Mixture design of starchy substrates hydrolysis by an immobilized glucoamylase from Aspergillus brasiliensis

Starch has great importance in human diet, since it is a heteropolymer of plants, mainly found in roots, as potato, cassava and arrowroots. This carbohydrate is composed by a highly-branched chain: amylopectin; and a linear chain: amylose. The proportion between the chains varies according to the botanical source. Starch hydrolysis is catalyzed by enzymes of the amilolytic system, named amylases. Among the various enzymes of this system, the glucoamylases (EC 3.2.1.3 glucan 1,4-alpha-glucosidases) are the majority because they hydrolyze the glycosidic linkages at the end of starch chains releasing glucose monomers. In this work, a glucoamylase secreted in the culture medium, by the ascomycete Aspergillus brasiliensis, was immobilized in Dietilaminoetil Sepharose-Polyethylene Glycol (DEAE-PEG), since immobilized biocatalysts are more stable in long periods of hydrolysis, and can be recovered from the final product and reused for several cycles. Glucoamylase immobilization has shown great thermal stability improvement over the soluble enzyme, reaching 66% more activity after 6?h at 60?°C, and 68% of the activity after 10 hydrolysis cycles. A simplex centroid experimental mixture design was applied as a tool to characterize the affinity of the immobilized enzyme for different starchy substrates. In assays containing several proportions of amylose, amylopectin and starch, the glucoamylase from A. brasiliensis mainly hydrolyzed the amylopectin chains, showing to have preference by branched substrates.     

Comparative proteomic analysis of Xanthomonas citri ssp. citri periplasmic proteins reveals changes in cellular envelope metabolism during in vitro pathogenicity induction

Citrus canker is a plant disease caused by Gram‐negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm‐enriched fraction was performed for XAC cells grown in pathogenicity‐inducing (XAM‐M) and pathogenicity‐non‐inducing (nutrient broth) media using two‐dimensional electrophoresis combined with liquid chromatography‐tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up‐regulated proteins related to cellular envelope metabolism included glucose‐1‐phosphate thymidylyltransferase, dTDP‐4‐dehydrorhamnose‐3,5‐epimerase and peptidyl‐prolyl cis–trans‐isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real‐time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up‐regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60‐kDa chaperonin and glyceraldehyde‐3‐phosphate dehydrogenase were identified, suggesting the presence of post‐translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence‐related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.     

Phytochemical Characterization of Cannabis sativa L. Roots from Northeastern Brazil

This article describes the phytochemical study of Cannabis sativa roots from northeastern Brazil. The dried plant material was pulverized and subjected to exhaustive maceration with ethanol at room temperature, obtaining the crude ethanolic extract (Cs-EEBR). The volatile compounds were analyzed by gas chromatography coupled with mass spectrometry (GC/MS), which allowed to identify 22 compounds by comparing the linear retention index (LRI), the similarity index (SI) and the fragmentation pattern of the constituents with the literature. By this technique the major compounds identified were: friedelan-3-one and β-sitosterol. In addition, two fractions were obtained from Cs-EEBR by classical column chromatography and preparative thin layer chromatography. These fractions were analyzed by NMR and IR and together with the mass spectrometry data allowed to identify the compounds: epifriedelanol, friedelan-3-one, β-sitosterol and stigmasterol. The study contributed to the phytochemical knowledge of Cannabis sativa, specifically the roots, as there are few reports on the chemical constituents of this part of the plant.     

One year of exercise training promotes distinct adaptations in right and left ventricle of female Sprague-Dawley rats

Journal of Physiology and Biochemistry - Aerobic exercise training induces a unique cardioprotective phenotype, but it is becoming clear that it does not promote the same structural, functional,...     

Intravoxel Incoherent Motion Diffusion Weighted MR Imaging at 3.0 T: Assessment of Steatohepatitis and Fibrosis Compared with Liver Biopsy in Type 2 Diabetic Patients

