A new training model using the self-healing properties of supramolecular hydrogels for endoscopic combined intrarenal surgery.

The combination of hydronephrosis formation, ureteroscopic imaging, and ultrasound delineation has not been included in any non-biological training model of percutaneous nephrolithotomy or endoscopic combined intrarenal surgery. We aimed to develop a realistic kidney phantom using the self-healing properties of supramolecular hydrogels for percutaneous nephrolithotomy and endoscopic combined intrarenal surgery and evaluate its suitability as a training model.Expert and resident urologists performed ultrasound-guided renal pelvic punctures and flexible ureteroscopies for endoscopic combined intrarenal surgery using a training model. Subsequently, the training model was evaluated using a 17-item Likert scale questionnaire (range, 1-5 points). After being filled with carrageenan, the collecting system was inflated, and the relationship between the collecting system volume and collecting system pressure was determined. The durability of the model was verified by repeatedly inserting a 16-Fr access sheath. Five novices and seven urology experts performed the procedure. The mean questionnaire score was 4.25 (standard deviation, 0.37). The model was able to hold 50 mL of air, and the pressure in the collecting system ranged from 6 to 33 mmHg. Repeated punctures were possible even when a 16-Fr access sheath was inserted. Our new training model included the self-healing properties of supramolecular hydrogels, which are tough and flexible and can be evaluated using ultrasonography. According to the questionnaire score, the model was highly satisfactory and has potential as a new educational tool.

A Fiber-Optic Non-Invasive Swallowing Assessment Device Based on a Wearable Pressure Sensor.

We developed a wearable swallowing assessment device using a hetero-core fiber-optic pressure sensor for the detection of laryngeal movement during swallowing. The proposed pressure sensor (comfortably attached to the skin of the neck) demonstrated a high sensitivity of 0.592 dB/kPa and a linearity of R2 = 0.995 within a 14 kPa pressure band, which is a suitable pressure for the detection of laryngeal movement. In addition, since the fabricated hetero-core fiber-optic pressure sensor maintains appreciable sensitivity over the surface of the sensor, the proposed wearable swallowing assessment device can accurately track the subtle pressure changes induced by laryngeal movements during the swallowing process. Sixteen male subjects and one female subject were evaluated in a variety of age groups ranging from 30 to 60 years old. For all subjects, characteristic swallowing waveforms (with two valleys based on laryngeal movements consisting of upward, forward, backward, and downward displacements) were acquired using the proposed wearable swallowing assessment device. Since the denoted time of the first valley in the acquired waveform determines the "aging effect", significant differences in swallowing functions among the different age groups were ultimately determined based on the time of the first valley. Additionally, by analyzing each age group using the proposed device, due to p-values being consistently less than 0.05, swallowing times were found to exhibit statistically significant differences within the same groups.

Rheological and mechanical properties of edible gel materials for 3D food printing technology.

3D food printing sectors require comprehensive knowledge on viscoelastic and mechanical properties of diverse food materials in order to effectively utilize them in rapid and customized 3D production for supply and manufacturing chains. In this work, we present mechanical and rheological properties of Agar and Konjac based edible gels at different Agar and Konjac weight ratio and discuss their 3D printing performance. Gel samples with higher Konjac content positively contributed to the viscoelastic properties of the gel samples which in return has been found viable for extrusion-based 3D printing. By choosing appropriate printing parameters, different shapes are printed to demonstrate printing resolution. We expect, this study will add potential scope for evaluating and optimizing soft-gel materials for 3D food printing sector.

Light Scattering and Rheological Studies of 3D/4D Printable Shape Memory Gels Based on Poly (N,N-Dimethylacrylamide-co-Stearyl Acrylate and/or Lauryl Acrylates).

In this work, we present the structural analysis of 3D/4D printable N,N-dimethylacrylamide (DMAAm)-co-stearyl acrylate (SA) and/or lauryl acrylate (LA)-based shape memory gels (SMGs). We characterized these gels by scanning microscopic light scattering technique (SMILS) where a time- and space-averaged correlation function is obtained to overcome the inhomogeneous media. Thus, the characteristic size of the gel internal network (mesh size, ξ) and crosslinking densities are estimated from the Einstein-Stokes formula. The rheological study of the SMGs revealed information about their mechanical strength and transition temperature. From the experimental storage modulus measured by rheological study, crosslinking density and mesh size of the network were also calculated. Both the techniques suggest that SMG with high crystalline content of SA monomer in the gel network contain smaller mesh size (1.13 nm for SMILS and 9.5 nm for rheology study) and high crosslinking density. The comparative study between the light scattering technique and rheological analysis through the quantitative analysis of crosslinking densities will be important to understand the structural properties of the SMGs.

