Open source platform for the execution and analysis of mechanical refolding experiments

Abstract Motivation: Single-molecule force spectroscopy has facilitated the experimental investigation of biomolecular force-coupled kinetics, from which the kinetics at zero force can be extrapolated via explicit theoretical models. The atomic force microscope (AFM) in particular is routinely used to study protein unfolding kinetics, but only rarely protein folding kinetics. The discrepancy arises because mechanical protein refolding studies are more technically challenging. Results: We developed software that can drive and analyse mechanical refolding experiments when used with the commercial AFM setup 'Picoforce AFM', Bruker (previously Digital Instruments). We expect the software to be easily adaptable to other AFM setups. We also developed an improved method for the statistical characterization of protein folding kinetics, and implemented it into an AFM-independent software module. Availability: Software and documentation are available at http://code.google.com/p/refolding under Apache License 2.0. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online

Polymorph Separation by Ordered Patterning

We herein address the problem of polymorph selection by introducing a general and straightforward concept based on their ordering. We demonstrated the concept by the ordered patterning of four compounds capable of forming different polymorphs when deposited on technologically relevant surfaces. Our approach exploits the fact that, when the growth of a crystalline material is confined within sufficiently small cavities, only one of the possible polymorphs is generated. We verify our method by utilizing several model compounds to fabricate micrometric "logic patterns" in which each of the printed pixels is easily identifiable as comprising only one polymorph and can be individually accessed for further operations

Comparison of separation methods for immunomodulatory extracellular vesicles from helminths

Helminths survive within their host by secreting immunomodulatory compounds, which hold therapeutic potential for inflammatory conditions. Helminth-derived extracellular vesicles (EVs) are one such component proposed to possess immunomodulatory activities. Due to the recent discovery of helminth EVs, standardised protocols for EV separation are lacking. Excretory/secretory products of the porcine helminth, Ascaris suum, were used to compare three EV separation methods: Size exclusion chromatography (SEC), ultracentrifugation (UC) and a combination of the two. Their performance was evaluated by EV yield, sample purity and the ability of EVs to suppress lipopolysaccharide (LPS)-induced inflammation in vitro. We found that all three separation methods successfully separated helminth EVs with a similar EV yield. Functional studies showed that EVs from all three methods reduced LPS-induced levels of tumour necrosis factor (TNF-α) in a dose-dependent manner. Overall, the three separation methods showed similar performance, however, the combination of UC+SEC presented with slightly higher purity than either method alone

Conformational equilibria in monomeric alpha-synuclein at the single molecule level

Natively unstructured proteins defy the classical "one sequence-one structure" paradigm of protein science. Monomers of these proteins in pathological conditions can aggregate in the cell, a process that underlies socially relevant neurodegenerative diseases such as Alzheimer and Parkinson. A full comprehension of the formation and structure of the so-called misfolded intermediates from which the aggregated states ensue is still lacking. We characterized the folding and the conformational diversity of alpha-synuclein (aSyn), a natively unstructured protein involved in Parkinson disease, by mechanically stretching single molecules of this protein and recording their mechanical properties. These experiments permitted us to directly observe directly and quantify three main classes of conformations that, under in vitro physiological conditions, exist simultaneously in the aSyn sample, including disordered and "beta-like" structures. We found that this class of "beta-like" structures is directly related to aSyn aggregation. In fact, their relative abundance increases drastically in three different conditions known to promote the formation of aSyn fibrils: the presence of Cu2+, the occurrence of the pathogenic A30P mutation, and high ionic strength. We expect that a critical concentration of aSyn with a "beta-like" structure must be reached to trigger fibril formation. This critical concentration is therefore controlled by a chemical equilibrium. Novel pharmacological strategies can now be tailored to act upstream, before the aggregation process ensues, by targeting this equilibrium. To this end, Single Molecule Force Spectroscopy can be an effective tool to tailor and test new pharmacological agents.Comment: 37 pages, 9 figures (including supplementary material

