Cross-linking Of Molecules Research Articles

Effects of Calcium Concentration in Potato Tuber Cells on the Formation of Cross-Links between Pectin Molecules by Ca2+

The formation of cross-links between pectin molecules via Ca2+ in the potato tuber cell wall is a determinant factor on processing properties of potato and the quality of its products such as French fries. Thus, in this study, potato tubers varying significantly in their calcium concentrations were analyzed to investigate whether an increased absorption of calcium by a potato tuber led to an increase in the calcium concentration in the cell wall and how the calcium concentration in the cell wall influenced on the formation of cross-links between pectin molecules via Ca2+. Correlation analysis revealed that calcium absorbed by a potato tuber was bound to the cell wall as a water insoluble form 99 days after planting or later. Furthermore, with an increase in the calcium concentration in the cell wall, the content of chelator soluble pectin increased throughout tuber bulking and maturation stages. However, the degree of methylation was not a limiting factor in the formation of cross-links between pectin chains via Ca2+. Atomic force microscopy images of parenchyma cell walls prepared from mature potato tubers indicated an increase in the amount of calcium cross-linked pectin molecules with an increase in the calcium concentration in the cell wall. The present study demonstrated that the calcium concentration of the cell wall of potato tubers significantly affected the formation of cross-linkages between pectin molecules and, consequently, contributed to an enhanced formation of pectin-calcium networks in the cell wall.

Bis-Azide Low-Band Gap Cross-Linkable Molecule N3-[CPDT(FBTTh2)2] to Fully Thermally Stabilize Organic Solar Cells Based on P3HT:PC61BM.

We synthesized a novel bis-azide low-band gap cross-linkable molecule N3-[CPDT(FBTTh2)2] with wide absorption. This compound is of interest as an additive in polymer/fullerene bulk heterojunction solar cells. In addition to providing efficient thermal stabilization of the morphology, the additive can harvest additional solar light compared with pristine poly(3-hexyl thiophene) to improve the power-conversion efficiency (PCE). The additional donor material was visualized from the appearance of additional external quantum efficiency contributions between 650 and 800 nm. An open-circuit voltage increase of ∼2% compensates the decrease in the short-circuit current of ∼2% to achieve a fully thermally stabilized PCE of 3.5% after 24 h of annealing at 150 °C.

Towards a Versatile Photoreactive Platform for Biosensing Applications

An original interfacial hybrid cross-linker molecule combined electrochemical and photochemical properties by substitution of two functional groups, pyrene, and diazirine. The first group enables anchoring by strong π-stacking interaction or by electropolymerization onto multi-walled carbon nanotubes (MWCNT). The other photoreactive group allows the covalent link with biomolecules under long-wavelength UV illumination. Diazirine was chosen due to its ability to photogenerate high reactive radicals under UV irradiation. The photoreactivity of these immobilized nanostructured conductive surfaces was tested towards the covalent attachment of tyrosinase which is well known to oxidize a large range of phenolic compounds and its yield and availability was evaluated by amperometric measurements of catechol by using molecular dioxygen. The architecture exhibiting the best analytical characteristics obtained for catechol was then chosen to detect dopamine.

Enzymatic Synthesis of Cellulose Oligomer Hydrogels Composed of Crystalline Nanoribbon Networks under Macromolecular Crowding Conditions.

Macromolecular crowding, a solution state with high macromolecular concentrations, was used to promote the crystallization-driven self-assembly of enzymatically synthesized cellulose oligomers. Cellulose oligomers were synthesized via cellodextrin phosphorylase-catalyzed enzymatic reactions in the concentrated solutions of water-soluble polymers, such as dextran, poly(ethylene glycol), and poly(N-vinylpyrrolidone). The reaction mixtures were transformed into cellulose oligomer hydrogels composed of well-grown crystalline nanoribbon networks irrespective of the polymer species. This method was successfully applied in the one-pot preparation of double network hydrogels composed of the nanoribbons and physically cross-linked gelatin molecules through the simple control of reaction temperatures, demonstrating the superior mechanical properties of the composite hydrogels. Our concept that promotes the growth of self-assembled architectures under macromolecular crowding conditions demonstrates a new avenue into developing novel hydrogel materials.

