Silencing the crowd: high-throughput functional genomics in Magnaporthe oryzae

A new high-throughput RNA-silencing system has been developed for use in the rice blast fungus Magnaporthe oryzae , allowing rapid generation of transformants in which individual genes have been silenced. Development of this system will allow large-scale functional analysis of genes in the fungus to define the cellular processes required for plant infection and disease symptoms. Functional analysis of 37 genes predicted to be involved in calcium signalling was carried out by RNA silencing to validate the new strategy and has provided new insight into the role of calcium-mediated signal transduction in plant pathogenic fungi.     

Synthesis of new 2-C-(2,3:5,6-di-O-isopropylidene)-beta-D-mannofuranosyldithioacetate derivatives

Various new C-glycosides have been synthesized through the thioacylation reaction of different amines by a 2-C-mannofuranosyldithioacetate. Amino acids, di- and polyamines (putresceine, spermine, spermidine) and one amino alcohol were in particular used to generate glycothiopeptidic or precursors of "bola" structures.     

Analysis of yeasts derived from natural fermentation in a Tokaj winery

The diversity of yeast flora was investigated in a spontaneously fermenting sweet white wine in a Tokaj winery. The non-Saccharomyces yeasts dominating the first phase of fermentation were soon replaced by a heterogeneous Saccharomycespopulation, which then became dominated by Saccharomyces bayanus. Three Saccharomyces sensu stricto strains isolated from various phases of fermentation were tested for genetic stability, optimum growth temperature, tolerance to sulphur dioxide, copper and ethanol as well as for the ability to produce hydrogen sulphide and various secondary metabolites known to affect the organoleptic properties of wines. The analysis of the single-spore cultures derived from spores of dissected asci revealed high stability of electrophoretic karyotypes and various degrees of heterozygosity for mating-types, the fermentation of galactose and the production of metabolic by-products. The production levels of the by-products did not segregate in a 2:2 fashion, suggesting that the synthesis of these compounds is under polygenic control.     

A comparative study on the beneficial effects of garlic (Allium sativum Linn), amla (Emblica Officinalis Gaertn)and onion (Allium cepa Linn) on the hyperlipidemia induced by butter fat and beef fat in rats

Three months feeding of butter fat (BUF) and beef (BF) separately as components of diet at a level of 21% by weight for albino rats, significantly raised their serum and tissue lipids, lipid peroxidation and activities of certain enzymes. BUF was found to be more atherogenic than BF. On incorporation of 5% garlic, amla or onion separately in the above diets, each of them ameliorated the deleterious effects of the animal fats. A higher hyperlipidemic effect of BUF as compared to that of BF may be due to the fact that the ratio of unsaturated to saturated fats is lower for the former (0.56) than for the latter (0.75) and also that the former is richer in cholesterol content than the latter. The order of the curative effects of the vegetables are garlic>amla>onion. The better hypolipidemic effects and correction of elevated levels of certain enzymes shown by garlic and amla may be due to the facts that they contain comparatively better active principles than that found in onions.     

Assembling a xylanase–lichenase chimera through all-atom molecular dynamics simulations

Multifunctional enzyme engineering can improve enzyme cocktails for emerging biofuel technology. Molecular dynamics through structure-based models (SB) is an effective tool for assessing the tridimensional arrangement of chimeric enzymes as well as for inferring the functional practicability before experimental validation. This study describes the computational design of a bifunctional xylanase–lichenase chimera (XylLich) using the xynA and bglS genes from Bacillus subtilis. In silico analysis of the average solvent accessible surface area (SAS) and the root mean square fluctuation (RMSF) predicted a fully functional chimera, with minor fluctuations and variations along the polypeptide chains. Afterwards, the chimeric enzyme was built by fusing the xynA and bglS genes. XylLich was evaluated through small-angle X-ray scattering (SAXS) experiments, resulting in scattering curves with a very accurate fit to the theoretical protein model. The chimera preserved the biochemical characteristics of the parental enzymes, with the exception of a slight variation in the temperature of operation and the catalytic efficiency (k_cat/K_m). The absence of substantial shifts in the catalytic mode of operation was also verified. Furthermore, the production of chimeric enzymes could be more profitable than producing a single enzyme separately, based on comparing the recombinant protein production yield and the hydrolytic activity achieved for XylLich with that of the parental enzymes.     

A comparison between two brine shrimp assays to detect in vitro cytotoxicity in marine natural products

Background  

The brine shrimp lethality assay is considered a useful tool for preliminary assessment of toxicity. It has also been suggested for screening pharmacological activities in plant extracts. However, we think that it is necessary to evaluate the suitability of the brine shrimp methods before they are used as a general bio-assay to test natural marine products for pharmacological activity.     

Inhibition of cholesterol transport impairs Cav-1 trafficking and small extracellular vesicles secretion,promoting amphisome formation in melanoma cells

Caveolin-1 (Cav-1) is a fundamental constituent of caveolae, whose functionality and structure are strictly dependent on cholesterol. In this work the U18666A inhibitor was used to study the role of cholesterol transport in the endosomal degradative-secretory system in a metastatic human melanoma cell line (WM266-4). We found that U18666A induces a shift of Cav-1 from the plasma membrane to the endolysosomal compartment, which is involved, through Multi Vesicular Bodies (MVBs), in the formation and release of small extracellular vesicles (sEVs). Moreover, this inhibitor induces an increase in the production of sEVs with chemical–physical characteristics similar to control sEVs but with a different protein composition (lower expression of Cav-1 and increase of LC3II) and reduced transfer capacity on target cells. Furthermore, we determined that U18666A affects mitochondrial function and also cancer cell aggressive features, such as migration and invasion. Taken together, these results indicate that the blockage of cholesterol transport, determining the internalization of Cav-1, may modify sEVs secretory pathways through an increased fusion between autophagosomes and MVBs to form amphisome, which in turn fuses with the plasma membrane releasing a heterogeneous population of sEVs to maintain homeostasis and ensure correct cellular functionality.