Identification of novel protein domain for sialyloligosaccharide binding essential to Mycoplasma mobile gliding.

Sialic acids, terminal structures of sialylated glycoconjugates, are widely distributed in animal tissues and are often involved in intercellular recognitions, including some bacteria and viruses. Mycoplasma mobile, a fish pathogenic bacterium, binds to sialyloligosaccharide (SO) through adhesin Gli349 and glides on host cell surfaces. The amino acid sequence of Gli349 shows no similarity to known SO-binding proteins. In the present study, we predicted the binding part of Gli349, produced it in Escherichia coli and proved its binding activity to SOs of fetuin using atomic force microscopy. Binding was detected with a frequency of 10.3% under retraction speed of 400 nm/s and was shown to be specific for SO, as binding events were competitively inhibited by the addition of free 3'-sialyllactose. The histogram of the unbinding forces showed 24 pN and additional peaks. These results suggested that the distal end of Gli349 constitutes a novel sialoadhesin domain and is directly involved in the gliding mechanism of M. mobile.

Carbon fiber doped thermosetting elastomer for flexible sensors: physical properties and microfabrication.

We have developed conductive microstructures using micropatternable and conductive hybrid nanocomposite polymer. In this method carbon fibers (CFs) were blended into polydimethylsiloxane (PDMS). Electrical conductivities of different compositions were investigated with various fiber lengths (50-250 µm), and weight percentages (wt%) (10-60 wt%). Sample composites of 2 cm × 1 cm × 500 µm were fabricated for 4-point probe conductivity measurements. The measured percolation thresholds varied with length of the fibers: 50 wt% (307.7 S/m) for 50 µm, 40 wt% (851.1 S/m) for 150 µm, and 30 wt% (769.23 S/m) for 250 µm fibers. The conductive composites showed higher elastic modulus when compared to that of PDMS.

3D printing of shape memory hydrogels with tunable mechanical properties.

Utilization of soft material like hydrogels for task-specific applications such as in soft robotics requires freedom in the manufacturing process and designability. Here, we have developed highly robust thermoresponsive poly(dimethyl acrylamide-co-stearyl acrylate and/or lauryl acrylate) (PDMAAm-co-SA and/or LA)-based shape memory gels (SMGs) using a customized optical 3D gel printer. This process enabled rapid and moldless fabrication of SMGs with a variety of shapes and sizes. By varying the compositions of the constituent monomers, a wide variety of SMGs with tunable mechanical, thermal, optical and swelling properties have been obtained. Printed SMGs with excellent fixity and recovery ratios have exhibited a wide range of values of Young's modulus (0.04-17.35 MPa) and strain (612-2363%) at room temperature when the acrylate co-monomer (SA and LA) content was varied and the value of strain has been found to be enhanced at elevated temperatures. Thermogravimetric analysis (TGA) of the SMGs shows one step peak degradation (407-417 °C) regardless of composition after an initial mass loss due to water evaporation. Dynamic mechanical analysis (DMA) and differential scanning calorimetry (DSC) revealed variable transition temperatures (29-49.5 °C) depending on the SA and LA content. SMGs with all of the composition ratios possess high transparency with variable swelling degrees in water and different organic solvents and exhibit refractive index values in the range of intraocular lenses, making them suitable for applications in the optical field. These unique properties of 3D printed SMGs with free formability and tunable properties are expected to generate rapid demand in a variety of sectors in biomedicine, robotics and sensing applications.

Evaluation of 3D Printer Accuracy in Producing Fractal Structure.

Hierarchical structures, also known as fractal structures, exhibit advantageous material properties, such as water- and oil-repellency as well as other useful optical characteristics, owing to its self-similarity. Various methods have been developed for producing hierarchical geometrical structures. Recently, fractal structures have been manufactured using a 3D printing technique that involves computer-aided design data. In this study, we confirmed the accuracy of geometrical structures when Koch curve-like fractal structures with zero to three generations were printed using a 3D printer. The fractal dimension was analyzed using a box-counting method. This analysis indicated that the fractal dimension of the third generation hierarchical structure was approximately the same as that of the ideal Koch curve. These findings demonstrate that the design and production of fractal structures can be controlled using a 3D printer. Although the interior angle deviated from the ideal value, the side length could be precisely controlled.

Optimizing the calculation of energy landscape parameters from single-molecule protein unfolding experiments.

