Cross-linked Regions Research Articles

Phosphorylation of Fibronectin Influences the Structural Stability of the Predicted Interchain Domain.

As a key player in cell adhesion, the glycoprotein fibronectin is involved in the complex mechanobiology of the extracellular matrix. Although the function of many modules in the fibronectin molecule has already been understood, the structure and biological relevance of the C-terminal cross-linked region (CTXL) still remains unclear. It is known that fibronectin is only phosphorylated in the CTXL domain, but no results have been presented to date, which indicate a biological function based on this phosphorylation. For the first time, we introduce a structural model of the CTXL region in fibronectin, which we obtained by exhaustive replica exchange molecular dynamics simulations (TIGER2hs). The sampling revealed a conformational landscape of the dimerization module, and the global minimum state showed an umbrella-like module body and conspicuous structural region with two feet. We observed that the CTXL foot region exhibits a structural stability in its physiological state, which disappears upon changes in the phosphorylation state. Thus, our in silico studies enabled us to show that the flexibility of the CTXL region is guided by phosphorylation. These results indicate an in vivo function of the CTXL domain in protein binding and cell adhesion, which is controlled by phosphorylation.

Synthesis and characterization of novel functionalized perarylated polysiloxane for proton exchange membrane fuel cells

ABSTRACTThis work presents the synthesis and characterization of a pearylated polysiloxane material (PAP) from a polycondensation reaction, followed by functionalization with HClSO3 by an electrophilic substitution reaction. According to the characterization techniques applied, a sulfonated pearylated polysiloxane was also obtained, (SPAP). The purpose of this sulfonated material is to obtain an ionomer able to be applied in hydrogen fuel cells of the proton exchange membrane kind (PEMFC). The reaction to produce the polysiloxane precursor was carried out with the commercial reagents: PhSiCl3, Ph2SiCl2 and Ph3SiCl in anhydrous THF at 75 °C and the SPAP material was obtained by sulfonation of the precursor with chlorosulfonic acid. PAP and SPAP were characterized by 1H, NMR for liquids, 29Si NMR for solids, IR-ATR, SEM, and cyclic voltammetry. The NMR 29Si spectra show that PAP and PAPS contain crosslinking regions due to PhSiCl3, growing chain zones due to Ph2SiCl2 and polymer termination zones due to Ph3SiCl, obtaining a mixture of siloxanes. The analysis by cyclic voltammetry indicates that by integrating the area under the curve of the adsorption peaks of H2, a value of 0.062 mC/cm2 is obtained, a value close to the commercial ionomer of Nafion®.

Metal-Catalyst-Free Growth of Patterned Graphene on SiO2 Substrates by Annealing Plasma-Induced Cross-Linked Parylene for Optoelectronic Device Applications.

A metal-catalyst-free method for the direct growth of patterned graphene on an insulating substrate is reported in this paper. Parylene N is used as the carbon source. The surface molecule layer of parylene N is cross-linked by argon plasma bombardment. Under high-temperature annealing, the cross-linking layer of parylene N is graphitized into nanocrystalline graphene, which is a process that transforms organic to inorganic and insulation to conduction, while the parylene N molecules below the cross-linking layer decompose and vaporize at high temperature. Using this technique, the direct growth of a graphene film in a large area and with good uniformity is achieved. The thickness of the graphene is determined by the thickness of the cross-linking layer. Patterned graphene films can be obtained directly by controlling the patterns of the cross-linking region (lithography-free patterning). Graphene-silicon Schottky junction photodetectors are fabricated using the as-grown graphene. The Schottky junction shows good performance. The application of direct-grown graphene in optoelectronics is achieved with a great improvement of the device fabrication efficiency compared with transferred graphene. When illuminated with a 792 nm laser, the responsivity and specific detectivity of the detector measured at room temperature are 275.9 mA/W and 4.93 × 109 cm Hz1/2/W, respectively.

