Impact Of Crosslinking Research Articles

DFF-ChIP: a method to detect and quantify complex interactions between RNA polymerase II, transcription factors, and chromatin.

Recently, we introduced a chromatin immunoprecipitation (ChIP) technique utilizing the human DNA Fragmentation Factor (DFF) to digest the DNA prior to immunoprecipitation (DFF-ChIP) that provides the precise location of transcription complexes and their interactions with neighboring nucleosomes. Here we expand the technique to new targets and provide useful information concerning purification of DFF, digestion conditions, and the impact of crosslinking. DFF-ChIP analysis was performed individually for subunits of Mediator, DSIF, and NELF that that do not interact with DNA directly, but rather interact with RNA polymerase II (Pol II). We found that Mediator was associated almost exclusively with preinitiation complexes (PICs). DSIF and NELF were associated with engaged Pol II and, in addition, potential intermediates between PICs and early initiation complexes. DFF-ChIP was then used to analyze the occupancy of a tight binding transcription factor, CTCF, and a much weaker binding factor, glucocorticoid receptor (GR), with and without crosslinking. These results were compared to those from standard ChIP-Seq that employs sonication and to CUT&RUN which utilizes MNase to fragment the genomic DNA. Our findings indicate that DFF-ChIP reveals details of occupancy that are not available using other methods including information revealing pertinent protein:protein interactions.

Facile fabrication of chitosan/polysialic acid composite hydrogels crosslinked with tannic acid to enhance wound healing after cesarean section

Infections and post-wound healing following a cesarean section are among the most challenging complications of surgery. In the current study, novel chitosan/polysialic acid (CTS/PSA) crosslinked hydrogels were designed and employed as superficial wound dressings to increase the healing ability of cesarean wounds and prevent bactericidal infections. The impact of crosslinking was more pronounced with a lower concentration of tannic acid (TA), which still formed a crosslinked network. The impact of TA crosslinking on CTS/PSA hydrogel system was 15 wt% TA. The morphology of the hydrogels was investigated by scanning electron microscopy (SEM) analysis. The pore sizes of CTS/PSA, TA@CTS/PSA-5%, TA@CTS/PSA-10%, and TA@CTS/PSA-15% were 96.7±6.3, 122.3±7.1, 166.8±9.3, and 271.2±20.3 μm, respectively. In addition, the swelling ratio, degradation, and mechanical performance of the hydrogels were studied. The crosslinked CTS/PSA hydrogels were shown to be effective against the gram-positive and gram-negative strains of S. aureus and E. coli. Furthermore, the TA@CTS/PSA hydrogels exhibited good cytocompatibility towards non-cancerous L929 fibroblast cells. The results demonstrated that TA@CTS/PSA hydrogels are potential dressings for post-cesarean-section wound care.

Impact of Crosslinking on the Characteristics of Pectin Monolith Cryogels

In this research, the pectin monoliths were prepared via the sol-gel process through different routes of crosslinking and additional freeze-drying. The crosslinking reaction was induced by the use of calcium ions in aqueous solutions and in alcohol/water solutions. The resulting pectin monoliths obtained by freeze-drying were macroporous with open cells, limited specific surface area, moderate mechanical stability and moderate biodegradation rate. The presence of alcohol in crosslinking solution significantly changed the morphology of final pectin monoliths, which was evidenced by the reduction of their pore size for one order. The specific surface area of pectin monoliths obtained through the calcium-water-alcohol route was 25.7 m2/g, the Young compressive modulus was 0.52 MPa, and the biodegradation rate was 45% after 30 days of immersion in compost media. Considering that pectin can be obtained from food waste, and its physical properties could be tailored by different crosslinking routes, the pectin monoliths could find wide application in the pharmaceutical, agricultural, medical and food industries, providing sustainable development concepts.

