Cross-linked Products Research Articles

Quantification of a Novel DNA-Protein Cross-Link Product Formed by Reacting Apurinic/Apyrimidinic Sites in DNA with Cysteine Residues in Protein by Liquid Chromatography-Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method.

Emerging evidence suggests that cross-links formed by reacting DNA lesions with proteins may play a significant role in the pathophysiology of human cancer and degenerative diseases. The goal of this study was to develop a method involving liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with the stable isotope-dilution method to quantify DNA-protein cross-link (DPC). A novel type of cross-link involving a S-glycosidic linkage formed by reacting an abasic site in DNA with the cysteine residues in protein was targeted in this study. The method entails hydrolysis of the cross-link to a 2'-deoxyribose-cysteine adduct, addition of isotopically labeled internal standard, and quantitation by LC-MS/MS analysis. The accuracy and precision of the method were evaluated with a synthetic peptide containing the cross-link. The validated method was then applied to quantitate the levels of the DNA-protein cross-link in vitro and in HeLa cells exposed to alkylating agent methylmethanesulfonate (MMS). The analysis detected dosage-dependent formation of the cross-link in both purified DNA (6.0 ± 0.6 DPC per 106 nt μM-1 MMS) and in human cells (7.8 ± 1.2 DPC per 106 nt mM-1 MMS). With the abasic site being one of the most common DNA lesions produced continuously by multiple pathways, the results provide significant new knowledge for better understanding the potential biological implications of its associated DNA-protein cross-link.

Modification of Novel Chitosan-Starch Cross-Linked Derivatives Polymers: Synthesis and Characterization

This paper reports on the synthesis and characterization of cross-linked chitosan products prepared from using crab, shrimp beads and ten different cross-linked polymers which includes; glutaraldehyde, formaldehyde, epichlorohydrine, maleic anhydride, p-benzoquinone, poly (ethylene) glycol diglycidyl ether (PEG diglycidyl ether), 1-vinyl-2-pyrrolidone, 1,3-dichloroaceone, acrylic acid and s-methyl-benzylamine. Characterization of the cross-liked chitosan products was done using Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM) to determine the structural morphology and the stretching frequencies of the products. The obtained FTIR stretching frequencies of the cross-linked chitosan products were matched against the starting material and the literature values to confirm the products. SEM analysis showed mixtures of regular and amorphous products. Different percentage yields (crab, 26–98% and shrimp, 30–88%) were obtained for the cross-linked chitosan products depending on the cross-linked polymer used.

Cytoskeleton, Transglutaminase and Gametophytic Self-Incompatibility in the Malinae (Rosaceae).

Self-incompatibility (SI) is a complex process, one out of several mechanisms that prevent plants from self-fertilizing to maintain and increase the genetic variability. This process leads to the rejection of the male gametophyte and requires the co-participation of numerous molecules. Plants have evolved two distinct SI systems, the sporophytic (SSI) and the gametophytic (GSI) systems. The two SI systems are markedly characterized by different genes and proteins and each single system can also be divided into distinct subgroups; whatever the mechanism, the purpose is the same, i.e., to prevent self-fertilization. In Malinae, a subtribe in the Rosaceae family, i.e., Pyrus communis and Malus domestica, the GSI requires the production of female determinants, known as S-RNases, which penetrate the pollen tube to interact with the male determinants. Beyond this, the penetration of S-RNase into the pollen tube triggers a series of responses involving membrane proteins, such as phospholipases, intracellular variations of cytoplasmic Ca2+, production of reactive oxygen species (ROS) and altered enzymatic activities, such as that of transglutaminase (TGase). TGases are widespread enzymes that catalyze the post-translational conjugation of polyamines (PAs) to different protein targets and/or the cross-linking of substrate proteins leading to the formation of cross-linked products with high molecular mass. When actin and tubulin are the substrates, this destabilizes the cytoskeleton and inhibits the pollen-tube’s growth process. In this review, we will summarize the current knowledge of the relationship between S-RNase penetration, TGase activity and cytoskeleton function during GSI in the Malinae.

Computational Study of the pH-Dependent Competition between Carbonate and Thymine Addition to the Guanine Radical.