Effect of gel diffusion on the frontal sorption and desorption of erythromycin by molecularly imprinted polymeric monoliths

ABSTRACTThe frontal dynamics of the sorption and desorption of the antibacterial antibiotic erythromycin on molecularly imprinted monoliths on the base of 2-hydroxyethyl methacrylate and ethylene glycol dimethacrylate and on non-imprinted analog was investigated. In contrast to large molecules, the sorption of the antibiotic was determined not only by convective mass transfer, but also by gel diffusion into weakly cross-linked regions of the polymer sorbents and molecular imprinting of monoliths polymer matrix resulted in an increase in gel diffusion. At desorption from molecularly imprinted polymeric monoliths, an individual front of erythromycin desorption from specific imprint sites was observed.

Cross-linker effect on solute adsorption in swollen thermoresponsive polymer networks.

The selective solute partitioning within a polymeric network is of key importance to applications in which controlled release or uptake of solutes in a responsive hydrogel is required. In this work we investigate the impact of cross-links on solute adsorption in a swollen polymer network by means of all-atom, explicit-water molecular dynamics simulations. We focus on a representative network subunit consisting of poly(N-isopropylacrylamide) (PNIPAM) and N,N'-methylenebisacrylamide (BIS/MBA) cross-linker types. Our studied system consists of one BIS-linker with four atactic PNIPAM chains attached in a tetrahedral geometry. The adsorption of several representative solutes of different polarity in the low concentration limit at the linker region is examined. We subdivide the solute adsorption regions and distinguish between contributions stemming from polymer chains and cross-link parts. In comparison to a single polymer chain, we observe that the adsorption of the solutes to the cross-link region can significantly differ, with details depending on the specific compounds' size and polarity. In particular, for solutes that have already a relatively large affinity to PNIPAM chains the dense cross-link region (where many-body attractions are at play) amplifies the local adsorption by an order of magnitude. We also find that the cross-link region can serve as a seed for the aggregation of mutually attractive solutes at higher solute concentrations. Utilizing the microscopic adsorption coefficients in a mean-field model of an idealized macroscopic polymer network, we extrapolate these results to the global solute partitioning in a swollen hydrogel and predict that these adsorption features may lead to non-monotonic partition ratios as a function of the cross-link density.

Insights into the Network Structure of Cross-Linked Polymers Synthesized via Miniemulsion Nitroxide-Mediated Radical Polymerization

The effect of reversible-deactivation radical polymerization on the structure of the network formed by copolymerization of mono- and divinyl monomers is studied. The nitroxide-mediated radical copolymerization (NMP) of butyl methacrylate and ethylene glycol dimethacrylate, initiated with the alkoxyamine 3-(((2-cyanopropan-2-yl)oxy)cyclohexylamino)-2,2-dimethyl-3-phenylpropanenitrile (Dispolreg 007) in an aqueous miniemulsion was used as a case study. Combination of asymmetric-flow field-flow fractionation and small-angle X-ray scattering clearly showed that NMP led to a more homogeneous network structure formed by regions of relatively open networks connected with short polymer chain segments, whereas that of the polymer formed by free radical polymerization consists of densely cross-linked regions linked by long polymer chain portions.

Exploring pH-Responsive, Switchable Crosslinking Mechanisms for Programming Reconfigurable Hydrogels Based on Aminopolysaccharides

Dynamic, stimuli-responsive, reconfigurable materials are finding growing interest and applications. A recent experiment (He et al., Adv Funct Matter., 2017) demonstrated a pH-responsive, reconfigurable hydrogel film based on a single biopolymer chitosan and two physical cross-linking mechanisms. Here, we use state-of-the-art molecular dynamics simulations to gain atomically detailed insights into the three salient features of this experimental system: (1) a pH-responsive switch between the crystalline network junctions formed by intermolecular hydrogen bonding between chitosan chains and electrostatic cross-links based on sodium dodecyl sulfate (SDS) micelles; (2) viscoelastic behavior of the SDS-crosslinked network; and (3) a stable but erasable gradient between the two cross-linked regions. Our simulations showed that the electrostatic cross-links are formed through the pH-dependent salt-bridge interactions between the chitosan glucosamines and SDS sulfates. Interestingly, the strength and directionali...

