Steady-State NTPase Activity of Dengue Virus NS3: Number of Catalytic Sites,Nucleotide Specificity and Activation by ssRNA

Dengue virus nonstructural protein 3 (NS3) unwinds double stranded RNA driven by the free energy derived from the hydrolysis of nucleoside triphosphates. This paper presents the first systematic and quantitative characterization of the steady-state NTPase activity of DENV NS3 and their interaction with ssRNA. Substrate curves for ATP, GTP, CTP and UTP were obtained, and the specificity order for these nucleotides - evaluated as the ratio (k_cat/K_M)- was GTPATPCTP UTP, which showed that NS3 have poor ability to discriminate between different NTPs. Competition experiments between the four substrates indicated that all of them are hydrolyzed in one and the same catalytic site of the enzyme. The effect of ssRNA on the ATPase activity of NS3 was studied using poly(A) and poly(C). Both RNA molecules produced a 10 fold increase in the turnover rate constant (k_cat) and a 100 fold decrease in the apparent affinity (K_M) for ATP. When the ratio [RNA bases]/[NS3] was between 0 and 20 the ATPase activity was inhibited by increasing both poly(A) and poly(C). Using the theory of binding of large ligands (NS3) to a one-dimensional homogeneous lattice of infinite length (RNA) we tested the hypothesis that inhibition is the result of crowding of NS3 molecules along the RNA lattices. Finally, we discuss why this hypothesis is consistent with the idea that the ATPase catalytic cycle is tightly coupled to the movement of NS3 helicase along the RNA.     

The Relationship between Gene Isoform Multiplicity,Number of Exons and Protein Divergence

At present we know that phenotypic differences between organisms arise from a variety of sources, like protein sequence divergence, regulatory sequence divergence, alternative splicing, etc. However, we do not have yet a complete view of how these sources are related. Here we address this problem, studying the relationship between protein divergence and the ability of genes to express multiple isoforms. We used three genome-wide datasets of human-mouse orthologs to study the relationship between isoform multiplicity co-occurrence between orthologs (the fact that two orthologs have more than one isoform) and protein divergence. In all cases our results showed that there was a monotonic dependence between these two properties. We could explain this relationship in terms of a more fundamental one, between exon number of the largest isoform and protein divergence. We found that this last relationship was present, although with variations, in other species (chimpanzee, cow, rat, chicken, zebrafish and fruit fly). In summary, we have identified a relationship between protein divergence and isoform multiplicity co-occurrence and explained its origin in terms of a simple gene-level property. Finally, we discuss the biological implications of these findings for our understanding of inter-species phenotypic differences.     

Clinical Impact of the Temporal Relationship between Depression and Type 2 Diabetes: The Fremantle Diabetes Study Phase II

Background

The clinical features of type 2 diabetes may differ depending on whether first depression episode precedes or follows the diagnosis of diabetes.

Methods

Type 2 patients from the observational community-based Fremantle Diabetes Study Phase II underwent assessment of lifetime depression using the Brief Lifetime Depression Scale (developed and validated for this study) supplemented by information on current depression symptoms (Patient Health Questionnaire, 9-item version) and use of antidepressants. Patients were categorized as never depressed (Group 1), having had depression before diabetes diagnosis (Group 2), diagnosed with depression and diabetes within 2 years of each other (Group 3) and having depression after diabetes diagnosis (Group 4).

Results

Of 1391 patients, 20.8% were assigned to Group 2, 6.0% to Group 3 and 14.5% to Group 4. In Group 2, depression occurred a median 15.6 years before diabetes onset at age 37.2±14.7 years. These patients had similar clinical characteristics to never depressed patients except for reduced self-care behaviours and having more symptomatic peripheral arterial disease. In Group 4, depression occurred a median 9.9 years after diabetes onset at age 59.8±13.0 years. These patients had long duration diabetes, poor glycaemic control, more intensive management and more diabetic complications. Group 4 patients had more current depression than Group 2 but were less likely to be receiving antidepressants.

Conclusions/Interpretation

The clinical features of depression and type 2 diabetes are heterogeneous depending on their temporal relationship. There may be corresponding differences in the pathogenesis of depression in diabetes that have implications for diagnosis and management.     

Screening Microalgae Strains for Biodiesel Production: Lipid Productivity and Estimation of Fuel Quality Based on Fatty Acids Profiles as Selective Criteria

The viability of algae-based biodiesel industry depends on the selection of adequate strains in regard to profitable yields and oil quality. This work aimed to bioprospecting and screening 12 microalgae strains by applying, as selective criteria, the volumetric lipid productivity and the fatty acid profiles, used for estimating the biodiesel fuel properties. Volumetric lipid productivity varied among strains from 22.61 to 204.91 mg l^?1 day^?1. The highest lipid yields were observed for Chlorella (204.91 mg l^?1 day¹) and Botryococcus strains (112.43 and 98.00 mg l^?1 day^?1 for Botryococcus braunii and Botryococcus terribilis, respectively). Cluster and principal components analysis analysis applied to fatty acid methyl esters (FAME) profiles discriminated three different microalgae groups according to their potential for biodiesel production. Kirchneriella lunaris, Ankistrodesmus fusiformis, Chlamydocapsa bacillus, and Ankistrodesmus falcatus showed the highest levels of polyunsaturated FAME, which incurs in the production of biodiesels with the lowest (42.47–50.52) cetane number (CN), the highest (101.33–136.97) iodine values (IV), and the lowest oxidation stability. The higher levels of saturated FAME in the oils of Chlamydomonas sp. and Scenedesmus obliquus indicated them as source of biodiesel with higher oxidation stability, higher CN (63.63–64.94), and lower IV (27.34–35.28). The third group, except for the Trebouxyophyceae strains that appeared in isolation, are composed by microalgae that generate biodiesel of intermediate values for CN, IV, and oxidation stability, related to their levels of saturated and monosaturated lipids. Thus, in this research, FAME profiling suggested that the best approach for generating a microalgae-biodiesel of top quality is by mixing the oils of distinct cell cultures.     