Beyond the 2D Field‐Effect Charge Transport Paradigm in Molecular Thin‐Film Transistors

Organic field-effect transistors (OFETs) are considered almost purely interfacial devices with charge current mainly confined in the first two semiconducting layers in contact with the dielectric with no active role of the film thickness exceeding six to eight monolayers (MLs). By a combined electronic, morphological, structural, and theoretical investigation, it is demonstrated that the charge mobility and source–drain current in 2,20-(2,20-bithiophene-5,50-diyl)bis(5-butyl-5H-thieno[2,3-c]pyrrole-4,6)-dione (NT4N) organic transistors directly correlate with the out-of-plane domain size and crystallite orientation in the vertical direction, well beyond the dielectric interfacial layers. Polycrystalline films with thickness as high as 75 nm (≈30 MLs) and 3D molecular architecture provide the best electrical and optoelectronic OFET characteristics, highlighting that the molecular orientational order in the bulk of the film is the key-enabling factor for optimum device performance. X-ray scattering analysis and multiscale simulations reveal the functional correlation between the thickness-dependent molecular packing, electron mobility, and vertical charge distribution. These results call for a broader view of the fundamental mechanisms that govern field-effect charge transport in OFETs beyond the interfacial 2D paradigm and demonstrate the unexpected role of the out-of-plane domain size and crystallite orientation in polycrystalline films to achieve optimum electronic and optoelectronic properties in organic transistors

Graphene oxide doped polysulfone membrane adsorbers for the removal of organic contaminants from water

This work explored polysulfone (PS) – graphene oxide (GO) based porous membranes (PS-GO) as adsorber of seven selected organic contaminants of emerging concern (EOCs) including pharmaceuticals, personal care products, a dye and a surfactant from water. PS-GO was prepared by phase inversion method starting from a PS and GO mixture (5% w/w of GO). The porous PS-GO membranes showed asymmetric and highly porous micrometer sized pores on membrane top (diameter ≈20 μm) and bottom (diameter ≈2–5 μm) surfaces and tens of microns length finger like pores in the section. Nanomechanical mapping reveals patches of a stiffer material with Young modules comprised in the range 15–25 GPa, not present in PS pure membranes that are compatible with the presence of GO flakes on the membrane surfaces. PS-GO was immersed in EOCs spiked tap water and the adsorbance efficiency at different contact times and pH evaluated by HPLC analysis. Ofloxacin (OFLOX), benzophenone-3 (BP-3), rhodamine b (Rh), diclofenac (DCF) and triton X-100 (TRX) were removed with efficiency higher than 90% after 4 h treatments. Regeneration of PS-GO and reuse possibilities were demonstrated by washing with ethanol. The adsorption efficiencies toward OFLOX, Rh, DCF and carbamazepine (CBZ) were significantly higher than those of pure PS membrane. Moreover, PS-GO outperformed a commercial granular activated carbon (GAC) at low contact times and compared well at longer contact time for OFLOX, Rh, BP-3 and TRX suggesting the suitability of the newly introduced material for drinking water treatment

Platelets as key cells in endometriosis patients: Insights from small extracellular vesicles in peritoneal fluid and endometriotic lesions analysis

Endometriosis is a chronic inflammatory condition characterized by the presence of endometrium-like tissue outside the uterus, primarily affecting pelvic organs and tissues. In this study, we explored platelet activation in endometriosis. We utilized the STRING database to analyze the functional interactions among proteins previously identified in small extracellular vesicles (EVs) isolated from the peritoneal fluid of endometriosis patients and controls. The bioinformatic analysis indicated enriched signaling pathways related to platelet activation, hemostasis, and neutrophil degranulation. Double immunohistochemistry analysis for CD61 and MPO revealed a significant presence of neutrophils and platelets in close contact infiltrating endometriotic lesions, suggesting potential cell–cell interactions. Subsequently, we isolated small EVs from the peritoneal fluid of women diagnosed with endometriosis and from women without endometriosis who underwent surgery for non-inflammatory benign diseases. We performed single-particle phenotyping analysis based on platelet biomarkers GPIIb/IIIa and PF4 using nanoflow cytometry, as well as single-particle morphological and nanomechanical characterization through atomic force microscopy. The study demonstrated that patients with endometriosis had a notably higher proportion of particles testing positive for platelet biomarkers compared to the total number of EVs. This finding implies a potential role for platelets in the pathogenesis of endometriosis. Further research is necessary to delve into the mechanisms underlying this phenomenon and its implications for disease progression