Impact of crosslinker on stereochemistry of a dynamic covalent polymer network: A molecular dynamics simulation

We studied the effect of crosslinking density, ϕc, crosslinker length, m, and crosslinker flexibility on endo to exo adduct ratio in Diels–Alder polymer networks. The fraction of endo attachments, ψendo, increased with an increase of ϕc due to suppression of exo adducts at the early time-limiting stage of the network formation, as also observed in our recently published experimental study [1]. Polymer networks formed with flexible crosslinkers demonstrated a linear growth of ψendo with ϕc for all values of m. ψexo of networks with stiff crosslinkers sharply grew in the range of lower ϕc (ϕc<0.6) and plateaued at ϕc>0.6.

Genipin cross-linked chitosan–PVA composite films: An investigation on the impact of cross-linking on accelerating wound healing

The applications of composite films in wound healing are immense, and the quest for suitable biomaterials drives research. Natural polymers lack mechanical strength; by cross-linking, their chemical nature can be changed through interlinking via intermolecular interactions rather than chemical bonds. This study explores the applications of cross-linked chitosan–polyvinyl alcohol–genipin films prepared using the solvent casting method. The cross-linked films were analyzed for their chemical structural changes, structural morphology, mechanical strength, and water retention capabilities. Comparative studies were performed with the un-crosslinked counterparts. Results indicated an increased tensile strength of 67.2% and improved water retention because of their compact structure. Further, Fourier-transform infrared spectroscopy confirmed that cross-linking occurred in the films, and scanning electron microscope micrographs showed that the films were uniform with a continuous morphology. Thereafter, an in vivo study was performed to assess the effectiveness of the films on wounds made on Albino Wistar rats. Histopathological analysis indicated quick fibroblast generation and angiogenesis using the cross-linked films, affirming their suitability for wound healing applications.

Impact of Cross-Linking of Collagen Matrices on Tissue Regeneration in a Rabbit Calvarial Bone Defect.

The cross-linking of collagen matrices (Cl_CM) may provide volume-stable enhanced defect regeneration when compared to non-cross-linked matrices (Ncl_CM). The aim of the present study was to investigate the bone forming potential of collagen matrices (CMs) and the effects of cross-linking CMs in a rabbit calvaria defect model. (1) Empty controls (n = 6), (2) Ncl_CM (n = 8), and (3) Cl_CM (n = 8) were selected to be observed for the healing in 10 mm critical-sized calvarial bone defects. The potential for the bone as well as the connective tissue formation were evaluated by micro-CT and histomorphometry at three months post-surgery. There were no statistically significant differences in terms of new bone volume in the defects between the groups. However, the Cl_CM induced significantly greater fibrous tissue regeneration (5.29 ± 1.57 mm2) when compared to the controls (3.51 ± 0.93 mm2) by histomorphometry. The remnants of collagen fibers with immune cells, including macrophages and giant cells, were occasionally observed in the Cl_CM group but not in the Ncl_CM group. In conclusion, the cross-linking of collagen did not influence the potential for bone formation. Nevertheless, Cl_CM might be advantageous for the maintenance of fibrous tissue volume without disturbing bone formation in the defects.

Impact of crosslinking on organic solvent nanofiltration performance in polydimethylsiloxane composite membrane: Probed by in-situ low-field nuclear magnetic resonance spectroscopy

Polydimethylsiloxane (PDMS) has been widely used in gas separation, pervaporation, and organic solvent nanofiltration. The crosslinking and penetration behavior of PDMS has been of great importance for fabricating composite membranes. In this study, an in-situ low-field nuclear magnetic resonance (LF-NMR) spectroscopy was employed to probe the crosslink density of PDMS during the crosslinking process. The relationship between crosslink density and nanostructure of PDMS selective layer was systematically investigated. It was found when the PDMS network had low crosslink density (υe < 1.32 × 10-3 mol cm-3), the un-crosslinked PDMS chains could easily infiltrate into the pores of the substrate, leading to a defective active layer. Therefore, the rejection for Evans blue from organic solvents was below 3.9%. With the increasing crosslink density to 1.57 × 10-3 mol cm-3, the integrity of the PDMS selective layer was remarkably reinforced with a rejection of 99.0%. Moreover, the as-prepared membrane exhibited good performance for concentrating trace vitamin B12 from different solvents. The swelling property was responsive monitored with T2 relaxation time using LF-NMR.