Collagen fiber crimping following in vivo UVA-induced corneal crosslinking

The purpose of this study was to measure collagen fiber crimping (CFC) using nonlinear optical imaging of second harmonic generated (SHG) signals to determine the effects of UVA-riboflavin induced corneal collagen crosslinking (UVA CXL) on collagen structure. Two groups, four rabbits each, were treated in the right eye with standard UVA CXL. In vivo confocal microscopy was performed at 1, 2, and 4 weeks after treatment for the first group and up to three months for the second group to measure epithelial/stromal thickness and corneal haze during recovery. Rabbits were sacrificed at one and three months, respectively, and their corneas fixed under pressure. Regions of crosslinking were identified by the presence of collagen autofluorescence (CAF) and then collagen structure was imaged using SHG microscopy. The degree of CFC was determined by measuring the percentage difference between the length of the collagen fiber and the linear distance traveled. CFC was measured in the central anterior and posterior CXL region, the peripheral non-crosslinked region in the same cornea, and the central cornea of the non-crosslinked contralateral eye. No change in corneal thickness was detected after one month, however the stromal thickness surpassed its original baseline thickness at three months by 25.9 μm. Corneal haze peaked at one month and then began to clear. Increased CAF was detected in all CXL corneas, localized to the anterior stroma and extending to 42.4 ± 3.4% and 47.7 ± 7.6% of the corneal thickness at one and three months. There was a significant (P < 0.05) reduction in CFC in the CAF region in all eyes averaging 1.007 ± 0.006 and 1.009 ± 0.005 in one and three month samples compared to 1.017 ± 0.04 and 1.016 ± 0.06 for controls. These results indicate that there is a significant reduction in collagen crimping following UVA CXL of approximately 1%. One possible explanation for this loss of crimping could be shortening of the collagen fibers over the CXL region.

Peculiar extensibility of swollen statistical hydrogels with structural nanoheterogeneities

An inherent structural nanoheterogeneity in statistical hydrogels leads to a peculiarity in macroscopic tensile properties. Some types of statistical hydrogels with considerable degrees of non-uniform distribution of cross-link, which are formed via free radical copolymerization of mono- and bi-functional monomers, are unusually extensible: The elongation at break before and after equilibrium swelling remains almost unchanged, although the gels undergo a significant increase in volume by swelling, i.e., a finite degree of pre-stretching of network strands. This peculiar extensibility emerges exclusively in uniaxial stretching whereas it is not observed in biaxial stretching including equi-biaxial and planar extension. The concentration ranges of the mono- and bi-functional monomers which result in the gels with the peculiar extensibility are revealed. The two-dimensional SAXS patterns of the uniaxially stretched swollen gels with marked extensibility show the presence of a spatially long-range structure of ca. 50 nm which results from the significantly non-affine deformation of the networks composed of densely and loosely cross-linked regions.

Controlling the kinetics of thiol-maleimide Michael-type addition gelation kinetics for the generation of homogenous poly(ethylene glycol) hydrogels.

The development of synthetic hydrogels analogs for the extracellular matrix has proven a useful and important tool to study the role of specific signals on biological outcomes invitro and to serve as scaffolds for tissue repair. Although the importance of physical properties (e.g. microstructure and stiffness) in the micro and nano scale on cell fate has been widely reported, bulk modulus measurements are typically used to characterize hydrogels. Thus, the physical properties of hydrogels have not been widely tested for their controlled physical properties in the nano and micron scales. In this report, we show that although fast Michael-type addition crosslinked hydrogels appear uniform by bulk modulus readings and visual inspection, they are non-uniform in the micron scale, with high and low crosslinking regions. Further, we show that these regions of high and low crosslinking result in differences in cellular behavior. Since these regions are random in density and shape, this leads to misleading cellular responses. These inconsistences are most widely observed when the gel forms faster than the material can be mixed. This study slows the gelation rate of thiol-maleimide cross-linked hydrogels in order to overcome the cellular response variability between batches.