Single-molecule force spectroscopy using an atomic force microscope (AFM) can be used to measure the average unfolding force of proteins in a constant velocity experiment. In combination with Monte Carlo simulations and through the application of the Zhurkov-Bell model, information about the parameters describing the underlying unfolding energy landscape of the protein can be obtained. Using this approach, we have completed protein unfolding experiments on the polyprotein (I27)(5) over a range of pulling velocities. In agreement with previous work, we find that the observed number of protein unfolding events observed in each approach-retract cycle varies between one and five, due to the nature of the interactions between the polyprotein, the AFM tip, and the substrate, and there is an unequal unfolding probability distribution. We have developed a Monte Carlo simulation that incorporates the impact of this unequal unfolding probability distribution on the median unfolding force and the calculation of the protein unfolding energy landscape parameters. These results show that while there is a significant, unequal unfolding probability distribution, the unfolding energy landscape parameters obtained from use of the Zhurkov-Bell model are not greatly affected. This result is important because it demonstrates that the minimum acceptance criteria typically used in force extension experiments are justified and do not skew the calculation of the unfolding energy landscape parameters. We further validate this approach by determining the error in the energy landscape parameters for two extreme cases, and we provide suggestions for methods that can be employed to increase the level of accuracy in single-molecule experiments using polyproteins.

Nanomechanics of HaloTag tethers.

The active site of the Haloalkane Dehydrogenase (HaloTag) enzyme can be covalently attached to a chloroalkane ligand providing a mechanically strong tether, resistant to large pulling forces. Here we demonstrate the covalent tethering of protein L and I27 polyproteins between an atomic force microscopy (AFM) cantilever and a glass surface using HaloTag anchoring at one end and thiol chemistry at the other end. Covalent tethering is unambiguously confirmed by the observation of full length polyprotein unfolding, combined with high detachment forces that range up to ∼2000 pN. We use these covalently anchored polyproteins to study the remarkable mechanical properties of HaloTag proteins. We show that the force that triggers unfolding of the HaloTag protein exhibits a 4-fold increase, from 131 to 491 pN, when the direction of the applied force is changed from the C-terminus to the N-terminus. Force-clamp experiments reveal that unfolding of the HaloTag protein is twice as sensitive to pulling force compared to protein L and refolds at a slower rate. We show how these properties allow for the long-term observation of protein folding-unfolding cycles at high forces, without interference from the HaloTag tether.

Variation in the mechanical unfolding pathway of p53DBD induced by interaction with p53 N-terminal region or DNA.

The tumor suppressor p53 plays a crucial role in the cell cycle checkpoints, DNA repair, and apoptosis. p53 consists of a natively unfolded N-terminal region (NTR), central DNA binding domain (DBD), C-terminal tetramerization domain, and regulatory region. In this paper, the interactions between the DBD and the NTR, and between the DBD and DNA were investigated by measuring changes in the mechanical unfolding trajectory of the DBD using atomic force microscopy (AFM)-based single molecule force spectroscopy. In the absence of DNA, the DBD (94-293, 200 amino acids (AA)) showed two different mechanical unfolding patterns. One indicated the existence of an unfolding intermediate consisting of approximately 60 AA, and the other showed a 100 AA intermediate. The DBD with the NTR did not show such unfolding patterns, but heterogeneous unfolding force peaks were observed. Of the heterogeneous patterns, we observed a high frequency of force peaks indicating the unfolding of a domain consisting of 220 AA, which is apparently larger than that of a sole DBD. This observation implies that a part of NTR binds to the DBD, and the mechanical unfolding happens not solely on the DBD but also accompanying a part of NTR. When DNA is bound, the mechanical unfolding trajectory of p53NTR+DBD showed a different pattern from that without DNA. The pattern was similar to that of the DBD alone, but two consecutive unfolding force peaks corresponding to 60 and 100 AA sub-domains were observed. These results indicate that interactions with the NTR or DNA alter the mechanical stability of DBD and result in drastic changes in the mechanical unfolding trajectory of the DBD.

A soft and transparent handleable protein model.

The field of structural biology currently relies on computer-generated graphical representations of three-dimensional (3D) structures to conceptualize biomolecules. As the size and complexity of the molecular structure increases, model generation and peer discussions become more difficult. It is even more problematic when discussing protein-protein interactions wherein large surface area contact is considered. This report demonstrates the viability of a new handleable protein molecular model with a soft and transparent silicone body similar to the molecule's surface. A full-color printed main chain structure embedded in the silicone body enables users to simultaneously feel the molecular surface, view through the main chain structure, and manually simulate molecular docking. The interactive, hands-on experience deepens the user's intuitive understanding of the complicated 3D protein structure and elucidates ligand binding and protein-protein interactions. This model would be an effective discussion tool for the classroom or laboratory that stimulates inspired learning in this study field.

