Structural Heterogeneity and Its Influence on Nonlinear Deformation and the Fracture of Ultrasoft Hydrogels

Understanding the deformation and fracture behavior of human organs exhibiting a very low stiffness (<1 kPa) is of vital importance to gain information on how these soft tissues react to the medical device during loading conditions in surgical robotics applications. We investigated the deformation and fracture behavior of model poly(vinyl alcohol) ultrasoft hydrogels by puncture tests with flat-ended indenters whose size is comparable to the elasto-capillary length, with a particular focus on the effect of structural heterogeneity. By tuning the polymer and cross-linker concentrations, gels with a modulus ranging from 56–2700 Pa were synthesized, with varied structural heterogeneity, examined by light scattering. We found that structural heterogeneity plays a key role in lowering the fracture resistance of the soft gels under large nonlinear deformations. This work provides insights into the mechanics and fracture of ultrasoft materials under extreme deformation conditions and opens the question of the interplay between elasticity and capillarity in such ultrasoft gels at small length scales

Residue-specific conformation and dynamics of the wild type prion-like domain in aqueous solution.

<p>(A) Residue specific (ΔCα-ΔCβ) chemical shifts of the prion-like domain at 25°C. Green arrows are used for indicating the regions populated with nascent helical conformations while red arrows for those with extended conformations. (B) Secondary structure score obtained by analyzing chemical shifts of the prion-like domain with the SSP program. A score of +1 is for the well-formed helix while a score of -1 for the well-formed extended strand. (C) {¹H}-¹⁵N heteronuclear steady-state NOE (hNOE) of the prion-like domain at 25°C. (D) Residue-specific temperature coefficients of the wild-type prion-like domain in Milli-Q water at pH 4.0 (blue), in 1 mM phosphate buffer at pH 5.0 (red) and in 1 mM phosphate buffer at pH 6.0 (cyan). (E) NOE connectivities defining secondary structures of the prion-like domain. NMR data for preparing the above figures are presented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002338#pbio.1002338.s001" target="_blank">S1 Data</a>.</p

Conformations of the wild type and three mutants in bicelle.

<p>(A) Far-UV CD spectra of the wild-type and three mutant domains acquired at 25°C in the presence of DMPC/DHPC bicelle at a ratio of 1:200 after 5 min and 1 d. Superimposition of HSQC spectra acquired at 25°C in the presence of the DMPC/DHPC bicelle at a ratio of 1:200 for the wild type (blue) and mutants (red) for A315E (B), Q331K (C) and M337V (D). The mutant residues with HSQC peaks significantly shifted from those of the corresponding wild-type residues are labeled.</p

ALS-Causing Mutations Significantly Perturb the Self-Assembly and Interaction with Nucleic Acid of the Intrinsically Disordered Prion-Like Domain of TDP-43

<div><p>TAR-DNA-binding protein-43 (TDP-43) C-terminus encodes a prion-like domain widely presented in RNA-binding proteins, which functions to form dynamic oligomers and also, amazingly, hosts most amyotrophic lateral sclerosis (ALS)-causing mutations. Here, as facilitated by our previous discovery, by circular dichroism (CD), fluorescence and nuclear magnetic resonance (NMR) spectroscopy, we have successfully determined conformations, dynamics, and self-associations of the full-length prion-like domains of the wild type and three ALS-causing mutants (A315E, Q331K, and M337V) in both aqueous solutions and membrane environments. The study decodes the following: (1) The TDP-43 prion-like domain is intrinsically disordered only with some nascent secondary structures in aqueous solutions, but owns the capacity to assemble into dynamic oligomers rich in β-sheet structures. By contrast, despite having highly similar conformations, three mutants gained the ability to form amyloid oligomers. The wild type and three mutants all formed amyloid fibrils after incubation as imaged by electron microscopy. (2) The interaction with nucleic acid enhances the self-assembly for the wild type but triggers quick aggregation for three mutants. (3) A membrane-interacting subdomain has been identified over residues Met311-Gln343 indispensable for TDP-43 neurotoxicity, which transforms into a well-folded Ω-loop-helix structure in membrane environments. Furthermore, despite having very similar membrane-embedded conformations, three mutants will undergo further self-association in the membrane environment. Our study implies that the TDP-43 prion-like domain appears to have an energy landscape, which allows the assembly of the wild-type sequence into dynamic oligomers only under very limited condition sets, and ALS-causing point mutations are sufficient to remodel it to more favor the amyloid formation or irreversible aggregation, thus supporting the emerging view that the pathologic aggregation may occur via the exaggeration of functionally important assemblies. Furthermore, the coupled capacity of TDP-43 in aggregation and membrane interaction may critically account for its high neurotoxicity, and therefore its decoupling may represent a promising therapeutic strategy to treat TDP-43 causing neurodegenerative diseases.</p></div

Unexpected Fracture Behavior of Ultrasoft Associative Hydrogels Due to Strain-Induced Crystallization

