Designer peptide-DNA cytoskeletons regulate the function of synthetic cells.

The bottom-up engineering of artificial cells requires a reconfigurable cytoskeleton that can organize at distinct locations and dynamically modulate its structural and mechanical properties. Here, inspired by the vast array of actin-binding proteins and their ability to reversibly crosslink or bundle filaments, we have designed a library of peptide-DNA crosslinkers varying in length, valency and geometry. Peptide filaments conjoint through DNA hybridization give rise to tactoid-shaped bundles with tunable aspect ratios and mechanics. When confined in cell-sized water-in-oil droplets, the DNA crosslinker design guides the localization of cytoskeletal structures at the cortex or within the lumen of the synthetic cells. The tunable spatial arrangement regulates the passive diffusion of payloads within the droplets and complementary DNA handles allow for the reversible recruitment and release of payloads on and off the cytoskeleton. Heat-induced reconfiguration of peptide-DNA architectures triggers shape deformations of droplets, regulated by DNA melting temperatures. Altogether, the modular design of peptide-DNA architectures is a powerful strategy towards the bottom-up assembly of synthetic cells.

Multiscale Microrheology Using Fluctuating Filaments as Stealth Probes.

The mechanical properties of soft materials can be probed on small length scales by microrheology. A common approach tracks fluctuations of micrometer-sized beads embedded in the medium to be characterized. This approach yields results that depend on probe size when the medium has structure on comparable length scales. Here, we introduce filament-based microrheology using high-aspect-ratio semiflexible filaments as probes. Such quasi-1D probes are much less invasive than beads due to their small cross sections. Moreover, by imaging transverse bending modes, we simultaneously determine the micromechanical response of the medium on multiple length scales corresponding to the mode wavelengths. We use semiflexible single-walled carbon nanotubes as probes that can be accurately and rapidly imaged based on their stable near-IR fluorescence. We find that the viscoelastic properties of sucrose, polyethylene oxide, and hyaluronic acid solutions measured in this way are in good agreement with those measured by conventional micro- and macrorheology.

Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues.

Whole-organ/body three-dimensional (3D) staining and imaging have been enduring challenges in histology. By dissecting the complex physicochemical environment of the staining system, we developed a highly optimized 3D staining imaging pipeline based on CUBIC. Based on our precise characterization of biological tissues as an electrolyte gel, we experimentally evaluated broad 3D staining conditions by using an artificial tissue-mimicking material. The combination of optimized conditions allows a bottom-up design of a superior 3D staining protocol that can uniformly label whole adult mouse brains, an adult marmoset brain hemisphere, an ~1 cm3 tissue block of a postmortem adult human cerebellum, and an entire infant marmoset body with dozens of antibodies and cell-impermeant nuclear stains. The whole-organ 3D images collected by light-sheet microscopy are used for computational analyses and whole-organ comparison analysis between species. This pipeline, named CUBIC-HistoVIsion, thus offers advanced opportunities for organ- and organism-scale histological analysis of multicellular systems.

Reply to the 'Comment on "A symmetrical method to obtain shear moduli from microrheology"' by M. Tassieri, Soft Matter, 2018, 14, DOI.

The Comment on our paper introducing "a symmetric method to obtain shear moduli from microrheology" proposes an interpolation method to generate oversampled data from an original time series that are then used to approximate shear moduli at frequencies "beyond the Nyquist frequency." The author states that this can be done without the use of "preconceived fitting functions," implying that the results are unique and reliable. We disagree with these assertions. While it is possible to generate reasonable looking transforms at frequencies above the Nyquist limit by interpolation, any results obtained above the Nyquist limit will be questionable at best. Moreover, while the cubic spline interpolation the author uses may be standard, it constitutes a particular "preconceived" fit and produces oversampled data that are not unique.

Ion Gel Network Formation in an Ionic Liquid Studied by Time-Resolved Small-Angle Neutron Scattering.

We report the time-resolved small-angle neutron scattering (SANS) study of tetra-arm poly(ethylene glycol) (TetraPEG) polymer network formation in a typical ionic liquid (IL), 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)amide ([C2mim][TFSA]). To observe time-dependent SANS profiles, the reaction rate for the AB-type cross-end coupling reaction of TetraPEG macromers was controlled by adding an analogous protic IL, 1-ethylimidazolium TFSA ([C2imH][TFSA]). At polymer concentrations higher than the overlap concentration ( c*), the SANS profile remained unchanged during the gelation reaction, indicating that the network structure was independent of macromer connectivity in a semidiluted solution. On the other hand, at low polymer concentrations, an increase in the SANS profile intensity was clearly observed. The correlation length (ξ), estimated by a fitting analysis based on the Ornstein-Zernike function, increased as the reaction proceeded. This result indicated that the sparsely dispersed macromers formed clusters during the cross-linking process and polymer size growth followed thereafter. We found that the network formation process and homogeneity of the network structure were strongly dependent on the polymer concentration in IL solutions.

