The Rab-binding profiles of bacterial virulence factors during infection

Legionella pneumophila, the causative agent of Legionnaire's disease, uses its type IV secretion system to translocate over 300 effector proteins into host cells. These effectors subvert host cell signaling pathways to ensure bacterial proliferation. Despite their importance for pathogenesis, the roles of most of the effectors are yet to be characterized. Key to understanding the function of effectors is the identification of host proteins they bind during infection. We previously developed a novel tandem-affinity purification (TAP) approach using hexahistidine and BirA-specific biotinylation tags for isolating translocated effector complexes from infected cells whose composition were subsequently deciphered by mass spectrometry. Here we further advanced the workflow for the TAP approach and determined the infection-dependent interactomes of the effectors SidM and LidA, which were previously reported to promiscuously bind multiple Rab GTPases in vitro. In this study we defined a stringent subset of Rab GTPases targeted by SidM and LidA during infection, comprising of Rab1A, 1B, 6, and 10; in addition, LidA targets Rab14 and 18. Taken together, this study illustrates the power of this approach to profile the intracellular interactomes of bacterial effectors during infection

Magnetization dynamics of magnetic domain wall imprinted magnetic films

The influence of micromagnetic objects on the dynamic magnetic excitation in magnetic thin films is studied by imprinting periodic domain wall patterns through selective ion irradiation in exchange biased Ni81Fe 19/IrMn structures. For high domain wall densities an increased precessional frequency is achieved. The zero field resonance of the domain wall state hereby depends directly on the stripe period, showing a pronounced increase with decrease of domain wall spacing. With the abrupt annihilation of magnetic domain walls with an applied bias field a jump-like decrease in precessional frequency takes place. The experimental data and micromagnetic simulations prove that the characteristic collective dynamic mode for the domain wall configurations is attributed to strongly coupled tilted magnetization structure. This is evidenced by an overlapping Néel wall structure for the narrowly spaced imprinted antiparallel unidirectional anisotropy state. The controlled introduction of high density frozen-in micromagnetic objects is a novel way to control the dynamic magnetic properties of continuous magnetic thin films

Doubly resonant optical nanoantenna arrays for polarization resolved measurements of surface-enhanced Raman scattering

We report that rhomb-shaped metal nanoantenna arrays support multiple plasmonic resonances, making them favorable bio-sensing substrates. Besides the two localized plasmonic dipole modes associated with the two principle axes of the rhombi, the sample supports an additional grating-induced surface plasmon polariton resonance. The plasmonic properties of all modes are carefully studied by far-field measurements together with numerical and analytical calculations. The sample is then applied to surface-enhanced Raman scattering measurements. It is shown to be highly efficient since two plasmonic resonances of the structure were simultaneously tuned to coincide with the excitation and the emission wave- length in the SERS experiment. The analysis is completed by measuring the impact of the polarization angle on the SERS signal.Comment: 13 pages, 5 figure

Characterization and quantification of postharvest losses of apple fruit stored under commercial conditions.

The objectives of this study were to characterize and quantify postharvest losses of apples under commercial conditions in Santa Catarina state, Brazil. Two experiments were conducted using ?Gala? and ?Fuji? apples. The first experiment was to characterize and quantify the most important causes of loss of fruit treated or not treated with 1-methylcyclopropene (1-MCP) then held in controlled atmosphere (CA) storage. This experiment was conducted in commercial storage facilities from 2007 to 2010. In each year, 10 samples of ≈380 kg each for ?Gala? and 400 kg each for ?Fuji? were collected from bins of commercially harvested fruit from each of 15 ?Gala? and 17 ?Fuji? orchards. Half of the samples from each orchard were treated with 1-MCP at harvest. Fruit were stored in CA, at 0.7 °C, for 150 to 300 days. After storage, one subsample of 100 disorder-free apples were selected from each sample and held at 22 °C for 7 days to simulate shelf-life conditions. The fruit were analyzed after CA storage and shelf life for the incidence of disorders. The second experiment was conducted in 2011 to identify the main fungi causing decay during storage. In this study, apples were stored in 10 commercial CA storage rooms at 0.7 °C for 180 to 240 days. After storage, fruit with decay symptoms were collected at the commercial sorting line. A total of 10 samples of 100 decayed apples were taken throughout the sorting period for each cultivar and storage room. The fungal decays were identified by visual symptoms on each fruit. Total apple losses during storage varied from 3.9% to 12.1% for ?Gala? and 6.6% to 8.4% for ?Fuji?, depending on the year and 1-MCP treatment. During storage, deterioration caused by fungal decay was ≈60% and 80% of total losses for ?Gala? and ?Fuji?, respectively. During shelf life, additional losses caused by fungal decay ranged from 8.4% to 17.6% for ?Gala? and 12.4% to 27.2% for ?Fuji?, depending on the year. Senescent breakdown and superficial scald were the major physiological disorders. 1-MCP treatment had no effect on losses due to decay. Bull?s-eye rot, blue mold, gray mold, and alternaria rot were the most prevalent fungal decay symptoms, accounting for 52%, 27%, 9% and 10% of ?Gala? losses and 42%, 25%, 18% and 5% of ?Fuji? losses, respectively. Sources of variability for losses among years and orchards is discussed