Strain-induced crystallization (SIC) is a well-known toughening strategy in elastomers, but is rarely observed in hydrogels due to their high-water content and limited deformability. Here we report a phenomenon of SIC in highly swollen and associative hydrogels by introducing an extremely large deformation by indentation with a needle. Using in situ birefringence imaging, we discovered that SIC occurs close to the needle tip upon large strain, displacing the nucleation of a crack from the needle tip to a position further away from the tip. The morphology of the fracture as well as the force to induce the gel fracture with the needle can be controlled by playing with temperature and cross-linking and hence triggering or not the SIC. Our discovery points to a future direction in creating SIC in highly swollen hydrogels, with potential implications for many biological material designs, and surgical injury prediction or prevention in associative tissues

Residue-specific conformation of the wild-type prion-like domain in DPC micelle.

<p>(A) Residue specific (ΔCα-ΔCβ) chemical shifts of the prion-like domain in DPC micelle (red) and in aqueous solution (blue). (B) Secondary structure scores of the prion-like domain in DPC micelle (red) and in aqueous solution (blue), which were obtained by analyzing their chemical shifts of the prion-like domain with the SSP program. (C) {¹H}-¹⁵N heteronuclear steady-state NOE (hNOE) of the prion-like domain in DPC micelle (red) and in aqueous solution (blue). (D) NOE connectivities defining secondary structures of the prion-like domain in DPC micelle. (E) Ratios of HSQC peak intensities of the prion-like domain in DPC micelle with and without 10 mM gadodiamide. Red line (0.59) representing the average value plus a standard deviation is set up as a cut-off. NMR data for preparing the above figures are presented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002338#pbio.1002338.s002" target="_blank">S2 Data</a>.</p

NMR characterization of the interactions with ssDNA.

<p>One-dimensional ¹H NMR spectra over 0.5–3.4 ppm at molar ratios (protein:ssDNA) of 1:0 (black), 1:0.2 (red), 1:0.5 (blue) and 1:1 (pink), as well as HSQC spectra at molar ratios of 1:0 (blue), 1:0.5 (blue), and 1:1 (green), respectively, acquired at 25°C for the wild type: (A)–(B); A315E: (C)–(D); Q331K: (E)–(F); and M337V: (G)–(H) at a protein concentration of 40 μM in 1 mM phosphate buffer (pH 5.0). Star is used to indicate the up-field NMR peaks manifested upon interacting with ssDNA only by the wild type.</p

NMR characterization of the self-association.

<p>One-dimensional ¹H NMR spectra over 0.6–0.96 ppm at different time points acquired at 25°C for the wild type (A), A315E (B), Q331K (C), and M337V (D), at a protein concentration of 40 μM in 1 mM phosphate buffer (pH 6.8). Stars are used to indicate the up-field NMR peaks manifested during the self-association only by the wild-type prion-like domain.</p

CD and fluorescence characterization of the self-association.

<p>Far-UV CD spectra acquired at 25°C at different time points of the incubation for the wild type (A), A315E (B), Q331K (C), and M337V (D) at a protein concentration of 20 μM in 1 mM phosphate buffer (pH 6.8). Emission spectra of the intrinsic visible fluorescence for the wild type (E), A315E (F), Q331K (G), and M337V (H) in water at pH 4.0, and in 1 mM phosphate buffer (pH 6.8) at different time points of the incubation. The wavelengths of the emission maxima are labeled for the spectra of the samples in water (pH 4.0), 5 min and 6 d after dilution into 1 mM phosphate buffer (pH 6.8). The wild type has an emission maximum very different from those of the three mutants.</p

Interactions of the wild-type prion-like domain with membranes.

<p>(A) Far-UV CD spectra of the wild-type prion-like domain acquired at 25°C in Milli-Q water at pH 4.0 (black), in the presence of the DMPC/DHPC bicelle (red) and DPC micelle (blue) at a ratio of 1:200. (B) Superimposition of HSQC spectra of the prion-like domain acquired at 25°C in aqueous solution (blue) and in the presence of the DMPC/DHPC bicelle at a ratio of 1:200 (red). The assignments of the disappeared HSQC peaks are labeled. Inlet: HSQC peaks of three Trp side chains in aqueous solution (blue) and in the presence of DMPC/DHPC bicelle at a ratio of 1:200 (red). (C) Far-UV CD spectra of the prion-like domain acquired at 25°C in the presence of DPC micelle (blue) at different ratios (0–200). (D) Superimposition of HSQC spectra of the prion-like domain acquired at 25°C in aqueous solution (blue) and in the presence of DPC micelle at a ratio of 1:50 (red). (E) Superimposition of HSQC spectra of the prion-like domain acquired at 25°C in aqueous solution (blue) and in the presence of DPC micelle at a ratio of 1:100 (red). (F) Superimposition of HSQC spectra of the prion-like domain acquired at 25°C in aqueous solution (blue), in the presence of DPC micelle at a ratio of 1:100 (red) and 1:400 (green).</p