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1.
Immobilization of Serratia plymuthica by ionic gelation and cross-linking with transglutaminase for the conversion of sucrose into isomaltulose.
Carvalho, PH, Kawaguti, HY, de Souza, WFC, Sato, HH
Bioprocess and biosystems engineering. 2021;(6):1109-1118
Abstract
Isomaltulose is an alternative sugar obtained from sucrose using some bacteria producing glycosyltransferase. This work aimed to optimize conditions for the immobilization of Serratia plymuthica through ionic gelation and cross-linking by transglutaminase using the sequential experimental strategy for the conversion of sucrose into isomaltulose. The effect of five variables (concentrations of cell mass, alginate, gelatin, transglutaminase, and calcium chloride) was studied, as well as the interactions between them on the matrix composition for the S. plymuthica immobilization. Three experimental designs were used to optimize the concentrations of each variable to obtain higher concentration of isomaltulose. A high conversion of sucrose into isomaltulose (71.04%) was obtained by the cells immobilized in a matrix composed of alginate (1.7%), CaCl2 (0.25 mol/L), gelatin (0.5%), transglutaminase (3.5%) and cell mass (33.5%). As a result, the transglutaminase application as a cross-linking agent improved the immobilization of Serratia plymuthica cells and the conversion of sucrose into isomaltulose.
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2.
Epigallocatechin-3-gallate plays more predominant roles than caffeine for inducing actin-crosslinking, ubiquitin/proteasome activity and glycolysis, and suppressing angiogenesis features of human endothelial cells.
Gallemit, PEM, Yoodee, S, Malaitad, T, Thongboonkerd, V
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. 2021;:111837
Abstract
A recent expression proteomics study has reported changes in cellular proteome (set of proteins) of human endothelial cells (ECs) induced by caffeine and epigallocatechin-3-gallate (EGCG), the most abundant bioactive compounds in coffee and green tea, respectively. Although both common and differential changes were highlighted by bioinformatics prediction, no experimental validation was performed. Herein, we reanalyzed these proteome datasets and performed protein-protein interactions network analysis followed by functional investigations using various assays to address the relevance of such proteome changes in human ECs functions. Protein-protein interactions network analysis revealed actin-crosslink formation, ubiquitin-proteasome activity and glycolysis as the three main networks among those significantly altered proteins induced by caffeine and EGCG. The experimental data showed predominant increases of actin-crosslink formation, ubiquitin-proteasome activity, and glycolysis (as reflected by increased F-actin and β-actin, declined ubiquitinated proteins and increased intracellular ATP, respectively) in the EGCG-treated cells. Investigations on angiogenesis features revealed that EGCG predominantly reduced ECs proliferation, migration/invasion, endothelial tube formation (as determined by numbers of nodes/junctions and meshes), barrier function (as determined by levels of VE-cadherin, zonula occludens-1 (ZO-1) and transendothelial resistance (TER)), and angiopoietin-2 secretion. However, both caffeine and EGCG had no effects on matrix metalloproteinase-2 (MMP-2) secretion. These data indicate that EGCG exhibits more potent effects on human ECs functions to induce actin-crosslink, ubiquitin-proteasome activity and glycolysis, and to suppress angiogenesis processes that commonly occur in various diseases, particularly cancers.
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Characterization of Cross-Linked Enzyme Aggregates of the Y509E Mutant of a Glycoside Hydrolase Family 52 β-xylosidase from G. stearothermophilus.
Romero, G, Contreras, LM, Aguirre, C, Wilkesman, J, Clemente-Jiménez, JM, Rodríguez-Vico, F, Las Heras-Vázquez, FJ
Molecules (Basel, Switzerland). 2021;(2)
Abstract
Cross-linked enzyme aggregates (CLEAs) of the Y509E mutant of glycoside hydrolase family 52 β-xylosidase from Geobacillus stearothermophilus with dual activity of β-xylosidase and xylanase (XynB2Y509E) were prepared. Ammonium sulfate was used as the precipitant agent, and glutaraldehyde as cross-linking agent. The optimum conditions were found to be 90% ammonium sulfate, 12.5 mM glutaraldehyde, 3 h of cross-linking reaction at 25 °C, and pH 8.5. Under these (most effective) conditions, XynB2Y509E-CLEAs retained 92.3% of their original β-xylosidase activity. Biochemical characterization of both crude and immobilized enzymes demonstrated that the maximum pH and temperature after immobilization remained unchanged (pH 6.5 and 65 °C). Moreover, an improvement in pH stability and thermostability was also found after immobilization. Analysis of kinetic parameters shows that the K m value of XynB2Y509E-CLEAs obtained was slightly higher than that of free XynB2Y509E (1.2 versus 0.9 mM). Interestingly, the xylanase activity developed by the mutation was also conserved after the immobilization process.
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4.
Transepithelial versus Epithelium-off Corneal Collagen Cross-linking for Corneal Ectasia: A Systematic Review and Meta-analysis.