When oligonucleotides are oxidized by carbonate radical, thymine and carbonate can add to guanine radical, yielding either a guanine-thymine cross-link product (G∧T) or 8-oxo-7,8-dehydroguanine (8oxoG) and its further oxidation products such as spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). The ratio of thymine addition to carbonate addition depends strongly on the pH. Details of the mechanism have been explored by density functional calculations using the ωB97XD/6-31+G(d,p) level of theory with the SMD implicit solvation method, augmented with a few explicit waters. Free energies of intermediates and transition states in aqueous solution have been calculated along the pathways for addition of thymine, CO32-/HCO3- and carbonate radical to guanine radical. The pH dependence was examined by using appropriate explicit proton donors/acceptors as computational models for buffers at pH 2.5, 7, and 10. Deprotonation of thymine is required for nucleophilic addition at C8 of guanine radical, and thus is favored at higher pH. The barrier for carbonate radical addition is lower than for bicarbonate or carbonate dianion addition; however, for low concentrations of carbonate radical, the reaction may proceed by addition of bicarbonate/carbonate dianion to guanine radical. Thymine and bicarbonate/carbonate dianion addition are followed by oxidation by O2, loss of a proton from C8 and decarboxylation of the carbonate adduct. At pH 2.5, guanine radical cation can be formed by oxidization with sulfate radical. Water addition to guanine radical cation is the preferred path for forming 8oxoG at pH 2.5.

Chitosan-graft-poly(N-hydroxy ethyl acrylamide) copolymers: Synthesis, characterization and preliminary blood compatibility in vitro

Poly(N-hydroxy ethyl acrylamide) was grafted onto chitosan in aqueous acidic medium using potassium per sulphate initiator to obtain polymer surfaces with blood compatibility for potential biomedical applications. Thermally cross-linked products were obtained upon drying at 60 °C overnight. Chemically cross-linked films were prepared using methylene bis acrylamide (MBA) cross-linker. The products were characterized by FTIR, XRD and SEM analyses. Protein adsorption onto the film surfaces gave, on the average, 30% bovine serum albumin (BSA) and 10% human serum albumin (HSA) removal from aqueous solution, in vitro. Blood compatibility was evaluated with respect to activated prothrombin time (PT), activated partial thromboplastin time (APTT), and platelet adhesion. PT and APTT values remained within normal ranges after blood-polymer contact, with chitosan-graft-polyHEAA films, in vitro. Chitosan-graft-MBA or chitosan-graft-(polyHEAA;MBA), and blank chitosan films produced higher PT and APTT values under similar experimental conditions with chitosan-graft-polyHEAA films, exhibiting blood anticoagulant activity. SEM pictures taken before and after contact with blood sample did not reveal any significant blood component adhesion on the chitosan-graft-(polyHEAA;MBA) film surface contrary to the observation made on the blank chitosan film.

Processing of Biopolymers-based, Efficiently Integrated Bilayer Membranes for Use in Guided Tissue Regeneration Procedures

Periodontitis is a chronic inflammatory disease affecting the tooth supporting structures - the alveolar bone in particular, to extent of eventual tooth loss. Among others, the guided tissue regeneration (GTR) procedure including placement of occlusive barrier membrane is very promising approach. Herein we develop chitosan (CHT) / gelatin (GEL) bilayer membranes via successive solvent– and freeze-casting procedures and genipin (GEN)-mediated cross-linking chemistry. By utilizing the auto-fluorescence signal from GEN cross-linking products (i.e. the secondary CHT (GEL) amines and GEN esters), the Confocal Fluorescent Microscopy (CFM) identifies the chemical as well as physical integration between layers´ interfaces, supported also by Scanning Electron Microscopy (SEM) data. The presence of non- and highly μ-porous and pore-interconnecting regions is demonstrated within cross-sections of GEL-prevalent membranes in contrast to the sheet-like organization in membrane with equal components presence. The constant processing conditions onto variable compositions did not significantly affect the pore size distributions (in 1-230 μm range), while pore wall thickness increases up to 220 μm with GEL increase, which also improves the yield stress at compression from 10 to 19 kPa and elastic modulus from 26 to 34 kPa. The further applied rapid mineralization resulted in deposition of non-regular to spherical minerals, containing nonstoichiometric carbonated apatite with Ca/P ration in 1.7-2 range, which demonstrates formation of osseointegrative interface. The intensive, composition-dependent swelling (up to 580%), as well as 67% to 100% weigh loss in 4 weeks in vitro degradation experiment imply on bilayer membranes GTR relevance.

