Amyloid Fibrils of Stefin B Show Anisotropic Properties.

Human stefin B, a member of the cystatin family of cysteine protease inhibitors, tends to form amyloid fibrils under relatively mild conditions, which is why it is used as a model protein to study amyloid fibrillation. Here, we show for the first time that bundles of amyloid fibrils, i.e., helically twisted ribbons, formed by human stefin B exhibit birefringence. This physical property is commonly observed in amyloid fibrils when stained with Congo red. However, we show that the fibrils arrange in regular anisotropic arrays and no staining is required. They share this property with anisotropic protein crystals, structured protein arrays such as tubulin and myosin, and other anisotropic elongated materials, such as textile fibres and liquid crystals. In certain macroscopic arrangements of amyloid fibrils, not only birefringence is observed, but also enhanced emission of intrinsic fluorescence, implying a possibility to detect amyloid fibrils with no labels by using optical microscopy. In our case, no enhancement of intrinsic tyrosine fluorescence was observed at 303 nm; instead, an additional fluorescence emission peak appeared at 425 to 430 nm. We believe that both phenomena, birefringence and fluorescence emission in the deep blue, should be further explored with this and other amyloidogenic proteins. This may allow the development of label-free detection methods for amyloid fibrils of different origins.

Supramolecular Polymorphism of (G4C2)n Repeats Associated with ALS and FTD.

Guanine-rich DNA sequences self-assemble into highly stable fourfold structures known as DNA-quadruplexes (or G-quadruplexes). G-quadruplexes have furthermore the tendency to associate into one-dimensional supramolecular aggregates termed G-wires. We studied the formation of G-wires in solutions of the sequences d(G4C2)n with n = 1, 2, and 4. The d(G4C2)n repeats, which are associated with some fatal neurological disorders, especially amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), represent a challenging research topic due to their extensive structural polymorphism. We used dynamic light scattering (DLS) to measure translational diffusion coefficients and consequently resolve the length of the larger aggregates formed in solution. We found that all three sequences assemble into longer structures than previously reported. The d(G4C2) formed extremely long G-wires with lengths beyond 80 nm. The d(G4C2)2 formed a relatively short stacked dimeric quadruplex, while d(G4C2)4 formed multimers corresponding to seven stacked intramolecular quadruplexes. Profound differences between the multimerization properties of the investigated sequences were also confirmed by the AFM imaging of surface films. We propose that π-π stacking of the basic G-quadruplex units plays a vital role in the multimerization mechanism, which might be relevant for transformation from the regular medium-length to disease-related long d(G4C2)n repeats.

Nematic Liquid-Crystal Necklace Structure Made by Microfluidics System.

We report a necklace structure made of liquid crystal dispersed in poly(vinyl alcohol) (PVA) aqueous solution, which is fabricated by a microfluidic device. In the necklace structure, liquid crystal droplets that are tens of micrometers in diameter are connected by microtethers, which are birefringent, are not penetrating the droplets, and can be elastically stretched by applying external force. The necklace structure was analyzed by fluorescent confocal microscopy, and the tethers were made of liquid crystal and PVA composite. The elastic constant of the tether was determined by using laser tweezers to stretch the tether. The Whispering Gallery Modes circulating inside individual droplets in the necklace structure were also observed.

Nanoparticles Can Wrap Epithelial Cell Membranes and Relocate Them Across the Epithelial Cell Layer.

Although the link between the inhalation of nanoparticles and cardiovascular disease is well established, the causal pathway between nanoparticle exposure and increased activity of blood coagulation factors remains unexplained. To initiate coagulation tissue factor bearing epithelial cell membranes should be exposed to blood, on the other side of the less than a micrometre thin air-blood barrier. For the inhaled nanoparticles to promote coagulation, they need to bind lung epithelial-cell membrane parts and relocate them into the blood. To assess this hypothesis, we use advanced microscopy and spectroscopy techniques to show that the nanoparticles wrap themselves with epithelial-cell membranes, leading to the membrane's disruption. The membrane-wrapped nanoparticles are then observed to freely diffuse across the damaged epithelial cell layer relocating epithelial cell membrane parts over the epithelial layer. Proteomic analysis of the protein content in the nanoparticles wraps/corona finally reveals the presence of the coagulation-initiating factors, supporting the proposed causal link between the inhalation of nanoparticles and cardiovascular disease.

