A striking feature of the alpha-hemolysin channel—a prime candidate for biotechnological applications—is the dependence of its ionic conductance on the magnitude and direction of the applied bias. Through a combination of lipid bilayer single-channel recording and molecular dynamics (MD) simulations, we characterized the current-voltage relationship of alpha-hemolysin for all alkali chloride salts at neutral pH. The rectification of the ionic current was found to depend on the type of cations and increase from Li+ to Cs+. Analysis of the MD trajectories yielded a simple quantitative model that related the ionic current to the electrostatic potential, the concentration and effective mobility of ions in the channel. MD simulations reveal that the major contribution to the current asymmetry and rectification properties originates from the cationic contribution to the current that is significantly reduced in a cationic dependent way when the membrane polarity is reversed. The variation of chloride current was found to be less important. We report that the differential affinity of cations for the charged residues positioned at the channel’s end modulates the number of ions inside the channel stem thus affecting the current properties. Through direct comparison of simulation and experiment, this study evaluates the accuracy of the MD method for prediction of the asymmetric, voltage dependent conductances of a membrane channel.

The measurement of physicochemical properties of polydisperse complex biological samples, for example, extracellular vesicles, is critical to assess their quality, for example, resulting from their production and isolation methods. The community is gradually becoming aware of the need to combine multiple orthogonal techniques to perform a robust characterization of complex biological samples. Three pillars of critical quality attribute characterization of EVs are sizing, concentration measurement and phenotyping. The repeatable measurement of vesicle concentration is one of the keychallenges that requires further efforts, in order to obtain comparable results by using different techniques and assure reproducibility. In this study, the performance of measuring the concentration of particles in the size range of 50-300 nm with complementary techniques is thoroughly investigated in a step-by step approach of incremental complexity. The six applied techniques include multi-angle dynamic light scattering (MADLS), asymmetric flow field flow fractionation coupled with multi-angle light scattering (AF4-MALS), centrifugal liquid sedimentation (CLS), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), and high-sensitivity nano flow cytometry (nFCM). To achieve comparability, monomodal samples and complex polystyrene mixtures were used as particles of metrological interest, in order to check the suitability of each technique in the size and concentration range of interest, and to develop reliable post-processing data protocols for the analysis. Subsequent complexity was introduced by testing liposomes as validation of the developed approaches with a known sample of physicochemical properties closer to EVs. Finally, the vesicles in EV containing plasma samples were analysed with all the tested techniques. The results presented here aim to shed some light into the requirements for the complex characterization of biological samples, as this is a critical need for quality assurance by the EV and regulatory community. Such efforts go with the view to contribute to both, setup reproducible and reliable characterization protocols, and comply with the Minimal Information for Studies of Extracellular Vesicles (MISEV) requirements.

This article explores the role of some geometrical factors on the electrophoretically driven translocations of macromolecules through nanopores. In the case of asymmetric pores, we show how the entry requirements and the direction of translocation can modify the information content of the blocked ionic current as well as the transduction of the electrophoretic drive into a mechanical force. To address these effects we studied the translocation of single-stranded DNA through an asymmetric alpha-hemolysin pore. Depending on the direction of the translocation, we measure the capacity of the pore to discriminate between both DNA orientations. By unzipping DNA hairpins from both sides of the pores we show that the presence of single strand or double strand in the pore can be discriminated based on ionic current levels. We also show that the transduction of the electrophoretic drive into a denaturing mechanical force depends on the local geometry of the pore entrance. Eventually we discuss the application of this work to the measurement of energy barriers for DNA unzipping as well as for protein binding and unfolding.

BackgroundFibrosis, which is characterized by the pathological accumulation of collagen, is recognized as an important feature of many chronic diseases, and as such, constitutes an enormous health burden. We need non-invasive specific methods for the early diagnosis and follow-up of fibrosis in various disorders. Collagen targeting molecules are therefore of interest for potential in vivo imaging of fibrosis. In this study, we developed a collagen-specific probe using a new approach that takes advantage of the inherent specificity of Glycoprotein VI (GPVI), the main platelet receptor for collagens I and III.Methodology/Principal FindingsAn anti-GPVI antibody that neutralizes collagen-binding was used to screen a bacterial random peptide library. A cyclic motif was identified, and the corresponding peptide (designated collagelin) was synthesized. Solid-phase binding assays and histochemical analysis showed that collagelin specifically bound to collagen (Kd 10−7 M) in vitro, and labelled collagen fibers ex vivo on sections of rat aorta and rat tail. Collagelin is therefore a new specific probe for collagen. The suitability of collagelin as an in vivo probe was tested in a rat model of healed myocardial infarctions (MI). Injecting Tc-99m-labelled collagelin and scintigraphic imaging showed that uptake of the probe occurred in the cardiac area of rats with MI, but not in controls. Post mortem autoradiography and histological analysis of heart sections showed that the labeled areas coincided with fibrosis. Scintigraphic molecular imaging with collagelin provides high resolution, and good contrast between the fibrotic scars and healthy tissues. The capacity of collagelin to image fibrosis in vivo was confirmed in a mouse model of lung fibrosis.Conclusion/SignificanceCollagelin is a new collagen-targeting agent which may be useful for non-invasive detection of fibrosis in a broad spectrum of diseases.