Influence of mixed organosilane coatings with variable RGD surface densities on the adhesion and proliferation of human osteosarcoma Saos-2 cells to magnesium alloy AZ31

In the last decade, the use of magnesium and its alloys as biodegradable implant materials has become increasingly accepted. However, surface modification of these materials to control the degradation rate in the early stages of healing and improve their biocompatibility is crucial to the successful implementation of magnesium alloy implants in medicine. Cell adhesion and proliferation at the implant surface is a vital factor for successful integration of a biomaterial within the body. Cells accomplish this task by binding to ligands such as the arginine-glycine-aspartic acid peptide sequence (RGD) commonly found on adhesive proteins present in the extracellular matrix. In this paper, we report a biomimetic surface modification strategy involving deposition of a mixed organosilane layer on Mg AZ31 followed by covalent immobilization of RGD peptides through a heterobifunctional cross-linker molecule. Our results indicate that with optimized deposition conditions uniform organosilane coatings were successfully deposited on the Mg AZ31 substrate. Furthermore, we have demonstrated that the surface density of immobilized RGD can be varied by depositing organosilane layers from solutions containing two different organosilanes in specified ratios. Increases in cell adhesion and cell proliferation were observed on the surface modified substrates.

Polymer-assisted formation of 3D Pd nanoassemblies: highly active catalysts for formic acid electrooxidation

Three-dimensional (3D) Pd nanoassemblies were synthesized by a facile hydrothermal procedure, in which polyallylamine hydrochloride (PAH) was simultaneously used as a cross-linking and scaffold molecule in order to construct the 3D interconnected nanoassemblies.

Alkaline phosphatase-fused repebody as a new format of immuno-reagent for an immunoassay

Enzyme-linked immunoassays based on an antibody-antigen interaction are widely used in biological and medical sciences. However, the conjugation of an enzyme to antibodies needs an additional chemical process, usually resulting in randomly cross-linked molecules and a loss of the binding affinity and enzyme activity. Herein, we present the development of an alkaline phosphatase-fused repebody as a new format of immuno-reagent for immunoassays. A repebody specifically binding to human TNF-α (hTNF-α) was selected through a phage display, and its binding affinity was increased up to 49 nM using a modular engineering approach. A monomeric alkaline phosphatase (mAP), which was previously isolated from a metagenome library, was genetically fused to the repebody as a signal generator, and the resulting repebody-mAP fusion protein was used for direct and sandwich immunoassays of hTNF-α. We demonstrate the utility and potential of the repebody-mAP fusion protein as an immuno-reagent by showing the sensitivity of 216 pg mL−1 for hTNF-α in a sandwich immunoassay. Furthermore, this repebody-mAP fusion protein enabled the detection of hTNF-α spiked in a serum-supplemented medium with high accuracy and reproducibility. It is thus expected that a mAP-fused repebody can be broadly used as an immuno-reagent in immunoassays.

Injectable hydrogels prepared from partially oxidized hyaluronate and glycol chitosan for chondrocyte encapsulation

Hyaluronate has attracted great attention in tissue engineering as a scaffolding material. However, hyaluronate typically requires chemical cross-linking molecules to form hydrogels, which may induce undesirable side effects in the body. In this study, hyaluronate was partially oxidized with sodium periodate to generate aldehyde groups in the backbone, and simply mixed with glycol chitosan to form gels via Schiff base formation. The effects of the degree of oxidization, polymer concentration, and polymer composition on the mechanical properties of oxidized hyaluronate/glycol chitosan hydrogels were investigated in vitro. Degradation behavior and biocompatibility of oxidized hyaluronate/glycol chitosan gels were also evaluated in vitro. This system may be potentially useful as an injectable system in many tissue engineering applications, including cartilage regeneration.

Label free ultrasensitive optical sensor decorated with polyaniline nanofibers: Characterization and immunosensing application

Conducting polymers have been studied for decades, and the use of such polymers in sensing applications has been explored extensively. Most of these have exploited the fact that protonation or de-protonation of these polymers, have an effect on their conductivity and conformational structure. We demonstrate a phenomenon, i.e. changes to the spectroscopic properties of conducting polymer polyaniline (PAni), without explicit protonation or de-protonation, due to immunological interactions happening on the surface. To explore the reason behind these spectroscopic changes, PAni was analyzed using Hydrogen Nuclear Magnetic Resonance (HNMR), and Fourier Transformed Infrared (FTIR) spectroscopy. It was further characterized using Scanning Electron Microscope (SEM) imaging and Selected Area Electron Diffraction (SAED). These optical alterations of polyaniline have been utilized to develop a simple, cost effective, yet highly sensitive immunosensor. Human Immunoglobulin G (HIgG) was immobilized on the polyaniline coated fiber-optic probe using cross linker molecules, and goat anti-Human Immunoglobulin G (GaHIgG) was used as the analyte. This study fundamentally utilizes optical properties of PAni to develop a simple immunosensor which is able to detect analyte concentrations as low as 37pM.