Particle profiling of EV‐lipoprotein mixtures by AFM nanomechanical imaging

The widely overlapping physicochemical properties of lipoproteins (LPs) and extracellular vesicles (EVs) represents one of the main obstacles for the isolation and characterization of these pervasive biogenic lipid nanoparticles. We herein present the application of an atomic force microscopy (AFM)-based quantitative morphometry assay to the rapid nanomechanical screening of mixed LPs and EVs samples. The method can determine the diameter and the mechanical stiffness of hundreds of individual nanometric objects within few hours. The obtained diameters are in quantitative accord with those measured via cryo-electron microscopy (cryo-EM); the assignment of specific nanomechanical readout to each object enables the simultaneous discrimination of co-isolated EVs and LPs even if they have overlapping size distributions. EVs and all classes of LPs are shown to be characterised by specific combinations of diameter and stiffness, thus making it possible to estimate their relative abundance in EV/LP mixed samples in terms of stoichiometric ratio, surface area and volume. As a side finding, we show how the mechanical behaviour of specific LP classes is correlated to distinctive structural features revealed by cryo-EM. The described approach is label-free, single-step and relatively quick to perform. Importantly, it can be used to analyse samples which prove very challenging to assess with several established techniques due to ensemble-averaging, low sensibility to small particles, or both, thus providing a very useful tool for quickly assessing the purity of EV/LP isolates including plasma- and serum-derived preparations

Helminth extracellular vesicles co-opt host monocytes to drive T cell anergy

Parasitic helminths secrete extracellular vesicles (EVs) into their host tissues to modulate immune responses, but the underlying mechanisms are poorly understood. We demonstrate that Ascaris EVs are efficiently internalised by monocytes in human peripheral blood mononuclear cells and increase the percentage of classical monocytes. Furthermore, EV treatment of monocytes induced a novel anti-inflammatory phenotype characterised by CD14+, CD16−, CC chemokine receptor 2 (CCR2−) and programmed death-ligand 1 (PD-L1)+ cells. In addition, Ascaris EVs induced T cell anergy in a monocyte-dependent mechanism. Targeting professional phagocytes to induce both direct and indirect pathways of immune modulation presents a highly novel and efficient mechanism of EV-mediated host-parasite communication. Intra-peritoneal administration of EVs induced protection against gut inflammation in the dextran sodium sulphate model of colitis in mice. Ascaris EVs were shown to affect circulating immune cells and protect against gut inflammation; this highlights their potential as a subject for further investigation in inflammatory conditions driven by dysregulated immune responses. However, their clinical translation would require further studies and careful consideration of ethical implications

Epitaxial multilayers of alkanes on two-dimensional black phosphorus as passivating and electrically insulating nanostructures

© The Royal Society of Chemistry. Mechanically exfoliated two-dimensional (2D) black phosphorus (bP) is epitaxially terminated by monolayers and multilayers of tetracosane, a linear alkane, to form a weakly interacting van der Waals heterostructure. Atomic force microscopy (AFM) and computational modelling show that epitaxial domains of alkane chains are ordered in parallel lamellae along the principal crystalline axis of bP, and this order is extended over a few layers above the interface. Epitaxial alkane multilayers delay the oxidation of 2D bP in air by 18 hours, in comparison to 1 hour for bare 2D bP, and act as an electrical insulator, as demonstrated using electrostatic force microscopy. The presented heterostructure is a technologically relevant insulator-semiconductor model system that can open the way to the use of 2D bP in micro-and nanoelectronic, optoelectronic and photonic applications