ObjectiveTo evaluate the capability of intravoxel incoherent motion (IVIM) diffusion-weighted imaging (DWI) to assess steatohepatitis and fibrosis determined by histopathology in type 2 diabetic patients.MethodsFifty-nine type 2 diabetic patients (49 women, 10 men; mean age, 54 ± 9 years) were submitted to liver biopsy for the evaluation of non-alcoholic fatty liver disease (NAFLD) and underwent DWI on a 3.0T MR system using 10 b values. Institutional approval and patient consent were obtained. Pure molecular-based (D), perfusion-related (D*), and vascular fraction (f) were calculated using a double exponential model and least squares curve fitting. D, D*, and f were compared between patients with and without steatohepatitis and between patients with and without fibrosis. The variables were compared by using the Ranksum test and Student t-test.ResultsSteatohepatitis was observed in 22 patients and fibrosis in 16 patients. A lower D median (0.70 s/mm² vs. 0.83 s/mm², p<0.05) and a lower D* median (34.39 s/mm² vs. 45.23 s/mm², p<0.05) were observed among those with steatohepatitis. A lower D median (0.70 s/mm² vs. 0.82 s/mm², p<0.05) and a lower D* median (35.01 s/mm² vs. 44.76 s/mm², p=0.05) were also observed among those with fibrosis.ConclusionIVIM-DWI has the potential to aid in the characterization of steatohepatitis and fibrosis.     

Denitrification of a landfill leachate with high nitrate concentration in an anoxic rotating biological contactor

The denitrification performance of a lab-scale anoxic rotating biological contactor (RBC) using landfill leachate with high nitrate concentration was evaluated. Under a carbon to nitrogen ratio (C/N) of 2, the reactor achieved N-NO₃ ⁻ removal efficiencies above 95% for concentrations up to 100 mg N-NO₃ ⁻ l⁻¹. The highest observed denitrification rate was 55 mg N-NO₃ ⁻ l⁻¹ h⁻¹ (15 g N-NO₃ ⁻ m⁻² d⁻¹) at a nitrate concentration of 560 mg N-NO₃ ⁻ l⁻¹. Although the reactor has revealed a very good performance in terms of denitrification, effluent chemical oxygen demand (COD) concentrations were still high for direct discharge. The results obtained in a subsequent experiment at constant nitrate concentration (220 mg N-NO₃ ⁻ l⁻¹) and lower C/N ratios (1.2 and 1.5) evidenced that the organic matter present in the leachate was non-biodegradable. A phosphorus concentration of 10 mg P-PO₄ ³⁻ l⁻¹ promoted autotrophic denitrification, revealing the importance of phosphorus concentration on biological denitrification processes.     

Clonal diversity and conservation genetics of the medicinal plant Carapichea ipecacuanha (Rubiaceae)

The roots of the understorey shrub Carapichea ipecacuanha (ipecac) have medicinal properties, and the uprooting of wild plants has supplied most of the world demand for this species. Although under severe population decline, C. ipecacuanha lacks legal protection. In the wild, the aerial stems of ipecac clump together to form clusters with well-defined borders. Cluster size may range from several to hundreds of aerial stems. To investigate the extent of clonality among aerial stems in ipecac clusters, we sampled 50 wild clusters (a total of 291 aerial stems) and screened them with 89 inter-simple sequence repeat (ISSR) markers. The 291 aerial stems were grouped into 42 putative clones. The clonal groups generally consisted of aerial stems from the same cluster, and there was little or no genetic differentiation among aerial stems at the cluster level. These findings suggest that strategies designed to conserve ipecac in situ should not rely upon census data, which are based on the number of aerial stems per cluster and the number of clusters per population, because such data greatly underestimate the species effective population size and genetic diversity. Our results also indicate that this species needs protection at a federal level.     

Clinical Profiles and Factors Associated with Death in Adults with Dengue Admitted to Intensive Care Units,Minas Gerais,Brazil

The purpose of our study was to describe the clinical profile of dengue-infected patients admitted to Brazilian intensive care units (ICU) and evaluate factors associated with death. A longitudinal, multicenter case series study was conducted with laboratory-confirmed dengue patients admitted to nine Brazilian ICUs situated in Minas Gerais state, southeastern Brazil from January 1, 2008, to December 31, 2013. Demographic, clinical and laboratory data; disease severity scores; and mortality were evaluated. A total of 97 patients were studied. The in-ICU and in-hospital mortality rates were 18.6% and 19.6%, respectively. Patients classified as having severe dengue according to current World Health Organization classifications showed an increased risk of death in a univariate analysis. Nonsurvivors were older, exhibited lower serum albumin concentrations and higher total leukocyte counts and serum creatinine levels. Other risk factors (vomiting, lethargy/restlessness, dyspnea/respiratory distress) were also associated with death in a univariate analysis. Multivariate analysis indicated that in-hospital mortality was significantly associated with Acute Physiology and Chronic Health Evaluation II and the Sequential Organ Failure Assessment score. The ICU and in-hospital mortality observed in this study were higher than values reported in similar studies. An increased frequency of ICU admission due to severe organ dysfunction, higher severity indices and scarcity of ICU beds may partially explain the higher mortality.