A cross-linking/mass spectrometry workflow based on MS-cleavable cross-linkers and the MeroX software for studying protein structures and protein–protein interactions

Chemical cross-linking in combination with mass spectrometric analysis of the created cross-linked products is an emerging technology aimed at deriving valuable structural information from proteins and protein complexes. The goal of our protocol is to obtain distance constraints for structure determination of proteins and to investigate protein-protein interactions. We present an integrated workflow for cross-linking/mass spectrometry (MS) based on protein cross-linking with MS-cleavable reagents, followed by enzymatic digestion, enrichment of cross-linked peptides by strong cation-exchange chromatography (SCX), and LC/MS/MS analysis. To exploit the full potential of MS-cleavable cross-linkers, we developed an updated version of the freely available MeroX software for automated data analysis. The commercially available, MS-cleavable cross-linkers (DSBU and CDI) used herein possess different lengths and react with amine as well as hydroxy groups. Owing to the formation of two characteristic 26-u doublets in their MS/MS spectra, many fewer false positives are found than when using classic, non-cleavable cross-linkers. The protocol, exemplified herein for BSA and the whole Escherichia coli ribosome, is robust and widely applicable, and it allows facile identification of cross-links for deriving spatial constraints from purified proteins and protein complexes. The cross-linking/MS procedure takes 2-3 days to complete.

Plasma d-dimer level correlated with advanced breast carcinoma in female patients.

BackgroundAdvanced breast cancer is a common disease among female in the world. There is a correlation between cancer and hyper coagulation. In a cancer state, there is an increase in the level of cross-linked fibrin degradation product (d-dimer)which indicates systemic activation of fibrinolysis and hemostasis. So, there is a relation between increase d-dimer value and advanced breast disease.ObjectiveTo confirm the relation between increase d-dimer levels and advanced breast carcinoma in female patients.Patients and methodsA prospective study (cohort study) done in Baghdad teaching hospital (department of surgery) from Jan 2014 to Jan 2016. Seventy patients were categorized intotwo equal groups, group 1 with breast carcinoma, group 2 with benign breast tumor. Plasma d-dimer levels compared for each group, and in relation to (tumor size, stage, grade, lympho-vascular invasion, and lymph nodes involvement).ResultsD-dimer level was normal in group two (<0.25) mg/l and high in group one in other words, d-dimer level was increasing in advanced breast carcinoma group with enlarge tumor size, high stage, grade, lympho-vascular invasion and lymph nodes involvement.ConclusionsPlasma d-dimer levels was good prognostic factor in breast carcinoma specially in advanced breast carcinoma and its considered factor clinical stage progression lympho-vascular invasion and metastasis.

PHBV-graft-GMA via reactive extrusion and its use in PHBV/nanocellulose crystal composites

Reactive extrusion was used for dicumyl peroxide (DCP)-initiated grafting of glycidyl methacrylate (GMA) to poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). The effects of GMA and DCP content and their weight ratio on the GMA grafting percentage (GP%), the polymer melt viscosity, and the PHBV molecular weight were investigated. FTIR spectroscopy determined that the DCP did indeed initiate GMA grafting. However, the changes in both the zero-shear viscosity (η0) and the molecular weight suggested the existence of crosslinking products in the extruded polymers. A negative correlation between the degree of crystallinity (χc) of the PHBV-g-GMA and the GP% suggested the influence of chain branching on crystallinity. In addition, the GMA content was found as a key factor determining the GP%. The PHBV-g-GMA was used as a matrix polymer in cellulose nanocomposites to evaluate its effects on CNC dispersion and CNC-matrix adhesion relative to the unmodified PHBV matrix. The SEM images and the change in crystallization temperature suggested enhanced dispersion of CNC in a PHBV-g-GMA matrix. However, little increase in strength properties were found with CNC addition suggesting inadequate stress transfer between the matrix and CNCs.