In situ laser-imprinted surface realignment of a nematic liquid crystal.

We present a new method for the in-plane realignment of nematic liquid crystals in already fully assembled cells with uni-directionally rubbed polyimide as an aligning layer. We use nematic liquid crystals (NLCs) with a relatively high nematic-isotropic transition temperature and we focus the IR laser beam of the laser tweezers selectively onto one or the other of the inner interfaces. The heat generated by the IR absorption locally melts the liquid crystal and creates an isotropic island with well-defined molecular anchoring at the nematic-isotropic interface. By scanning the laser beam along a pre-defined line, the moving isotropic-nematic interface leaves behind a well oriented LC domain, with LC molecules aligned at 45° to the rubbing direction. If we in addition move the sample with respect to this scanning line, we would be able to selectively realign micro-domains of the liquid crystal with respect to the original alignment induced by the PI rubbing. The realignment can be performed independently on each LC-glass interface, thereby producing predefined domains with customized and controllable alignment within an otherwise uniformly aligned cell.

The effect of tyrosine residues on α-synuclein fibrillation.

Aggregation of the intrinsically disordered protein α-synuclein into ordered amyloid fibrils is implicated in the pathogenesis of Parkinson's disease. To unravel the role of Tyr residues in α-synuclein fibrillation, we prepared recombinant N-terminal (Y39A) and C-terminal (Y(125,133,136)A) mutants of α-synuclein and examined their fibrillation propensities by thioflavin T and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescent probes, SDS-PAGE and atomic force microscopy. We demonstrate that in contrast to wild-type α-synuclein, both mutants show large, but comparable delays in the fibrillation process and exhibit enhanced hydrophobicity during fibril-like assembly. Both Tyr mutants form fibril-like structures after prolonged incubation periods, which are morphologically distinct from those of the wild-type protein. Our results suggest that the N-terminal and C-terminal Tyr residues of α-synuclein are important primarily for the initiation of the fibrillation process.

Synthesis of bioactive ß-TCP coatings with tailored physico-chemical properties on zirconia bioceramics.

The objective of this work was to develop a synthesis procedure for the deposition of ß-TCP coatings with tailored physico-chemical properties on zirconia bioceramics. The synthesis procedure involved two steps: (i) a rapid wet-chemical deposition of a biomimetic CaP coating and (ii) a subsequent post-deposition processing of the biomimetic CaP coating, which included a heat treatment between 800 and 1200 °C, followed by a short sonication in a water bath. By regulating the heating temperature the topography of the ß-TCP coatings could be controlled. The average surface roughness (Ra) ranged from 42 nm for the coating that was heated at 900 °C (TCP-900) to 630 nm for the TCP-1200 coating. Moreover, the heating temperature also affected the dissolution rate of the coatings in a physiological solution, their protein-adsorption capacity and their bioactivity in a simulated body fluid.

The role of initial oligomers in amyloid fibril formation by human stefin B.

Oligomers are commonly observed intermediates at the initial stages of amyloid fibril formation. They are toxic to neurons and cause decrease in neural transmission and long-term potentiation. We describe an in vitro study of the initial steps in amyloid fibril formation by human stefin B, which proved to be a good model system. Due to relative stability of the initial oligomers of stefin B, electrospray ionization mass spectrometry (ESI MS) could be applied in addition to size exclusion chromatography (SEC). These two techniques enabled us to separate and detect distinguished oligomers from the monomers: dimers, trimers, tetramers, up to decamers. The amyloid fibril formation process was followed at different pH and temperatures, including such conditions where the process was slow enough to detect the initial oligomeric species at the very beginning of the lag phase and those at the end of the lag phase. Taking into account the results of the lower-order oligomers transformations early in the process, we were able to propose an improved model for the stefin B fibril formation.

Neural networks determination of material elastic constants and structures in nematic complex fluids.

Supervised machine learning and artificial neural network approaches can allow for the determination of selected material parameters or structures from a measurable signal without knowing the exact mathematical relationship between them. Here, we demonstrate that material nematic elastic constants and the initial structural material configuration can be found using sequential neural networks applied to the transmmited time-dependent light intensity through the nematic liquid crystal (NLC) sample under crossed polarizers. Specifically, we simulate multiple times the relaxation of the NLC from a random (qeunched) initial state to the equilibirum for random values of elastic constants and, simultaneously, the transmittance of the sample for monochromatic polarized light. The obtained time-dependent light transmittances and the corresponding elastic constants form a training data set on which the neural network is trained, which allows for the determination of the elastic constants, as well as the initial state of the director. Finally, we demonstrate that the neural network trained on numerically generated examples can also be used to determine elastic constants from experimentally measured data, finding good agreement between experiments and neural network predictions.

