Membrane Chaperone SecDF Plays a Role in the Secretion of Listeria monocytogenes Major Virulence Factors

Listeria monocytogenes is a Gram-positive human intracellular pathogen that infects diverse mammalian cells. Upon invasion, L. monocytogenes secretes multiple virulence factors that target host cellular processes and promote infection. It has been presumed, but was not empirically established, that the Sec translocation system is the primary mediator of this secretion. Here, we validate an important role for SecDF, a component of the Sec system, in the secretion of several critical L. monocytogenes virulence factors. A ΔsecDF mutant is demonstrated to exhibit impaired membrane translocation of listeriolysin O (LLO), PlcA, PlcB, and ActA, factors that mediate L. monocytogenes phagosomal escape and spread from cell to cell. This impaired translocation was monitored by accumulation of the factors on the bacterial membrane and by reduced activity upon secretion. This defect in secretion is shown to be associated with a severe intracellular growth defect of the ΔsecDF mutant in macrophages and a less virulent phenotype in mice, despite normal growth in laboratory medium. We further show that SecDF is upregulated when the bacteria reside in macrophage phagosomes and that it is necessary for efficient phagosomal escape. Taken together, these data support the premise that SecDF plays a role as a chaperone that facilitates the translocation of L. monocytogenes virulence factors during infection.     

Learning to Attend to Threat Accelerates and Enhances Memory Consolidation

Practice on a procedural task involves within-session learning and between-session consolidation of learning, with the latter requiring a minimum of about four hours to evolve due to involvement of slower cellular processes. Learning to attend to threats is vital for survival and thus may involve faster memory consolidation than simple procedural learning. Here, we tested whether attention to threat modulates the time-course and magnitude of learning and memory consolidation effects associated with skill practice. All participants (N = 90) practiced in two sessions on a dot-probe task featuring pairs of neutral and angry faces followed by target probes which were to be discriminated as rapidly as possible. In the attend-threat training condition, targets always appeared at the angry face location, forming an association between threat and target location; target location was unrelated to valence in a control training condition. Within each attention training condition, duration of the between-session rest interval was varied to establish the time-course for emergence of consolidation effects. During the first practice session, we observed robust improvement in task performance (online, within-session gains), followed by saturation of learning. Both training conditions exhibited similar overall learning capacities, but performance in the attend-threat condition was characterized by a faster learning rate relative to control. Consistent with the memory consolidation hypothesis, between-session performance gains (delayed gains) were observed only following a rest interval. However, rest intervals of 1 and 24 hours yielded similar delayed gains, suggesting accelerated consolidation processes. Moreover, attend-threat training resulted in greater delayed gains compared to the control condition. Auxiliary analyses revealed that enhanced performance was retained over several months, and that training to attend to neutral faces resulted in effects similar to control. These results provide a novel demonstration of how attention to threat can accelerate and enhance memory consolidation effects associated with skill acquisition.     

Conveying Advanced Li‐ion Battery Materials into Practice The Impact of Electrode Slurry Preparation Skills

Li‐ion batteries (LIB's) are of the greatest practical utility for portable electronics and electric vehicles (EV's). LIB energy, power and cycle life performances depend on cathode and anode compositions and morphology, electrolyte composition and the overall cell design. Electrode morphology is influenced by the shape and size of the active material (AM), conductive additive (CA) particles, the polymeric binder properties, and also on the AM/CA/binder mass ratio. At the same time, it also substantially depends on the electrode preparation process. This process is usually comprised of mixing a solvent, a binder, AM and CA powders, and casting the resulting slurry onto a current collector foil followed by a drying process. Whereas the problems of electrode morphology and their influence on the LIB‐electrode performance always receive a proper attention, the influence of slurry properties and slurry preparation techniques on the electrode morphology is often overlooked or at least underrated. The present work summarizes the current state‐of‐the‐art in the field of LIB‐electrode precursor slurries preparation, characterized by multicomponent compounds and large variations in sizes and shapes of the solid components. Approaches to LIB‐electrode slurry preparation are outlined and discussed in the context of the ultimate LIB‐electrode morphology and performance.     

Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

1. Download high-res image (195KB)
2. Download full-size image

     

Recent advances in the structure and function of isopenicillin N synthase

Isopenicillin N synthase is a key enzyme in the biosynthesis of penicillin and cephalosporin antibiotics, catalyzing the oxidative ring closure of -(L--aminoadipoyl)-L-cysteinyl-D-valine to form isopenicillin N. Recent advances in our understanding of the unique chemistry of this enzyme have come through the combined application of spectroscopic, molecular genetic and crystallographic approaches and led to important new insights into the structure and function of this enzyme. Here we review new information on the nature of the endogenous ligands that constitute the ferrous iron active site, sequence evidence for a novel structural motif involved in iron binding in this and related non-heme iron dependent dioxygenases, crystal structure studies on the enzyme and its substrate complex and the impact of these and site-directed mutagenesis studies for unraveling the mechanism of the isopenicillin N synthase reaction.     

