Rad51-Rad52 Mediated Maintenance of Centromeric Chromatin in Candida albicans

Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromere-specific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI) proximal to an early replicating centromere (CEN7) in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR) proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-A(CaCse4) levels at centromeres, as determined by chromatin immunoprecipitation (ChIP) experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENP-A(CaCse4) in vivo. Thus, the HR proteins Rad51 and Rad52 epigenetically maintain centromere functioning by regulating CENP-A(CaCse4) levels at the programmed stall sites of early replicating centromeres.

A new microscopic insight into membrane penetration and reorganization by PETIM dendrimers

Dendrimers are highly branched polymeric nanoparticles whose structure and topology, largely, have determined their efficacy in a wide range of studies performed so far. An area of immense interest is their potential as drug and gene delivery vectors. Realizing this potential, depending on the nature of cell surface-dendrimer interactions, here we report controlled model membrane penetration and reorganization, using a model supported lipid bilayer and poly(ether imine) (PETIM) dendrimers of two generations. By systematically varying the areal density of the lipid bilayers, we provide a microscopic insight, through a combination of high resolution scattering, atomic force microscopy and atomistic molecular dynamics simulations, into the mechanism of PETIM dendrimer membrane penetration, pore formation and membrane re-organization induced by such interactions. Our work represents the first systematic observation of a regular barrel-like membrane spanning pore formation by dendrimers, tunable through lipid bilayer packing, without membrane disruption.

Structural and functional analysis of two universal stress proteins YdaA and YnaF from Salmonella typhimurium: possible roles in microbial stress tolerance

In many organisms ``Universal Stress Proteins'' CUSPS) are induced in response to a variety of environmental stresses. Here we report the structures of two USPs, YnaF and YdaA from Salmonella typhimurium determined at 1.8 angstrom and 2.4 angstrom resolutions, respectively. YnaF consists of a single USP domain and forms a tetrameric organization stabilized by interactions mediated through chloride ions. YdaA is a larger protein consisting of two tandem USP domains. Two protomers of YdaA associate to form a structure similar to the YnaF tetramer. YdaA showed ATPase activity and an ATP binding motif G-2X-G-9X-G(S/T/N) was found in its C-terminal domain. The residues corresponding to this motif were not conserved in YnaF although YnaF could bind ATP. However, unlike YdaA, YnaF did not hydrolyse ATP in vitro. Disruption of interactions mediated through chloride ions by selected mutations converted YnaF into an ATPase. Residues that might be important for ATP hydrolysis could be identified by comparing the active sites of native and mutant structures. Only the C-terminal domain of YdaA appears to be involved in ATP hydrolysis. The structurally similar N-terminal domain was found to bind a zinc ion near the segment equivalent to the phosphate binding loop of the C-terminal domain. Mass spectrometric analysis showed that YdaA might bind a ligand of approximate molecular weight 800 daltons. Structural comparisons suggest that the ligand, probably related to an intermediate in lipid A biosynthesis, might bind at a site close to the zinc ion. Therefore, the N-terminal domain of YdaA binds zinc and might play a role in lipid metabolism. Thus, USPs appear to perform several distinct functions such as ATP hydrolysis, altering membrane properties and chloride sensing. (C) 2015 Elsevier Inc. All rights reserved.

Effect of Particle Concentration on Shape Deformation and Secondary Atomization Characteristics of a Burning Nanotitania Dispersion Droplet

Secondary atomization characteristics of burning bicomponent (ethanol-water) droplets containing titania nanoparticles (NPs) in dilute (0.5% and 1 wt.%) and dense concentrations (5% and 7.5 wt.%) are studied experimentally at atmospheric pressure under normal gravity. It is observed that both types of nanofuel droplets undergo distinct modes of secondary breakup, which are primarily responsible for transporting particles from the droplet domain to the flame zone. For dilute nanosuspensions, disruptive response is characterized by low intensity atomization modes that cause small-scale localized flame distortion. In contrast, the disruption behavior at dense concentrations is governed by high intensity bubble ejections, which result in severe disruption of the flame envelope.