One-Pot Synthesis of Sustainable High-Performance Thermoset by Exploiting Eugenol Functionalized 1,3-Dioxolan-4-one

1,3-Dioxolan-4-one (DOX) chemistry was explored for production of “one-pot” biobased polyester thermosets. DOX monomer was first functionalized by naturally occurring eugenol to introduce a structural element, which could induce cross-linking reaction through cationic polymerization of the double bond. The feasibility of polymerizing DOX monomers bearing bulky side groups was proven by model phenol-substituted DOX monomer (PhDOX). Once the reaction was shown to be effective, the same protocol was applied to eugenol-substituted monomer (EuDOX). A brief screening of the optimal catalyst concentration was performed, to obtain a highly cross-linked product. The synthesized thermoset showed good thermal resistance and high mechanical strength probably due to the rich aromatic content. The obtained thermoset was further subjected to microwave-assisted hydrothermal degradation test, which demonstrated complete recyclability to water or methanol soluble products. NMR and matrix-assisted laser desorption/ionizatio...

Configuration of Ten Light-Harvesting Chlorophyll a/b Complex I Subunits in Chlamydomonas reinhardtii Photosystem I

In plants, the photosystem I (PSI) core complex stably associates with its light-harvesting chlorophyll a/b complex I (LHCI) to form the PSI-LHCI supercomplex. The vascular plant PSI core complex associates with four distinct LHCI subunits, whereas that of the green alga Chlamydomonas reinhardtii binds nine distinct LHCI subunits (LHCA1-LHCA9). The stoichiometry and configuration of these LHCI subunits in the PSI-LHCI supercomplex of C. reinhardtii remain controversial. Here, we determined the stoichiometry of the nine distinct LHCI subunits relative to PSI subunits through uniform labeling of total proteins using 14C. We separated the nine LHCI polypeptides by three different sodium dodecyl sulfate-polyacrylamide gel electrophoresis systems. Our data revealed that the PSI-LHCI supercomplex contains two LHCA1 proteins and one of each of the other eight LHCI subunits. Subsequently, we identified their cross-linked products by immunodetection and mass spectrometry to determine the configuration of the 10 LHCI subunits within the PSI-LHCI supercomplex. Furthermore, analyses of PSI-LHCI complexes isolated from ΔLHCA2 and ΔLHCA5 mutants and oligomeric LHCI from a PSI-deficient (ΔpsaA/B) mutant provided supporting evidence for the LHCI subunit configuration. In conclusion, eight LHCI subunits bind to the PSI core at the site of PSAF subunit in two layers: LHCA1-LHCA8-LHCA7-LHCA3 from PSAG to PSAK, in the inner layer, and LHCA1-LHCA4-LHCA6-LHCA5 in the outer layer. The other two LHCI subunits, LHCA2 and LHCA9, bind PSAB between PSAG and PSAH, PSAG-LHCA9-LHCA2-PSAH. Our study provides new insights into the LHCI configuration linked to the PSI core.

Reverse Binding Mode of Phosphotyrosine Peptides with SH2 Protein.

Discerning the different interaction states during dynamic protein-ligand binding is difficult. Here we apply site-specific cysteine-α-chloroacetyl cross-linking to scrutinize the binding between the Src homology 2 (SH2) domain and phosphotyrosine (pY) peptides, a highly dynamic interaction that is a key to cellular signal transduction. From a model SH2 protein to a set of representative SH2 domains, we showed here that a proximity-induced cysteine-α-chloroacetyl reaction cross-linked two spatially adjacent chemical groups as a result of the binding interaction, and reciprocally, the information about the interaction states can be deduced from the cross-linked products. To our surprise, we found SH2 domains can adopt a reverse binding mode with "single-pronged", "two-pronged", and "half" pY peptides. This finding was further supported by a set of 500 ns molecular dynamics simulations. This serendipitous finding defies the canonical theory of SH2 binding, suggests a possible answer about the source of the versatility of SH2 signaling, and sets a model for other protein binding interactions.

Cross-Linked Polyethylene for Total Hip Arthroplasty: A Model for Evaluation of a Medical Device: Commentary on an article by R. de Steiger, MBBS, FRACS, FAOrthA, et al.: "Cross-Linked Polyethylene for Total Hip Arthroplasty Markedly Reduces Revision Surgery at 16 Years".