Fabrication of native silica, cross-linked, and hybrid aerogel monoliths with customized geometries

In this work, a previously developed (White et al 2015 J. Mater. Chem. A 3 762) rapid synthesis approach is used to fabricate native and cross-linked aerogel monoliths with customized geometries. This technique does not require solvent exchange, therefore fabrication times do not depend on part size. To prove this, parts with a smallest dimension of approximately 3.6 cm were fabricated within the same time scale as that of small cylinders with a diameter of 7 mm. In addition, monoliths with customized geometries exhibiting physical detail on the order of 1 mm were produced to demonstrate the versatility of this technique. Furthermore, hybrid materials consisting of native silica aerogel integrated with selected regions of polymer cross-linking were produced. The cross-linked regions allow for adhesion to other surfaces or labeling while the majority of the material retains the physical characteristics of a native silica aerogel. The physical and thermal properties of all aerogel components were examined. All aerogel materials produced in this work exhibited characteristics that were within the range of aerogel materials produced using more conventional methods. For native silica materials, this includes densities in the range of 0.03–0.116 g cm−3, surface areas between 342–799 m2 g−1, mode pore sizes in the range of 30–39 nm, and thermal conductivities in the range of 0.020–0.026 W m−1 K−1. For cross-linked aerogel materials, densities ranged between 0.154–0.340 g cm−3, surface areas were between 291–388 m2 g−1, mode pore sizes were in the range of 29–41 nm, and thermal conductivities were in range of 0.038–0.066 W m−1 K−1.

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.

Effect of chondroitin sulfate on the type I collagen metabolism in the compact bone in alloxan-induced rats

Aim. To study the type I collagen metabolism in the compact bone in rats with alloxan-induced diabetes receiving sulfated glycosaminoglycans.&#x0D; Methods. The study was performed on 67 white outbred male rats with body weight of 180-220 g. Mortality at diabetes reproduction was 44.8%. To clarify the role of exogenous glycosaminoglycans on bone collagen metabolism at diabetes mellitus, 16 animals with alloxan-induced diabetes received 1 mg/kg of chondroitin sulfate intramuscularly every second day. The second group (21 animals) with alloxan-induced diabetes did not received any chondroitin sulfate. Control group included 10 intact animals who were administered a single injection on 0.5% ml of normal saline. The levels of type I collagen metabolism markers (PINP - aminoterminal propeptide of type I procollagen, a marker of bone formation; β-CrossLaps - β-isomerized carboxy-terminal cross-linking region of type I collagen, a marker of bone resorption) and the amount of total collagen were determined in homogenates of femoral shaft.&#x0D; Results. Administration of alloxan to the animals has induced the development of diabetes mellitus. The levels of PINP and β-CrossLaps was significantly higher in alloxan-induced rats which were administered chondroitin sulfate compared to rats with «isolated» alloxan-induced diabetes by 21 (p=0.001) and 28 (p=0.01) days of follow-up, the level of total collagen was higher at 70% at 28 day of the experiment (p=0.0004).&#x0D; Conclusion. Effect of sulfated glycosaminoglycans on type I collagen metabolism of the compact bone in animals with «isolated» alloxan-induced diabetes is manifested by intensified catabolic and anabolic processes with a predominance of the latter over the control and alloxan-induced rats at 21 and 28 days of the experiment.

Structural Insights into Cargo Recognition by the Yeast PTS1 Receptor

The peroxisomal matrix protein import is facilitated by cycling import receptors that shuttle between the cytosol and the peroxisomal membrane. The import receptor Pex5p mediates the import of proteins harboring a peroxisomal targeting signal of type I (PTS1). Purified recombinant Pex5p forms a dimeric complex with the PTS1-protein Pcs60p in vitro with a KD of 0.19 μm. To analyze the structural basis for receptor-cargo recognition, the PTS1 and adjacent amino acids of Pcs60p were systematically scanned for Pex5p binding by an in vitro site-directed photo-cross-linking approach. The cross-linked binding regions of the receptor were subsequently identified by high resolution mass spectrometry. Most cross-links were found with TPR6, TPR7, as well as the 7C-loop of Pex5p. Surface plasmon resonance analysis revealed a bivalent interaction mode for Pex5p and Pcs60p. Interestingly, Pcs60p lacking its C-terminal tripeptide sequence was efficiently cross-linked to the same regions of Pex5p. The KD value of the interaction of truncated Pcs60p and Pex5p was in the range of 7.7 μm. Isothermal titration calorimetry and surface plasmon resonance measurements revealed a monovalent binding mode for the interaction of Pex5p and Pcs60p lacking the PTS1. Our data indicate that Pcs60p contains a second contact site for its receptor Pex5p, beyond the C-terminal tripeptide. The physiological relevance of the ancillary binding region was supported by in vivo import studies. The bivalent binding mode might be explained by a two-step concept as follows: first, cargo recognition and initial tethering by the PTS1-receptor Pex5p; second, lock-in of receptor and cargo.