Does the Current State of Evidence Justify the Broad Use of Cross-Links in Dorsal Instrumentation? A Systematic Review

Cross-links increase the stability of screw-rod systems in biomechanical testing. The aim of this systemic review was to find evidence pertaining to the additional benefit of the implantation of cross-links in clinical practice in regard to different spinal diseases. Therefore, a systematic literature analysis of two online databases was performed according to the PRISMA statement. Inclusion criteria were prospective and retrospective studies investigating the use of cross-links in dorsal instrumentation. Biomechanical studies and case series were excluded. A total of seven retrospective studies remained for final full-text evaluation. In total, two studies each address the use of cross-links in adolescent idiopathic scoliosis, neuromuscular scoliosis or atlantoaxial fusion, one study in congenital scoliosis. In atlantoaxial fusion the additional use of cross-links may provide earlier bony fusion. In surgical treatment for pediatric scoliosis the additional use of cross-links does not provide additional benefit. Radiological outcome and complication rate did not differ in between groups. No study addressed the use of cross-links in short- or long-segment fusion due to degenerative or traumatic disorders of the spine. There is a deficiency in published literature towards the impact of cross-links in spinal surgery. The current clinical evidence data do not confirm the biomechanical advantages of cross-links in clinical practice. Further studies are needed to warrant the use of cross-links in the future.

The impact of crosslinking and non-crosslinking fixatives on antigen retrieval and immunohistochemistry

Pre-analytical factors can greatly influence the outcome of molecular analyses in medical diagnostics and research. This also applies to in situ staining techniques such as immunohistochemistry (IHC), where different types of tissue fixation methods lead to different modifications of proteins and thus can affect differently the detection by antibodies. For formalin-fixed paraffin-embedded (FFPE) tissue, antigen retrieval is applied in order to reverse the negative effects of formalin and re-establish immunoreactivity. Most antibodies and protocols used in IHC are optimized for FFPE tissue, but not for paraffin-embedded tissue treated with other fixatives such as non-crosslinking fixatives.We report results from systematic studies on distinct pre-analytical conditions in IHC, immunofluorescence and electron microscopy. Parameters investigated are the impact of crosslinking and non-crosslinking fixatives (comparing formalin and PAXgene Tissue fixation) on whole tissue, subcellular structures and organelles, as well as on ultrastructure. The results generated show that minor changes in antigen retrieval conditions may have a major impact on IHC results and that protocols optimized for crosslinking fixatives may not be used for other fixatives without re-validation. Key antigen retrieval parameters such as buffers with different pH and duration of microwave treatment must be tested systematically for each antibody and fixation protocol.

Structural and mechanical characterization of crosslinked and sterilised nanocellulose-based hydrogels for cartilage tissue engineering

Nanocellulose is a natural biopolymer derived from cellulose. Combined with sodium alginate, it is used to 3D print hydrogels for articular and nasal cartilage engineering and shows good integration, promising cartilage regeneration and mechanical stability over 60 days of implantation in mice. Yet, little is known about their structural and mechanical properties, particularly the impact of crosslinking and sterilisation methods. This study investigates the impact of different calcium chloride crosslinker concentrations and sterilization methods on the structural and mechanical properties of nanocellulose-based hydrogels containing plant-derived cellulose nanofibrils, cellulose nanocrystals or a blend of the two. Crosslinking significantly alters the overall network distribution, surface morphology, pore size and porosity of the hydrogels. Sterilisation has a striking effect on pore size and affects swelling depending on the sterilisation method. The effect of crosslinker and sterilisation on the overall properties of the hydrogels was reliant on the form of nanocellulose that comprised them.