Commentary The article by Professor de Steiger and associates describes an important study providing clinical confirmation of the promise of improved survivorship with highly cross-linked polyethylene (XLPE) liners in total hip arthroplasty (THA). The authors report the results of their survivorship analysis comparing 41,171 THAs performed with ultra-high molecular weight conventional polyethylene (CPE) and 199,131 performed with gamma, or electron beam, irradiated XLPE between 1999 and 2016. The 16-year cumulative percentage of revisions was 11.7% (95% confidence interval [CI] = 11.1% to 12.3%) for CPE compared with 6.2% (95% CI = 5.7% to 6.7%) for XLPE. The hazard ratio at 9 years was 3.02. Reduced in vitro wear of XLPE liners had previously been reported with preclinical testing1, but laboratory testing of earlier polyethylene enhancements, such as Hylamer, did not accurately predict clinical performance2. Early clinical experience with XLPE was encouraging as the devices were not identified as outliers in large national registries such as the Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR). Reduced wear of XLPE liners in vivo was reported in radiostereometric analysis (RSA) studies3, resulting in further optimism for this bearing surface. Improved survivorship with XLPE liners was recently reported in a randomized controlled trial (RCT) with a 10-year follow-up that demonstrated a revision rate of 1.9% with Marathon liners compared with 14.6% with Enduron liners4. This was a single-center study of 122 THAs performed with cemented stems and cementless Duraloc cups by 2 experienced surgeons in 1 medical center. The Kaiser Permanente Total Joint Replacement Registry previously reported improved survivorship of XLPE liners compared with CPE liners5. The cumulative incidence of revision was 5.4% of 1,815 THAs with a CPE liner compared with 2.8% of 25,008 with an XLPE liner at 7 years; the hazard ratio for aseptic failure was 1.9. The study by de Steiger et al. utilized cases from the AOANJRR and expanded on this earlier report with longer follow-up, larger sample sizes, and a greater variety of hip implants. The AOANJRR was initiated in 1999, about the time that several highly cross-linked irradiated polyethylene products became commercially available. This timely coincidence allowed for a longitudinal clinical study of a large number of THA cases representing the gradual transition from CPE to XLPE. The registry quickly collected a large volume of cases performed by many surgeons, in many different hospitals, with a variety of implants. However, unlike prospective trials, in which interventions are performed concurrently, there are temporal differences between cohorts in registry analyses. At the beginning of the study period examined by de Steiger et al., most THAs were done with a CPE liner, whereas at the end of the study period most were performed with XLPE. During the 17-year study period, there may have been differences in patient selection, perioperative care protocols, and possibly surgeon experience. Although the authors speculated that any patient selection likely favored the use of XLPE in younger individuals, the opposite may be true because the study samples represented only 74% of the THAs performed for osteoarthritis. Until the AOANJRR formally recalled the ASR (Articular Surface Replacement) in 2009, surgeons may have chosen metal-on-metal or ceramic-on-ceramic bearings—not necessarily XLPE liners—for younger and more active patients. Despite this limitation, the subanalysis performed by de Steiger et al. showed a reduced risk of revision with XLPE cups even in patients under the age of 55 years. The strengths of this observational registry study include data that are generalizable to the “real world,” unlike the findings from an RCT, which may involve limited numbers of patients, few implant types, and limited numbers of surgeons in a single center. In the community, THA procedures are performed by a variety of surgeons with varying skills and specializations. Additionally, in the community, multiple implant combinations are utilized and procedures are performed in a variety of hospital settings. The story of XLPE liners is a good example of how new implants should be introduced and monitored: with careful preclinical testing, rigorous early clinical studies, widespread surveillance, and carefully performed mid-term clinical investigations. The current study is an important, and reassuring, contribution to the orthopaedic literature as XLPE has been widely used throughout the world in millions of patients.

