Search

A versatile and robust procedure is developed that allows the identification of individual target molecules using antibodies bound to a DeepTipTM functionalized atomic-force microscopy probe. The model system used for the validation of this process consists of a biotinylated anti-lactate dehydrogenase antibody immobilized on a streptavidin-decorated AFM probe. Lactate dehydrogenase (LDH) is employed as target molecule and covalently immobilized on functionalized MicroDeckTM substrates. The interaction between sensor and target molecules is explored by recording force–displacement (F–z) curves with an atomic-force microscope. F–z curves that correspond to the genuine sensor–target molecule interaction are identified based on the following three criteria: (i) number of peaks, (ii) value of the adhesion force, and (iii) presence or absence of the elastomeric trait. The application of these criteria leads to establishing seven groups, ranging from no interaction to multiple sensor–target molecule interactions, for which force–displacement curves are classified. The possibility of recording consistently single-molecule interaction events between an antibody and its specific antigen, in combination with the high proportion of successful interaction events obtained, increases remarkably the possibilities offered by affinity atomic-force microscopy for the characterization of biological and biomimetic systems from the molecular to the tissue scales. [ABSTRACT FROM AUTHOR]

The interaction between the plant lipid transfer protein Pru p 3 and phytosphingosine was assessed using an atomic force microscope. Phytosphingosine was covalently immobilized on DeepTipTM probes and Pru p 3 on MicroDeckTM functionalized substrates. Single-molecular interaction events between both molecules were retrieved and classified and the distribution for each one of the identified types was calculated. A success rate of over 70% was found by comparing the number of specific Pru p 3-phytosphingosine interaction events with the total number of recorded curves. The analysis of the distribution established among the various types of curves was further pursued to distinguish between those curves that can mainly be used for assessing the recognition between phytosphingosine (sensor molecule) and Pru p 3 (target molecule) in the context of affinity atomic force microscopy, and those that entail details of the interaction and might be employed in the context of force spectroscopy. The successful application of these functionalized probes and substrates to the characterization of the low-intensity hydrophobic interaction characteristic of this system is a clear indication of the potential of exploiting this approach with an extremely wide range of different biological molecules of interest. The possibility of characterizing molecular assembly events with single-molecule resolution offers an advantageous procedure to plough into the field of molecular biomimetics. [ABSTRACT FROM AUTHOR]

Titanium (Ti-6Al-4V) substrates were functionalized through the covalent binding of fibronectin, and the effect of the existence of this extracellular matrix protein on the surface of the material was assessed by employing mesenchymal stem cell (MSC) cultures. The functionalization process comprised the usage of the activation vapor silanization (AVS) technique to deposit a thin film with a high surface density of amine groups on the material, followed by the covalent binding of fibronectin to the amine groups using the N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) crosslinking chemistry. The biological effect of the fibronectin on murine MSCs was assessed in vitro. It was found that functionalized samples not only showed enhanced initial cell adhesion compared with bare titanium, but also a three-fold increase in the cell area, reaching values comparable to those found on the polystyrene controls. These results provide compelling evidence of the potential to modulate the response of the organism to an implant through the covalent binding of extracellular matrix proteins on the prosthesis. [ABSTRACT FROM AUTHOR]

The trends exhibited by the parameters that describe the mechanical behaviour of major ampullate gland silk fibers spun by Argiope bruennichi spiders is explored by performing a series of loading-unloading tests at increasing values of strain, and by the subsequent analysis of the true stress-true strain curves obtained from these cycles. The elastic modulus, yields stress, energy absorbed, and energy dissipated in each cycle are computed in order to evaluate the evolution of these mechanical parameters with this cyclic straining. The elastic modulus is observed to increase steadily under these loading conditions, while only a moderate variation is found in the yield stress. It is also observed that a significant proportion of the energy initially absorbed in each cycle is not only dissipated, but that the material may recover partially from the associated irreversible deformation. This variation in the mechanical performance of spider silk is accounted for through a combination of irreversible and reversible deformation micromechanisms in which the viscoelasticity of the material plays a leading role. [ABSTRACT FROM AUTHOR]

