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Biomechanical effect of ultraviolet-A-riboflavin cross-linking on simulated human corneal stroma model and its correlation with changes in corneal stromal microstructure.
Li, H, Liu, T, Mu, B, Zhao, X, Xue, C, Shen, M, Jhanji, V, Wang, Y
Experimental eye research. 2020;:108109
Abstract
In this study, we established an experimental human corneal stroma model of simulated cornea tissue composed of thin anterior cornea strips layers obtained from small incision lenticular extraction (SMILE) surgery. We investigated the biomechanical effect of ultraviolet-A- riboflavin cross-linking at different depths of corneal stroma model and correlated it with stromal microstructural changes examined by transmission electron microscopy (TEM). Corneal strips were harvested from fresh human corneal lenticules obtained after SMILE surgery. Experimental models (n = 34) were established by superimposing the corneal lenticule strips until their thickness reached close to 500 μm. Corneal cross-linking (CXL) was performed subsequently using standard or accelerated protocol. Elasticity and viscosity were quantified using stress-strain extensometer. TEM was used to visualize the collagen fiber diameter and interfibrillar spacing. The relative change in Young's modulus (rel. ΔE) decreased nonlinearly with increasing stromal depth both in the standard and accelerated groups. Compared to the sham controls, the rel. ΔE in standard and accelerated CXL groups increased significantly in the anterior 400 μm and 275 μm depth, respectively. Also, the relative change in stress (rel. ΔS) was significantly lower after standard and accelerated CXL compared to sham controls. Depth analysis showed similar results for the elastic effect. TEM images showed a small, non-significant increase in fibril diameter. The interfibrillar spacing decreased significantly after standard and accelerated CXL in the anterior-mid stromal region. We noted that the increase of corneal stiffness correlated with decrease in interfibrillar spacing after CXL. The stiffening effect was depth dependent. The effect of accelerated CXL was less in the deep corneal stromal regions compared to standard CXL.
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Effects of cross linking and/or acetylation on sorghum starch and film characteristics.
Mehboob, S, Ali, TM, Sheikh, M, Hasnain, A
International journal of biological macromolecules. 2020;:786-794
Abstract
In the present study, starch was modified by a) cross-linking through addition of 3% mixture of sodium trimetaphosphate (STMP) and sodium tripolyphosphate (STPP) based on starch weight b) acetylation through addition of 2%, 4% and 6% acetic anhydride based on starch weight c) dual modification i.e. cross-linking using 3% mixture of STMP and STPP followed by acetylation at 2%, 4% and 6% levels based on starch weight. These starches were then used to develop edible films followed by their characterization. Experimental results revealed that films with increased thickness were obtained from acetylated and cross linked starches whereas, dual modified starch films had reduced thickness compared to native starch film. Films from acetylated and dual modified starches retained more moisture compared to native and cross linked starch films while dual modified starches were more water soluble than acetylated and native starch films. All modified starch films were more clear and transparent compared to native starch films. Films from acetylated starches depicted lower tensile strength but they were more elastic and flexible than native and cross linked starch films. In contrast, dual modified starch films showed excellent mechanical properties and lower water permeability compared to native starch film.
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High-density chemical cross-linking for modeling protein interactions.
Mintseris, J, Gygi, SP
Proceedings of the National Academy of Sciences of the United States of America. 2020;(1):93-102
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Abstract
Detailed mechanistic understanding of protein complex function is greatly enhanced by insights from its 3-dimensional structure. Traditional methods of protein structure elucidation remain expensive and labor-intensive and require highly purified starting material. Chemical cross-linking coupled with mass spectrometry offers an alternative that has seen increased use, especially in combination with other experimental approaches like cryo-electron microscopy. Here we report advances in method development, combining several orthogonal cross-linking chemistries as well as improvements in search algorithms, statistical analysis, and computational cost to achieve coverage of 1 unique cross-linked position pair for every 7 amino acids at a 1% false discovery rate. This is accomplished without any peptide-level fractionation or enrichment. We apply our methods to model the complex between a carbonic anhydrase (CA) and its protein inhibitor, showing that the cross-links are self-consistent and define the interaction interface at high resolution. The resulting model suggests a scaffold for development of a class of protein-based inhibitors of the CA family of enzymes. We next cross-link the yeast proteasome, identifying 3,893 unique cross-linked peptides in 3 mass spectrometry runs. The dataset includes 1,704 unique cross-linked position pairs for the proteasome subunits, more than half of them intersubunit. Using multiple recently solved cryo-EM structures, we show that observed cross-links reflect the conformational dynamics and disorder of some proteasome subunits. We further demonstrate that this level of cross-linking density is sufficient to model the architecture of the 19-subunit regulatory particle de novo.
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Tear function and ocular surface changes following corneal collagen cross-linking treatment in keratoconus patients: 18-month results.