Surface alignment of nematic liquid crystals by direct laser writing of photopolymer alignment layers.

We demonstrate the fabrication of good quality surface alignment layers on glass by Direct Laser Writing method using a 2-photon polymerisation technique. We use commercially available photosensitive resins to print alignment layers by scanning the focal point of a femtosecond laser near the resin-glass interface. This results in down to ~ 100 nm thin alignment layers that provide good planar anchoring of 5CB and MLC13300, with the easy axis of alignment along the scanning direction. The azimuthal anchoring strength is ~ 5 × 10-6 J/m2 and is an order of magnitude weaker compared to commercial rubbed polyimide alignment layer. The threshold voltage for Fréedericksz transition in a 90° twisted nematic cell is slightly increased compared to conventional rubbed polyimide for printed alignment layers. The turn-on switching time is longer for printed layers compared to polyimide alignment layers, whereas the turn-off time is shorter for printed alignment layers. The advantage of this new method is in its flexibility, as we demonstrate printing of complex surface alignment patterns with alignment layer thickness below 100 nm.

Immunochemical properties and pathological relevance of anti-ß2-glycoprotein I antibodies of different avidity.

Despite available treatment, there is still significant morbidity and mortality present among patients with the autoimmune thrombophilic condition termed 'antiphospholipid syndrome' (Espinosa, G. and Cervera, R. 2009. Morbidity and mortality in the antiphospholipid syndrome. Curr. Opin. Pulm. Med. 15:413.). High-avidity (HAv) anti-ß(2)-glycoprotein I (anti-ß(2)GPI) antibodies, shown to correlate with thrombotic events in patients, could represent the much needed improved prognostic marker. By studying their effect on crystalline annexin A5 shield on phospholipid surfaces (one of proposed pathogenic mechanisms), with the use of atomic force microscopy, the pathogenic potential of HAv anti-ß(2)GPI antibodies was confirmed. Furthermore, by using surface plasmon resonance and enzyme-linked immunosorbent assays, unique binding characteristics of HAv antibodies in comparison with low avidity antibodies were established. HAv anti-ß(2)GPI were confirmed to (i) recognize ß(2)-glycoprotein I in a solution, (ii) interact predominantly monovalently (much lower dependency on the antigen density) and (iii) form more stable complexes with the antigen. Since enzyme-linked immunosorbent assays currently used in routine diagnostics detect anti-ß(2)GPI antibodies of unknown avidity, our observations are potentially useful for the development of improved diagnostic tests capable of detecting clinically relevant antibodies.

Flexoelectric Polarization in a Nematic Liquid Crystal Enhanced by Dopants with Different Molecular Shape Polarities.

Flexoelectricity may have an important impact on the switching properties of nematic and cholesteric liquid crystals due to the linear coupling between the flexoelectric polarization of the liquid crystal and the applied electric field. This coupling is the origin of the extraordinary electro-optic effect in cholesterics aligned in the uniform lying helix texture, resulting in fast switching and field control of both rise and fall times. Therefore, the flexoelectric properties of the liquid crystals have become an important issue when designing and synthesizing liquid crystal materials and/or preparing their mixtures with appropriate flexoelectric compounds (dopants). Here, we report on the flexoelectric polarization of a highly polar nematic liquid crystal host enhanced by doping it with two newly synthesized dopants SK 1-6 and SK 1-8, possessing a hockey stick molecular shape, and comparing their doping effect with the one of the dimeric dopants CB7CB possessing a symmetric bend molecular shape. All dopants were dissolved in small concentration (5 wt %) in the nematic host so that the linear approximation of the dependence of the difference between splay e s and bend e b flexoelectric constants, that is, (e s - e b), on the concentration of the dopant in the host material can be applied. In this way, (e s - e b) was estimated for the hockey stick dopants SK 1-6 and SK 1-8 to be 0.182 and 0.204 nC/m, respectively. The obtained flexoelectric polarization of these dopants is among the highest reported in the literature so far.

The use of atomic force microscopy to study the pathologic effects of anti-annexin autoantibodies.