After an injury, the limited regenerative capacity of the central nervous system makes the reconnection and functional recovery of the affected nervous tissue almost impossible. To address this problem, biomaterials appear as a promising option for the design of scaffolds that promote and guide this regenerative process. Based on previous seminal works on the ability of regenerated silk fibroin fibers spun through the straining flow spinning (SFS) technique, this study is intended to show that the usage of functionalized SFS fibers allows an enhancement of the guidance ability of the material when compared with the control (nonfunctionalized) fibers. It is shown that the axons of the neurons not only tend to follow the path marked by the fibers, in contrast to the isotropic growth observed on conventional culture plates, but also that this guidance can be further modulated through the biofunctionalization of the material with adhesion peptides. Establishing the guidance ability of these fibers opens the possibility of their use as implants for spinal cord injuries, so that they may represent the core of a therapy that would allow the reconnection of the injured ends of the spinal cord. [ABSTRACT FROM AUTHOR]

Single molecule interactions between biotin and streptavidin were characterized with functionalized DeepTipTM probes and used as a model system to develop a comprehensive methodology for the high-yield identification and analysis of single molecular events. The procedure comprises the covalent binding of the target molecule to a surface and of the sensing molecule to the DeepTipTM probe, so that the interaction between both chemical species can be characterized by obtaining force–displacement curves in an atomic force microscope. It is shown that molecular resolution is consistently attained with a percentage of successful events higher than 90% of the total number of recorded curves, and a very low level of unspecific interactions. The combination of both features is a clear indication of the robustness and versatility of the proposed methodology. [ABSTRACT FROM AUTHOR]

Two different polyglycine-rich fragments were selected as representatives of major ampullate gland spidroins (MaSp) 1 and 2 types, and their behavior in a water-saturated environment was simulated within the framework of molecular dynamics (MD). The selected fragments are found in the sequences of the proteins MaSp1a and MaSp2.2a of Argiope aurantia with respective lengths of 36 amino acids (MaSp1a) and 50 amino acids (MaSp2.2s). The simulation took the fully extended β-pleated conformation as reference, and MD was used to determine the equilibrium configuration in the absence of external forces. Subsequently, MD were employed to calculate the variation in the distance between the ends of the fragments when subjected to an increasing force. Both fragments show an elastomeric behavior that can be modeled as a freely jointed chain with links of comparable length, and a larger number of links in the spidroin 2 fragment. It is found, however, that the maximum recovery force recorded from the spidroin 2 peptide (Fmax ≈ 400 pN) is found to be significantly larger than that of the spidroin 1 (Fmax ≈ 250 pN). The increase in the recovery force of the spidroin 2 polyglycine-rich fragment may be correlated with the larger values observed in the strain at breaking of major ampullate silk fibers spun by Araneoidea species, which contain spidroin 2 proteins, compared to the material produced by spider species that lack these spidroins (RTA-clade). [ABSTRACT FROM AUTHOR]

Age‐associated changes in T‐cell function play a central role in immunosenescence. The role of aging in the decreased T‐cell repertoire, primarily because of thymic involution, has been extensively studied. However, increasing evidence indicates that aging also modulates the mechanical properties of cells and the internal ordering of diverse cell components. Cellular functions are generally dictated by the biophysical phenotype of cells, which itself is also tightly regulated at the molecular level. Based on previous evidence suggesting that the relative nuclear size contributes to variations of T‐cell stiffness, here we examined whether age‐associated changes in T‐cell migration are dictated by biophysical parameters, in part through nuclear cytoskeleton organization and cell deformability. In this study, we first performed longitudinal analyses of a repertoire of 111 functional, biophysical and biomolecular features of the nucleus and cytoskeleton of mice CD4+ and CD8+ T cells, in both naive and memory state. Focusing on the pairwise correlations, we found that age‐related changes in nuclear architecture and internal ordering were correlated with T‐cell stiffening and declined interstitial migration. A similarity analysis confirmed that cell‐to‐cell variation was a direct result of the aging process and we applied regression models to identify biomarkers that can accurately estimate individuals' age. Finally, we propose a biophysical model for a comprehensive understanding of the results: aging involves an evolution of the relative nuclear size, in part through DNA‐hypomethylation and nuclear lamin B1, which implies an increased cell stiffness, thus inducing a decline in cell migration. [ABSTRACT FROM AUTHOR]