Conductivity studies on microwave synthesized glasses

Conductivity measurements have been made on x V O-2(5) - (100-x) 0.5 Na2O + 0.5 B2O3] (where 10 a parts per thousand currency sign x a parts per thousand currency sign 50) glasses prepared by using microwave method. DC conductivity (sigma) measurements exhibit temperature-and compositional-dependent trends. It has been found that conductivity in these glasses changes from the predominantly `ionic' to predominantly `electronic' depending upon the chemical composition. The dc conductivity passes through a deep minimum, which is attributed to network disruption. Also, this nonlinear variation in sigma (dc) and activation energy can be interpreted using ion-polaron correlation effect. Electron paramagnetic resonance (EPR) and impedance spectroscopic techniques have been used to elucidate the nature of conduction mechanism. The EPR spectra reveals, in least modified (25 Na2O mol%) glasses, conduction is due to the transfer of electrons via aliovalent vanadium sites, while in highly modified (45 Na2O mol%) glasses Na+ ion transport dominates the electrical conduction. For highly modified glasses, frequency-dependent conductivity has been analysed using electrical modulus formalism and the observations have been discussed.

Interplay of electrostatics and lipid packing determines the binding of charged polymer coated nanoparticles to model membranes

Understanding of nanoparticle-membrane interactions is useful for various applications of nanoparticles like drug delivery and imaging. Here we report on the studies of interaction between hydrophilic charged polymer coated semiconductor quantum dot nanoparticles with model lipid membranes. Atomic force microscopy and X-ray reflectivity measurements suggest that cationic nanoparticles bind and penetrate bilayers of zwitterionic lipids. Penetration and binding depend on the extent of lipid packing and result in the disruption of the lipid bilayer accompanied by enhanced lipid diffusion. On the other hand, anionic nanoparticles show minimal membrane binding although, curiously, their interaction leads to reduction in lipid diffusivity. It is suggested that the enhanced binding of cationic QDs at higher lipid packing can be understood in terms of the effective surface potential of the bilayers which is tunable through membrane lipid packing. Our results bring forth the subtle interplay of membrane lipid packing and electrostatics which determine nanoparticle binding and penetration of model membranes with further implications for real cell membranes.

Modelling staphylococcal pneumonia in a human 3D lung tissue model system delineates toxin-mediated pathology

Staphylococcus aureus necrotizing pneumonia is recognized as a toxin-mediated disease, yet the tissue-destructive events remain elusive, partly as a result of lack of mechanistic studies in human lung tissue. In this study, a three-dimensional (3D) tissue model composed of human lung epithelial cells and fibroblasts was used to delineate the role of specific staphylococcal exotoxins in tissue pathology associated with severe pneumonia. To this end, the models were exposed to the mixture of exotoxins produced by S. aureus strains isolated from patients with varying severity of lung infection, namely necrotizing pneumonia or lung empyema, or to purified toxins. The necrotizing pneumonia strains secreted high levels of alpha-toxin and Panton-Valentine leukocidin (PVL), and triggered high cytotoxicity, inflammation, necrosis and loss of E-cadherin from the lung epithelium. In contrast, the lung empyema strain produced moderate levels of PVL, but negligible amounts of alpha-toxin, and triggered limited tissue damage. alpha-toxin had a direct damaging effect on the epithelium, as verified using toxin-deficient mutants and pure alpha-toxin. Moreover, PVL contributed to pathology through the lysis of neutrophils. A combination of alpha-toxin and PVL resulted in the most severe epithelial injury. In addition, toxin-induced release of pro-inflammatory mediators from lung tissue models resulted in enhanced neutrophil migration. Using a collection of 31 strains from patients with staphylococcal pneumonia revealed that strains producing high levels of alpha-toxin and PVL were cytotoxic and associated with fatal outcome. Also, the strains that produced the highest toxin levels induced significantly greater epithelial disruption. Of importance, toxin-mediated lung epithelium destruction could be inhibited by polyspecific intravenous immunoglobulin containing antibodies against alpha-toxin and PVL. This study introduces a novel model system for study of staphylococcal pneumonia in a human setting. The results reveal that the combination and levels of alpha-toxin and PVL correlate with tissue pathology and clinical outcome associated with pneumonia.