A Facile Route for Patterned Growth of Metal-Insulator Carbon Lateral Junction through One-Pot Synthesis.

Precise graphene patterning is of critical importance for tailor-made and sophisticated two-dimensional nanoelectronic and optical devices. However, graphene-based heterostructures have been grown by delicate multistep chemical vapor deposition methods, limiting preparation of versatile heterostructures. Here, we report one-pot synthesis of graphene/amorphous carbon (a-C) heterostructures from a solid source of polystyrene via selective photo-cross-linking process. Graphene is successfully grown from neat polystyrene regions, while patterned cross-linked polystyrene regions turn into a-C because of a large difference in their thermal stability. Since the electrical resistance of a-C is at least 2 orders of magnitude higher than that for graphene, the charge transport in graphene/a-C heterostructure occurs through the graphene region. Measurement of the quantum Hall effect in graphene/a-C lateral heterostructures clearly confirms the reliable quality of graphene and well-defined graphene/a-C interface. The direct synthesis of patterned graphene from polymer pattern could be further exploited to prepare versatile heterostructures.

Studying RNA-Protein Interactions of Pre-mRNA Complexes by Mass Spectrometry.

RNA-protein interactions play a crucial role in gene expression. These interactions take place in so-called ribonucleoprotein (RNP) complexes. To investigate which proteins interact with RNA in these complexes, and how they do so, UV-light-induced cross-linking has proven to be a valuable, yet straightforward technique. UV irradiation induces a covalent bond between the RNA and the proteins, whereafter cross-linked proteins can be identified by mass spectrometric (MS) approaches. Moreover, the cross-linked region of the protein, and often the actual cross-linked amino acid, can be identified by state-of-the-art MS, as can the cross-linked RNA moiety. This protocol describes in detail how to isolate peptide-RNA oligonucleotide cross-links from UV-irradiated human pre-mRNA RNPs and to perform the subsequent MS investigation of these peptide-RNA conjugates in combination with a dedicated computational analysis, in order to obtain sequence information about the cross-linked peptide and oligoribonucleotide. The described workflow can be applied to any RNP, irrespective of its origin, e.g., RNPs assembled in vitro (as described here) or RNPs isolated from UV-irradiated cells, either ex vivo or in vivo.

High vitamin E content, impact resistant UHMWPE blend without loss of wear resistance.

Antioxidant stabilization of radiation cross-linked ultrahigh molecular weight polyethylene (UHMWPE) has been introduced to improve the oxidative stability of total joint implant bearing surfaces. Blending of antioxidants (most commonly vitamin E) with UHMWPE resin powder followed by consolidation and uniform radiation cross-linking is currently available for use in both total hips and total knees. It was previously shown that the fatigue resistance of vitamin E-blended and irradiated UHMWPEs could be further improved by spatially manipulating the vitamin E concentration throughout the implant and limiting cross-linking to the surface of the implant where it is necessary for wear resistance. This was possible by designing a low concentration of vitamin E on the surface and higher concentration in the bulk of the implant because cross-linking is hindered in UHMWPE as a function of increasing vitamin E concentration. In this study, we hypothesized that such a surface cross-linked UHMWPE with low wear rate and high fatigue strength could be obtained by limiting the penetration of radiation into UHMWPE with uniform vitamin E concentration. Our hypothesis tested positive; we were able to obtain control of the surface cross-linked region by manipulating the energy of the irradiation, resulting in extremely low wear, and high impact strength. In addition, we discussed alternatives of improving the oxidation resistance of such a material by using additional vitamin E reservoirs. These results are significant because this material may allow increased use of antioxidant-stabilized, cross-linked UHMWPEs in high stress applications and in more active patients.