A symmetrical method to obtain shear moduli from microrheology.

Passive microrheology typically deduces shear elastic loss and storage moduli from displacement time series or mean-squared displacements (MSD) of thermally fluctuating probe particles in equilibrium materials. Common data analysis methods use either Kramers-Kronig (KK) transformation or functional fitting to calculate frequency-dependent loss and storage moduli. We propose a new analysis method for passive microrheology that avoids the limitations of both of these approaches. In this method, we determine both real and imaginary components of the complex, frequency-dependent response function χ(ω) = χ'(ω) + iχ''(ω) as direct integral transforms of the MSD of thermal particle motion. This procedure significantly improves the high-frequency fidelity of χ(ω) relative to the use of KK transformation, which has been shown to lead to artifacts in χ'(ω). We test our method on both model and experimental data. Experiments were performed on solutions of worm-like micelles and dilute collagen solutions. While the present method agrees well with established KK-based methods at low frequencies, we demonstrate significant improvement at high frequencies using our symmetric analysis method, up to almost the fundamental Nyquist limit.

Defect-free network formation and swelling behavior in ionic liquid-based electrolytes of tetra-arm polymers synthesized using a Michael addition reaction.

The gelation mechanism of tetra-arm poly(ethylene glycol) (TetraPEG) prepolymers via a Michael addition reaction was investigated from the viewpoint of chemical reaction kinetics. The polymer network was formed by mixing two different TetraPEGs functionalized with maleimide and thiol terminal groups (TetraPEG-MA and TetraPEG-SH) in aqueous solutions, and the gelation rate was strongly dependent on the solution pH. We found that the gelation reaction can be a second-order reaction when the acid-base equilibrium of the terminal SH groups (-SH ⇆ -S- + H+) was taken into account, resulting in a quantitative estimation of the rate constant (kgel) in the current polymer solution system. Based on the kgel value, the network connectivity (p), which corresponds to efficiency at the linking point, was evaluated to be p > 95% at the end of the reaction; thus, the resulting TetraPEG hydrogels have a homogeneous polymer network without network defects. We used the TetraPEG network as a polymer matrix in a lithium-ion battery gel electrolyte: dried TetraPEG gels were swollen with ionic liquid-based electrolytes containing Li salts to prepare TetraPEG ion gel electrolytes. Swelling behaviors of the TetraPEG network were characterized from the swelling rate and the equilibrium swelling ratio, and we found that these swelling behaviors were significantly affected by the Li-ion component. We concluded that an intermolecular interaction between Li-ions and the polymer (Li-ion coordination with the O atoms within the PEG chains) plays a key role in the fundamental physical properties of the gel electrolyte.

Decisive test of the ideal behavior of tetra-PEG gels.

The objective of this work is to investigate the thermodynamic and scattering behavior of tetra-poly(ethylene glycol) (PEG) gels. Complementary measurements, including osmotic swelling pressure, elastic modulus, and small angle neutron scattering (SANS), are reported for a series of tetra-PEG gels made from different molecular weight precursor chains at different concentrations. Analysis of the osmotic swelling pressure vs polymer volume fraction curves makes it possible to separate the elastic and mixing contributions of the network free energy. It is shown that in tetra-PEG gels these free energy components are additive. The elastic term varies with the one-third power of the polymer volume fraction and its numerical value is equal to the shear modulus obtained from independent mechanical measurements. The mixing pressure of the cross-linked polymer is slightly smaller than that of the corresponding solution of the uncross-linked polymer of infinite molecular weight but it exhibits similar dependence on the polymer concentration. The observed deviation between the osmotic mixing pressures of the gel and the solution can be attributed to the presence of small amount of structural inhomogeneities frozen-in by the cross-links. SANS reveals that the scattering response of tetra-PEG gel is mainly governed by the thermodynamic concentration fluctuations of the network, i.e., the contribution from static inhomogeneities to the SANS signal is small.

2D pair distribution function analysis of anisotropic small-angle scattering patterns from elongated nano-composite hydrogels.