Stabilized electrospinning of heat stimuli/in situ crosslinkable nanofibers and their self‐same nanocomposites

ABSTRACTWe present a strategy for stabilizing the morphological integrity of electrospun polymeric nanofibers by heat stimuli in situ crosslinking. Amorphous polymer nanofibers, such as polystyrene (PS) and its co‐polymers tend to lose their fiber morphology during processing at temperatures above their glass transition temperature (Tg) typically bound to happen in nanocomposite/structural composite applications. As an answer to this problem, incorporation of the crosslinking agents, phthalic anhydride (PA) and tributylamine (TBA), into the electrospinning polymer solution functionalized by glycidylmethacrylate (GMA) copolymerization, namely P(St‐co‐GMA), is demonstrated. Despite the presence of the crosslinker molecules, the electrospinning polymer solution is stable and its viscosity remains unaffected below 60 °C. Crosslinking reaction stands‐by and can be thermally stimulated during post‐processing of the electrospun P(St‐co‐GMA)/PA‐TBA fiber mat at intermediate temperatures (below the Tg). This strategy enables the preservation of the nanofiber morphology during subsequent high temperature processing. The crosslinking event leads to an increase in Tg of the base polymer by 30 °C depending on degree of crosslinking. Crosslinked nanofibers are able to maintain their nanofibrous morphology above the Tg and upon exposure to organic solvents. In situ crosslinking in epoxy matrix is also reported as an example of high temperature demanding application/processing. Finally, a self‐same fibrous nanocomposite is demonstrated by dual electrospinning of P(St‐co‐GMA) and stabilized P(St‐co‐GMA)/PA‐TBA, forming an intermingled nanofibrous mat, followed by a heating cycle. The product is a composite of crosslinked P(St‐co‐GMA)/PA‐TBA fibers fused by P(St‐co‐GMA) matrix. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 44090.

Gel Point Suppression in RAFT Polymerization of Pure Acrylic Cross-Linker Derived from Soybean Oil.

Here we report the reversible addition-fragmentation chain transfer (RAFT) polymerization of acrylated epoxidized soybean oil (AESO), a cross-linker molecule, to high conversion (>50%) and molecular weight (>100 kDa) without macrogelation. Surprisingly, gelation is suppressed in this system far beyond the expectations predicated both on Flory-Stockmeyer theory and multiple other studies of RAFT polymerization featuring cross-linking moieties. By varying AESO and initiator concentrations, we show how intra- versus intermolecular cross-linking compete, yielding a trade-off between the degree of intramolecular linkages and conversion at gel point. We measured polymer chain characteristics, including molecular weight, chain dimensions, polydispersity, and intrinsic viscosity, using multidetector gel permeation chromatography and NMR to track polymerization kinetics. We show that not only the time and conversion at macrogelation, but also the chain architecture, is largely affected by these reaction conditions. At maximal AESO concentration, the gel point approaches that predicted by the Flory-Stockmeyer theory, and increases in an exponential fashion as the AESO concentration decreases. In the most dilute solutions, macrogelation cannot be detected throughout the entire reaction. Instead, cyclization/intramolecular cross-linking reactions dominate, leading to microgelation. This work is important, especially in that it demonstrates that thermoplastic rubbers could be produced based on multifunctional renewable feedstocks.

Temozolomide induces the expression of the glioma Big Potassium (gBK) ion channel, while inhibiting fascin-1 expression: possible targets for glioma therapy

ABSTRACTObjective: Temozolomide (TMZ) improves Glioblastoma Multiforme (GBM) patient survival. The invasive behavior of the glioma cells is the cause of GBM relapse. The glioma BK ion channel (gBK) may provide glioma cells with a mechanism to invade surrounding tissue. gBK contains epitopes that cytolytic T lymphocytes (CTLs) can recognize and kill glioma cells. Fascin-1 is an actin crosslinking molecule that supports microvilli; these membrane protrusions provide a physical defense against CTLs. TMZ was investigated to determine its effect on gBK and fascin-1 expression.Research design and methods: Human glioma cells cultured in TMZ were analyzed for their altered mRNA and gBK protein levels by using quantitative real time PCR, immunostaining and cellular functional assays.Results: TMZ slowed glioma cell growth and inhibited their transmigratory properties due to loss of fascin-1. TMZ induced increased gBK and HLA expression and allowed these TMZ-treated cells to become better targets for gBK-specific CTLs.Conclusions: Besides its traditional chemotherapeutic effect, TMZ can have four other targeted pathways: 1) slowed glioma cell growth; 2) inhibited glioma cell transmigration; 3) increased HLA-A2 and gBK tumor antigen production; 4) increased CTL-mediated cytolysis of the TMZ treated glioma cells due to the loss of their defensive membrane protrusions supported by fascin-1.