Autofluorescence-aided assessment of integration and μ-structuring in chitosan/gelatin bilayer membranes with rapidly mineralized interface in relevance to guided tissue regeneration

Beyond providing barrier function, the advanced materials in guided tissue regeneration (GTR) concept are further prompt to foster regeneration of distinct interfacing tissues. Herein we develop chitosan (CHT)/gelatin (GEL) bilayer membranes via successive solvent– and freeze–casting procedures and genipin (GEN) cross-linking chemistry. By utilizing the autofluorescence signal from GEN cross-linking products (i.e. the secondary CHT (GEL) amines and GEN esters), the Confocal Fluorescent Microscopy (CFM) identifies the chemical inter-linking as well as physical integration between interface layers. The presence of non- and highly μ-porous and pore-interconnecting regions is demonstrated within cross-sections of membranes with (by weight) prevalent GEL contribution in contrast to the sheet-like organization in membrane with equal presence of components. The constant processing conditions on variable compositions did not significantly affect the pore size distributions (in 1–230 μm range), while pore wall thickness increase up to 220 μm with GEL increase, which also improves the yield stress at compression (from 10 kPa to 19 kPa) and elastic modulus (from 26 kPa to 34 kPa). The rapid mineralization procedure resulted in deposition of non-regular to spherical minerals, containing nonstoichiometric carbonated apatite with Ca/P ration in 1.7–2 range, which demonstrates formation of osseointegrative interface. The fast and high (up to 580%), composition-dependent swelling, as well as 67% to 100% weight loss in 4 weeks in vitro degradation experiment point on membranes' relevance in GTR.

Direct identification of Cu(II) species adsorbed on rosin-derived resins using electron paramagnetic resonance (EPR) spectroscopy

Resins derived from natural rosin have been described recently as environmentally-friendly adsorbents with potential uses in the clean-up of wastewaters and the treatment of drinking waters. However, in order to improve the design of these products it is important to understand the chemical mechanisms through which they act. We now describe the combined use of EPR spectroscopy and Cu(II) to probe directly the metal coordination environment after uptake by various rosin-derived products. Clear distinctions are demonstrated between the copper bonding in different adsorbent preparations. Furthermore, in the case of nitrogen cross-linked products, the spectral parameters also provide information on the number of nitrogen atoms in the immediate atomic environment of the copper atom when covalent bonding is involved. This methodology works well for adsorption from solutions containing multiple components, because only those that are paramagnetic are detected, and different types of paramagnetic metal ion produce distinctly different EPR spectra.

Settling volume and morphology changes in cross-linked and unmodified starches from wheat, waxy wheat, and waxy maize in relation to their pasting properties

Normal wheat, waxy wheat and waxy maize starches were cross-linked with 0.01, 0.03 and 0.06% (sb) phosphorus oxychloride. The objective of this study was to correlate the morphology changes and settling volume to the pasting properties of those cross-linked and unmodified starches. Pasting and microscopic data for waxy maize starch and its cross-linked products was similar to waxy wheat starch, except changes occurred at ∼5 °C higher in temperature. At 6% solids, waxy wheat starch cross-linked with 0.01% POCl3 had a greater settling volume and a higher pasting viscosity than the cross-linked waxy maize starch, but at 7 and 8% solids, waxy maize starch cross-linked with 0.03% and 0.06% POCl3 had a higher pasting viscosity. At 6% starch solids, particle volume fraction appeared to be the dominant factor controlling consistency, but at higher starch solids contents, the deformability (rigidity) of swollen granules became important in controlling viscosity.

Contribution of Factor VIII A2 Domain Residues 400-409 to a Factor X-Interactive Site in the Factor Xase Complex.

The link between factor (F)VIII and FX is essential for optimum activity of the tenase complex. The interactive site(s) in FVIII for FX remains to be completely clarified, however. We investigated the FVIII A2 domain-FX association that was speculated from inhibitory mechanism(s) by an anti-A2 autoantibody. SDS-PAGE demonstrated that the purified inhibitor IgG recognizing residues 373-562 blocked FXa cleavage at Arg372 in FVIII, and surface-plasmon resonance (SPR)-based assays showed that intact A2 subunit directly bound to FX (Kd; 63 nM). The FVIII structure model indicated possible FX-binding site(s) in residues 400-429 in A2. One peptide corresponding to residues 400-409 competitively inhibited both the A2-FX binding and FVIIIa/FIXa-dependent FXa generation. Covalent cross-linking was observed between this peptide and FX following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) using SDS-PAGE. K408 and S409 were not evident in N-terminal sequence analysis of the cross-linked product, suggesting that two residues participated in cross-link formation. SPR-based assays using recombinant FVIII mutants with one or both residues substituted to alanine demonstrated that K408A and K408A/S409A had approximately fourfold high Kd values of wild-type (WT-)FVIII. FXa cleavages at Arg372 in both mutants were significantly delayed, suggesting a contribution of K408 for FXa cleavage at Arg372. Furthermore, FXa generation assays with these mutants demonstrated that the Km values were 1.4- to 1.7-fold greater, and overall catalytic efficiency (kcat/Km) was 0.49- to 0.89-fold lower than with WT-FVIII, suggesting a significant contribution of K408 for FVIII-FX interaction in tenase assembly. We concluded that the K408 in the A2 domain provided an interactive-site for FX.