Small angle scattering (SAS) on polymer nanocomposites under elongation or shear flow is an important experimental method to investigate the reinforcement effects of the mechanical properties by fillers. However, the anisotropic scattering patterns that appear in SAS are very complicated and difficult to interpret. A representative example is a four-spot scattering pattern observed in the case of polymer materials containing silica nanoparticles, the origin of which is still in debate because of the lack of quantitative analysis. The difficulties in the interpretation of anisotropic scattering patterns mainly arise from the abstract nature of the reciprocal space. Here, we focus on the 2D pair distribution function (PDF) directly evaluated from anisotropic scattering patterns. We applied this method to elongated poly(N,N-dimethylacrylamide) gels containing silica nanoparticles (PDAM-NP gel), which show a four-spot scattering pattern under elongation. From 2D PDFs, we obtained detailed and concrete structural information about the elongated PDAM-NP gel, such as affine and non-affine displacements of directly attached and homogeneously dispersed silica nanoparticles, respectively. We proposed that nanoparticles homogeneously dispersed in the perpendicular direction are not displaced due to the collision of the adsorbed polymer layer during elongation, while those in the parallel direction are displaced in an affine way. We assumed that this suppression of the lateral compression is the origin of the four-spot pattern in this study. These results strongly indicate that our 2D PDF analysis will provide deep insight into the internal structure of polymer nanocomposites hidden in the anisotropic scattering patterns.

Experimental Observation of Two Features Unexpected from the Classical Theories of Rubber Elasticity.

Although the elastic modulus of a Gaussian chain network is thought to be successfully described by classical theories of rubber elasticity, such as the affine and phantom models, verification experiments are largely lacking owing to difficulties in precisely controlling of the network structure. We prepared well-defined model polymer networks experimentally, and measured the elastic modulus G for a broad range of polymer concentrations and connectivity probabilities, p. In our experiment, we observed two features that were distinct from those predicted by classical theories. First, we observed the critical behavior G∼|p-p_{c}|^{1.95} near the sol-gel transition. This scaling law is different from the prediction of classical theories, but can be explained by analogy between the electric conductivity of resistor networks and the elasticity of polymer networks. Here, p_{c} is the sol-gel transition point. Furthermore, we found that the experimental G-p relations in the region above C^{*} did not follow the affine or phantom theories. Instead, all the G/G_{0}-p curves fell onto a single master curve when G was normalized by the elastic modulus at p=1, G_{0}. We show that the effective medium approximation for Gaussian chain networks explains this master curve.

Probe-SAXS on hydrogels under elongation.

We have investigated the effect of polymer/filler interaction on the displacements of silica nanoparticles in gels by introducing them into poly(N,N-dimethylacrylamide) gel (PDAM-NP gel) and polyacrylamide gel (PAM-NP gel). It is well known that PDAM chains are strongly adsorbed onto silica nanoparticles, while PAM chains are not. We carried out SAXS measurements on these gels under uniaxial elongation. Interestingly, we found that the SAXS scattering profiles of PDAM-NP and PAM-NP gels were totally different. A four-spot pattern was observed in the 2D structure factors of the PDAM-NP gel and was assigned to the movement of the nanoparticles in an affine way. On the other hand, as for the PAM-NP gel, a sharp peak was observed in the much lower q region than the prediction of affine deformation, indicating that the peak corresponds to the correlation peak of the high cross-linking region. These experimental findings may lead to the development of "probe-SAXS", which is a new technology for detecting nano-order inhomogeneity in hydrogels.

Rubber elasticity for percolation network consisting of Gaussian chains.

A theory describing the elastic modulus for percolation networks of Gaussian chains on general lattices such as square and cubic lattices is proposed and its validity is examined with simulation and mechanical experiments on well-defined polymer networks. The theory was developed by generalizing the effective medium approximation (EMA) for Hookian spring network to Gaussian chain networks. From EMA theory, we found that the ratio of the elastic modulus at p, G to that at p = 1, G0, must be equal to G/G0 = (p - 2/f)/(1 - 2/f) if the position of sites can be determined so as to meet the force balance, where p is the degree of cross-linking reaction. However, the EMA prediction cannot be applicable near its percolation threshold because EMA is a mean field theory. Thus, we combine real-space renormalization and EMA and propose a theory called real-space renormalized EMA, i.e., REMA. The elastic modulus predicted by REMA is in excellent agreement with the results of simulations and experiments of near-ideal diamond lattice gels.

