Impact of N-Terminal Tags on De Novo Vimentin Intermediate Filament Assembly

Vimentin, a type III intermediate filament protein, is found in most cells along with microfilaments and microtubules. It has been shown that the head domain folds back to associate with the rod domain and this association is essential for filament assembly. The N-terminally tagged vimentin has been widely used to label the cytoskeleton in live cell imaging. Although there is previous evidence that EGFP tagged vimentin fails to form filaments but is able to integrate into a pre-existing network, no study has systematically investigated or established a molecular basis for this observation. To determine whether a tag would affect de novo filament assembly, we used vimentin fused at the N-terminus with two different sized tags, AcGFP (239 residues, 27 kDa) and 3 × FLAG (22 residues; 2.4 kDa) to assemble into filaments in two vimentin-deficient epithelial cells, MCF-7 and A431. We showed that regardless of tag size, N-terminally tagged vimentin aggregated into globules with a significant proportion co-aligning with β-catenin at cell–cell junctions. However, the tagged vimentin aggregates could form filaments upon adding untagged vimentin at a ratio of 1:1 or when introduced into cells containing pre-existing filaments. The resultant filament network containing a mixture of tagged and untagged vimentin was less stable compared to that formed by only untagged vimentin. The data suggest that placing a tag at the N-terminus may create steric hinderance in case of a large tag (AcGFP) or electrostatic repulsion in case of highly charged tag (3 × FLAG) perhaps inducing a conformational change, which deleteriously affects the association between head and rod domains. Taken together our results shows that a free N-terminus is essential for filament assembly as N-terminally tagged vimentin is not only incapable of forming filaments, but it also destabilises when integrated into a pre-existing network

Sequential Acquisition of Virulence and Fluoroquinolone Resistance Has Shaped the Evolution of Escherichia coli ST131

UNLABELLED: Escherichia coli ST131 is the most frequently isolated fluoroquinolone-resistant (FQR) E. coli clone worldwide and a major cause of urinary tract and bloodstream infections. Although originally identified through its association with the CTX-M-15 extended-spectrum β-lactamase resistance gene, global genomic epidemiology studies have failed to resolve the geographical and temporal origin of the ST131 ancestor. Here, we developed a framework for the reanalysis of publically available genomes from different countries and used this data set to reconstruct the evolutionary steps that led to the emergence of FQR ST131. Using Bayesian estimation, we show that point mutations in chromosomal genes that confer FQR coincide with the first clinical use of fluoroquinolone in 1986 and illustrate the impact of this pivotal event on the rapid population expansion of ST131 worldwide from an apparent origin in North America. Furthermore, we identify virulence factor acquisition events that predate the development of FQR, suggesting that the gain of virulence-associated genes followed by the tandem development of antibiotic resistance primed the successful global dissemination of ST131. IMPORTANCE: Escherichia coli sequence type 131 (ST131) is a recently emerged and globally disseminated multidrug-resistant clone frequently associated with human urinary tract and bloodstream infections. In this study, we have used two large publically available genomic data sets to define a number of critical steps in the evolution of this important pathogen. We show that resistance to fluoroquinolones, a class of broad-spectrum antibiotic used extensively in human medicine and veterinary practice, developed in ST131 soon after the introduction of these antibiotics in the United States, most likely in North America. We also mapped the acquisition of several fitness and virulence determinants by ST131 and demonstrate these events occurred prior to the development of fluoroquinolone resistance. Thus, ST131 has emerged by stealth, first acquiring genes associated with an increased capacity to cause human infection, and then gaining a resistance armory that has driven its massive population expansion across the globe

Repeated local emergence of carbapenem-resistant Acinetobacter baumannii in a single hospital ward