Optical band gap of cross-linked, curved, and radical polyaromatic hydrocarbons.

In this work, the optical band gaps of polycyclic aromatic hydrocarbons (PAHs) crosslinked via an aliphatic bond, curved via pentagon integration and with radical character were computed using density functional theory. A variety of different functionals were benchmarked against optical band gaps (OBGs) measured by ultraviolet-visible spectroscopy with HSE06 being most accurate with a percentage error of 6% for a moderate basis set. Pericondensed aromatics with different symmetries were calculated with this improved functional providing new scaling relationships for the OBG versus size. Further calculations showed crosslinks cause a small decrease in the OBG of the monomers which saturates after 3-4 crosslinks. Curvature in PAHs was shown to increase the optical band gap due to the resulting change in hybridisation of the system, but this increase saturated at larger sizes. The increase in OBG between a flat PAH and a strained curved one was shown to be equivalent to a difference of several rings in size for pericondensed aromatic systems. The effect of σ-radicals on the optical band gap was also shown to be negligible, however, π-radicals were found to decrease the band gap by ∼0.5 eV. These findings have applications in understanding the molecular species involved in soot formation.

Waterborne epoxy-thiol decorated silica sol-gel coatings: impact of crosslinking on corrosion prevention

Organic–inorganic hybrid coatings of crosslinked epoxy and thiol silicates (TGST) were synthesized for the corrosion protection of low carbon steel via a waterborne sol-gel route. The coatings were synthesized in two stages; in the first step two separate aqueous formulations of epoxy and thiol decorated silica colloids were prepared and subsequently crosslinked in a 1:1 ratio in the second stage. The structure and rate of crosslinking was monitored using Raman spectroscopy. Formulation stability was measured based on viscosity changes and pot-life assessment. The novel material was characterized for its particle size using dynamic light scattering. Viscosity average molecular weight was calculated using Fikentscher’s equation and using polyethylene oxide molecular weights as a calibration standard. The covalently bonded epoxy-thiol silicate thin-film coating was uniform and defect free. Accelerated corrosion resistance was tested by exposing the panels to standard salt solution tests. TGST showed a 96% improvement in corrosion inhibition over the non-crosslinked formulations in a corrosive environment. The synthesis and characterization of TGST using a simple approach, revealed key synthetic parameters for generating optimized sol-gel corrosion prevention materials.

Кросслинкинг в комплексном лечении язв роговицы и трансплантата

Purpose: to study the impact of crosslinking (CL) on the healing of corneal ulcers of various etiologies. Materials and methods. Treatment results of 15 patients with corneal and transplant ulcers by CL and its combination with other surgical interventions were analyzed. Results. For the first time three options of combined treatment of corneal ulcers were proposed: 1) CL as an independent method; 2) CL combined with transplantation of amniotic membrane (ТАМ) and temporary tarsorrhaphy; 3) corneal grafting combined with CL. The main diagnostic technique of treatment result evaluation was OCT of the anterior segment of the eye. Results. CL was found to contribute to the cleaning of the bottom and the edges of the ulcer surface, to promote the resorption of the hypopyon and the edema, leads to rapid healing of the ulcer and the reduction of the corneal syndrome and increases visual acuity. Conclusion. The obtained data indicate the need for further study of the impact of CL on the healing process of corneal and transplant ulcers. For citations: Chentsova E.V., Verigo E.N., Makarov P.V., Khazamova A.I. Crosslinking in the complex treatment of corneal ulceration and corneal grafting. Russian ophthalmological journal. 2017; 10 (3): 93-100. doi: 10.21516/2072-0076-2017-10-3-93-100 (in Russian).

