182 research outputs found
Curli-only FSB binding assay.
No full text(A) A standard curve of the FSB absorbance versus concentration was obtained in order to determine the molar absorptivity of FSB at 365 nm (ε365nm = 33.68 mM-1cm-1; r2 = 0.892). (B) Incubation of FSB with increasing amounts of curli resulted in increasing depletion of FSB from solution. (C) A saturation binding curve obtained by varying the FSB concentration. The curve was fit to obtain binding parameters, where y = (Bmax•x)/(Kd+x).</p
The spectral properties of curli-bound FSB.
No full text(A) The UV-Vis spectra indicate that binding of FSB to curli results in a red shift in the absorbance spectrum. Specifically, the absorbance spectrum of curli-bound FSB is red-shifted from a λmax of 361 nm to 386 nm (red shift = 25 nm), which is greater than the 10 nm red shift observed for CR bound to curli. (B) The fluorescence emission spectra of each dye alone and in the curli-bound samples were obtained upon excitation at 540 nm for CR and at 390 nm for FSB. FSB exhibits significantly enhanced fluorescence when bound to curli as compared to Congo red. In fact, the curli-bound FSB sample was diluted 400-fold relative to the curli-bound CR sample to prevent exceeding the detection range of the spectrophotometer.</p
Antimicrobial surfaces based on self-assembled nanoreactors : from block copolymer synthesis to bacterial adhesion study
No full textThe aim of this work is to develop a new strategy for the prevention of biofilm growth. For this purpose, we prepared bioactive surfaces resulting from the surface-immobilization of nanoreactors self-assembled from amphiphilic poly(isobutylene)-block-oligonucleotide copolymers. The block copolymer was synthesized and characterized via appropriate complementary techniques. Self-assembly into vesicles allowed the functional encapsulation of enzymes, as assayed through enzyme activity monitoring, leading to a prodrug-drug system. The self-assembled structures were specifically immobilized on surfaces via base pairing between the oligonucleotide block of the copolymer and the surface tethered complementary nucleotide sequence.
Using E.coli strains, we first observed an influence of the two density of oligonucleotides immobilized on the surface on the number of adherent bacteria. This influence may be due to an effect of surface charge density. We then confirmed the well-known role of curli in biofilm cohesion, and we showed gene over-expression associated with curli production on oligonucleotide-modified surfaces. We demonstrated that gene over-expression does not depend on the topographical features of the surface or on the composition of the nucleotide sequences used in this study. Finally, we demonstrated that the presence of the vesicular structure is able to produce strong anti-adhesive properties of the surface. We assume, from observations of bacterial response in dynamic conditions, that this effect is due to increased bacterial motility on the surface, leading to a high detachment rate. Which is further confirms by a comparable bacterial response observed on agar hydrogel of different hardnesses. This result provides a preliminary outcome, paving the way to new approaches to antimicrobial strategies
The Suez Canal and Italian Port Cities: Local Competition and Global Ambitions Within a Nation in the Making (1850s–1870s)
No full textInteractions of CR with isolated curli.
No full text<p>(A) Surface plasmon resonance experiments performed with immobilized curli revealed that CR binding is relatively weak; CR binds to curli with an approximate k<sub>d</sub> of 2.8 μM. (B) A CR depletion assay allowed facile examination of CR binding to isolated curli, similar to the whole-cell assay performed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140388#pone.0140388.g001" target="_blank">Fig 1</a>. The data demonstrate that CR binding to curli is pH dependent and is not dependent on histidine in the CsgA sequence. (C) Curli produced by wild-type MC4100 are comparable to those formed by the histidine mutants and are not influenced by changes in pH, as demonstrated by electron microscopy.</p
LL-37 binds to recombinant polymerized CsgA and isolated wild-type curli.
No full text<p>(<b>A</b>) Western blot analysis of supernatants after precipitation of LL-37 with curli. By adding polymeric CsgA (pol CsgA) or wild-type curli (wt curli) to a solution of 0.1 µM LL-37, the levels of LL-37 decreased in the supernatants after centrifugation. (<b>B</b>) Surface plasmon resonance. LL-37 exhibits a stronger association and lower dissociation rates to both polymeric (upper panel) and monomeric CsgA (lower panel) compared to the control peptides sLL-37 and VIP.</p
Curli increase the resistance to the antimicrobial peptide LL-37.