High-performance regenerated silkworm (Bombyx mori) silk fibers can be produced efficiently through the straining flow spinning (SFS) technique. In addition to an enhanced biocompatibility that results from the removal of contaminants during the processing of the material, regenerated silk fibers may be functionalized conveniently by using a range of different strategies. In this work, the possibility of implementing various functionalization techniques is explored, including the production of fluorescent fibers that may be tracked when implanted, the combination of the fibers with enzymes to yield fibers with catalytic properties, and the functionalization of the fibers with cell-adhesion motifs to modulate the adherence of different cell lineages to the material. When considered globally, all these techniques are a strong indication not only of the high versatility offered by the functionalization of regenerated fibers in terms of the different chemistries that can be employed, but also on the wide range of applications that can be covered with these functionalized fibers. [ABSTRACT FROM AUTHOR]

The expression of spidroins in the major ampullate, minor ampullate, flagelliform, and tubuliform silk glands of Trichonephila clavipes spiders was analyzed using proteomics analysis techniques. Spidroin peptides were identified and assigned to different gene products based on sequence concurrence when compared with the whole genome of the spider. It was found that only a relatively low proportion of the spidroin genes are expressed as proteins in any of the studied glands. In addition, the expression of spidroin genes in different glands presents a wide range of patterns, with some spidroins being found in a single gland exclusively, while others appear in the content of several glands. The combination of precise genomics, proteomics, microstructural, and mechanical data provides new insights both on the design principles of these materials and how these principles might be translated for the production of high‐performance bioinspired artificial fibers. Research highlights: ‐Proteomics was used to analyse the spidroin content of MA, mi, Flag and Tub glands.‐Peptides were compared with spidroin genes.‐Only a fraction of the genes is expressed in the glands.‐Two proteins were identified in the four glands. [ABSTRACT FROM AUTHOR]

The possibility of spinning regenerated silkworm (Bombyx mori) fibers from a range of aqueous dopes by using a biomimetic approach based on straining flow spinning is explored in this work. It is found that spinning conditions can be established that allow fibers to be produced from environmentally friendly dopes with fibroin concentrations even one order of magnitude lower than that of the natural system. However, it is also found that the spinning process is favored and that the mechanical properties of the fibers are improved when dopes with higher fibroin concentration are employed. Since highly concentrated fibroin solutions in water are unstable, a stabilizing agent is required in order to obtain a spinnable dope at such large protein concentrations. CaCl2 is found to be an adequate stabilizing agent compatible with the straining flow spinning process. The optimization of the spinning parameters leads to the production of high-performance fibers with a work to fracture comparable to that of the natural material. [ABSTRACT FROM AUTHOR]

In the last few decades there has been increasing interest in the spinning of regenerated silk fibers. Although this research started with the use of harsh chemicals to dissolve and then coagulate silk proteins, it has shifted to biomimetic approaches that allow the use of environmentally friendly chemistries. This shift is not surprising since silk fibers in Nature are spun under extremely mild conditions. In this work we use a biomimetic spinning process called straining flow spinning to spin regenerated silkworm silk fibers. Our results show that straining flow spinning is a versatile technique that allows the spinning of silk fibers using different coagulating systems and provides the possibility of developing an industrially scalable and environmentally friendly spinning process. [ABSTRACT FROM AUTHOR]