α- and β-casein aggregation induced by riboflavin-sensitized photo-oxidation occurs via di-tyrosine cross-links and is oxygen concentration dependent

Type I photo-oxidation generates Trp-(TrpN) and Tyr-derived (TyrO) radicals in proteins which can dimerize producing cross-links, or alternatively react with O2. It was therefore hypothesized that the O2 concentration may have a significant effect on dye-photosensitized reactions. We studied photo-oxidation of α- and β-caseins induced by riboflavin (RF), a photosensitizing vitamin present in milk, under aerobic and anaerobic conditions. Triplet-state RF induced oxidative modifications on both caseins, and significant levels of cross-links. The extent of damage, and the yield of cross-links versus oxidized products, was dependent on the O2 concentration. In the absence of O2, the overall extent of damage was decreased, but the yield of cross-linked products was significantly elevated. These cross-links are consistent with inter- and intra-molecular di-Tyr or di-Trp bridges. Alternative cross-links were detected in the presence of O2, consistent with pathways involving the reaction of protein radicals with O2 or O2−.

Carboxyl-Photo-Reactive MS-Cleavable Cross-Linkers: Unveiling a Hidden Aspect of Diazirine-Based Reagents.

A major challenge in cross-linking/mass spectrometry (MS) is targeting carboxyl functions in proteins under physiological conditions that do not disturb the protein's conformation. Cross-linking of glutamic acid and aspartic acid residues in proteins will greatly expand the scope of structural mass spectrometry. We discovered that carboxyl-reactive cross-linkers have already been employed for many years in cross-linking/MS studies, yet in a completely different context. Diazirine-based cross-linkers, such as photomethionine and succinimidyldiazirine cross-linkers, are currently considered to react nonspecifically upon UV-A photoactivation with all 20 proteinogenic amino acids through a reactive carbene that inserts mainly into C-H bonds. We discovered that the cross-linking capability of diazirines based on X-H (X = C, N, O) insertion is in fact only the tip of the iceberg. Diazirines isomerize to linear diazo compounds that can react with carboxylic acids to yield esters. On top of that, the resulting cross-linked products are MS-cleavable allowing an automated analysis of cross-links via customized software tools. Therefore, diazirines open an entirely new route for photo-cross-linking of carboxylic acids. Previous cross-linking studies using diazirines have to be revisited in the light of these findings.

Interface conjugation enhances the interface adhesion performance of carbon fiber-reinforced phthalonitrile composites by π-π stacking.

There has been little research focus on the interface problems of phthalonitrile (PN) resin and carbon fiber. However, interface performance is related to the overall mechanical properties of composites and is very important. This study focused on the interfacial performance and adhesion mechanism of a carbon fiber Cf/PN composite. Micro-composites of Cf/PN and Cf/epoxy resins were prepared, and their interfacial shear strengths (IFSS) were tested by micro-droplet testing. The result showed that the IFSS of Cf/PN was higher than that of Cf/epoxy resin, indicating that the interfacial adhesion of the PN matrix composite must be more effective. To explain the obtained results, a number of tests, including SEM, SEM-EDS, FTIR, and TGA, were carried out. From the SEM analyses, cured PN polymer films were found on the surface of de-bonded carbon fibers. With the aid of SEM-EDS, the elements on the de-bonded carbon fiber surface of the Cf/PN composite were detected in situ. An interesting synchronous relationship was observed in the IFSS and SEM-EDS results. Through the FTIR spectra, the chemical structures of the PN polymers were identified. From the detailed analyses and discussion in this work, the effective interfacial bond function in the Cf/PN composite appears to be a complex result for all relative functions. The functional advantage of the PN composite may be the interface conjugation between the PN polymers and the graphene layer on the surface of the carbon fiber.