Patients with recurrent pregnancy loss and a history of thrombotic events have often been noted to have autoantibodies directed at annexin A5. However, the relationship of these autoantibodies to immunopathology is still unknown, although it has been proposed that they have a direct effect on the function of annexin A5. Annexin A5 may be a significant immunological target with pathologic implications. Essentially, annexin A5 is an anticoagulant protein that crystallizes over negatively charged phospholipid surfaces and thereby blocks them from availability for coagulation reactions. To address this issue, we have taken advantage of our expertise with atomic force microscopy and studied anti-annexin A5 autoantibodies isolated from patients and focused on the ability of these antibodies to influence annexin A5 crystallization on planar mica-supported phospholipid bilayers. We report herein that such antibodies from patients, but not controls, produced a significant disruption of incomplete annexin A5 crystalline shield on phospholipid bilayer. In addition, the IgG fraction isolated from such patients significantly decreased the velocity of annexin A5 crystallization. Atomic force microscopy is a powerful tool to study the pathologic mechanisms of autoantibodies and the data herein reflect the potential of anti-annexin A5 antibodies that produce pathology in a number of varied but overlapping clinical conditions, including autoimmune thrombosis and antiphospholipid syndrome.

Lamellar liquid crystals maintain keratinocytes' membrane fluidity: An AFM qualitative and quantitative study.

Despite extensive investigations of lamellar liquid crystals for dermal application, the effects of these systems at the cellular level are still not well elucidated. The key aim of this study was to determine the elasticity and morphological features of keratinocytes after exposure to a lamellar liquid crystal system (LLCS) using atomic force microscopy (AFM) as the method of choice. Prior to AFM assessment, a cell proliferation test and light plus fluorescence imaging were applied to determine the sub-toxic concentration of LLCS. According to the AFM results, slightly altered morphology was observed in the case of fixed keratinocytes, while an intact morphology was visualized on live cells. From the quantitative study, decreased Young's moduli were determined for fixed cells (i.e., 8.6 vs. 15.2 MPa and 1.3 vs. 2.9 MPa for ethanol or PFA-fixed LLCS-treated vs. control cells, respectively) and live cells (i.e., ranging from 0.6 to 2.8 for LLCS-treated vs. 1.1-4.5 MPa for untreated cells), clearly demonstrating increased cell elasticity. This is related to improved membrane fluidity as a consequence of interactions between the acyl chains of cell membrane phosphatidylcholine and those of LLCS. What seems to be of major importance is that the study confirms the potential clinical relevance of such systems in treatment of aged skin with characteristically more rigid epithelial cells.

Self-assembly of nematic colloids.

Dispersions of colloidal particles in nematic liquid crystals show new classes of interparticle forces, which are anisotropic, long range, and several thousand times stronger than van der Waals forces in water-based colloids. These forces are responsible for a variety of new self-assembled colloidal microstructures, which cannot be observed in isotropic solvents, such as chains of microspheres and cellular soft solid materials. Basic principles of particle self-assembly in 2D nematic colloids are discussed, showing this is a novel paradigm in colloid science, which could lead to new approaches of colloidal self-assembly for photonic devices.

Amyloid fibril formation by human stefin B: influence of pH and TFE on fibril growth and morphology.

As shown before, human stefin B (cystatin B) populates two partly unfolded species, a native-like state at pH 4.8 and a structured molten globule state at pH 3.3 (high ionic strength), from each of which amyloid fibrils grow. Here, we show that the fibrils obtained at pH 3.3 differ from those at pH 4.8 and that those obtained at pH 3.3 (protofibrils) do not transform readily to mature fibrils. In addition we show that amorphous aggregates are also a source of fibrils. The kinetics of amyloid fibril formation at different trifluoroethanol (TFE) concentrations were measured. TFE accelerates fibril growth at predenaturational concentrations of the alcohol. At concentrations higher than 10%, the fibrillar yield decreases proportionately as the population of an all alpha-helical, denatured form of the protein increases. At an optimum TFE concentration, the lag and the growth phases are observed, similarly to some other amyloidogenic proteins. Morphology of the protein species at the beginning and the end of the reactions was observed using atomic force microscopy and transmission electron microscopy. Final fibril morphologies differ depending on solvent conditions.

Human stefin B readily forms amyloid fibrils in vitro.