Mechanical deformability of cells is an important property for their function and development, as well as a useful marker of cell state. The classical technique of micropipette aspiration allows single-cell studies and we provide here a method to measure the two basic mechanical parameters, elastic modulus and Poisson's ratio. The proposed method, developed from finite-element analysis of micropipette aspiration experiments, may be implemented in future technologies for the automated measurement of mechanical properties of cells, based on the micropipette aspiration technique or on the cell transit through flow constrictions. We applied this method to measure the elastic parameters of lymphocytes, in which the mechanical properties depend on their activation state. Additionally, we discuss in this work the accuracy of previous models to estimate the elastic modulus of cells, in particular the analytical model by Theret et al., widely used in the field. We show the necessity of using an improved model, taking into account the finite size of the cells, to obtain new insights that may remain hidden otherwise. [ABSTRACT FROM AUTHOR]

Spider silk fibers were produced through an alternative processing route that differs widely from natural spinning. The process follows a procedure traditionally used to obtain fibers directly from the glands of silkworms and requires exposure to an acid environment and subsequent stretching. The microstructure and mechanical behavior of the so-called spider silk gut fibers can be tailored to concur with those observed in naturally spun spider silk, except for effects related with the much larger cross-sectional area of the former. In particular spider silk gut has a proper ground state to which the material can revert Independently from its previous loading history by supercontraction. A larger cross-sectional area implies that spider silk gut outperforms the natural material in terms of the loads that the fiber can sustain. This property suggests that it could substitute conventional spider silk fibers in some intended uses, such as sutures and scaffolds in tissue engineering. [ABSTRACT FROM AUTHOR]

True stress-true strain curves of naturally spun viscid line fibres retrieved directly from the spiral of orb-webs built by Argiope trifasciata spiders were measured using a novel methodology. This new procedure combines a method for removing the aqueous coating of the fibres and a technique that allows the accurate measurement of their cross-sectional area. Comparison of the tensile behaviour of different samples indicated that naturally spun viscid lines show a large variability, comparable to that of other silks, such as major ampullate gland silk and silkworm silk. Nevertheless, application of a statistical analysis allowed the identification of two independent parameters that underlie the variability and characterize the observed range of true stress-true strain curves. The combination of this result with previous mechanical and microstructural data suggested the assignment of these two independent effects to the degree of alignment of the protein chains and to the local relative humidity, which, in turn, depends on the composition of the viscous coating and on the external environmental conditions. [ABSTRACT FROM AUTHOR]

Regenerated silkworm fibers spun through a wet-spinning process followed by an immersion postspinning drawing step show a work to fracture comparable with that of natural silkworm silk fibers in a wide range of spinning conditions. The mechanical behavior and microstructure of these high performance fibers have been characterized, and compared with those fibers produced through conventional spinning conditions. The comparison reveals that both sets of fibers share a common semicrystalline microstructure, but significant differences are apparent in the amorphous region. Besides, high performance fibers show a ground state and the possibility of tuning their tensile behavior. These properties are characteristic of spider silk and not of natural silkworm silk, despite both regenerated and natural silkworm silk share a common composition different from that of spider silk. © 2011 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2012 [ABSTRACT FROM AUTHOR]

The purpose of this paper is to provide information on the behaviour of steel prestressing wires under likely conditions that could be expected during a fire or impact loads. Four loadings were investigated: a) the influence of strain rate - from 10-3 to 600 s-1 - at room temperature, b) the influence of temperature - from 24 to 600 °C - at low strain rate, c) the influence of the joint effect of strain rate and temperature, and d) damage after three plausible fire scenarios. At room temperature it was found that using 'static' values is a safe option. At high temperatures our results are in agreement with design codes. Regarding the joint effect of temperature and strain rate, mechanical properties decrease with increasing temperature, although for a given temperature, yield stress and tensile strength increase with strain rate. The data provided can be used profitably to model the mechanical behaviour of steel wires under different scenarios. [ABSTRACT FROM AUTHOR]

The possible deleterious effects of coiling and long-term storage of coiled wires on the stress relaxation behaviour of prestressing steel wires has been checked by means of experimental work and a simple analytical model. The results show that if the requirements of standards are fulfilled (minimum coiling diameters), these effects can be neglected. However, some other factors, such as previous residual stresses, long-term storage or storage at high temperatures, can trigger or emphasize this damage to the material. In the authors' opinion, checking the final curvature of the wires after uncoiling prior to prestressing, as required in some standards, is to be recommended. [ABSTRACT FROM AUTHOR]

The article discusses a study which examined the effect of water on regenerated silkworm silk fibers and compared with that of water on natural silkworm silk fibers (SF). The study aimed to clarify some aspects of the microstructure and the relationship between microstructure and mechanical properties of regenerated SF fibers when submerged in water. Also explored are the possibility that water can be used to modify, in a predictable and reproducible way, the properties of SF regenerated fibers.