Cytokine polymorphisms and histologic expression in autopsy studies: contribution of TNF-alpha and TGF-beta 1 to the pathogenesis of autoimmune-associated congenital heart block

Although Abs to SSA/Ro-SSB/La are necessary for the development of congenital heart block (CHB), the low frequency suggests that fetal factors are contributory. Because CHB involves a cascade from inflammation to scarring, polymorphisms of the TNF-alpha promoter region and codons 10 and 25 of the TGF-beta gene were evaluated in 88 children (40 CHB, 17 rash, 31 unaffected siblings) and 74 mothers from the Research Registry for Neonatal Lupus (NL). Cytokine expression was assessed in autopsy material from two fetuses with CHB. Significantly increased frequency of the -308A (high-producer) allele of TNF-alpha was observed in all NL groups compared with controls. In contrast, the TGF-beta polymorphism Leu(10) (associated with increased fibrosis) was significantly higher in CHB children (genotypic frequency 60%, allelic frequency 78%) than unaffected offspring (genotypic frequency 29%, p = 0.016; allelic frequency 56%, p = 0.011) and controls, while there were no significant differences between controls and other NL groups. For the TGF-beta polymorphism, Arg(25), there were no significant differences between NL groups and controls. In fetal CHB hearts, protein expression of TGF-beta, but not TNF-alpha, was demonstrated in septal regions, extracellularly in the fibrous matrix, and intracellularly in macrophage infiltrates. Age-matched fetal hearts from voluntary terminations expressed neither cytokine. TNF-alpha may be one of several factors that amplify susceptibility; however, the genetic studies, backed by the histological data, more convincingly link TGF-beta to the pathogenesis of CHB. This profibrosing cytokine and its secretion/activation circuitry may provide a novel direction for evaluating fetal factors in the development of a robust animal model of CHB as well as therapeutic strategies in humans.     

The human islet amyloid polypeptide forms transient membrane-active prefibrillar assemblies

The formation of amyloid fibrils by the human islet amyloid polypeptide is associated with type II diabetes. While it was previously suggested that the formed fibrils are toxic to pancreatic beta-cells due to membrane permeation activity, more recent studies suggested that protofibrillar assemblies have significantly higher potency in permeating lipid bilayers. Here, we specifically studied the membrane interaction activity of soluble and insoluble islet amyloid polypeptide assemblies at high temporal resolution. A colorimetric analysis using lipid/polydiacetylene (PDA) biomimetic vesicles clearly demonstrated the transient formation of soluble assemblies that strongly interact with the lipid vesicles. A peak in the level of membrane binding of the soluble fraction, as reflected by the colorimetric assay, was observed after incubation for approximately 1 h, followed by a decrease in the level of membrane interaction of the assemblies. The transient nature of the membrane-active assemblies was independently confirmed by a fluorescence quenching assay. Ultrastructural analysis using transmission electron microscopy provided morphological evidence of prefibrillar assemblies, supported the transient existence of membrane interacting soluble species, and facilitated observation of the non-membrane-active filaments in the solution. Taken together, our results provide experimental evidence for the formation of transient soluble prefibrillar assemblies which are highly membrane-active. The implications of these observations are discussed in light of designed fibrillization inhibitors.     

Speciation and the establishment of zonation in an intertidal barnacle: specific settlement vs. selection

The tropical barnacle Tetraclita forms a belt on hard substrates in the intertidal zone of the Red Sea. Based on morphological data, three distinct species were suggested to exist, occupying different vertical levels - T. barnesorum, T. rufotincta and T. achituvi. In this study we used molecular (12S mitochondrial ribosomal DNA) and ecological data to examine whether this morphological variability reflects genetic differences, or is a result of environmental factors. Adults and spats, collected from settlement plates, were censused and screened genotypically using single strand conformation polymorphism (SSCP) analysis, and settlement dynamics was recorded. We provide evidence for the existence of only two distinct species, and point out both phenotypic plasticity and convergence within and between the proposed species. Cyprids of T. achituvi settle specifically at the lower part of the Tetraclita belt, and feature one phenotype. In contrast, T. rufotincta, occupying the upper and middle portions of the Tetraclita belt, settles throughout the range, shows phenotypic plasticity (three variants), and presumably undergoes selection at the lower part. Thus, the vertical zonation of Tetraclita is produced by the combination of pre-settlement and post-settlement factors, in T. achituvi and T. rufotincta, respectively. The examined system may offer a model in which to study the mechanisms underlying sympatric speciation.