Crosslinking of extracellular matrix scaffolds derived from pluripotent stem cell aggregates modulates neural differentiation

At various developmental stages, pluripotent stem cells (PSCs) and their progeny secrete a large amount of extracellular matrices (ECMs) which could interact with regulatory growth factors to modulate stem cell lineage commitment. ECMs derived from PSC can be used as unique scaffolds that provide broad signaling capacities to mediate cellular differentiation. However, the rapid degradation of ECMs can impact their applications as the scaffolds for in vitro cell expansion and in vivo transplantation. To address this issue, this study investigated the effects of crosslinking on the ECMs derived from embryonic stem cells (ESCs) and the regulatory capacity of the crosslinked ECMs on the proliferation and differentiation of reseeded ESC-derived neural progenitor cells (NPCs). To create different biological cues, undifferentiated aggregates, spontaneous embryoid bodies, and ESC-derived NPC aggregates were decellularized. The derived ECMs were crosslinked using genipin or glutaraldehyde to enhance the scaffold stability. ESC-derived NPC aggregates were reseeded on different ECM scaffolds and differential cellular compositions of neural progenitors, neurons, and glial cells were observed. The results indicate that ESC-derived ECM scaffolds affect neural differentiation through intrinsic biological cues and biophysical properties. These scaffolds have potential for in vitro cell culture and in vivo tissue regeneration study. Statement of significanceDynamic interactions of acellular extracellular matrices and stem cells are critical for lineage-specific commitment and tissue regeneration. Understanding the synergistic effects of biochemical, biological, and biophysical properties of acellular matrices would facilitate scaffold design and the functional regulation of stem cells.The present study assessed the influence of crosslinked embryonic stem cell-derived extracellular matrix on neural differentiation and revealed the synergistic interactions of various matrix properties.While embryonic stem cell-derived matrices have been assessed as tissue engineering scaffolds, the impact of crosslinking on the embryonic stem cell-derived matrices to modulate neural differentiation has not been studied.The results from this study provide novel knowledge on the interface of embryonic stem cell-derived extracellular matrix and neural aggregates. The findings reported in this manuscript are significant for stem cell differentiation toward the applications in stem cell-based drug screening, disease modeling, and cell therapies.

Crosslinks and replication: the show must go on

Crosslinks and replication: the show must go on

Permittivity and Loss Characterization of SU-8 Films for mmW and Terahertz Applications

The dielectric permittivity measurement of thick SU-8 film is presented for the entire frequency band of 1 GHz to 1 THz. SU-8 is a high-resolution UV-patternable photoresist that can be used for fabrication of high-aspect-ratio 3-D structures for millimeter-wave and terahertz devices. Here, we report the measured dielectric constant and loss tangent of SU-8 films using terahertz time-domain spectroscopy. A quadratic polynomial model is established for the accurate calculation of complex permittivity up to 1 THz. The loss tangent of fully cross-linked 430- $\mu\hbox{m}$ -thick SU-8 film was measured to be 0.015, 0.027, and 0.055 at 1, 200, and 1000 GHz, respectively. Similarly, relative permittivity was found to be 3.24, 3.23, and 2.92. The fabrication process and level of cross-linking were demonstrated to have significant impact on the loss behavior of this material and the impact of cross-linking on dielectric permittivity is quantified across a wide frequency band. The characterization results reported in this letter are a platform for developing next-generation millimeter-wave and terahertz devices.

Copolymerization and properties of multicomponent crosslinked hydrogels

A series of multicomponent hydrogels were prepared by the copolymerization of hydrophobic silicon-containing monomer 3-bis(trimethylsilyloxy) methylsilylpropyl glycerol methacrylate (SiMA) with the solvent-responsive monomers 2-hydroxyethyl methacrylate (HEMA) and N-vinyl pyrrolidone (NVP) and thermosensitive monomer N,N-dimethyl acrylamide (DMA). 2-Hydroxy-2-methyl phenyl acetone (D-1173) was chosen as UV initiator and five different dienes/triene monomers were selected as crosslinking agent in order to select the best crosslinker. The ethanol extraction experiments as well as the FTIR, DSC and TG results showed that the copolymerization was effective. The optical, permeability, and mechanical analysis results demonstrated that the obtained hydrogels were highly transparent with good oxygen permeability and mechanical properties. And the impact of crosslinker on the mechanical properties of the hydrogels was also discussed in detail. The basic results demonstrated that the obtained hydrogels had good stimuli-responsive effects to both pH value and solvent.