We recently reported a dramatic increase in the prevalence of carbapenem-resistant Acinetobacter baumannii infections in the intensive care unit (ICU) of a Vietnamese hospital. This upsurge was associated with a specific oxa23-positive clone that was identified by multilocus VNTR analysis. Here, we used whole-genome sequence analysis to dissect the emergence of carbapenem-resistant A. baumannii causing ventilator-associated pneumonia (VAP) in the ICU during 2009-2012. To provide historical context and distinguish microevolution from strain introduction, we compared these genomes with those of A. baumannii asymptomatic carriage and VAP isolates from this same ICU collected during 2003-2007. We identified diverse lineages co-circulating over many years. Carbapenem resistance was associated with the presence of oxa23, oxa40, oxa58 and ndm1 genes in multiple lineages. The majority of resistant isolates were oxa23-positive global clone GC2; fine-scale phylogenomic analysis revealed five distinct GC2 sublineages within the ICU that had evolved locally via independent chromosomal insertions of oxa23 transposons. The increase in infections caused by carbapenem-resistant A. baumannii was associated with transposon-mediated transmission of a carbapenemase gene, rather than clonal expansion or spread of a carbapenemase-harbouring plasmid. Additionally, we found evidence of homologous recombination creating diversity within the local GC2 population, including several events resulting in replacement of the capsule locus. We identified likely donors of the imported capsule locus sequences amongst the A. baumannii isolated on the same ward, suggesting that diversification was largely facilitated via reassortment and sharing of genetic material within the localized A. baumannii population

Effects of Zn/B nanofertilizer on biophysical characteristics and growth of coffee seedlings in a greenhouse

[[abstract]]The effects of Zn/B nanofertilizer on the biophysical characteristics and growth of coffee seedlings in a greenhouse were investigated. Zn/B nanofertilizer was prepared by loading ZnSO4 and H3BO3 on a chitosan nanoparticles emulsion that was prepared by ionic gelation with tripolyphosphate. The nanofertilizer was characterized by TEM, SEM, zeta potential value and size distribution. The nanofertilizer was sprayed on the leaves of coffee seedlings in five different doses of 0, 10, 20, 30 and 40 ppm. Application of the nanofertilizer enhanced the uptake of zinc, nitrogen and phosphorus. The results were found to increase the chlorophyll content and photosynthesis of the coffee. Finally, the nanofertilizer promoted growth of the coffee plants in the leaf area, height of plant and stem diameter. The nanofertilizer seems to be a great potential foliar feed for the growth of coffee and other plants.[[sponsorship]]科技部[[notice]]補正完

Preparation of NPK nanofertilizer based on chitosan nanoparticles and its effect on biophysical characteristics and growth of coffee in green house

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Inorganic Niobium and Tantalum Octahedral Cluster Halide Compounds with Three-dimensional Frameworks: A Review on their Crystallographic and Electronic Structures

International audienceThis review summarizes the development of the rich crystal and bonding chemistry of face-capped and edge-bridged inorganic niobium and tantalum octahedral cluster halide compounds, with a particular emphasis on those showing three-dimensional cluster frameworks. Discussion is made on varied structures and bonding which are intimately linked to the valence electron concentration, i.e., the number of electrons that held the octahedral metal cluster architecture. Exploration of the literature indicates that apart from Nb6I11 and derivatives, which show electron-deficient face-capped M6Xi8Xa6 units, compounds containing edge-bridged M6Xi12Xa6 motifs are the most largely encountered. Closed-shell compounds with a valence electron concentration of 16 are predominant, although a few 15-electron open-shell magnetic compounds or even 14-electron closed-shell species have also been reported. Particularly interesting from a structural point of view is the fashion in which these face-capped and edge-bridged clusters “pack” in crystals. The astonishing diversity of structural types, which are observed, is mainly due to the flexibility of the halogen ligands to coordinate in various manners to metal atoms. However, a rigorous structural analysis of these compounds reveals no close relationship between the valence electron concentration and the variability of the intercluster connections and/or the nature of the counterions. Indeed, the main bonding features of these compounds can be understood from the delocalized bonding picture of isolated “molecular-like” M6Xi8Xa6 or M6Xi12Xa6 clusters