Regional actorness and interregional relations:ASEAN, the EU and Mercosur

The European Union (EU) has a long tradition of interregional dialogue mechanisms with other regional organisations and is using these relations to project its own model of institutionalised actorness. This is partly motivated by the emerging actorness of the EU itself, which benefits from fostering capable regional counterparts in other parts of the world. This article advances the argument that actorness, which we conceptualise in terms of institutions, recognition and identity, is a relational concept, dependent on context and perception. Taking the Association of Southeast Asian Nations (ASEAN) and the Common Market of the South (Mercosur) and their relations with the EU as case studies, this article demonstrates that the actorness capabilities of all three organisations have been enhanced as result of ASEAN-EU and Mercosur-EU relations. However, there are clear limits to the development of the three components of regional actorness and to the interregional relations themselves. These limits stem both from the type of interregionalism at play and from the different regional models the actors incorporate. While there is evidence of institutional enhancement in ASEAN and Mercosur, these formal changes have been grafted on top of firmly entrenched normative underpinnings. Within the regional organisations, interactions with the EU generate centrifugal forces concerning the model to pursue, thus limiting their institutional cohesion and capacity. In addition, group-to-group relations have reinforced ASEAN and Mercosur identities in contrast to the EU. The formation of such differences has narrowed the scope of EU interregionalism despite the initial success of improved regional actorness

Functional characterization of a melon alcohol acyl-transferase gene family involved in the biosynthesis of ester volatiles. Identification of the crucial role of a threonine residue for enzyme activity

Volatile esters, a major class of compounds contributing to the aroma of many fruit, are synthesized by alcohol acyl-transferases (AAT). We demonstrate here that, in Charentais melon (Cucumis melo var. cantalupensis), AAT are encoded by a gene family of at least four members with amino acid identity ranging from 84% (Cm-AAT1/Cm-AAT2) and 58% (Cm-AAT1/Cm-AAT3) to only 22% (Cm-AAT1/Cm-AAT4). All encoded proteins, except Cm-AAT2, were enzymatically active upon expression in yeast and show differential substrate preferences. Cm-AAT1 protein produces a wide range of short and long-chain acyl esters but has strong preference for the formation of E-2-hexenyl acetate and hexyl hexanoate. Cm-AAT3 also accepts a wide range of substrates but with very strong preference for producing benzyl acetate. Cm-AAT4 is almost exclusively devoted to the formation of acetates, with strong preference for cinnamoyl acetate. Site directed mutagenesis demonstrated that the failure of Cm-AAT2 to produce volatile esters is related to the presence of a 268-alanine residue instead of threonine as in all active AAT proteins. Mutating 268-A into 268-T of Cm-AAT2 restored enzyme activity, while mutating 268-T into 268-A abolished activity of Cm-AAT1. Activities of all three proteins measured with the prefered substrates sharply increase during fruit ripening. The expression of all Cm-AAT genes is up-regulated during ripening and inhibited in antisense ACC oxidase melons and in fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. The data presented in this work suggest that the multiplicity of AAT genes accounts for the great diversity of esters formed in melon

Freiburg RNA tools: a central online resource for RNA-focused research and teaching.

The Freiburg RNA tools webserver is a well established online resource for RNA-focused research. It provides a unified user interface and comprehensive result visualization for efficient command line tools. The webserver includes RNA-RNA interaction prediction (IntaRNA, CopraRNA, metaMIR), sRNA homology search (GLASSgo), sequence-structure alignments (LocARNA, MARNA, CARNA, ExpaRNA), CRISPR repeat classification (CRISPRmap), sequence design (antaRNA, INFO-RNA, SECISDesign), structure aberration evaluation of point mutations (RaSE), and RNA/protein-family models visualization (CMV), and other methods. Open education resources offer interactive visualizations of RNA structure and RNA-RNA interaction prediction as well as basic and advanced sequence alignment algorithms. The services are freely available at http://rna.informatik.uni-freiburg.de