Human stefin B (cystatin B) is an intracellular cysteine proteinase inhibitor broadly distributed in different tissues. Here, we show that recombinant human stefin B readily forms amyloid fibrils in vitro. It dimerises and further oligomerises, starting from the native-like acid intermediate, I(N), populated at pH 5. On standing at room temperature it produces regular (over 4 microm long) fibrils over a period of several months. These have been visualised by transmission electron microscopy and atomic force microscopy. Their cross-sectional diameter is about 14 nm and blocks of 27 nm repeat longitudinally. The fibrils are smooth, of unbranched surface, consistent with findings of other amyloid fibrils. Thioflavin T fluorescence spectra as a function of time were recorded and Congo red dye binding to the fibrils was demonstrated. Adding 10% (v/v) trifluoroethanol resulted in an increased rate of fibrillation with a typical lag phase. The finding that human stefin B, in contrast to the homologue stefin A, forms amyloid fibrils rather easily should promote further studies of the protein's behaviour in vivo, and/or as a model system for fibrillogenesis.

Different propensity to form amyloid fibrils by two homologous proteins-Human stefins A and B: searching for an explanation.

By using ThT fluorescence, X-ray diffraction, and atomic force microscopy (AFM), it has been shown that human stefins A and B (subfamily A of cystatins) form amyloid fibrils. Both protein fibrils show the 4.7 A and 10 A reflections characteristic for cross beta-structure. Similar height of approximately 3 nm and longitudinal repeat of 25-27 nm were observed by AFM for both protein fibrils. Fibrils with a double height of 5.6 nm were only observed with stefin A. The fibril's width for stefin A fibrils, as observed by transmission electron microscopy (TEM), was in the same range as previously reported for stefin B (Zerovnik et al., Biochem Biophys Acta 2002;1594:1-5). The conditions needed to undergo fibrillation differ, though. The amyloid fibrils start to form at pH 5 for stefin B, whereas in stefin A, preheated sample has to be acidified to pH < 2.5. In both cases, adding TFE, seeding, and alignment in a strong magnetic field accelerate the fibril growth. Visual analysis of the three-dimensional structures of monomers and domain-swapped dimers suggests that major differences in stability of both homologues stem from arrangement of specific salt bridges, which fix alpha-helix (and the alpha-loop) to beta-sheet in stefin A monomeric and dimeric forms.

The design trend in tissue-engineering scaffolds based on nanomechanical properties of individual electrospun nanofibers.

This paper especially highlights the finding that the mechanical properties of polymeric nanofibers can be tuned by changing the fiber size as well as the composition. For this purpose, the bending Young's modulus was determined using atomic force microscope by involving single-material (polyvinyl alcohol (PVA), polyethylene oxide (PEO 400K)) and composite nanofibers (polyvinyl alcohol/hyaluronic acid (PVA/HA), polyethylene oxide/chitosan (PEO 400K/CS)). The mechanical property, namely the bending Young's modulus, increases as the diameter of the fibers decreases from the bulk down to the nanometer regime (less than 200 nm). The ranking of increasing stiffness according to the AFM measurements of the three-point beam bending test are in agreement, and can be ranked: PEO 400K

Addressing potent single molecule AFM study in prediction of swelling and dissolution rate in polymer matrix tablets.

Our goal was to understand and thus be able to predict the swelling behavior of xanthan matrix tablets in media of various pH and ionic strengths using data obtained from single xanthan molecules and films with atomic force microscopy. Imaging was performed in 1-butanol using contact mode AFM in order to characterize single xanthan chains prepared from various solutions. Image analysis was used to calculate the molecular contour, persistence length, and radius of gyration. Nanoindentation measurements of xanthan films were carried out to evaluate their mechanical properties. Increasing the ionic strength of solutions induced reductions in chain parameters such as molecular contour, persistence length, and radius of gyration. Nanomechanical measurements demonstrated that Young's moduli of xanthan films prepared from solutions with higher ionic strengths are twice as large as those prepared at lower ionic strengths. This may help explain xanthan matrix tablets' reduced degree of swelling and faster dissolution rate in the presence of salts or ions. We successfully come to conclusion that microscopic polymer properties such as radius of gyration and persistence length are responsible for the macroscopic polymer behavior. For instance, longer persistence lengths and radius of gyration of xanthan's chains result in a higher degree of swelling, corresponding to softer polymer films, increased gel layers in matrix, and a slower release rate of the incorporated drug from the tablets.