Nath, S, Shen, C, Koziarz, A, Banfield, L, Nowrouzi-Kia, B, Fava, MA, Hodge, WG
Ophthalmology. 2021;(8):1150-1160
Abstract
TOPIC To evaluate the safety and efficacy of transepithelial corneal cross-linking in comparison with the established epithelium-off technique for corneal ectasia. CLINICAL RELEVANCE Considerable debate exists regarding whether transepithelial and epithelium-off cross-linking are comparable in their safety and efficacy. METHODS We searched 16 electronic databases, including Medline, Embase, Web of Science, and the grey literature, current to July 8, 2020, for randomized controlled trials comparing transepithelial and epithelium-off cross-linking for corneal ectasia. We excluded studies evaluating cross-linking for nonectatic indications, as well as non-randomized controlled trials. Our primary outcome was the change in maximal keratometry (Kmax) at 12 months after cross-linking, and we considered additional topographic, visual, and safety outcomes. We summarized our analyses by calculating weighted mean differences (MDs) with associated 95% confidence intervals (CIs) for continuous outcomes and relative risks (RRs) with corresponding 95% CIs for dichotomous outcomes. We conducted trial sequential analysis to determine whether the required information size was met for each outcome. The quality of individual trials was evaluated using the Cochrane Collaboration's risk of bias assessment tool, and the evidence was assessed at an outcome level using the Grading of Recommendations Assessment, Development, and Evaluation methodology. RESULTS Twelve studies totaling 966 eyes were eligible. A significant difference was found between transepithelial and epithelium-off cross-linking groups in the change in Kmax at 12 months (MD, 0.75; 95% CI, 0.23-1.28; P = 0.004; primary outcome) and at longest follow-up (MD, 1.20; 95% CI, 0.62-1.77; P < 0.001; secondary outcome) after treatment. No significant difference was found between the 2 groups when examining uncorrected distance visual acuity (MD, 0.04; 95% CI, -0.06 to 0.14; P = 0.386) or corrected distance visual acuity (MD, 0.01; 95% CI, -0.06 to 0.09; P = 0.732). Transepithelial cross-linking was associated with significantly fewer complications than the epithelium-off approach (RR, 0.22; 95% CI, 0.06-0.79; P = 0.020), although it was associated with an increased rate of disease progression at 12 months after treatment (RR, 4.49; 95% CI, 1.24-16.25; P = 0.022). The required information size was met for our primary outcome and trial sequential analysis supported the conventional meta-analysis. The quality of evidence was rated as moderate using the Grading of Recommendations Assessment, Development, and Evaluation methodology. DISCUSSION The efficacy of transepithelial cross-linking remains inferior to the epithelium-off approach, although it is significantly safer.
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Diffuse Lamellar Keratitis in a Patient Undergoing Collagen Corneal Cross-Linking 18 Years After Laser In Situ Keratomileusis Surgery.
Grassmeyer, JJ, Goertz, JG, Baartman, BJ
Cornea. 2021;(7):917-920
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Abstract
PURPOSE To report a case of diffuse lamellar keratitis (DLK) after corneal collagen cross-linking in an eye with a remote history of laser in situ keratomileusis (LASIK) surgery. METHODS This is a case report and literature review. RESULTS This report describes the development of unilateral stage IV DLK in a patient who underwent bilateral corneal cross-linking for corneal ectasia 18 years after LASIK surgery. The patient was treated with high-dose topical steroids that were tapered over 1 month and multiple flap lifts. The ultimate best-corrected visual outcome was 20/60. CONCLUSIONS DLK is a potential sight-threatening complication of refractive surgery that can occur at any time in the postoperative period, even years after the procedure. Undergoing a subsequent corneal procedure that may disrupt or promote inflammation within the surgical flap-stromal interface, such as corneal collagen cross-linking, is a recognized risk factor for the development of DLK. This case suggests that patients with any history of LASIK surgery undergoing corneal cross-linking or other lamellar corneal surgeries may benefit from closer follow-up (eg, daily) than patients with no history of LASIK.
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Cross-Linking Assisted Infection Reduction (CLAIR): A Randomized Clinical Trial Evaluating the Effect of Adjuvant Cross-Linking on Bacterial Keratitis.
Prajna, NV, Radhakrishnan, N, Lalitha, P, Rajaraman, R, Narayana, S, Austin, AF, Liu, Z, Keenan, JD, Porco, TC, Lietman, TM, et al
Cornea. 2021;(7):837-841
Abstract
PURPOSE To determine whether there is a benefit to adjuvant corneal cross-linking (CXL) for bacterial keratitis. METHODS This is an outcome-masked, randomized controlled clinical trial. Consecutive patients presenting with a smear-positive bacterial ulcer at Aravind Eye Hospitals at Madurai, Pondicherry, and Coimbatore in India were enrolled. Study eyes were randomized to topical moxifloxacin 0.5% or topical moxifloxacin 0.5% plus CXL. The primary outcome of the trial was microbiological cure at 24 hours on repeat culture. Secondary outcomes included best spectacle corrected visual acuity at 3 weeks and 3 months, percentage of study participants with epithelial healing at 3 weeks and 3 months, infiltrate and/or scar size at 3 weeks and 3 months, 3-day smear and culture, and adverse events. RESULTS Those randomized to CXL had 0.60 decreased odds of culture positivity at 24 hours (95% confidence interval [CI]: 0.10-3.50; P = 0.65), 0.9 logarithm of the minimum angle of resolution lines worse visual acuity (95% CI: -2.8 to 4.6; P = 0.63), and 0.41-mm larger scar size (95% CI: -0.48 to 1.30; P = 0.38) at 3 months. We note fewer corneal perforations or need for therapeutic penetrating keratoplasty in the CXL group. CONCLUSIONS We were unable to confirm a benefit to adjuvant CXL in the primary treatment of moderate bacterial keratitis. However, CXL may reduce culture positivity and complication rates; therefore, a larger trial to fully evaluate this is warranted. TRIAL REGISTRATION NCT02570321.
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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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9.
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.