In prestressed concrete elements, the stress relaxation losses in steel tendons are of paramount importance to structural design. In this work, experimental and numerical results show a clear relationship between stress relaxation and the presence of residual stresses in steel wires for prestressing concrete. Stress relaxation tests were performed at different initial loads, from 50 to 98% of the rupture load, with three types of wire differing only in their distribution of residual stresses. Cold-drawn wires having the same mechanical properties required by standards (ASTM E421, ISO15630/3) but with different residual stresses will exhibit different stress relaxation losses; such losses increase with increasing surface tensile residual stresses (as a consequence of cold drawing) or increasing inner residual tensile stresses (as a consequence of mechanical postdrawing treatments). Actual expressions for prediction of stress relaxation losses do not take into account residual stresses. Better performance of the wires can be achieved by changing the profile of residual stresses according to the requirements of the structures. This note shows how to deal with this challenge. [ABSTRACT FROM AUTHOR]

The outstanding mechanical properties of silk fibers from the spider Argiope trifasciata are reviewed in this article, particularly the tensile behavior under controlled humidity and temperature. Samples obtained by forced silking showed a remarkable reproducibility. A novel procedure, wet stretching, developed by the authors, promises to shed light on the spinning of artificial silk fibers. [ABSTRACT FROM AUTHOR]

A test developed for ambient temperatures is described which may be used to measure the tensile and compressive strengths of concrete at temperatures of liquefied natural gas (LNG) and lower. Tests were performed on specimens of different geometries, ages, and moisture contents. Results have been analyzed and compared with those from two available empirical expressions. The double-punch test is proposed as a simple and reliable test to determine the tensile strength of concrete at very low temperatures. [ABSTRACT FROM AUTHOR]

The prominence of spider silk as a hallmark in biomimetics relies not only on its unrivalled mechanical properties, but also on how these properties are the result of a set of original design principles. In this sense, the study of spider silk summarizes most of the main topics relevant to the field and, consequently, offers a nice example on how these topics could be considered in other biomimetic systems. This review is intended to present a selection of some of the essential design principles that underlie the singular microstructure of major ampullate gland silk, as well as to show how the interplay between them leads to the outstanding tensile behavior of spider silk. Following this rationale, the mechanical behavior of the material is analyzed in detail and connected with its main microstructural features, specifically with those derived from the semicrystalline organization of the fibers. Establishing the relationship between mechanical properties and microstructure in spider silk not only offers a vivid image of the paths explored by nature in the search for high performance materials, but is also a valuable guide for the development of new artificial fibers inspired in their natural counterparts. [ABSTRACT FROM AUTHOR]

The conditions required for the emergence of supercontraction in regenerated silkworm (Bombyx mori) silk fibers are assessed through an experimental approach that combines the spinning of regenerated fibers with controlled properties and their characterization by 13C solid-state nuclear magnetic resonance (NMR). Both supercontracting and non-supercontracting regenerated fibers are produced using the straining flow spinning (SFS) technique from 13C labeled cocoons. The short-range microstructure of the fibers is assessed through 13C CP/MAS in air and 13C DD/MAS in water, and the main microstructural features are identified and quantified. The mechanical properties of the regenerated fibers and their microstructures are compared with those of natural silkworm silk. The combined analysis highlights two possible key elements as responsible for the emergence of supercontraction: (1) the existence of an upper and a lower limit of the amorphous phase compatible with supercontraction, and (2) the existence of two ordered phases, β-sheet A and B, which correspond to different packing arrangements of the protein chains. [ABSTRACT FROM AUTHOR]