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1.
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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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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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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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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5.
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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6.
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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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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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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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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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.