Fluctuation driven height reduction of crosslinked polymer brushes: A Monte Carlo study

We study the changes in the conformations of brushes upon the addition of crosslinks between the chains using the bond fluctuation model. The Flory-Rehner model applied to uniaxially swollen networks predicts a collapse for large degrees of crosslinking q proportional to q(-1∕3) in disagreement with our simulation data. We show that the height reduction of the brushes is driven by monomer fluctuations in a direction perpendicular to the grafting plane and not due to network elasticity. We observe that the impact of crosslinking is different for reactions between monomers of the same or on different chains. If the length reduction of the effective chain length due to both types of reactions is accounted for in a function β(q), the height of the brush can be derived from a Flory approach for the equilibrium brush height leading to H(q) ≈ Hbβ(q)(1∕3), whereby Hb denotes the height of the non-crosslinked brush.

Model thrombi formed under flow reveal the role of factorXIII-mediated cross-linking in resistance to fibrinolysis.

SummaryBackground: Activated factor XIII (FXIIIa), a transglutaminase, introduces fibrin–fibrin and fibrin–inhibitor cross-links, resulting in more mechanically stable clots. The impact of cross-linking on resistance to fibrinolysis has proved challenging to evaluate quantitatively. Methods:We used a whole blood model thrombus system to characterize the role of cross-linking in resistance to fibrinolytic degradation. Model thrombi, which mimic arterial thrombi formed in vivo, were prepared with incorporated fluorescently labeled fibrinogen, in order to allow quantification of fibrinolysis as released fluorescence units per minute. Results:A site-specific inhibitor of transglutaminases, added to blood from normal donors, yielded model thrombi that lysed more easily, either spontaneously or by plasminogen activators. This was observed both in the cell/platelet-rich head and fibrin-rich tail. Model thrombi from an FXIII-deficient patient lysed more quickly than normal thrombi; replacement therapy with FXIII concentrate normalized lysis. In vitro addition of purified FXIII to the patient's preprophylaxis blood, but not to normal control blood, resulted in more stable thrombi, indicating no further efficacy of supraphysiologic FXIII. However, addition of tissue transglutaminase, which is synthesized by endothelial cells, generated thrombi that were more resistant to fibrinolysis; this may stabilize mural thrombi in vivo. Conclusions:Model thrombi formed under flow, even those prepared as plasma ‘thrombi’, reveal the effect of FXIII on fibrinolysis. Although very low levels of FXIII are known to produce mechanical clot stability, and to achieve γ-dimerization, they appear to be suboptimal in conferring full resistance to fibrinolysis.

Impact of cross-linker on alginate matrix integrity and drug release

Sodium alginate, a biopolymer, was employed in the formulation of matrix tablets. They cracked or laminated at acidic pH, compromising their dissolution performance. Improved mechanical strength and reduced barrier permeability of calcium alginate gel provided the rationale for cross-linking the alginate matrix to sustain drug release. Studies had suggested that the incorporation of soluble calcium salts in alginate matrix tablets could sustain drug release at near-neutral pH due to in situ cross-linking. However, results from the present study showed otherwise when gastrointestinal pH conditions were simulated. Significant reduction in drug release rate was only observed when an external calcium source was utilized at low concentration. High calcium ion concentrations caused matrix disintegration. In contrast, matrices pre-coated by calcium alginate could sustain drug release at pH 1.2 followed by pH 6.8 for over 12 h. The presence of cross-linked barrier impeded matrix lamination and preserved matrix structure, contributing to at least three-fold reduction in drug release at pH 1.2. Zero order release as well as delayed burst release could be achieved by employing appropriate grade of alginate and cross-linking conditions.