Direct Covalent Grafting of Phytate to Titanium Surfaces through Ti-O-P Bonding Shows Bone Stimulating Surface Properties and Decreased Bacterial Adhesion.

Myo-inositol hexaphosphate, also called phytic acid or phytate (IP6), is a natural molecule abundant in vegetable seeds and legumes. Among other functions, IP6 inhibits bone resorption. It is adsorbed on the surface of hydroxyapatite, inhibiting its dissolution and decreasing the progressive loss of bone mass. We present here a method to directly functionalize Ti surfaces covalently with IP6, without using a cross-linker molecule, through the reaction of the phosphate groups of IP6 with the TiO2 layer of Ti substrates. The grafting reaction consisted of an immersion in an IP6 solution to allow the physisorption of the molecules onto the substrate, followed by a heating step to obtain its chemisorption, in an adaptation of the T-Bag method. The reaction was highly dependent on the IP6 solution pH, only achieving a covalent Ti-O-P bond at pH 0. We evaluated two acidic pretreatments of the Ti surface, to increase its hydroxylic content, HNO3 30% and HF 0.2%. The structure of the coated surfaces was characterized by X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectrometry, and ellipsometry. The stability of the IP6 coating after three months of storage and after sterilization with γ-irradiation was also determined. Then, we evaluated the biological effect of Ti-IP6 surfaces in vitro on MC3T3-E1 osteoblastic cells, showing an osteogenic effect. Finally, the effect of the surfaces on the adhesion and biofilm viability of oral microorganisms S. mutans and S. sanguinis was also studied, and we found that Ti-IP6 surfaces decreased the adhesion of S. sanguinis. A surface that actively improves osseointegration while decreasing the bacterial adhesion could be suitable for use in bone implants.

Investigation of Ultraviolet Light Curable Polysilsesquioxane Gate Dielectric Layers for Pentacene Thin Film Transistors.

Polysilsesquioxane (PSQ) comprising 3-methacryloxypropyl groups was investigated as an ultraviolet (UV)-light curable gate dielectric-material for pentacene thin film transistors (TFTs). The surface of UV-light cured PSQ films was smoother than that of thermally cured ones, and the pentacene layers deposited on the UV-Iight cured PSQ films consisted of larger grains. However, carrier mobility of the TFTs using the UV-light cured PSQ films was lower than that of the TFTs using the thermally cured ones. It was shown that the cross-linker molecules, which were only added to the UV-light cured PSQ films, worked as a major mobility-limiting factor for the TFTs.

Biodistribution and PET Imaging of Labeled Bispecific T Cell-Engaging Antibody Targeting EpCAM.

AMG 110, a bispecific T cell engager (BiTE) antibody construct, induces T cell-mediated cancer cell death by cross-linking epithelial cell adhesion molecule (EpCAM) on tumor cells with a cluster of differentiation 3 ε (CD3ε) on T cells. We labeled AMG 110 with (89)Zr or near-infrared fluorescent dye (IRDye) 800CW to study its tumor targeting and tissue distribution. Biodistribution and tumor uptake of (89)Zr-AMG 110 was studied up to 6 d after intravenous administration to nude BALB/c mice bearing high EpCAM-expressing HT-29 colorectal cancer xenografts. Tumor uptake of (89)Zr-AMG 110 was compared with uptake in head and neck squamous cell cancer FaDu (intermediate EpCAM) and promyelocytic leukemia HL60 (EpCAM-negative) xenografts. Intratumoral distribution in HT-29 tumors was studied using 800CW-AMG 110. Tumor uptake of (89)Zr-AMG 110 can be clearly visualized using small-animal PET imaging up to 72 h after injection. The highest tumor uptake of (89)Zr-AMG 110 at the 40-μg dose level was observed at 6 and 24 h (respectively, 5.35 ± 0.22 and 5.30 ± 0.20 percentage injected dose per gram; n = 3 and 4). Tumor uptake of (89)Zr-AMG 110 was EpCAM-specific and correlated with EpCAM expression. 800CW-AMG 110 accumulated at the tumor cell surface in viable EpCAM-expressing tumor tissue. PET and fluorescent imaging provided real-time information about AMG 110 distribution and tumor uptake in vivo. Our data support using (89)Zr and IRDye 800CW to evaluate tumor and tissue uptake kinetics of bispecific T cell engager antibody constructs in preclinical and clinical settings.