No full text<p>(<b>A</b>) Bladder epithelial cells T24 were infected with bacteria for 30 min and adherent bacteria were subjected to LIVE/DEAD staining. Curli and cellulose expression enhanced bacterial resistance to antimicrobial properties (<sup>### </sup><i>P</i><0.001 for curliated strains versus non-curliated strains, <sup>*</sup><i>P</i> = 0.023 and <sup>**</sup><i>P</i> = 0.003 for cellulose expressing strains with or without curli, <i>t</i>-test). Combined data from four experiments are shown. (<b>B+C</b>) Bacteria were exposed to conditioned medium of bladder epithelial cells T24 stimulated with phenylbutyrate to enhance LL-37 production. Curli expression enhanced bacterial survival over 30 min (<sup>## </sup><i>P</i> = 0.006, <i>t</i>-test) (<b>B</b>). Results from three experiments in triplicate are shown. Conditioned medium was incubated with neutralizing anti-LL-37-antibodies (nAb) or isotype control antibodies (Co) prior to bacterial inoculation. Neutralizing of LL-37 had no effect on viability of the curliated strain (left) but enhanced viability of the double knockout (right, <sup>*</sup><i>P</i> = 0.047, <i>t</i>-test) (<b>C</b>). Results from four experiments in triplicate are shown. (<b>D–G</b>) The susceptibility to LL-37 and mCRAMP of <i>E. coli</i> strains expressing or lacking curli or cellulose was tested by the broth dilution method. The expression of curli increased the resistance to both LL-37 (<b>D+E</b>) and mCRAMP (<b>F+G</b>). A significant difference of bacterial growth was observed at 10 µM LL-37 between curliated and non-curliated strains (<sup>### </sup><i>P</i><0.001, <i>t</i>-test). The curliated strains were also significantly more resistant to 5 µM mCRAMP than bacteria not producing curli (<sup># </sup><i>P</i><0.05, <i>t</i>-test). An increased resistance to both cathelicidins was not observed for cellulose. Mean and standard deviation from data of two separate experiments in triplicates are shown. The IC<sub>50</sub> is indicated by a broken line.</p
Discourses on Europe and their political value in Restoration France
No full textThe chapter focuses on the different political uses of discourses on Europe in French debates during the Restoration. In the first years, from the Congress of Vienna to the second Bourbon dynasty's second return to the throne, both the opponents and the defenders of the French Revolution used different depictions of Europe to justify their political stances. The moderate-royalists imagined the material reorganization of Europe on principles of legitimacy and religion; the Ultra-royalists painted Europe as governed by chaos, arguing that only Catholicism, aristocracy and the value of the family could restore peace and morality; the liberals talked about a set of free nations and representative governments; for Bonapartists, instead, Europe was a continent set along the path to freedom, which the Allied Powers (especially England) wanted to bring back to a new slavery. Around the various and antithetical discourses on Europe the battle for or against the French Revolution was fought. But a few years later, during the Greek independence war against the Ottoman Empire, these different depictions of Europe seemed to lose importance: in front of Muslim enemy, both the Christian religion and the right of nations to self-determination contributed to development of the idea of Europe's superiority over the rest of the world
Characterization of the Curli Nucleator CsgB.
No full textAmyloid biogenesis is the underlying cause of many neurological disorders such as Alzheimer’s disease. During the progression of Alzheimer’s disease it is thought that normally soluble Aβ peptides self assemble into β-sheet rich, detergent resistant amyloid fibers via a template mediated-nucleation dependent polymerization reaction. Although the polymerization of Aβ is well characterized in vitro, the molecular events that govern nucleation in vivo are poorly described. Curli fibers are extracellular functional amyloid fibers utilized by enteric bacteria for the formation of biofilms. The fibers are composed of two proteins, CsgA, the major subunit, and CsgB, the minor subunit. CsgB is required for nucleating CsgA polymerization in vivo. CsgA and CsgB are 50% similar at the amino level and in silico structural predictions suggest both proteins are β-sheet rich. We hypothesized that CsgB drives the polymerization of CsgA by acting as a β-sheet rich folding template at the cell surface, and predicted that CsgB could self-polymerize into amyloid fibers in vitro. We found that CsgB and mutant variants polymerized into amyloid fibers in vitro, and that these fibers seeded CsgA polymerization. Perturbing the C-terminal portion of CsgB caused CsgB to be mislocalized in vivo and greatly reduced CsgB function. Our results are consistent with the hypothesis that CsgB templates CsgA polymerization by adopting a β-sheet rich fold, and that disruption of the CsgB-cell surface interaction leads to impaired CsgB function. Using the methods established in the course of this work, I have also begun to identify protein- and small molecule inhibitors of curli nucleation and polymerization. For example, CsgE, an accessory protein required for curli biogenesis, and rationally-designed small molecule inhibitors of Aβ polymerization inhibit CsgA polymerization in vitro. Together these results suggest curli biogenesis may be a powerful model for elucidating the initial molecular events involved in amyloid biogenesis.PhDMicrobiology & ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/63820/1/nhammer_1.pd
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