Neonatal exposure to 17 beta-estradiol down-regulates the expression of synaptogenesis related genes in selected brain regions of adult female rats

Aims: Administration of estradiol or compounds with estrogenic activity to newborn female rats results in irreversible masculinization as well as defeminization in the brain and the animals exhibit altered reproductive behavior as adults. The cellular and molecular mechanism involved in inducing the irreversible changes is largely unknown. In the present study, we have monitored the changes in the expression of selected synaptogenesis related genes in the sexually dimorphic brain regions such as POA, hypothalamus and pituitary following 17 beta-estradiol administration to neonatal female rats. Main methods: Female Wistar rats which were administered 17 beta-estradiol on day 2 and 3 after birth were sacrificed 120 days later and the expression levels of genes implicated in synaptogenesis were monitored by semi-quantitative reverse transcription PCR. Since estradiol induced up-regulation of COX-2 in POA is a marker for estradiol induced masculinization as well as defeminization, in the present study only animals in which the increase in expression of COX-2 gene was observed in POA were included in the study. Key findings: Down-regulation of genes such as NMDA-2B, NETRIN-1, BDNF, MT-5 MMP and TNF-alpha was observed in the pre-optic area of neonatally E2 treated female rat brain but not in hypothalamus and pituitary compared to the vehicle- treated controls as assessed by RT-PCR and Western blot analysis. Significance: Our results suggest a possibility that down-regulation of genes associated with synaptogenesis in POA, may be resulting in disruption of the cyclical regulation of hormone secretion by pituitary the consequence of which could be infertility and altered reproductive behavior. (C) 2015 Elsevier Inc. All rights reserved.

Designer porous antibacterial membranes derived from thermally induced phase separation of PS/PVME blends decorated with an electrospun nanofiber scaffold

We report the development of porous membranes by thermally induced phase separation of a PS/PVME (polystyrene/polyvinylmethyl ether]) blend, which is a typical LCST mixture. The morphology of the membrane after etching out the PVME phase was characterized by scanning electron microscopy. To give the membrane an antibacterial surface, polystyrene (PS) and polyvinyl(methyl ether)]-alt-maleic anhydride (PVME-MAH) with silver nanoparticles (nAg) were electrospun on the membrane surface. Pure water flux was evaluated by using a cross-flow membrane setup. The microgrooved fibers changed the flux across the membrane depending on the surface properties. The antibacterial properties of the membrane were confirmed by the reduction in the colony count of E. coli. The SEM images show the disruption of the bacterial cell membrane and the antibacterial mechanism was discussed.

Case studies on the formation of chalcogenide self-assembled monolayers on surfaces and dissociative processes

This report examines the assembly of chalcogenide organic molecules on various surfaces, focusing on cases when chemisorption is accompanied by carbon-chalcogen atom-bond scission. In the case of alkane and benzyl chalcogenides, this induces formation of a chalcogenized interface layer. This process can occur during the initial stages of adsorption and then, after passivation of the surface, molecular adsorption can proceed. The characteristics of the chalcogenized interface layer can be significantly different from the metal layer and can affect various properties such as electron conduction. For chalcogenophenes, the carbon-chalcogen atombond breaking can lead to opening of the ring and adsorption of an alkene chalcogenide. Such a disruption of the pi-electron system affects charge transport along the chains. Awareness about these effects is of importance from the point of view of molecular electronics. We discuss some recent studies based on X-ray photoelectron spectroscopy that shed light on these aspects for a series of such organic molecules.

Extract of Vernonia condensata, Inhibits Tumor Progression and Improves Survival of Tumor-allograft Bearing Mouse

Medicinal plants are considered as one of the ideal sources for cancer therapy due to their bioactive contents and low toxicity to humans. Vernonia genus is one of the common medicinal plants, which has wide spread usage in food and medicine. However, there are limited studies to explore its anticancer properties. In the current study, we have used Vernonia condensata, to explore its anticancer activity using various approaches. Here, we show that extract prepared from Vernonia condensata (VCE) exhibits cytotoxic properties against various cancer cells in a dose- and time-dependent manner. Interestingly, when treated with VCE, there was no significant cytotoxicity in peripheral blood mononuclear cells (PBMCs). Flow cytometry analysis revealed that although VCE induced cell death, arrest was not observed. VCE treatment led to disruption of mitochondrial membrane potential in a concentration dependent manner resulting in activation of apoptosis culminating in cell death. Immunoblotting studies revealed that VCE activated intrinsic pathway of apoptosis. More importantly, VCE treatment resulted in tumor regression leading to significant enhancement in life span in treated mice, without showing any detectable side effects. Therefore, for the first time our study reveals the potential of extract from Vernonia condensata to be used as an anticancer agent.