Automated Cross-Links-Based Conformational Sampling of Protein Assemblies: The Geometrical Challenge of Cross-Links

Cross-linking mass spectrometry (XL-MS) is increasingly used for structural modeling of multi-subunit protein complexes. Similar to NMR, chemical cross-linking captures conformational heterogeneity, which typically results in conflicting cross-links that cannot be satisfied in a single structural model. Here we introduce log-harmonic potential as a formulation of spatial restraints that realistically reflects the experimentally observed cross-linking distances in solution. In combination with a Bayesian modeling framework that iteratively adjusts weights during optimization, conflicting restraints and conformational heterogeneity are appropriately dealt with.In addition, we developed a new approach to quickly estimate the Solvent Accessible Surface Distance (SASD), which is computed at each step of the procedure to enhance the restraints weighting. We show that the SASD efficiently eliminates false positives by describing the geometrical path that is required by the cross-linking molecule. We demonstrated the relevance of SASD and power of our method, by building atomic models of several protein complexes. Overall, sampling with SASD allows a faster convergence towards meaningful conformations.

From nano- to macro-engineering of oxide-encapsulated-nanoparticles for harsh reactions: one-step organization via cross-linking molecules.

A strategy of "macro-micro-nano" organization is reported for embedding oxide-encapsulated-nanoparticles onto monolithic substrates in one-step with the aid of molecularly defined cross-linking agents. Such catalysts, with enhanced heat/mass transfer and high permeability, are qualified for several harsh reaction processes such as CH4/VOC abatement, gas-phase hydrogenation of dimethyl oxalate and oxidative dehydrogenation of ethane.

Response of biopolymer networks governed by the physical properties of cross-linking molecules.

In this study, we examine how the physical properties of cross-linking molecules affect the bulk response of bio-filament networks, an outstanding question in the study of biological gels and the cytoskeleton. We show that the stress-strain relationship of such networks typically undergoes linear increase - strain hardening - stress serration - total fracture transitions due to the interplay between the bending and stretching of individual filaments and the deformation and breakage of cross-linkers. Interestingly, the apparent network modulus is found to scale with the linear and rotational stiffness of the crosslinks to a power exponent of 0.78 and 0.13, respectively. In addition, the network fracture energy will reach its minimum at intermediate rotational compliance values, reflecting the fact that most of the strain energy will be stored in the distorted filaments with rigid cross-linkers while the imposed deformation will be "evenly" distributed among significantly more crosslinking molecules with high rotational compliance.

Deuteron NMR resolved mesogen vs. crosslinker molecular order and reorientational exchange in liquid single crystal elastomers.

Differences in the temperature behaviour of orientational ordering of structurally equivalent side-chain liquid single crystal elastomers (LSCEs) with (2)H-labelled crosslinker and mesogen have been studied by deuteron quadrupole-perturbed NMR. The impact of nematic director reorientations on the deuteron NMR spectral shapes was analyzed in terms of a discrete reorientational exchange model. This provided for the determination of the degree of nematic director alignment and for the quantification of the influence of the reorientational exchange on the (2)H NMR spectra in terms of two parameters, the nematic director orientational dispersion parameter σθ and the motional effectiveness parameter α. A comparative analysis of model simulations and experimental spectra reveals that mesogenic molecules in LSCEs exhibit faster reorientational dynamics as compared to crosslinker molecules and that mesogens and crosslinkers exhibit a similar and rather substantial static director orientational disorder.

Ultrastable BSA-capped gold nanoclusters with a polymer-like shielding layer against reactive oxygen species in living cells.

The prevalence of reactive oxygen species (ROS) production and the enzyme-containing intracellular environment could lead to the fluorescence quenching of bovine serum albumin (BSA)-capped gold nanoclusters (AuNCs). Here we report an efficient strategy to address this issue, where a polymer-like shielding layer is designed to wrap around the Au core to significantly improve the stability of AuNCs against ROS and protease degradation. The key of our design is to covalently incorporate a thiolated AuNC into the BSA-AuNC via carbodiimide-activated coupling, leading to the formation of a AuNC pair inside the cross-linked BSA molecule. The as-designed paired AuNCs in BSA (or BSA-p-AuNCs for short) show improved performances in living cells.