Cations Modulate Actin Bundle Mechanics, Assembly Dynamics, and Structure

Actin bundles are key factors in the mechanical support and dynamic reorganization of the cytoskeleton. High concentrations of multivalent counterions promote bundle formation through electrostatic attraction between actin filaments that are negatively charged polyelectrolytes. In this study, we evaluate how physiologically relevant divalent cations affect the mechanical, dynamic, and structural properties of actin bundles. Using a combination of total internal reflection fluorescence microscopy, transmission electron microscopy, and dynamic light scattering, we demonstrate that divalent cations modulate bundle stiffness, length distribution, and lateral growth. Molecular dynamics simulations of an all-atom model of the actin bundle reveal specific actin residues coordinate cation-binding sites that promote the bundle formation. Our work suggests that specific cation interactions may play a fundamental role in the assembly, structure, and mechanical properties of actin bundles

A Rapid Blood Test To Determine the Active Status and Duration of Acute Viral Infection

The ability to rapidly detect and diagnose acute viral infections is crucial for infectious disease control and management. Serology testing for the presence of virus-elicited antibodies in blood is one of the methods used commonly for clinical diagnosis of viral infections. However, standard serology-based tests have a significant limitation: they cannot easily distinguish active from past, historical infections. As a result, it is difficult to determine whether a patient is currently infected with a virus or not, and on an optimal course of action, based off of positive serology testing responses. Here, we report a nanoparticle-enabled blood test that can help overcome this major challenge. The new test is based on the analysis of virus-elicited immunoglobulin G (IgG) antibody present in the protein corona of a gold nanoparticle surface upon mixing the gold nanoparticles with blood sera. Studies conducted on mouse models of influenza A virus infection show that the test gives positive responses only in the presence of a recent acute viral infection, approximately between day 14 and day 21 following the infection, and becomes negative thereafter. When used together with the traditional serology testing, the nanoparticle test can determine clearly whether a positive serology response is due to a recent or historical viral infection. This new blood test can provide critical clinical information needed to optimize further treatment and/or to determine if further quarantining should be continued

Inhibition of Cholera Toxin and Other AB Toxins by Polyphenolic Compounds

<div><p>Cholera toxin (CT) is an AB-type protein toxin that contains a catalytic A1 subunit, an A2 linker, and a cell-binding B homopentamer. The CT holotoxin is released into the extracellular environment, but CTA1 attacks a target within the cytosol of a host cell. We recently reported that grape extract confers substantial resistance to CT. Here, we used a cell culture system to identify twelve individual phenolic compounds from grape extract that inhibit CT. Additional studies determined the mechanism of inhibition for a subset of the compounds: two inhibited CT binding to the cell surface and even stripped CT from the plasma membrane of a target cell; two inhibited the enzymatic activity of CTA1; and four blocked cytosolic toxin activity without directly affecting the enzymatic function of CTA1. Individual polyphenolic compounds from grape extract could also generate cellular resistance to diphtheria toxin, exotoxin A, and ricin. We have thus identified individual toxin inhibitors from grape extract and some of their mechanisms of inhibition against CT.</p></div

Compound-induced alterations to host-toxin interactions involving CT.

<p>Compound-induced alterations to host-toxin interactions involving CT.</p

Polyphenolic compounds disrupt CT adherence to the host plasma membrane.

<p>(A) Vero cells were incubated at 4°C for 30 min with 1 μg/mL of FITC-CTB. Unbound toxin was removed from the medium and replaced with 100 μg/mL of grape seed extract, 100 μg/mL of a cocktail containing all 12 CT hit compounds (12C), 17 μg/mL of a cocktail containing PB2 and EGCG (2C), 10 μg/mL of PB2, or 10 μg/mL of EGCG. After an additional 30 min at 4°C, FITC-CTB fluorescence was recorded with a plate reader. Values were standardized to the FITC-CTB signal from control cells incubated in the absence of grape compounds. (B) Vero cells were incubated at 4°C for 1 h in the combined presence of FITC-CTB and 100 μg/mL of grape seed extract, various concentrations of the 12C cocktail, or various concentrations of the 2C cocktail. FITC-CTB fluorescence was then recorded, with values standardized to the FITC-CTB signal from control cells incubated in the absence of grape compounds. Data from both panels represent the means ± SEMs of 4 independent experiments with 6 replicate samples per condition.</p

Phenolic compounds do not affect the thermal unfolding or ER-to-cytosol translocation of CTA1.

<p>(A) A purified CTA1/CTA2 heterodimer was placed in 20 mM sodium phosphate buffer (pH 7.4) containing 10 mM β-mercaptoethanol. Aliquots (1 μg) of the toxin were either left untreated (lanes 1–2), treated with 100 μg/mL of grape seed extract (lane 3), or treated with 10 μg/mL of a specific grape compound: caftaric acid (lane 4), quercitrin (lane 5), gallic acid (lane 6), or PB1 (lane 7). All samples were incubated at 37°C for 1 h. The samples were then shifted to 4°C and exposed to the protease thermolysin for 1 h, with the exception of the untreated toxin sample in lane 1. Samples were visualized by SDS-PAGE with Coomassie staining. Previous control experiments demonstrated that grape seed extract does not directly inhibit the proteolytic activity of thermolysin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166477#pone.0166477.ref015" target="_blank">15</a>]. (B) Using a plasmid-based transfection system, CTA1 was co-translationally inserted into the ER lumen before export back into the cytosol. The intracellular distribution of CTA1 was determined by immunoprecipitation of organelle (O) and cytosol (C) fractions from transfected cells radiolabeled for 1 h in the absence of compound (control), in the presence of a phenolic cocktail (100 μg/mL) containing all CT hit compounds other than petunidin and resveratrol (10C), or in the presence of 0.1 μM GA. The percentage of radiolabeled CTA1 found in the cytosol was calculated from two independent experiments (averages ± ranges).</p

Inhibition of toxin activity by purified phenolic compounds.

<p>Inhibition of toxin activity by purified phenolic compounds.</p