Atomistic Simulation of Stacked Nucleosome Core Particles: Tail Bridging, the H4 Tail, and Effect of Hydrophobic Forces

We report the first atomistic simulation of two stacked nucleosome core particles (NCPs), with an aim to understand, in molecular detail, how they interact, the effect of salt concentration, and how different histone tails contribute to their interaction, with a special emphasis on the H4 tail, known to have the largest stabilizing effect on the NCP-NCP interaction. We do not observe specific K16-mediated interaction between the H4 tail and the H2A-H2B acidic patch, in contrast with the findings from crystallographic studies, but find that the stacking was stable even in the absence of this interaction. We perform simulations with the H4 tail (partially/completely) removed and find that the region between LYS-16 and LYS-20 of the H4 tail holds special importance in mediating the inter-NCP interaction. Performing similar tail-clipped simulations with the H3 tail removed, we compare the roles of the H3 and H4 tails in maintaining the stacking. We discuss the relevance of our simulation results to the bilayer and other liquid-crystalline phases exhibited by NCPs in vitro and, through an analysis of the histone-histone interface, identify the interactions that could possibly stabilize the inter-NCP interaction in these columnar mesophases. Through the mechanical disruption of the stacked nucleosome system using steered molecular dynamics, we quantify the strength of inter-NCP stacking in the presence and absence of salt. We disrupt the stacking at some specific sites of internucleosomal tail-DNA contact and perform a comparative quantification of the binding strengths of various tails in stabilizing the stacking. We also examine how hydrophobic interactions may contribute to the overall stability of the stacking and find a marked difference in the role of hydrophobic forces as compared with electrostatic forces in determining the stability of the stacked nucleosome system.

Unimpeded permeation of water through biocidal graphene oxide sheets anchored on to 3D porous polyolefinic membranes

3D porous membranes were developed by etching one of the phases (here PEO, polyethylene oxide) from melt-mixed PE/PEO binary blends. Herein, we have systematically discussed the development of these membranes using X-ray micro-computed tomography. The 3D tomograms of the extruded strands and hot-pressed samples revealed a clear picture as to how the morphology develops and coarsens over a function of time during post-processing operations like compression molding. The coarsening of PE/PEO blends was traced using X-ray micro-computed tomography and scanning electron microscopy (SEM) of annealed blends at different times. It is now understood from X-ray micro-computed tomography that by the addition of a compatibilizer (here lightly maleated PE), a stable morphology can be visualized in 3D. In order to anchor biocidal graphene oxide sheets onto these 3D porous membranes, the PE membranes were chemically modified with acid/ethylene diamine treatment to anchor the GO sheets which were further confirmed by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and surface Raman mapping. The transport properties through the membrane clearly reveal unimpeded permeation of water which suggests that anchoring GO on to the membranes does not clog the pores. Antibacterial studies through the direct contact of bacteria with GO anchored PE membranes resulted in 99% of bacterial inactivation. The possible bacterial inactivation through physical disruption of the bacterial cell wall and/or reactive oxygen species (ROS) is discussed herein. Thus this study opens new avenues in designing polyolefin based antibacterial 3D porous membranes for water purification.

Perturbation of the stability of amyloid fibrils through alteration of electrostatic interactions.

The self-assembly of proteins and peptides into polymeric amyloid fibrils is a process that has important implications ranging from the understanding of protein misfolding disorders to the discovery of novel nanobiomaterials. In this study, we probe the stability of fibrils prepared at pH 2.0 and composed of the protein insulin by manipulating electrostatic interactions within the fibril architecture. We demonstrate that strong electrostatic repulsion is sufficient to disrupt the hydrogen-bonded, cross-β network that links insulin molecules and ultimately results in fibril dissociation. The extent of this dissociation correlates well with predictions for colloidal models considering the net global charge of the polypeptide chain, although the kinetics of the process is regulated by the charge state of a single amino acid. We found the fibrils to be maximally stable under their formation conditions. Partial disruption of the cross-β network under conditions where the fibrils remain intact leads to a reduction in their stability. Together, these results support the contention that a major determinant of amyloid stability stems from the interactions in the structured core, and show how the control of electrostatic interactions can be used to characterize the factors that modulate fibril stability.

Analysis of the legislative framework in the Brazilian policymaking process

Analisa os aspectos da estrutura política brasileira que contribuem para falhas nos processos decisórios e ineficácia nos resultados finais de políticas de governo, com ênfase nos problemas institucionais do poder legislativo. Aborda os seguintes tópicos: fragmentação do sistema partidário, infidelidade partidária, modelo de federalismo, preenchimento de cargos públicos, larga utilização de medidas provisórias, disfunções do processo legislativo.