanobacteria (11.9%), and unclassified bacteria (44.5%). Almost all archaeal clones were related to uncultivated Crenarchaeota species, which were shared between high impacted and low impacted waters. Rarefaction curves showed that bacterial groups are more diverse than archaeal groups while the overall prokaryotic biodiversity is lower in high metal impacted environments than in less polluted habitats. Knowledge of this microbial community structure will help in understanding prokaryotic diversity, biogeography, and the role of microorganisms in zinc smelting AMD generation and perhaps it may be exploited for environmental remediation procedures in this area.     

Gradients within gradients: The mesoscale distribution patterns of palms in a central Amazonian forest

Questions: What are the relative contributions of environmental factors and geographic distance to palm community structure at the mesoscale, and how do they depend on the length of the environmental gradient covered? How do soil and topography affect variation of the canopy and understory palm community structure at the mesoscale? How does fine‐scale variation within the broad edaphic/topographic classes affect palm community composition? Location: Reserva Ducke, terra‐firme forest, Manaus, Brazil. Methods: Palms were sampled in 72 plots 250 m × 4 m, systematically distributed over an area of 100 km². Soil, topography and distance to watercourses were measured for all plots. The relationship between community structure axes, summarized by NMDS ordinations, and environmental predictors, was analysed with multivariate regressions. Matrix regressions were used to determine the proportions of variance explained by environmental and geographic predictors. Results: Floristic variation at the mesoscale was mostly related to environmental variation, and the proportion of variance explained depended on the amplitude of the environmental gradient. Soil was the main predictor of floristic change, but its effects differed between life forms, with the understory palm community structured within one of the edaphic/topographic classes, in association with distance to watercourses. Conclusions: Dispersal limitation does not explain palm composition at the mesoscale, and the amplitude of environmental gradients covered by the analysis can be as important as the scale of analysis, in determining the relative contributions of environmental and geographical components to community structure. Soil and topography can predict a large proportion of palm composition, but gradients differ in scale, with some environmental gradients being nested within others. Therefore, although all environmental gradients are nested within distance, they do not necessarily coincide.     

Marked upregulation of cholesterol 25-hydroxylase expression by lipopolysaccharide

During screening of genes upregulated by lipopolysaccharide (LPS; endotoxin) treatment of bone marrow-derived mouse macrophages, it was unexpectedly found that cholesterol 25-hydroxylase (Ch25h) was strongly upregulated. Treatment of macrophages with 10 ng/ml of LPS for 2 h resulted in a 35-fold increase in the expression of Ch25h. In contrast, LPS treatment did not increase the expression of Cyp27a1 or Cyp7b1. The increased Ch25h expression was found to be independent of Myeloid differentiation protein 88 signaling but dependent on Toll-like receptor 4 signaling. LPS treatment of macrophages caused a 6- to 7-fold increase in cellular 25-hydroxycholesterol concentration. When macrophages were treated with increasing concentrations of 25-hydroxycholesterol, a dose-dependent release of CCL5 into the culture medium was observed. Intravenous injection of LPS in eight healthy volunteers resulted in an increase in plasma 25-hydroxycholesterol concentration. The possibility is discussed that 25-hydroxycholesterol may have a role in the inflammatory response, in addition to its more established role in the regulation of cholesterol homeostasis.     

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax antibodies and its use in epidemiological surveys

There are data indicating that the distribution of Trypanosoma vivax in the Brazilian territory is expanding with potential to reach other areas, where the vectors are present. The detection of anti-trypanosomal antibodies in serum provides important information of the trypanosomal status in cattle herds. For this reason, an enzyme-linked immunosorbent assay (Tv-ELISA-Ab) with crude antigen from one Brazilian isolate of T. vivax was developed and evaluated. The sensitivity and specificity were respectively 97.6 and 96.9%. In the evaluation of cross-reactions, three calves inoculated with T. evansi trypimastigotes blood forms showed optical densities (OD) under the cut-off during the whole experimental period, except one at 45 days post-inoculation. With relation to Babesia bovis, B. bigemina, and Anaplasma marginale, which are endemic hemoparasites in the studied area, the cross-reactions were shown to be 5.7, 5.3, and 1.1%, respectively. The first serological survey of Pantanal and state of Pará showed that T. vivax is widespread, although regions within both areas had significantly different prevalences. Therefore, this Tv-ELISA-Ab may be a more appropriate test for epidemiological studies in developing countries because the diagnostic laboratories in most countries may be able to perform an ELISA, which is not true for polymerase chain reaction.