Fabrication and sensing behavior of one-dimensional ZnO-Zn2GeO4 heterostructures.

Well-crystalline one-dimensional ZnO-Zn2GeO4 (ZGO) heterostructures were successfully synthesized using a high-temperature solid-state reaction between the ZnO and Ge layers of ZnO-Ge core-shell nanostructures. The polycrystalline ZGO crystallites had a thickness in the range of 17 to 26 nm. The high-temperature solid-state reaction induced grooves and crystal defects on the surfaces of the ZGO crystallites. The sensors made from the ZnO-ZGO heterostructures exhibited a marked photocurrent response to UV light at room temperature and a gas sensing response to acetone gas at 325°C. The observed sensing properties are attributed to the rugged surface of the ZGO heterointerfaces between ZnO and ZGO, surface crystal defects of ZGO, and cross-linked contact regions of ZnO-ZGO.

Reply to comment on “Polymerization, bending, tension: What happens at the leading edge of motile cells?” by Falko Ziebert and Igor S. Aranson

We are grateful to Falko Ziebert and Igor Aranson, who continue with their comment on our publication [5] a discussion on modelling concepts. Ziebert and Aranson present in their contribution to this volume [14] a model concept for cell motility and morphodynamics focusing on gel flow and its determinants. This type of models is particularly useful for describing slow dynamics on the length scale of the whole cell and modelling of cell shape [1,8,11,13,14]. Our approach is set apart from the gel models by taking into account a weakly cross-linked F-actin network region close to the location of polymerization in the lamellipodia of motile cells (semi-flexible region) in addition to the gel in the bulk. This addition explains a variety of non-linear dynamic regimes in cellular and reconstituted systems, and the force-velocity relation of fish keratocytes. Ziebert and Aranson point out in their comment that 1) a more detailed modelling of gel processes may be required to capture large cell deformations, 2) the dynamics of adhesion strength and distribution may be relevant for understanding the relation between cell shape, the dynamic regime of motion and cell velocity, 3) coarse grained models may allow for unifying both concepts, and 4) fluctuations are important in morphodynamics.

Assembly of Nsp1 Nucleoporins Provides Insight into Nuclear Pore Complex Gating

Nuclear pore complexes (NPCs) form gateways for material transfer across the nuclear envelope of eukaryotic cells. Disordered proteins, rich in phenylalanine-glycine repeat motifs (FG-nups), form the central transport channel. Understanding how nups are arranged in the interior of the NPC may explain how NPC functions as a selectivity filter for transport of large molecules and a sieve-like filter for diffusion of small molecules (< or ). We employed molecular dynamics to model the structures formed by various assemblies of one kind of nup, namely the 609-aa-long FG domain of Nsp1 (Nsp1-FG). The simulations started from different initial conformations and geometrical arrangements of Nsp1-FGs. In all cases Nsp1-FGs collectively formed brush-like structures with bristles made of bundles of 2–27 nups, however, the bundles being cross-linked through single nups leaving one bundle and joining a nearby one. The degree of cross-linking varies with different initial nup conformations and arrangements. Structural analysis reveals that FG-repeats of the nups not only involve formation of bundle structures, but are abundantly present in cross-linking regions where the epitopes of FG-repeats are highly accessible. Large molecules that are assisted by transport factors (TFs) are selectively transported through NPC apparently by binding to FG-nups through populated FG-binding pockets on the TF surface. Therefore, our finding suggests that TFs bind concertedly to multiple FGs in cross-linking regions and break-up the bundles to create wide pores for themselves and their cargoes to pass. In addition, the cross-linking between Nsp1-FG bundles, arising from simulations, is found to set a molecular size limit of < for passive diffusion of molecules. Our simulations suggest that the NPC central channel, near the periphery where tethering of nups is dominant, features brush-like moderately cross-linked bundles, but in the central region, where tethering loses its effect, features a sieve-like structure of bundles and frequent cross-links.