Prying open single GroES ring complexes by force reveals cooperativity across domains.

Understanding how the mechanical properties of a protein complex emerge from the interplay of intra- and interchain interactions is vital at both fundamental and applied levels. To investigate whether interdomain cooperativity affects protein mechanical strength, we employed single-molecule force spectroscopy to probe the mechanical stability of GroES, a homoheptamer with a domelike quaternary stucture stabilized by intersubunit interactions between the first and last ß-strands of adjacent domains. A GroES variant was constructed in which each subunit of the GroES heptamer is covalently linked to adjacent subunits by tripeptide linkers and folded domains of protein L are introduced to the heptamer's termini as handle molecules. The force-distance profiles for GroES unfolding showed, for the first time that we know of, a mechanical phenotype whereby seven distinct force peaks, with alternating behavior of unfolding force and contour length (ΔL(c)), were observed with increasing unfolding-event number. Unfolding of (GroES)(7) is initiated by breakage of the interface between domains 1 and 7 at low force, which imparts a polarity to (GroES)(7) that results in two distinct mechanical phenotypes of these otherwise identical protein domains. Unfolding then proceeds by peeling domains off the domelike native structure by sequential repetition of the denaturation of mechanically weak (unfoldon 1) and strong (unfoldon 2) units. These results indicate that domain-domain interactions help to determine the overall mechanical strength and unfolding pathway of the oligomeric structure. These data reveal an unexpected richness in the mechanical behavior of this homopolyprotein, yielding a complex with greater mechanical strength and properties distinct from those that would be apparent for GroES domains in isolation.

Mechanical unfolding studies of protein molecules.

Atomic force microscopy (AFM) enables the pick up of a single protein molecule to apply a mechanical force. This technique, called "force spectroscopy," provides unique information about the intermediates and free energy landscape of the mechanical unfolding of proteins. In this review, we introduce the AFM-based single molecule force spectroscopy of proteins and describe recent studies that answer some fundamental questions such as "is the mechanical resistance of proteins isotropic?", "what is the structure of the transition state in mechanical unfolding?", and "is mechanical unfolding related to biological functions?"

Dynamics of the coiled-coil unfolding transition of myosin rod probed by dissipation force spectrum.

The motor protein myosin II plays a crucial role in muscle contraction. The mechanical properties of its coiled-coil region, the myosin rod, are important for effective force transduction during muscle function. Previous studies have investigated the static elastic response of the myosin rod. However, analogous to the study of macroscopic complex fluids, how myosin will respond to physiological time-dependent loads can only be understood from its viscoelastic response. Here, we apply atomic force microscopy using a magnetically driven oscillating cantilever to measure the dissipative properties of single myosin rods that provide unique dynamical information about the coiled-coil structure as a function of force. We find that the friction constant of the single myosin rod has a highly nontrivial variation with force; in particular, the single-molecule friction constant is reduced dramatically and increases again as it passes through the coiled-uncoiled transition. This is a direct indication of a large free-energy barrier to uncoiling, which may be related to a fine-tuned dynamic mechanosignaling response to large and unexpected physiological loads. Further, from the critical force at which the minimum in friction occurs we determine the asymmetry of the bistable landscape that controls uncoiling of the coiled coil. This work highlights the sensitivity of the dissipative signal in force unfolding to dynamic molecular structure that is hidden to the elastic signal.

Application of HaloTag protein to covalent immobilization of recombinant proteins for single molecule force spectroscopy.

We have developed the HaloTag system for the covalent immobilization of polyproteins onto a mica substrate for single molecule force spectroscopy using the atomic force microscope. A recombinant fusion polyprotein of titin I27 with HaloTag7 protein was produced, and the covalent and site-specific attachment on a HaloTag-ligand-modified mica surface was confirmed by force-extension measurements. Two mechanical unfolding intermediates of HaloTag7 protein were found by contour length analysis. This tethering method allows site-specific covalent immobilization of a protein that complements the standard method utilizing thiol-gold interaction, thus facilitating force-extension measurements for cysteine-containing proteins.

Reduction of the damping on an AFM cantilever in fluid by the use of micropillars.