Calmodulin Inhibitors from Aspergillus stromatoides

An organic extract was prepared from the culture medium and mycelia of the marine fungus Aspergillus stromatoides Raper & Fennell . The extract was fractionated via column chromatography, and the resulting fractions were tested for their abilities to quench the fluorescence of the calmodulin (CaM) biosensor hCaM M124C‐mBBr. From the active fraction, emodin ( 1 ) and ω‐hydroxyemodin ( 2 ) were isolated as CaM inhibitors. Anthraquinones 1 and 2 quenched the fluorescence of the hCaM M124C‐mBBr biosensor in a concentration‐dependent manner with K_d values of 0.33 and 0.76 μM , respectively. The results were compared with those of chlorpromazine (CPZ), a classical inhibitor of CaM, with a K_d value of 1.25 μM . Docking analysis revealed that 1 and 2 bind to the same pocket of CPZ. The CaM inhibitor properties of 1 and 2 were correlated with some of their reported biological properties. Citrinin ( 3 ), methyl 8‐hydroxy‐6‐methyl‐9‐oxo‐9H‐xanthene‐1‐carboxylate ( 4 ), and coniochaetone A ( 5 ) were also isolated in the present study. The X‐ray structure of 5 is reported for the first time.     

CO2 adsorption on polar surfaces of ZnO

Physical and chemical adsorption of CO₂ on ZnO surfaces were studied by means of two different implementations of periodic density functional theory. Adsorption energies were computed and compared to values in the literature. In particular, it was found that the calculated equilibrium structure and internuclear distances are in agreement with previous work. CO₂ adsorption was analyzed by inspection of the density of states and electron localization function. Valence bands, band gap and final states of adsorbed CO₂ were investigated and the effect of atomic displacements analyzed. The partial density of states (PDOS) of chemical adsorption of CO₂ on the ZnO(0001) surface show that the p orbitals of CO₂ were mixed with the ZnO valence band state appearing at the top of the valence band and in regions of low-energy conduction band.

Jatropha curcas L. (Euphorbiaceae) exhibits a mixed mating system, high correlated mating and apomixis

The hierarchical mating system among and within fruits of Jatropha curcas was investigated in a base population using five microsatellite loci, employing mixed mating and correlated mating models. Open-pollinated fruits were collected from 15 randomly selected seed trees, sampling seven fruits per tree for a total of 21 seeds from each tree. We detected multilocus genotypes identical to the mother tree in 13 % of offspring, implying the occurrence of apomixis in J. curcas. The presumed apomictic individuals were excluded from the analysis of the remaining results. Evidence of substantial selfing was provided by the average multilocus outcrossing rate (t _m?=?0.683), showing that the species exhibits a mixed mating system. The outcrossing rate showed a large variation among seed trees, ranging from 0.21 to 1.0, indicating that the species is not self-incompatible. Significant differences were detected between the multilocus and the single locus outcrossing rates (t _m???t _s?=?0.347) that suggested mating among related individuals, possibly because of the presence of individuals from the same progeny (sibs) in the base population. The multilocus paternity correlation was extremely high for the population (r _p(m)?=?0.999), indicating that the progenies were manly composed of full-sibs. As a consequence of selfing and a high paternity correlation, the co-ancestry coefficient within the progeny was higher (Θ?=?0.369) than expected for panmictic populations. Our results indicated that J. curcas produces seeds asexually by apomixis and sexually by a mixed mating system, combining selfing and outcrossing.     

Morphological changes, chitinolytic enzymes and hydrophobin-like proteins as responses of Lecanicillium lecanii during growth with hydrocarbon

Lecanicillium lecanii, Verticillium chlamydosporium, V. fungicola var flavidum and Beauveria bassiana were evaluated on their growth with pure n-hexane, toluene and n-hexane:toluene 17:83 (v:v) mixture. Another set of treatments were conducted with colloidal chitin as additional carbon source. All the strains of Lecanicillium were able to grow using hydrocarbons with or without the addition of chitin, although the presence of hydrocarbons showed significant inhibition evidenced by measured biomass, radial growth and microscopic analyses. Degradation of n-hexane ranged within 43 and 62 % and it was higher than that with toluene. The strains L460, L157 and L2149, which presented the highest growth, were further selected for determinations of hydrocarbon consumptions in microcosms. Strain L157 showed the highest consumption of n-hexane (55.6 %) and toluene (52.9 %) as sole carbon source and it also displayed activities of endochitinases, N-acetylhexosaminidase and production of hydrophobins class I and II.     

Bruton's Tyrosine Kinase (BTK) and Vav1 Contribute to Dectin1-Dependent Phagocytosis of Candida albicans in Macrophages

Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton''s Tyrosine Kinase (BTK) and Vav1. BTK and Vav1 are recruited to phagocytic cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid PI(3,4,5)P₃ and F-actin at the phagocytic cup, but not with diacylglycerol (DAG) which marks more mature phagosomal membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the subsequent recruitment of PKCε. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C. albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans, comparable to Dectin1-deficient cells. BTK- or Vav1-deficient mice are more susceptible to systemic C. albicans infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.