Uysal, BS, Akcay, E, Kilicarslan, A, Mutlu, M, Hondur, G, Kosekahya, P, Cagil, N
International ophthalmology. 2020;(1):169-177
Abstract
PURPOSE To investigate the long-term effects that CXL has on the tear function and ocular surface in keratoconus. METHODS Twenty-one consecutive patients (24 eyes) with progressive keratoconus scheduled for CXL were included. All patients underwent the following procedures: conjunctival impression cytology analysis, ocular surface disease index (OSDI) score, tear osmolarity test, Schirmer test, tear film breakup time (TBUT), ophthalmic surface fluorescein (Fl) staining, and topographical corneal evaluation before as well as 3 and 18 months after accelerated CXL. RESULTS There was no change in the median OSDI score, tear osmolarity test, Schirmer test, and the Fl staining score after CXL. The median TBUT increased from 9.00 s at baseline to 12.00 s at 18 months postoperative (P < 0.001). The cytological features of the temporal and superior bulbar conjunctiva deteriorated at 3 months post-CXL (P < 0.001). An improvement in impression cytology analysis of the temporal conjunctiva was noted at 18-month follow-up (P < 0.001). Significant improvements in the median maximum keratometry and mean keratometry (K-mean) readings were also noted 18 months after CXL (P < 0.001). The changes in the K-mean correlated significantly with the changes in TBUT levels at 18-month follow-up as compared to baseline (r = - 0.688, P < 0.001). CONCLUSIONS The improvement in TBUT, conjunctival squamous metaplasia, and the goblet cell density indicates a favorable effect of CXL on the ocular surface and tear film in keratoconus, presumably due to the reduced corneal irregularity after CXL.
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A Genetically Encoded Diazirine Analogue for RNA-Protein Photo-crosslinking.
Dziuba, D, Hoffmann, JE, Hentze, MW, Schultz, C
Chembiochem : a European journal of chemical biology. 2020;(1-2):88-93
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Abstract
Ultraviolent crosslinking is a key experimental step in the numerous protocols that have been developed for capturing and dissecting RNA-protein interactions in living cells. UV crosslinking covalently stalls dynamic interactions between RNAs and the directly contacting RNA-binding proteins and enables stringent denaturing downstream purification conditions needed for the enrichment and biochemical analysis of RNA-protein complexes. Despite its popularity, conventional 254 nm UV crosslinking possesses a set of intrinsic drawbacks, with the low photochemical efficiency being the central caveat. Here we show that genetically encoded photoreactive unnatural amino acids bearing a dialkyl diazirine photoreactive group can address this problem. Using the human iron regulatory protein 1 (IRP1) as a model RNA-binding protein, we show that the photoreactive amino acids can be introduced into the protein without diminishing its RNA-binding properties. A sevenfold increase in the crosslinking efficiency compared to conventional 254 nm UV crosslinking was achieved using the diazirine-based unnatural amino acid DiAzKs. This finding opens an avenue for new applications of the unnatural amino acids in studying RNA-protein interactions.
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Improving Identification of In-organello Protein-Protein Interactions Using an Affinity-enrichable, Isotopically Coded, and Mass Spectrometry-cleavable Chemical Crosslinker.
Makepeace, KAT, Mohammed, Y, Rudashevskaya, EL, Petrotchenko, EV, Vögtle, FN, Meisinger, C, Sickmann, A, Borchers, CH
Molecular & cellular proteomics : MCP. 2020;(4):624-639
Abstract
An experimental and computational approach for identification of protein-protein interactions by ex vivo chemical crosslinking and mass spectrometry (CLMS) has been developed that takes advantage of the specific characteristics of cyanurbiotindipropionylsuccinimide (CBDPS), an affinity-tagged isotopically coded mass spectrometry (MS)-cleavable crosslinking reagent. Utilizing this reagent in combination with a crosslinker-specific data-dependent acquisition strategy based on MS2 scans, and a software pipeline designed for integrating crosslinker-specific mass spectral information led to demonstrated improvements in the application of the CLMS technique, in terms of the detection, acquisition, and identification of crosslinker-modified peptides. This approach was evaluated on intact yeast mitochondria, and the results showed that hundreds of unique protein-protein interactions could be identified on an organelle proteome-wide scale. Both known and previously unknown protein-protein interactions were identified. These interactions were assessed based on their known sub-compartmental localizations. Additionally, the identified crosslinking distance constraints are in good agreement with existing structural models of protein complexes involved in the mitochondrial electron transport chain.
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Bifunctional Non-Canonical Amino Acids: Combining Photo-Crosslinking with Click Chemistry.
Hoffmann, JE
Biomolecules. 2020;(4)
Abstract
Genetic code expansion is a powerful tool for the study of protein interactions, as it allows for the site-specific incorporation of a photoreactive group via non-canonical amino acids. Recently, several groups have published bifunctional amino acids that carry a handle for click chemistry in addition to the photo-crosslinker. This allows for the specific labeling of crosslinked proteins and therefore the pulldown of peptides for further analysis. This review describes the properties and advantages of different bifunctional amino acids, and gives an overview about current and future applications.