Electrophoretic mobility of semi-flexible double-stranded DNA in defect-controlled polymer networks: Mechanism investigation and role of structural parameters.

Our previous studies have reported an empirical model, which explains the electrophoretic mobility (µ) of double-stranded DNA (dsDNA) as a combination of a basic migration term (Rouse-like or reptation) and entropy loss term in polymer gels with ideal network structure. However, this case is of exception, considering a large amount of heterogeneity in the conventional polymer gels. In this study, we systematically tune the heterogeneity in the polymer gels and study the migration of dsDNA in these gels. Our experimental data well agree with the model found for ideal networks. The basic migration mechanism (Rouse-like or reptation) persists perfectly in the conventional heterogeneous polymer gel system, while the entropy loss term continuously changes with increase in the heterogeneity. Furthermore, we found that in the limit where dsDNA is shorter than dsDNA persistence length, the entropy loss term may be related to the collisional motions between DNA fragments and the cross-links.

Gelation mechanism of tetra-armed poly(ethylene glycol) in aprotic ionic liquid containing nonvolatile proton source, protic ionic liquid.

We report the gelation mechanism of tetra-armed prepolymer chains in typical aprotic ionic liquid (aIL), i.e., A-B type cross-end coupling reaction of tetra-armed poly(ethylene glycol)s with amine and activated ester terminals (TetraPEG-NH2 and TetraPEG-NHS, respectively) in 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)amide ([C2mIm][TFSA]). In the ion gel system, we focused on the pH (or H(+) concentration) dependence of the gelation reaction. We thus applied the protic ionic liquid (pIL), 1-ethylimidazolium TFSA ([C2ImH][TFSA]), as a nonvolatile H(+) source, and added it into the solvent aIL. It was found that the gelation time of TetraPEG ion gel can be successfully controlled from 1 min to 3 h depending on the concentration of pIL (cpIL = 0-3 mM). This suggests that the acid-base properties of TetraPEG-NH2 showing acid-base equilibrium (-NH2 + H(+) ⇆ -NH3(+)) in the solutions play a key role in the gelation process. The acid dissociation constants, pKa's of TetraPEG-NH3(+) and C2ImH(+) (cation of pIL) in aIL were directly determined by potentiometric titration to be 16.4 and 13.7, respectively. This indicates that most of the H(+) ions bind to TetraPEG-NH2 and then C2ImH(+) exists as neutral C2Im. The reaction efficiency of amide bond (cross-linked point) systematically decreased with increasing cpIL, which was reflected to the mechanical strength of the ion gels. From these results, we discuss the gelation mechanism of TetraPEG in aIL to point out the relationship between polymer network structure and [H(+)] in the solutions.

Rubber elasticity for incomplete polymer networks.

We investigated the relationship between the elastic modulus, G and the reaction probability, p for polymer networks. First, we pointed out that the elastic modulus is expressed by G = {(fp∕2 - 1) + O((p - 1)(2))} Nk(B)T∕V (percolated network law), which does not depend on the local topology of the network structure or the existence of the loops. Here, N is the number of lattice point, V is the system volume, f is the functionality of the cross-link, k(B) is the Boltzmann constant, and T is the absolute temperature. We also conducted simulations for polymer networks with triangular and diamond lattices, and mechanical testing experiments on tetra-poly(ethylene glycol) (PEG) gel with systematically tuning the reaction probability. Here, the tetra-PEG gel was confirmed to be a potential candidate for ideal polymer networks consisting of unimodal strands free from defects and entanglements. From the results of simulations and experiments, it was revealed, for the first time, that the elastic modulus obeys this law in the wide range of p (p(c) ≪ p ≤ 1), where p(c) is the reaction probability at gelation threshold.

Muir-Torre syndrome associated with endometrial carcinoma.

Muir-Torre syndrome (MTS) is an autosomaldominant skin condition of genetic origin, characterized by tumors of the sebaceous glands or keratoacanthomas that are associated with malignant visceral diseases. MTS associated with gynecologic malignancy has rarely been reported. Here we report a woman with no family history of colorectal cancer who developed endometrial carcinoma, stage 3a, at 49 years of age and at age 51 years, developed two skin tumors, a nasal squamous cell carcinoma and a sebaceous carcinoma of the right eyelid. The appearance pattern of these skin tumors suggested MTS. Although MTS associated with endometrial carcinoma is rare, patients with endometrial carcinoma should undergo evaluation for visceral malignancies (mainly colon cancer) and sebaceous skin lesions, regardless of whether or not there is a family history of colorectal cancer.