High-pressure treatment of polytene chromosomes improves structural resolution

The exceptional cytology provided by polytene chromosomes has made Drosophila melanogaster a premier model for chromosome studies, but full exploitation of polytene cytology is impeded by the difficulty in preparing high-quality chromosome spreads. Here we describe use of high pressure to produce formaldehyde-fixed chromosome spreads, which upon light-microscopy examination reveal structural detail previously observed only in electron microscopy preparations. We demonstrate applications to immunofluorescence and in situ hybridization.     

The studies on substrate,product and inhibitor binding to a wild-type and neuronopathic form of human acid-β-glucosidase

Gaucher disease is a lysosomal storage disorder caused by deficiency of human acid β-glucosidase. Recent x-ray structural elucidation of the enzyme alone and in the presence of its inhibitor was done, which provided an excellent template for further studies on the binding of substrate, product and inhibitor. To draw correlations between the clinical manifestation of the disease driven by point mutations, L444P and L444R, and the placement and function of putative S-binding sites, the presented theoretical studies were undertaken, which comprised of molecular dynamics and molecular docking methods. The obtained results indicate the D443 and D445 residues as extremely important for physiological functionality of an enzyme. They also show, although indirectly, that binding of the substrate is influenced by an interplay of E235 and E334 residues, constituting putative substrate binding site, and the region flanked by D435 and D445 residues. Figure The binding of an arbitrarily chosen structure of glucosylceramide (A), conduritol-β-epoxide (B), glucose (C) to the active site D443/D445 (A1, B1, C1) and E320/E340 (A2, B2, C2) of the wild-type structure of human acid-β-glucosidase. A1, B1, C1 blue mask represents the residues D443-D445; red mask represents the residue D444; A2, B2, C2 blue mask represents loop1 (Ser³⁴⁵-Glu³⁴⁹) and loop2 (Val³⁹⁴-Asp³⁹⁹), whereas red mask the residues E235 and 340     

Leaf functional traits and monodominance in Southern Amazonia tropical forests

Tropical monodominant forests are rare communities with low tree species diversity. Species monodominance is not the product of a single mechanism, but the result of a set of not yet fully understood integrated ecological factors acting together. We compared populations of Brosimum rubescens in monodominant and mixed forests in Southern Amazonia to test whether leaf functional traits are ecological factors related to monodominance. Individuals of B. rubescens in the mixed forest invest in conservative strategies, while those in the monodominant forest invest in acquisitive strategies. Leaf functional traits, such as petiole length and adaxial cuticle thickness, could be associated with the monodominance of B. rubescens. Our study highlights for the first time the power of integrating leaf functional traits as a component of the set of ecological conditions to explain species monodominance. B. rubescens showed different functional strategies to establish and maintain its population in different forests, which makes it a strong competitor for resources, such as water and light, through variation in its leaf functional traits. We also suggest that such high plasticity can be an important condition for the persistence of the species over time.

     

Tranexamic acid suppresses the release of mitochondrial DNA,protects the endothelial monolayer and enhances oxidative phosphorylation

Damage-associated molecular patterns, including mitochondrial DNA (mtDNA) are released during hemorrhage resulting in the development of endotheliopathy. Tranexamic acid (TXA), an antifibrinolytic drug used in hemorrhaging patients, enhances their survival despite the lack of a comprehensive understanding of its cellular mechanisms of action. The present study is aimed to elucidate these mechanisms, with a focus on mitochondria. We found that TXA inhibits the release of endogenous mtDNA from granulocytes and endothelial cells. Furthermore, TXA attenuates the loss of the endothelial monolayer integrity induced by exogenous mtDNA. Using the Seahorse XF technology, it was demonstrated that TXA strongly stimulates mitochondrial respiration. Studies using Mitotracker dye, cells derived from mito-QC mice, and the ActivSignal IPAD assay, indicate that TXA stimulates biogenesis of mitochondria and inhibits mitophagy. These findings open the potential for improvement of the strategies of TXA applications in trauma patients and the development of more efficient TXA derivatives.     

Factors affecting yield and survival of cells when suspensions are subjected to centrifugation

The goals of the centrifugation of cell suspensions are to obtain the maximum yield of cells with minimum adverse effects of centrifugation. In the case of mechanically sensitive cells such as mouse sperm, the two goals are somewhat contradictory in that g-forces sufficient to achieve high yields are damaging, and g-forces that yield high viability produce low yields. This paper mathematically analyzes the factors contributing to each goal. The total yield of pelleted cells is determined by the sedimentation rate governed by Stokes' Law, and depends on the relative centrifugal force, centrifugation time, size and shape of the cells, density of the cells and medium, viscosity of the medium, and the length of the column of suspension. Because in the situation analyzed the column is short relative to the rotor radius, the analysis considers the centrifugal field to be quasi-homogeneous. The assumption is that cells are not damaged during sedimentation, but that they become injured at an exponential rate once they are pelleted, a rate that will depend on the specific cell type. The behavior is modeled by the solution of coupled differential equations. The predictions of the analysis are in good agreement with experimental data on the centrifugation of mouse sperm.