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Safety and efficacy of riboflavin-assisted collagen cross-linking of cornea in progressive keratoconus patients: A prospective study in North East India.
Bhattacharyya, A, Sarma, P, Das, K, Agarwal, B, Medhi, J, Das Mohapatra, SS
Indian journal of pharmacology. 2019;(3):157-167
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Abstract
INTRODUCTION Riboflavin- and ultraviolet (UV)-A-mediated collagen cross-linking of the cornea is a frequently used therapeutic measure for the treatment of progressive keratoconus (PK). First, riboflavin increases cross-linking and second, it serves as a protective shield to other deep ocular structures. However, pharmacogenomic variation in riboflavin efficacy is reported. As the Northeast Indian population represents a genetically diverse group of population compared to mainstream India, we have assessed the efficacy of the procedure in a northeastern population with PK. METHODS In this study, 78 eyes with PK were included (n = 39 in the treatment arm and n = 39 in the control arm). The primary objective was to evaluate the effect of corneal collagen cross-linking using riboflavin (C3R) (epithelium off) on maximum keratometry. The secondary objectives were evaluation of change in corneal topography parameters, i.e., minimum keratometry (Kmin), simulated keratometry (Sim K), subjective refraction (cylinder power, spherical power, and spherical equivalent [SE]), uncorrected visual acuity (UCVA), best-corrected visual acuity (BCVA), and contrast sensitivity (CS) and safety (intraocular pressure, endothelial cell density, and percentage hexagonality) at 1, 3, and 6 months following C3R procedure. RESULTS Statistically significant improvement was noted in Kmin (6 months), Sim K (3 and 6 months), cylinder power (3 and 6 months), spherical power (3 and 6 months), SE (3 and 6 months), BCVA (6 months), and UCVA (1, 6 months) in the C3R group (n = 39) when compared to the control group (n = 39). The mean CS decreased at 1 month and gradually improved to achieve statistically significant value at 6 months in the C3R group (P < 0.05). CONCLUSION Riboflavin-assisted C3R treatment showed promising efficacy in the treatment of PK patients in our population. As the collagen turnover rate of cornea is 2-3 years and the progression of PK is highly variable, we need long-term studies to evaluate the efficacy of C3R over time, requirement of repeat therapy, and safety of repeat cross-linking.
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Application of diethylenetriamine grafted on glyoxal cross-linked chitosan composite for the effective removal of metal ions in batch system.
Igberase, E, Osifo, PO
International journal of biological macromolecules. 2019;:1145-1155
Abstract
This investigation studied the removal of Cu2+, Pb2+, Cd2+, Zn2+, Ni2+ and Cr6+ ions from synthesised wastewater using modified chitosan macromolecules. On this note, chitosan beads (CS) were prepared and cross-linked with glyoxal solution. It was found that cross-linking increased the beads mechanical strength and chemical stability in acid solution and also increased the crystallinity of the beads in the process, which is a shortcoming, as the beads tend to have reduced adsorption capacity. To reduce this shortcoming, the cross-linked chitosan beads (DCS) were grafted with diethylenetriamine. The beads were characterised prior to adsorption studies. The amine concentration of the grafted cross-linked beads (GDCS) was observed to be nearly the same as the adsorption capacity (qmax); this concludes that the amine group of chitosan are the main reactive group. Also, the qmax was found to be 6.3 mmol/g with a 44.2% degree of grafting. The kinetics of the adsorption process was described reasonably well with the Swan model, where the experimental and simulated data were in close agreement. The effective diffusion coefficients (Deff) obtained by fitting the model to experimental data were found to be between 2.25·10-10 to 2.50·10-10 for Cu2+, Pb2+, Cd2+, Zn2+, Ni2+ and Cr6+ adsorption unto GDCS.
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Corneal crosslinking: Current protocols and clinical approach.
Beckman, KA, Gupta, PK, Farid, M, Berdahl, JP, Yeu, E, Ayres, B, Chan, CC, Gomes, JAP, Holland, EJ, Kim, T, et al
Journal of cataract and refractive surgery. 2019;(11):1670-1679
Abstract
Members of the ASCRS Cornea Clinical Committee performed a review of the current literature on the corneal crosslinking (CXL) procedure for treating corneal ectasia. The members explored the data on the techniques currently in use and under investigation, including their advantages, safety profiles, risks, and cost analyses, compared with data on corneal transplantation. They concluded that CXL limits the progression of keratoconus, thus reducing the need for transplantation. They also found that compared with permitting the disease to progress naturally, CXL techniques carry significant and long-term cost and safety benefits, primarily by reducing the need for corneal transplantation. Studies of various CXL techniques (eg, epithelium-on treatment, changes in ultraviolet light parameters, riboflavin composition) continue with the ultimate goal of improving the procedure's safety and efficacy.