In single molecule force measurements with soft atomic force microscope (AFM) cantilevers, the force sensitivity is limited by the Brownian motion of the cantilever. When a cantilever is close to the surface, the hydrodynamic interaction between the cantilever beam and the surface, called the "squeezing effect", becomes significant, and the resonance peak of the thermal oscillation of the cantilever is heavily broadened and shifted to lower frequency which makes it difficult to eliminate the thermal noise by low-pass filtering. In this study, we propose an easy and low-cost method to improve the force sensitivity. We demonstrate that by bringing a tip of a cantilever onto the edge of a micropillar structure a significant reduction of the damping and an enhancement of force sensitivity are achieved.

Identification of a mechanical rheostat in the hydrophobic core of protein L.

The ability of proteins and their complexes to withstand or respond to mechanical stimuli is vital for cells to maintain their structural organisation, to relay external signals and to facilitate unfolding and remodelling. Force spectroscopy using the atomic force microscope allows the behaviour of single protein molecules under an applied extension to be investigated and their mechanical strength to be quantified. protein L, a simple model protein, displays moderate mechanical strength and is thought to unfold by the shearing of two mechanical sub-domains. Here, we investigate the importance of side-chain packing for the mechanical strength of protein L by measuring the mechanical strength of a series of protein L variants containing single conservative hydrophobic volume deletion mutants. Of the five thermodynamically destabilized variants characterised, only one residue (I60 V) close to the interface between two mechanical sub-domains was found to differ in mechanical properties to wild type (Delta F(I60 V-WT)=-36 pN at 447 nm s(-1), Delta x(uI60V-WT)=0.2 nm). Phi-value analysis of the unfolding data revealed a highly native transition state. To test whether the number of hydrophobic contacts across the mechanical interface does affect the mechanical strength of protein L, we measured the mechanical properties of two further variants. protein L L10F, which increases core packing but does not enhance interfacial contacts, increased mechanical strength by 13+/-11 pN at 447 nm s(-1). By contrast, protein L I60F, which increases both core and cross-interface contacts, increased mechanical strength by 72+/-13 pN at 447 nm s(-1). These data suggest a method by which nature can evolve a varied mechanical response from a limited number of topologies and demonstrate a generic but facile method by which the mechanical strength of proteins can be rationally modified.

Internal friction of single polypeptide chains at high stretch.

Experiments that measure the viscoelasticity of single molecules from the Brownian fluctuations of an atomic force microscope (AFM) have provided a new window onto their internal dynamics in an underlying conformational landscape. Here we develop and apply these methods to examine the internal friction of unfolded polypeptide chains at high stretch. The results reveal a power law dependence of internal friction with tension (exponent 1.3 +/- 0.5) and a relaxation time approximately independent of force. To explain these results we develop a frictional worm-like chain (FWLC) model based on the Rayleigh dissipation function of a stiff chain with dynamical resistance to local bending. We analyse the dissipation rate integrated over the chain length by its Fourier components to calculate an effective tension-dependent friction constant for the end-to-end vector of the chain. The result is an internal friction that increases as a power law with tension with an exponent 3/2, consistent with experiment. Extracting the intrinsic bending friction constant of the chain it is found to be approximately 7 orders of magnitude greater than expected from solvent friction alone; a possible explanation we offer is that the underlying energy landscape for bending amino acids and/or peptide bond is rough, consistent with recent results on both proteins and polysaccharides.

The effect of temperature on mechanical resistance of the native and intermediate states of I27.

We investigated the effect of temperature on the mechanical unfolding of I27 from human cardiac titin, employing a custom-built temperature control device for single-molecule atomic force microscopy measurement. A sawtooth pattern was observed in the force curves where each force peak reports on the unfolding of an I27 domain. In early unfolding events, we observed a "hump-like" deviation due to the detachment of beta-strand A from each I27 domain. The force at which the humps appear was approximately 130 pN and showed no temperature dependence, at least in the temperature range of 2 degrees C-30 degrees C. The hump structure was successfully analyzed with a two-state worm-like chain model, and the Gibbs free energy difference of the detachment reaction was estimated to be 11.6 +/- 0.58 kcal/mol and found to be temperature independent. By contrast, upon lowering the temperature, the mean unfolding force from the partly unfolded intermediate state was found to markedly increase and the unfolding force distribution to broaden significantly, suggesting that the distance (x(u)) between the folded and transition states in the energy landscape along the pulling direction is decreased. These results suggest that the local structure of beta-strand A are stabilized by topologically simple local hydrogen-bond network and that the temperature does not affect the detachment reaction thermodynamically and kinetically, whereas the interaction between the beta-strands A' and G, which is a critical region for its mechanical stability, is strongly dependent on the temperature.